Spread of Enterobacteriaceae carrying the PER-1 extended-spectrum beta-lactamase gene as a chromosomal insert: a report from Italy.
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Anti-Bacterial Agents↗
Biomedical subjects
Mariagrazia Perilli
Publications and source records attributed to Mariagrazia Perilli.
Spread of bla(CTX-M-type) and bla(PER-2) beta-lactamase genes in clinical isolates from Bolivian hospitals.
OBJECTIVES: To assess the prevalence and types of genes encoding extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. from Bolivia. METHODS: A total of 642 clinical isolates were collected consecutively during a 4 month period (September to December 2004). Resistance or reduced susceptibility to cefotaxime and/or ceftazidime and/or aztreonam was assessed using double disc synergy tests using clavulanic acid, cefotaxime, ceftazidime and aztreonam to identify putative ESBL-producing isolates. The ESBL determinants were characterized by colony blot hybridization, PCR and DNA sequencing. RESULTS: Of the 642 isolates, 220 (34.3%) showed resistance or reduced susceptibility to cefotaxime and/or ceftazidime and/or aztreonam, and 150 (23.4%) were putative ESBL producers. A total of 106 ESBL-producing isolates contained the bla(CTX-M-2) gene, and 32 isolates had a novel allele, bla(CTX-M-43). bla(CTX-M) alleles were detected in all P. aeruginosa and Acinetobacter spp. studied. In contrast, only 12 ESBL-producing isolates had bla(PER-2), mainly Enterobacteriaceae, although it was also found in a strain of P. aeruginosa. CONCLUSIONS: This is the first study on ESBL-producing strains in Bolivia and it reveals a high prevalence of bla(CTX-M) genes. The PER-2 enzyme was less prevalent, but its gene was detected in several species, including P. aeruginosa, which is consistent with horizontal transfer.
Acinetobacter↗
Occurrence of extended spectrum beta-lactamases among isolates of Enterobacteriaceae from urinary tract infections in southern Italy.
The present investigation was undertaken to assess the prevalence of extended-spectrum beta-lactamases (ESbetaLs) among urinary tract infection (UTI) isolates. During 4 months in 2004, a total of 650 Enterobacteriaceae strains from UTIs was collected by five clinical microbiology laboratories located in southern Italy and the beta-lactamase production was investigated. A total of 50 of the 650 isolates were double-disk positive and suspected of producing an ESbetaL; Escherichia coli (36.0%) and Klebsiella pneumoniae (32.0%) were the most common species among all ESbetaL producers. Characterization of ESbetaL determinants was carried out by the colony blot hybridization method, and polymerase chain reaction (PCR) and DNA sequencing in order to identify the presence of bla (TEM), bla (SHV), bla (PER), and bla (CTX-M) determinants. The ESbetaL variants found in this study were the following: TEM-15, TEM-24, TEM-52, TEM-134, SHV-12, CTX-M-1, CTX-M-3, CTX-M-15, and PER-1. As expected, the majority of the isolates were found to be susceptible to imipenem (94%), cefepime (54%) and piperacillin-tazobactam (54%). The results of this survey show the prevalence of ESbetaL enzymes among enterobacterial pathogens causing UTIs in southern Italy.
Anti-Bacterial Agents↗
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.
Aminoglycosides↗
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.
Data Collection↗
Evolution of CTX-M-type beta-lactamases in isolates of Escherichia coli infecting hospital and community patients.
Escherichia coli isolates collected at our Institution from 1999 to 2003 (n=20,258) were studied to evaluate the production of CTX-M-type extended-spectrum beta-lactamases (ESBL). Isolates suspected of producing CTX-M enzymes were analyzed by the double-disk synergy test, hybridization with specific probes, PCR and direct DNA sequencing. Overall, 53 ESBL-positive isolates were found to carry CTX-M-type genes (blaCTX-M-1, n=51; blaCTX-M-15, n=2). The isolation of CTX-M-positive strains increased from 1 per year (1999) to 26 per year (2003). The first isolate carrying the blaCTX-M-15 gene appeared in 2003 and was obtained from a patient previously treated with ceftazidime. CTX-M-positive isolates were characterized by multi-drug resistance and were obtained both from inpatients (n=29) and outpatients (n=24). Most patients were over 60-year-old (n=45), had underlying chronic diseases (n=32), and had been hospitalized more than once (n=33). Strains were frequently isolated from the urinary tract, often after recurrent infections. Our study demonstrates that CTX-M-producing isolates are increasing among E. coli strains. Adequate laboratory detection may help in choosing appropriate treatment and in limiting the spread of this resistance trait.
Aged↗
Ciprofloxacin, salicylate, and 2,4-dinitrophenol decrease production of AmpC-type beta-lactamase in two Citrobacter freundii clinical isolates.
The effect of ciprofloxacin and two mar RAB inducers on the susceptibility to beta-lactam antibiotics in two AmpC beta-lactamase semi-constitutive producer Citrobacter freundii clinical isolates (the DM 1 and DM 2 strains) was studied. Possible changes in outer membrane protein expression, permeability to cephaloridine, active efflux, and hydrolytic activity of beta-lactamase-crude extracts were evaluated under the influence of ciprofloxacin, sodium salicylate, and 2,4-dinitrophenol. Results were compared with those of the effect of the same three chemicals on a normally beta-lactamase-inducible wild-type C. freundii strain. The three assayed compounds decreased beta-lactamase hydrolysis on cephaloridine in both the two clinical isolates as well as in the wild-type strain. However, only the DM 1 and DM 2 strains showed increased susceptibility to beta-lactams. Sodium salicylate and 2,4-dinitrophenol, but not ciprofloxacin, reduced the expression of a 45-kDa outer membrane protein in the three studied strains, which was accompanied by a 4- to 20-fold diminution in permeability to cephaloridine. In conclusion, two mar RAB inducers and ciprofloxacin induced the Mar phenotype and repressed AmpC beta-lactamase synthesis in the DM 1 and DM 2 clinical isolates.
2,4-Dinitrophenol↗
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.
Anti-Bacterial Agents↗
Proteus mirabilis bloodstream infections: risk factors and treatment outcome related to the expression of extended-spectrum beta-lactamases.
Bloodstream infection (BSI) due to Proteus mirabilis strains is a relatively uncommon clinical entity, and its significance has received little attention. This study was initiated to evaluate risk factors and treatment outcome of BSI episodes due to P. mirabilis producing extended-spectrum beta-lactamases (ESBLs). Twenty-five BSI episodes caused by P. mirabilis occurred at our hospital (Ospedale di Circolo e Fondazione Macchi, Varese, Italy) over a 7.5-year period. Phenotypic and molecular methods were used to assess ESBL production. Clinical records of BSI patients were examined retrospectively. Demographic data, underlying diseases (according to McCabe and Jackson classification and Charlson weighted index), risk factors, and treatment outcome were investigated by comparing cases due to ESBL-positive strains to cases due to ESBL-negative strains. Eleven isolates were found to express ESBLs (TEM-52 or TEM-92). The remaining 14 isolates were ESBL negative and were uniformly susceptible to extended-spectrum cephalosporins and monobactams. Comparison of the two groups showed that previous hospitalization in a nursing home (P = 0.04) and use of bladder catheter (P = 0.01) were significant risk factors for infections due to ESBL-positive strains. In addition, cases due to ESBL-positive strains showed a significantly higher mortality attributable to BSI (P = 0.04). BSI cases due to ESBL-negative isolates uniformly responded to therapy, whereas 5/11 cases due to ESBL-positive isolates failed to respond (P < 0.01). Use of carbapenems was associated with complete response independently of ESBL production. Therapeutic failure and mortality may occur in BSI episodes caused by ESBL-positive P. mirabilis isolates. Thus, recognition of ESBL-positive strains appears to be critical for the clinical management of patients with systemic P. mirabilis infections.
Aged↗
Overexpression system and biochemical profile of CTX-M-3 extended-spectrum beta-lactamase expressed in Escherichia coli.
An efficient over-expression system was developed for CTX-M-3 extended-spectrum-beta-lactamase. The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme. The N-terminal amino acid sequence was determined to be NH2-QTADVQ... Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates. Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme.
Biotechnology↗
Salicylate decreases production of AmpC type beta-lactamases and increases susceptibility to beta-lactams in a Morganella morganii clinical isolate.
The effect of salicylate, a marRAB inducer, on the resistance to beta-lactams was characterized in an AmpC beta-lactamase hyperproducer Morganella morganii clinical isolate (the M1 strain). Results were compared with those of the effect of salicylate in a wild-type M. morganii strain. Salicylate induced a decreased susceptibility to nalidixic acid, norfloxacin and tetracycline and simultaneously increased the susceptibility to beta-lactams apparently due to the repression of AmpC beta-lactamase synthesis in the M1 strain. Likewise, salicylate only repressed 46 kDa outer membrane protein expression in the wild-type strain, since the clinical isolate M1 did not express it.
Anti-Bacterial Agents↗
Antimicrobial susceptibility of clinical isolates of Enterobacteriaceae producing complex beta-lactamase patterns including extended-spectrum enzymes.
The antimicrobial susceptibility of 103 clinical isolates of Enterobacteriaceae to 11 antibiotics, was investigated, using a conventional inoculum size (5 x 10(5) CFU) and a higher inoculum size (5 x 10(8) CFU). All the isolates produced complex beta-lactamase patterns, including an extended-spectrum beta-lactamase (ESBL) of the TEM- or SHV-type plus other enzymes (a TEM-type or an SHV-type non-ESBL and/or a class C enzyme). The following repertoire of ESBLs was produced by the isolates: TEM-15, TEM-19, TEM-26, TEM-52, TEM-72, TEM-87, TEM-92, SHV-2a, SHV-5 and SHV-12, as assessed by sequencing. Production of the other enzymes was showed by analytical isoelectric focusing. Overall, meropenem was the most active agent and less influenced by inoculum size, while other beta-lactams showed a lower activity and a significant inoculum size effect. In conclusion, from its in vitro performance, meropenem could be considered as the last resource drug against strains producing complex beta-lactamase patterns including an ESBL.
Anti-Bacterial Agents↗
Enhanced active efflux, repression of porin synthesis and development of Mar phenotype by diazepam in two enterobacteria strains.
The aim of this work was to determine whether diazepam could induce the multiple antibiotic resistance (Mar) phenotype in Klebsiella pneumoniae and Escherichia coli strains. The Mar phenotype is characterized by decreased susceptibility to multiple antibiotics due to the loss of porins and/or increased expression of active efflux systems. The effect of subinhibitory concentrations of diazepam on the susceptibility of different antimicrobial agents, outer-membrane protein expression and norfloxacin intracellular accumulation was studied. The results revealed that diazepam concentrations equal or twice adult dosage induced the same Mar phenotype as two well known E. coli marRAB inducers, sodium salicylate and sodium benzoate. Susceptibility to norfloxacin in a K. pneumoniae clinical isolate and E. coli strain Ag100 decreased due to enhanced active efflux and loss of porin expression. A decreased susceptibility to chloramphenicol, tetracycline, nalidixic acid and beta-lactam antibiotics was also observed. In conclusion, like sodium salicylate or sodium benzoate, diazepam may induce the Mar phenotype.
Anti-Bacterial Agents↗
Biochemical characterization of laboratory mutants of extended-spectrum beta-lactamase TEM-60.
Three mutants of the extended-spectrum beta-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.
Chemical Phenomena↗
Bacteremia due to Klebsiella pneumoniae isolates producing the TEM-52 extended-spectrum beta-lactamase: treatment outcome of patients receiving imipenem or ciprofloxacin.
The treatment outcome of 35 cases of bacteremia due to Klebsiella pneumoniae isolates producing TEM-52 extended-spectrum beta-lactamase was studied. Twenty-eight cases, classified as "nonfatal disease" using the McCabe and Jackson classification, were investigated with regard to ciprofloxacin and imipenem response. Because ciprofloxacin was active in vitro against 21 of 28 isolates, only the treatment outcome of the ciprofloxacin-susceptible subgroup was evaluated. Eight of 10 cases occurred in patients who experienced a complete response to imipenem; 2 of 10 failed to respond. In contrast, only 2 of 7 cases had a partial response to ciprofloxacin, and, in 5 of 7 cases, the treatment failed. Statistical analysis revealed a significant difference in the treatment outcome of the 2 groups (P=.03). Because the isolates had minimum inhibitory concentrations of ciprofloxacin close to the susceptibility breakpoint, treatment failure could be ascribed to the inability of the drug to reach therapeutic concentrations at infected sites.
Bacteremia↗
Prevalence of extended spectrum beta-lactamases among Enterobacteriaceae: an Italian survey.
The prevalence of extended-spectrum beta-lactamase (ESBL) production by consecutive non-repeated isolates of Enterobacteriaceae was determined over a 6-month period. A total of 8015 strains were isolated from ten Italian laboratories and 509 (6.3%) of these were designated ESBL producers from the results of a double-disk synergy test. Escherichia coli was the most isolated microrganism, followed by Klebsiella pneumoniae and Proteus mirabilis. Providencia stuartii (28.1%) was the most frequently isolated ESBL producer, followed by K. pneumoniae and Enterobacter aerogenes (20.5%). However, amongst all ESBL producers, K. pneumoniae (38.2%) was the most represented followed by P. mirabilis (25.7%). All the strains positive to DD tests were confirmed for the carriage of TEM and SHV genes using colony-blot hybridisation (CH). A total of 447 strains (88.0%) were CH-positive, of which 42.3% hybridised with the TEM-type probe, 30.1% with the SHV-type probe and 15.6% with both probes. In conclusion, our findings indicate that 6.3% of all Enterobacteriaceae tested produced ESBLs, 42.3% of which were TEM-derived enzymes. More than 20% of P. stuartii, K. pneumoniae and E. aerogenes harbour these enzymes. The double-disk test seems to be a useful test to identify ESBL producing strains.
Anti-Bacterial Agents↗
Biochemical characterization of TEM-92 extended-spectrum beta-lactamase, a protein differing from TEM-52 in the signal peptide.
A bla(TEM-92) gene was cloned from a Proteus mirabilis isolate and expressed in Escherichia coli. Production of the enzyme caused reduction of susceptibility to penicillins and narrow- to expanded-spectrum cephalosporins but not to moxalactam and cephamycins. Determination of kinetic parameters with the purified enzyme revealed hydrolysis of expanded-spectrum cephalosporins, while cephamycins, moxalactam, and aztreonam were very poorly or not hydrolyzed. Clavulanate and penicillanic acid sulfones acylated TEM-92 slowly, and deacylation occurred at measurable rates.
Anti-Bacterial Agents↗
Characterization of a new extended-spectrum beta-lactamase (TEM-87) isolated in Proteus mirabilis during an Italian survey.
A new natural TEM derivative, named TEM-87, was identified in a Proteus mirabilis isolate from an Italian hospital. Compared to TEM-1, TEM-87 contains the following mutations: E104K, R164C, and M182T. Kinetic analysis of TEM-87 revealed extended-spectrum activity against oxyimino cephalosporins (preferentially ceftazidime) and aztreonam. Expression of blaTEM-87 in Escherichia coli decreased the host susceptibility to these drugs.
Aztreonam↗