Elisabetta Nucleo

Nosocomial Outbreak Caused by Multidrug-Resistant Pseudomonas aeruginosa Producing IMP-13 Metallo-β-Lactamase

ABSTRACT An outbreak of Pseudomonas aeruginosa showing a multidrug-resistant (MDR) phenotype (including carbapenems, ceftazidime, cefepime, gentamicin, tobramycin, and fluoroquinolones) was observed, during a 5-month period, in a general intensive care unit of a large tertiary care and clinical research hospital in southern Italy. The outbreak involved 15 patients, with a total of 87 isolates, mostly from lower respiratory tract specimens. Analysis of isolates involved in the outbreak revealed production of metallo-β-lactamase (MBL) activity, and genotyping by pulsed-field gel electrophoresis of genomic DNA digested by SpeI revealed clonal relatedness among isolates. Molecular analysis of the MBL determinant showed the presence of a blaIMP-13 gene carried on a gene cassette inserted in a class 1 integron which also contained an aacA4 aminoglycoside resistance cassette encoding an AAC(6′)-Ib enzyme. The blaIMP-13-containing integron and its genetic environment appeared to be similar to those found in P. aeruginosa isolates producing IMP-13 from a hospital in Rome. The blaIMP-13 gene was not transferable by conjugation and was apparently carried on the chromosome. The outbreak was coincidental with a shortage of nursing personnel, and resolution was apparently associated with reinstatement of nursing personnel and reinforcement of general infection control practices within the intensive care unit. To our best knowledge this is the first description of a nosocomial outbreak of relatively large size caused by an IMP-producing gram-negative pathogen in Europe.

Multifocal Detection of Multidrug-Resistant Pseudomonas aeruginosa Producing the PER-1 Extended-Spectrum β-Lactamase in Northern Italy

ABSTRACT Forty-four nonreplicate clinical isolates of Pseudomonas aeruginosa that were resistant to extended-spectrum cephalosporins (ceftazidime and cefepime) and aztreonam, that putatively produced an acquired extended- spectrum β-lactamase (ESBL), according to the results of a double-disk synergy test, and that had been involved in nosocomial outbreaks were obtained from six different hospitals in northern Italy and screened for the presence of blaPER ESBL determinants. Twenty isolates, associated with nine independent outbreaks that occurred in five hospitals in the Milan area and its surroundings during 1995-2000, were found to carry an acquired blaPER-1 gene. PER-1 producers representative of the nine outbreaks exhibited a multidrug resistance (MDR) phenotype, including resistance to extended-spectrum cephalosporins, aztreonam, meropenem, aminoglycosides, and in most cases, imipenem and ciprofloxacin. An analysis of macrorestriction profiles of their genomic DNAs by pulsed-field gel electrophoresis revealed an overall clonal diversity of the PER-1 producers, although interhospital clonal spread was also observed. The blaPER-1 gene was not transferable and appeared to be chromosomally located. An analysis of the EcoRI and EcoRV restriction fragment length polymorphisms of the blaPER-1 locus revealed identical patterns for all isolates, and the characterization of a 1.9-kb region containing blaPER-1 revealed a conserved structure in representatives of the various clonal lineages. The present findings indicate that MDR P. aeruginosa clones producing the PER-1 ESBL are endemic to this area of northern Italy, where they have been circulating since the mid-1990s and have been associated with several nosocomial outbreaks.

CMY-16, a Novel Acquired AmpC-Type β-Lactamase of the CMY/LAT Lineage in Multifocal Monophyletic Isolates of Proteus mirabilis from Northern Italy

ABSTRACT We report multifocal detection (four different cities in northern Italy) of Proteus mirabilis isolates resistant to both oxyimino- and 7-α-methoxy-cephalosporins and producing a novel acquired AmpC-like β-lactamase. The enzyme, named CMY-16, is a variant of the CMY/LAT lineage, which differs from the closest homologues, CMY-4 and CMY-12, by a single amino acid substitution (A171S or N363S, respectively) and from CMY-2 by two substitutions (A171S and W221R). Expression of the cloned blaCMY-16 gene in Escherichia coli decreased susceptibility to penicillins, cephalosporins, and aztreonam. Tazobactam was more effective than clavulanate at antagonizing the enzyme activity. Genotyping, by random amplification of polymorphic DNA and pulsed-field gel electrophoresis of genomic DNA digested with SfiI, showed that isolates were clonally related to each other, although not identical. The blaCMY-16 gene was not transferable to E. coli by conjugation or transformation. In all isolates, it was chromosomally located and inserted in a conserved genetic environment. PCR mapping experiments revealed that the blaCMY-16 was flanked by ISEcp1 and the blc gene, similar to other genes of this lineage from plasmids of Salmonella enterica, Klebsiella spp., and E. coli. Overall, these results revealed multifocal spreading of a CMY-16-producing P. mirabilis clone in northern Italy. This finding represents the first report of an acquired AmpC-like β-lactamase in Proteus mirabilis from Italy and underscores the emergence of similar resistance determinants in the European setting.

Growth in glucose-based medium and exposure to subinhibitory concentrations of imipenem induce biofilm formation in a multidrug-resistant clinical isolate of Acinetobacter baumannii

BackgroundAcinetobacter baumannii is emerging as an important nosocomial pathogen. Multidrug resistance, as well as ability to withstand environmental stresses, makes eradication of A. baumannii difficult, particularly from hospital settings.ResultsOver a six-year period, 73 isolates of A. baumannii were collected from infected patients in two hospitals in Italy. While 69 out of the 73 isolates displayed identical multidrug antibiotic resistance pattern, they were susceptible to carbapenems. Genetic profiles of these 69 isolates, determined by Pulsed Field Gel Electrophoresis (PFGE), indicated that they were genetically related and could be clustered in a specific clone, called SMAL. We tested the ability of the SMAL clone to form biofilm, an important determinant for bacterial colonization of the human host and for persistence in the hospital environment. Biofilm formation by A. baumannii SMAL, measured as surface adhesion to polystyrene, is strongly affected by growth conditions, being impaired in rich growth media such as LB, while being favoured in glucose-based medium. Surface adhesion in glucose-based media is inhibited by treatment with cellulase, suggesting that it depends on production of cellulose or of a chemically related extracellular polysaccharide. Exposure of A. baumannii SMAL to subinhibitory concentrations of imipenem resulted in biofilm stimulation and increased production of iron uptake proteins. Growth in iron-supplemented medium also stimulated surface adhesion, thus suggesting that increased intracellular iron concentrations might act as an environmental signal for biofilm formation in A. baumannii SMAL.ConclusionsOur results indicate that exposure to subinhibitory concentrations of imipenem can stimulate biofilm formation and induce iron uptake in a pathogenic strain of A. baumannii, with potential implications on antibiotic susceptibility and ability to persist in the human host.

Epidemiological characterization and distribution of carbapenem-resistant Acinetobacter baumannii clinical isolates in Italy

This study was aimed at tracing the molecular characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates in Italy with both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Two hundred and two CRAB isolates were collected during 2004-2009, in two different surveillance periods, from 22 Italian hospitals that were representative for both distribution and infection. PFGE was performed, and the MLST scheme used was based on the gene sequence as published on the MLST Pasteur website http://www.pasteur.fr/mlst. Representatives of the major European clones I (RUH 875) and II (RUH 134) were used as controls. The two groups of isolates were characterized for their carbapenem resistance genes: 154 of 202 carried bla(OXA-58) alone, 21 of 202 also carried bla(OXA-23) , and 27 of 202 carried bla(OXA-23) alone. No isolates were positive for bla(OXA-24) . Genotype analysis of all isolates identified four distinct patterns by PFGE, which correlated with four distinct sequence types (STs) by MLST. The distribution of these four clusters in Italy confirmed the propensity of A. baumannii for nosocomial cross-transmission in a vast geographical area. We observed that clones A and B had similarities with European clone II and I respectively. By MLST, clone A was ST2, like European clone II, and clone B was ST1, like European clone I. PFGE and MLST showed the same discriminatory power and reproducibility. In addition, the two methods were concordant in defining CRAB Italian clones and in correlating them with the two pan-European clones.

Molecular epidemiology of KI and WU polyomaviruses in infants with acute respiratory disease and in adult hematopoietic stem cell transplant recipients

Polyomaviruses KI (KIPyV) and WU (WUPyV) were described recently in children with acute respiratory disease. The pathogenic potential of these human viruses has not been determined completely, but a correlation between immunosuppression and virus reactivation has been suggested. In the present study, the association between KI/WUPyV infection and immunosuppression was investigated using sequential nasopharyngeal aspirates from asymptomatic adult hematopoietic stem cell transplant recipients. In parallel, an investigation on the WU/KIPyV prevalence in children with acute respiratory disease was also carried out. Two of the 126 samples obtained from the 31 hematopoietic transplant recipients were positive for KIPyV (1 sample, 0.79%) and WUPyV (1 sample, 0.79%). Both samples were obtained 15 days after allogeneic transplantation and virus persistence was not observed in subsequent samples. In symptomatic children, 7 of the 486 nasopharyngeal aspirates were positive for WUPyV (1.4%) and 1 for KIPyV (0.2%). Single polyomavirus infection was detected in four patients, whereas the remaining patients were co‐infected with respiratory syncityal virus (three patients) or adenovirus (one patient). The results suggest that WU/KIPyVs have a limited circulation in Italy and a low pathogenic potential in young children. Brief and asymptomatic infection can occur in hematopoietic transplant recipients. J. Med. Virol. 82:153–156, 2010.

Epidemic Diffusion of OXA-23-Producing Acinetobacter baumannii Isolates in Italy: Results of the First Cross-Sectional Countrywide Survey

ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAb) is emerging worldwide as a public health problem in various settings. The aim of this study was to investigate the prevalence of CRAb isolates in Italy and to characterize their resistance mechanisms and genetic relatedness. A countrywide cross-sectional survey was carried out at 25 centers in mid-2011. CRAb isolates were reported from all participating centers, with overall proportions of 45.7% and 22.2% among consecutive nonreplicate clinical isolates of A. baumannii from inpatients (n = 508) and outpatients (n = 63), respectively. Most of them were resistant to multiple antibiotics, whereas all remained susceptible to colistin, with MIC50 and MIC90 values of ≤0.5 mg/liter. The genes coding for carbapenemase production were identified by PCR and sequencing. OXA-23 enzymes (found in all centers) were by far the most common carbapenemases (81.7%), followed by OXA-58 oxacillinases (4.5%), which were found in 7 of the 25 centers. In 6 cases, CRAb isolates carried both bla OXA-23-like and bla OXA-58-like genes. A repetitive extragenic palindromic (REP)-PCR technique, multiplex PCRs for group identification, and multilocus sequence typing (MLST) were used to determine the genetic relationships among representative isolates (n = 55). Two different clonal lineages were identified, including a dominant clone of sequence type 2 (ST2) related to the international clone II (sequence group 1 [SG1], SG4, and SG5) and a clone of ST78 (SG6) previously described in Italy. Overall, our results demonstrate that OXA-23 enzymes have become the most prevalent carbapenemases and are now endemic in Italy. In addition, molecular typing profiles showed the presence of international and national clonal lineages in Italy.

Emergence and spread of a multidrug-resistant Acinetobacter baumannii clone producing both the carbapenemase OXA-23 and the 16S rRNA methylase ArmA

Acinetobacter baumannii is a Gram-negative organism reported worldwide as a cause of health-care-associated infections, particularly in intensive care units (ICUs). The aim of this study is to describe the emergence and spread of carbapenem-resistant A. baumannii (CRAB) isolates in hospitalized patients. From March to November 2009, multidrug-resistant CRAB isolates were obtained from 21 patients hospitalized in different wards (mostly ICUs). Antimicrobial susceptibility was determined by using the Etest method. Carbapenem and aminoglycoside resistance determinants were studied by PCR and sequencing. Genetic relatedness was investigated by pulsed-field gel electrophoresis and multiplex PCR identification of sequence groups. Clinical records of patients were examined retrospectively. CRAB isolates were consistently resistant to multiple drugs including fluoroquinolones and aminoglycosides, whereas they retained a susceptibility to colistin. Molecular analysis revealed that 19 of the 21 CRAB isolates belonged to a single clone producing both the carbapenemase OXA-23 and the 16S rRNA methylase ArmA. Based on clinical data, the patients included in the study were classified as infected (n=13) or colonized (n=8). Colistin alone or in combination with ampicillin-sulbactam was administered to 11 of the 13 infected patients. A complete or partial response was obtained in eight cases, whereas a failure to respond was observed in one patient and a relapse was observed in two patients. An A. baumannii clone producing both OXA-23 and ArmA has been identified as an emerging and rapidly spreading pathogen. To our knowledge, this is the first report of the ArmA enzyme in A. baumannii in Italy and is the first report of hospital dissemination of A. baumannii carrying both bla(OXA-23) and armA genes.

Characterization of resistance mechanisms and genetic relatedness of carbapenem-resistant Acinetobacter baumannii isolated from blood, Italy☆

The aim of this study was to characterize the resistance mechanisms and genetic relatedness of 21 carbapenem-resistant Acinetobacter baumannii blood isolates collected in Italy during a 1-year multicenter prospective surveillance study. Genes coding for carbapenemase production were identified by polymerase chain reaction (PCR) and sequencing. Pulsed-field gel electrophoresis (PFGE), multiplex PCRs for group identification, and multilocus sequence typing (MLST) were used to determine genetic relationships. Carbapenem resistance was consistently related to the production of oxacillinases, mostly the plasmid-mediated OXA-58 enzyme. Strains producing the OXA-23 enzyme (chromosomally mediated) were also detected. Seven PFGE clones were identified, some of which being related to international (ICL- I and ICL-II) or national clonal lineages. Multiplex PCRs identified 4 different groups (group 2 being dominant), further distinguishable in 6 sequence types by MLST. The heterogeneity of profiles highlights the diffusion of international and national clonal lineages in Italy. Continuous surveillance is needed for monitoring the spread of these worrisome strains equipped with multiple drug resistance mechanisms.

Evolving beta-lactamase epidemiology in Enterobacteriaceae from Italian nationwide surveillance, October 2013: KPC-carbapenemase spreading among outpatients

Extended-spectrum beta-lactamases (ESBLs), AmpC-type beta-lactamases (ACBLs) and carbapenemases are among the most important resistance mechanisms in Enterobacteriaceae. This study investigated the presence of these resistance mechanisms in consecutive non-replicate isolates of Escherichia coli (n = 2,352), Klebsiella pneumoniae (n = 697), and Proteus mirabilis (n = 275) from an Italian nationwide cross-sectional survey carried out in October 2013. Overall, 15.3% of isolates were non-susceptible to extended-spectrum cephalosporins but susceptible to carbapenems (ESCR-carbaS), while 4.3% were also non-susceptible to carbapenems (ESCR-carbaR). ESCR-carbaS isolates were contributed by all three species, with higher proportions among isolates from inpatients (20.3%) but remarkable proportions also among those from outpatients (11.1%). Most ESCR-carbaS isolates were ESBL-positive (90.5%), and most of them were contributed by E. coli carrying blaCTX-M group 1 genes. Acquired ACBLs were less common and mostly detected in P. mirabilis. ESCR-carbaR isolates were mostly contributed by K. pneumoniae (25.1% and 7.7% among K. pneumoniae isolates from inpatients and outpatients, respectively), with blaKPC as the most common carbapenemase gene. Results showed an increasing trend for both ESBL and carbapenemase producers in comparison with previous Italian surveys, also among outpatients.