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MICROBIAL DRUG RESISTANCEVolume 10, Number 2, 2004© Mary Ann Liebert, Inc.

Involvement of SHV-12 and SHV-2a Encoding Plasmids inOutbreaks of Extended-Spectrum �-Lactamase-Producing

Klebsiella pneumoniae in a Tunisian Neonatal Ward

THOURAYA BEN-HAMOUDA,1 THIERRY FOULON,2 and KAMEL BEN-MAHREZ1

ABSTRACT

Previous genotypic investigations of extended-spectrum �-lactamase (ESBL)-producing Klebsiella pneumoniaerecovered in a Tunisian neonatal ward revealed the spread of two epidemic strains and a high number of ge-netically unrelated isolates. The aim of the present study was to determine the role of the dissemination ofself-transferrable plasmids harboring bla genes in the outbreaks experienced by the ward. The 49 previouslyidentified clinical isolates of ESBL-producing K. pneumoniae were examined for relationships between theirenzymes and plasmids. Analysis of crude extracts by isoelectric focusing showed four �-lactamase-activitiesat pI 8.2, 7.6, 6, and 5.4. Clinical isolates contained large plasmids that could be transferred by conjugationand transformation conferring resistance to expanded-spectrum cephalosporins. DNA amplification and se-quencing were performed to confirm the identities of transferred �-lactamases. Nucleotide sequence analysisof SHV-specific PCR products from six isolates identified two blaSHV genes corresponding to SHV derivedESBLs, SHV-12 and SHV-2a. PstI digestion of plasmid DNA from transformants revealed six restriction pat-terns. The occurrence of the prevalent plasmid pattern in both epidemic strains and unrelated isolates indi-cated that diffusion and endemic persistence of the blaSHV-ESBL genes in the ward were due to concomitantspread of epidemic strains and plasmid dissemination among unrelated strains.

INTRODUCTION

THE PRODUCTION OF �-LACTAMASES is the most commonmechanism of resistance to �-lactam antibiotics among

Gram-negative bacteria. Extended-spectrum �-lactamases (ESBLs) are enzymes manifesting considerable hydrolyzing ac-tivity on a wide variety of �-lactam antibiotics including oxy-iminocephalosporins and aztreonam.8 Among enterobacteria,Klebsiella pneumoniae strains and, to a lesser extent, Esch-erichia coli, producing ESBLs are frequently implicated in hos-pital outbreaks.18 The nosocomial pathogen K. pneumoniae hasthe potential to cause severe morbidity and mortality in inten-sive care units, in medical and surgical wards and is an impor-tant cause of infection acquired in neonatal wards, particularlyin developing countries. The frequent use of antibiotics inneonatal wards increases the likelihood of infection with resis-tant strains by selection in the neonate microflora.17

The genes encoding for ESBLs are usually carried by plas-mids, which strongly facilitates their spread among strains ofmany bacterial species. Due to their global presence, plasmid-mediated ESBLs are a matter of great concern.13 The epidemi-ology of ESBL-producing K. pneumoniae is complex and in-volves the spread of self-transferable plasmids among unrelatedisolates as well as clonal dissemination of the enzyme-produc-ing microorganisms.2,15,22,26,31,37 Thus, often, more than oneESBL variant is present in the microflora of a given hospitalenvironment.14

Early studies reported a particular prevalence of SHV-2 typeESBL in clinical isolates from Tunisian hospitals.16 Until now,no further information on the characterization of other ESBLshas been available although the intensive use of newer broad-spectrum �-lactams and the increasing frequency of noso-comial infections caused by ESBL-producing species inTunisia.5,6,16 Clinical isolates of ESBL-producing K. pneumo-

1Laboratoire de Biochimie et de Biotechnologie, Faculté des Sciences de Tunis, Université Tunis El Manar, Tunis, Tunisia.2Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, Université Pierre et Marie Curie—Centre National de la Recherche Scien-

tifique, F-75006 Paris, France.

OUTBREAKS OF SHV-12- AND SHV-2a-PRODUCING K. pneumoniae 133

niae recovered during 1994 and 1996 from the neonatal wardof the “Maternité Wassila Bourguiba” in Tunis, were previ-ously investigated for epidemiological relatedness by genotypictyping methods, pulsed-field gel electrophoresis (PFGE), ran-dom amplification of polymorphic DNA (RAPD), and enter-obacterial repetitive intergenic consensus (ERIC)-PCR.4 In thatstudy, we have shown that two epidemic strains and a highnumber of sporadic isolates were responsible for the outbreaksof ESBL-producing K. pneumoniae.4 The diversity of these iso-lates could be associated with the horizontal transfer of plas-mids carrying blaESBL genes between unrelated strains. In the present work, the complexity of the epidemiological situa-tion was further investigated by the combination of plasmidprofile analysis, �-lactamase isoelectric focusing and blaESBL

gene sequencing.

MATERIALS AND METHODS

Bacterial strains

The 49 isolates of K. pneumoniae collected in the neonatalward of “Maternité Wassila Bourguiba” in Tunis have been pre-viously described.4 Thirty-two isolates corresponded to a ma-jor outbreak observed in the first quarter of 1996 and the 17other isolates were recovered during 1995 (Table 1). These iso-lates were resistant to penicillins (amoxicillin and ticarcillin),

cephalothin, and extended-spectrum cephalosporins (ceftriax-one and cefotaxime). A double-synergy test between amoxi-cillin-clavulanic acid and cefotaxime was positive for all iso-lates indicating ESBL production.4

The streptomycin-resistant Escherchia coli HB101 and E.coli DH5� were used as recipients in mating and transforma-tion experiments. E. coli K12 transformed with plasmids har-boring different bla genes, p453 (blaSHV-1), R111 (blaTEM-1),RP4 (blaTEM-2), and pCFF04 (blaTEM-3) were used as referencesfor �-lactamase studies.7

Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MICs) of various �-lac-tam antibiotics were determined using the broth microdilutionmethod and interpreted according to the guidelines of the An-tibiogram Committee of the Microbiology.10 The following an-tibiotics were used: ampicillin (Medochimie LTD, Chypre), cefotaxime (Laboratoires Roussel, France), ceftazidime (Glaxo-operations UK LTD, England), and aztreonam (SanofiWinthrop, France).

Isoelectric focusing of �-lactamases

Crude �-lactamase extracts of the isolates were prepared andsubjected to isoelectric focusing (IEF) as previously described3

utilizing a model 111 Mini IEF Cell (Bio-Rad, Hercules, CA).The �-lactamase activities were detected on the gels by a starch-

TABLE 1. �-LACTAMASE AND PLASMID-DIGESTION PROFILES OF K. pneumoniae ISOLATES AND THEIR TRANSFORMANTS

K. pneumoniae Transformants K. pneumoniae Transformants

Isolate pI of pI of Plasmid Isolate pI of pI of Plasmidnumber �-lactamase �-lactamase type number �-lactamase �-lactamase type

1 7.6, 8.2 8.2 III 26 7.6, 8.2 7.6 ND2a 7.6, 8.2 7.6, 8.2 III 27b 7.6, 8.2 8.2 ND3a 7.6, 8.2 8.2 III 28b 7.6, 8.2 8.2 III4a 7.6, 8.2 8.2 III 29 5.4, 7.6, 8.2 5.4 ND5 7.6, 8.2 — ND 30 7.6, 8.2 8.2 III6a 7.6, 8.2 8.2 III 31 7.6, 8.2 — ND7 7.6, 8.2 — ND 32b 7.6, 8.2 — ND8a 7.6, 8.2 7.6, 8.2 III 33 7.6, 8.2 7.6, 8.2 ND9 6, 7.6, 8.2 6 ND 34a 7.6 7.6 III10a 7.6, 8.2 8.2 III 35 6 6 ND11a 5.4, 7.6, 8.2 5.4 ND 36 7.6, 8.2 8.2 ND12a 7.6, 8.2 — ND 37 7.6, 8.2 8.2 III13 7.6 — ND 38 7.6, 8.2 8.2 III14a 7.6, 8.2 8.2 III 39 7.6, 8.2 8.2 ND15 7.6, 8.2 8.2 III 40 7.6, 8.2 — ND16b 5.4, 7.6, 8.2 5.4, 8.2 V 41 7.6, 8.2 8.2 ND17b 7.6, 8.2 8.2 III 42 7.6, 8.2 8.2 ND18b 7.6, 8.2 8.2 ND 43 7.6, 8.2 8.2 VI19a 7.6, 8.2 8.2 ND 44 7.6, 8.2 7.6 III20b 7.6, 8.2 8.2 ND 45 6, 7.6, 8.2 7.6 ND21 5.4, 7.6, 8.2 5.4 V 46a 7.6, 8.2 8.2 I22 7.6 7.6 IV 47 7.6, 8.2 7.6, 8.2 ND23 7.6, 8.2 8.2 ND 48a 7.6, 8.2 8.2 II24 7.6, 8.2 8.2 ND 49 7.6 — ND25b 7.6, 8.2 8.2 III

Strains 1–32 were isolated during an outbreak in 1996; strains 33–49 were isolated in 1995.a,b Isolates belonging to two major PFGE types; others are sporadic strains.ND, Not determined.

iodine procedure using cephalotin, cefotaxime, or ceftazidime,respectively.30 �-Lactamases with known isoelectric points(TEM-1, pI 5.4; TEM-2, pI 5.6; TEM-3, pI 6.4; SHV-1, pI 7.6)were focused in parallel as pI markers.

Plasmid DNA preparation and resistance transfer

Plasmid DNA of isolates was extracted by the alkaline-lysismethod, analyzed by electrophoresis on 0.8% agarose gels, andtransferred into E. coli DH5� by the calcium chloride methodas described by Sambrook et al.32 Transformants were selectedon Luria-Bertani (LB)-agar plates supplemented with ampicillin(100 �g/ml).

Mating was carried out by mixing equal volumes (1 ml) ofexponentially growing cultures of the donor and the recipientstrains. The mixture was incubated statically overnight at 37°Cand the transconjugants were then selected on LB-agar platessupplemented with ampicillin (100 �g/ml) and streptomycin(256 �g/ml; SERVA Feinbiochemica, Germany).

Plasmid DNA fingerprinting

For fingerprinting analysis, PstI endonuclease (New EnglandBioLabs, Beverly, MA) was used according to the manufac-turer’s instructions. Digested DNA samples were analyzed byelectrophoresis on 1% agarose gels and detected with ethidiumbromide under UV light. HindIII- (Bio-Rad) and BstEII- (NewEngland BioLabs) digested � phage DNA and pUC Mix (MBIFermentas, Lithuania) were used as molecular weight markers.

Digested DNA was transferred from agarose gels onto ny-lon membranes by the procedure of Southern35 and probed forhybridization under stringent conditions32 with an 885-bp se-quence of the blaSHV-1 gene. The probe was obtained by PCRamplification from the blaSHV-1 gene (p453 plasmid) and radio-labeled using the rediprime™ random prime labeling system(Amersham Pharmacia Biotech, UK).

PCR amplification of blaSHV genes

Detection of blaSHV genes was performed by PCR using plas-mid DNAs extracted from transformed cells as templates andthe SHV-A, -B, -C, and -D primers. SHV-A (5�-CACT-CAAGGATGTATTGTG-3�) and SHV-B (5�-TTAGCGTTG-CCAGTGCTCG-3�) were used for amplification of entireblaSHV genes28 and SHV-C (5�-ACTGAATGAGGCGCTTCC-3�) and SHV-D (5�-ATCCCGCAGATAAATCACC-3�) forpartial amplification.14 PCR reactions were performed in aDNA thermal cycler (TRIO-thermoblock, Biometra) in 50-�lmixtures containing 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2,5% dimethylsulfoxide (DMSO), 200 �M of each dNTP, 100ng of each primer, 50 ng of plasmid DNA, and 1 U of Dy-nazyme™ EXT DNA polymerase (Finnzymes, Finlande) ac-cording to the following conditions: 94°C for 1 min, 52°C for30 sec, and 72°C for 1 min (30 cycles). The amplified productswere analyzed by electrophoresis of 5-�l aliquots on 1%agarose gels in TBE.

Cloning and sequencing of blaSHV genes

The 885-bp-long blaSHV PCR products were purified withthe QIAquick gel extraction kit (QiagenTM, Gmbh, Germany)according to the manufacturer’s recommendations. Then, thePCR product was cloned into the pTAG plasmid (Invitrogen

NC, The Netherlands) followed by transformation of E. coliDH5� as described by Sambrook et al.32 Sequencing reactionswere performed with SHV-A and SHV-B primers using thedideoxy chain-termination method33 and the modified phage T7DNA polymerase (Sequenase 2.0, Amersham PharmaciaBiotech). Sequencing was conducted on both DNA strands andwith multiple PCR products. Data were confirmed by the Cus-tom DNA sequencing service of MWG-Biotech AG (Ebersberg,Germany) and nucleotide sequences were compared to theknown SHV sequences using the BLAST algorithm and theGenBank data base (http://www.infobiogen.fr).

RESULTS

IEF analysis of �-lactamases expressed by K. pneumoniae isolates

The �-lactamase contents of all strains were examined byanalytical IEF and the results are summarized in Table 1. Iodo-metric development with cephalotin revealed four enzymes ofpIs 8.2, 7.6, 6, and 5.4, respectively. The pI 7.6 �-lactamasewas identified in all isolates but one (no. 35), and probably cor-responds to the specific K. pneumoniae chromosomal �-lacta-mase SHV-1. The pI 8.2 �-lactamase was also a major activ-ity detected in 44 strains. These two �-lactamases areaccompanied by the pI 6 �-lactamase in two isolates (nos. 9and 45). The pI 5.4 �-lactamase, which is consistent with TEM-1, was found in four isolates (nos. 11, 16, 21 and 29). Finally,only five isolates exhibited only one �-lactamase with a pI of7.6 (nos. 13, 22, 34 and 49) or pI 6 (no. 35).

Transfer of �-lactam resistance and �-lactamasecontent of transformants

All K. pneumoniae isolates contained large plasmids (30–60 kb), in some cases in association with lower molecularweight plasmids (Fig. 1). Plasmid DNA of each isolate was

BEN-HAMOUDA ET AL.134

FIG. 1. Agarose gel electrophoresis of plasmids from K.pneumoniae isolates, their transformants, and their transconju-gants. E. coli p453 (lane 1); E. coli DH5� (lane 2); K. pneu-moniae 33 and its transformant (lanes 3 and 4); K. pneumoniae15 and its transformant (lanes 5 and 6); E. coli HB101 (lane 7);E. coli RP4 (lane 8); K. pneumoniae 33 and its transconjugant(lanes 9 and 10); K. pneumoniae 8 and its transconjugant (lanes11 and 12).

OUTBREAKS OF SHV-12- AND SHV-2a-PRODUCING K. pneumoniae 135

extracted and used to transform E. coli DH5�. Resistance toESBLs was successfully transferred to E. coli DH5� from 41of 49 K. pneumoniae isolates. �-Lactamases produced by thetransformants were then analyzed by IEF. Results are presentedin Table 1.

The transfer of �-lactam resistance was associated with thetransfer of the pI 8.2 �-lactamase in the case of 31 isolates.Four transformants were found to acquire both activities of pI7.6 and 8.2 and one transformant co-expressed the pI 5.4 andpI 8.2 �-lactamases. The remaining transformants expressed ei-ther the pI 7.6 (isolates nos. 22, 26, 34, 44, and 45), the pI 5.4(isolates nos. 11, 21, and 29), or the pI 6 (isolates nos. 9 and35) �-lactamase activity. In all cases, the pI 6, 7.6 and 8.2 �-lactamases were revealed also with both ceftazidime and cefo-taxime demonstrating that these enzymes are ESBLs.

Mating and plasmid contents of transconjugants

Clinical isolates numbers 3, 4, 8, 24, 33, 39, 43, and 44among those exhibiting the predominant �-lactamase pattern(with pIs 7.6 and 8.2) were subjected to conjugation experi-ments with E. coli as recipient. Conjugal transfer of ESBL resistance was observed with a frequency of 10�3 to 10�5/donor. Table 2 shows the �-lactam antibiotic susceptibility with regard to the ESBL profiles and indicates that the sus-ceptibility patterns of transconjugants correlated with those ofparent isolates although MICs for the transconjugants strainswere substantially lower. On the other hand, the MIC valuesof oxyimino-cephalosporins and aztreonam were higher for the transconjugants expressing the �-lactamase of pI 8.2 than those producing the pI 7.6 enzyme. Confirmation that resistance markers reside on self-transferrable plasmids wasachieved by agarose-gel electrophoresis analysis of plasmidDNAs. This analysis showed the presence of large plasmidsthat were transferred to E. coli transconjugants and transfor-mants (Fig. 1).

Plasmid fingerprinting and blaSHV detection

PstI restriction patterns of the �-lactamase-encoding plas-mids from 23 transformants were analyzed by electrophoresison agarose gels. Similarities between the restriction patternswere detected allowing identification of at least six distinct pat-terns (I–VI). The most prevalent plasmid of type III (Fig. 2,lanes 3, 5, and 6) was observed with 17 out of 23 isolates. Plas-mids of this type did not show however absolute identity toeach other. The transformants of isolates 46 and 48 containedplasmids of types I and II, respectively (Fig. 2, lanes 1 and 2).These latter plasmids were distinct from each other and fromtype III. The plasmid of type V (Fig. 2, lanes 8 and 9) exhib-ited by transformants of isolates 16 and 21 was also differentfrom the other patterns. The plasmid types IV and VI (Fig. 2,lanes 7 and 12) were observed with DNA from the strains num-bers 22 and 43, respectively.

To obtain a better characterization of the SHV plasmids oftype III, hybridization analysis was performed using a radiola-beled blaSHV specific probe. Each plasmid was found to pro-duce two hybridization bands of about 850 bp (data not shown).PstI is known to cleave the blaSHV gene, thus explaining thepresence of two bands.

Identification of ESBLs by PCR and sequence analysis

Because almost all isolates studied here produced ESBLswith basic pIs, plasmid DNA preparations of all transformantsexpressing pI 7.6 and/or 8.2 ESBLs were used as templates inspecific PCR to confirm the molecular identity of their �-lac-tamase genes. In all cases, PCR products of the expected sizeof about 880 bp and 300 bp were obtained using SHV-A andSHV-B, SHV-C, and SHV-D primers, respectively. These re-actions indicated that these enzymes belonged to the SHV fam-ily of �-lactamases.

We performed nucleotide sequencing of SHV-specific PCRproducts yielded from six E. coli transformants correspond-

TABLE 2. ANTIMICROBIAL SUSCEPTIBILITIES OF K. pneumoniae ISOLATES AND THEIR TRANSCONJUGANTS

MIC (�g/ml)

Isolates (�-lactamase profile) AMP CTX CAZ ATM

Isolate 3 (7.6 and 8.2) �1024 512 �1024 �1024Transconjugant 3 (8.2) �1024 256 �1024 �1024Isolate 4 (7.6 and 8.2) �1024 1024 �1024 �1024Transconjugant 4 (8.2) �1024 128 512 1024Isolate 8 (7.6 and 8.2) �1024 512 1024 1024Transconjugant 8 (7.6 and 8.2) �1024 256 512 1024Isolate 24 (7.6 and 8.2) �1024 512 1024 1024Transconjugant 24 (8.2) �1024 128 512 1024Isolate 33 (7.6 and 8.2) �1024 512 512 �512Transconjugant 33 (7.6, 8.2) �1024 64 64 512Isolate 39 (7.6 and 8.2) �1024 512 1024 �1024Transconjugant 39 (8.2) �1024 256 1024 �1024Isolate 43 (7.6 and 8.2) �1024 512 1024 �1024Transconjugant 43 (8.2) �1024 8 64 64Isolate 44 (7.6 and 8.2) �1024 512 64 64Transconjugant 44 (7.6) �1024 128 32 32

Amp, Ampicillin; CTX, cefotaxime; CAZ, ceftazidime; ATM, aztreonam.

ing to clinical isolates numbers 22, 30, 37, 39, 43, and 44. Sequences were used to perform a BLAST search in the GenBank database, allowing the identification of these genes.The SHV gene of the pI 8.2 �-lactamase was found to be iden-tical to the sequences encoding SHV-12, and the gene of thepI 7.6 �-lactamase was identical to the sequences encodingSHV-2a.23,29

DISCUSSION

Resistance to the expanded-spectrum cephalosporins amongmembers of the family Enterobacteriaceae is often associatedin the transmissible plasmid-encoded ESBLs.27 In this study,we investigated the ESBLs and the plasmids encoding them in49 clinical isolates of K. pneumoniae recovered in a neonatalward in Tunis during 1995 and 1996.

�-lactamases were typed primarily by IEF, which showedthree kinds of enzymes exhibiting ESBL activity with pIs 8.2,7.6, and 6 and a fourth enzyme of pI 5.4 consistent with TEM-1. All the isolates, but one, produced also a �-lactamase of pI7.6, which was likely to be the chromosomal �-lactamase SHV-1. Enzymes focusing in the range typical for SHV variants out-numbered those focusing in the range typical of TEM variants.SHV-derived ESBLs were indeed detected in all isolates, butone (isolate number 35), and 44 of them harbored ESBL witha pI of 8.2. A preponderance of SHV variants was also foundin European and American ESBL surveys and in countries ofthe Far East.9,19,34,37,38 Our study shows agreement with theconclusion that SHV-type resistance genes are ubiquitous in K.pneumoniae.1 Therefore, we concentrated on these SHV vari-ants to define the types of SHV �-lactamases circulating in the

ward in an attempt to determine the evolution of the resistancein the Tunisian clinical environment.

Transformation and conjugation experiments with E. colidemonstrated that the �-lactamases were encoded on conjuga-tive plasmids. Enzyme contents of transconjugants and trans-formants indicated that each plasmid carried one or two differ-ent bla genes. Coexistence of two �-lactamases encoding geneson the same plasmid of K. pneumoniae was also reported inothers studies.9,11,37

The analysis of PstI restriction patterns of plasmids from 23transformants revealed the heterogeneity of these plasmids withat least six identified fingerprints (I–VI). The most prevalentpattern III found in 17 clinical isolates presented a distributionthat did not correlate with the distribution of �-lactamase pat-terns or with that of the previously identified PFGE types.4

However, Southern blot hybridization studies demonstrated sig-nificant similarities between these plasmids of type III, sug-gesting that the molecular environment surrounding the blaSHV-

ESBL genes was similar within the type III plasmids.Specific DNA amplification followed by sequencing of PCR

products from six isolates revealed nucleotide sequence identi-cal to those of SHV-12 and SHV-2a, which were consistentwith the pI 8.2 and 7.6 ESBLs, respectively.23,29

Studies on the distribution of SHV ESBLs in worldwide haveshown that SHV-2, SHV-4, and SHV-5 enzymes are the mostprevalent in the United States and Europe.19 In the Far East,ESBL-producing strains have been reported with a prevalenceof SHV-12 and SHV-2a in Korea and SHV-12 in Taiwan andJapan. In Thailand, SHV-12 and SHV-5 were the dominanttypes of SHV-derived ESBL.9,20,37,36

Our results can not exclude the presence of other blaSHV

genes coding for SHV-12- and SHV-2-like in isolates whosePCR products were not sequenced, and may not correctly re-flect the distribution of ESBLs in other clinical environmentsin Tunisia. We believe this study represent the first report ofthe presence of SHV-12 and SHV-2a in our country and de-scribes an ESBL distribution probably different from that re-ported for the other Mediterraneen countries where SHV-2 andSHV-5 were commonly found.11 A recent investigation re-vealed differences in the SHV-ESBL distribution in another ge-ographic area, with SHV-12 being the most prevalent one in K.pneumoniae from Italian hospitals.25

The conclusion of our previous study based on genotypictyping methods, which suggested that the outbreak of ESBL-producing K. pneumoniae was due to concomitant spread oftwo strains with the persistence of one of them and dissemina-tion of unrelated isolates,4 was confirmed by the present study.

Resistance-transfer experiments showed, indeed, that vari-ous plasmids carrying �-lactamase genes can readily spreadfrom one isolate to another among unrelated K. pneumoniae.The occurrence of the predominant plasmid type in unrelatedstrains, but also in the two epidemic strains, and its persistencein the ward during 1995 and 1996, suggested that the outbreakinvolved the dissemination of both plasmids harboring blagenes and the isolates themselves, as shown for ESBL-produc-ing K. pneumoniae in a Portuguese hospital.2

Through stepwise mutation, SHV-1 may form SHV-2(Gly238Ser), and then SHV-2a (Gly238Ser and Leu35Gln),SHV-5 (Gly238Ser and Glu240Lys), or SHV-12 (Gly238Ser,Glu240Lys and Leu35Gln).8 Because most SHV-ESBLs differ

BEN-HAMOUDA ET AL.136

FIG. 2. Representative PstI-digestion profiles of plasmidsfrom E. coli transformants. Plasmid type I (lane 1); type II (lane2); type III (lanes 3, 5 and 6); type IV (lane 7); type V (lanes8 and 9); type VI (lane 12). � DNA digested by BstEII (lanes4 and 10: 8.45; 7.24; 6.36; 5.68; 4.82; 4.32; 3.67; 2.32; 1.92;1.37; 1.26; 0.70 kb) and HindIII (lane 11: 23.13; 9.41; 6.55;4.36; 2.32; 2.02; 0.56; 0.12 kb).

OUTBREAKS OF SHV-12- AND SHV-2a-PRODUCING K. pneumoniae 137

from one another by only one amino acid,12 mutations may ex-plain why plasmids of the type III carried the blaSHV-12 genewhereas others had the blaSHV-2a gene. The SHV-12 ESBL mayhave evolved from SHV-2a ESBL within one of the epidemicstrains (Table 1) identified previously.4 The direct evolution ofSHV-12 from SHV-2a, but not from SHV-2 to -5, was sug-gested and considered to be the reason of the absence of SHV-5 besides SHV-2a and SHV-12 in clinical isolates collected inKorea.20,21 Gene duplication followed by a single mutationcould have produced a derivative plasmid harboring blaSHV-12

and blaSHV-2a simultaneously.The diversity of plasmid types may have resulted from var-

ious DNA rearrangements or from the bla gene transfer to plas-mids of different origins, as was suggested in another study.24

The fact that one plasmid of type V carried TEM-encoding genein addition to the blaSHV-12 lends support to the second alter-native.

In conclusion, the complexity and extent of the nosocomialinfections due to ESBL-producing K. pneumoniae could be ex-plained by the diffusion and persistence of the blaSHV-12 andbla SHV-2a in the ward. At least six different plasmids carryingthese genes were able to spread both by occurring in epidemicstrains and transferring among unrelated isolates. Our studyconfirms that the epidemiology of ESBL-producing K. pneu-moniae is often complex.9,24,37,38

ACKNOWLEDGMENTS

This work was funded by grants from the Tunisian “Min-istère de la Recherche Scientifique et de la Technologie,” the“Centre National de la Recherche Scientifique,” and the “Uni-versité Pierre et Marie Curie.” T. B-H was supported by a fel-lowship from the Tunisian “Ministère de l’EnseignementSupérieur.”

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Address reprint requests to:Dr. Kamel Ben-Mahrez

Laboratoire de Biochimie et de BiotechnologieFaculté des Sciences de Tunis

Université Tunis El Manar2092 El-Manar II

Tunis, Tunisia

E-mail: Kamel.BenMahrez@fst.rnu.tn

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