Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France

We analyzed 19 clinical isolates of the family Enterobacteriaceae (16 Escherichia coli isolates and 3 Klebsiella pneumoniae isolates) collected from four different hospitals in Paris, France, from 2000 to 2002. These strains had a particular extended-spectrum cephalosporin resistance profile characterized by a higher level of resistance to cefotaxime and aztreonam than to ceftazidime. The bla(CTX-M) genes encoding these beta-lactamases were involved in this resistance, with a predominance of bla(CTX-M-15). Ten of the 19 isolates produced both TEM-1- and CTX-M-type enzymes. One strain (E. coli TN13) expressed CMY-2, TEM-1, and CTX-M-14. bla(CTX-M) genes were found on large plasmids. In 15 cases the same insertion sequence, ISEcp1, was located upstream of the 5' end of the bla(CTX-M) gene. In one case we identified an insertion sequence designated IS26. Examination of the other three bla(CTX-M) genes by cloning, sequencing, and PCR analysis revealed the presence of a complex sul1-type integron that includes open reading frame ORF513, which carries the bla gene and the surrounding DNA. Five isolates had the same plasmid DNA fingerprint, suggesting clonal dissemination of CTX-M-15-producing strains in the Paris area.     

Characterization of the highly variable region surrounding the bla(CTX-M-9) gene in non-related Escherichia coli from Barcelona

OBJECTIVES: The dispersion of a clone, a plasmid or a mobile element carrying the bla(CTX-M-9) gene was evaluated in 30 Escherichia coli strains isolated in Barcelona between 1996 and 1999. The presence of the previously described orf513-bearing class 1 integron, In60, carrying the bla(CTX-M-9) gene, was also studied. METHODS: The clonality was analysed by pulsed-field gel electrophoresis. Plasmid analysis was performed by S1 digestion and hybridization with the CTX-M-9 probe. PCR mapping using specific designed primers was used to study the presence of In60 and In60-like structures. RESULTS: The clonality between the 30 strains was minor. The size of bla(CTX-M-9) carrying plasmids ranged between approximately 80 and 430 kb. One strain produced only a chromosome-encoded CTX-M-9 beta-lactamase. Thirty-six per cent of the strains showed differences with respect to the In60 structure due to an insertion or deletion events. CONCLUSIONS: These findings suggest that the bla(CTX-M-9) gene may be carried by a mobile element that disperses it between plasmids. The fast dispersion of the CTX-M-9 enzyme could therefore be due to both diffusion of plasmids and mobile elements.     

Molecular epidemiology of orf513-bearing class 1 integrons in multiresistant clinical isolates from Argentinean hospitals

The spread of orf513-bearing class 1 integrons is associated with bla(CTX-M-2) in gram-negative clinical isolates in Argentina, with In35 being the most frequently found integron (74%). Among 65 isolates without bla(CTX-M-2), only one harbored a novel orf513-bearing class 1 integron with the dfrA3b gene. The finding of orf513 not associated with class 1 integrons in two gram-positive strains indicates the widespread occurrence of this putative site-specific recombinase.     

Description of In116, the first blaCTX-M-2-containing complex class 1 integron found in Morganella morganii isolates from Buenos Aires, Argentina

OBJECTIVES: We analysed the architecture and probable origin of a class 1 integron from cefotaxime-resistant Morganella morganii isolates. METHODS: bla genes and class 1 integron elements were detected by PCR and DNA-DNA hybridization in a M. morganii strain isolated in 1996. PCR-mapping and sequencing of different fragments were carried out to determine the integron's architecture. RESULTS AND CONCLUSIONS: A class 1 integron (In116), strongly related to the In6/In7 family, was detected in a plasmid from an oxyimino-cephalosporin-resistant M. morganii strain, producing CTX-M-2 beta-lactamase. The variable region of In116 contains aacA4, bla(OXA-2) and orfD cassettes. Downstream of the 3'-conserved-segment (3'-CS), an orf513-containing common region is followed by bla(CTX-M-2) and flanking regions, having 96-99% nucleotide identity with Kluyvera ascorbata's kluA-1 and neighbouring sequences. Some of the evidence supporting the incorporation of foreign DNA is as follows: a partial deletion in a second 3'-CS (3'-CS2), and the absence of 59-base element or IS-like structures upstream of bla(CTX-M-2).     

CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum beta-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel

The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was bla(CTX-M); 16 isolates carried bla(CTX-M-2) and three carried a new ESBL gene designated bla(CTX-M-39). Three strains carried bla(SHV) (two bla(SHV-12) and one bla(SHV-5)), and two strains carried inhibitor-resistant ESBL genes, bla(TEM-33) and bla(TEM-30); 18 strains carried bla(TEM-1) and eight strains carried bla(OXA-2). Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that bla(CTX-M-2) is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated bla(CTX-M-39), which revealed 99% homology with bla(CTX-M-26), with a substitution of arginine for glutamine at position 225.     

Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum beta-lactamase gene blaCTX-M-3

Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum beta-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla(CTX-M-3), it also bears the bla(TEM-1), aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of beta-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla(CTX-M-3), accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.     

Emergence of CTX-M-12 extended-spectrum beta-lactamase-producing Escherichia coli in Korea

OBJECTIVES: To characterize CTX-M-12 extended-spectrum beta-lactamase (ESBL) produced by clinical Escherichia coli isolates and to investigate its genetic environment. METHODS: Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the double-disc synergy test was carried out. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the genetic environments of the bla(CTX-M-12) genes were investigated by PCR and sequencing of the regions surrounding the genes. Kinetic parameters were determined from purified CTX-M-12. RESULTS: Sequence data for the CTX-M-1 cluster from three clinical E. coli isolates indicated the presence of CTX-M-12. An ISEcp1 insertion sequence was located 49 bp upstream of bla(CTX-M-12) in all three E. coli isolates. CTX-M-12 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and was encoded on a self-transferable approximately 18 kbp plasmid. CONCLUSIONS: This work shows that CTX-M-12, which confers high-level resistance to cefotaxime but not to ceftazidime, has emerged in Korea. The bla(CTX-M-12) gene was associated with an upstream ISEcp1 insertion sequence.     

Detection and characterization of extended-spectrum beta-lactamases in Salmonella enterica strains of healthy food animals in Spain

OBJECTIVES: To carry out the characterization of the genes encoding extended-spectrum beta-lactamases (ESBLs) and their genetic environments in four expanded-spectrum cephalosporin-resistant Salmonella enterica isolates (serovars: two Virchow, one Enteritidis, one Rissen) recovered during the monitoring programmes performed in Spain by the VAV Network from faecal samples of pigs, poultry and laying hens at the slaughterhouse level. METHODS: The presence and characterization of ESBL genes as well as their genetic environments in the four S. enterica isolates were investigated by PCR and sequencing. The presence of other resistance genes was also analysed by PCR and sequencing. RESULTS: Three avian S. enterica isolates (two Virchow and one Enteritidis) harboured the bla(CTX-M-9) gene combined with bla(TEM-1b). The bla(CTX-M-9) gene was included in these three isolates in a class 1 integron with the following 5'-->3' structure: integron 1 variable region (dfrA16-aadA2 gene cassettes)-qacEDelta1-sul1-orf513-bla(CTX-M-9)-orf3-like-orf1005. The sul2 gene was also detected in these three bla(CTX-M-9)-containing isolates and tet(A) in one of them. The two serovar Virchow isolates showed an indistinguishable PFGE pattern, although they were recovered from different animal species (broiler and laying hen). A porcine ESBL-positive isolate (serovar Rissen) harboured the bla(SHV-12) gene combined with bla(TEM-1b). This bla(SHV-12)-containing isolate also harboured the tet(A), aadA and sul1 genes. CONCLUSIONS: The emergence of ESBL-producing S. enterica isolates among food animals is described for the first time in Spain, with those of the CTX-M group being the predominant ESBLs detected.     

Vibrio cholerae InV117, a class 1 integron harboring aac(6')-Ib and blaCTX-M-2, is linked to transposition genes

A ca. 150-kbp Vibrio cholerae O1 biotype El Tor plasmid includes bla(CTX-M-2) and a variant of aac(6')-Ib within InV117, an orf513-bearing class 1 integron. InV117 is linked to a tnp1696 module in which IRl carries an insertion of IS4321R. The complete structure could be a potential mobile element.     

Comparative analysis of IncHI2 plasmids carrying blaCTX-M-2 or blaCTX-M-9 from Escherichia coli and Salmonella enterica strains isolated from poultry and humans

Salmonella enterica bla(CTX-M-2) and bla(CTX-M-9) plasmid backbones from isolates from Belgium and France were analyzed. The bla(CTX-M-2-)plasmids from both human and poultry isolates were related to the IncHI2 pAPEC-O1-R plasmid, previously identified in the United States in avian Escherichia coli strains; the bla(CTX-M-9) plasmids were closely related to the IncHI2 R478 plasmid.     

Novel complex class 1 integron bearing an ISCR1 element in an Escherichia coli isolate carrying the blaCTX-M-14 gene

This work identifies an ISCR1-related bla(CTX-M-14) gene, which has never been reported before, from a clinical isolate of Escherichia coli. The bla(CTX-M-14) gene was preceded by an ISCR1 element that was followed by a class 1 integron containing three different insert gene cassettes, i.e., dfrA12, orfF, and aadA2.     

A novel bla(CTX-M-14) gene-harboring complex class 1 integron with an In4-like backbone structure from a clinical isolate of Escherichia coli

Escherichia coli BDE0502 contained 3 beta-lactamase genes including bla(DHA-1), bla(SHV-12), and bla(CTX-M-14). The bla(CTX-M-14) gene was found on a complex class 1 integron with an In4-like backbone structure. The bla(CTX-M-14) gene was proceeded by a partial copy of ISEcp1 in addition to the complete copy of ISCR1 element.     

Molecular characterization of extended-spectrum-β-lactamase-producing and plasmid-mediated AmpC β-lactamase-producing Escherichia coli isolated from stray dogs in South Korea

A total of 47 extended-spectrum-cephalosporin-resistant Escherichia coli strains isolated from stray dogs in 2006 and 2007 in the Republic of Korea were investigated using molecular methods. Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase phenotypes were identified in 12 and 23 E. coli isolates, respectively. All 12 ESBL-producing isolates carried bla(CTX-M) genes. The most common CTX-M types were CTX-M-14 (n = 5) and CTX-M-24 (n = 3). Isolates producing CTX-M-3, CTX-M-55, CTX-M-27, and CTX-M-65 were also identified. Twenty-one of 23 AmpC β-lactamase-producing isolates were found to carry bla(CMY-2) genes. TEM-1 was associated with CTX-M and CMY-2 β-lactamases in 4 and 15 isolates, respectively. In addition to bla(TEM-1), two isolates carried bla(DHA-1), and one of them cocarried bla(CMY-2). Both CTX-M and CMY-2 genes were located on large (40 to 170 kb) conjugative plasmids that contained the insertion sequence ISEcp1 upstream of the bla genes. Only in the case of CTX-M genes was there an IS903 sequence downstream of the gene. The spread of ESBLs and AmpC β-lactamases occurred via both horizontal gene transfer, accounting for much of the CTX-M gene dissemination, and clonal spread, accounting for CMY-2 gene dissemination. The horizontal dissemination of bla(CTX-M) and bla(CMY-2) genes was mediated by IncF and IncI1-Iγ plasmids, respectively. The clonal spread of bla(CMY-2) was driven mainly by E. coli strains of virulent phylogroup D lineage ST648. To our knowledge, this is the first report of bla(DHA-1) in E. coli strains isolated from companion animals. This study also represents the first report of CMY-2 β-lactamase-producing E. coli isolates from dogs in the Republic of Korea.     

beta-Lactamases among extended-spectrum beta-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in The Netherlands

OBJECTIVES: The purpose of this work was to study the genetic determinants responsible for extended-spectrum beta-lactamase (ESBL) resistance of Salmonella isolated from Dutch poultry, poultry meat and hospitalized humans. METHODS: Thirty-four ESBL-resistant Salmonella isolates from The Netherlands were tested towards 21 antimicrobial agents. PCR and sequencing were used to determine the underlying genetic determinants responsible for the ESBL phenotypes. The transferability of the ESBL phenotypes was tested by conjugation to a susceptible Salmonella enterica serovar Dublin and plasmid purification, restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) were employed to further characterize a subset of the isolates. RESULTS: A great genetic diversity was seen among the isolates. The bla(TEM-52) gene was most predominant and was found among Salmonella enterica serovars Blockley, Thomson, London, Enteritidis phage type 14b, Paratyphi B, Virchow and Typhimurium phage types 11 and 507. We also found the bla(TEM-20) gene in S. Paratyphi B var. Java and the bla(TEM-63) gene in S. Isangi. Furthermore, we detected the bla(CTX-M-28) gene in S. Isangi and the bla(CTX-M-3) gene in S. Typhimurium phage type 507. The bla(CTX-M-2) gene was identified in S. Virchow, which also contained a copy of the bla(SHV-2) gene and a copy of the bla(TEM-1) gene. The bla(SHV-12) gene was found alone in S. Concord and together with the bla(TEM-52) gene in S. Typhimurium. Finally, the bla(ACC-1) gene was cloned from a S. Bareilly isolate and was found to be present on indistinguishable plasmids in all S. Bareilly isolates examined as well as in a S. Braenderup isolate and a S. Infantis isolate. CONCLUSIONS: Our data underscore the diversity of ESBL genes in Salmonella enterica isolated from animals, food products and human patients.     

Recombination in IS26 and Tn2 in the evolution of multiresistance regions carrying blaCTX-M-15 on conjugative IncF plasmids from Escherichia coli

CTX-M-15 now appears to be the dominant extended-spectrum β-lactamase worldwide, and a number of different factors may contribute to this success. These include associations between bla(CTX-M-15) and particular plasmids (IncF) and/or strains, such as Escherichia coli ST131, as well as the genetic contexts in which this gene is found. We previously identified bla(CTX-M-15) as the dominant ESBL gene in the western Sydney area, Australia, and found that it was carried mainly on IncF or IncI1 plasmids. Here, we have mapped the multiresistance regions of the 11 conjugative plasmids with one or more IncF replicons obtained from that survey and conducted a limited comparison of plasmid backbones. Two plasmids with only an IncFII replicon appear to be very similar to the published plasmids pC15-1a and pEK516. The remaining nine plasmids, with multiple IncF replicons, have multiresistance regions related to those of pC15-1a and pEK516, but eight contain additional modules previously found in resistance plasmids from different geographic locations that carry a variety of different resistance genes. Differences between the multiresistance regions are largely due to IS26-mediated deletions, insertions, and/or rearrangements, which can explain the observed variable associations between bla(CTX-M-15) and certain other resistance genes. We found no evidence of independent movement of bla(CTX-M-15) or of a large multiresistance region between different plasmid backbones. Instead, homologous recombination between common components, such as IS26 and Tn2, appeared to be more important in creating new multiresistance regions, and this may be coupled with recombination in plasmid backbones to reassort multiple IncF replicons as well as components of multiresistance regions.     

Extended-spectrum beta-lactamases among Enterobacter isolates obtained in Tel Aviv, Israel

The extended-spectrum beta-lactamase (ESBL)-producing phenotype is frequent among Enterobacter isolates at the Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. We examined the clonal relatedness and characterized the ESBLs of a collection of these strains. Clonal relatedness was determined by pulsed-field gel electrophoresis. Isoelectric focusing (IEF) and transconjugation experiments were performed. ESBL gene families were screened by colony hybridization and PCR for bla(TEM), bla(SHV), bla(CTX-M), bla(IBC), bla(PER), bla(OXA), bla(VEB), and bla(SFO); and the PCR products were sequenced. The 17 Enterobacter isolates studied comprised 15 distinct genotypes. All isolates showed at least one IEF band (range, one to five bands) whose appearance was suppressed by addition of clavulanate; pIs ranged from 5.4 to > or = 8.2. Colony hybridization identified at least one family of beta-lactamase genes in 11 isolates: 10 harbored bla(TEM) and 9 harbored bla(SHV). PCR screening and sequence analysis of the PCR products for bla(TEM), bla(SHV), and bla(CTX-M) identified TEM-1 in 11 isolates, SHV-12 in 7 isolates, SHV-1 in 1 isolate, a CTX-M-2-like gene in 2 isolates, and CTX-M-26 in 1 isolate. In transconjugation experiments with four isolates harboring bla(TEM-1) and bla(SHV-12), both genes were simultaneously transferred to the recipient strain Escherichia coli HB101. Plasmid mapping, PCR, and Southern analysis with TEM- and SHV-specific probes demonstrated that a single transferred plasmid carried both the TEM-1 and the SHV-12 genes. The widespread presence of ESBLs among Enterobacter isolates in Tel Aviv is likely due not to clonal spread but, rather, to plasmid-mediated transfer, at times simultaneously, of genes encoding several types of enzymes. The dominant ESBL identified was SHV-12.     

Molecular characterization of plasmids encoding CTX-M-15 beta-lactamases from Escherichia coli strains in the United Kingdom

OBJECTIVES: The UK, like other countries worldwide, has a growing problem with CTX-M beta-lactamase-producing Escherichia coli. Five major clonally related strains have been identified among CTX-M-15 producers. We characterize here the plasmids from clonal strains A and D. METHODS: Plasmids were extracted and transformed into E. coli DH5alpha; conjugative mating was attempted on agar. MICs were determined by agar dilution. beta-Lactamases were typed by isoelectric focusing; antibiotic resistance genes and integrons were identified by PCR and sequenced. Plasmid incompatibility groups were determined by replicon PCR. RESULTS: bla(CTX-M-15) was carried by a 150 kb plasmid in strain A and a 70 kb plasmid in strain D. Conjugative transfer of cefotaxime resistance was only achieved from strain D; plasmids from both strains were transferred by transformation. The plasmid from strain A additionally carried bla(TEM-1) (variably), bla(OXA-1), aac(6')-Ib-cr and tet(A), as well as a class 1 integron with the gene cassettes aadA5 and dfr(17); the plasmid from strain D carried bla(TEM-1) consistently, also bla(OXA-1), aac(6')-Ib-cr, aac3-IIa and tet(A). Both plasmids belonged to incompatibility group FII. CONCLUSIONS: bla(CTX-M-15) was plasmid-mediated in both strains A and D and was linked to other antibiotic resistance genes including aac(6')-Ib-cr, which encodes an acetyltransferase, not previously found in Europe, acting on both aminoglycosides and some fluoroquinolones. Although the plasmids from the two strains differed in size, both were related and conferred similar multi-drug resistance phenotypes, suggesting that they may share a similar genetic scaffold. Both shared features with plasmids encoding CTX-M-15 beta-lactamases in E. coli from Canada and India.