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.

Colonization of the tip of a thoracic catheter by Enterococcus faecalis resistant to vancomycin and linezolid.

We report the isolation of Enterococcus faecalis resistant to vancomycin and linezolid from the tip of a thoracic drainage catheter in an elderly patient. He was treated with vancomycin for a pleural empyema due to a meticillin-resistant Staphylococcus aureus but never received linezolid. A surveillance rectal swab yielded both linezolid-susceptible and -resistant strains, and the two isolates were not genotypically related. Careful monitoring for linezolid-resistance is critical to avoid potential therapy failure and transmission of resistant E. faecalis.

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.

Epidemic Dissemination of a Carbapenem-Resistant Acinetobacter baumannii Clone Carrying armA Two Years After Its First Isolation in an Italian Hospital.

This study describes the dissemination of a carbapenem-resistant Acinetobacter baumannii (CRAB) strain in a university hospital in Northeast Italy. Characterization of the outbreak strain was combined with a retrospective analysis of all CRAB isolates collected in the same hospital during the 5 years preceding the outbreak, with the aim of elucidating the origin of the epidemic spread. The outbreak strain was shown to belong to the International Clone II and carry the blaOXA-23 gene, flanked by two ISAba1 sequences in opposite orientation (Tn2006 arrangement). The epidemic clone harbored also the blaOXA-66 allele of the carbapenemase intrinsic to A. baumannii, the determinant of ArmA 16S rRNA methylase and a class 1 integron, with the aacA4, catB8, and aadA1 cassette array. Genotype analysis, performed by macrorestriction analysis and VRBA, revealed that isolates related to outbreak strain had been sporadically collected from inpatients in the 2 years preceding outbreak start. Carriage of blaOXA-66, armA, and the integron further supported relatedness of these isolates to the outbreak clone. Outbreak initially involved three medical wards, typically hosting elderly patients with a history of prolonged hospitalization. The study highlights the need to adopt strict infection control measures also when CRAB isolation appears to be a sporadic event.

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.

Molecular evolution of metallo-beta-lactamase-producing Pseudomonas aeruginosa in a nosocomial setting of high-level endemicity.

An outbreak of multidrug-resistant Pseudomonas aeruginosa strains producing VIM-type metallo-beta-lactamases (MBLs) has occurred in an Italian hospital since 2000 (C. Lagatolla, E. A. Tonin, C. Monti-Bragadin, L. Dolzani, F. Gombac, C. Bearzi, E. Edalucci, F. Gionechetti, and G. M. Rossolini, Emerg. Infect. Dis. 10:535-538, 2004). In this work, using molecular methods, we characterized 128 carbapenem-resistant isolates (including 98 VIM-positive isolates) collected from that hospital from 2000 to 2002 to investigate the dynamics of the dissemination of MBL producers in the clinical setting. Genotyping by random amplification of polymorphic DNA and pulsed-field gel electrophoresis showed that most VIM-positive isolates belonged to two different clonal lineages, producing either a VIM-1- or a VIM-2-like MBL, whose ancestors were detected for the first time in the hospital in 1999, suggesting that clonal expansion played a predominant role in the dissemination of these isolates. The 86 clonally related isolates carrying a blaVIM-1-like gene on an In70-like integron were clearly related to a VIM-1-positive P. aeruginosa clone circulating in various Italian hospitals since the late 1990s. VIM-negative P. aeruginosa strains related to the VIM-1-positive clone were detected during the same period, suggesting that the latter strain was derived from a clonal lineage already circulating in the hospital. In the VIM-2-like positive clone, the MBL gene was carried by an unusual class 1 integron, named In71, lacking the 3' conserved sequence region typical of sul1-associated integrons. A different class 1 integron with an original structure carrying a blaVIM-2 determinant, named In74, was detected in a sporadic isolate. A retrospective investigation did not reveal the presence of strains related to any of the VIM-producing isolates earlier than 1997.

Helicobacter pylori expresses an autolytic enzyme: gene identification, cloning, and theoretical protein structure.

Helicobacter pylori is an important pathogen of the gastric system. The clinical outcome of infection is thought to be correlated with some genetic features of the bacterium. However, due to the extreme genetic variability of this organism, it is hard to draw definitive conclusions concerning its virulence factors. Here we describe a novel H. pylori gene which expresses an autolytic enzyme that is also capable of degrading the cell walls of both gram-positive and gram-negative bacteria. We designated this gene lys. We found this gene and observed its expression in a number of unrelated clinical strains, a fact that suggests that it is well conserved in the species. A comparison of the nucleotide sequences of lys and the hypothetical gene HP0339 from H. pylori strain ATCC 26695 revealed almost total identity, except for the presence of an insertion consisting of 24 nucleotides in the lys sequence. The coding sequences of lys and HP0339 show a high degree of homology with the coding sequence of bacteriophage T4 lysozyme. Because of this similarity, it was possible to model the three-dimensional structures of both the lys and HP0339 products.

Molecular characterization of integrons in epidemiologically unrelated clinical isolates of Acinetobacter baumannii from Italian hospitals reveals a limited diversity of gene cassette arrays.

Integron carriage by 36 epidemiologically unrelated Acinetobacter baumannii isolates collected over an 11-year period from patients in six different Italian hospitals was investigated. Sixteen type 1 integron-positive isolates (44%) were found, 13 of which carried the same array of cassettes, i.e., aacC1, orfX, orfX', and aadA1a. As ribotype analysis of the isolates demonstrated a notable genetic diversity, horizontal transfer of the entire integron structure or ancient acquisition was hypothesized.

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.