Int J Mol Epidemiol Genet 2014;5(3):125-134www.ijmeg.org
/ISSN:1948-1756/IJMEG0001465Original Article Molecular detection of
virulence genes as markers in Pseudomonas aeruginosa isolated
fromurinary tract infections Neha Sabharwal, Shriya Dhall, Sanjay
Chhibber, Kusum HarjaiDepartment of Microbiology, Basic Medical
Sciences Block-I, South Campus, Panjab University, Chandigarh,
India, 160014Received July 19, 2014; Accepted September 10, 2014;
Epub October 22, 2014; Published October 30,
2014Abstract:CatheterassociatedurinarytractinfectionsbyP.aeruginosaarerelatedtovarietyofcomplications.
Quorumsensingandrelatedcircuitryguarditsvirulencepotential.ThoughP.aeruginosaaccountsforanappre-ciable
amount of virulence factors, this organism is highly unstable
phenotypically. Thus, genotyping of clinical iso-lates of P.
aeruginosa is of utmost importance for understanding the
epidemiology of infection. This may contribute towards development
of immunotherapeutic approaches against this multi drug resistant
pathogen. Moreover, no epidemiological study has been reported yet
on uroisolates of P. aeruginosa. Thus this study was planned to
obtain information regarding presence, distribution and rate of
occurrence of quorum sensing and some associated viru-lence genes
at genetic level. The profling of quorum sensing genes lasI, lasR,
rhlI, rhlR and virulence genes like toxA, aprA, rhlAB, plcH, lasB
and fiC of twelve strains of P. aeruginosaisolated from patients
with UTIs was done by direct PCR. The results showed variable
distribution of quorum sensing genes and virulence genes. Their
percent-age occurrence may be specifcally associated with different
levels of intrinsic virulence and pathogenicity in urinary
tract.SuchinformationcanhelpinidentifyingthesevirulencegenesasusefuldiagnosticmarkersforclinicalP.
aeruginosa strains isolated from UTIs. Keywords: Epidemiology, PCR,
Pseudomonas aeruginosa, urinary tract infections, quorum
sensingIntroductionP. aeruginosa is one of the most important
nos-ocomialpathogenresponsibleforavarietyof
infectionswithlimitedtherapeuticoptions
becauseofitsantibioticresistance[1].Itpro-ducesimpressivearrayofvirulencefactors,
whosecoordinatedexpressionisregulatedby
differentregulatorysystems.Arecentsurvey
showedthatP.aeruginosaisoneofthemost frequent pathogen isolated
from ICU (Intensive careunit)-acquiredinfections[2].Catheter
associated urinary tract infections (CAUTIs) are
responsiblefor40%ofnosocomialinfections.
P.aeruginosawithinthecatheterfrequently
developsasbioflmbydirectlyattachingtoits
surface.Thesesurface-associated,matrix-enclosed, microbial
communities are responsi-ble for chronicity and recurrence of such
infec-tionsleadingtohighmorbidityandmortality
[3].Onceestablished,bacteriacommunicate with each other to
coordinate the expression of specifc genes in a cell
density-dependent fash-ion.Quorumsensing(QS)viaacyl-homoserine
lactone(HSL),controlstheexpressionofan
arrayofvirulencegenesinP.aeruginosa.The
autoinducersynthase,LasI,synthesisesN-(3-oxododecanoyl)homoserinelactone(3OC12-HSL),whichregulatestheproductionofelas-tase,exotoxinAandalkalineprotease,while
RhlIsynthesizestheautoinducerN-butyryl
homoserinelactone(C4-HSL),whichregulates
theproductionofrhamnolipid,alkalineprote-ase,elastase,cyanideandpyocyanin[4,5].
Because of the regulatory control of production of virulence
factors, QS mechanisms are being proposed as a novel target for
development of innovativestrategiestocontrolinfections.
Moreover,importanceofQSintheestablish-ment of a successful
infection has been shown in different types of animal model studies
such asacutepulmonaryinfection,burnwound infection, microbial
keratitis, chronic lung infec-tion and urinary tract infections
[6-10]. Virulence gene distribution in P. aeruginosa UTI126Int J
Mol Epidemiol Genet 2014;5(3):125-134Table 1. The conditions used
for the PCR amplifcation of QS and virulence genes in P. aeruginosa
strainsThe amplifcation programGene Initial DenaturationNo. of
cyclesDenaturation AnnealingPrimerextensionFinalextensionlasI 95C,
2 min. 30 95C, 40 sec. 50C/60C, 1 min. 72C, 2 min. 72C, 10 min.lasR
60C, 1 min.rhlI 60C, 1 min.rhlR 60C, 1 min.toxA 95C, 2 min. 30 95C,
40 sec. 50C, 1 min. 72C, 2 min.aprA 65C, 1 min.rhlAB 95C, 5 min. 35
95C, 30 sec. 52C, 30 sec. 72C, 30 sec.plcH 55C, 30 sec.lasB 55C, 30
sec.fiC 95C, 2 min. 30 95C, 40 sec. 55C, 1 min. 72C, 2
min.Phenotypictypingmethodssuchasbiotyping, serotyping, bacteriocin
typing and anti-microbi-al testing are available in literature.
However, P. aeruginosa is phenotypically very unstable and
therefore genotyping tend to be of great value. Infact, an
understanding about the distribution of virulence genes in clinical
strains can help in understandingtheepidemiologyofinfections.
DetectionandidentifcationofQSsignalspro-vided information about the
expression of viru-lencecomponentsoftheinfectingpathogen [9].
Scarcity of literature exists regarding genotypic analysis of P.
aeruginosa responsible for
caus-ingCAUTIs.Dependinguponthesiteandtype of infection, importance
and role of a particular virulence factor may differ in a
particular strain. However,distributionofvirulencegenesmay be
different in UTI strains of P. aeruginosa, as no specifc virulence
genotype has been asso-ciatedwithsuchstrains,rathercontradictory
reports exist in nature. Role of QS is well estab-lished in P.
aeruginosa induced microbial kera-titis, cystic fbrosis [8],
pneumonia [6] as well as ithasbeenfoundtobenecessaryduringUTI [11].
Its role in UTI is controversial as some QS defcient clinical
strains have been shown to be
capableofcausingclinicalinfectionsofthe
humansaswell[10,12].Thus,thepresent study was planned to identify
specifc virulence genotypeinP.aeruginosaclinicaluroisolates.
Virulence genes in this study were selected on
thebasisoftheirimportance.Isolateswere
screenedforQSsystems,alkalineprotease
(aprA),rhamnolipidAB(rhlAB),phospholipase
(plcH)andelastase(lasB).Theywerealso
screenedforexotoxinA(toxA)whichishighly
conservedinP.aeruginosaandnotinother species of this genus. Highly
conserved fagel-linbandheterologousfagellinawerealso screened in
the uroisolates. The study provides
informationaboutthepercentageoccurrence
anddistributionofQSandsomeassociated
virulencegenes.Informationobtainedfrom such studies may provide an
insight into identi-fyingvirulencegenesasusefuldiagnostic markers
which may further contribute towards development of
immunotherapeutic approach-es for treating UTIs caused by P.
aeruginosa. MethodsClinical strains and phenotypic
identifcationTwelve clinical strains of P. aeruginosa isolated
frompatientswithUTIsobtainedfrom
GovernmentMedicalCollegeandHospital, Chandigarh, India, were
examined. The isolates werecheckedforpurity,identifedbycolony
morphology,oxidasetestandbiochemical
reactionsspecifctoPseudomonas.Inthis
study,onewelldefnedlaboratorystrain Pseudomonas aeruginosa PAO1 was
also used as a positive control. Standard strain PAO1 was
generouslyprovidedbyProf.BarbaraH.Iglewski,UniversityofRochester,NewYork,
U.S.A. The strains were grown in Luria Bertani (LB) (HiMedia) and
maintained in 50% glycerol and stored at -20C. Gradient PCR
amplifcationFor the molecular characterization of the genet-ic
support of QS (las and rhl), extracellular viru-Virulence gene
distribution in P. aeruginosa UTI127Int J Mol Epidemiol Genet
2014;5(3):125-134lencefactors(exotoxinA,alkalineproteaseA,
rhamnolipid,phospholipase,elastase)andini-tialcolonizationfactorfagellin(fiC),genomic
DNAwasextractedfromtwelveselectedP. aeruginosa strains and from P.
aeruginosa ref-erencestrainPAO1.Onecolonyofeachstrain cultured on
solid medium was inoculated into 5 mlofLBandgrownovernightat37Cwith
shaking.Fromthesecultures,DNAextraction was performed by using DNA
extraction mini kit (Favorgen) according to the manufacturers
rec-ommendations. At different annealing
tempera-tures(50C,55C,60C,65C),amplifcation of above mentioned
genes was carried out with PAO1DNA.Bestannealingtemperature
(intenseampliconbandwithnoprimerdim-mers)waschosenfortheproflingofclinical
strains. PCR
protocolAmplifcationswerecarriedoutin25lvol-umescontainingtemplateDNA(50ng),Taq
buffer(1X),DMSO(Dimethylsulfoxide),
Magnesiumchloride(2mM),eachprimer(10
pM/l),nucleotides(dATP,dCTP,dGTP,dTTP)
(200M,ThermoScientifc)andTaqpoly-phospholipaseH(plcH),elastase(lasB)[7].A
setofconservedoligonucleotideprimers CW45, CW46 [16] was also used
to analyse fa-gellinsubtypesintheclinicalstrains.The
sequencesofspecifcprimersusedinPCR
reactionsandthemolecularweightofthe
obtainedampliconsarepresentedinTable2. After amplifcation, 10 l
sample was subjected toelectrophoresisonastandard1%agarose gel for
1 h at 100 V, stained with ethidium
bro-mide(Sigma)anddetectedbyUVtransillu- mination.Results PCR assay
for QS genesTheresultsshoweddifferentdistributionof lasI, lasR,
rhlI and rhlR genes in P. aeruginosa
strains.TheprimersusedfordetectionofQS
genesallowedtheamplifcationofthewhole
genes.Nine(75%)strainsnamelyB1,B2,B3,
B4,B5,B6,B7,B9andB12werepositivefor lasI gene giving amplifcation
at 60C at 295 bp (Figure 1). Similarly B2, B3, B5, B6, B7, B8, B9,
B11 and B12 were positive (75%) for lasR gene
whichgaveamplifcationat60Cat130bp Table 2. The primer sequences
used in PCR assays for QS and virulence genes detection in P.
aeruginosa strainsGene Primer Nucleotide Sequence Amplicon size
lasI forward 5 CGTGCTCAAGTGTTCAAGG 3 295 bpreverse 5
TACAGTCGGAAAAGCCCAG 3lasR forward 5 AAGTGGAAAATTGGAGTGGAG 3 130
bpreverse 5 GTAGTTGCCGACGACGATGAAG 3rhlI forward 5
TTCATCCTCCTTTAGTCTTCCC 3 155 bpreverse 5 TTCCAGCGATTCAGAGAGC 3rhlR
forward 5 TGCATTTTATCGATCAGGGC 3 133 bpreverse 5
CACTTCCTTTTCCAGGACG 3toxA forward 5 GGAGCGCAACTATCCCACT 3 150
bpreverse 5 TGGTAGCCGACGAACACATA 3aprA forward 5
GTCGACCAGGCGGCGGAGCAGATA 3 993 bpreverse 5 GCCGAGGCCGCCGTAGAGGATGTC
3rhlAB forward 5 TCATGGAATTGTCACAACCGC 3 151 bpreverse 5
ATACGGCAAAATCATGGCAAC 3plcH forward 5 GAAGCCATGGGCTACTTCAA 3 307
bpreverse 5 AGAGTGACGAGGAGCGGTAG 3lasB forward 5
TTCTACCCGAAGGACTGATAC 3 153 bpreverse 5 AACACCCATGATCGCAAC 3fiC
CW45 forward 5 GGCAGCTGGTTNGCCTG 3 1.02 kb (type a)CW46 reverse 5
GGCCTGCAGATCNCCAA 3 1.25 kb (type b)merase(1U/l,FIR-
Epol).Amplifcations werecarriedoutina BioradThermalCycler
for30cyclesconsist-ingofpre-denatur-ation, denaturation, an-
nealing,extensionand postelongation.The parameters for the am-
plifcationcyclesused ineachPCRexperi-mentarerepresented in Table 1.
A set of oli-gonucleotideprimers (EurofnsGenomics) that allowed to
amplify wholeQSgenes(lasI, lasR, rhlI and rhlR) [13]
wereselected.Also, PCR assays were used
todetecttheextracel-lularvirulencegenes
encodingalkalinepro-tease(aprA)[14],exo-toxinA(toxA)[15]and
rhamnolipid AB (rhlAB), Virulence gene distribution in P.
aeruginosa UTI128Int J Mol Epidemiol Genet 2014;5(3):125-134(Figure
2). Five out of twelve strains (41.6%) B3, B6, B7, B8 and B11 were
positive for the pres-enceofrhlIgene,givingamplifcationat60C
at155bp(Figure3).rhlRgenewasfoundin
sevenstrains(58.3%)B2,B3,B4,B5,B7,B8 and B11, giving amplifcation
at 60C at 133 bp (Figure 4 and Table 3). PCR assay for virulence
genes (fiC, toxA, aprA, lasB, plcH,
rhlAB)Inthepresentstudy,clinicalstrainswere
screenedfortheprevalenceofdifferentviru-lence genes of P.
aeruginosa. Surprisingly, aprA gene had lowest occurrence of 16.6%
in only 2 strains,B4andB8(Figure5).Theprevalence
oflasBandplcHwasfoundin75%ofstrains (Figures 6 and 7 respectively).
lasB was found
Figure1.AgarosegelelectrophoresisofPCRprod-uctsafteramplifcationoflasIgene.MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different
strainsofP.aeruginosa(lasIgeneproductsat295
bp).Figure2.AgarosegelelectrophoresisofPCRprod-uctsafteramplifcationoflasRgene.MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different strains of P.
aeruginosa (lasR gene products at 130
bp).Figure3.AgarosegelelectrophoresisofPCRprod-uctsafteramplifcationofrhlIgene.MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different
strainsofP.aeruginosa(rhlIgeneproductsat155
bp).Figure4.AgarosegelelectrophoresisofPCRprod-uctsafteramplifcationofrhlRgene.MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different strains of P.
aeruginosa (rhlR gene products at 133 bp).Virulence gene
distribution in P. aeruginosa UTI129Int J Mol Epidemiol Genet
2014;5(3):125-134strainsnamelyB1,B2,B3,B6,B7andB8 (Figure 8). 100%
prevalence of toxA gene was found in all analyzed strains (Figure
9). P. aeru-ginosa PAO1 was found to have b-type fagel-lin, giving
PCR amplifcation at 1.25 kb. Strains B1, B5 and B6 showed b-type
fagellin. Strains Table 3. The distribution of virulence genes
among P. aeruginosa strains Source of strain isolationThe percent
(%) of positive strains for genes encoding different virulence
factorsUrinary Tract lasI lasR rhlI rhlR toxA aprA rhlAB plcH lasB
fiC75% 75% 41.6% 58.3% 100% 16.6% 50% 75% 75% 58.3%a bB1 + - - - +
- + + + - +B2 + + - + + - + + + - -B3 + + + + + - + + + - -B4 + - -
+ + + - + - + -B5 + + - + + - - - + - +B6 + + + - + - + + - - +B7 +
+ + + + - + - + - -B8 - + + + + + + + + + -B9 + + - - + - - + - -
-B10 - - - - + - - + + + -B11 - + + + + - - + + + -B12 + + - - + -
- - + -
-Figure5.AgarosegelelectrophoresisofPCRprod-uctsafteramplifcationofaprAgene.MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different strains of P.
aeruginosa (aprA gene products at 993 bp).positive for strains B1,
B2, B3, B5, B7, B8, B10, B11andB12.plcHwasfoundpositivefor
strainsB1,B2,B3,B4,B6,B8,B9,B10and B11. rhlAB was found to be
positive in 50% of
Figure6.AgarosegelelectrophoresisofPCRprod-uctsafteramplifcationoflasBgene.MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different strains of P.
aeruginosa (lasB gene products at 153 bp).Virulence gene
distribution in P. aeruginosa UTI130Int J Mol Epidemiol Genet
2014;5(3):125-134B4, B8, B10 and B11 showed a-type fagellin
givingPCRamplifcationat1.02kb.Non-fagellar strains were: B2, B3,
B7, B9 and B12, whichsurprisinglyshowednoamplifcation
(Figure10).PercentagefiCoccurrencewas found to be 58.3% (25% b-type
fagellin, 33% a-type) (Table 3).
Figure7.AgarosegelelectrophoresisofPCRprod-uctsafteramplifcationofplcHgene.MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different strains of P.
aeruginosa (plcH gene products at 307
bp).Figure8.AgarosegelelectrophoresisofPCRprod-ucts after
amplifcation of rhlAB gene.
MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different strains of P.
aeruginosa (rhlAB gene products at 151
bp).Figure9.AgarosegelelectrophoresisofPCRprod-uctsafteramplifcationoftoxAgene.MWM-molec-ularweightmarker(100bpDNAladder,#DL004,
Geneaid),+C-PAO1controlDNA,B1-B12-different strains of P.
aeruginosa (toxA gene products at 150 bp). Figure 10. Agarose gel
electrophoresis of PCR prod-ucts after amplifcation of fiC gene.
MWM-molecular weightmarker(ORangeRuler200bpDNAladder,
#SM0633,Fermentas),+C-PAO1controlDNA,B1-B12-differentstrainsofP.aeruginosa(fiCgene
products at 1.02 kb and 1.25 kb).Virulence gene distribution in P.
aeruginosa UTI131Int J Mol Epidemiol Genet
2014;5(3):125-134DiscussionThelargegenomeanditsgeneticcomplexity
allow P. aeruginosa to thrive in diverse ecologic conditions.
Multiple bacterial virulence factors impact the pathogenesis of P.
aeruginosa infec-tions.Thecombinationofvirulencefactors expressed
by each P. aeruginosa strain tend to
determinetheoutcomeofaninfectiouspro-cess. However, in the clinical
cases, it is often diffcult to distinguish between simple
coloniza-tionandinfection,andnodiagnostictoolis available to assess
the virulence potential of a given isolate [17]. Taking into
account the poor availabilityofinformationonthepatternsof
virulencefactorspossessedbyP.aeruginosa strains isolated from
patients with CAUTIs, the genetic profling of virulence
determinants was carried out. P. aeruginosa possesses two QS
systems, Las andRhl.Isolatesobtainedfrompatientswith lower
respiratory tract, non-surgical or surgical
woundinfectionsandsputaofcysticfbrosis patients showed high
percentage of functional QS systems (97.5%) [12]. Similarly, all
analysed isolatesfromrespiratorytract,woundsecre-tions and from
patients with cardiovascular
sur-geryassociatedinfectionspossessedQS
genes(100%)[7].Inthepresentstudy,allthe strains were found to have
varied distribution of individualQSgenes.Theprimersusedfor
detection of QS genes allowed the amplifcation of the whole genes.
All the four QS genes were notdetectedinanyofthestrainstested.
Senturk et al. revealed that lasI, lasR, rhlI, rhlR genes may be
differently distributed in clinical
isolates.However,presenceofallfourQS genes may not be necessarily
indicative of phe-notypicproductionofC4-HSLandC12-HSL. These
defciencies were linked to combinations of point mutation [10]. The
two QS systems do notoperateindependently.LasR-C12-HSL
complexpositivelyregulatestranscriptionof
RhlRandRhlI.ExpressionofrhlRisnotonly
dependentonLasR,butonRhlRitself[18]. Since the circuitry is
interlinked, the absence of any of the component in clinical
strains did not compromise their ability for phenotypic expres-sion
of QS system [10, 12]. AnotherimportantP.aeruginosavirulence
determinantisalkalineprotease.Theproteas-es promote the development
of bacteria within theinfectedhostandinterferewiththehost immune
system. Only 30 - 40% of strains from
ear,bloodandlungsshowedhighprotease activity [19]. Uroisolates of
P. aeruginosa in our study showed least presence of alkaline
prote-ase(16.6%),indicatingthatalkalineprotease may not be playing
a very important role in the pathogenesis of UTI. This was also
indicated in anotherstudyfromourlaboratorywhere50%
ofstrainspossessingalkalineproteasewere
althoughabletocolonizekidneytissue,but
wereunabletomultiplyandshowedverylow bacterial count [20]. Since
aprA is encoded by bothLasandRhlsystems,itspercentage
occurrencemaycorroboratewiththelower
presenceofRhlsystem.Althoughlessimpor-tant in establishing UTI, its
negligible presence maymakethesestrainsdifferentfromstrains
isolated from other infectious sites and hence
animportantmarkerinstrainscausingUTIs.
OurresultscorroboratethefndingofCotaret
al.whoshowedthatlowerpresenceofapar-ticular virulence factor makes
it a more impor-tantfactorinthatparticularinfection.Inthis regard,
PCR results of an earlier study showed the presence of gene
encoding ExoS in 64.87% ofstrainsandExoUin56.54%strains.Itwas
suggested that ExoU could be a marker of viru-lence for strains
isolated from respiratory tract and wound secretions [7]. Analysis
of P. aerugi-nosaclinicalisolatesfromdifferentsitesalso
highlightedthatboththeinfectionsiteaswell as the duration of
infection infuenced the
viru-lenceofthebacteriabyalteringproductionof
extracellularvirulencefactors[21].Thus
expressionofaprAmayalsoberelatedtoan
infectionsitewhereabundantsubstrateis
availableforitsgrowth.Theurinarytractmay not be providing the
substrate for expression of aprA, which needs to be exploited.
Elastase (encoded by lasB) is a powerful T2SS secreted proteolytic
enzyme. This enzyme has a wide range of substrates, including
elements of connective tissue such as elastin, collagen,
fbronectinandlaminen.Phospholipidsare hydrolysed by phospholipase C
which is encod-edbyplcHgene.Rhamnolipid,beingarham-nose-containing
glycolipid biosurfactant, has a
detergent-likestructureandisconsideredto solubilize the
phospholipids of lung surfactant,
makingthemmoreaccessibletocleavageby
phospholipaseC[22].ItsencodedbyrhlAB
gene.ProductionoftheQS-dependentviru-lence factor, rhamnolipid by
P. aeruginosa iso-latesisassociatedwithdevelopmentofVAP
(ventilator-associatedpneumonia)[6].Infew studies, all isolates
from burn, wound and pul-Virulence gene distribution in P.
aeruginosa UTI132Int J Mol Epidemiol Genet
2014;5(3):125-134monarytractinfectionsharboredlasB,plcH and rhlAB
gene. Prevalence of a gene in all the environmental and clinical
isolates implies the importance of a factor for survival of P.
aerugi-nosainvarioussettings[7,23,24].Inthe present study the
prevalence of lasB and plcH wasfoundin75%ofstrains.Thepresenceof
lasB can be corroborated with the percentage occurrence of las
genes (lasI and lasR) since it isencodedbybothLasandRhlsystems.
Similarly rhamnolipid is encoded by Rhl system. The presence of
rhlAB (50%) corroborated with
thepercentageoccurrenceofRhlgenes(rhlI; 41.6% and rhlR; 58.3%).
100%prevalenceoftoxAgeneinallanalyzed strains was found to be
related to the presence oflasIandlasRgenes.Itplaysanimportant role
as a virulence factor of P. aeruginosa with-in catheter associated
UTIs [15] and in one of the study, over 80% of isolates from urine
were found to possess toxA gene [24]. Its presence was also
observed in high percentage among P.
aeruginosastrainsisolatedfromrespiratory and burn infections [7].
In case of bacteraemia, 56.7%ofP.aeruginosastrainsproducedexo-toxin
[25]. Khan and Cerniglia developed a PCR to detect P. aeruginosa by
amplifying the toxA geneandreportedthat96%ofP.aeruginosa
isolatescontainedthetoxAgene,whereas other species of bacteria did
not yield any posi-tiveresults[26].Inanotherstudy,90.7%ofP.
aeruginosaisolatestestedfromburn,wound
andpulmonarytractinfections,harboredtoxA gene [23]. The ptxR gene,
expression enhancer of toxA gene, was only detected in P.
aerugino-saisolates;whereasotherspeciesof Pseudomonas did not yield
any positive results [27], indicating importance of toxA in P.
aerugi-nosa and in UTI. Apartfromextracellularfactors,theinitial
attachmentmediator(fagella)playsasignif-cant role in initiation of
infection. Two types of fagellinproteinshavebeenidentifedinP.
aeruginosa, type a and type b, which can be
distinguishedonthebasisofmolecularsize and reactions with
type-specifc polyclonal and monoclonal antibodies [28]. Type a and
b fa-gellinofP.aeruginosadonotexhibitphase variation; a single
strain produces single type of fagellin,andnoswitchingbetweentypesa
andbhasbeenobserved.Oligonucleotide
primersspecifcforN-terminal(CW46)and C-terminal (CW45) conserved
regions of fagel-lin gene were used for PCR amplifcation of the
fagellin gene of P. aeruginosa PAO1. In a
physi-calgenomeanalysisofthevirulence-associat-ed fiC locus in P.
aeruginosa strains, mapping
andsequencingrevealedgroupsofheterolo-gous a-type (1164 bp; 1185
bp) and highly con-servedb-type(1467bp)fagellingenes[29].
PercentagefiCoccurrencewasfoundtobe
58.3%(25%b-typefagellin,33%a-type).
Absenceoffagellinwasobservedinalmost 40% of the uroisolates
obtained from patients withUTIsinourstudy.Theorganismmust
becomenon-motiletochronicallypersist.
Phagocyticcellsresponddirectlytofagellar motility. This represents
a novel mechanism by whichtheinnateimmunesystemfacilitates
clearance of bacterial pathogens, and provides an explanation for
how selective pressure may result in bacteria with down-regulated
fagellar geneexpressionandmotilityasisevidentin
isolatescausingchronicinfections[30].Thus,
variationinthefagellingenedistribution among P. aeruginosa isolates
from UTI patients maybeduetotheselectivepressureofthe
disease.Differencesinthedistributionsofvirulence
genesinthepopulationstrengthenstheprob-ability that some P.
aeruginosa strains are bet-ter adapted to the specifc conditions
found in specifcinfectioussites.Althoughgenotypic role of
extracellular products such as protease
andexotoxinAhavebeenshownincorneal infection, respiratory infection
and burn wound [7, 14, 24, 25], no genotypic reports are avail-able
on P. aeruginosa induced UTIs, apart from
somephenotypicstudies[10,20].Deter- mination of different virulence
genes of P. aeru-ginosa isolates suggest that they are
associat-edwithdifferentlevelsofintrinsicvirulence and
pathogenicity. This may have different
con-sequencesontheoutcomeofinfection.The
presentstudyisfrstofitskindtoshowthe presence and distribution of
four QS genes and sixvirulencegenesviz:lasI,lasR,rhlI,rhlR, toxA,
aprA, rhlAB, plcH, lasB and fiC across the
genomeofP.aeruginosauroisolates.These virulence factors could
represent a useful
diag-nosticmarkerfortheinvestigationofuroiso-latesofP.aeruginosa.Thus,simultaneous
detection of genes by PCR provides more
conf-dentdetectionofP.aeruginosafromUTIs, gives an idea of
percentage and rate of occur-Virulence gene distribution in P.
aeruginosa UTI133Int J Mol Epidemiol Genet
2014;5(3):125-134renceofsomevirulencegenesandsourceof infection
(urinary tract), which further can
con-tributetothederivationofanimmunotherapy against
UTI.AcknowledgementsWegratefullyacknowledgeDr.BarbaraH. Iglewski,
University of Rochester, New York USA
forprovidingthestandardstrainofPseudo-
monasaeruginosa.Thefnancialassistance provided in the form of a
research fellowship by University Grant Commission (UGC), New
Delhi, India is gratefully acknowledged. Disclosure of confict of
interestNone to declare.Address correspondence to: Kusum Harjai,
Depart- mentofMicrobiology,BasicMedicalSciencesBlo- ck-I, South
Campus, Panjab University, Chandigarh,
India,160014.Tel:+91-1722534142;E-mail:ku-
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