Outbreak of linezolid-resistant Staphylococcus haemolyticus in an Italian intensive care unit.

We report an outbreak of linezolid-resistant Staphylococcus haemolyticus strains (MIC 32 mg/L) in patients admitted to the Verona University Hospital Intensive Care Unit. The strains proved to be clonally related at pulsed field gel electrophoresis. All the strains showed the G2576T mutation responsible for linezolid-resistance and retained their resistance even after several passages on antibiotic-free medium. After a decade of linezolid use, multifocal emergence of linezolid resistance in coagulase-negative staphylococci has become an important matter of concern and mandates stricter control over the use of this antibiotic in order to preserve its clinical utility.

Klebsiella pneumoniae carbapenemase (KPC) producer resistant to ceftazidime-avibactam due to a deletion in the blaKPC3 gene.

OBJECTIVES: Carbapenemase-producing strains of Klebsiella pneumoniae (KPC) are a great health concern, and therapy with ceftazidime-avibactam represents a choice for the treatment of infections involving these strains. We report a strain resistant to ceftazidime-avibactam due to a deletion of six nucleotides in the blaKPC gene sequence. METHODS: Two strains, namely AMP920 and AMP2009, were isolated from the same patient a month apart. Antimicrobial susceptibility testing was performed both by broth microdilution and by Etest. Immunoenzymatic assay to detect carbapenemase was performed for both strains. The blaKPC gene of both strains was amplified by PCR and sequenced. Enzyme activity towards carbapenems was tested by the CarbaNP test and hydrolysis spectrophotometer assay. RESULTS: The two isolates differed in antimicrobial susceptibility. AMP920 showed meropenem and imipenem resistance (MIC 32 and 32 mg/mL). A month later the carbapenem MIC decreased to 8 and 1 mg/mL respectively, while the ceftazidime-avibactam MIC increased from 1 to 16 mg/mL. Both isolates showed a positive immunoenzymatic test for the KPC enzyme, but only AMP920 showed a positive CarbaNP test hydrolysing imipenem. The BlaKPC gene was amplified in both strains. After sequencing, the two amplicons showed a KPC3 variant. The gene of the second isolate showed a deletion of six nucleotides at 498-503, resulting in a mutant variant with the deletion of glutamic acid and leucine residues at positions 167 and 168. CONCLUSIONS: We detected a new deletion in the blaKPC gene of a clinical strain of K. pneumoniae which resulted in resistance to ceftazidime-avibactam. The amino acids deleted are in the Ω loop (amino acids 165-179) of the KPC enzyme, enhancing ceftazidime affinity and preventing avibactam binding.

Susceptibilities of Streptococcus pyogenes and Streptococcus pneumoniae to macrolides and telithromycin: data from an Italian multicenter study.

687 isolates of Streptococcus pyogenes and 600 isolates of Streptococcus pneumoniae , isolated over the period 2002-2003 from specimens of different human origin obtained in 16 different Italian centres, were assayed for their susceptibilities to different macrolides and to telithromycin, and were investigated by PCR to detect their different erythromycin resistance genes. 25.5% of the S. pyogenes isolates proved resistant to erythromycin, as well as to clarithromycin and azithromycin. 6.6% of the isolates proved non-susceptible to clindamycin. 4.9% of the isolates were non-susceptible to telithromycin. 22.3% of all erythromycin-resistant isolates exhibited cMLS B resistance, 50.3% iMLS B resistance, and 27.4% Mtype resistance. All cMLS B strains had the erm(B) gene, all M strains had the mef (A) gene, and no resistance genes were found in the erythromycin-susceptible strains. Roughly one quarter of the iMLS(B) strains had erm(A) and roughly three quarters erm(B). 35.2% of the S. pneumoniae isolates proved resistant to erythromycin, and virtually all of them also proved resistant to clarithromycin and azithromycin, too. Only 6.0% of the pneumococcal isolates were resistant to penicillin and a further 11.0% were intermediate. Only 0.2% of the isolates were nonsusceptible to telithromycin. 65.9% of all erythromycin-resistant S. pneumoniae isolates had cMLS B resistance, 18.0% had iMLS B resistance, and 16.1% had M-type resistance. All the MLS B-resistant isolates had an erm(B) gene, and all the M-type isolates had a mef gene. We conclude that macrolide resistance of streptococci still persists in Italy with incidences as high as 40%, more often than not being characterised by the MLS B phenotype. The ketolide telithromycin, structurally related to macrolides and most likely to substitute for them in a number of clinical uses, is confirmed as being extremely active even against recent clinical streptococcal isolates.

Diffusion of carbapenems through the outer membrane of enterobacteriaceae and correlation of their activities with their periplasmic concentrations.

Scarce information is available on the real mechanism by which carbapenemes penetrate in Enterobacteriaceae, although a considerable amount of evidence suggests that in many species of this family the lack of certain outer membrane proteins is associated with the acquisition of resistance to these antibiotics. The existance of specific pathways for the carbapenems has never been demonstrated, although at times it has been postulated in both wild and mutant strains, on the basis of evident discordances between permeability patterns and suceptibility data. By using the Zimmerman and Rosselet technique, which requires the strain under investigation to harbor a suitable beta-lactamase, the permeability of intact Escherichia coli and Enterobacter cloacae cells to meropenem and imipenem was investigated by transferring a constructed vector carrying the carbapenem hydrolyzing CphA metallo-beta-lactamase gene into the parental strains and their porin-deficient mutants. Reduced amounts of nonspecific porins significantly reduced the penetration of both carbapenems. The virtual absence of porins caused the MICs of meropenem to increase, mostly in Enterobacter cloacae, while it did not affected the MICs of imipenem. No evidence of specific porin pathways of the type described in Pseudomonas aeruginosa was found.

High incidence of erythromycin-resistant Streptococcus pyogenes in Monza (North Italy) in untreated children with symptoms of acute pharyngo-tonsillitis: an epidemiological and molecular study.

A retrospective analysis of susceptibility data available for Group A streptococcal isolates collected between January 1990 and January 1996 at the Hospital Microbiology Laboratory of Monza (North Italy), showed a sharp rise in the erythromycin resistance rates during the last 3 years. Streptococcus pyogenes resistant to erythromycin accounted for approximately 1% of strains isolated between 1990 and 1992; the percentage then rose from 5% in 1993 to almost 39% in 1995. In January 1996, the resistance rates peaked to 81%. A prospective controlled study performed between March and May of 1996 to determine the percentage of erythromycin-resistant Group A streptococci isolated in Monza from untreated children with acute pharyngo-tonsillitis, gave further confirmation of a high rate of erythromycin resistance (47%) in this area. Molecular characterization by T-serotyping and pulse-field gel electrophoresis analysis of 25 erythromycin-resistant Group A streptococcal isolates, showed a relatively high degree of heterogeneity among these strains, demonstrating that the increased resistance is not caused by the spread of a single clone.

Diversity of carbapenemases in clinical isolates of Enterobacteriaceae in Croatia--the results of a multicentre study.

Since the first carbapenem-resistant Klebsiella pneumoniae strain was isolated in 2008, Enterobacteriaceae with reduced susceptibility to one or more carbapenems have emerged sporadically in different geographical regions in Croatia. These observations gave rise to a multicenter study on carbapenem resistance in Enterobacteriaceae from Croatia. Fifty-seven carbapenem-non-susceptible strains of Enterobacteriaceae were collected during 2011-2012 from four large hospital centres in Croatia. Overall, 36 strains produced VIM-1 ß-lactamase, three produced NDM-1, and one produced KPC-2. A high degree of clonal relatedness was observed in Enterobacter cloacae and Citrobacter freundii strains, in contrast to K. pneumoniae strains. BlaVIM genes were located within class1 integron which contained genes encoding resistance to aminoglycosides (aacA4 ). The study found strong association between blaVIM and qnrB6 and between blaNDM and qnrA6 genes.

Klebsiella pneumoniae harbouring OXA-48 carbapenemase in a Libyan refugee in Italy.

A carbapenem-resistant Klebsiella pneumoniae was isolated from a blood-culture of an inpatient from Libya, hospitalized in the intensive-care unit of Negrar Hospital, Italy. The clinical isolate carried the following ß-lactamase genes, bla(TEM -1), bla(SHV -11), bla(OXA -1), bla(CTX -M-15) and bla(OXA -48), respectively. The bla(OXA -48) gene was inserted in the Tn1999.2 transposon type, carried on a conjugative, 60-kilobase plasmid, that presented an L/M backbone, hosted by a multidrug-resistant ST 101 K. pneumoniae strain. Our report highlights the international transfer of bla(OXA -48) gene and the importance of screening measures of multidrug-resistant Enterobacteriaceae.

Prevalence of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae strains isolated in North-East Italy.

We investigated the prevalence of plasmid-mediated quinolone resistance genes in 756 clinical isolates of Enterobacteriaceae originating from Microbiology Diagnostic Laboratories of North-East Italy. Five point zero two percent of isolates carried a qnr determinant while the aac(6')-Ib-cr determinant was detected in 9·25% of isolates. We also investigated the association between the plasmid-mediated quinolone resistance and the beta-lactamase genes, and characterized the plasmids carrying these determinants of resistance.

Performance of different commercial methods for determining minimum inhibitory concentrations of glycopeptides and linezolid against blood isolates of Staphylococcus aureus.

The aim of this study was to determine the accuracy of commercial systems (VITEK® 2, Etest and Sensititre®) in determining the minimum inhibitory concentrations of vancomycin, teicoplanin and linezolid of Staphylococcus aureus strains and to evaluate the reproducibility of each system in a clinical microbiology laboratory. In total, 115 strains of S. aureus isolated from blood cultures were tested with all three commercial methods as well as the broth microdilution method, which is designated as the standard for glycopeptides and linezolid. Fourteen different S. aureus strains were included in a reproducibility test for all methods and antibiotics. For these strains, antimicrobial susceptibility testing was repeated 10 times on different days with all four methods, each time using the same inoculum. All three commercial methods exhibited similar performance in categorisation of nearly all of the meticillin-susceptible S. aureus (MSSA) isolates. Discrepancies were registered for meticillin-resistant S. aureus (MRSA); 2.5% of the strains in the intermediate or resistant category with the VITEK 2 system were not recognised as resistant by Etest and Sensititre. Moreover, none of the three commercial methods provided accurate results compared with homemade broth microdilution. Reproducibility of vancomycin and teicoplanin was 100% with VITEK 2 and Sensititre and 98.75% with Etest. Microdilution showed a reproducibility of 95.6% with vancomycin and 83.1% with teicoplanin. In contrast to previous reports, the best agreement with microdilution was exhibited by VITEK 2 both for MSSA and MRSA. For the antibiotics tested, the best reproducibility was obtained with the VITEK 2 and Sensititre systems.

Description and plasmid characterization of qnrD determinants in Proteus mirabilis and Morganella morganii.

We investigated the presence of qnrC and qnrD among 756 non-replicate Enterobacteriaceae isolated in Italy, selected for being non-susceptible to fluoroquinolones and/or resistant to third-generation cephalosporins. Four Proteus mirabilis and one Morganella morganii (0.66% of the total) presented a qnrD gene, located in a 2687-base-pair plasmid that was entirely sequenced. The plasmid is un-typable, and contains no known coding region other than qnrD. That the qnrD gene was found in four unrelated P. mirabilis and in one M. morganii isolate might suggest a frequent association of this gene with the tribe Proteeae.

Rapid molecular technique analysis of a KPC-3-producing Klebsiella pneumoniae outbreak in an Italian surgery unit.

The rapid emergence of KPC-producing Klebsiella pneumoniae has become a serious problem in health-care settings, increasing in frequency worldwide. These infections are worrisome, since the antimicrobial treatment options for infections due to multidrug-resistant strains are very limited, and outbreaks must be rapidly detected and controlled. A semi-automated, repetitive-sequence-based PCR (rep-PCR) instrument (DiversiLab system) was evaluated in comparison with the pulse-field gel electrophoresis (PFGE) and multilocus sequence typing to investigate the outbreak of KPC-producing K. pneumoniae in a surgery unit at the University Hospital of Verona, Italy, as a rapid method for outbreak investigations. A selection of seven epidemiologically related K. pneumoniae showing resistance to carbapenem and three epidemiologically unrelated K. pneumoniae isolates were collected from patient with hospital-acquired infection. Among the epidemiologically related isolates, PFGE and Rep-PCR identified a unique pattern with more than 90% of homology. The concordance between DiversiLab and PFGE results confirmed the usefulness of rapid molecular techniques to investigate outbreaks due to multidrug-resistant bacteria. Moreover, this result could meet the international need for a harmonised typing tool, allowing the implementation of strict control measures to prevent dissemination of these organisms in health-care settings.

A novel VIM-type metallo-beta-lactamase (VIM-14) in a Pseudomonas aeruginosa clinical isolate from a neonatal intensive care unit.

A Pseudomonas aeruginosa highly resistant to carbapenems was isolated in a neonatal intensive care unit in Palermo, Italy. The strain was found to carry a novel VIM-type enzyme, classified as VIM-14. The novel enzyme differs from VIM-4 in a G31S mutation. VIM-14 was harboured in a class 1 integron with a new organization. The integron carried the genes aac7, blaVIM-14, blaOXA-20 and aac4 in that order.

Detection of a new SHV-type extended-spectrum beta-lactamase, SHV-31, in a Klebsiella pneumoniae strain causing a large nosocomial outbreak in The Netherlands.

A Klebsiella pneumoniae strain resistant to third-generation cephalosporins was isolated in the eastern Netherlands. The strain was found to carry a novel extended-spectrum beta-lactamase, namely, SHV-31. The combination of the two mutations by which SHV-31 differs from SHV-1, namely, L35Q and E240K, had previously only been described in association with one or more additional mutations.

A strain of Salmonella enterica serovar Virchow isolated in Turkey and carrying a CTX-M-3 extended-spectrum beta-lactamase.

A multiply resistant strain of Salmonella enterica subsp. enterica serovar Virchow was isolated in November 2002 from a catheterized patient admitted to the SSK Training Hospital in Ankara, Turkey. This isolate showed an antimicrobial susceptibility pattern compatible with the presence of a CTX-M-type ESBL, namely resistance to cefotaxime, aztreonam and cefepime, and intermediate susceptibility to ceftazidime. On checking for the presence of the bla(TEM), bla(SHV), and bla(CTX-M )resistance genes by PCR, negative results were obtained with the primers specific for SHV and TEM genes, while positive results were obtained with those specific for CTX-M-type genes. After sequencing, the beta-lactamase was identified as CTX-M-3. This is the first report of this enzyme in Salmonella Virchow and represents a further disquieting threat to the therapy of infections caused by Salmonella isolates.

Contributions of the AmpC beta-lactamase and the AcrAB multidrug efflux system in intrinsic resistance of Escherichia coli K-12 to beta-lactams.

The roles of the AmpC chromosomal beta-lactamase and the AcrAB efflux system in levels of intrinsic resistance and susceptibility of Escherichia coli to beta-lactams were studied with a set of isogenic strains. MICs of ureidopenicillins, carbenicillin, oxacillin, and cloxacillin were drastically reduced by the inactivation of AcrAB, whereas those of the earlier cephalosporins were affected mostly by the loss of AmpC beta-lactamase.

High-level fluoroquinolone-resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA.

Immunoblotting with antibody against AcrA, an obligatory component of the AcrAB multidrug efflux system, showed that this protein was overexpressed by >/=170% in 9 of 10 clinical isolates of Esherichia coli with high-level ciprofloxacin resistance (MICs, >/=32 microg/ml) but not in any of the 15 isolates for which the MIC was

Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-1, a novel transferable metallo-beta-lactamase.

A total of 8 Pseudomonas aeruginosa isolates was collected from 7 different patients in different wards of the University Hospital of Verona, Italy, from February 1997 to February 1998. The high level of resistance to carbapenems (imipenem minimum inhibitory concentration was always >128 microg/mL) and other broad-spectrum beta-lactams and the rate of imipenem hydrolysis and its inhibition by ethylenediamine-tetra-acetic acid were all suggestive of production of a carbapenem-hydrolyzing metallo-beta-lactamase. A specific DNA probe derived from the recently cloned bla(VIM-1) gene hybridized to all the isolates. A genomic DNA fingerprinting profile revealed clonal relatedness for 7 of 8 isolates. A description of this hospital outbreak is reported, the occurrence of which confirms that proliferation of metallo-beta-lactamase-producing strains multiply resistant to beta-lactams is already a reality outside Japan. These findings emphasize the need for early recognition of similar isolates.