To perform the first multinational Enterobacter cloacae clonality study, using the MLST scheme newly developed in Japan. The analysis included 195 rectal carriage E. cloacae isolates resistant to expanded-spectrum cephalosporins (ESCs), collected from patients in 12 hospital units across Europe and Israel. All of the isolates were typed by PFGE and 173 isolates were subjected to MLST. ESC resistance was analysed phenotypically; genes encoding ESBLs and carbapenemases were identified by PCR and sequencing. MLST distinguished 88 STs, which correlated with the PFGE data. PFGE was more discriminatory, producing 129 pulsotypes (169 patterns). Numerous STs were observed in several countries each. The most widespread were ST66, ST78, ST108 and ST114, each having at least 10 isolates from three to five countries, diversified into multiple pulsotypes, with clusters of related isolates in one or more centres. Analysis of the STs against the MLST database revealed several epidemic clonal complexes, such as those with central genotypes ST74 (including ST78) or ST114 (including ST66). ESC resistance was equally related to overexpression of the AmpC cephalosporinase and to ESBL production. Among ESBL producers some spreading subclones were identified, including specific ST66, ST78 and ST114 pulsotypes, associated with CTX-M-15 production. Several isolates produced carbapenemase VIM-1 or KPC-2. Together with the information available in the MLST database, our results suggest that, like Escherichia coli and Klebsiella pneumoniae, E. cloacae harbours clonal lineages of increased epidemic potential that may be associated with resistance spread.

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 β-lactamase in clinical populations of the familyEnterobacteriaceaein Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besidesbla_CTX-M-3, it also bears thebla_TEM-1,aacC2, andarmAgenes, as well as integronicaadA2,dfrA12, andsul1, which altogether confer resistance to the majority of β-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks,traandtrb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Onlybla_CTX-M-3, accompanied by an ISEcp1element, is placed separately, in a DNA fragment previously identified as a fragment of theKluyvera ascorbatachromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogenErwinia amylovoraby 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.

The objective of this study was to characterize New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae isolates reported in Poland in 2012–14. Representative isolates were typed by PFGE and MLST. NDM and other β-lactamase genes were amplified and sequenced. Plasmids with bla_NDM genes were analysed by nuclease S1 plus hybridization profiling, by transfer assays and by PCR-based replicon typing. The bla_NDM genetic context was studied by PCR mapping assays. Of 374 cases of infection/colonization with NDM-positive Enterobacteriaceae identified in 2012–14, 370 cases in 40 hospitals, 10 outpatient clinics and 1 nursing home were associated with a Klebsiella pneumoniae outbreak with epicentres in Poznań and Warsaw. The outbreak strain of K. pneumoniae ST11 was similar to an isolate from the Czech Republic from 2013. Like the Czech strain, many of the isolates had two bla_NDM-1-carrying IncFII- and IncR-type plasmids of variable size, sharing a bla_NDM-1-containing segment. The early isolates also produced CTX-M-15 co-encoded by the IncR-type plasmids, and differentiated later by extensive plasmid rearrangements. Four other NDM cases were reported in 2013, three being associated with arrivals from Montenegro, India or Afghanistan. The Indian Escherichia coli ST448 NDM-5 isolate revealed similarity to a recent isolate from Spain, including the bla_NDM genetic context observed previously in E. coli strains in Poland and France (of Congolese and Indian origins, respectively). The Afghani Proteus mirabilis was the second isolate of this species with a chromosomal bla_NDM-1 location. The largest NDM outbreak in a non-endemic country has been observed, being an alarming phenomenon in resistance epidemiology in Poland.

The CTX-M-15 extended-spectrum β-lactamase (ESBL) was recently identified in Enterobacteriaceae isolates in India, and demonstrated significant hydrolytic activity against ceftazidime, in contrast to the majority of CTX-M enzymes. CTX-M-15 differs from CTX-M-3, which is one of the most prevalent ESBLs in Poland, by only a single amino acid change (Asp-240→Gly). Three cefotaxime- and ceftazidime-resistant Enterobacteriaceae isolates, recovered during 1998–2000 in two Polish hospitals, were found to produce CTX-M-15. Similar to those from India, the isolates contained the ISEcp1 insertion sequence located upstream of the bla_CTX-M-15 gene, which has been recently demonstrated to mobilize 3′-adjacent genes to transfer between DNA replicons. However, its different position with respect to the β-lactamase gene indicated the independent selection of the ESBL gene in the two countries.

Eighty-four clinical isolates of the family Enterobacteriaceae , recovered from 1998 to 2000 in 15 hospitals in 10 Polish cities, were analyzed. All the isolates produced β-lactamases with pIs of 8.4 and 5.4, and the pI 8.4 enzymes were demonstrated to hydrolyze cefotaxime but not ceftazidime in the in vitro bioassay. PCR analysis and DNA sequencing have revealed that in all cases the pI 8.4 β-lactamase was probably the CTX-M-3 extended-spectrum β-lactamase (ESBL) variant, which was originally identified in 1996 in Praski Hospital in Warsaw. In the majority of isolates, bla _CTX-M-3 genes resided within large conjugative plasmids with similar fingerprints, which, in the context of the high degree of diversity of the randomly amplified polymorphic DNA types of the isolates, suggested that horizontal transfer of plasmids was likely the main mechanism of CTX-M-3 spread. The dissemination of plasmids was probably preceded by the center-to-center transmission of several strains, as indicated by the identification by pulsed-field gel electrophoresis of closely related or possibly related Klebsiella pneumoniae , Escherichia coli , and Citrobacter freundii isolates in five different hospitals. CTX-M-3-producing organisms revealed a very high degree of diversity in β-lactam resistance levels and patterns. This was attributed to several factors, such as the production of other β-lactamases including additional ESBLs, possible quantitative variations in CTX-M-3 expression, segregation of AmpC derepressed mutants, and permeability alterations.

After the first report in May 2008, the National Reference Center for Susceptibility Testing confirmed 113 cases of infection or colonization by KPC-producing members of the family Enterobacteriaceae in Poland by the end of 2009. The vast majority of patients were found in 18 hospitals; three patients were diagnosed at outpatient clinics. Most of the institutions were in the Warsaw area, including three hospitals with the highest numbers of cases. When available, the data on previous hospitalizations often indicated that these hospitals were the probable acquisition sites; one patient arrived from New York. The group of 119 unique isolates consisted of Klebsiella pneumoniae (n = 114), followed by Klebsiella oxytoca (n = 3), and Escherichia coli (n = 2). The K. pneumoniae isolates were dominated by the clone sequence type 258 (ST258) (n = 111); others were ST11 and ST23. The ST258 group was heterogeneous, with 28 pulsed-field gel electrophoresis (PFGE) subtypes, ∼25 plasmid profiles, and nine β-lactamase patterns differing by KPC variants (KPC-2 mainly), and SHV-12, CTX-M-3, and TEM-1-like enzymes. Plasmids carrying bla_KPC genes varied in size (∼48 to 250 kb), structure, and conjugation potential. Transferable IncFII_K plasmids of ∼110 to 160 kb, probably pKpQIL or its derivatives, were observed in all K. pneumoniae clones and in K. oxytoca. Also prevalent were nontypeable pETKp50-like plasmids of ∼50 kb, found in K. pneumoniae ST258 and E. coli isolates (ST93 and ST224). Two K. pneumoniae-E. coli pairs from single patients might represent the in vivo transfer of such plasmids. The striking diversity of KPC producers at the early stage of dissemination could result from several introductions of these bacteria into the country, their multidirectional evolution during clonal spread, and transfer of the plasmids.