Mosaic Structure of p1658/97, a 125-Kilobase Plasmid Harboring an Active Amplicon with the Extended-Spectrum {beta}-Lactamase Gene blaSHV-5

Escherichia coli isolates recovered from patients during a clonaloutbreak in a Warsaw, Poland, hospital in 1997 produced differentlevels of an extended-spectrum ß-lactamase (ESBL)of the SHV type. The ß-lactamase hyperproduction correlatedwith the multiplication of ESBL gene copies within a plasmid.Here, we present the complete nucleotide sequence of plasmidp1658/97 carried by the isolates recovered during the outbreak.The plasmid is 125,491 bp and shows a mosaic structure in whichall modules constituting the plasmid core are homologous tothose found in plasmids F and R100 and are separated by segmentsof homology to other known regions (plasmid R64, Providenciarettgeri genomic island R391, Vibrio cholerae STX transposon,Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97bears two replication systems, IncFII and IncFIB; we demonstratedthat both are active in E. coli. The presence of an active partitionsystem (sopABC locus) and two postsegregational killing systems(pemIK and hok/sok) indicates that the plasmid should be stablymaintained in E. coli populations. The conjugative transferis ensured by the operons of the tra and trb genes. We alsodemonstrate that the plasmidic segment undergoing amplificationcontains the bla_SHV-5 gene and is homologous to a 7.9-kb fragmentof the K. pneumoniae chromosome. The amplicon displays the structureof a composite transposon of type I.

INTRODUCTION

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Introduction

Bacteria have an amazing ability to adapt to changes in theirenvironment. They exchange genetic material via horizontal genetransfer, which is considerably enhanced by mobile genetic elements,such as plasmids, phages, transposons (Tn), insertion sequences(ISs), and gene cassettes. The majority of known gene cassettesand transposons, as well as numerous plasmids (R plasmids),contain antibiotic resistance determinants (38). Many of thesehave been demonstrated to originate from bacterial chromosomes,as is the case for several ß-lactamase gene families,including those coding for the SHV-type enzymes. The spreadof the bla_SHV genes throughout the bacterial world has probablybeen initiated by repeated events of the IS26-mediated transpositionthat mobilizes these genes to plasmids from the chromosome ofKlebsiella pneumoniae (6, 7, 13). The plasmid-encoded SHV ß-lactamasesare frequently found in most of the clinically important enterobacterialspecies, but they have also been observed in other gram-negativeorganisms, like Pseudomonas aeruginosa (12, 18, 26, 32). Oneof the SHV enzymes, SHV-5, belongs to the most commonly encounteredextended-spectrum ß-lactamases (ESBLs) that hydrolyzethe vast majority of ß-lactam antibiotics, includingthe newer cephalosporins and monobactams. It has been identifiedworldwide in almost all countries in which ESBLs have been studiedat the molecular level (12, 18).

The plasmidic bla_SHV-5 gene was identified in 12 Escherichiacoli isolates recovered during a clonal outbreak in a Warsaw,Poland, hospital in 1997 (25). All of the isolates exhibitedidentical levels of resistance to non-ß-lactam antibiotics(aminoglycosides); but many of the isolates differed in theirlevels of resistance to expanded-spectrum cephalosporins andcould be separated into two groups, "resistant" and "susceptible,"for which the MICs of ceftazidime were >128 µg/ml and4 µg/ml, respectively. PstI restriction analysis of plasmidsisolated from the "resistant" and "susceptible" groups revealedidentical restriction patterns except for some fragments presentin nonequimolar amounts in the "resistant" plasmid (25). Twoof these fragments were subsequently found by hybridizationwith the gene-specific probe to contain the bla_SHV gene. Thesefacts indicated that the "resistant" phenotype correlated withthe multiplication of a plasmid region comprising bla_SHV. Astudy was commenced in order to understand the phenomenon ofthe plasmid fragment amplification. The first step of the projectwas to reveal the complete sequence of one of plasmids of the"susceptible" group, p1658/97, which is a presumed initial moleculein the amplification process. The results of the nucleotidesequence analysis and identification of the region within thebla_SHV-5 gene that undergoes amplification are presented inthis paper.

MATERIALS AND METHODS

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Materials and Methods

Plasmids and bacterial strains. Plasmids p1658/97 and p1657/97 were purified from "susceptible"and "resistant" E. coli isolates 1658/97 and 1657/97, respectively,which were recovered from patients in the neonatal ward in aWarsaw hospital in October 1997 (25). pSGMU2 is a cloning vectorand is a derivative of pUC13, which carries the cat gene. Thecat gene confers resistance to ampicillin and chloramphenicol(8). pACYC184 is a cloning vector with ori P15A and genes causingresistance to tetracycline and chloramphenicol (5). PlasmidspFBAMP and pR were constructed in this study and are derivativesof p1658/97. pFBAMP (23,870 bp; positions 85091 to 108960 ofp1658/97) was obtained after BcuI digestion of p1658/97 andsubsequent religation, and it contains the IncFIB replicon andthe bla_SHV-5 gene. pR (13,590 bp) was obtained upon StuI digestionof p1658/97, followed by ligation of its fragment (positions29356 to 42120) with the PCR-generated cat gene of pACYC184.It contains the IncFII replicon. Experiments in this study wereperformed with E. coli strains DH5 ${alpha}$ and A15 (3, 25).

Conjugation assay. One-milliliter volumes of cultures of the donor and recipientstrains (10⁹ CFU of each strain per ml) grown in Luria-Bertani(LB) medium (Biocorp, Warsaw, Poland) were mixed and incubatedfor 30 min at 37°C. E. coli A15 or DH5 ${alpha}$ , both of which areresistant to rifampin, was used as the recipient strain. Transconjugantswere selected on LB agar (Biocorp) supplemented with ceftazidime(10 µg/ml; GlaxoSmithKline, Stevenage, United Kingdom)and rifampin (100 µg/ml; Polfa Tarchomin, Warsaw, Poland).

Selection of E. coli "resistant" clones carrying p1658/97 with the amplified bla_SHV-5 gene. One-hundred microliters of the E. coli DH5 ${alpha}$ (p1658/97) overnightculture or its appropriate dilution was plated on LB agar, andthen a sterile disk (diameter, 10 mm; Whatman 3MM paper) with50 µg of ceftazidime was placed on it. The plates wereincubated overnight at 37°C. The E. coli clones able toform colonies inside the zone of growth inhibition were collectedas "resistant" colonies.

DNA cloning and sequencing. The vectors were purified by using a Plasmid Midi AX DNA purificationkit (A&A Biotechnology, Gdañsk, Poland). A largeamount of p1658/97 DNA for random library construction, directsequencing, and digestions was prepared by the alkaline lysisprocedure (2), followed by CsCl-ethidium bromide gradient ultracentrifugation(36). The library of random fragments of p1658/97 was preparedby sonication by the procedure used for sequencing of the Haemophilusinfluenzae Rd genome (42). Fragments of between 1.6 and 2.0kb were cloned into a unique SmaI site of the pSGMU2 vector(8). The plasmid DNA from the resulting subclones was purifiedprior to sequencing by using DNA purification kits (Macherey& Nagel, Düren, Germany; QIAGEN, Hilden, Germany).Cycle sequencing was performed with the use of BigDye (version1.0 or 2.0; Applied Biosystems, Foster City, CA) in a GeneAmp9700 PCR system (Applied Biosystems). Samples were subsequentlypurified on CentriSep columns (Applied Biosystems) or subjectedto sodium acetate-ethanol precipitation, according to the protocolof the BigDye kit manufacturer. Extension products were separatedon an ABI 377 automated sequencer (Applied Biosystems). Sequencingwas performed with random library clones, the PCR products,and p1658/97 as templates. In the last case, a BAC sequencingprotocol was used (Applied Biosystems). All enzymes used forDNA manipulations were from MBI Fermentas, Vilnius, Lithuania.

PCR amplification of the cat cassette. Primers chlL (5'-ATCCGCTTATTATCACTTATTCAGG-3') and chlP (5'-GGTGTCCCTGTTGATACCGG-3')were used for amplification of the cat gene of pACYC184 (5).The reaction conditions were as follows: 3 min at 95°C;25 cycles of 30 s at 94°C, 30 s at 48°C, and 30 s at72°C; and finally, 7 min at 72°C. The resulting PCRproducts were run in 1% agarose gels (SeaKem; FMC Bioproducts,Rockland, ME) and purified for cloning by using a DNA Gel-Outgel extraction kit (A&A Biotechnology).

Plasmid sequence analysis. The resulting nucleotide sequences were assembled by using theDNA Lasergene 99 package (DNASTAR, Madison, WI); and analysisand annotation of the sequence were performed by additionallyusing Glimmer 2.0 (The Institute for Genomic Research, 1999[35]; RBSFinder.pl [The Institute for Genomic Research]; SignalP[CBS; www.cbs.dtu.dk/services/SignalP-2.0/] [24]), InteroScan(Expasy; www.ebi.ac.uk/InteroScan/ [45]), and TMHMM Server (version2.0; CBS; www.cbs.dtu.dk/services/) software.

Nucleotide sequence accession number. The complete nucleotide sequence of plasmid p1658/97 has beensubmitted to the GenBank database under accession no. AF550679.

RESULTS AND DISCUSSION

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Results and Discussion

Preliminary plasmid analysis. E. coli 1658/97, one of the ESBL-producing outbreak isolatesof the "susceptible" group (25), was chosen as a donor of plasmidp1658/97 for conjugal transfer to E. coli A15 and then to DH5 ${alpha}$ .The transconjugants of A15 (RecA⁺), as well as those of DH5 ${alpha}$ (RecA^–), were obtained; and subsequently, 10 of them weresubjected to the ceftazidime disk test. All clones tested wereable to form colonies inside the growth inhibition zone. Restrictionanalysis of the p1658/97 plasmids recovered from these colonieswith the rare cutting enzyme, SwaI, revealed that one band wasnonproportionally more intense when compared with p1658/97 isolatedfrom the "susceptible" colony taken from outside the inhibitionzone (Fig. 1). Although similar data were previously shown forplasmids p1657/97 and p1658/97 (extracted from "resistant" and"susceptible" clinical isolates, respectively) (25), this resultindicated that acquisition of the "resistance" phenotype canproceed in a RecA-independent manner. Moreover, as shown inFig. 1, comparison of the SwaI digestion of p1657/97 isolatedfrom the E. coli DH5 ${alpha}$ strain cultured in the presence of 128µg/ml ceftazidime with that of p1658/97 isolated fromthe "resistant" DH5 ${alpha}$ colony revealed similar restriction patterns,with the overrepresented DNA fragments isolated from the plasmidsbeing of identical sizes and similar intensities. This indicatedthat in both p1658/97 from the "resistant" DH5 ${alpha}$ colony and p1657/97the same region underwent amplification, pointing to the identicalmechanism of this event. Therefore, the p1658/97 plasmid isolatedfrom the "susceptible" DH5 ${alpha}$ clone was subjected to DNA sequenceanalysis.

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FIG. 1. SwaI digestion of p1658/97 and p1657/97. The results of SwaI digestion of p1658/97 DNA with the "nonamplified" and the "amplified" regions isolated from E. coli colonies collected from outside and inside the growth inhibition zone ("susceptible" and "resistant," respectively) and p1657/97 isolated from DH5 ${alpha}$ cultured with the presence of 128 µg/ml ceftazidime are shown. Equal amounts of all plasmids were loaded. The lanes are described at the top. The arrow shows the amplified fragment of 8,817 bp. Lane M, GeneRuler DNA Ladder Mix (MBI Fermentas). Numbers on the left are in base pairs.

Overall gene content and G+C content of p1658/97. The complete nucleotide sequence of plasmid p1658/97 was determinedby sequencing of its random fragments cloned into the pSGMU2vector. It was found to be a circular molecule of 125,491 bp.Bioinformatic analysis of the sequence has revealed the presenceof 142 putative genes, 7 of which (4.93%) had no homologs inpublic databases and thus could be considered p1658/97 specific,while 52 (36.62%) and 83 open reading frames (ORFs) (58.45%)had homologs of unknown and known functions, respectively.

Two main parts can be distinguished in the p1658/97 sequence,based on the over 90% similarity to other DNA molecules at thenucleotide level. The first part, which covers about 61 kb,i.e., 48% of p1658/97 (Fig. 2), demonstrates homology with sequencesof conjugative plasmids F (sex factor; 99 kb; GenBank accessionno. AP001918) and R100 (resistance to mercuric ions, streptomycin,chloramphenicol, and tetracycline; 94 kb; GenBank accessionno. AP000342). The F/R100-like sequence comprises one largecluster and two smaller clusters of genes. The large cluster(positions 114495 to 43552) is interrupted by orf62 and containsgenes responsible for conjugative transfer, postsegregationalkilling (two systems), and plasmid replication (system IncFII).Of the two smaller regions, the first one spans 2 kb (positions99420 to 101435) and encompasses another plasmid replicationsystem (IncFIB), whereas the second one, which is 3.5 kb (positions109127 to 112552), encodes the active partition system, as wellas orf53, which is of unknown function. The latter segment isflanked by two IS1 copies, suggesting that it was acquired byp1658/97 via an IS1-mediated recombination event.

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FIG. 2. Graphical overview of p1658/97. ORFs are shown by arrows: p1658/97-specific ORFs are in black, ORFs homologous to genes of known function are in white, and ORFs homologous to genes of unknown function are in gray. The positions of mobile elements are additionally shown as white boxes. The orientations of the mobile elements are marked with black arrows; incomplete mobile elements are marked by a ${Delta}$ symbol. Regions of over 90% similarity (at the nucleotide level) to the following DNA molecules are shown by shadings according to the legend: 1, plasmid F; 2, plasmid R100; 3, both plasmids F and R100; 4, the chromosomes of both E. coli and S. enterica serovar Typhimurium; 5, a region found in the K. pneumoniae chromosome and plasmids pHNM1, pSEM, and pACM1; 6, P. rettgeri genomic island R391 and the Vibrio cholerae STX transposon; 7, plasmid R64; 8, p1658/97-specific regions; 9, a region shared with pSEM, pACM1, and some other plasmids containing an integron; and 10, DNA molecules other than those mentioned above. The functional modules of p1658/97 outside the circle are indicated as follows: PAR, partition; PSK, postsegregational killing; REP, replication. The G+C contents of the sequence blocks are denoted in the inner circle.

The second major part of the p1658/97 sequence emphasizes itsmosaic character; it is composed of six subregions displayinghomology to molecules other than F and R100: (i) two subregionshomologous to plasmid R64 (GenBank accession no. AP005147; Salmonellaenterica serovar Typhimurium); (ii) a subregion homologous tothe R391 genomic island (GenBank accession no. U13633l; Providenciarettgeri) and the STX transposon (GenBank accession no. AY055428;Vibrio cholerae); (iii) a subregion with the bla_SHV-5 gene homologousto a fragment of the K. pneumoniae chromosome (http://genome.wustl.edu/projects/bacterial/kpneumoniae),as well as plasmids pSEM (GenBank accession no. AJ245670; S.enterica serovar Typhimurium), pACM1 (GenBank accession no.AY081221; Klebsiella oxytoca), and pHNM1 (GenBank accessionno. AY532647; Enterobacter cloacae); (iv) a subregion with anintegron shared with pSEM (GenBank accession nos. AJ245670 andAJ009820), pACM1 (GenBank accession nos. U90945, AY309067, andAY309066), and some other plasmids; and (v) a subregion homologousto the chromosomes of E. coli and S. enterica serovar Typhimurium(GenBank accession nos. AF155222 and AL627276, respectively).This part of the p1658/97 molecule also contains two small p1658/97-specificsubregions and two other regions that display homology to DNAmolecules different from those mentioned above.

The mean G+C content of the p1658/97 nucleotide sequence is50.5%, which is consistent with the average amount of G+C foundin the genomes of members of the family Enterobacteriaceae.However, the G+C content among the particular sequence blocksof p1658/97 varies from 30% to 69%. Four blocks with mean G+Ccontents different from the average by more than 10% are markedin Fig. 2, and their borders are superimposed with some of themosaic subregions homologous to DNA molecules other than F andR100.

Plasmid backbone. The functional modules of the plasmid backbone coding for replication,conjugative transfer, and the stable maintenance of p1658/97have homologs in either F or R100, or both, suggesting thatall three of these plasmids evolved from a common ancestor.

The functionalities of two replication systems found in p1658/97,IncFIB (positions 99420 to 101435) and IncFII (positions 37402to 39441), were tested in E. coli. The replicons were separatedby construction of two p1658/97 derivatives, pR and pFBAMP,which contain the IncFII and IncFIB replication systems, respectively.These plasmids were introduced into E. coli DH5 ${alpha}$ cells. In bothcases transformants were obtained, and the reisolation of theintroduced plasmids (data not shown) demonstrated that the tworeplication systems of p1658/97 are functional in E. coli.

The stability of p1658/97 in a bacterial population might beensured by two mechanisms: active partition determined by thesopABC operon (positions 109127 to 111994) and homologous tothat of the plasmid F (11, 15) and two postsegregational killingsystems, pemI/pemK (downstream of the IncFII replication module;positions 40555 to 41158) (40) and hok/sok (positions 124314to 124583) (37). Both of these systems are identical to thosein their R100 counterparts.

The conjugative transfer of plasmid p1658/97 is ensured by theproducts of the tra and trb genes. These genes are organizedin a 33.3-kb operon (positions 2103 to 35307) that is almostidentical at the nucleotide level to the tra operon found inthe F plasmid (U01159) and that consists of 36 genes: 24 tragenes, 10 trb genes, artA, and finO (10). Nevertheless, twodifferences between these tra operons have been found. The firstone is a frame shift in the trbF gene, creating the stop codonthat results in shortening of the TrbF protein of p1658/97 by25 amino acids. The TrbF function is unknown, but it is predictedto be an integral membrane protein (10). The second differenceconcerns the finO sequence, which codes for a conjugation regulatoryfunction. In the F plasmid, this gene is interrupted by IS3a,resulting in an increase in transfer frequency in broth, evaluatedas one transconjugant per donor cell (10, 38). In contrast tothe sequence of the F plasmid, no IS(s) has been found withinfinO of p1658/97. The conjugative transfer frequency of p1658/97in liquid medium was measured to be 4.5 x 10^–3 ±4.1 x 10^–4 transconjugants per donor cell. This valueseemed to be significantly lower than the transfer frequencyof the F plasmid and could have been caused by the presenceof the functional finO sequence (10, 38).

Mobile elements. Nineteen mobile elements identified in p1658/97 constitutedabout 14% of the plasmid sequence and are listed in Table 1.All of them (e.g., IS26, IS1, and IS100), except for the remnantof Tn21, are situated within the region of similarity to otherthan F or R100 molecules. The fragment of Tn21 is localizeddownstream of the IncFII replicon; thus, it has the same localizationas that in the R100 plasmid, in which its complete version isfound. The mobile elements of p1658/97 separate subregions ofdifferent homologies, which suggests their involvement in themobilization of these subregions to the plasmid molecule.

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TABLE 1. Mobile elements found in p1658/97^a

Antibiotic resistance determinants in p1658/97. Apart from the bla_SHV-5 gene (see below), all other antimicrobialresistance determinants of p1658/97 were found to be locatedin a class 1 integron (Fig. 3). These are genes aadA1, aacC1,and aacA4, which altogether confer resistance to the entirespectrum of clinically used aminoglycosides. The integron (positions78683 to 85188) is flanked by one complete IS26 sequence andone incomplete IS26 sequence in the direct orientation (IS26cp2and ${Delta}$ IS26, respectively). The aadA1, aacC1, and aacA4 gene cassettesform the variable region, together with two ORFs, orfX and orfX',which code for polypeptides of unknown function. The identicalcomposition of the gene cassette array has been found in integronINTCZD37 from K. pneumoniae (GenBank accession no. AF282595);integron In-t3, which is present in plasmid pSEM (20, 39); anintegron from plasmid pACM1 (20, 29, 30, 31); and an integronfrom Acinetobacter baumannii (GenBank accession no. DQ370505).The sequence identities of the variable regions of these integronsvary between 99 and 100%. The p1658/97 integron contains thecomplete 3' conserved sequence (3'-CS) with the left invertedrepeat (IRt), whereas its 5'-CS is truncated by 113 bp at the5' end (89 bp from the stop codon of intI1) due to the IS26insertion (IS26/ ${Delta}$ 5'-CS). The insertion produced the incompletecopy of the element ( ${Delta}$ IS26) and caused the lack of the integron'sright inverted repeat (IRi). An identical IS26/ ${Delta}$ 5'-CS has beenreported in integrons in three plasmids belonging to the IncL/Mgroup, the aforementioned plasmids pSEM (20, 39), pACM1 (20,29, 30, 31), and pAK33 from K. pneumoniae (In111; GenBank accessionno. AY260546) (20, 44), and also in plasmid p541 from E. coli,which belongs to the IncN group (In-e541; GenBank accessionno. AY340637) (20, 21, 43). The integrons of p1658/97, pSEM,and pACM1 are associated with the almost identical region encompassingthe bla_SHV-5 gene and are flanked by two IS26 copies (IS26cp3and IS26cp4; see below); however, the mutual localization ofthe integron and this region is different in all three molecules(20, 29). In p1658/97 and pACM1, the integron is localized downstreamof the bla_SHV-5 gene-containing region, but in pSEM it is invertedand placed upstream. In both pACM1 and pSEM, the complete IS26copy was found at the 5'-CS of the integrons, whereas in p1658/97it is truncated ( ${Delta}$ IS26). Moreover, in pACM1 the integron andthe bla_SHV-5-containing region are separated by another unrelatedintegron that is adjacent to IS1, and the IS6100 element wasidentified in the 3'-CS of the pSEM integron.

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FIG. 3. Schematic representation of the multiresistance locus of p1658/97 with the amplicon and the integron. Black arrows indicate IS26, gray arrows represent genes localized within the integron and the amplicon, and white arrows indicate the remnant of IS26 ( ${Delta}$ IS26) and the bla_SHV-5 gene. The position of SwaI and the sizes of the restriction fragments are indicated.

Evolutionary history of the p1658/97 structure. A region flanked by identical copies of an IS(s) can be considereda composite transposon type I that, as a consequence, shouldbe able to transpose (38). Most of the transposons duplicatetheir target sequence of a specific size during transposition(19), and such duplicated sequences (direct repeats [DRs]) thatflank segments of more complex structures allow these to beidentified as integrated mobile elements; e.g., IS100 generates5-bp DRs (1), whereas IS26 produces 8-bp DRs (22). In p1658/97,the perfect duplication of the target site was observed onlyin the case of IS100 (Fig. 2). The sequences surrounding otherpotentially mobile elements are different; however, IS26cp1and IS26cp4 are each flanked at one end by DR-like sequencesthat are identical in six of eight nucleotides (underlined):GAAAATAT and GAAAATCG, respectively (Fig. 4). The 48-kb "IS26cp1-spacingsequence-IS26cp4" segment encompasses almost all of the mosaicsubregions of the plasmid, e.g., IS100, the "IS26cp2-integron- ${Delta}$ IS26"region, and the "IS26cp3-bla_SHV-5-IS26cp4" region. This findingsuggests that the 48-kb segment of p1658/97 could have originatedfrom the IS26-mediated transposition of a composite elementfrom an unidentified molecule into the site adjacent to theincomplete Tn1 copy (Fig. 4). On the other hand, the structureof this segment itself probably arose from a variety of separateintegration events that implanted other mobile elements presentwithin this region. The "IS26cp2-integron- ${Delta}$ IS26" and "IS26cp3-bla_SHV-5-IS26cp4"regions that are localized within the 48-kb segment of p1658/97have the structure of a composite transposon type I themselves.However, the lack of DRs at their ends suggests, rather, recombinationas a mechanism of their acquisition by p1658/97, similar tothat for the IS26-flanked DNA fragments observed in plasmidspSEM, pAK33, p541, pACM1 (20), and pHNM1. It is impossible toelucidate whether the full structure of the 48-kb segment wasformed before or after the recruitment of the putative compositetransposon into p1658/97.

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FIG. 4. Hypothetical events leading to the present structure of p1658/97. The partial p1658/97 sequence (positions 35000 to 114500) is drawn to scale; the remaining part (including the tra region) is out of scale. The module "IS26cp1-spacing region-IS26cp4" (positions 47137 to 95371) is marked at the top. Open boxes represent the mobile elements, and arrows represent ORFs. The orientations of the mobile elements are marked with black arrows; incomplete mobile elements are marked with a ${Delta}$ symbol. Dotted lines show hypothetical mobilization events that implant different submodules. The regions of the plasmid backbone are marked: REP, replication; PAR, partition; PSK, postsegregational killing. DRs, when they have been identified, are shown. Regions of over 90% similarity (at the nucleotide level) to DNA molecules other than F and R100 are shown.

Identification of the region that undergoes amplification. In order to determine the DNA region that undergoes amplificationin plasmid p1658/97, the results of restriction analysis obtainedin silico were compared with those obtained in vitro. The SwaIrestriction enzyme recognizes only seven sites within the plasmidmolecule, and five of them are localized within the IS26 copies,one site per each copy (Fig. 3). The increased intensity ofone band of approximately 9 kb in the lane representing plasmidDNA from the "resistant" colony indicated the amplificationof that region (Fig. 1). The only SwaI fragment correspondingin size to the observed band was that of 8,817 bp, which containedthe bla_SHV-5 region flanked by the parts of IS26cp3 and IS26cp4delimited by the SwaI sites. Numerous reports concerning DNAamplification showed that amplifying segments are most frequentlyflanked by homologous sequences (repeats) at their ends (27,28, 33, 34). The two IS26 elements, each of which is 820 bp,probably played the role of such long homologous repeats. Therefore,the region of 9,637 bp, including the complete IS26cp3 and IS26cp4sequences, was identified as an amplicon. Restriction analysisof plasmid p1658/97 isolated from the "resistant" colony performedwith other enzymes confirmed that amplification was limitedonly to the region of the amplicon (data not shown). The amplificationof the bla_SHV gene copy number in association with the presenceof IS26 has also been observed in K. pneumoniae clinical isolatesby Hammond et al. (14).

The details of gene amplification in plasmid p1658/97 are thesubject of a further study. Preliminary results, which haveshown no difference in the amplification frequencies of RecA-proficientand RecA-deficient E. coli isogenic strains bearing p1658/97(tested on medium with 30 µg/ml ceftazidime), suggest,rather, the RecA independence of the process studied. However,when high antibiotic pressure was applied (128 µg/ml ceftazidime),approximately 100-fold more "resistant" colonies were obtainedfor the RecA-proficient strain. Simultaneously, "resistant"clones selected with 128 µg/ml ceftazidime harbored plasmidswith significantly more copies of the amplified bla_SHV-5 regionthan those isolated with 30 µg/ml ceftazidime, irrespectiveof the RecA content (data not shown). These results led to thespeculation that the RecA protein could be involved in amplificationonly subsequent to the RecA-independent amplification, whenseveral amplicon copies are available. Similar results havebeen demonstrated for plasmid NR1, in which the initial duplicationsof the amplifiable region flanked by IS1 occurred in the RecA-independentbut IS1-mediated manner, and these duplications were a limitingstep for further RecA-mediated multiplication (28).

Amplicon structure. The organization of the amplicon is shown in Fig. 3. Apart frombla_SHV-5, the region contains an incomplete operon involvedin sugar metabolism (truncated ygbM and genes fucA, ygbK, andygbJ) and its regulatory gene (ygbI) in p1658/97, named ORFs36 to 39 and 40, respectively (41, 46); a gene coding for aputative RecF protein with the incomplete catalytic domain (orf42)(9); and a truncated lacY gene, which encodes the lactose transportprotein. As mentioned above, the DNA fragment located betweenthe two IS26 elements was found to be a derivative of the K.pneumoniae chromosome (20, 39), which could be expected, consideringthe earlier data indicating that this species is a source ofbla_SHV genes (4, 6, 7, 13, 16). The presence of the same segmentof K. pneumoniae DNA flanked by two IS26 elements in p1658/97,pSEM, pACM1, and pHNM1 indicates that it probably arose froma single escape from the K. pneumoniae chromosome and laterbecame disseminated into a variety of plasmid molecules. Interestingly,as already mentioned, pHNM1, pSEM, and also pACM1 belong tothe IncL/M incompatibility group and are able to replicate ina broad range of hosts (38). This fact emphasizes the possibilityof the spread of the amplicon among various DNA replicons andbacterial species. Similar fragments of the K. pneumoniae chromosomewith bla_SHV genes have also been found within other partiallysequenced plasmids: pZMP1 (GenBank accession no. X53817; K.pneumoniae), pPa-1 (GenBank accession no. AF074954; P. aeruginosa),pNSF-1 (GenBank accession no. AF282921; Shigella flexneri) (9),and pPAG-KE (GenBank accession no. AF096930; P. aeruginosa).The structures of plasmids pZMP1 and pPa-1 show the associationof SHV-type ß-lactamase genes with IS26 as well. Theresults presented and discussed above strongly support the hypothesisthat IS26 plays a major role in mobilization of the bla_SHV genesfrom the K. pneumoniae genome and their further disseminationto a wide range of enterobacterial strains (6).

Conclusions. We report here the complete nucleotide sequence of a plasmidin which ESBL gene bla_SHV-5 undergoes amplification. This processresults in the high-level resistance of clinical isolates toß-lactam antibiotics, including expanded-spectrumcephalosporins. The amplicon originated by mobilization fromthe chromosome of K. pneumoniae and was later spread into severaldifferent plasmids and, by means of their horizontal transfer,to a variety of bacterial species over a wide geographic area.Although the amplicon is flanked by two IS26 elements and thushas a structure of a type I composite transposon, the majorway of its dissemination among DNA replicons is probably mediatedby recombination by IS26 copies, which is a subject of our furtherstudies. This work, together with several other investigations(7, 14, 17, 20, 23), strongly underlines the important roleof IS26 in the spread of ß-lactamase genes acquiredby clinical bacterial populations, especially those coding forthe SHV-type enzymes.

ACKNOWLEDGMENTS

This work was started and conducted by Piotr Ceg $l$ owski untilhe passed away in 2004.

We thank Alessandra Carattoli for kindly providing plasmid pSEM.

This work was supported by a grant in the Fifth EU Frame Projectfor PC (grant ICA-CT-2000-70010).

FOOTNOTES

* Corresponding author. Present address: Department of Plant Physiology, Warsaw University, 02-096 Warsaw, Poland. Phone: (48 22) 554 39 12. Fax: (48 22) 554 39 10. E-mail: maximus{at}biol.uw.edu.pl

Published ahead of print on 12 January 2007.

Present address: Department of Biotechnology, Faculty of Biologyand Earth Sciences, Nicolaus Copernicus University, Toruñ,Poland.

Present address: Department of Molecular Genetics and Microbiology,Box 3054, Duke University, Durham, NC 27710.

REFERENCES

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