Mariagrazia Perilli

Occurrence of Extended-Spectrum β-Lactamases in Members of the Family Enterobacteriaceae in Italy: Implications for Resistance to β-Lactams and Other Antimicrobial Drugs

ABSTRACT An Italian nationwide survey was carried out to assess the prevalences and the antimicrobial susceptibilities of members of the family Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs). Over a 6-month period, 8,015 isolates were obtained from hospitalized patients and screened for resistance to extended-spectrum cephalosporins and monobactams. On the basis of a synergistic effect between clavulanate and selected β-lactams (ceftazidime, aztreonam, cefotaxime, cefepime, and ceftriaxone), 509 isolates were found to be ESBL positive (6.3%). Colony blot hybridization with blaTEM and blaSHV DNA probes allowed one to distinguish four different genotypes: TEM-positive, SHV-positive, TEM- and SHV-positive, and non-TEM, non-SHV ESBL types. MICs for each isolate (E-test) were obtained for widely used β-lactams, combinations of β-lactams with β-lactamase inhibitors, aminoglycosides, and fluoroquinolones. Among ESBL-positive strains, Klebsiella pneumoniae, Proteus mirabilis, and Escherichia coli accounted for 73.6% of isolates. Overall, TEM-type ESBLs were more prevalent than SHV-type enzymes (234 versus 173), whereas the prevalence of strains producing both TEM- and SHV-type ESBLs was similar to that of isolates producing non-TEM, non-SHV enzymes (55 and 38, respectively). In vitro, all but one of the ESBL-producing isolates remained susceptible to imipenem. Susceptibility to other drugs varied: piperacillin-tazobactam, 91%; amoxicillin-clavulanic acid, 85%; cefoxitin, 78%; amikacin, 76%; ampicillin-sulbactam, 61%; ciprofloxacin, 58%; and gentamicin, 56%. Associated resistance to aminoglycosides and ciprofloxacin was observed most frequently among TEM-positive strains. Since therapeutic options for multiresistant Enterobacteriaceae are limited, combinations of β-lactams and β-lactamase inhibitors appear to represent an important alternative for treating infections caused by ESBL-producing Enterobacteriaceae.

Molecular Characterization of Extended-Spectrum β-Lactamases Produced by Nosocomial Isolates of Enterobacteriaceae from an Italian Nationwide Survey

ABSTRACT Extended-spectrum β-lactamases (ESBLs) are widespread in hospital settings worldwide. The present investigation was undertaken to assess the distribution and prevalence of ESBLs belonging to the TEM and SHV families in 448 ESBL-producing clinical isolates of Enterobacteriaceae collected from 10 different Italian hospitals. The natures of TEM and SHV determinants were identified by direct sequencing of PCR-amplified genes. TEM-52 and SHV-12 were the most common variants, and they were found in most hospitals and in several different species. Other less frequent variants included TEM-5, TEM-12, TEM-15, TEM-19, TEM-20, TEM-24, TEM-26, TEM-43, TEM-60, TEM-72, TEM-87, SHV-2a, SHV-5, and SHV-11. Proteus mirabilis was the most common producer of TEM-type ESBLs, while Klebsiella pneumoniae was the most common producer of SHV-type ESBLs. The distribution of TEM- and SHV-type ESBL variants in Enterobacteriaceae from Italian hospitals exhibited notable differences from those from other geographical settings.

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

ABSTRACT Results of a 2003 survey carried out in Italy to evaluate the prevalence of extended-spectrum β-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 β-lactam/β-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.

CTX-M-Type Extended-Spectrum β-Lactamases in Italy: Molecular Epidemiology of an Emerging Countrywide Problem

ABSTRACT A nationwide survey of extended-spectrum β-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 blaCTX-M-1 and blaCTX-M-32 than of blaCTX-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.

Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer.

The gene encoding a class A β‐lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high‐level amoxicillin‐resistant mutant that produces large amounts of β‐lactamase (blaF). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high‐level production of the β‐lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N‐terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or biaF) to the E. coli gene phoA resulted in the production of BlaF‐PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.

Proteus mirabilis Bloodstream Infections: Risk Factors and Treatment Outcome Related to the Expression of Extended-Spectrum β-Lactamases

ABSTRACT 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 β-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.

Nosocomial Infections Caused by Multidrug-Resistant Isolates of Pseudomonas putida Producing VIM-1 Metallo-β-Lactamase

ABSTRACT Successful carbapenem-based chemotherapy for the treatment of Pseudomonas infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-β-lactamases, which confer high-level resistance to carbapenems and most other β-lactams. Recently, multidrug-resistant Pseudomonas putida isolates for which carbapenem MICs were ≥32 μg/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy. Enzyme assays revealed production of a metallo-β-lactamase activity, while molecular analysis detected in each isolate a blaVIM-1 determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the β-lactamase determinant to Escherichia coli or Pseudomonas aeruginosa. Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin. PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the blaVIM-1 determinant (named In110) showed that the blaVIM-1-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a blaVIM-1-containing plasmid-borne integron from an Achromobacter xylosoxidans isolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.

Dynamics of a Nosocomial Outbreak of Multidrug-Resistant Pseudomonas aeruginosa Producing the PER-1 Extended-Spectrum β-Lactamase

ABSTRACT From November 1998 to August 1999, a large outbreak occurred in the general intensive care unit of the Ospedale di Circolo in Varese (Italy), caused by Pseudomonas aeruginosa producing the PER-1 extended-spectrum β-lactamase. A total of 108 clinical isolates of P. aeruginosa resistant to broad-spectrum cephalosporins were recovered from 18 patients. Epidemic isolates were characterized by synergy between clavulanic acid and ceftazidime, cefepime, and aztreonam. Isoelectric focusing of crude bacterial extracts detected two nitrocefin-positive bands with pI values of 8.0 and 5.3. PCR amplification and characterization of the amplicons by restriction analysis and direct sequencing indicated that the epidemic isolates carried a blaPER-1 determinant. The outbreak was of clonal origin as shown by pulsed-field gel electrophoresis analysis. This technique also indicated that the epidemic strain was not related to three other PER-1-positive isolates obtained at the same hospital in 1997. Typing by enterobacterial repetitive intergenic consensus-PCR showed that minor genetic variations occurred during the outbreak. The epidemic strain was characterized by a multiple-drug-resistance phenotype that remained unchanged over the outbreak, including extended-spectrum cephalosporins, monobactams, aminoglycosides, and fluoroquinolones. Isolation of infected patients and appropriate carbapenem therapy were successful in ending the outbreak. Our report indicates that theblaPER-1 resistance determinant may become an emerging therapeutic problem in Europe.

Parp1 localizes within the Dnmt1 promoter and protects its unmethylated state by its enzymatic activity

Background Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found. Methodology/Principal Findings Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic over-expression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation. Conclusions/Significance In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression.

ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites.

PARylation [poly(ADP-ribosyl)ation] is involved in the maintenance of genomic methylation patterns through its control of Dnmt1 [DNA (cytosine-5)-methyltransferase 1] activity. Our previous findings indicated that Ctcf (CCCTC-binding factor) may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Parp1 [poly(ADP-ribose) polymerase 1], which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. The results of the present study showed Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Parp1 at specific Ctcf target sequences and that PARylation is responsible for the maintenance of the unmethylated status of some Ctcf-bound CpGs. We suggest a mechanism by which Parp1, tethered and activated at specific DNA target sites by Ctcf, preserves their methylation-free status.