pKBuS13, a KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 833, carrying Tn4401b inserted into an Xer site-specific recombination locus.

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported.

Spread of Vancomycin-Resistant Enterococcus faecium Isolates Despite Validated Infection Control Measures in an Italian Hospital: Antibiotic Resistance and Genotypic Characterization of the Endemic Strain.

An alarming increase of vancomycin-resistant Enterococcus faecium (VREfm) isolates was detected in an Italian referral hospital subjected to policies of infection control validated by the Joint Commission International. Analysis of the population structure of 122 consecutive, nonreplicate VREfm isolates collected over an 18-month period identified a single major clone that spread around the whole hospital, rapidly establishing an endemic state. It belonged to sequence type (ST) 17 and showed a highly multidrug-resistant phenotype, being resistant to all antimicrobial classes for the carriage of several resistance determinants. Furthermore, some strains with decreased susceptibility to daptomycin were detected. Eighteen out of the 122 isolates did not group in the major clone. They showed a low spreading potential inside the hospital wards, even if most of them displayed a multidrug-resistant phenotype and belonged to a hospital-adapted lineage. Causes that led to the VREfm endemic state have not been fully elucidated. However, it is conceivable that the increase in systemic antibiotic consumption and the use of selective digestive tract decontamination, including vancomycin in critically ill patients during the period before 2014, may have played a role in the ST17 clone dissemination, but additional traits conferring high fitness in hospital environment cannot be excluded.

Microbiological characterisation of Burkholderia cepacia isolates from cystic fibrosis patients: investigation of the exopolysaccharides produced.

Eleven strains of Burkholderia cepacia were isolated directly from clinical specimens: 10 from sputum of cystic fibrosis patients, and one from a vaginal swab. They were biochemically identified using API20NE and confirmed by a PCR-based assay. The genomovar characterisation obtained by specific PCR amplification revealed seven strains belonging to genomovar I, three belonging to genomovar IIIA and one belonging to genomovar IV. All isolates were also typed by ribotyping and random amplification of polymorphic DNA analysis. Some of the characterised strains were examined for the ability to produce exopolysaccharides, with the aim of correlating the genomovar with the exopolysaccharide structure. The polysaccharides were analysed by means of methylation analysis and 1H-NMR spectroscopy in order to determine structural similarities. It was shown that different strains are capable of producing chemically different polysaccharides.

Metallo-ß-lactamase expression confers an advantage to Pseudomonas aeruginosa isolates compared with other ß-lactam resistance mechanisms, favoring the prevalence of metallo-ß-lactamase producers in a clinical environment.

The Pseudomonas aeruginosa isolate TS-832035 was responsible for an outbreak that occurred in an Italian hospital between 1999 and 2002. It exhibited a high-level resistance to carbapenems due to the contemporary presence of two independent mechanisms: the production of a carbapenemase, coded by a bla(VIM-1) determinant carried by the chromosomal class 1 integron In70.2 (containing also the aacA4, aphA15, and aadA1 genes in its cassette array), and the lack of the OprD porin. We compared TS-832035 with a strictly related isolate, TS-103, whose resistance to carbapenems was due to the lack of the OprD porin only, as it did not carry In70.2. We evaluated their growth kinetics, in both separate cultures and competition assays, under permissive conditions. These experiments highlighted a significant in vitro fitness cost associated with the integron. On the contrary, none of the resistance determinants other than the bla(VIM-1) seemed to confer a real selective advantage to its host. Comparison of these results with the in vivo behavior, showing that the In70.2-carrying isolates largely prevailed over the In70.2-lacking ones, besides the detection of similar integrons in other Italian clinical isolates, evidenced the need to investigate accurately the causes of their large distribution, as possible soft spots could exist in the ability of their hosts to adapt to the hospital settings.

Endemic carbapenem-resistant Pseudomonas aeruginosa with acquired metallo-beta-lactamase determinants in European hospital.

Acquired metallo-beta-lactamases (MBLs) can confer broad-spectrum beta-lactam resistance (including carbapenems) not reversible by conventional beta-lactamase inhibitors and are emerging resistance determinants of remarkable clinical importance. In 2001, multidrug-resistant Pseudomonas aeruginosa carrying bla(VIM) MBL genes were found to be widespread (approximately 20% of all P. aeruginosa isolates and 70% of the carbapenem-resistant isolates) at Trieste University Hospital. Clonal diversity and heterogeneity of resistance determinants (either bla(VIM-1)-like or bla(VIM-2)-like) were detected among MBL producers. This evidence is the first that acquired MBLs can rapidly emerge and establish a condition of endemicity in certain epidemiologic settings.