First countrywide survey of acquired metallo-beta-lactamases in gram-negative pathogens in Italy.

Metallo-beta-lactamases (MBLs) can confer resistance to most beta-lactams, including carbapenems. Their emergence in gram-negative pathogens is a matter of major concern. Italy was the first European country to report the presence of acquired MBLs in gram-negative pathogens and is one of the countries where MBL producers have been detected repeatedly. Here, we present the results of the first Italian nationwide survey of acquired MBLs in gram-negative pathogens. Of 14,812 consecutive nonreplicate clinical isolates (12,245 Enterobacteriaceae isolates and 2,567 gram-negative nonfermenters) screened for reduced carbapenem susceptibility during a 4-month period (September to December 2004), 30 isolates (28 Pseudomonas aeruginosa isolates, 1 Pseudomonas putida isolate, and 1 Enterobacter cloacae isolate) carried acquired MBL determinants. MBL producers were detected in 10 of 12 cities, with a predominance of VIM-type enzymes over IMP-type enzymes (4:1). Although having an overall low prevalence (1.3%) and significant geographical differences, MBL-producing P. aeruginosa strains appeared to be widespread in Italy, with a notable diversity of clones, enzymes, and integrons carrying MBL gene cassettes.

Chronic infection sustained by a Pseudomonas aeruginosa High-Risk clone producing the VIM-1 metallo-ß-lactamase in a cystic fibrosis patient after lung transplantation.

BACKGROUND: The significance of chronic lung infection by multidrug-resistant (MDR) pathogens in Cystic Fibrosis (CF) transplanted patients remains controversial, and the available information is overall limited. Here we describe the case of a chronic infection, sustained by a metallo-ß-lactamase (MBL)-producing P. aeruginosa strain, in a CF patient following lung transplantation. METHODS: Twelve P. aeruginosa isolates collected from a CF patient over a 15-years follow-up period after lung transplantation were analysed for their antibiotic susceptibility profile, MBL production and clonal relatedness. Available clinical and microbiological records were reviewed. RESULTS: The transplanted CF patient was chronically infected by an MBL-producing P. aeruginosa strain which harboured a blaVIM-1 determinant inserted into a novel class 1 integron. The strain exhibited an MDR phenotype and belonged to the globally widespread ST235 epidemic clonal lineage, which however is not a typical CF-associated epidemic clone. Despite the chronic infection, the long-term outcome of this patient during the post-transplant period was characterized by the absence of acute exacerbations and by a mostly stable pulmonary function. CONCLUSIONS: This report provides one of the few descriptions of MBL-producing P. aeruginosa infections in CF patients, and the first description of such an infection after lung transplantation in these patients. Infection with the MBL-producing strain apparently did not significantly affect the patient pulmonary function.

Prevalence and characterization of metallo-beta-lactamases in clinical isolates of pseudomonas aeruginosa.

The prevalence and the type(s) of metallo-beta-lactamases (MBLs) produced by isolates of Pseudomonas aeruginosa were investigated. During 2001, 506 nonduplicate isolates were obtained from hospitalized patients. Eighty-two strains were selected because of resistance to carbapenems and/or ceftazidime. Screening for MBL production was performed in the latter isolates by the Etest MBL strips (AB Biodisk, Solna, Sweden) and by a broth microdilution method measuring minimum inhibitory concentrations (MICs) of imipenem alone and in the presence of metal-chelating agents (EDTA and o-phenanthroline). Specific DNA probes were used to investigate the presence of genes coding for IMP- or VIM-type enzymes. Overall, four isolates of P. aeruginosa (obtained from independent patients) were found to carry a blaVIM gene. Polymerase chain reaction (PCR) experiments and DNA sequencing revealed that the VIM-2 determinant was present in three cases, whereas VIM-1 was detected in one isolate. Surveillance programs should be adopted to avoid the spread of these worrisome resistance genes.

Persistence of TEM-52/TEM-92 and SHV-12 extended-spectrum ß-lactamases in clinical isolates of Enterobacteriaceae in Italy.

Extended-spectrum ß-lactamases (ESBLs) belonging to the TEM and SHV families were investigated in 583 ESBL-producing Enterobacteriaceae collected at the clinical microbiology laboratories of 11 teaching Italian hospitals. By molecular analysis TEM-type and SHV-type ESBLs were confirmed on 154 and 74 isolates, respectively. High variability was found among TEM-types ß-lactamases with the following variants: TEM-5, TEM-6, TEM-12, TEM-15, TEM-24, TEM-26, TEM-29, TEM-52, TEM-92, TEM-134, and TEM-149. Among SHV variants, SHV-2a, SHV-5, SHV-12, and SHV-28 have been detected. The most widespread variants are TEM-52/92 and SHV-12.

CTX-M-type extended-spectrum beta-lactamases in Italy: molecular epidemiology of an emerging countrywide problem.

A nationwide survey of extended-spectrum beta-lactamase (ESBL) production among Enterobacteriaceae, carried out in 2003, showed that CTX-M-type enzymes have achieved a sizeable prevalence among ESBL producers in Italy, mostly in Escherichia coli and, to a lesser extent, in Klebsiella pneumoniae. In this work, we report on the molecular epidemiology of the CTX-M-producing isolates from that survey and on the mechanisms of dissemination of these emerging resistance determinants. The CTX-M-producing isolates were detected in 10 of the 11 participating centers distributed across the Italian national territory, although at remarkably variable rates in different centers (1.2 to 49.5% of the ESBL producers). All CTX-M determinants were of group 1, with CTX-M-15 and CTX-M-1 being the most prevalent variants (60% and 35%, respectively) and CTX-M-32 carried by a minority (5%) of isolates. Each variant was detected both in E. coli and in K. pneumoniae. Genotyping of the CTX-M-producing isolates by random amplification of polymorphic DNA revealed a notable diversity, especially among those producing CTX-M-1, while clonal expansion was evident with some CTX-M-15-producing strains. Mating experiments revealed a higher overall transferability of bla(CTX-M-1) and bla(CTX-M-32) than of bla(CTX-M-15). Coresistance to quinolones and aminoglycosides was overall higher with the CTX-M-15-producing isolates. The present results indicate that CTX-M-producing strains are now widespread across the Italian territory and underscore the emerging role of these ESBL determinants in the European setting. They also reveal notable differences in the dissemination mechanisms of genes encoding different CTX-M variants of the same lineage.

Trends in production of extended-spectrum beta-lactamases among enterobacteria of medical interest: report of the second Italian nationwide survey.

Results of a 2003 survey carried out in Italy to evaluate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing enterobacteria are presented. Eleven Italian Microbiology Laboratories investigated 9,076 consecutive nonreplicate isolates (inpatients, 6,850; outpatients, 2,226). ESBL screening was performed by MIC data analysis. Confirmation was obtained using the double-disk synergy test and the combination disk test based on CLSI methodology. ESBL determinants were investigated by colony blot hybridization and confirmed by sequencing. Results were compared to those of the 1999 Italian survey (8,015 isolates). The prevalence of ESBL producers was 7.4% among isolates from inpatients (in 1999, 6.3%) and 3.5% among outpatients (no data were available for 1999). Among hospitalized patients, the most prevalent ESBL-positive species was Escherichia coli (Klebsiella pneumoniae in 1999). Proteus mirabilis was the most prevalent ESBL-positive species among outpatients. In both groups, most ESBL-positive pathogens were obtained from urinary tract infections. TEM-type ESBLs were the most prevalent enzymes (45.4%). Non-TEM, non-SHV determinants emerged: CTX-M-type in E. coli and K. pneumoniae, and PER-type in P. mirabilis, Providencia spp., and E. coli. With the exception of 3/163 P. mirabilis isolates and 1/44 Providencia stuartii isolate (all of which were intermediate for imipenem), carbapenems were active against all ESBL-positive enterobacteria. Susceptibility to other drugs was as follows: 84.7% for amikacin, 84.4% for piperacillin-tazobactam, 48.0% for gentamicin, and 32.8% for ciprofloxacin. Carbapenems appear to be the drug of choice. Amikacin and beta-lactam/beta-lactamase inhibitor combinations represent an alternative in non-life-threatening infections. The appearance of ESBL-positive enterobacteria in the community makes it mandatory that family physicians learn how to treat these pathogens.

Novel TEM-type extended-spectrum beta-lactamase, TEM-134, in a Citrobacter koseri clinical isolate.

A new natural TEM derivative with extended-spectrum beta-lactamase activity, TEM-134, was identified in a ceftazidime-resistant clinical isolate of Citrobacter koseri. Compared to TEM-1, TEM-134 contains the following mutations: Q39K, E104K, R164H, and G238S. The bla(TEM-134) gene was not transferable by conjugation and, apparently, was chromosomally encoded. Expression studies with Escherichia coli revealed efficient cefotaximase and ceftazidimase activity for TEM-134.

Dissemination of CTX-M-type extended-spectrum beta-lactamase genes to unusual hosts.

A Citrobacter amalonaticus and a Morganella morganii producing the CTX-M-1 extended-spectrum beta-lactamase (ESBL) were isolated from an area where this enzyme is now widespread in Escherichia coli. This is the first report of CTX-M-1 in the former species. In both cases the ESBL determinant was possibly acquired by these unusual hosts in vivo, after coinfection with E. coli strains carrying conjugative plasmids encoding CTX-M-1.

Simple microdilution test for detection of metallo-beta-lactamase production in Pseudomonas aeruginosa.

A microdilution test measuring imipenem MICs in the presence or absence of a mixture of EDTA plus 1,10-phenanthroline was developed and tested on 190 Pseudomonas aeruginosa isolates, including 18 VIM- and 4 IMP-type metallo-beta-lactamase (MBL) producers. The chelator mixture reduced by fourfold or more the imipenem MICs for MBL producers, while a lower effect or no effect was usually observed with MBL nonproducers.

IMP-12, a new plasmid-encoded metallo-beta-lactamase from a Pseudomonas putida clinical isolate.

A Pseudomonas putida strain showing broad-spectrum resistance to beta-lactams, including expanded-spectrum cephalosporins and carbapenems, was isolated from a patient with a urinary tract infection at the University Hospital of Varese in northern Italy. The isolate was found to produce metallo-beta-lactamase activity and to harbor a 50-kb plasmid, named pVA758, carrying a new bla(IMP) determinant, named bla(IMP-12). Plasmid pVA758 was not self-transferable by conjugation to either Escherichia coli or Pseudomonas aeruginosa but could be introduced by electroporation and maintained in the latter host, where it conferred resistance or decreased susceptibility to various beta-lactams. The IMP-12 enzyme is quite divergent from other IMP variants: its closest relatives are IMP-8 and IMP-2 (89 and 88% sequence identity, respectively), and IMP-1 is 85% identical to IMP-12. The bla(IMP-12) determinant is carried on an integron-borne gene cassette whose attC recombination site is related to those present in cassettes containing bla(IMP-1), bla(IMP-6), bla(IMP-7), bla(IMP-10), and bla(IMP-11) and unrelated to that present in cassettes containing bla(IMP-2) and bla(IMP-8). IMP-12 was overproduced in E. coli by using a T7-based expression system and was purified by cation-exchange chromatography followed by gel filtration. Kinetic analysis revealed that, like other IMP variants, IMP-12 exhibits an overall preference for cephalosporins and carbapenems rather than for penicillins and does not hydrolyze temocillin and aztreonam. However, IMP-12 also exhibits some notable functional differences from other IMP variants, including uniformly poor activity toward penicillins (k(cat)/K(m) values, around 10(4) M(-1). s(-1)) and a remarkably high K(m) (around 900 micro M) for imipenem.