Izabela Kern-Zdanowicz

Complete Nucleotide Sequence of the pCTX-M3 Plasmid and Its Involvement in Spread of the Extended-Spectrum β-Lactamase Gene blaCTX-M-3

ABSTRACT 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 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 blaCTX-M-3, it also bears the blaTEM-1, aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, 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, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only blaCTX-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 Klebsiella pneumoniae ST258 with KPC-2 in Poland

Plasmidic KPC β-lactamases in gram-negative pathogens are of the highest clinical and epidemiologic concern, conferring resistance to all β-lactams, including carbapenems (15, 18). The main KPC producer is Klebsiella pneumoniae, but other species are being identified as well (15). Strains with KPCs spread rapidly and cause outbreaks; in hospitals on the east coast of the United States and in Israel, they have already become endemic (2, 3, 10, 14, 17). Recently, more countries have reported the presence of these organisms, in some cases as a result of importation (15).

International Clones of Klebsiella pneumoniae and Escherichia coli with Extended-Spectrum β-Lactamases in a Czech Hospital

ABSTRACT A 2-month survey of extended-spectrum β-lactamase (ESBL) producers was performed in a Czech hospital. Klebsiella pneumoniae produced SHV-2, -5, or -12, Escherichia coli produced CTX-M-9 or -15, and other species produced TEM-92 or -132. All K. pneumoniae and E. coli isolates belonged to sequence types (STs) or clonal complexes (CCs) spread across the world (K. pneumoniae clonal complex 11 [CC11], CC14, and sequence type 101 [ST101] and E. coli CC31, CC73, CC131, and CC405) and carried various plasmids (mainly with A/C- and FII-type replicons).

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

ABSTRACT Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum β-lactamase (ESBL) of the SHV type. The β-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

High-level fluoroquinolone resistant Salmonella enterica serovar Kentucky ST198 epidemic clone with IncA/C conjugative plasmid carrying blaCTX-M-25 gene

Multidrug resistant Salmonella Kentucky strains have been isolated from turkeys in Poland since 2009. Multiple mutations within chromosomal genes gyrA and parC were responsible for high-level ciprofloxacin resistance. One of the isolates was extended spectrum β-lactamase- (ESBL) positive: the strain 1643/2010 carried a conjugative 167,779 bps plasmid of IncA/C family. The sequence analysis revealed that it carried a blaCTX-M-25 gene and an integron with another β-lactamase encoding gene-blaOXA-21. This is the first known report of a CTX-M-25 encoding gene both in Poland and in Salmonella Kentucky world-wide, as well as in the IncA/C plasmid. Analysis of the integron showed a novel arrangement of gene cassettes-aacA4, aacC-A1 and blaOXA-21 where the latter might result from an intergeneric gene transfer. The study confirmed Salmonella Kentucky population isolated in Poland belongs to global epidemics of high level fluoroquinolone resistant clone ST198 that can carry rare β-lactamase genes.

DHA-1-Producing Klebsiella pneumoniae in a Teaching Hospital in the Czech Republic

Thirty-eight AmpC-producing Klebsiella pneumoniae isolates identified from January to October 2006 in a large teaching hospital in the Czech Republic were analyzed. The AmpC cephalosporinase was identified as DHA-1, encoded by a plasmid-located complex class 1 integron, previously observed in a K. pneumoniae isolate from the Parisian region. The DHA-1 expression was inducible, and although in two isolates with higher resistance, the induction effect was masked at the phenotypic level. All of the isolates belonged to the international K. pneumoniae clone sequence type 11, split into two disseminated pulsed-field gel electrophoresis types. This is the first report on enterobacteriaceae with acquired AmpCs in the Czech Republic and possibly the first description of organisms with DHA-1 in the Central and Eastern Europe.

Tandem multiplication of the IS26‐flanked amplicon with the blaSHV‐5 gene within plasmid p1658/97

The IncF plasmid p1658/97 (c. 125 kb) from Escherichia coli isolates recovered during a clonal outbreak in a hospital in Warsaw, Poland, in 1997 contains the extended-spectrum β-lactamase (ESBL) gene bla(SHV-5), originated from the Klebsiella pneumoniae chromosome. A region containing the bla(SHV-5) gene is flanked by two IS26 copies and its copy number multiplies spontaneously within p1658/97 and RecA-deficient E. coli strains. Here, we demonstrate that the amplified IS26-bla(SHV-5) units were arranged in tandems, containing up to more than 10 units, which could raise ceftazidime MICs for host strains from 4 μg mL(-1) to more than 128 μg mL(-1). Successive deletions within p1658/97, located outside the amplifiable module and encompassing even as little as c. 15% of the plasmid, blocked the amplification. Moreover, the complementing re-introduction of the deleted fragments in trans did not restore the process. Similarly, insertions of a 1-kb DNA fragment into the amplicon inhibited its self-multiplication ability. The module was able to transmit into another IS26-containing plasmid by recombination. The results prompted us to speculate that local DNA structure, especially favorable in p1658/97, might have been responsible for the IS26-bla(SHV-5) multiplication ability.