Research ArticleMolecular Characterization and PhylogeneticAnalysis of Listeria monocytogenes Isolated fromMilk and Milk Products in Kaduna Nigeria

U B Usman1 J K P Kwaga2 J Kabir2 O S Olonitola3 S Radu4 and F Bande15

1Department of Veterinary Services Ministry of Animal Health and Fisheries Development Usman Faruk Secretariat SokotoSokoto State 840221 Nigeria2Department of Veterinary Public Health and Preventive Medicine Faculty of Veterinary Medicine Ahmadu Bello UniversityZaria 810004 Nigeria3Department of Microbiology Faculty of Science Ahmadu Bello University Zaria 810004 Nigeria4Center of Excellence for Food Safety Research Faculty of Food Science and Technology Universiti Putra Malaysia 43400 SerdangSelangor Darul Ehsan Malaysia5Department of Pathology and Microbiology Faculty of Veterinary Medicine Universiti Putra Malaysia 43400 SerdangSelangor Darul Ehsan Malaysia

Correspondence should be addressed to U B Usman usmanbashirymailcom

Received 28 February 2016 Revised 28 June 2016 Accepted 11 July 2016

Academic Editor Giordano Dicuonzo

Copyright copy 2016 U B Usman et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In this study Listeria (L) monocytogenes isolated from milk and milk products in Kaduna Nigeria were subjected to a multiplexPCR assay to identify virulence-associated genes (such as prf A inlA hlyA actA and iap) Of the 36 isolates 9 (25) were positivefor one or two virulence-associated genes Based on the sample type 6 (169) of the isolates that possessed virulence-associatedgenes were obtained from rawmilk 2 (32) from ldquoManshanurdquo and 1 (28) from ldquoKindrimordquo Sequence and phylogenetic analysisbased on the 16S rRNA revealed that Nigerian L monocytogenes isolates (NGA 34A NGA 35A NGA 41A and NGA 38A) whencompared with reference L monocytogenes were grouped into two distinct clusters A and B with sequence (NGA 34A NGA35A and NGA 41A) phylogenetically closer to J1776 N1-011A R2-502 J1816 and J2-031 whereas L monocytogenes isolate (NGA38A) clustered with EDG J1-220 J1926 J1817 and J2-1091 The separation of the Nigerian L monocytogenes isolates into linage A(responsible for epidemic listeriosis) and lineage B (responsible for sporadic cases of listeriosis) is of public health concern and thatlocal isolates might have potentials for human food borne listeriosis based on the virulence factors so far identified

1 Introduction

Listeriosis is a food borne infection caused by opportunisticbacterial pathogen Listeria (L) monocytogenes that is abun-dant in the environment [1] The bacteria have been isolatedfrom the soil water plants faeces decaying vegetables meatsea food dairy products and asymptomatic human andanimal carriers [2] It has been isolated from sheep goatand cow milk [3] L monocytogenes is a Gram-positive fac-ultative intracellular food borne pathogen causing listeriosisa rare but severe infection in humans and animals with a

mortality rate of 25ndash30 [4] Listeriosis is either noninvasiveself-limiting and gastrointestinal occurring in healthy indi-viduals or invasive and systemic occurring in immuno-compromised individuals such as pregnant women andnewborns resulting in meningitis encephalitis septicaemiamother-to-foetus infection and abortion [5] There are sev-eral virulence genes so far identified in L monocytogenesThese include the internalins (encoded by inl A inl C andinl J) listeriolysin O (LLO encoded by hly A) actin (actA) phosphatidylinositol-phospholipase C (PI-PLC encodedby plc A) iap (invasion associated protein encoded by iap)

Hindawi Publishing CorporationCanadian Journal of Infectious Diseases and Medical MicrobiologyVolume 2016 Article ID 4313827 7 pageshttpdxdoiorg10115520164313827

2 Canadian Journal of Infectious Diseases and Medical Microbiology

and virulence regulator (encoded by prf A) These virulencefactors play significant role in the bacterial pathogenicity andinfection outcome [6 7]

It has been observed that Listeria monocytogenes employthese virulence factors that act synergistically in the intra-cellular pathogenicity of the bacteria [8] For exampleexpressions of virulence genes such as listeriolysin O (LLO)phospholipases (plc A and plc B) and internalins A and B(inl A and inl B) are regulated by prf A and these genesfacilitate the intracellular growth and spread of the bacteriumwithin themammalian host [9] Pointmutation is responsiblefor low virulence of Listeria monocytogenes strains in anumber of virulence genes and as a result of spontaneousmutations some L monocytogenes strains may lose one ormore virulence determinants [10 11] A virulence factor ofphospholipase called PI-PLC is expressed by pathogenicspecies of L monocytogenes and Listeria ivanovii only andthis virulence factor is an important marker for the dis-crimination between pathogenic and nonpathogenic Listeriaspecies [12] There is growing evidence that virulence is nota stable property but can be influenced by environmentalconditions For example it has been shown that acid and saltstress increase the expression of virulence genes and in vitropathogenicity of the bacteria [13] Virulence potentials arealso influenced by temperature the presence or absence ofoxygen osmotic stress and pH [14] L monocytogenes has13 serotypes of these only four serotypes (12a 12b 12cand 4b) are known to cause human listeriosis [15] Basedon the data generated from molecular subtyping methodsL monocytogenes isolates have been grouped into two majorgenetic divisions or lineages termed as lineage I and lineage II[15] A third lineage predominantly associated with animalshas been described by some studies as well [16] Serotypes12a 3b 3c and 4b belong to lineage I while serotypes 12a12c and 3a belong to lineage II [17] Studies have shown thatlineage I strains are significantly higher among human clini-cal listeriosis cases and contaminated foods [1] while lineageII strains show a significantly higher prevalence among foodisolates and animal clinical cases than among human clinicallisteriosis [16] Pathogenic potentials of lineage I isolates aregreater than those of lineage II as determined by their abilityto spread to neighbouring host cells in a cell culture plaqueassay [1] It will be observed that there is a consensus thatlineage I strainsmay represent a human host-adaptive lineagewhile lineage II strains may represent an environmentaladapted lineage [15]The application ofmultiplex PCR for thedetection of more than one virulence gene in a single tubeis desirable because it reduces cost and labour and will beuseful in a large scale detection of virulent strains of Listeria[18]

The prevalence and molecular characteristics of Listeriamonocytogenes are generally unknown in Nigeria and thusthis present study was carried out with the aim of charac-terizing L monocytogenes isolates from raw milk and milkproducts using PCR and to determine the virulence and thephylogenetic characteristics of the isolates

2 Materials and Methods

21 Sample Collection

211 Bacterial Strains and Biochemical The isolation of Liste-riamonocytogeneswas carried out according to the procedureof Roberts et al [19] Briefly about 10mL of the incubatedhomogenate was added to 90mL Listeria enrichment broth(Oxoid CM 0862) which contains selective Listeria enrich-ment supplements (Oxoid SR 0141E) and incubated at 30∘Cfor 48 hrs A loop full of the 48 hrs broth was cultured ontoChromogenic Listeria agar (Oxoid CM 1080) plates whichcontains brilliance Listeria differential supplement (OxoidSR 0228E) and brilliance Listeria selective supplement(Oxoid SR 00227) and the plates was incubated at 37∘Cfor 24ndash48 hrs Colonies of Listeria monocytogenes havinga greenish-blue coloration were picked and streaked onnutrient agar (Oxoid CM 0003) slants and incubated at 37∘Cfor 24 hrs before storage at 4-5∘CThe suspected isolates werefurther subjected to conventional biochemical test [Gramstaining oxidase catalase 120573-hemolysis bile esculin and car-bohydrate fermentation test (xylose Mannitol RhamnoseMaltose Inositol and Sucrose)] as described previously [20]Isolates found to ferment mannitol and xylose and positiveto rhamnose were considered as L monocytogenes Furthercharacterization usingMicrobact 12L Listeria identificationkit (Oxoid MB 1128) was carried out

Following cultural identification and biochemical assaysstated above 36 Listeria monocytogenes isolates were identi-fied (Table 1)

22 Polymerase Chain Reaction and Gel Electrophoresis Bac-terial isolates obtained locally as well as reference strainswere subjected to PCR assay The reference strain designatedATCC 19155 used as positive control was kindly provided byProfessor Radu of the Center of Excellence for Food SafetyResearch Faculty of Food Science and Technology UniversitiPutra Malaysia where the molecular studies were carriedout Bacterial genomic DNA was extracted from both localand reference isolates using the Quick-g DNAminiprep kit(Zymo Research SA) following the manufacturerrsquos instruc-tions

Multiplex PCR assay was used to detect L monocytogenesharbouring hly A gene and 16S rRNA of L monocytogenes[21] The primer pairs designated as LM1 and LM2 (LM1 51015840ndashCCTAAGACGCCAATCGAA-31015840 and LM2 ndash 51015840-AAGCGCTTG CAA CTG CTC-31015840) were used for the detection of Lmonocytogenes harbouring hly A gene On the other handprimer pairs designated as U1 and LI1 (LI1 51015840ndashCTC CATAAA GGT GAC CCT-31015840 and U1 51015840ndashCAG CMG CCG CGGTAATWC-31015840) were used to amplify 938 bp region in the 16SrRNA gene specific for the detection of Listeria genus [21]These primerswere synthesized by InvitrogenUSA andwere18ndash20 bp in length L monocytogenesATCC 19155 was used asa positive control

The PCR amplification was carried out in a 25 120583L reactionmixture that consisted of 5 120583L of 5x PCR buffer dNTPs(05 120583L) MgCl

2(2 120583L) Taq DNA polymerase (05 120583L) 05 120583L

of each 20 pM primer [LM1 LM2 U1 and LI1] 13120583L of

Canadian Journal of Infectious Diseases and Medical Microbiology 3

Table 1 Listeria monocytogenes characteristics

Snumber Isolate name (code) Isolated from Tip name in tree(1) LMFR151 Raw milk 49A(2) ZKMM83 Manshanu 48A(3) MDGR115 Raw milk 47A(4) MMM103 Manshanu 46A(5) ZKMM84 Manshanu 34A(6) LSNK136 Kindrimo 44A(7) SGMK77 Kindrimo 43A(8) MGDR100 Raw milk 42A(9) ZKMR75 Raw milk 35A(10) SGHR39 Raw milk 38A(11) ZKJR55 Raw milk 40A(12) SGHR7 Raw milk 39A(13) SGHR12 Raw milk 37A(14) SGHR30 Raw milk 41A(15) ZKMM87 Manshanu 36A(16) ZKMM84 Manshanu 45A(17) ZKJR5 Raw milk 33A(18) SGMM14 Manshanu 32A(19) SGMM34 Manshanu 31A(20) SGHR45 Raw milk 30A(21) LMFR161 Raw milk 29A(22) MDGR109 Raw milk 28A(23) SGHR32 Raw milk 27A(24) MDGR10 Raw milk 25A(25) ZKJR10 Raw milk 24A(26) SGSM9 Manshanu 23A(27) LMFR162 Raw milk 15A(28) SGHR55 Raw milk 22A(29) MDGM123 Manshanu 21A(30) SGSK1 Kindrimo 26A(31) SGHR15 Raw milk 20A(32) SGHR37 Raw milk 19A(33) SGSK21 Kindrimo 18A(34) LMFK153 Kindrimo 17A(35) SGMM37 Manshanu 16A(36) SGMM11 Manshanu 14A

distilled water and 2120583L of DNA template PCR amplificationwas carried out in a programmed thermocycler with thefollowing thermal conditions hot start PCR plate at 95∘C for3mins followed by 35 cycles each of 30 sec denaturation at94∘C 15 sec annealing at 53∘C 90 sec extension for 72∘C andfinal extension at 72∘C for 7mins

Following PCR amplification about 5120583L of the PCRproduct was mixed with DNA loading dye (6x) and elec-trophoresed in 10 Agarose gel in TAE buffer using aminitank at 80V 400 amp and 45min The electrophoresedproduct on the gel was stained with ethidium bromidefor 30min destained for 20min and visualized under UVilluminator (SYNGENE Biosystems UK) A 100 bp DNA

ladder (PromegaCorporationUSA)was included to estimatethe size of the amplified products

23 Detection of L monocytogenes Virulence-AssociatedGenes The obtained DNA product was used in performinganother multiplex PCR assay with the aim of detectingthe presence of five virulence-associated genes [hemolysinA gene (hly A) regulatory gene (prf A) actin gene (actA) invasion associated protein p60 gene (iap) and surfaceprotein (inl A)] of L monocytogenes as described by Rawoolet al [18] The primers used for the detection of thesevirulence geneswere synthesized by SigmaAldrich USAThedetails of the primer sequences are shown in Table 2 TheDNA obtained using the Quick-g DNA miniprep-uncappedcolumn kit (Zymo Research) was used for the detection ofvirulence-associated genes

The PCR was standardized for the detection of virulence-associated genes of L monocytogenes by optimizing theannealing temperatures between 50 and 60∘C Based onoptimization trials the standardized PCRprotocol for a 25120583Lreactionmixture included 5120583LPCRbuffer (100mMTrisndashHClbuffer pH 83 containing 500mM KCl 15mM MgCl

2 and

001 gelatine) 1 120583L dNTP mix 3 120583L MgCl2and 05 120583L of

forward and reverse primers 1 120583L of 5 units of Taq DNApolymerase 5 120583LDNA template and 5120583L nuclease free watertomake up the reaction volumeTheDNAamplification reac-tion was performed in aMaster Cycler gradient thermocycler(Eppendorf Hamburg Germany) with a preheated lid inPCR tubes (02mL)The cycling conditions for PCR includedan initial denaturation of DNA at 95∘C for 2min followed by35 cycles each of 15 sec denaturation at 95∘C 30 sec annealingat 53∘C and 90 sec extension at 72∘C Final was carried out in10min at 72∘C and then PCR product held at 4∘C

The resultant PCR products were separated by elec-trophoresis in 15 Agarose gel for 45min at 100V inTris-acetate EDTA buffer stained with ethidium bromide(05 120583gmL) and visualized with a UV transilluminator (Syn-gene Frederick MD)The gel image was documented by a geldocumentation apparatus and DNA size was determined bya 100 bp DNA ladder (Promega)

24 Purification of PCR Products 5 120583L volume PCR productfor each of the 9 hly A and 16S rRNA harbouring L mono-cytogenes positive isolates was analysed on a 15 Agarosegel to confirm successful amplification of the 16S rRNAThe remaining 20 120583L of each of the PCR product was laterrun and each corresponding 16S rRNA band was cut andpurified by using the MEGA quick-spin Total FragmentDNA purification kit (iNtRON Biotechnology Gyeonggi-doSouth Korea)

25 DNA Sequencing and Phylogenetic Analysis Purified gelwas used for DNA sequencing All sequencing reactionswere commercially done by iNtRON (iNtRONBiotechnologyGyeonggi-do South Korea) using the Sangers sequencingmethod Both forward and reverse primers were used toensure adequate sequencing coverage For sequencing andphylogenetic tree only 15 samples were randomly selectedand tested

4 Canadian Journal of Infectious Diseases and Medical Microbiology

Table 2 Primer sequences used for the amplification of virulence-associated genes of Listeria monocytogenes

Target gene Primer sequence (51015840-31015840) Product size (bp)

inl A Fwd 51015840-AGATCTAGACCAAGTTACAACGCTTCAG-31015840Rev 51015840-TAA TAT CAT TTG CTG TTT TAT CTG TC-31015840 255 bp

act A Fwd 510158401015840-ACG TGA AGT AAG TCACGT GAT ATT G 31015840Fwd 510158401015840-ACG TGAAGTAAGCTCACGT GAT ATT G-31015840 268 bp

prf A Fwd 51015840-ACC GCT CAG AAA AGT TCT TC-31015840Rev 51015840-TCT TGT TCT ATT ATGTCT AGC-31015840 1060 bp

iap Fwd 51015840-ACA AGC TGC ACC TGT TGC AG-31015840Rev 5-TGA CAG CGT TGT TAG TAG CA-31015840 131 bp

hly A Fwd 51015840-CCT AAG ACG CCA ATC GAA-31015840Rev 51015840-AAG CGC TTG CAA GTC CTC-31015840 702 bp

The obtained partial 16S rRNA sequences were proof-read and assembled in Chromas Lite version 211 Multiplesequence alignment of the DNA sequences was performedusing ClustalW in Bioedit software version 725 (BiologicalSequence Alignment Editor for Win 9598NT2KXP7)as described by Hall 1999 [22] To compare the sequenceof local L monocytogenes with reference isolates availablein the GenBank database sequence similarity search wasperformedusing theNCBI nucleotide BLAST search to deter-mine the similarity of the local L monocytogenes sequence(httpblastncbinlmnihgov)

Phylogenetic analysis was carried out based on partialsequence of 16S rRNA gene of L monocytogenes In thisregard the aligned sequence was subjected to a MaximumLikelihood (ML) method in MEGA 60 software [23]However prior to performing ML analysis best substitutionmodel that described the sequence data set was obtainedtaking into considerations the lowest Bayesian inferencesEqually 1000 bootstraps values were used to determine theconfidence interval of the resultant tree In order to root thetree a 16S rRNA sequence from Mycobacterium tuberculosiswas used as an out-group

All local L monocytogenes sequences used in the phy-logenetic study were submitted to GenBank for archivingThe accession numbers of these sequences together with thereference isolates are summarised in Table 3

3 Result

31 Detection of hly A Harbouring Listeria monocytogenes Iso-lates The PCR results for the detection of hly A gene amongthe 36 L monocytogenes isolates showed that only 9 (250)(ZKMM84 SGHR30 SGSK1 ZKJR56 ZKJR39 ZKMR75SGMM87 SGHR15 and LMFR162) L monocytogenes isolatesharboured the hly A gene (Figure 1)

32 Detection of Virulence-Associated Genes of Listeria mono-cytogenes Isolates Analysis of multiplex PCR used for thedetection of virulence-associated genes revealed that of the36 L monocytogenes isolates 9 (250) possessed at leastone or more virulence-associated genes (Figure 2) Of the5 virulence-associated genes screened for only 3 virulence-associated genes (hly A iap and prf A) genes were detected

Table 3 Local and reference L monocytogenes isolates used in thephylogenetic analysis

Snumber Strain GenBankaccession number

(1) Listeria monocytogenes NGA 34A KX358070(2) Listeria monocytogenes NGA 35A KX358071(3) Listeria monocytogenes NGA 38A KX358072(4) Listeria monocytogenes NGA 41A KX358073(5) Listeria monocytogenes N1-011A CP00659(6) Listeria monocytogenes J1816 CP006047(7) Listeria monocytogenes J2-031 CP006593(8) Listeria monocytogenes J1-220 CP0060461(9) Listeria monocytogenes J1926 CP006600(10) Listeria monocytogenes J1817 CP006599(11) Listeria monocytogenes R2-502 CP006594(12) Listeria monocytogenes J1776 CP006598(13) Listeria monocytogenes EDG HG421741(14) Listeria monocytogenes FW040025 CP011345(15) Mycobacterium bovis AY360331

each of 5 (138) isolates possessed the iap and prf A genesBased on the sample types 6 (169) of the L monocytogenesisolates that possessed virulence-associated genes were fromraw milk 2 (32) ldquoManshanurdquo and 1 (28) ldquoKindrimordquo

33 Phylogenetic Tree Analysis Phylogenetic analysis wascarried out based on the 16S rRNA sequences encompassing938 bp obtained for 4 of the L monocytogenes isolates(Nigerian isolates) Interestingly maximum likelihood (ML)analysis revealed two different clusters A and B with clusterA comprising of Nigeria L monocytogenes NGA 35A NGA34A andNGA41A and reference Lmonocytogenes J1776 N1-011A R2-502 J1816 and J2-031 while cluster B is comprised ofNigerian L monocytogenes sequence NGA 38A and referenceL monocytogenes sequence EDG J1-220 J1926 J1817 andFW040025 Sequence in cluster A was supported by a strongbootstrap values of 100 in contrast with cluster Bwhich showsa very low bootstrap value of 16 (Figure 3)

Canadian Journal of Infectious Diseases and Medical Microbiology 5

1000 bp700 bp500 bp

1M 2 3 4 5 6 7 8 9 10 11 12 13 14

16S rRNA (938 bp)hly A (702 bp)

Figure 1 Agarose gel electrophoresis showing electrophoretic mobility of PCR products obtained following amplification of the 16S rRNAgene (938 bp) and listeriolysin O encoding gene (hly A) (702 bp) from different Listeria monocytogenes isolated from raw milk and milkproducts Lane M 100 bp ladder Lane 1 positive control (Listeria monocytogenes ATCC 19155) Lane 3 Listeria monocytogenes (LM) isolateZKMM84 Lane 5 LMSGHR30 Lane 6 LMSGSK1 Lane 9 LMZKJR56 Lane 10 LMSGHR39 Lane 11 LMZKMR75 Lane 12 LMSGMM87Lane 13 LM SGHR15 Lane 14 LM LMFR162 corresponding to 938 bp and 702 bp amplicons

1M 2 3 4 5 6 7 8 9 10 11 12

1000 bp

100 bp

500 bp

bp)060bp)702

prf A (1hly A (

bp)131iap (

Figure 2 Agarose gel electrophoresis result for multiplex PCR following amplification of prf A hly A and iap virulence-associated genesLane M molecular weight marker (Promega) in base pairs Lane 1 Listeria monocytogenes (LM) ATTC 19155 Lane 2 LM isolate ZKMM84Lane 3 LM SGHRR30 Lane 4 LM SGSK1 Lane 5 negative control Lane 6 LM ZKJR55 Lane 7 LM SGHR39 Lane 8 LM ZKMR75= Lane9 LM SGMM37 Lane 11 LM SGHR15 Lane 12 LMFR162

L monocytogenes J1776L monocytogenes N1-011A7L monocytogenes R2-502L monocytogenes J1816L monocytogenes J2-031L monocytogenes NGA 41AL monocytogenes NGA 34AL monocytogenes NGA 35A

L monocytogenes NGA 38AL monocytogenes J1-220L monocytogenes EGDL monocytogenes J1926L monocytogenes J1817L monocytogenes FW040025

Mycobacterium bovis

16 7719

100

01

B

A

Figure 3 Molecular phylogenetic tree showing relationship between Nigerian L monocytogenes isolates (red color) with reference sequencesfromGenBank databaseThe evolutionary history was inferred by using theMaximum Likelihoodmethod based on the Tamura 3-parametermodelThe tree with the highest log likelihood (minus27602483) is shownThe percentage of trees in which the associated taxa clustered togetheris shown next to the branch and 1000 bootstrap value was used to assess the tree confidence interval All analyses were performed inMEGA6software

4 Discussion

To ensure food quality and safety detection of pathogenicbacteria should be a fundamental objective The applicationof molecular techniques has facilitated the identification

and characterization of major virulence-associated genes inL monocytogenes [24] In this study the multiplex PCRidentified 9 Listeria monocytogenes isolates harbouring hlyA gene The target genes for L monocytogenes and Listeriagenus produced PCR products of 702 bp and 938 bp in size

6 Canadian Journal of Infectious Diseases and Medical Microbiology

respectively The PCR protocol used in this study was basedon the amplification of hly A gene by using a set of primersLM1 and LM2 Border et al [21] recommended LM1 andLM2 primers as the best primer pair to be used for thedetection of L monocytogenes because they are designed toamplify specific fragments in L monocytogenes strains thatare genetically and biochemically assessed as belonging tothis specie on the basis of the presence of the 702 bp and938 bp amplicons [21]

Kuan et al [25] were also able to amplify the DNA attheir expected amplification site The result of this studyis not in agreement with the result of Jallewar et al [26]who reported the carriage of hly A gene by all the isolatesof L monocytogenes tested The inability of some of the Lmonocytogenes isolates in this study to harbour the hly Agene may be due to the nonvirulent characteristics of some Lmonocytogenes isolates because some may be environmentalisolates or it may be possible that some L monocytogenesstrains may lack one or more virulence determinants becauseof spontaneous mutations [11]

The ability to survive and multiply in the macrophagesand other host cells is key to the pathogenicity of Lmonocytogenes [27] Rapid methods for the isolation andconfirmation of L monocytogenes in foods are still beingexplored [28] Because of the importance of Lmonocytogenesepidemiology to human health and the notable diversity inthe pathogenicity among strains subtyping and virulencecharacterization are of importance [7] Due to the diversestrains of L monocytogenes of varying pathogenicity theability to precisely track the strains involved in listeriosisoutbreaks and rapidly determine their pathogenic potentialsis critical for the control and prevention of further occur-rences of this deadly disease [24] The phenotypic subtypingmethods are generally less sensitive have low differentiationability and are not easy to reproduce whereas the genotypicapproaches are more sensitive and reliable [29] Virulence-associated genes of L monocytogenes namely prf A hlyA and iap were amplified according to their respectivefragment sizes of 1060 bp 702 bp and 131 bp respectivelyThis study revealed that of the 36 L monocytogenes isolatesscreened for virulence-associated genes only 9 (250) iso-lates harboured one or more of such genes and none ofthem possessed all the five genes It is plausible that someL monocytogenes strains may lack one or more virulencedeterminants because of spontaneous mutations [11]

Henriques et al [30] detected hly A genes from Lmonocytogenes isolates from Portuguese ready-to-eat meatbased food industry which is similar to the findings ofthis study where hly A was detected in all the nine Lmonocytogenes isolates However the detection of act A genein the work of Henriques et al [30] differs from this studywhich failed to detect act A Listeriolysin O and act Aare associated with the bacteriumrsquos capability of passing theintestinal barrier cell to cell spread andmotility cell invasionand intracellular parasitism [7 18] Therefore simultaneousdetection of virulence genes in a single step will be desirableas it reduces time and labour and it will be useful in largescale survey for detecting virulent strains of Listeria [18]

The separation of Nigerian Listeria monocytogenes iso-lates sequence (NGA34ANGA35ANGA38 andNGA41A)as well as the L monocytogenes sequences from the GenBank(N1-011A R2-502 J1816 J2-031 EDG J1-220 J1926 J1817 andPW040025) into two major lineages A and B is consistentwith the major lineages described previously by Oris et al[31] who reported that L monocytogenes isolates

L monocytogenes consist of at least 4 evolutionary lin-eages (I II III and IV) Even though only cluster A isstatistically supported with a bootstrap value of 100 clusterB is not statistically supported

Most L monocytogenes isolates seem to belong to lineagesI and II which include the serotypes more commonlyassociated with human clinical cases including serotypes12b and 4b (lineage I) and serotype 12a (lineage II) Sinceit is a rooted tree we can infer evolutionary relationshipinformation regarding the common ancestors and the pathof evolution from the rooted phylogenetic tree

Since lineage I L monocytogenes strains are more preva-lent in human clinical listeriosis cases and contaminated food[31] it can be said that Nigerian L monocytogenes isolates(NGA 34A NGA 35A and NGA41A) are from contaminatedfoods (ie milk and milk products) From the phylogenictree Nigerian L monocytogenes isolates (NGA 34A NGA35A and NGA 41A) were classified as lineage I which areknown to be of higher pathogenic potentials due to intrinsicvirulence features as compared to lineage II strains of Lmonocytogenes [32]Morobe et al [33] in their study observedthat most of the L monocytogenes isolates belong to lineageIII (4a and 4c) which are known to be more prevalent inanimals with clinical listeriosis [31] as opposed to this studywhere none of the L monocytogenes isolate were found tobelong to lineage III Isolate NGA 35a was found to belong tolineage II which is opposed to the findings of Morobe et al[33] where they observed that none of the L monocytogenesisolates belong to lineage II

The implication of the present findings is that L mono-cytogenes of lineage 1 originating from animal milk and milkproduct could be associated with increased human risk to Lmonocytogenes infection in Nigeria and thus effort to controlthe disease should be targeted at improving the food safetyand quality

Competing Interests

The authors declare that they have no competing interests

References

[1] M J Gray N E Freitag and K J Boor ldquoHow the bacterialpathogen Listeria monocytogenes mediates the switch fromenvironmental Dr Jekyll to pathogenicMr Hyderdquo Infection andImmunity vol 74 no 5 pp 2506ndash2512 2006

[2] T Hain C Steinweg and T Chakraborty ldquoComparative andfunctional genomics of Listeria spprdquo Journal of Biotechnologyvol 126 no 1 pp 37ndash51 2006

[3] E Rahimi M Ameri and H Momtaz ldquoPrevalence and antimi-crobial resistance of Listeria species isolated from milk and

Canadian Journal of Infectious Diseases and Medical Microbiology 7

dairy products in Iranrdquo Food Control vol 21 no 11 pp 1448ndash1452 2010

[4] B Karen ListeriaMonocytogenes InfectionMedscape LLCNewYork NY USA 2015 httpemedicinemedscapecomarticle220684-overview

[5] F Stavru F Bouillaud A Sartori D Ricquier and P Cos-sart ldquoListeria monocytogenes transiently alters mitochon-drial dynamics during infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no9 pp 3612ndash3617 2011

[6] J A Vazquez-Boland M Kuhn P Berche et al ldquoListeriapathogenesis and molecular virulence determinantsrdquo ClinicalMicrobiology Reviews vol 14 no 3 pp 584ndash640 2001

[7] D Liu M L Lawrence F W Austin and A J AinsworthldquoA multiplex PCR for species- and virulence-specific determi-nation of Listeria monocytogenesrdquo Journal of MicrobiologicalMethods vol 71 no 2 pp 133ndash140 2007

[8] A Camejo F Carvalho O Reis E Leitao S Sousa and DCabanes ldquoThe arsenal of virulence factors deployed by Listeriamonocytogenes to promote its cell infection cyclerdquo Virulencevol 2 no 5 pp 379ndash394 2011

[9] B Joseph and W Goebel ldquoLife of Listeria monocytogenes in thehost cellsrsquo cytosolrdquo Microbes and Infection vol 9 no 10 pp1188ndash1195 2007

[10] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[11] K J Cooray T Nishibori H Xiong T Matsuyama M FujitaandM Mitsuyama ldquoDetection of multiple virulence associatedgenes of Listeriamonocytogenes by PCR in artificially contam-inated milk samplesrdquo Applied Microbiology vol 60 pp 3023ndash3026 1994

[12] S H W Notermans J Dufrenne M Leimeister-Wachter EDomann and T Chakraborty ldquoPhosphatidy-linositol-specificphospholipase C activity as a marker to distinguish betweenpathogenic and non-pathogenic Listeria speciesrdquo Applied andEnvironmental Microbiology vol 57 no 9 pp 2666ndash2670 1991

[13] J E Olsen ldquoDNA-based methods for detection of food-bornebacterial pathogensrdquo Food Research International vol 33 no 3-4 pp 257ndash266 2000

[14] E Walecka J Molenda R Karpıskova and J Bania ldquoEffect ofosmotic stress and culture density on invasiveness of Listeriamonocytogenes strainsrdquo International Journal of Food Microbi-ology vol 144 no 3 pp 440ndash445 2011

[15] M Wiedmann ldquoMolecular subtyping methods for Listeriamonocytogenesrdquo Journal of AOAC International vol 85 no 2pp 524ndash531 2002

[16] G T Jeffers J L Bruce P L McDonough J Scarlett K J Boorand M Wiedmann ldquoComparative genetic characterizationof Listeria monocytogenes isolates from human and animallisteriosis casesrdquo Microbiology vol 147 no 5 pp 1095ndash11042001

[17] C A Nadon D L Woodward C Young F G Rodgers andM Wiedmann ldquoCorrelations between molecular subtypingand serotyping of Listeria monocytogenesrdquo Journal of ClinicalMicrobiology vol 39 no 7 pp 2704ndash2707 2001

[18] D B Rawool SV SMalik I Shakuntala AM Sahare and S BBarbuddhe ldquoDetection of multiple virulence-associated genesin Listeria monocytogenes isolated from bovine mastitis casesrdquoInternational Journal of Food Microbiology vol 113 no 2 pp201ndash207 2007

[19] D Roberts W Hoper and M Greenwood Practical FoodMicrobiology Public Health Laboratory Services London UK1995

[20] M M Janzten J Navas A Corujo R Moreno V Lopez andJ V Martınez-Suarez ldquoReview Specific detection of Listeriamonocytogenes in foods using commercial methods fromchromogenic media to real-time PCRrdquo Spanish Journal ofAgricultural Research vol 4 no 3 pp 235ndash247 2006

[21] P M Border J J Howard G S Plastow and K W SiggensldquoDetection of Listeria species and Listeria monocytogenes usingpolymerase chain reactionrdquo Letters in AppliedMicrobiology vol11 no 3 pp 158ndash162 1990

[22] T A Hall ldquoBioedit a user friendly biological sequence align-ment editor and analysis programme for windows 9598NTrdquoin Nucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[23] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2002

[24] D Liu ldquoIdentification subtyping and virulence determinationof Listeria monocytogenes an important foodborne pathogenrdquoJournal of Medical Microbiology vol 55 no 6 pp 645ndash6592006

[25] C H Kuan S G Goh Y Y Loo et al ldquoPrevalence andquantification of Listeria monocytogenes in chicken offal at theretail level in Malaysiardquo Poultry Science vol 92 no 6 pp 1664ndash1669 2013

[26] P K Jallewar D R Kalorey N V Kurkure V V Pande andS B Barbuddhe ldquoGenotypic characterization of Listeria sppisolated from fresh water fishrdquo International Journal of FoodMicrobiology vol 114 no 1 pp 120ndash123 2007

[27] J Kreft and J A Vazquez-Boland ldquoRegulation of virulencegenes in Listeriardquo International Journal of Medical Microbiologyvol 291 no 2 pp 145ndash157 2001

[28] R R Beumer and W C Hazeleger ldquoListeria monocytogenesdiagnostic problemsrdquo FEMS Immunology and Medical Micro-biology vol 35 no 3 pp 191ndash197 2003

[29] Y H Shuckken Y T Grohn and M Wiedmann ldquoPart VIlisteria monocytogenes epidemiology of listeriosisrdquo in Micro-bial Food Safety in Animal Agriculture Current Topics M ETorrence and R E Isaacson Eds pp 221ndash232Wiley-BlackwellHoboken NJ USA 2003

[30] A R Henriques A S Barreto and M J Fraqueza ldquoDetectionof virulence-associated genes in Listeria monocytogenes isolatesfrom Portuguese ready-to- eat meat-based food industryrdquoApplied and Environmental Microbiology vol 61 pp 817ndash8191998

[31] R H Orsi H C den Bakker and M Wiedmann ldquoListeriamonocytogenes lineages genomics evolution ecology andphenotypic characteristicsrdquo International Journal of MedicalMicrobiology vol 301 no 2 pp 79ndash96 2011

[32] B Swaminathan and P Gerner-Smidt ldquoThe epidemiology ofhuman listeriosisrdquo Microbes and Infection vol 9 no 10 pp1236ndash1243 2007

[33] I C Morobe C L Obi M A Nyila M I Matsheka andB A Gashe ldquoMolecular characterization and serotyping ofListeria monocytogenes with a focus on food safety and diseasepreventionrdquo in Biochemical Testing J C Jimenez-Lopez Edchapter 8 pp 197ndash216 InTech Rijeka Croatia 2012

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2 Canadian Journal of Infectious Diseases and Medical Microbiology

and virulence regulator (encoded by prf A) These virulencefactors play significant role in the bacterial pathogenicity andinfection outcome [6 7]

It has been observed that Listeria monocytogenes employthese virulence factors that act synergistically in the intra-cellular pathogenicity of the bacteria [8] For exampleexpressions of virulence genes such as listeriolysin O (LLO)phospholipases (plc A and plc B) and internalins A and B(inl A and inl B) are regulated by prf A and these genesfacilitate the intracellular growth and spread of the bacteriumwithin themammalian host [9] Pointmutation is responsiblefor low virulence of Listeria monocytogenes strains in anumber of virulence genes and as a result of spontaneousmutations some L monocytogenes strains may lose one ormore virulence determinants [10 11] A virulence factor ofphospholipase called PI-PLC is expressed by pathogenicspecies of L monocytogenes and Listeria ivanovii only andthis virulence factor is an important marker for the dis-crimination between pathogenic and nonpathogenic Listeriaspecies [12] There is growing evidence that virulence is nota stable property but can be influenced by environmentalconditions For example it has been shown that acid and saltstress increase the expression of virulence genes and in vitropathogenicity of the bacteria [13] Virulence potentials arealso influenced by temperature the presence or absence ofoxygen osmotic stress and pH [14] L monocytogenes has13 serotypes of these only four serotypes (12a 12b 12cand 4b) are known to cause human listeriosis [15] Basedon the data generated from molecular subtyping methodsL monocytogenes isolates have been grouped into two majorgenetic divisions or lineages termed as lineage I and lineage II[15] A third lineage predominantly associated with animalshas been described by some studies as well [16] Serotypes12a 3b 3c and 4b belong to lineage I while serotypes 12a12c and 3a belong to lineage II [17] Studies have shown thatlineage I strains are significantly higher among human clini-cal listeriosis cases and contaminated foods [1] while lineageII strains show a significantly higher prevalence among foodisolates and animal clinical cases than among human clinicallisteriosis [16] Pathogenic potentials of lineage I isolates aregreater than those of lineage II as determined by their abilityto spread to neighbouring host cells in a cell culture plaqueassay [1] It will be observed that there is a consensus thatlineage I strainsmay represent a human host-adaptive lineagewhile lineage II strains may represent an environmentaladapted lineage [15]The application ofmultiplex PCR for thedetection of more than one virulence gene in a single tubeis desirable because it reduces cost and labour and will beuseful in a large scale detection of virulent strains of Listeria[18]

The prevalence and molecular characteristics of Listeriamonocytogenes are generally unknown in Nigeria and thusthis present study was carried out with the aim of charac-terizing L monocytogenes isolates from raw milk and milkproducts using PCR and to determine the virulence and thephylogenetic characteristics of the isolates

2 Materials and Methods

21 Sample Collection

211 Bacterial Strains and Biochemical The isolation of Liste-riamonocytogeneswas carried out according to the procedureof Roberts et al [19] Briefly about 10mL of the incubatedhomogenate was added to 90mL Listeria enrichment broth(Oxoid CM 0862) which contains selective Listeria enrich-ment supplements (Oxoid SR 0141E) and incubated at 30∘Cfor 48 hrs A loop full of the 48 hrs broth was cultured ontoChromogenic Listeria agar (Oxoid CM 1080) plates whichcontains brilliance Listeria differential supplement (OxoidSR 0228E) and brilliance Listeria selective supplement(Oxoid SR 00227) and the plates was incubated at 37∘Cfor 24ndash48 hrs Colonies of Listeria monocytogenes havinga greenish-blue coloration were picked and streaked onnutrient agar (Oxoid CM 0003) slants and incubated at 37∘Cfor 24 hrs before storage at 4-5∘CThe suspected isolates werefurther subjected to conventional biochemical test [Gramstaining oxidase catalase 120573-hemolysis bile esculin and car-bohydrate fermentation test (xylose Mannitol RhamnoseMaltose Inositol and Sucrose)] as described previously [20]Isolates found to ferment mannitol and xylose and positiveto rhamnose were considered as L monocytogenes Furthercharacterization usingMicrobact 12L Listeria identificationkit (Oxoid MB 1128) was carried out

Following cultural identification and biochemical assaysstated above 36 Listeria monocytogenes isolates were identi-fied (Table 1)

22 Polymerase Chain Reaction and Gel Electrophoresis Bac-terial isolates obtained locally as well as reference strainswere subjected to PCR assay The reference strain designatedATCC 19155 used as positive control was kindly provided byProfessor Radu of the Center of Excellence for Food SafetyResearch Faculty of Food Science and Technology UniversitiPutra Malaysia where the molecular studies were carriedout Bacterial genomic DNA was extracted from both localand reference isolates using the Quick-g DNAminiprep kit(Zymo Research SA) following the manufacturerrsquos instruc-tions

Multiplex PCR assay was used to detect L monocytogenesharbouring hly A gene and 16S rRNA of L monocytogenes[21] The primer pairs designated as LM1 and LM2 (LM1 51015840ndashCCTAAGACGCCAATCGAA-31015840 and LM2 ndash 51015840-AAGCGCTTG CAA CTG CTC-31015840) were used for the detection of Lmonocytogenes harbouring hly A gene On the other handprimer pairs designated as U1 and LI1 (LI1 51015840ndashCTC CATAAA GGT GAC CCT-31015840 and U1 51015840ndashCAG CMG CCG CGGTAATWC-31015840) were used to amplify 938 bp region in the 16SrRNA gene specific for the detection of Listeria genus [21]These primerswere synthesized by InvitrogenUSA andwere18ndash20 bp in length L monocytogenesATCC 19155 was used asa positive control

The PCR amplification was carried out in a 25 120583L reactionmixture that consisted of 5 120583L of 5x PCR buffer dNTPs(05 120583L) MgCl

2(2 120583L) Taq DNA polymerase (05 120583L) 05 120583L

of each 20 pM primer [LM1 LM2 U1 and LI1] 13120583L of

Canadian Journal of Infectious Diseases and Medical Microbiology 3

Table 1 Listeria monocytogenes characteristics

Snumber Isolate name (code) Isolated from Tip name in tree(1) LMFR151 Raw milk 49A(2) ZKMM83 Manshanu 48A(3) MDGR115 Raw milk 47A(4) MMM103 Manshanu 46A(5) ZKMM84 Manshanu 34A(6) LSNK136 Kindrimo 44A(7) SGMK77 Kindrimo 43A(8) MGDR100 Raw milk 42A(9) ZKMR75 Raw milk 35A(10) SGHR39 Raw milk 38A(11) ZKJR55 Raw milk 40A(12) SGHR7 Raw milk 39A(13) SGHR12 Raw milk 37A(14) SGHR30 Raw milk 41A(15) ZKMM87 Manshanu 36A(16) ZKMM84 Manshanu 45A(17) ZKJR5 Raw milk 33A(18) SGMM14 Manshanu 32A(19) SGMM34 Manshanu 31A(20) SGHR45 Raw milk 30A(21) LMFR161 Raw milk 29A(22) MDGR109 Raw milk 28A(23) SGHR32 Raw milk 27A(24) MDGR10 Raw milk 25A(25) ZKJR10 Raw milk 24A(26) SGSM9 Manshanu 23A(27) LMFR162 Raw milk 15A(28) SGHR55 Raw milk 22A(29) MDGM123 Manshanu 21A(30) SGSK1 Kindrimo 26A(31) SGHR15 Raw milk 20A(32) SGHR37 Raw milk 19A(33) SGSK21 Kindrimo 18A(34) LMFK153 Kindrimo 17A(35) SGMM37 Manshanu 16A(36) SGMM11 Manshanu 14A

distilled water and 2120583L of DNA template PCR amplificationwas carried out in a programmed thermocycler with thefollowing thermal conditions hot start PCR plate at 95∘C for3mins followed by 35 cycles each of 30 sec denaturation at94∘C 15 sec annealing at 53∘C 90 sec extension for 72∘C andfinal extension at 72∘C for 7mins

Following PCR amplification about 5120583L of the PCRproduct was mixed with DNA loading dye (6x) and elec-trophoresed in 10 Agarose gel in TAE buffer using aminitank at 80V 400 amp and 45min The electrophoresedproduct on the gel was stained with ethidium bromidefor 30min destained for 20min and visualized under UVilluminator (SYNGENE Biosystems UK) A 100 bp DNA

ladder (PromegaCorporationUSA)was included to estimatethe size of the amplified products

23 Detection of L monocytogenes Virulence-AssociatedGenes The obtained DNA product was used in performinganother multiplex PCR assay with the aim of detectingthe presence of five virulence-associated genes [hemolysinA gene (hly A) regulatory gene (prf A) actin gene (actA) invasion associated protein p60 gene (iap) and surfaceprotein (inl A)] of L monocytogenes as described by Rawoolet al [18] The primers used for the detection of thesevirulence geneswere synthesized by SigmaAldrich USAThedetails of the primer sequences are shown in Table 2 TheDNA obtained using the Quick-g DNA miniprep-uncappedcolumn kit (Zymo Research) was used for the detection ofvirulence-associated genes

The PCR was standardized for the detection of virulence-associated genes of L monocytogenes by optimizing theannealing temperatures between 50 and 60∘C Based onoptimization trials the standardized PCRprotocol for a 25120583Lreactionmixture included 5120583LPCRbuffer (100mMTrisndashHClbuffer pH 83 containing 500mM KCl 15mM MgCl

2 and

001 gelatine) 1 120583L dNTP mix 3 120583L MgCl2and 05 120583L of

forward and reverse primers 1 120583L of 5 units of Taq DNApolymerase 5 120583LDNA template and 5120583L nuclease free watertomake up the reaction volumeTheDNAamplification reac-tion was performed in aMaster Cycler gradient thermocycler(Eppendorf Hamburg Germany) with a preheated lid inPCR tubes (02mL)The cycling conditions for PCR includedan initial denaturation of DNA at 95∘C for 2min followed by35 cycles each of 15 sec denaturation at 95∘C 30 sec annealingat 53∘C and 90 sec extension at 72∘C Final was carried out in10min at 72∘C and then PCR product held at 4∘C

The resultant PCR products were separated by elec-trophoresis in 15 Agarose gel for 45min at 100V inTris-acetate EDTA buffer stained with ethidium bromide(05 120583gmL) and visualized with a UV transilluminator (Syn-gene Frederick MD)The gel image was documented by a geldocumentation apparatus and DNA size was determined bya 100 bp DNA ladder (Promega)

24 Purification of PCR Products 5 120583L volume PCR productfor each of the 9 hly A and 16S rRNA harbouring L mono-cytogenes positive isolates was analysed on a 15 Agarosegel to confirm successful amplification of the 16S rRNAThe remaining 20 120583L of each of the PCR product was laterrun and each corresponding 16S rRNA band was cut andpurified by using the MEGA quick-spin Total FragmentDNA purification kit (iNtRON Biotechnology Gyeonggi-doSouth Korea)

25 DNA Sequencing and Phylogenetic Analysis Purified gelwas used for DNA sequencing All sequencing reactionswere commercially done by iNtRON (iNtRONBiotechnologyGyeonggi-do South Korea) using the Sangers sequencingmethod Both forward and reverse primers were used toensure adequate sequencing coverage For sequencing andphylogenetic tree only 15 samples were randomly selectedand tested

4 Canadian Journal of Infectious Diseases and Medical Microbiology

Table 2 Primer sequences used for the amplification of virulence-associated genes of Listeria monocytogenes

Target gene Primer sequence (51015840-31015840) Product size (bp)

inl A Fwd 51015840-AGATCTAGACCAAGTTACAACGCTTCAG-31015840Rev 51015840-TAA TAT CAT TTG CTG TTT TAT CTG TC-31015840 255 bp

act A Fwd 510158401015840-ACG TGA AGT AAG TCACGT GAT ATT G 31015840Fwd 510158401015840-ACG TGAAGTAAGCTCACGT GAT ATT G-31015840 268 bp

prf A Fwd 51015840-ACC GCT CAG AAA AGT TCT TC-31015840Rev 51015840-TCT TGT TCT ATT ATGTCT AGC-31015840 1060 bp

iap Fwd 51015840-ACA AGC TGC ACC TGT TGC AG-31015840Rev 5-TGA CAG CGT TGT TAG TAG CA-31015840 131 bp

hly A Fwd 51015840-CCT AAG ACG CCA ATC GAA-31015840Rev 51015840-AAG CGC TTG CAA GTC CTC-31015840 702 bp

The obtained partial 16S rRNA sequences were proof-read and assembled in Chromas Lite version 211 Multiplesequence alignment of the DNA sequences was performedusing ClustalW in Bioedit software version 725 (BiologicalSequence Alignment Editor for Win 9598NT2KXP7)as described by Hall 1999 [22] To compare the sequenceof local L monocytogenes with reference isolates availablein the GenBank database sequence similarity search wasperformedusing theNCBI nucleotide BLAST search to deter-mine the similarity of the local L monocytogenes sequence(httpblastncbinlmnihgov)

Phylogenetic analysis was carried out based on partialsequence of 16S rRNA gene of L monocytogenes In thisregard the aligned sequence was subjected to a MaximumLikelihood (ML) method in MEGA 60 software [23]However prior to performing ML analysis best substitutionmodel that described the sequence data set was obtainedtaking into considerations the lowest Bayesian inferencesEqually 1000 bootstraps values were used to determine theconfidence interval of the resultant tree In order to root thetree a 16S rRNA sequence from Mycobacterium tuberculosiswas used as an out-group

All local L monocytogenes sequences used in the phy-logenetic study were submitted to GenBank for archivingThe accession numbers of these sequences together with thereference isolates are summarised in Table 3

3 Result

31 Detection of hly A Harbouring Listeria monocytogenes Iso-lates The PCR results for the detection of hly A gene amongthe 36 L monocytogenes isolates showed that only 9 (250)(ZKMM84 SGHR30 SGSK1 ZKJR56 ZKJR39 ZKMR75SGMM87 SGHR15 and LMFR162) L monocytogenes isolatesharboured the hly A gene (Figure 1)

32 Detection of Virulence-Associated Genes of Listeria mono-cytogenes Isolates Analysis of multiplex PCR used for thedetection of virulence-associated genes revealed that of the36 L monocytogenes isolates 9 (250) possessed at leastone or more virulence-associated genes (Figure 2) Of the5 virulence-associated genes screened for only 3 virulence-associated genes (hly A iap and prf A) genes were detected

Table 3 Local and reference L monocytogenes isolates used in thephylogenetic analysis

Snumber Strain GenBankaccession number

(1) Listeria monocytogenes NGA 34A KX358070(2) Listeria monocytogenes NGA 35A KX358071(3) Listeria monocytogenes NGA 38A KX358072(4) Listeria monocytogenes NGA 41A KX358073(5) Listeria monocytogenes N1-011A CP00659(6) Listeria monocytogenes J1816 CP006047(7) Listeria monocytogenes J2-031 CP006593(8) Listeria monocytogenes J1-220 CP0060461(9) Listeria monocytogenes J1926 CP006600(10) Listeria monocytogenes J1817 CP006599(11) Listeria monocytogenes R2-502 CP006594(12) Listeria monocytogenes J1776 CP006598(13) Listeria monocytogenes EDG HG421741(14) Listeria monocytogenes FW040025 CP011345(15) Mycobacterium bovis AY360331

each of 5 (138) isolates possessed the iap and prf A genesBased on the sample types 6 (169) of the L monocytogenesisolates that possessed virulence-associated genes were fromraw milk 2 (32) ldquoManshanurdquo and 1 (28) ldquoKindrimordquo

33 Phylogenetic Tree Analysis Phylogenetic analysis wascarried out based on the 16S rRNA sequences encompassing938 bp obtained for 4 of the L monocytogenes isolates(Nigerian isolates) Interestingly maximum likelihood (ML)analysis revealed two different clusters A and B with clusterA comprising of Nigeria L monocytogenes NGA 35A NGA34A andNGA41A and reference Lmonocytogenes J1776 N1-011A R2-502 J1816 and J2-031 while cluster B is comprised ofNigerian L monocytogenes sequence NGA 38A and referenceL monocytogenes sequence EDG J1-220 J1926 J1817 andFW040025 Sequence in cluster A was supported by a strongbootstrap values of 100 in contrast with cluster Bwhich showsa very low bootstrap value of 16 (Figure 3)

Canadian Journal of Infectious Diseases and Medical Microbiology 5

1000 bp700 bp500 bp

1M 2 3 4 5 6 7 8 9 10 11 12 13 14

16S rRNA (938 bp)hly A (702 bp)

Figure 1 Agarose gel electrophoresis showing electrophoretic mobility of PCR products obtained following amplification of the 16S rRNAgene (938 bp) and listeriolysin O encoding gene (hly A) (702 bp) from different Listeria monocytogenes isolated from raw milk and milkproducts Lane M 100 bp ladder Lane 1 positive control (Listeria monocytogenes ATCC 19155) Lane 3 Listeria monocytogenes (LM) isolateZKMM84 Lane 5 LMSGHR30 Lane 6 LMSGSK1 Lane 9 LMZKJR56 Lane 10 LMSGHR39 Lane 11 LMZKMR75 Lane 12 LMSGMM87Lane 13 LM SGHR15 Lane 14 LM LMFR162 corresponding to 938 bp and 702 bp amplicons

1M 2 3 4 5 6 7 8 9 10 11 12

1000 bp

100 bp

500 bp

bp)060bp)702

prf A (1hly A (

bp)131iap (

Figure 2 Agarose gel electrophoresis result for multiplex PCR following amplification of prf A hly A and iap virulence-associated genesLane M molecular weight marker (Promega) in base pairs Lane 1 Listeria monocytogenes (LM) ATTC 19155 Lane 2 LM isolate ZKMM84Lane 3 LM SGHRR30 Lane 4 LM SGSK1 Lane 5 negative control Lane 6 LM ZKJR55 Lane 7 LM SGHR39 Lane 8 LM ZKMR75= Lane9 LM SGMM37 Lane 11 LM SGHR15 Lane 12 LMFR162

L monocytogenes J1776L monocytogenes N1-011A7L monocytogenes R2-502L monocytogenes J1816L monocytogenes J2-031L monocytogenes NGA 41AL monocytogenes NGA 34AL monocytogenes NGA 35A

L monocytogenes NGA 38AL monocytogenes J1-220L monocytogenes EGDL monocytogenes J1926L monocytogenes J1817L monocytogenes FW040025

Mycobacterium bovis

16 7719

100

01

B

A

Figure 3 Molecular phylogenetic tree showing relationship between Nigerian L monocytogenes isolates (red color) with reference sequencesfromGenBank databaseThe evolutionary history was inferred by using theMaximum Likelihoodmethod based on the Tamura 3-parametermodelThe tree with the highest log likelihood (minus27602483) is shownThe percentage of trees in which the associated taxa clustered togetheris shown next to the branch and 1000 bootstrap value was used to assess the tree confidence interval All analyses were performed inMEGA6software

4 Discussion

To ensure food quality and safety detection of pathogenicbacteria should be a fundamental objective The applicationof molecular techniques has facilitated the identification

and characterization of major virulence-associated genes inL monocytogenes [24] In this study the multiplex PCRidentified 9 Listeria monocytogenes isolates harbouring hlyA gene The target genes for L monocytogenes and Listeriagenus produced PCR products of 702 bp and 938 bp in size

6 Canadian Journal of Infectious Diseases and Medical Microbiology

respectively The PCR protocol used in this study was basedon the amplification of hly A gene by using a set of primersLM1 and LM2 Border et al [21] recommended LM1 andLM2 primers as the best primer pair to be used for thedetection of L monocytogenes because they are designed toamplify specific fragments in L monocytogenes strains thatare genetically and biochemically assessed as belonging tothis specie on the basis of the presence of the 702 bp and938 bp amplicons [21]

Kuan et al [25] were also able to amplify the DNA attheir expected amplification site The result of this studyis not in agreement with the result of Jallewar et al [26]who reported the carriage of hly A gene by all the isolatesof L monocytogenes tested The inability of some of the Lmonocytogenes isolates in this study to harbour the hly Agene may be due to the nonvirulent characteristics of some Lmonocytogenes isolates because some may be environmentalisolates or it may be possible that some L monocytogenesstrains may lack one or more virulence determinants becauseof spontaneous mutations [11]

The ability to survive and multiply in the macrophagesand other host cells is key to the pathogenicity of Lmonocytogenes [27] Rapid methods for the isolation andconfirmation of L monocytogenes in foods are still beingexplored [28] Because of the importance of Lmonocytogenesepidemiology to human health and the notable diversity inthe pathogenicity among strains subtyping and virulencecharacterization are of importance [7] Due to the diversestrains of L monocytogenes of varying pathogenicity theability to precisely track the strains involved in listeriosisoutbreaks and rapidly determine their pathogenic potentialsis critical for the control and prevention of further occur-rences of this deadly disease [24] The phenotypic subtypingmethods are generally less sensitive have low differentiationability and are not easy to reproduce whereas the genotypicapproaches are more sensitive and reliable [29] Virulence-associated genes of L monocytogenes namely prf A hlyA and iap were amplified according to their respectivefragment sizes of 1060 bp 702 bp and 131 bp respectivelyThis study revealed that of the 36 L monocytogenes isolatesscreened for virulence-associated genes only 9 (250) iso-lates harboured one or more of such genes and none ofthem possessed all the five genes It is plausible that someL monocytogenes strains may lack one or more virulencedeterminants because of spontaneous mutations [11]

Henriques et al [30] detected hly A genes from Lmonocytogenes isolates from Portuguese ready-to-eat meatbased food industry which is similar to the findings ofthis study where hly A was detected in all the nine Lmonocytogenes isolates However the detection of act A genein the work of Henriques et al [30] differs from this studywhich failed to detect act A Listeriolysin O and act Aare associated with the bacteriumrsquos capability of passing theintestinal barrier cell to cell spread andmotility cell invasionand intracellular parasitism [7 18] Therefore simultaneousdetection of virulence genes in a single step will be desirableas it reduces time and labour and it will be useful in largescale survey for detecting virulent strains of Listeria [18]

The separation of Nigerian Listeria monocytogenes iso-lates sequence (NGA34ANGA35ANGA38 andNGA41A)as well as the L monocytogenes sequences from the GenBank(N1-011A R2-502 J1816 J2-031 EDG J1-220 J1926 J1817 andPW040025) into two major lineages A and B is consistentwith the major lineages described previously by Oris et al[31] who reported that L monocytogenes isolates

L monocytogenes consist of at least 4 evolutionary lin-eages (I II III and IV) Even though only cluster A isstatistically supported with a bootstrap value of 100 clusterB is not statistically supported

Most L monocytogenes isolates seem to belong to lineagesI and II which include the serotypes more commonlyassociated with human clinical cases including serotypes12b and 4b (lineage I) and serotype 12a (lineage II) Sinceit is a rooted tree we can infer evolutionary relationshipinformation regarding the common ancestors and the pathof evolution from the rooted phylogenetic tree

Since lineage I L monocytogenes strains are more preva-lent in human clinical listeriosis cases and contaminated food[31] it can be said that Nigerian L monocytogenes isolates(NGA 34A NGA 35A and NGA41A) are from contaminatedfoods (ie milk and milk products) From the phylogenictree Nigerian L monocytogenes isolates (NGA 34A NGA35A and NGA 41A) were classified as lineage I which areknown to be of higher pathogenic potentials due to intrinsicvirulence features as compared to lineage II strains of Lmonocytogenes [32]Morobe et al [33] in their study observedthat most of the L monocytogenes isolates belong to lineageIII (4a and 4c) which are known to be more prevalent inanimals with clinical listeriosis [31] as opposed to this studywhere none of the L monocytogenes isolate were found tobelong to lineage III Isolate NGA 35a was found to belong tolineage II which is opposed to the findings of Morobe et al[33] where they observed that none of the L monocytogenesisolates belong to lineage II

The implication of the present findings is that L mono-cytogenes of lineage 1 originating from animal milk and milkproduct could be associated with increased human risk to Lmonocytogenes infection in Nigeria and thus effort to controlthe disease should be targeted at improving the food safetyand quality

Competing Interests

The authors declare that they have no competing interests

References

[1] M J Gray N E Freitag and K J Boor ldquoHow the bacterialpathogen Listeria monocytogenes mediates the switch fromenvironmental Dr Jekyll to pathogenicMr Hyderdquo Infection andImmunity vol 74 no 5 pp 2506ndash2512 2006

[2] T Hain C Steinweg and T Chakraborty ldquoComparative andfunctional genomics of Listeria spprdquo Journal of Biotechnologyvol 126 no 1 pp 37ndash51 2006

[3] E Rahimi M Ameri and H Momtaz ldquoPrevalence and antimi-crobial resistance of Listeria species isolated from milk and

Canadian Journal of Infectious Diseases and Medical Microbiology 7

dairy products in Iranrdquo Food Control vol 21 no 11 pp 1448ndash1452 2010

[4] B Karen ListeriaMonocytogenes InfectionMedscape LLCNewYork NY USA 2015 httpemedicinemedscapecomarticle220684-overview

[5] F Stavru F Bouillaud A Sartori D Ricquier and P Cos-sart ldquoListeria monocytogenes transiently alters mitochon-drial dynamics during infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no9 pp 3612ndash3617 2011

[6] J A Vazquez-Boland M Kuhn P Berche et al ldquoListeriapathogenesis and molecular virulence determinantsrdquo ClinicalMicrobiology Reviews vol 14 no 3 pp 584ndash640 2001

[7] D Liu M L Lawrence F W Austin and A J AinsworthldquoA multiplex PCR for species- and virulence-specific determi-nation of Listeria monocytogenesrdquo Journal of MicrobiologicalMethods vol 71 no 2 pp 133ndash140 2007

[8] A Camejo F Carvalho O Reis E Leitao S Sousa and DCabanes ldquoThe arsenal of virulence factors deployed by Listeriamonocytogenes to promote its cell infection cyclerdquo Virulencevol 2 no 5 pp 379ndash394 2011

[9] B Joseph and W Goebel ldquoLife of Listeria monocytogenes in thehost cellsrsquo cytosolrdquo Microbes and Infection vol 9 no 10 pp1188ndash1195 2007

[10] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[11] K J Cooray T Nishibori H Xiong T Matsuyama M FujitaandM Mitsuyama ldquoDetection of multiple virulence associatedgenes of Listeriamonocytogenes by PCR in artificially contam-inated milk samplesrdquo Applied Microbiology vol 60 pp 3023ndash3026 1994

[12] S H W Notermans J Dufrenne M Leimeister-Wachter EDomann and T Chakraborty ldquoPhosphatidy-linositol-specificphospholipase C activity as a marker to distinguish betweenpathogenic and non-pathogenic Listeria speciesrdquo Applied andEnvironmental Microbiology vol 57 no 9 pp 2666ndash2670 1991

[13] J E Olsen ldquoDNA-based methods for detection of food-bornebacterial pathogensrdquo Food Research International vol 33 no 3-4 pp 257ndash266 2000

[14] E Walecka J Molenda R Karpıskova and J Bania ldquoEffect ofosmotic stress and culture density on invasiveness of Listeriamonocytogenes strainsrdquo International Journal of Food Microbi-ology vol 144 no 3 pp 440ndash445 2011

[15] M Wiedmann ldquoMolecular subtyping methods for Listeriamonocytogenesrdquo Journal of AOAC International vol 85 no 2pp 524ndash531 2002

[16] G T Jeffers J L Bruce P L McDonough J Scarlett K J Boorand M Wiedmann ldquoComparative genetic characterizationof Listeria monocytogenes isolates from human and animallisteriosis casesrdquo Microbiology vol 147 no 5 pp 1095ndash11042001

[17] C A Nadon D L Woodward C Young F G Rodgers andM Wiedmann ldquoCorrelations between molecular subtypingand serotyping of Listeria monocytogenesrdquo Journal of ClinicalMicrobiology vol 39 no 7 pp 2704ndash2707 2001

[18] D B Rawool SV SMalik I Shakuntala AM Sahare and S BBarbuddhe ldquoDetection of multiple virulence-associated genesin Listeria monocytogenes isolated from bovine mastitis casesrdquoInternational Journal of Food Microbiology vol 113 no 2 pp201ndash207 2007

[19] D Roberts W Hoper and M Greenwood Practical FoodMicrobiology Public Health Laboratory Services London UK1995

[20] M M Janzten J Navas A Corujo R Moreno V Lopez andJ V Martınez-Suarez ldquoReview Specific detection of Listeriamonocytogenes in foods using commercial methods fromchromogenic media to real-time PCRrdquo Spanish Journal ofAgricultural Research vol 4 no 3 pp 235ndash247 2006

[21] P M Border J J Howard G S Plastow and K W SiggensldquoDetection of Listeria species and Listeria monocytogenes usingpolymerase chain reactionrdquo Letters in AppliedMicrobiology vol11 no 3 pp 158ndash162 1990

[22] T A Hall ldquoBioedit a user friendly biological sequence align-ment editor and analysis programme for windows 9598NTrdquoin Nucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[23] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2002

[24] D Liu ldquoIdentification subtyping and virulence determinationof Listeria monocytogenes an important foodborne pathogenrdquoJournal of Medical Microbiology vol 55 no 6 pp 645ndash6592006

[25] C H Kuan S G Goh Y Y Loo et al ldquoPrevalence andquantification of Listeria monocytogenes in chicken offal at theretail level in Malaysiardquo Poultry Science vol 92 no 6 pp 1664ndash1669 2013

[26] P K Jallewar D R Kalorey N V Kurkure V V Pande andS B Barbuddhe ldquoGenotypic characterization of Listeria sppisolated from fresh water fishrdquo International Journal of FoodMicrobiology vol 114 no 1 pp 120ndash123 2007

[27] J Kreft and J A Vazquez-Boland ldquoRegulation of virulencegenes in Listeriardquo International Journal of Medical Microbiologyvol 291 no 2 pp 145ndash157 2001

[28] R R Beumer and W C Hazeleger ldquoListeria monocytogenesdiagnostic problemsrdquo FEMS Immunology and Medical Micro-biology vol 35 no 3 pp 191ndash197 2003

[29] Y H Shuckken Y T Grohn and M Wiedmann ldquoPart VIlisteria monocytogenes epidemiology of listeriosisrdquo in Micro-bial Food Safety in Animal Agriculture Current Topics M ETorrence and R E Isaacson Eds pp 221ndash232Wiley-BlackwellHoboken NJ USA 2003

[30] A R Henriques A S Barreto and M J Fraqueza ldquoDetectionof virulence-associated genes in Listeria monocytogenes isolatesfrom Portuguese ready-to- eat meat-based food industryrdquoApplied and Environmental Microbiology vol 61 pp 817ndash8191998

[31] R H Orsi H C den Bakker and M Wiedmann ldquoListeriamonocytogenes lineages genomics evolution ecology andphenotypic characteristicsrdquo International Journal of MedicalMicrobiology vol 301 no 2 pp 79ndash96 2011

[32] B Swaminathan and P Gerner-Smidt ldquoThe epidemiology ofhuman listeriosisrdquo Microbes and Infection vol 9 no 10 pp1236ndash1243 2007

[33] I C Morobe C L Obi M A Nyila M I Matsheka andB A Gashe ldquoMolecular characterization and serotyping ofListeria monocytogenes with a focus on food safety and diseasepreventionrdquo in Biochemical Testing J C Jimenez-Lopez Edchapter 8 pp 197ndash216 InTech Rijeka Croatia 2012

Submit your manuscripts athttpwwwhindawicom

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Disease Markers

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Canadian Journal of Infectious Diseases and Medical Microbiology 3

Table 1 Listeria monocytogenes characteristics

Snumber Isolate name (code) Isolated from Tip name in tree(1) LMFR151 Raw milk 49A(2) ZKMM83 Manshanu 48A(3) MDGR115 Raw milk 47A(4) MMM103 Manshanu 46A(5) ZKMM84 Manshanu 34A(6) LSNK136 Kindrimo 44A(7) SGMK77 Kindrimo 43A(8) MGDR100 Raw milk 42A(9) ZKMR75 Raw milk 35A(10) SGHR39 Raw milk 38A(11) ZKJR55 Raw milk 40A(12) SGHR7 Raw milk 39A(13) SGHR12 Raw milk 37A(14) SGHR30 Raw milk 41A(15) ZKMM87 Manshanu 36A(16) ZKMM84 Manshanu 45A(17) ZKJR5 Raw milk 33A(18) SGMM14 Manshanu 32A(19) SGMM34 Manshanu 31A(20) SGHR45 Raw milk 30A(21) LMFR161 Raw milk 29A(22) MDGR109 Raw milk 28A(23) SGHR32 Raw milk 27A(24) MDGR10 Raw milk 25A(25) ZKJR10 Raw milk 24A(26) SGSM9 Manshanu 23A(27) LMFR162 Raw milk 15A(28) SGHR55 Raw milk 22A(29) MDGM123 Manshanu 21A(30) SGSK1 Kindrimo 26A(31) SGHR15 Raw milk 20A(32) SGHR37 Raw milk 19A(33) SGSK21 Kindrimo 18A(34) LMFK153 Kindrimo 17A(35) SGMM37 Manshanu 16A(36) SGMM11 Manshanu 14A

distilled water and 2120583L of DNA template PCR amplificationwas carried out in a programmed thermocycler with thefollowing thermal conditions hot start PCR plate at 95∘C for3mins followed by 35 cycles each of 30 sec denaturation at94∘C 15 sec annealing at 53∘C 90 sec extension for 72∘C andfinal extension at 72∘C for 7mins

Following PCR amplification about 5120583L of the PCRproduct was mixed with DNA loading dye (6x) and elec-trophoresed in 10 Agarose gel in TAE buffer using aminitank at 80V 400 amp and 45min The electrophoresedproduct on the gel was stained with ethidium bromidefor 30min destained for 20min and visualized under UVilluminator (SYNGENE Biosystems UK) A 100 bp DNA

ladder (PromegaCorporationUSA)was included to estimatethe size of the amplified products

23 Detection of L monocytogenes Virulence-AssociatedGenes The obtained DNA product was used in performinganother multiplex PCR assay with the aim of detectingthe presence of five virulence-associated genes [hemolysinA gene (hly A) regulatory gene (prf A) actin gene (actA) invasion associated protein p60 gene (iap) and surfaceprotein (inl A)] of L monocytogenes as described by Rawoolet al [18] The primers used for the detection of thesevirulence geneswere synthesized by SigmaAldrich USAThedetails of the primer sequences are shown in Table 2 TheDNA obtained using the Quick-g DNA miniprep-uncappedcolumn kit (Zymo Research) was used for the detection ofvirulence-associated genes

The PCR was standardized for the detection of virulence-associated genes of L monocytogenes by optimizing theannealing temperatures between 50 and 60∘C Based onoptimization trials the standardized PCRprotocol for a 25120583Lreactionmixture included 5120583LPCRbuffer (100mMTrisndashHClbuffer pH 83 containing 500mM KCl 15mM MgCl

2 and

001 gelatine) 1 120583L dNTP mix 3 120583L MgCl2and 05 120583L of

forward and reverse primers 1 120583L of 5 units of Taq DNApolymerase 5 120583LDNA template and 5120583L nuclease free watertomake up the reaction volumeTheDNAamplification reac-tion was performed in aMaster Cycler gradient thermocycler(Eppendorf Hamburg Germany) with a preheated lid inPCR tubes (02mL)The cycling conditions for PCR includedan initial denaturation of DNA at 95∘C for 2min followed by35 cycles each of 15 sec denaturation at 95∘C 30 sec annealingat 53∘C and 90 sec extension at 72∘C Final was carried out in10min at 72∘C and then PCR product held at 4∘C

The resultant PCR products were separated by elec-trophoresis in 15 Agarose gel for 45min at 100V inTris-acetate EDTA buffer stained with ethidium bromide(05 120583gmL) and visualized with a UV transilluminator (Syn-gene Frederick MD)The gel image was documented by a geldocumentation apparatus and DNA size was determined bya 100 bp DNA ladder (Promega)

24 Purification of PCR Products 5 120583L volume PCR productfor each of the 9 hly A and 16S rRNA harbouring L mono-cytogenes positive isolates was analysed on a 15 Agarosegel to confirm successful amplification of the 16S rRNAThe remaining 20 120583L of each of the PCR product was laterrun and each corresponding 16S rRNA band was cut andpurified by using the MEGA quick-spin Total FragmentDNA purification kit (iNtRON Biotechnology Gyeonggi-doSouth Korea)

25 DNA Sequencing and Phylogenetic Analysis Purified gelwas used for DNA sequencing All sequencing reactionswere commercially done by iNtRON (iNtRONBiotechnologyGyeonggi-do South Korea) using the Sangers sequencingmethod Both forward and reverse primers were used toensure adequate sequencing coverage For sequencing andphylogenetic tree only 15 samples were randomly selectedand tested

4 Canadian Journal of Infectious Diseases and Medical Microbiology

Table 2 Primer sequences used for the amplification of virulence-associated genes of Listeria monocytogenes

Target gene Primer sequence (51015840-31015840) Product size (bp)

inl A Fwd 51015840-AGATCTAGACCAAGTTACAACGCTTCAG-31015840Rev 51015840-TAA TAT CAT TTG CTG TTT TAT CTG TC-31015840 255 bp

act A Fwd 510158401015840-ACG TGA AGT AAG TCACGT GAT ATT G 31015840Fwd 510158401015840-ACG TGAAGTAAGCTCACGT GAT ATT G-31015840 268 bp

prf A Fwd 51015840-ACC GCT CAG AAA AGT TCT TC-31015840Rev 51015840-TCT TGT TCT ATT ATGTCT AGC-31015840 1060 bp

iap Fwd 51015840-ACA AGC TGC ACC TGT TGC AG-31015840Rev 5-TGA CAG CGT TGT TAG TAG CA-31015840 131 bp

hly A Fwd 51015840-CCT AAG ACG CCA ATC GAA-31015840Rev 51015840-AAG CGC TTG CAA GTC CTC-31015840 702 bp

The obtained partial 16S rRNA sequences were proof-read and assembled in Chromas Lite version 211 Multiplesequence alignment of the DNA sequences was performedusing ClustalW in Bioedit software version 725 (BiologicalSequence Alignment Editor for Win 9598NT2KXP7)as described by Hall 1999 [22] To compare the sequenceof local L monocytogenes with reference isolates availablein the GenBank database sequence similarity search wasperformedusing theNCBI nucleotide BLAST search to deter-mine the similarity of the local L monocytogenes sequence(httpblastncbinlmnihgov)

Phylogenetic analysis was carried out based on partialsequence of 16S rRNA gene of L monocytogenes In thisregard the aligned sequence was subjected to a MaximumLikelihood (ML) method in MEGA 60 software [23]However prior to performing ML analysis best substitutionmodel that described the sequence data set was obtainedtaking into considerations the lowest Bayesian inferencesEqually 1000 bootstraps values were used to determine theconfidence interval of the resultant tree In order to root thetree a 16S rRNA sequence from Mycobacterium tuberculosiswas used as an out-group

All local L monocytogenes sequences used in the phy-logenetic study were submitted to GenBank for archivingThe accession numbers of these sequences together with thereference isolates are summarised in Table 3

3 Result

31 Detection of hly A Harbouring Listeria monocytogenes Iso-lates The PCR results for the detection of hly A gene amongthe 36 L monocytogenes isolates showed that only 9 (250)(ZKMM84 SGHR30 SGSK1 ZKJR56 ZKJR39 ZKMR75SGMM87 SGHR15 and LMFR162) L monocytogenes isolatesharboured the hly A gene (Figure 1)

32 Detection of Virulence-Associated Genes of Listeria mono-cytogenes Isolates Analysis of multiplex PCR used for thedetection of virulence-associated genes revealed that of the36 L monocytogenes isolates 9 (250) possessed at leastone or more virulence-associated genes (Figure 2) Of the5 virulence-associated genes screened for only 3 virulence-associated genes (hly A iap and prf A) genes were detected

Table 3 Local and reference L monocytogenes isolates used in thephylogenetic analysis

Snumber Strain GenBankaccession number

(1) Listeria monocytogenes NGA 34A KX358070(2) Listeria monocytogenes NGA 35A KX358071(3) Listeria monocytogenes NGA 38A KX358072(4) Listeria monocytogenes NGA 41A KX358073(5) Listeria monocytogenes N1-011A CP00659(6) Listeria monocytogenes J1816 CP006047(7) Listeria monocytogenes J2-031 CP006593(8) Listeria monocytogenes J1-220 CP0060461(9) Listeria monocytogenes J1926 CP006600(10) Listeria monocytogenes J1817 CP006599(11) Listeria monocytogenes R2-502 CP006594(12) Listeria monocytogenes J1776 CP006598(13) Listeria monocytogenes EDG HG421741(14) Listeria monocytogenes FW040025 CP011345(15) Mycobacterium bovis AY360331

each of 5 (138) isolates possessed the iap and prf A genesBased on the sample types 6 (169) of the L monocytogenesisolates that possessed virulence-associated genes were fromraw milk 2 (32) ldquoManshanurdquo and 1 (28) ldquoKindrimordquo

33 Phylogenetic Tree Analysis Phylogenetic analysis wascarried out based on the 16S rRNA sequences encompassing938 bp obtained for 4 of the L monocytogenes isolates(Nigerian isolates) Interestingly maximum likelihood (ML)analysis revealed two different clusters A and B with clusterA comprising of Nigeria L monocytogenes NGA 35A NGA34A andNGA41A and reference Lmonocytogenes J1776 N1-011A R2-502 J1816 and J2-031 while cluster B is comprised ofNigerian L monocytogenes sequence NGA 38A and referenceL monocytogenes sequence EDG J1-220 J1926 J1817 andFW040025 Sequence in cluster A was supported by a strongbootstrap values of 100 in contrast with cluster Bwhich showsa very low bootstrap value of 16 (Figure 3)

Canadian Journal of Infectious Diseases and Medical Microbiology 5

1000 bp700 bp500 bp

1M 2 3 4 5 6 7 8 9 10 11 12 13 14

16S rRNA (938 bp)hly A (702 bp)

Figure 1 Agarose gel electrophoresis showing electrophoretic mobility of PCR products obtained following amplification of the 16S rRNAgene (938 bp) and listeriolysin O encoding gene (hly A) (702 bp) from different Listeria monocytogenes isolated from raw milk and milkproducts Lane M 100 bp ladder Lane 1 positive control (Listeria monocytogenes ATCC 19155) Lane 3 Listeria monocytogenes (LM) isolateZKMM84 Lane 5 LMSGHR30 Lane 6 LMSGSK1 Lane 9 LMZKJR56 Lane 10 LMSGHR39 Lane 11 LMZKMR75 Lane 12 LMSGMM87Lane 13 LM SGHR15 Lane 14 LM LMFR162 corresponding to 938 bp and 702 bp amplicons

1M 2 3 4 5 6 7 8 9 10 11 12

1000 bp

100 bp

500 bp

bp)060bp)702

prf A (1hly A (

bp)131iap (

Figure 2 Agarose gel electrophoresis result for multiplex PCR following amplification of prf A hly A and iap virulence-associated genesLane M molecular weight marker (Promega) in base pairs Lane 1 Listeria monocytogenes (LM) ATTC 19155 Lane 2 LM isolate ZKMM84Lane 3 LM SGHRR30 Lane 4 LM SGSK1 Lane 5 negative control Lane 6 LM ZKJR55 Lane 7 LM SGHR39 Lane 8 LM ZKMR75= Lane9 LM SGMM37 Lane 11 LM SGHR15 Lane 12 LMFR162

L monocytogenes J1776L monocytogenes N1-011A7L monocytogenes R2-502L monocytogenes J1816L monocytogenes J2-031L monocytogenes NGA 41AL monocytogenes NGA 34AL monocytogenes NGA 35A

L monocytogenes NGA 38AL monocytogenes J1-220L monocytogenes EGDL monocytogenes J1926L monocytogenes J1817L monocytogenes FW040025

Mycobacterium bovis

16 7719

100

01

B

A

Figure 3 Molecular phylogenetic tree showing relationship between Nigerian L monocytogenes isolates (red color) with reference sequencesfromGenBank databaseThe evolutionary history was inferred by using theMaximum Likelihoodmethod based on the Tamura 3-parametermodelThe tree with the highest log likelihood (minus27602483) is shownThe percentage of trees in which the associated taxa clustered togetheris shown next to the branch and 1000 bootstrap value was used to assess the tree confidence interval All analyses were performed inMEGA6software

4 Discussion

To ensure food quality and safety detection of pathogenicbacteria should be a fundamental objective The applicationof molecular techniques has facilitated the identification

and characterization of major virulence-associated genes inL monocytogenes [24] In this study the multiplex PCRidentified 9 Listeria monocytogenes isolates harbouring hlyA gene The target genes for L monocytogenes and Listeriagenus produced PCR products of 702 bp and 938 bp in size

6 Canadian Journal of Infectious Diseases and Medical Microbiology

respectively The PCR protocol used in this study was basedon the amplification of hly A gene by using a set of primersLM1 and LM2 Border et al [21] recommended LM1 andLM2 primers as the best primer pair to be used for thedetection of L monocytogenes because they are designed toamplify specific fragments in L monocytogenes strains thatare genetically and biochemically assessed as belonging tothis specie on the basis of the presence of the 702 bp and938 bp amplicons [21]

Kuan et al [25] were also able to amplify the DNA attheir expected amplification site The result of this studyis not in agreement with the result of Jallewar et al [26]who reported the carriage of hly A gene by all the isolatesof L monocytogenes tested The inability of some of the Lmonocytogenes isolates in this study to harbour the hly Agene may be due to the nonvirulent characteristics of some Lmonocytogenes isolates because some may be environmentalisolates or it may be possible that some L monocytogenesstrains may lack one or more virulence determinants becauseof spontaneous mutations [11]

The ability to survive and multiply in the macrophagesand other host cells is key to the pathogenicity of Lmonocytogenes [27] Rapid methods for the isolation andconfirmation of L monocytogenes in foods are still beingexplored [28] Because of the importance of Lmonocytogenesepidemiology to human health and the notable diversity inthe pathogenicity among strains subtyping and virulencecharacterization are of importance [7] Due to the diversestrains of L monocytogenes of varying pathogenicity theability to precisely track the strains involved in listeriosisoutbreaks and rapidly determine their pathogenic potentialsis critical for the control and prevention of further occur-rences of this deadly disease [24] The phenotypic subtypingmethods are generally less sensitive have low differentiationability and are not easy to reproduce whereas the genotypicapproaches are more sensitive and reliable [29] Virulence-associated genes of L monocytogenes namely prf A hlyA and iap were amplified according to their respectivefragment sizes of 1060 bp 702 bp and 131 bp respectivelyThis study revealed that of the 36 L monocytogenes isolatesscreened for virulence-associated genes only 9 (250) iso-lates harboured one or more of such genes and none ofthem possessed all the five genes It is plausible that someL monocytogenes strains may lack one or more virulencedeterminants because of spontaneous mutations [11]

Henriques et al [30] detected hly A genes from Lmonocytogenes isolates from Portuguese ready-to-eat meatbased food industry which is similar to the findings ofthis study where hly A was detected in all the nine Lmonocytogenes isolates However the detection of act A genein the work of Henriques et al [30] differs from this studywhich failed to detect act A Listeriolysin O and act Aare associated with the bacteriumrsquos capability of passing theintestinal barrier cell to cell spread andmotility cell invasionand intracellular parasitism [7 18] Therefore simultaneousdetection of virulence genes in a single step will be desirableas it reduces time and labour and it will be useful in largescale survey for detecting virulent strains of Listeria [18]

The separation of Nigerian Listeria monocytogenes iso-lates sequence (NGA34ANGA35ANGA38 andNGA41A)as well as the L monocytogenes sequences from the GenBank(N1-011A R2-502 J1816 J2-031 EDG J1-220 J1926 J1817 andPW040025) into two major lineages A and B is consistentwith the major lineages described previously by Oris et al[31] who reported that L monocytogenes isolates

L monocytogenes consist of at least 4 evolutionary lin-eages (I II III and IV) Even though only cluster A isstatistically supported with a bootstrap value of 100 clusterB is not statistically supported

Most L monocytogenes isolates seem to belong to lineagesI and II which include the serotypes more commonlyassociated with human clinical cases including serotypes12b and 4b (lineage I) and serotype 12a (lineage II) Sinceit is a rooted tree we can infer evolutionary relationshipinformation regarding the common ancestors and the pathof evolution from the rooted phylogenetic tree

Since lineage I L monocytogenes strains are more preva-lent in human clinical listeriosis cases and contaminated food[31] it can be said that Nigerian L monocytogenes isolates(NGA 34A NGA 35A and NGA41A) are from contaminatedfoods (ie milk and milk products) From the phylogenictree Nigerian L monocytogenes isolates (NGA 34A NGA35A and NGA 41A) were classified as lineage I which areknown to be of higher pathogenic potentials due to intrinsicvirulence features as compared to lineage II strains of Lmonocytogenes [32]Morobe et al [33] in their study observedthat most of the L monocytogenes isolates belong to lineageIII (4a and 4c) which are known to be more prevalent inanimals with clinical listeriosis [31] as opposed to this studywhere none of the L monocytogenes isolate were found tobelong to lineage III Isolate NGA 35a was found to belong tolineage II which is opposed to the findings of Morobe et al[33] where they observed that none of the L monocytogenesisolates belong to lineage II

The implication of the present findings is that L mono-cytogenes of lineage 1 originating from animal milk and milkproduct could be associated with increased human risk to Lmonocytogenes infection in Nigeria and thus effort to controlthe disease should be targeted at improving the food safetyand quality

Competing Interests

The authors declare that they have no competing interests

References

[1] M J Gray N E Freitag and K J Boor ldquoHow the bacterialpathogen Listeria monocytogenes mediates the switch fromenvironmental Dr Jekyll to pathogenicMr Hyderdquo Infection andImmunity vol 74 no 5 pp 2506ndash2512 2006

[2] T Hain C Steinweg and T Chakraborty ldquoComparative andfunctional genomics of Listeria spprdquo Journal of Biotechnologyvol 126 no 1 pp 37ndash51 2006

[3] E Rahimi M Ameri and H Momtaz ldquoPrevalence and antimi-crobial resistance of Listeria species isolated from milk and

Canadian Journal of Infectious Diseases and Medical Microbiology 7

dairy products in Iranrdquo Food Control vol 21 no 11 pp 1448ndash1452 2010

[4] B Karen ListeriaMonocytogenes InfectionMedscape LLCNewYork NY USA 2015 httpemedicinemedscapecomarticle220684-overview

[5] F Stavru F Bouillaud A Sartori D Ricquier and P Cos-sart ldquoListeria monocytogenes transiently alters mitochon-drial dynamics during infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no9 pp 3612ndash3617 2011

[6] J A Vazquez-Boland M Kuhn P Berche et al ldquoListeriapathogenesis and molecular virulence determinantsrdquo ClinicalMicrobiology Reviews vol 14 no 3 pp 584ndash640 2001

[7] D Liu M L Lawrence F W Austin and A J AinsworthldquoA multiplex PCR for species- and virulence-specific determi-nation of Listeria monocytogenesrdquo Journal of MicrobiologicalMethods vol 71 no 2 pp 133ndash140 2007

[8] A Camejo F Carvalho O Reis E Leitao S Sousa and DCabanes ldquoThe arsenal of virulence factors deployed by Listeriamonocytogenes to promote its cell infection cyclerdquo Virulencevol 2 no 5 pp 379ndash394 2011

[9] B Joseph and W Goebel ldquoLife of Listeria monocytogenes in thehost cellsrsquo cytosolrdquo Microbes and Infection vol 9 no 10 pp1188ndash1195 2007

[10] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[11] K J Cooray T Nishibori H Xiong T Matsuyama M FujitaandM Mitsuyama ldquoDetection of multiple virulence associatedgenes of Listeriamonocytogenes by PCR in artificially contam-inated milk samplesrdquo Applied Microbiology vol 60 pp 3023ndash3026 1994

[12] S H W Notermans J Dufrenne M Leimeister-Wachter EDomann and T Chakraborty ldquoPhosphatidy-linositol-specificphospholipase C activity as a marker to distinguish betweenpathogenic and non-pathogenic Listeria speciesrdquo Applied andEnvironmental Microbiology vol 57 no 9 pp 2666ndash2670 1991

[13] J E Olsen ldquoDNA-based methods for detection of food-bornebacterial pathogensrdquo Food Research International vol 33 no 3-4 pp 257ndash266 2000

[14] E Walecka J Molenda R Karpıskova and J Bania ldquoEffect ofosmotic stress and culture density on invasiveness of Listeriamonocytogenes strainsrdquo International Journal of Food Microbi-ology vol 144 no 3 pp 440ndash445 2011

[15] M Wiedmann ldquoMolecular subtyping methods for Listeriamonocytogenesrdquo Journal of AOAC International vol 85 no 2pp 524ndash531 2002

[16] G T Jeffers J L Bruce P L McDonough J Scarlett K J Boorand M Wiedmann ldquoComparative genetic characterizationof Listeria monocytogenes isolates from human and animallisteriosis casesrdquo Microbiology vol 147 no 5 pp 1095ndash11042001

[17] C A Nadon D L Woodward C Young F G Rodgers andM Wiedmann ldquoCorrelations between molecular subtypingand serotyping of Listeria monocytogenesrdquo Journal of ClinicalMicrobiology vol 39 no 7 pp 2704ndash2707 2001

[18] D B Rawool SV SMalik I Shakuntala AM Sahare and S BBarbuddhe ldquoDetection of multiple virulence-associated genesin Listeria monocytogenes isolated from bovine mastitis casesrdquoInternational Journal of Food Microbiology vol 113 no 2 pp201ndash207 2007

[19] D Roberts W Hoper and M Greenwood Practical FoodMicrobiology Public Health Laboratory Services London UK1995

[20] M M Janzten J Navas A Corujo R Moreno V Lopez andJ V Martınez-Suarez ldquoReview Specific detection of Listeriamonocytogenes in foods using commercial methods fromchromogenic media to real-time PCRrdquo Spanish Journal ofAgricultural Research vol 4 no 3 pp 235ndash247 2006

[21] P M Border J J Howard G S Plastow and K W SiggensldquoDetection of Listeria species and Listeria monocytogenes usingpolymerase chain reactionrdquo Letters in AppliedMicrobiology vol11 no 3 pp 158ndash162 1990

[22] T A Hall ldquoBioedit a user friendly biological sequence align-ment editor and analysis programme for windows 9598NTrdquoin Nucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[23] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2002

[24] D Liu ldquoIdentification subtyping and virulence determinationof Listeria monocytogenes an important foodborne pathogenrdquoJournal of Medical Microbiology vol 55 no 6 pp 645ndash6592006

[25] C H Kuan S G Goh Y Y Loo et al ldquoPrevalence andquantification of Listeria monocytogenes in chicken offal at theretail level in Malaysiardquo Poultry Science vol 92 no 6 pp 1664ndash1669 2013

[26] P K Jallewar D R Kalorey N V Kurkure V V Pande andS B Barbuddhe ldquoGenotypic characterization of Listeria sppisolated from fresh water fishrdquo International Journal of FoodMicrobiology vol 114 no 1 pp 120ndash123 2007

[27] J Kreft and J A Vazquez-Boland ldquoRegulation of virulencegenes in Listeriardquo International Journal of Medical Microbiologyvol 291 no 2 pp 145ndash157 2001

[28] R R Beumer and W C Hazeleger ldquoListeria monocytogenesdiagnostic problemsrdquo FEMS Immunology and Medical Micro-biology vol 35 no 3 pp 191ndash197 2003

[29] Y H Shuckken Y T Grohn and M Wiedmann ldquoPart VIlisteria monocytogenes epidemiology of listeriosisrdquo in Micro-bial Food Safety in Animal Agriculture Current Topics M ETorrence and R E Isaacson Eds pp 221ndash232Wiley-BlackwellHoboken NJ USA 2003

[30] A R Henriques A S Barreto and M J Fraqueza ldquoDetectionof virulence-associated genes in Listeria monocytogenes isolatesfrom Portuguese ready-to- eat meat-based food industryrdquoApplied and Environmental Microbiology vol 61 pp 817ndash8191998

[31] R H Orsi H C den Bakker and M Wiedmann ldquoListeriamonocytogenes lineages genomics evolution ecology andphenotypic characteristicsrdquo International Journal of MedicalMicrobiology vol 301 no 2 pp 79ndash96 2011

[32] B Swaminathan and P Gerner-Smidt ldquoThe epidemiology ofhuman listeriosisrdquo Microbes and Infection vol 9 no 10 pp1236ndash1243 2007

[33] I C Morobe C L Obi M A Nyila M I Matsheka andB A Gashe ldquoMolecular characterization and serotyping ofListeria monocytogenes with a focus on food safety and diseasepreventionrdquo in Biochemical Testing J C Jimenez-Lopez Edchapter 8 pp 197ndash216 InTech Rijeka Croatia 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Canadian Journal of Infectious Diseases and Medical Microbiology

Table 2 Primer sequences used for the amplification of virulence-associated genes of Listeria monocytogenes

Target gene Primer sequence (51015840-31015840) Product size (bp)

inl A Fwd 51015840-AGATCTAGACCAAGTTACAACGCTTCAG-31015840Rev 51015840-TAA TAT CAT TTG CTG TTT TAT CTG TC-31015840 255 bp

act A Fwd 510158401015840-ACG TGA AGT AAG TCACGT GAT ATT G 31015840Fwd 510158401015840-ACG TGAAGTAAGCTCACGT GAT ATT G-31015840 268 bp

prf A Fwd 51015840-ACC GCT CAG AAA AGT TCT TC-31015840Rev 51015840-TCT TGT TCT ATT ATGTCT AGC-31015840 1060 bp

iap Fwd 51015840-ACA AGC TGC ACC TGT TGC AG-31015840Rev 5-TGA CAG CGT TGT TAG TAG CA-31015840 131 bp

hly A Fwd 51015840-CCT AAG ACG CCA ATC GAA-31015840Rev 51015840-AAG CGC TTG CAA GTC CTC-31015840 702 bp

The obtained partial 16S rRNA sequences were proof-read and assembled in Chromas Lite version 211 Multiplesequence alignment of the DNA sequences was performedusing ClustalW in Bioedit software version 725 (BiologicalSequence Alignment Editor for Win 9598NT2KXP7)as described by Hall 1999 [22] To compare the sequenceof local L monocytogenes with reference isolates availablein the GenBank database sequence similarity search wasperformedusing theNCBI nucleotide BLAST search to deter-mine the similarity of the local L monocytogenes sequence(httpblastncbinlmnihgov)

Phylogenetic analysis was carried out based on partialsequence of 16S rRNA gene of L monocytogenes In thisregard the aligned sequence was subjected to a MaximumLikelihood (ML) method in MEGA 60 software [23]However prior to performing ML analysis best substitutionmodel that described the sequence data set was obtainedtaking into considerations the lowest Bayesian inferencesEqually 1000 bootstraps values were used to determine theconfidence interval of the resultant tree In order to root thetree a 16S rRNA sequence from Mycobacterium tuberculosiswas used as an out-group

All local L monocytogenes sequences used in the phy-logenetic study were submitted to GenBank for archivingThe accession numbers of these sequences together with thereference isolates are summarised in Table 3

3 Result

31 Detection of hly A Harbouring Listeria monocytogenes Iso-lates The PCR results for the detection of hly A gene amongthe 36 L monocytogenes isolates showed that only 9 (250)(ZKMM84 SGHR30 SGSK1 ZKJR56 ZKJR39 ZKMR75SGMM87 SGHR15 and LMFR162) L monocytogenes isolatesharboured the hly A gene (Figure 1)

32 Detection of Virulence-Associated Genes of Listeria mono-cytogenes Isolates Analysis of multiplex PCR used for thedetection of virulence-associated genes revealed that of the36 L monocytogenes isolates 9 (250) possessed at leastone or more virulence-associated genes (Figure 2) Of the5 virulence-associated genes screened for only 3 virulence-associated genes (hly A iap and prf A) genes were detected

Table 3 Local and reference L monocytogenes isolates used in thephylogenetic analysis

Snumber Strain GenBankaccession number

(1) Listeria monocytogenes NGA 34A KX358070(2) Listeria monocytogenes NGA 35A KX358071(3) Listeria monocytogenes NGA 38A KX358072(4) Listeria monocytogenes NGA 41A KX358073(5) Listeria monocytogenes N1-011A CP00659(6) Listeria monocytogenes J1816 CP006047(7) Listeria monocytogenes J2-031 CP006593(8) Listeria monocytogenes J1-220 CP0060461(9) Listeria monocytogenes J1926 CP006600(10) Listeria monocytogenes J1817 CP006599(11) Listeria monocytogenes R2-502 CP006594(12) Listeria monocytogenes J1776 CP006598(13) Listeria monocytogenes EDG HG421741(14) Listeria monocytogenes FW040025 CP011345(15) Mycobacterium bovis AY360331

each of 5 (138) isolates possessed the iap and prf A genesBased on the sample types 6 (169) of the L monocytogenesisolates that possessed virulence-associated genes were fromraw milk 2 (32) ldquoManshanurdquo and 1 (28) ldquoKindrimordquo

33 Phylogenetic Tree Analysis Phylogenetic analysis wascarried out based on the 16S rRNA sequences encompassing938 bp obtained for 4 of the L monocytogenes isolates(Nigerian isolates) Interestingly maximum likelihood (ML)analysis revealed two different clusters A and B with clusterA comprising of Nigeria L monocytogenes NGA 35A NGA34A andNGA41A and reference Lmonocytogenes J1776 N1-011A R2-502 J1816 and J2-031 while cluster B is comprised ofNigerian L monocytogenes sequence NGA 38A and referenceL monocytogenes sequence EDG J1-220 J1926 J1817 andFW040025 Sequence in cluster A was supported by a strongbootstrap values of 100 in contrast with cluster Bwhich showsa very low bootstrap value of 16 (Figure 3)

Canadian Journal of Infectious Diseases and Medical Microbiology 5

1000 bp700 bp500 bp

1M 2 3 4 5 6 7 8 9 10 11 12 13 14

16S rRNA (938 bp)hly A (702 bp)

Figure 1 Agarose gel electrophoresis showing electrophoretic mobility of PCR products obtained following amplification of the 16S rRNAgene (938 bp) and listeriolysin O encoding gene (hly A) (702 bp) from different Listeria monocytogenes isolated from raw milk and milkproducts Lane M 100 bp ladder Lane 1 positive control (Listeria monocytogenes ATCC 19155) Lane 3 Listeria monocytogenes (LM) isolateZKMM84 Lane 5 LMSGHR30 Lane 6 LMSGSK1 Lane 9 LMZKJR56 Lane 10 LMSGHR39 Lane 11 LMZKMR75 Lane 12 LMSGMM87Lane 13 LM SGHR15 Lane 14 LM LMFR162 corresponding to 938 bp and 702 bp amplicons

1M 2 3 4 5 6 7 8 9 10 11 12

1000 bp

100 bp

500 bp

bp)060bp)702

prf A (1hly A (

bp)131iap (

Figure 2 Agarose gel electrophoresis result for multiplex PCR following amplification of prf A hly A and iap virulence-associated genesLane M molecular weight marker (Promega) in base pairs Lane 1 Listeria monocytogenes (LM) ATTC 19155 Lane 2 LM isolate ZKMM84Lane 3 LM SGHRR30 Lane 4 LM SGSK1 Lane 5 negative control Lane 6 LM ZKJR55 Lane 7 LM SGHR39 Lane 8 LM ZKMR75= Lane9 LM SGMM37 Lane 11 LM SGHR15 Lane 12 LMFR162

L monocytogenes J1776L monocytogenes N1-011A7L monocytogenes R2-502L monocytogenes J1816L monocytogenes J2-031L monocytogenes NGA 41AL monocytogenes NGA 34AL monocytogenes NGA 35A

L monocytogenes NGA 38AL monocytogenes J1-220L monocytogenes EGDL monocytogenes J1926L monocytogenes J1817L monocytogenes FW040025

Mycobacterium bovis

16 7719

100

01

B

A

Figure 3 Molecular phylogenetic tree showing relationship between Nigerian L monocytogenes isolates (red color) with reference sequencesfromGenBank databaseThe evolutionary history was inferred by using theMaximum Likelihoodmethod based on the Tamura 3-parametermodelThe tree with the highest log likelihood (minus27602483) is shownThe percentage of trees in which the associated taxa clustered togetheris shown next to the branch and 1000 bootstrap value was used to assess the tree confidence interval All analyses were performed inMEGA6software

4 Discussion

To ensure food quality and safety detection of pathogenicbacteria should be a fundamental objective The applicationof molecular techniques has facilitated the identification

and characterization of major virulence-associated genes inL monocytogenes [24] In this study the multiplex PCRidentified 9 Listeria monocytogenes isolates harbouring hlyA gene The target genes for L monocytogenes and Listeriagenus produced PCR products of 702 bp and 938 bp in size

6 Canadian Journal of Infectious Diseases and Medical Microbiology

respectively The PCR protocol used in this study was basedon the amplification of hly A gene by using a set of primersLM1 and LM2 Border et al [21] recommended LM1 andLM2 primers as the best primer pair to be used for thedetection of L monocytogenes because they are designed toamplify specific fragments in L monocytogenes strains thatare genetically and biochemically assessed as belonging tothis specie on the basis of the presence of the 702 bp and938 bp amplicons [21]

Kuan et al [25] were also able to amplify the DNA attheir expected amplification site The result of this studyis not in agreement with the result of Jallewar et al [26]who reported the carriage of hly A gene by all the isolatesof L monocytogenes tested The inability of some of the Lmonocytogenes isolates in this study to harbour the hly Agene may be due to the nonvirulent characteristics of some Lmonocytogenes isolates because some may be environmentalisolates or it may be possible that some L monocytogenesstrains may lack one or more virulence determinants becauseof spontaneous mutations [11]

The ability to survive and multiply in the macrophagesand other host cells is key to the pathogenicity of Lmonocytogenes [27] Rapid methods for the isolation andconfirmation of L monocytogenes in foods are still beingexplored [28] Because of the importance of Lmonocytogenesepidemiology to human health and the notable diversity inthe pathogenicity among strains subtyping and virulencecharacterization are of importance [7] Due to the diversestrains of L monocytogenes of varying pathogenicity theability to precisely track the strains involved in listeriosisoutbreaks and rapidly determine their pathogenic potentialsis critical for the control and prevention of further occur-rences of this deadly disease [24] The phenotypic subtypingmethods are generally less sensitive have low differentiationability and are not easy to reproduce whereas the genotypicapproaches are more sensitive and reliable [29] Virulence-associated genes of L monocytogenes namely prf A hlyA and iap were amplified according to their respectivefragment sizes of 1060 bp 702 bp and 131 bp respectivelyThis study revealed that of the 36 L monocytogenes isolatesscreened for virulence-associated genes only 9 (250) iso-lates harboured one or more of such genes and none ofthem possessed all the five genes It is plausible that someL monocytogenes strains may lack one or more virulencedeterminants because of spontaneous mutations [11]

Henriques et al [30] detected hly A genes from Lmonocytogenes isolates from Portuguese ready-to-eat meatbased food industry which is similar to the findings ofthis study where hly A was detected in all the nine Lmonocytogenes isolates However the detection of act A genein the work of Henriques et al [30] differs from this studywhich failed to detect act A Listeriolysin O and act Aare associated with the bacteriumrsquos capability of passing theintestinal barrier cell to cell spread andmotility cell invasionand intracellular parasitism [7 18] Therefore simultaneousdetection of virulence genes in a single step will be desirableas it reduces time and labour and it will be useful in largescale survey for detecting virulent strains of Listeria [18]

The separation of Nigerian Listeria monocytogenes iso-lates sequence (NGA34ANGA35ANGA38 andNGA41A)as well as the L monocytogenes sequences from the GenBank(N1-011A R2-502 J1816 J2-031 EDG J1-220 J1926 J1817 andPW040025) into two major lineages A and B is consistentwith the major lineages described previously by Oris et al[31] who reported that L monocytogenes isolates

L monocytogenes consist of at least 4 evolutionary lin-eages (I II III and IV) Even though only cluster A isstatistically supported with a bootstrap value of 100 clusterB is not statistically supported

Most L monocytogenes isolates seem to belong to lineagesI and II which include the serotypes more commonlyassociated with human clinical cases including serotypes12b and 4b (lineage I) and serotype 12a (lineage II) Sinceit is a rooted tree we can infer evolutionary relationshipinformation regarding the common ancestors and the pathof evolution from the rooted phylogenetic tree

Since lineage I L monocytogenes strains are more preva-lent in human clinical listeriosis cases and contaminated food[31] it can be said that Nigerian L monocytogenes isolates(NGA 34A NGA 35A and NGA41A) are from contaminatedfoods (ie milk and milk products) From the phylogenictree Nigerian L monocytogenes isolates (NGA 34A NGA35A and NGA 41A) were classified as lineage I which areknown to be of higher pathogenic potentials due to intrinsicvirulence features as compared to lineage II strains of Lmonocytogenes [32]Morobe et al [33] in their study observedthat most of the L monocytogenes isolates belong to lineageIII (4a and 4c) which are known to be more prevalent inanimals with clinical listeriosis [31] as opposed to this studywhere none of the L monocytogenes isolate were found tobelong to lineage III Isolate NGA 35a was found to belong tolineage II which is opposed to the findings of Morobe et al[33] where they observed that none of the L monocytogenesisolates belong to lineage II

The implication of the present findings is that L mono-cytogenes of lineage 1 originating from animal milk and milkproduct could be associated with increased human risk to Lmonocytogenes infection in Nigeria and thus effort to controlthe disease should be targeted at improving the food safetyand quality

Competing Interests

The authors declare that they have no competing interests

References

[1] M J Gray N E Freitag and K J Boor ldquoHow the bacterialpathogen Listeria monocytogenes mediates the switch fromenvironmental Dr Jekyll to pathogenicMr Hyderdquo Infection andImmunity vol 74 no 5 pp 2506ndash2512 2006

[2] T Hain C Steinweg and T Chakraborty ldquoComparative andfunctional genomics of Listeria spprdquo Journal of Biotechnologyvol 126 no 1 pp 37ndash51 2006

[3] E Rahimi M Ameri and H Momtaz ldquoPrevalence and antimi-crobial resistance of Listeria species isolated from milk and

Canadian Journal of Infectious Diseases and Medical Microbiology 7

dairy products in Iranrdquo Food Control vol 21 no 11 pp 1448ndash1452 2010

[4] B Karen ListeriaMonocytogenes InfectionMedscape LLCNewYork NY USA 2015 httpemedicinemedscapecomarticle220684-overview

[5] F Stavru F Bouillaud A Sartori D Ricquier and P Cos-sart ldquoListeria monocytogenes transiently alters mitochon-drial dynamics during infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no9 pp 3612ndash3617 2011

[6] J A Vazquez-Boland M Kuhn P Berche et al ldquoListeriapathogenesis and molecular virulence determinantsrdquo ClinicalMicrobiology Reviews vol 14 no 3 pp 584ndash640 2001

[7] D Liu M L Lawrence F W Austin and A J AinsworthldquoA multiplex PCR for species- and virulence-specific determi-nation of Listeria monocytogenesrdquo Journal of MicrobiologicalMethods vol 71 no 2 pp 133ndash140 2007

[8] A Camejo F Carvalho O Reis E Leitao S Sousa and DCabanes ldquoThe arsenal of virulence factors deployed by Listeriamonocytogenes to promote its cell infection cyclerdquo Virulencevol 2 no 5 pp 379ndash394 2011

[9] B Joseph and W Goebel ldquoLife of Listeria monocytogenes in thehost cellsrsquo cytosolrdquo Microbes and Infection vol 9 no 10 pp1188ndash1195 2007

[10] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[11] K J Cooray T Nishibori H Xiong T Matsuyama M FujitaandM Mitsuyama ldquoDetection of multiple virulence associatedgenes of Listeriamonocytogenes by PCR in artificially contam-inated milk samplesrdquo Applied Microbiology vol 60 pp 3023ndash3026 1994

[12] S H W Notermans J Dufrenne M Leimeister-Wachter EDomann and T Chakraborty ldquoPhosphatidy-linositol-specificphospholipase C activity as a marker to distinguish betweenpathogenic and non-pathogenic Listeria speciesrdquo Applied andEnvironmental Microbiology vol 57 no 9 pp 2666ndash2670 1991

[13] J E Olsen ldquoDNA-based methods for detection of food-bornebacterial pathogensrdquo Food Research International vol 33 no 3-4 pp 257ndash266 2000

[14] E Walecka J Molenda R Karpıskova and J Bania ldquoEffect ofosmotic stress and culture density on invasiveness of Listeriamonocytogenes strainsrdquo International Journal of Food Microbi-ology vol 144 no 3 pp 440ndash445 2011

[15] M Wiedmann ldquoMolecular subtyping methods for Listeriamonocytogenesrdquo Journal of AOAC International vol 85 no 2pp 524ndash531 2002

[16] G T Jeffers J L Bruce P L McDonough J Scarlett K J Boorand M Wiedmann ldquoComparative genetic characterizationof Listeria monocytogenes isolates from human and animallisteriosis casesrdquo Microbiology vol 147 no 5 pp 1095ndash11042001

[17] C A Nadon D L Woodward C Young F G Rodgers andM Wiedmann ldquoCorrelations between molecular subtypingand serotyping of Listeria monocytogenesrdquo Journal of ClinicalMicrobiology vol 39 no 7 pp 2704ndash2707 2001

[18] D B Rawool SV SMalik I Shakuntala AM Sahare and S BBarbuddhe ldquoDetection of multiple virulence-associated genesin Listeria monocytogenes isolated from bovine mastitis casesrdquoInternational Journal of Food Microbiology vol 113 no 2 pp201ndash207 2007

[19] D Roberts W Hoper and M Greenwood Practical FoodMicrobiology Public Health Laboratory Services London UK1995

[20] M M Janzten J Navas A Corujo R Moreno V Lopez andJ V Martınez-Suarez ldquoReview Specific detection of Listeriamonocytogenes in foods using commercial methods fromchromogenic media to real-time PCRrdquo Spanish Journal ofAgricultural Research vol 4 no 3 pp 235ndash247 2006

[21] P M Border J J Howard G S Plastow and K W SiggensldquoDetection of Listeria species and Listeria monocytogenes usingpolymerase chain reactionrdquo Letters in AppliedMicrobiology vol11 no 3 pp 158ndash162 1990

[22] T A Hall ldquoBioedit a user friendly biological sequence align-ment editor and analysis programme for windows 9598NTrdquoin Nucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[23] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2002

[24] D Liu ldquoIdentification subtyping and virulence determinationof Listeria monocytogenes an important foodborne pathogenrdquoJournal of Medical Microbiology vol 55 no 6 pp 645ndash6592006

[25] C H Kuan S G Goh Y Y Loo et al ldquoPrevalence andquantification of Listeria monocytogenes in chicken offal at theretail level in Malaysiardquo Poultry Science vol 92 no 6 pp 1664ndash1669 2013

[26] P K Jallewar D R Kalorey N V Kurkure V V Pande andS B Barbuddhe ldquoGenotypic characterization of Listeria sppisolated from fresh water fishrdquo International Journal of FoodMicrobiology vol 114 no 1 pp 120ndash123 2007

[27] J Kreft and J A Vazquez-Boland ldquoRegulation of virulencegenes in Listeriardquo International Journal of Medical Microbiologyvol 291 no 2 pp 145ndash157 2001

[28] R R Beumer and W C Hazeleger ldquoListeria monocytogenesdiagnostic problemsrdquo FEMS Immunology and Medical Micro-biology vol 35 no 3 pp 191ndash197 2003

[29] Y H Shuckken Y T Grohn and M Wiedmann ldquoPart VIlisteria monocytogenes epidemiology of listeriosisrdquo in Micro-bial Food Safety in Animal Agriculture Current Topics M ETorrence and R E Isaacson Eds pp 221ndash232Wiley-BlackwellHoboken NJ USA 2003

[30] A R Henriques A S Barreto and M J Fraqueza ldquoDetectionof virulence-associated genes in Listeria monocytogenes isolatesfrom Portuguese ready-to- eat meat-based food industryrdquoApplied and Environmental Microbiology vol 61 pp 817ndash8191998

[31] R H Orsi H C den Bakker and M Wiedmann ldquoListeriamonocytogenes lineages genomics evolution ecology andphenotypic characteristicsrdquo International Journal of MedicalMicrobiology vol 301 no 2 pp 79ndash96 2011

[32] B Swaminathan and P Gerner-Smidt ldquoThe epidemiology ofhuman listeriosisrdquo Microbes and Infection vol 9 no 10 pp1236ndash1243 2007

[33] I C Morobe C L Obi M A Nyila M I Matsheka andB A Gashe ldquoMolecular characterization and serotyping ofListeria monocytogenes with a focus on food safety and diseasepreventionrdquo in Biochemical Testing J C Jimenez-Lopez Edchapter 8 pp 197ndash216 InTech Rijeka Croatia 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Canadian Journal of Infectious Diseases and Medical Microbiology 5

1000 bp700 bp500 bp

1M 2 3 4 5 6 7 8 9 10 11 12 13 14

16S rRNA (938 bp)hly A (702 bp)

Figure 1 Agarose gel electrophoresis showing electrophoretic mobility of PCR products obtained following amplification of the 16S rRNAgene (938 bp) and listeriolysin O encoding gene (hly A) (702 bp) from different Listeria monocytogenes isolated from raw milk and milkproducts Lane M 100 bp ladder Lane 1 positive control (Listeria monocytogenes ATCC 19155) Lane 3 Listeria monocytogenes (LM) isolateZKMM84 Lane 5 LMSGHR30 Lane 6 LMSGSK1 Lane 9 LMZKJR56 Lane 10 LMSGHR39 Lane 11 LMZKMR75 Lane 12 LMSGMM87Lane 13 LM SGHR15 Lane 14 LM LMFR162 corresponding to 938 bp and 702 bp amplicons

1M 2 3 4 5 6 7 8 9 10 11 12

1000 bp

100 bp

500 bp

bp)060bp)702

prf A (1hly A (

bp)131iap (

Figure 2 Agarose gel electrophoresis result for multiplex PCR following amplification of prf A hly A and iap virulence-associated genesLane M molecular weight marker (Promega) in base pairs Lane 1 Listeria monocytogenes (LM) ATTC 19155 Lane 2 LM isolate ZKMM84Lane 3 LM SGHRR30 Lane 4 LM SGSK1 Lane 5 negative control Lane 6 LM ZKJR55 Lane 7 LM SGHR39 Lane 8 LM ZKMR75= Lane9 LM SGMM37 Lane 11 LM SGHR15 Lane 12 LMFR162

L monocytogenes J1776L monocytogenes N1-011A7L monocytogenes R2-502L monocytogenes J1816L monocytogenes J2-031L monocytogenes NGA 41AL monocytogenes NGA 34AL monocytogenes NGA 35A

L monocytogenes NGA 38AL monocytogenes J1-220L monocytogenes EGDL monocytogenes J1926L monocytogenes J1817L monocytogenes FW040025

Mycobacterium bovis

16 7719

100

01

B

A

Figure 3 Molecular phylogenetic tree showing relationship between Nigerian L monocytogenes isolates (red color) with reference sequencesfromGenBank databaseThe evolutionary history was inferred by using theMaximum Likelihoodmethod based on the Tamura 3-parametermodelThe tree with the highest log likelihood (minus27602483) is shownThe percentage of trees in which the associated taxa clustered togetheris shown next to the branch and 1000 bootstrap value was used to assess the tree confidence interval All analyses were performed inMEGA6software

4 Discussion

To ensure food quality and safety detection of pathogenicbacteria should be a fundamental objective The applicationof molecular techniques has facilitated the identification

and characterization of major virulence-associated genes inL monocytogenes [24] In this study the multiplex PCRidentified 9 Listeria monocytogenes isolates harbouring hlyA gene The target genes for L monocytogenes and Listeriagenus produced PCR products of 702 bp and 938 bp in size

6 Canadian Journal of Infectious Diseases and Medical Microbiology

respectively The PCR protocol used in this study was basedon the amplification of hly A gene by using a set of primersLM1 and LM2 Border et al [21] recommended LM1 andLM2 primers as the best primer pair to be used for thedetection of L monocytogenes because they are designed toamplify specific fragments in L monocytogenes strains thatare genetically and biochemically assessed as belonging tothis specie on the basis of the presence of the 702 bp and938 bp amplicons [21]

Kuan et al [25] were also able to amplify the DNA attheir expected amplification site The result of this studyis not in agreement with the result of Jallewar et al [26]who reported the carriage of hly A gene by all the isolatesof L monocytogenes tested The inability of some of the Lmonocytogenes isolates in this study to harbour the hly Agene may be due to the nonvirulent characteristics of some Lmonocytogenes isolates because some may be environmentalisolates or it may be possible that some L monocytogenesstrains may lack one or more virulence determinants becauseof spontaneous mutations [11]

The ability to survive and multiply in the macrophagesand other host cells is key to the pathogenicity of Lmonocytogenes [27] Rapid methods for the isolation andconfirmation of L monocytogenes in foods are still beingexplored [28] Because of the importance of Lmonocytogenesepidemiology to human health and the notable diversity inthe pathogenicity among strains subtyping and virulencecharacterization are of importance [7] Due to the diversestrains of L monocytogenes of varying pathogenicity theability to precisely track the strains involved in listeriosisoutbreaks and rapidly determine their pathogenic potentialsis critical for the control and prevention of further occur-rences of this deadly disease [24] The phenotypic subtypingmethods are generally less sensitive have low differentiationability and are not easy to reproduce whereas the genotypicapproaches are more sensitive and reliable [29] Virulence-associated genes of L monocytogenes namely prf A hlyA and iap were amplified according to their respectivefragment sizes of 1060 bp 702 bp and 131 bp respectivelyThis study revealed that of the 36 L monocytogenes isolatesscreened for virulence-associated genes only 9 (250) iso-lates harboured one or more of such genes and none ofthem possessed all the five genes It is plausible that someL monocytogenes strains may lack one or more virulencedeterminants because of spontaneous mutations [11]

Henriques et al [30] detected hly A genes from Lmonocytogenes isolates from Portuguese ready-to-eat meatbased food industry which is similar to the findings ofthis study where hly A was detected in all the nine Lmonocytogenes isolates However the detection of act A genein the work of Henriques et al [30] differs from this studywhich failed to detect act A Listeriolysin O and act Aare associated with the bacteriumrsquos capability of passing theintestinal barrier cell to cell spread andmotility cell invasionand intracellular parasitism [7 18] Therefore simultaneousdetection of virulence genes in a single step will be desirableas it reduces time and labour and it will be useful in largescale survey for detecting virulent strains of Listeria [18]

The separation of Nigerian Listeria monocytogenes iso-lates sequence (NGA34ANGA35ANGA38 andNGA41A)as well as the L monocytogenes sequences from the GenBank(N1-011A R2-502 J1816 J2-031 EDG J1-220 J1926 J1817 andPW040025) into two major lineages A and B is consistentwith the major lineages described previously by Oris et al[31] who reported that L monocytogenes isolates

L monocytogenes consist of at least 4 evolutionary lin-eages (I II III and IV) Even though only cluster A isstatistically supported with a bootstrap value of 100 clusterB is not statistically supported

Most L monocytogenes isolates seem to belong to lineagesI and II which include the serotypes more commonlyassociated with human clinical cases including serotypes12b and 4b (lineage I) and serotype 12a (lineage II) Sinceit is a rooted tree we can infer evolutionary relationshipinformation regarding the common ancestors and the pathof evolution from the rooted phylogenetic tree

Since lineage I L monocytogenes strains are more preva-lent in human clinical listeriosis cases and contaminated food[31] it can be said that Nigerian L monocytogenes isolates(NGA 34A NGA 35A and NGA41A) are from contaminatedfoods (ie milk and milk products) From the phylogenictree Nigerian L monocytogenes isolates (NGA 34A NGA35A and NGA 41A) were classified as lineage I which areknown to be of higher pathogenic potentials due to intrinsicvirulence features as compared to lineage II strains of Lmonocytogenes [32]Morobe et al [33] in their study observedthat most of the L monocytogenes isolates belong to lineageIII (4a and 4c) which are known to be more prevalent inanimals with clinical listeriosis [31] as opposed to this studywhere none of the L monocytogenes isolate were found tobelong to lineage III Isolate NGA 35a was found to belong tolineage II which is opposed to the findings of Morobe et al[33] where they observed that none of the L monocytogenesisolates belong to lineage II

The implication of the present findings is that L mono-cytogenes of lineage 1 originating from animal milk and milkproduct could be associated with increased human risk to Lmonocytogenes infection in Nigeria and thus effort to controlthe disease should be targeted at improving the food safetyand quality

Competing Interests

The authors declare that they have no competing interests

References

[1] M J Gray N E Freitag and K J Boor ldquoHow the bacterialpathogen Listeria monocytogenes mediates the switch fromenvironmental Dr Jekyll to pathogenicMr Hyderdquo Infection andImmunity vol 74 no 5 pp 2506ndash2512 2006

[2] T Hain C Steinweg and T Chakraborty ldquoComparative andfunctional genomics of Listeria spprdquo Journal of Biotechnologyvol 126 no 1 pp 37ndash51 2006

[3] E Rahimi M Ameri and H Momtaz ldquoPrevalence and antimi-crobial resistance of Listeria species isolated from milk and

Canadian Journal of Infectious Diseases and Medical Microbiology 7

dairy products in Iranrdquo Food Control vol 21 no 11 pp 1448ndash1452 2010

[4] B Karen ListeriaMonocytogenes InfectionMedscape LLCNewYork NY USA 2015 httpemedicinemedscapecomarticle220684-overview

[5] F Stavru F Bouillaud A Sartori D Ricquier and P Cos-sart ldquoListeria monocytogenes transiently alters mitochon-drial dynamics during infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no9 pp 3612ndash3617 2011

[6] J A Vazquez-Boland M Kuhn P Berche et al ldquoListeriapathogenesis and molecular virulence determinantsrdquo ClinicalMicrobiology Reviews vol 14 no 3 pp 584ndash640 2001

[7] D Liu M L Lawrence F W Austin and A J AinsworthldquoA multiplex PCR for species- and virulence-specific determi-nation of Listeria monocytogenesrdquo Journal of MicrobiologicalMethods vol 71 no 2 pp 133ndash140 2007

[8] A Camejo F Carvalho O Reis E Leitao S Sousa and DCabanes ldquoThe arsenal of virulence factors deployed by Listeriamonocytogenes to promote its cell infection cyclerdquo Virulencevol 2 no 5 pp 379ndash394 2011

[9] B Joseph and W Goebel ldquoLife of Listeria monocytogenes in thehost cellsrsquo cytosolrdquo Microbes and Infection vol 9 no 10 pp1188ndash1195 2007

[10] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[11] K J Cooray T Nishibori H Xiong T Matsuyama M FujitaandM Mitsuyama ldquoDetection of multiple virulence associatedgenes of Listeriamonocytogenes by PCR in artificially contam-inated milk samplesrdquo Applied Microbiology vol 60 pp 3023ndash3026 1994

[12] S H W Notermans J Dufrenne M Leimeister-Wachter EDomann and T Chakraborty ldquoPhosphatidy-linositol-specificphospholipase C activity as a marker to distinguish betweenpathogenic and non-pathogenic Listeria speciesrdquo Applied andEnvironmental Microbiology vol 57 no 9 pp 2666ndash2670 1991

[13] J E Olsen ldquoDNA-based methods for detection of food-bornebacterial pathogensrdquo Food Research International vol 33 no 3-4 pp 257ndash266 2000

[14] E Walecka J Molenda R Karpıskova and J Bania ldquoEffect ofosmotic stress and culture density on invasiveness of Listeriamonocytogenes strainsrdquo International Journal of Food Microbi-ology vol 144 no 3 pp 440ndash445 2011

[15] M Wiedmann ldquoMolecular subtyping methods for Listeriamonocytogenesrdquo Journal of AOAC International vol 85 no 2pp 524ndash531 2002

[16] G T Jeffers J L Bruce P L McDonough J Scarlett K J Boorand M Wiedmann ldquoComparative genetic characterizationof Listeria monocytogenes isolates from human and animallisteriosis casesrdquo Microbiology vol 147 no 5 pp 1095ndash11042001

[17] C A Nadon D L Woodward C Young F G Rodgers andM Wiedmann ldquoCorrelations between molecular subtypingand serotyping of Listeria monocytogenesrdquo Journal of ClinicalMicrobiology vol 39 no 7 pp 2704ndash2707 2001

[18] D B Rawool SV SMalik I Shakuntala AM Sahare and S BBarbuddhe ldquoDetection of multiple virulence-associated genesin Listeria monocytogenes isolated from bovine mastitis casesrdquoInternational Journal of Food Microbiology vol 113 no 2 pp201ndash207 2007

[19] D Roberts W Hoper and M Greenwood Practical FoodMicrobiology Public Health Laboratory Services London UK1995

[20] M M Janzten J Navas A Corujo R Moreno V Lopez andJ V Martınez-Suarez ldquoReview Specific detection of Listeriamonocytogenes in foods using commercial methods fromchromogenic media to real-time PCRrdquo Spanish Journal ofAgricultural Research vol 4 no 3 pp 235ndash247 2006

[21] P M Border J J Howard G S Plastow and K W SiggensldquoDetection of Listeria species and Listeria monocytogenes usingpolymerase chain reactionrdquo Letters in AppliedMicrobiology vol11 no 3 pp 158ndash162 1990

[22] T A Hall ldquoBioedit a user friendly biological sequence align-ment editor and analysis programme for windows 9598NTrdquoin Nucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[23] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2002

[24] D Liu ldquoIdentification subtyping and virulence determinationof Listeria monocytogenes an important foodborne pathogenrdquoJournal of Medical Microbiology vol 55 no 6 pp 645ndash6592006

[25] C H Kuan S G Goh Y Y Loo et al ldquoPrevalence andquantification of Listeria monocytogenes in chicken offal at theretail level in Malaysiardquo Poultry Science vol 92 no 6 pp 1664ndash1669 2013

[26] P K Jallewar D R Kalorey N V Kurkure V V Pande andS B Barbuddhe ldquoGenotypic characterization of Listeria sppisolated from fresh water fishrdquo International Journal of FoodMicrobiology vol 114 no 1 pp 120ndash123 2007

[27] J Kreft and J A Vazquez-Boland ldquoRegulation of virulencegenes in Listeriardquo International Journal of Medical Microbiologyvol 291 no 2 pp 145ndash157 2001

[28] R R Beumer and W C Hazeleger ldquoListeria monocytogenesdiagnostic problemsrdquo FEMS Immunology and Medical Micro-biology vol 35 no 3 pp 191ndash197 2003

[29] Y H Shuckken Y T Grohn and M Wiedmann ldquoPart VIlisteria monocytogenes epidemiology of listeriosisrdquo in Micro-bial Food Safety in Animal Agriculture Current Topics M ETorrence and R E Isaacson Eds pp 221ndash232Wiley-BlackwellHoboken NJ USA 2003

[30] A R Henriques A S Barreto and M J Fraqueza ldquoDetectionof virulence-associated genes in Listeria monocytogenes isolatesfrom Portuguese ready-to- eat meat-based food industryrdquoApplied and Environmental Microbiology vol 61 pp 817ndash8191998

[31] R H Orsi H C den Bakker and M Wiedmann ldquoListeriamonocytogenes lineages genomics evolution ecology andphenotypic characteristicsrdquo International Journal of MedicalMicrobiology vol 301 no 2 pp 79ndash96 2011

[32] B Swaminathan and P Gerner-Smidt ldquoThe epidemiology ofhuman listeriosisrdquo Microbes and Infection vol 9 no 10 pp1236ndash1243 2007

[33] I C Morobe C L Obi M A Nyila M I Matsheka andB A Gashe ldquoMolecular characterization and serotyping ofListeria monocytogenes with a focus on food safety and diseasepreventionrdquo in Biochemical Testing J C Jimenez-Lopez Edchapter 8 pp 197ndash216 InTech Rijeka Croatia 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Canadian Journal of Infectious Diseases and Medical Microbiology

respectively The PCR protocol used in this study was basedon the amplification of hly A gene by using a set of primersLM1 and LM2 Border et al [21] recommended LM1 andLM2 primers as the best primer pair to be used for thedetection of L monocytogenes because they are designed toamplify specific fragments in L monocytogenes strains thatare genetically and biochemically assessed as belonging tothis specie on the basis of the presence of the 702 bp and938 bp amplicons [21]

Kuan et al [25] were also able to amplify the DNA attheir expected amplification site The result of this studyis not in agreement with the result of Jallewar et al [26]who reported the carriage of hly A gene by all the isolatesof L monocytogenes tested The inability of some of the Lmonocytogenes isolates in this study to harbour the hly Agene may be due to the nonvirulent characteristics of some Lmonocytogenes isolates because some may be environmentalisolates or it may be possible that some L monocytogenesstrains may lack one or more virulence determinants becauseof spontaneous mutations [11]

The ability to survive and multiply in the macrophagesand other host cells is key to the pathogenicity of Lmonocytogenes [27] Rapid methods for the isolation andconfirmation of L monocytogenes in foods are still beingexplored [28] Because of the importance of Lmonocytogenesepidemiology to human health and the notable diversity inthe pathogenicity among strains subtyping and virulencecharacterization are of importance [7] Due to the diversestrains of L monocytogenes of varying pathogenicity theability to precisely track the strains involved in listeriosisoutbreaks and rapidly determine their pathogenic potentialsis critical for the control and prevention of further occur-rences of this deadly disease [24] The phenotypic subtypingmethods are generally less sensitive have low differentiationability and are not easy to reproduce whereas the genotypicapproaches are more sensitive and reliable [29] Virulence-associated genes of L monocytogenes namely prf A hlyA and iap were amplified according to their respectivefragment sizes of 1060 bp 702 bp and 131 bp respectivelyThis study revealed that of the 36 L monocytogenes isolatesscreened for virulence-associated genes only 9 (250) iso-lates harboured one or more of such genes and none ofthem possessed all the five genes It is plausible that someL monocytogenes strains may lack one or more virulencedeterminants because of spontaneous mutations [11]

Henriques et al [30] detected hly A genes from Lmonocytogenes isolates from Portuguese ready-to-eat meatbased food industry which is similar to the findings ofthis study where hly A was detected in all the nine Lmonocytogenes isolates However the detection of act A genein the work of Henriques et al [30] differs from this studywhich failed to detect act A Listeriolysin O and act Aare associated with the bacteriumrsquos capability of passing theintestinal barrier cell to cell spread andmotility cell invasionand intracellular parasitism [7 18] Therefore simultaneousdetection of virulence genes in a single step will be desirableas it reduces time and labour and it will be useful in largescale survey for detecting virulent strains of Listeria [18]

The separation of Nigerian Listeria monocytogenes iso-lates sequence (NGA34ANGA35ANGA38 andNGA41A)as well as the L monocytogenes sequences from the GenBank(N1-011A R2-502 J1816 J2-031 EDG J1-220 J1926 J1817 andPW040025) into two major lineages A and B is consistentwith the major lineages described previously by Oris et al[31] who reported that L monocytogenes isolates

L monocytogenes consist of at least 4 evolutionary lin-eages (I II III and IV) Even though only cluster A isstatistically supported with a bootstrap value of 100 clusterB is not statistically supported

Most L monocytogenes isolates seem to belong to lineagesI and II which include the serotypes more commonlyassociated with human clinical cases including serotypes12b and 4b (lineage I) and serotype 12a (lineage II) Sinceit is a rooted tree we can infer evolutionary relationshipinformation regarding the common ancestors and the pathof evolution from the rooted phylogenetic tree

Since lineage I L monocytogenes strains are more preva-lent in human clinical listeriosis cases and contaminated food[31] it can be said that Nigerian L monocytogenes isolates(NGA 34A NGA 35A and NGA41A) are from contaminatedfoods (ie milk and milk products) From the phylogenictree Nigerian L monocytogenes isolates (NGA 34A NGA35A and NGA 41A) were classified as lineage I which areknown to be of higher pathogenic potentials due to intrinsicvirulence features as compared to lineage II strains of Lmonocytogenes [32]Morobe et al [33] in their study observedthat most of the L monocytogenes isolates belong to lineageIII (4a and 4c) which are known to be more prevalent inanimals with clinical listeriosis [31] as opposed to this studywhere none of the L monocytogenes isolate were found tobelong to lineage III Isolate NGA 35a was found to belong tolineage II which is opposed to the findings of Morobe et al[33] where they observed that none of the L monocytogenesisolates belong to lineage II

The implication of the present findings is that L mono-cytogenes of lineage 1 originating from animal milk and milkproduct could be associated with increased human risk to Lmonocytogenes infection in Nigeria and thus effort to controlthe disease should be targeted at improving the food safetyand quality

Competing Interests

The authors declare that they have no competing interests

References

[1] M J Gray N E Freitag and K J Boor ldquoHow the bacterialpathogen Listeria monocytogenes mediates the switch fromenvironmental Dr Jekyll to pathogenicMr Hyderdquo Infection andImmunity vol 74 no 5 pp 2506ndash2512 2006

[2] T Hain C Steinweg and T Chakraborty ldquoComparative andfunctional genomics of Listeria spprdquo Journal of Biotechnologyvol 126 no 1 pp 37ndash51 2006

[3] E Rahimi M Ameri and H Momtaz ldquoPrevalence and antimi-crobial resistance of Listeria species isolated from milk and

Canadian Journal of Infectious Diseases and Medical Microbiology 7

dairy products in Iranrdquo Food Control vol 21 no 11 pp 1448ndash1452 2010

[4] B Karen ListeriaMonocytogenes InfectionMedscape LLCNewYork NY USA 2015 httpemedicinemedscapecomarticle220684-overview

[5] F Stavru F Bouillaud A Sartori D Ricquier and P Cos-sart ldquoListeria monocytogenes transiently alters mitochon-drial dynamics during infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no9 pp 3612ndash3617 2011

[6] J A Vazquez-Boland M Kuhn P Berche et al ldquoListeriapathogenesis and molecular virulence determinantsrdquo ClinicalMicrobiology Reviews vol 14 no 3 pp 584ndash640 2001

[7] D Liu M L Lawrence F W Austin and A J AinsworthldquoA multiplex PCR for species- and virulence-specific determi-nation of Listeria monocytogenesrdquo Journal of MicrobiologicalMethods vol 71 no 2 pp 133ndash140 2007

[8] A Camejo F Carvalho O Reis E Leitao S Sousa and DCabanes ldquoThe arsenal of virulence factors deployed by Listeriamonocytogenes to promote its cell infection cyclerdquo Virulencevol 2 no 5 pp 379ndash394 2011

[9] B Joseph and W Goebel ldquoLife of Listeria monocytogenes in thehost cellsrsquo cytosolrdquo Microbes and Infection vol 9 no 10 pp1188ndash1195 2007

[10] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[11] K J Cooray T Nishibori H Xiong T Matsuyama M FujitaandM Mitsuyama ldquoDetection of multiple virulence associatedgenes of Listeriamonocytogenes by PCR in artificially contam-inated milk samplesrdquo Applied Microbiology vol 60 pp 3023ndash3026 1994

[12] S H W Notermans J Dufrenne M Leimeister-Wachter EDomann and T Chakraborty ldquoPhosphatidy-linositol-specificphospholipase C activity as a marker to distinguish betweenpathogenic and non-pathogenic Listeria speciesrdquo Applied andEnvironmental Microbiology vol 57 no 9 pp 2666ndash2670 1991

[13] J E Olsen ldquoDNA-based methods for detection of food-bornebacterial pathogensrdquo Food Research International vol 33 no 3-4 pp 257ndash266 2000

[14] E Walecka J Molenda R Karpıskova and J Bania ldquoEffect ofosmotic stress and culture density on invasiveness of Listeriamonocytogenes strainsrdquo International Journal of Food Microbi-ology vol 144 no 3 pp 440ndash445 2011

[15] M Wiedmann ldquoMolecular subtyping methods for Listeriamonocytogenesrdquo Journal of AOAC International vol 85 no 2pp 524ndash531 2002

[16] G T Jeffers J L Bruce P L McDonough J Scarlett K J Boorand M Wiedmann ldquoComparative genetic characterizationof Listeria monocytogenes isolates from human and animallisteriosis casesrdquo Microbiology vol 147 no 5 pp 1095ndash11042001

[17] C A Nadon D L Woodward C Young F G Rodgers andM Wiedmann ldquoCorrelations between molecular subtypingand serotyping of Listeria monocytogenesrdquo Journal of ClinicalMicrobiology vol 39 no 7 pp 2704ndash2707 2001

[18] D B Rawool SV SMalik I Shakuntala AM Sahare and S BBarbuddhe ldquoDetection of multiple virulence-associated genesin Listeria monocytogenes isolated from bovine mastitis casesrdquoInternational Journal of Food Microbiology vol 113 no 2 pp201ndash207 2007

[19] D Roberts W Hoper and M Greenwood Practical FoodMicrobiology Public Health Laboratory Services London UK1995

[20] M M Janzten J Navas A Corujo R Moreno V Lopez andJ V Martınez-Suarez ldquoReview Specific detection of Listeriamonocytogenes in foods using commercial methods fromchromogenic media to real-time PCRrdquo Spanish Journal ofAgricultural Research vol 4 no 3 pp 235ndash247 2006

[21] P M Border J J Howard G S Plastow and K W SiggensldquoDetection of Listeria species and Listeria monocytogenes usingpolymerase chain reactionrdquo Letters in AppliedMicrobiology vol11 no 3 pp 158ndash162 1990

[22] T A Hall ldquoBioedit a user friendly biological sequence align-ment editor and analysis programme for windows 9598NTrdquoin Nucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[23] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2002

[24] D Liu ldquoIdentification subtyping and virulence determinationof Listeria monocytogenes an important foodborne pathogenrdquoJournal of Medical Microbiology vol 55 no 6 pp 645ndash6592006

[25] C H Kuan S G Goh Y Y Loo et al ldquoPrevalence andquantification of Listeria monocytogenes in chicken offal at theretail level in Malaysiardquo Poultry Science vol 92 no 6 pp 1664ndash1669 2013

[26] P K Jallewar D R Kalorey N V Kurkure V V Pande andS B Barbuddhe ldquoGenotypic characterization of Listeria sppisolated from fresh water fishrdquo International Journal of FoodMicrobiology vol 114 no 1 pp 120ndash123 2007

[27] J Kreft and J A Vazquez-Boland ldquoRegulation of virulencegenes in Listeriardquo International Journal of Medical Microbiologyvol 291 no 2 pp 145ndash157 2001

[28] R R Beumer and W C Hazeleger ldquoListeria monocytogenesdiagnostic problemsrdquo FEMS Immunology and Medical Micro-biology vol 35 no 3 pp 191ndash197 2003

[29] Y H Shuckken Y T Grohn and M Wiedmann ldquoPart VIlisteria monocytogenes epidemiology of listeriosisrdquo in Micro-bial Food Safety in Animal Agriculture Current Topics M ETorrence and R E Isaacson Eds pp 221ndash232Wiley-BlackwellHoboken NJ USA 2003

[30] A R Henriques A S Barreto and M J Fraqueza ldquoDetectionof virulence-associated genes in Listeria monocytogenes isolatesfrom Portuguese ready-to- eat meat-based food industryrdquoApplied and Environmental Microbiology vol 61 pp 817ndash8191998

[31] R H Orsi H C den Bakker and M Wiedmann ldquoListeriamonocytogenes lineages genomics evolution ecology andphenotypic characteristicsrdquo International Journal of MedicalMicrobiology vol 301 no 2 pp 79ndash96 2011

[32] B Swaminathan and P Gerner-Smidt ldquoThe epidemiology ofhuman listeriosisrdquo Microbes and Infection vol 9 no 10 pp1236ndash1243 2007

[33] I C Morobe C L Obi M A Nyila M I Matsheka andB A Gashe ldquoMolecular characterization and serotyping ofListeria monocytogenes with a focus on food safety and diseasepreventionrdquo in Biochemical Testing J C Jimenez-Lopez Edchapter 8 pp 197ndash216 InTech Rijeka Croatia 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Canadian Journal of Infectious Diseases and Medical Microbiology 7

dairy products in Iranrdquo Food Control vol 21 no 11 pp 1448ndash1452 2010

[4] B Karen ListeriaMonocytogenes InfectionMedscape LLCNewYork NY USA 2015 httpemedicinemedscapecomarticle220684-overview

[5] F Stavru F Bouillaud A Sartori D Ricquier and P Cos-sart ldquoListeria monocytogenes transiently alters mitochon-drial dynamics during infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no9 pp 3612ndash3617 2011

[6] J A Vazquez-Boland M Kuhn P Berche et al ldquoListeriapathogenesis and molecular virulence determinantsrdquo ClinicalMicrobiology Reviews vol 14 no 3 pp 584ndash640 2001

[7] D Liu M L Lawrence F W Austin and A J AinsworthldquoA multiplex PCR for species- and virulence-specific determi-nation of Listeria monocytogenesrdquo Journal of MicrobiologicalMethods vol 71 no 2 pp 133ndash140 2007

[8] A Camejo F Carvalho O Reis E Leitao S Sousa and DCabanes ldquoThe arsenal of virulence factors deployed by Listeriamonocytogenes to promote its cell infection cyclerdquo Virulencevol 2 no 5 pp 379ndash394 2011

[9] B Joseph and W Goebel ldquoLife of Listeria monocytogenes in thehost cellsrsquo cytosolrdquo Microbes and Infection vol 9 no 10 pp1188ndash1195 2007

[10] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[11] K J Cooray T Nishibori H Xiong T Matsuyama M FujitaandM Mitsuyama ldquoDetection of multiple virulence associatedgenes of Listeriamonocytogenes by PCR in artificially contam-inated milk samplesrdquo Applied Microbiology vol 60 pp 3023ndash3026 1994

[12] S H W Notermans J Dufrenne M Leimeister-Wachter EDomann and T Chakraborty ldquoPhosphatidy-linositol-specificphospholipase C activity as a marker to distinguish betweenpathogenic and non-pathogenic Listeria speciesrdquo Applied andEnvironmental Microbiology vol 57 no 9 pp 2666ndash2670 1991

[13] J E Olsen ldquoDNA-based methods for detection of food-bornebacterial pathogensrdquo Food Research International vol 33 no 3-4 pp 257ndash266 2000

[14] E Walecka J Molenda R Karpıskova and J Bania ldquoEffect ofosmotic stress and culture density on invasiveness of Listeriamonocytogenes strainsrdquo International Journal of Food Microbi-ology vol 144 no 3 pp 440ndash445 2011

[15] M Wiedmann ldquoMolecular subtyping methods for Listeriamonocytogenesrdquo Journal of AOAC International vol 85 no 2pp 524ndash531 2002

[16] G T Jeffers J L Bruce P L McDonough J Scarlett K J Boorand M Wiedmann ldquoComparative genetic characterizationof Listeria monocytogenes isolates from human and animallisteriosis casesrdquo Microbiology vol 147 no 5 pp 1095ndash11042001

[17] C A Nadon D L Woodward C Young F G Rodgers andM Wiedmann ldquoCorrelations between molecular subtypingand serotyping of Listeria monocytogenesrdquo Journal of ClinicalMicrobiology vol 39 no 7 pp 2704ndash2707 2001

[18] D B Rawool SV SMalik I Shakuntala AM Sahare and S BBarbuddhe ldquoDetection of multiple virulence-associated genesin Listeria monocytogenes isolated from bovine mastitis casesrdquoInternational Journal of Food Microbiology vol 113 no 2 pp201ndash207 2007

[19] D Roberts W Hoper and M Greenwood Practical FoodMicrobiology Public Health Laboratory Services London UK1995

[20] M M Janzten J Navas A Corujo R Moreno V Lopez andJ V Martınez-Suarez ldquoReview Specific detection of Listeriamonocytogenes in foods using commercial methods fromchromogenic media to real-time PCRrdquo Spanish Journal ofAgricultural Research vol 4 no 3 pp 235ndash247 2006

[21] P M Border J J Howard G S Plastow and K W SiggensldquoDetection of Listeria species and Listeria monocytogenes usingpolymerase chain reactionrdquo Letters in AppliedMicrobiology vol11 no 3 pp 158ndash162 1990

[22] T A Hall ldquoBioedit a user friendly biological sequence align-ment editor and analysis programme for windows 9598NTrdquoin Nucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[23] S Kumar K Tamura I B Jakobsen and M Nei ldquoMEGA2molecular evolutionary genetics analysis softwarerdquo Bioinfor-matics vol 17 no 12 pp 1244ndash1245 2002

[24] D Liu ldquoIdentification subtyping and virulence determinationof Listeria monocytogenes an important foodborne pathogenrdquoJournal of Medical Microbiology vol 55 no 6 pp 645ndash6592006

[25] C H Kuan S G Goh Y Y Loo et al ldquoPrevalence andquantification of Listeria monocytogenes in chicken offal at theretail level in Malaysiardquo Poultry Science vol 92 no 6 pp 1664ndash1669 2013

[26] P K Jallewar D R Kalorey N V Kurkure V V Pande andS B Barbuddhe ldquoGenotypic characterization of Listeria sppisolated from fresh water fishrdquo International Journal of FoodMicrobiology vol 114 no 1 pp 120ndash123 2007

[27] J Kreft and J A Vazquez-Boland ldquoRegulation of virulencegenes in Listeriardquo International Journal of Medical Microbiologyvol 291 no 2 pp 145ndash157 2001

[28] R R Beumer and W C Hazeleger ldquoListeria monocytogenesdiagnostic problemsrdquo FEMS Immunology and Medical Micro-biology vol 35 no 3 pp 191ndash197 2003

[29] Y H Shuckken Y T Grohn and M Wiedmann ldquoPart VIlisteria monocytogenes epidemiology of listeriosisrdquo in Micro-bial Food Safety in Animal Agriculture Current Topics M ETorrence and R E Isaacson Eds pp 221ndash232Wiley-BlackwellHoboken NJ USA 2003

[30] A R Henriques A S Barreto and M J Fraqueza ldquoDetectionof virulence-associated genes in Listeria monocytogenes isolatesfrom Portuguese ready-to- eat meat-based food industryrdquoApplied and Environmental Microbiology vol 61 pp 817ndash8191998

[31] R H Orsi H C den Bakker and M Wiedmann ldquoListeriamonocytogenes lineages genomics evolution ecology andphenotypic characteristicsrdquo International Journal of MedicalMicrobiology vol 301 no 2 pp 79ndash96 2011

[32] B Swaminathan and P Gerner-Smidt ldquoThe epidemiology ofhuman listeriosisrdquo Microbes and Infection vol 9 no 10 pp1236ndash1243 2007

[33] I C Morobe C L Obi M A Nyila M I Matsheka andB A Gashe ldquoMolecular characterization and serotyping ofListeria monocytogenes with a focus on food safety and diseasepreventionrdquo in Biochemical Testing J C Jimenez-Lopez Edchapter 8 pp 197ndash216 InTech Rijeka Croatia 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom