Journal of Cell and Molecular Biology 10(1):39-51, 2013 Research Article 39 Haliç University, Printed in Turkey. http://jcmb.halic.edu.tr

Study of Klebsiella pneumoniae isolates with ESBL activity, from ICU and Nurseries, on the island of Mauritius

Salima Khurshid MUNGLOO-RUJUBALI1, Mohamed Iqbal ISSACK2, Yasmina JAUFEERALLY-FAKIM1*

1Department of Biotechnology, University of Mauritius, Reduit, Mauritius. 2 Victoria Hospital, Ministry of Health and Quality of Life, Mauritius. (* author for correspondence; yasmina@uom.ac.mu)

Received: 11 August 2011, Accepted: 2 November 2012

Abstract

Klebsiella pneumoniae with extended spectrum beta lactamase activity circulate widely among nosocomial environments. Isolates were collected from hospitalised patients in ICU and nursery units on the island of Mauritius, over a two year period. They were tested for their resistance/susceptibility to various antibiotics using the double disc diffusion assay. Following the API biochemical assay their genus/species were confirmed. Their genetic diversity was determined using RAPD, REP and BOX-PCR. Among fifty isolates, most were highly diverse by these methods with a clustering into three groups. No correlation was found among isolates of the same hospital or unit or with dates of collection. Sequence analysis of parts of the TEM, SHV and CTX-M beta lactamase genes confirmed the beta lactamase domain and the presence of SHV-11 and SHV-28. CTX-M 15 was found in some isolates. Furthermore, integrase was PCR-amplified from a few isolates, showing the presence of an integron-borne gene cassette. This study shows diverse clones of Klebsiella pneumoniae circulating with ESBL activity.

Keywords: Klebsiella pneumoniae, ESBL, TEM, SHV, genetic diversity, beta lactamase

Özet

Mauritius adasında kreş ve yoğun bakım ünitesinden elde edilen ESBL aktiviteli Klebsiella pneumoniae izolatlarının çalışması

Geniş spektrumlu beta laktamaz aktivitesi olan Klebsiella pneumoniae nosokomiyal çevrelerde büyük ölçüde bulunmaktadır. İzolatlar, Mauritius adasındaki yoğun bakım ünitesinde yatan hastalardan ve kreşlerden iki yıl boyunca toplanmıştır. Çift disk difüzyon testi kullanılarak izolatların çeşitli antibiyotikler için dirençleri/duyarlılıkları incelenmiştir. Sonrasında API biyokimyasal test ile cins/tür teyidi yapılmıştır. Genetik farklılıkları RAPD, REP ve BOX-PCR kullanılarak belirlenmiştir. Test edilen elli kadar izolatın çoğu, üç grupta sınıflandırılan bu metodlar tarafından oldukça farklı bulunmuştur. Aynı hastane, birimler, ya da aynı tarihlerde toplanan izolatlar arasında herhangi bir korelasyon bulunmamıştır. TEM, SHV ve CTX-M beta laktamaz genlerinin dizi analizleri beta laktamaz domeynini ve SHV-11 ile SHV-28 varlığını onaylamıştır. Bazı izolatlarda CTX-M 15 bulunmuştur. Bundan başka, birkaç izolattan PCR-amplifikasyonu yapılmış olan integraz ile, integron-kaynaklı gen kasetinin bulunduğu gösterilmiştir. Bu çalışma ESBL aktivitesiyle dolaşan Klebsiella pneumoniae’nın farklı klonlarını göstermektedir.

Anahtar Kelimeler: Klebsiella pneumoniae, ESBL, TEM, SHV, genetik çeşitlilik, beta-laktamaz

40 Salima Khurshid MUNGLOO-RUJUBALI et al.

Introduction

Klebsiella pneumoniae is a very common source of infections in hospital environments and are particularly of threat to immune-compromised patients. It is a gram-negative bacillus of the Enterobacteriaceae family. It is mostly treated with beta lactams and fluoroquinolones, but over the years the bacteria have accumulated significant resistance to many of those. Beta lactamases, encoded in bacterial sequences, are able to efficiently hydrolyse the beta lactams of many antibiotics, thus rendering them inactive. A large number of studies have demonstrated their wide occurrence and rapid spread during the era of widespread antibiotic use for medical applications culminating in the appearance of extended spectrum beta lactamases (ESBLs), which are resistant to third generation cephalosporins (Nteimam, 2005).

The mode of action of beta lactamases relies on their ability to bind to enzymes of the bacterial cell wall biosynthesis process, thereby killing the cells. They have an active site serine residue. They act on peptidases that cross-link the penultimate D-alanine of one peptidoglycan unit to a free amino acid of diamino pimelic acid (gram negative) or a lysine residue (Gram positive). The D-alanine-D-alanine substrate of the peptidoglycan unit has a similar stereochemistry to the beta lactam moiety thus competing for binding at the active site of the peptidases. TEM and SHV beta lactamases were characterised first, followed by CTX-M. They are of the Class A enzymes (Ambler, 1980). According to Hall and Barlow (2004), TEM enzymes have experienced larger phenotypic evolution, than SHV and CTX-M. CTX-M ESBLs provide resistance to Cefotaxime, but usually not to Ceftazidime. The double disc synergy method using Cefotaxime and Ceftazidime for detection of ESBLs is the recommended one by the British Society for Anti Microbial Chemotherapy (BSAC). ESBLs can hydrolyse the so-called modern β-lactams that are cefotaxime, cefuroxime, aztreonam and ceftazidime.

The last two decades have seen the appearance of a large number of mutant forms of beta lactamase genes from diverse organisms. Most importantly, there seems to be a positive selection for non-synonymous point mutations, which alter

the binding affinity of the enzyme for its substrate. Key amino acid substitutions bring changes in the substrate and inhibitor binding affinities. Sequence comparisons of the genes have identified the homology within the TEM or within the SHV groups and revealed codon alterations which lead to different phenotypic profiles. Structural comparison is useful in understanding the outcome of nucleotide changes on the 3-dimensional features of the protein. Alleles TEM 1A and 1F differ by silent mutations; however derivative variants can arise by further point mutations or by crossing-over between two alleles. Examples of substitutions can be found at www.lahey.org which lists all the reported variants and their codon differences. Recent work has identified key amino acid replacements which are responsible for phenotypic changes. Mutations that cause the ESBL phenotype are known to be R164H (Arg to His), R164S (Arg to Ser) and G238S (Gly to Ser). TEM-68, which has a decreased sensitivity to inhibitors, has a R275L (Arg to Leu) replacement compared to TEM-1. Similarly other mutations lead to modulating effects such as increasing or decreasing MIC’s (minimum inhibitory concentrations). β -lactam antibiotics penetrate the outer

membrane of many gram-negative bacteria through porins, hence antibiotic resistance can also result from porin loss or deficiency (Nikaido, 1989). Several reports have demonstrated that beta lactamase expression is not the only form of resistance in ESBL bacteria. Other mechanisms such as porin loss have been described in many species (Tsu-Lan et al., 2001).

An understanding of the bacterial populations carrying ESBLs is essential to characterise the molecular features associated with the genes for β-lactamases and understand their evolution. This study describes the characterisation of Klebsiella pneumoniae isolates from hospital sources to assess their ESBL phenotype and identify the corresponding gene sequences. The genetic diversity of the strains carrying this phenotype was determined to gauge the

K. pneumonia isolates with ESBL activity 41

extent of occurrence of the ESBL feature.

Materials and Methods

Isolation and characterisation of K. pneumoniae

About fifty isolates of K. pneumoniae were obtained between March 2003 and January 2005 from six different hospitals (Table 1). These were collected by the Central Analytical Laboratory of

the Ministry of Health, Mauritius. They were pre-selected for their ESBL phenotype by double disk assay. An enlargement of the inhibition zone of 5 mm in the presence of clavulanic acid is a confirmation of an ESBL presence. The isolates were characterised biochemically using the Analytical Profile Index (API) system.

Table 1. List of K.pneumoniae isolates

ISOLATE DATE ISOLATED SEX AGE HOSPITAL WARD SOURCE

SS01 2.06.04 M 36 A ICU BLOOD

SS02 28.08.04 M 37 A ICU BLOOD

SS03 11.11.04 F 5 DAYS D 2-3 N/A

SS04 3.08.04 M 37 A ICU BLOOD

SS05 9.11.04 F 73 B D4 N/A

SS06 5.12.04 M NEWBORN A NURSERY BLOOD

SS07 23.08.04 F 60 C MICU BLOOD

SS08 27.01.04 F 69 C 1-3 N/A

SS09 14.11.04 F 4 DAYS A NURSERY N/A

SS10 26.03.03 NEONATES E NURSERY N/A

SS11 9.11.04 M 15 DAYS C NICU RECTALS

SS12 9.09.03 F 3 WEEKS C NICU N/A

SS13 24.10.04 NEONATES D NURSERY N/A

SS14 6.02.04 A NURSERY ENV(SINK)

SS15 29.11.04 F 6 DAYS A NURSERY N/A

SS16 23.06.04 M 29 B 04 BLOOD

SS17 19.11.04 M 62 B D4 N/A

SS18 20.10.04 M 11 B RDU VAS.CAT.FLUID

SS19 14.02.04 F 2 C S21 N/A

SS20 28.02.03 7 DAYS F N/A

SS21 22.11.02 4 DAYS B NICU N/A

SS22 3.06.04 M 40 A ICU URINE

SS23 3.06.04 M 40 A ICU URINE

42 Salima Khurshid MUNGLOO-RUJUBALI et al.

SS24 4.06.04 M 40 A ICU URINE

SS25 29.10.03 M 59 ICU CSF

SS26 19.09.04 M 53 D 4 N/A

SS27 11.11.04 M 5 DAYS C NICU RECTALS

SS28 27.10.03 M 67 ICU CSF

SS29 23.01.04 F 15 A ICU N/A

SS30 1.09.04 F 53 E DIALYSIS BLOOD

SS31 31.07.04 M 50 C MICU ET-SECRETION

SS32 26.11.04 F 11 DAYS A NURSERY BLOOD

SS33 14.01.05 M 44 A 2-4 BLOOD

SS34 19.02.04 F 61 E F69 BLOOD

SS35 10.12.04 F 5 WEEKS D NURSERY N/A

SS36 27.09.04 M 37 A 2-4 BLOOD

SS37 26.11.04 F 2 DAYS NURSERY N/A

SS38 14.01.05 M 57 E B1 BLOOD

SS39 20.09.04 M 53 D 4 BLOOD-DIALYSIS

SS40 22.10.03 M 50 C ICU BLOOD

SS41 2.10.03 M 42 C N/A

SS42 21.06.04 F 58 A 1-2 BLOOD

SS 43 18.11.03 F 68 B ICU BURN UNIT N/A

SS44 13.03.03 F 12 DAYS B NURSERY CSF

SS45 27.09.03 F 56 C ICU BLOOD

SS46 21.06.04 F 3 WEEKS D NURSERY BLOOD

SS47 16.06.04 M 44 C MICU N/A

SS48 4.10.04 C CARDIAC ICU ET SECRETION

SS49 30.11.04 M 34 A ICU BLOOD

SS50 29.07.04 M 34 D 1-3 BLOOD

RDU: Renal Dialysis unit, NICU: Neurological intensive care unit, MICU: Medical Intensive care unit CSF: Cerebro spinal fluid , ET: endotracheal secretion; N/A: Not available

Their sensitivity to the following antibiotics either alone or in the presence of clavulanic acid was assessed on Mueller-Hinton agar using the pairs of

Oxoid combination discs: (ceftazidime-30 µg and ceftazidime/clavulanate-30/10 µg; cefotaxime-30 µg and

K. pneumonia isolates with ESBL activity 43

cefotaxime/clavulanate - 30/10 µg; cefpodoxime-30 µg and cefpodoxime/clavulanate-30/10µg; cefpirome-30 µg and cefpirome/clavulanate-30/10µg).

BOX-PCR and RAPD PCR amplification

For the BOX-PCR, the primer 5’-CTACGGCAAGGCGACGCTGACG-3’ (Martin et al, 1992) was used. PCR was carried out in a total reaction mixture of 25 µl, containing 50 mM buffer, 2 mM MgCl2, 200 µM each deoxynucleoside triphosphate, 25 pmol of primer, 25 ng of template DNA and 1 U of Taq DNA polymerase . Amplifications were performed with a DNA thermocycler (BIO-RAD) as follows: 1 cycle of 95º C for 4 minutes, 30 cycles of 94º C for 30 s, 92º C for 30 s, 50º C for 1 minute, 65º C for 8 minutes and 1 cycle of 65º C for 8 minutes. The PCR

products were run in 1.5 % agarose gel stained with EtBr and observed to score for markers. Similar reaction was set up for RAPD except that the annealing temperature was 32º C. The gel banding patterns were scored and the data was analysed by NTSYS; and the tree was generated with Darwin 5.0.

Amplification and sequence analysis of TEM, SHV and CTX-M genes

Standard PCR reactions were done for the amplification of beta-lactamase genes using published primer sequences (Table 2). Products were purified after amplification and sequenced. Sequence results were manually checked and edited.

Table 2. List of primer sequences

Primer Name Primer Sequence Reference

SHV A 5- ACT GAA TGA GGC GCT TCC-3

Babini & Livermore, 2000 SHV B 5- ATC CCG CAG ATA AAT CAC C-3

SHV A1 5- TCA GCG AA AAC ACC TTG-3 SHV A2 5 –TCC CGC AGA TAA ATC ACC A-3 SHV A11 5- ATG CGT TAT ATT CGC CTG TG-3

Gniadkowski et al., 1998

SHV A12 5- GTT AGC GTT GCC AGT GCT CG-3

SHV S1 5- TGG TTA TGC GTT ATA TTC GCC-

3 SHV S2 5- GGT TAG CGT TGC CAG TGC T-3 SHV B1 5-ATG CGT TAT ATT CGC CTG TG-3 SHV B2 5- GTT AGC GTT GCC AGT GCT CG-3

TEM D1 5- GGG AAT TCT CGG GGA AAT

GTG CGC GGA AC-3

Bou et al., 2000 TEM D2 5- GGG ATC CGA GTA AAC TTG GTC

TGA CAG-3 TEM A1 5- TAA AAT TCT TGA AGA CG-3 TEM A2 5- TTA CCA ATG CTT AAT CA-3 CTX-M1 5- CGC TTT GCG ATG TGC AG-3

Jungmin Kim et al., 2005 CTX-M2 5- ACC GCG ATA TCG TTG GT-3 INT 2F 5-TCTCGGGTAACATCAAGG -3

Mazel et al., 2000 INT 2R 5- AAGCAGACTTGACCTGA -3

Sequence Analysis SHV, TEM and CTX-M homologue DNA sequences were identified with tblastx (www.ncbi.nlm.nih.gov/blast) search of the non-redundant National Center for Biotechnology Information (NCBI) sequence database. First of all

the nucleotide sequence were converted into contigs using the following web page http://pbil.univ-lyon1.fr/cap3.php. Tblastx was carried out with the contigs in NCBI.

The contigs were translated into amino acids by using the following program: http://www.biochem.ucl.ac.uk/cgi-

44 Salima

bin/mcdoThe a

was selecorresponlow E-va

The aCTX-M MultAlig

Results

Biochem

The API isolates whowever groups baGroup I: Group II:Group IIIGroup IVGroup V:Group VI

ESBL ac

Four out were isodifferencemm for isolates w

The dfew, mosextended

Figure 1Lane 44: 50: SS50

Khurshid MUN

onald/cgina2aaamino acid seected. This wnding sequenlue. amino acid seand their ho

gn.

mical characte

20E system were identifiethese isolates ased on the pr Profile 52157: Profile 52153I: Profile 5207

V: Profile 5205 Profile 52557I: Profile 5225

ctivity

of fifty isolatelates SS22, Se in the zone each one o

were confirmedouble disc assst of the isola

spectrum

. DNA fingerSS44, Lane 4. C: Control, H

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rprint analysis45: SS45, LanH: Hyperladde

UBALI et al.

no stop codigned with ted well with

SHV, TEM are aligned w

at 45g negatilla pneumoniassified into thrd:

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presence odoxime, SS 4eptible, whileSS 09 showeda slight inc

ence of inhibitas considered

X and RAPD P

X-PCR is rprinting baers targeting genic region

wed that isola, SS44, SS48,isolated duri

rent hospitalser (Figures

4, SS45, SS47shared the samures 2 and 5).

X-PCR. Lane 7, Lane 48: S

es SS 35 anda zone of i25 mm, resp

s zone by > 5lanate. Three S 42 were suno increase in

of inhibi42, SS 45 and

e with Cefpird some susce

crease of 4 tor clavulanata non-ESBL.

PCR

commonly acterial geno

repetitive ns. The oveates SS49, S, SS46, SS47,ing the same s, were group1, 2, 3). Iso7, SS48, SS4me API profile

42: SS42, LanS48, Lane 49

d SS 42 were inhibition of

pectively, and 5 mm in the isolates, SS

usceptible to n diameter in itor. With d SS 47 were rome SS 42 eptibility and mm in the

te. Isolate SS

used for omes using elements in erall results SS42, SS45,, SS50 which year, but in

ped into one olates SS42, 49 and SS50 e i.e profile 1

ne 43: SS43, : SS49, Lane

Figure 2joining clof 0.9529

Figure 3.3: SS03, Lane10: control, H

. Dendogramlustering for t9.

. RAPD fingeLane 4: SS0SS10, Lane

H:Hyperladder

generated frothe 50 isolates

erprint of K. p04, Lane 5: S11: SS11, Lar(11) Bioline

om BOX-PCRs of Klebsiella

neumoniae wS05, Lane6:

ane12: SS12,

K

R based on Dia pneumoniae

ith primer OPSS06, Lane 7Lane13: SS1

K. pneumonia is

ice similarity e with a cophe

PA12. Lane 1: 7: SS07, Lane13, Lane14: S

solates with ES

coefficient anenetic correlat

SS01, Lane 2e 8: SS08, LaSS14, Lane 1

BL activity 45

nd neighbour tion (r) value

2:SS02, Lane ane 9: SS09, 15: SS15. C:

46 Salima

Figure 4Lane 44: SS50.C: c

Figure 5joining clBOX and

Khurshid MUN

. RAPD fingeSS44, Lane45control, H:Hy

. Dendogramlustering of R

d RAPDs, the

NGLOO-RUJU

erprint of K .p5: SS45, Lane

yperladder(1) B

generated froRAPD for the 5

clustering of i

UBALI et al.

pneumoniae we 46: SS46, LaBioline.

om RAPD-PCR50 isolates of isolates was n

with primer Oane47: SS47,

R based on Df Klebsiella pnnearly the sam

OPB 01. Lane4Lane48: SS48

Dice similarity neumonia. For

me, with three d

42: SS42, Lan8, Lane 49: SS

coefficient anr both sets of discernable gr

ne 43: SS43, S49, Lane50:

nd neighbour data with the roups.

TEM, SH

Differentlonger thaand SHVfor SHV was achieThere we

Figure 6.

Figure 7.with isola

HV and CTX

t size productsan 1 kb. The C

V B1 and B2 gA11 and SHVeved with tw

ere only twelv

. Protein blast

. Alignment oate SS25. Ami

-M amplifica

s were obtaineCTX-M primegave a producV A12; twentyenty-one isolae isolates, wh

t of translated

of amino acid ino acid seque

ation

ed for each seers amplified ct of 865 bp. Ay two isolates ates for TEM

hich amplified

product from

sequence fromence from isol

K

t of primers. Ta product of 5Amplificationfor SHVB1 a

M A1/A2 and with CTX-M

m isolate SS11

m isolate SS1late SS47 alig

K. pneumonia is

The TEM A1/585 bp. Both sn were detecteand SHVB2, retwenty eight

M to give a pro

with primer S

1 with SHV-2gned well with

solates with ES

/A2 gave prodsets of SHV Aed for thirty sespectively. Aisolates for Tduct of 585 bp

SHV A11/A12

28. The same wh CTX-M 15.

BL activity 47

ducts slightly A11 and A12, even isolates

Amplification TEM D1/D2. p.

2

was obtained

48 Salima Khurshid MUNGLOO-RUJUBALI et al.

For one isolate, the presence of integrons was assessed using integrase specific primers. It was found that the INT amplicon from isolate SS02 had the sequence for integrase. The BLAST result returned a match with Corynebacterium diphtheriae integrase. The results for integrase from isolate SS02 are shown below. >2R.INT

QAMKT

ATAPLPPLRS VKVLDQLRER IRYLHYSLRT EQAYVHWVRA FIRFHGVRHP

ATLGSSEVEA FLSWLANERK VSVSTHRQAL AALLFFYGKV LCTDLPWLQE

IGRPRPSRRL PVVLTPDEVV RILGFLEGEH RLFAQLLYGT GMRISEGLQL

RVKDLDFDHG TII

ref|NP_940279.1| integrase [Corynebacterium diphtheriae NCTC ... 331 2e-89

REFSEQ: accession NC_002935.2

KEYWORDS complete genome.

SOURCE Corynebacterium diphtheriae NCTC 13129

ORGANISM Corynebacterium diphtheriae NCTC 13129

PUBMED 14602910

Amino acid sequence

>2int

mktataplpp lrsvkvldql rerirylhys lrteqayvhw vrafirfhgv rhpatlgsse

veaflswlan erkvsvsthr qalaallffy gkvlctdlpw lqeigrprps rrlpvvltpd

evvrilgfle gehrlfaqll ygtgmriseg lqlrvkdldf dhgtiivreg kgskdralml

peslapslre qlsrglcckd wrqsevgcrs apirrllrng g

DiscussioThe Klebto determcame froby classiidentificaDNA- baAPI 20 E

The pthe anti-isolates thin fact EKlebsiellarecent sitESBL froresistant b

OPB primers (so primeproducedtwo and aOPL 07 tSS40, SSSS48, SSof sizes ssame isoltwo primresults oproducedsame grou

No codates of

on bsiella pneumomine their gem ICU (42%ical biochemiation and Oxoiased methods.E system identiphenotypic cha-microbials inhat were colleESBLs. API a pneumoniaetuation regardom Klebsiellabacterial path01, OPL 07

(Fig 3&4), chers that wered more polymoall the isolatethe same profiS41, SS42, SSS49, and SS50sizes 700bp, lates gave dif

mers. The denof BOX-PCRd three groupsup for both m

orrelation wasf isolation su

Figure

oniae isolateenetic related

%). These isolaical (growth id Combinatio. Biochemicalified two grouaracterisation ndicated thatected and preconfirmed th

e. There are nding the extea pneumoniaogens in Mauand OPA12 w

hosen from thee screened. Porphic profiles were differeile was obtain43, SS44, SS40. They all ha1000 bp and

fferent patternndrograms obR and that o

. Several of thmethods. s found betweeuggesting tha

e 8. Conserve

s were analysdness and moates were teston media, Aon disk test) al profiles by tups. of sensitivityt most of t

e-screened, weheir identity no reports of tnt of spread

ae or any othuritius. were the RAPe twenty five Primer OPA es than the othent. With primned from isolat45, SS46, SS4ad only 3 ban1200 bp. The

ns with the othbtained with tof RAPD bohem were in t

en hospitals aat the ESBL

K

ed domain of i

sed ost ted

API and the

to the ere as

the of

her

PD or 12

her mer tes 47, nds ese her the oth the

and L -

carrydiverno cosame there ESBLcentredone beenpneumPFGEconcobetwestrain

Thwere speciM. Aand/oproduspeciAmplBLAand harboS25 This foundCTX-and enterogener

K. pneumonia is

ntegrases.

ing strains hse sources. Siorrelation fou

hospitals, phad not be

L carrying se, although thfrom patients

used for moniae (ThouE typing ordant resuleen groups ofns could be mahe presence odetected by

fic primers foAll fifty isolor SHV genuced a prodfic primerslicons were ST results indSS25 carried oured SHV 1and S41-49, variant is k

d worldwide. -M enzymes,

25. ESBLobacteria arration ceph

solates with ES

had originatedimilarly, there

und among samprobably ind

een significanstrains withinhe sampling ws. RAPD and

typing of uraya et al., and RAPDlts and dif epidemiologiade.

of the beta-lactPCR amplifi

for TEM, SHVates amplifienes while oduct with ts (data no

purified anddicated that i

SHV 28 wh1. For isolateCTX-M 15

known to beThere are fivnamely CTX

L carrying re resistant halosporins

BL activity 49

d from very e was little or mples of the

dicating that nt spread of n the same

was primarily d PFGE have f Klebsiella

2003). Both D produced iscrimination

ically related

tamase genes ication using V and CTX-ed the TEM only twelve the CTX-M ot shown).

d sequenced. solates SS11 hereas SS27 es S01, S02, was present. e commonly ve groups of

X-M 1, 2,3, 4 strains of

to third such as

50 Salima Khurshid MUNGLOO-RUJUBALI et al.

cefotaxime, cefdazidime and ceftriaxone; and are important threats. These enzymes do not affect cephamycins or imipenems. CTX-M is widely found in E. coli and Klebsiella isolates worldwide. CTX-M 15 in Salmonella enterica has also been shown to be present in stool samples of patients suffering from acute diarrhoea (Rotimi et al., 2008). The CTX-M 15 is encoded on large incompatibility plasmids of sizes varying between 145,5 and 242,5 kb. Many are found as IncFII together with IncFIA and IncFIB, with one report showing IncFI not associated with any IncFII (Mshana et al., 2009). This suggests that the plasmids contribute to the lateral transfer of the resistance genes. CTX-M ESBL hydrolyse cefepime more efficiently than other ESBLs (Mendonca et al., 2007).

TEM and SHV enzymes have evolved significantly over the last decade or so and there is some evidence of positive selection. A list of over hundred different combinations of amino acid substitutions have been reported for the 278 long polypeptide. It is likely that the evolution of the protein is directed towards finding the mutants that have enhanced catalytic efficiency and can better compete with others. Experimental prediction of how resistance genes evolve in response to selection pressure has shown that alleles of the genes will mutate in a similar way as in nature. Several in vitro experiments using gene shuffling, or mutagenesis with Taq Polymerase under error-prone or the use of nucleoside analogs have led to the appearance of mutants with extreme resistance (> 64X or to beta-lactamase inhibitor). Interestingly these in vitro experiments have recovered amino acid substitutions that are naturally found among the TEM beta-lactamases. Hall and Barlow (2002) have used error-prone PCR for assessing the evolution of TEM-1 under selection pressure. Mutations were introduced into the genes and the mutated genes cloned into E.coli. Growth in increasing concentrations of antibiotic led to the identification of mutations in the resistance genes which allowed survival in the highest antibiotic concentrations. The cycles of mutations and selection are repeated until there is no further increase in resistance.

The Barlow-Hall in vitro method recovered seven out of the nine amino acid substitutions that had arisen in nature. The same approach has been used to isolate an allele giving a resistance with a

MIC of 256 µg/ml compared to 0.5 µg ml for reported, natural form of TEM. Those resistant alleles had between 2 to 6 amino acid substitutions. Three of these were identified that increased the MIC from 0.5 to 2 µg /ml then to 32 µg /ml and finally from 32 µg /ml to 256µg /ml. This would strongly suggest that there is a “natural evolutionary” series of replacement leading to the most resistant allele (Ford and Avison, 2004).

Analysis of a K. pneumoniae genome sequence (strainMGH78578) reported two chromosomal blaSHV genes. Closer comparison of the surrounding sequence (GC content and other genes) of the two genes indicated that one had evolved from within that strain itself. On the other hand the second one was accompanied by insertion sequences IS26 suggesting that it was mobilised most likely from a different K. pneumoniae strain. Nucleotide sequence comparison and amino acid replacement at the active site have pointed to convergent evolution of ESBLs during the period of intense cefotaxime use and later that of ceftazidime.

Beta lactamase genes have been shown to be vehicled as gene cassettes within integron sequences. VEB-1 (Vietnamese Extended Spectrum) and GES-1 (Guyanese Extended Spectrum) were first described associated with the intI1 integrase (Poirel et al 1999., 2000). Being part of the integron ensures that the resistance genes are rapidly mobilised for lateral transfer intra- and inter-species. In this study, one CTX-M 15 from isolate SS02, was found together with an integrase (Figure 3c). This could explain its rapid spread since it was first described.

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