Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like ß-Lactamase.

OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most ß-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of blaOXA-535 and blaOXA-436 genetic environments suggests that an ISCR1 may be responsible for blaOXA-436 gene mobilization from the chromosome of Shewanella spp. to plasmids.

Inventory of Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae in France as Assessed by a Multicenter Study.

The objective of this study was to perform an inventory of the extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae isolates responsible for infections in French hospitals and to assess the mechanisms associated with ESBL diffusion. A total of 200 nonredundant ESBL-producing Enterobacteriaceae strains isolated from clinical samples were collected during a multicenter study performed in 18 representative French hospitals. Antibiotic resistance genes were identified by PCR and sequencing experiments. The clonal relatedness between isolates was investigated by the use of the DiversiLab system. ESBL-encoding plasmids were compared by PCR-based replicon typing and plasmid multilocus sequence typing. CTX-M-15, CTX-M-1, CTX-M-14, and SHV-12 were the most prevalent ESBLs (8% to 46.5%). The three CTX-M-type EBSLs were significantly observed in Escherichia coli (37.1%, 24.2%, and 21.8%, respectively), and CTX-M-15 was the predominant ESBL in Klebsiella pneumoniae (81.1%). SHV-12 was associated with ESBL-encoding Enterobacter cloacae strains (37.9%). qnrB, aac(6')-Ib-cr, and aac(3)-II genes were the main plasmid-mediated resistance genes, with prevalences ranging between 19.5% and 45% according to the ESBL results. Molecular typing did not identify wide clonal diffusion. Plasmid analysis suggested the diffusion of low numbers of ESBL-encoding plasmids, especially in K. pneumoniae and E. cloacae However, the ESBL-encoding genes were observed in different plasmid replicons according to the bacterial species. The prevalences of ESBL subtypes differ according to the Enterobacteriaceae species. Plasmid spread is a key determinant of this epidemiology, and the link observed between the ESBL-encoding plasmids and the bacterial host explains the differences observed in the Enterobacteriaceae species.

Long-term control of vancomycin-resistant Enterococcus faecium at the scale of a large multihospital institution: a seven-year experience.

Repeated outbreaks of vancomycin-resistant Enterococcus faecium (VRE) occurred between 2004 and 2010 in Assistance Publique--Hôpitaux de Paris (AP-HP), a 23,000-bed multi-hospital institution. From August 2004 to December 2005, the French guidelines for preventing cross-transmission of multiresistant bacteria were applied. Because the number of VRE cases continued to increase, an institutional control programme was implemented from January 2006 onwards: it foresees stopping transfer of VRE and contact patients, separating VRE and contact patients in distinct cohorts, intervention of a central infection control team to support local teams, and quick application of measures as soon as first VRE cases are identified. Between August 2004 and December 2010, 45 VRE outbreaks occurred in 21 of the 38 AP-HP hospitals, comprising 533 cases. Time series analysis showed that the mean number of cases increased by 0.8 cases per month (95% confidence interval (CI): 0.3 to 1.3, p=0.001) before, and decreased by 0.7 cases per month after implementation of the programme (95% CI: -0.9 to -0.5, p<0.001), resulting in a significant trend change of -1.5 cases per month (95% CI: -2.1 to -0.9, p<0.001). The number of cases per outbreak was significantly lower after implementation of the programme. A sustained and coordinated strategy can control emerging bacteria at the level of a large regional multihospital institution.

Control of a multi-hospital outbreak of KPC-producing Klebsiella pneumoniae type 2 in France, September to October 2009.

An outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae type 2 was detected in September 2009 in two hospitals in a suburb south of Paris, France. In total, 13 KPC-producing K. pneumoniae type 2 cases (four with infections and nine with digestive-tract colonisations) were identified, including a source case transferred from a Greek hospital. Of the 13 cases, seven were secondary cases associated with use of a contaminated duodenoscope used to examine the source case (attack rate: 41%) and five were secondary cases associated with patient-to-patient transmission in hospital. All isolated strains from the 13 patients: (i) exhibited resistance to all antibiotics except gentamicin and colistin, (ii) were more resistant to ertapenem (minimum inhibitory concentration (MIC) always greater than 4 mg/L) than to imipenem (MIC: 1­8 mg/L, depending on the isolate), (iii) carried the blaKPC-2 and blaSHV12 genes and (iv) had an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern. These cases occurred in three hospitals: some were transferred to four other hospitals. Extended infection control measures implemented in the seven hospitals included: (i) limiting transfer of cases and contact patients to other wards, (ii) cohorting separately cases and contact patients, (iii) reinforcing hand hygiene and contact precautions and (iv) systematic screening of contact patients. Overall, 341 contact patients were screened. A year after the outbreak, no additional case has been identified in these seven hospitals. This outbreak emphasises the importance of rapid identification and notification of emerging highly resistant K. pneumoniae strains in order to implement reinforced control measures.

[Cat scratch disease with bone involvement: a case report and literature review]. / Maladie des griffes du chat avec localisations osseuses: une observation et revue de la littérature.

INTRODUCTION: Cat scratch disease is an infectious disease caused by Bartonella henselae. Most of the patients present with a lymphadenopathy associated with a local infection at the site of the cat scratch. Disseminated infection is uncommon. CASE REPORT: We report an immunocompetent 61-year-old woman who presented with a systemic cat scratch disease including a multifocal osteomyelitis. Diagnosis was confirmed by PCR on the adenopathy. A literature review identified 51 other cases of osteomyelitis associated with cat scratch disease, 14 of those confirmed by PCR. CONCLUSION: Bone involvement in cat scratch disease is rare, especially in adults. The diagnosis should be suspected on the basis of patient questioning. The antibiotherapy and the place of surgery are discussed.

Spread of novel expanded-spectrum beta-lactamases in Enterobacteriaceae in a university hospital in the Paris area, France.

In 2002, 28 non-duplicate enterobacterial isolates producing extended-spectrum beta-lactamases (ESBLs) were collected from infected patients at the Bicêtre Hospital in Paris, France. Escherichia coli was the predominant ESBL-positive enterobacterial species, comprising ten (36%) of the isolates. CTX-M enzymes (CTX-M-3, CTX-M-10, CTX-M-14 and CTX-M-15) were produced by 11 (39%) of the isolates (six E. coli, two Enterobacter cloacae, one Enterobacter aerogenes, one Proteus mirabilis and one Citrobacter freundii). Other ESBLs, such as VEB-1 and PER-1, were also detected, but less frequently.

Three-year survey of community-acquired methicillin-resistant Staphylococcus aureus producing Panton-Valentine leukocidin in a French university hospital.

A retrospective survey was conducted at Bicêtre Hospital, France from January 2001 to September 2003 to screen for S. aureus isolates with a typical phenotype previously involved in necrotizing pneumonia in France. They were resistant to oxacillin and kanamycin, of intermediate susceptibility to fusidic acid, and susceptible to tobramycin and fluoroquinolones. Seventeen isolates were found and 16 were viable. The Panton-Valentine leukocidin (PVL) genes, various toxin genes and SCCmec IV and agr3 alleles were detected in all isolates. The clonal origin of these isolates was demonstrated by pulsed-field gel electrophoresis. Fourteen isolates were community-acquired methicillin-resistant Staphylococcus (CA-MRSA) isolated from previously healthy patients with skin or soft tissue infections. Three infections were of nosocomial origin, underlining that these PVL-producing CA-MRSA strains may also be hospital acquired. Five CA-MRSA isolates with an identical resistance phenotype collected in a neighbouring teaching hospital (Hôpital Pitié-Salpétrière, Paris, France) were also PVL positive. Three isolates were clonally related to those of the Bicêtre Hospital whereas two were not. This retrospective study identified PVL-producing CA-MRSA in two Parisian hospitals. The incidence at Bicêtre Hospital was 0.8% of all S. aureus and 2% of all MRSA isolated. Our data indicate that these MRSA isolates might become hospital acquired.

Nosocomial outbreak of staphylococcal scalded skin syndrome in neonates: epidemiological investigation and control.

Over a three-month period, 13 neonates developed staphylococcal scalded skin syndrome (SSSS) in a maternity unit, between four and 18 days after their birth. An epidemiological and descriptive study followed by a case-control study was performed. A case was defined as a neonate with blistering or peeling skin, and exfoliative toxin A Staphylococcus aureus positive cultures. Controls were selected at random from the asymptomatic, non-colonized neonates born on the same day as the cases. All staff members and all neonates born during the outbreak period were screened for carriage by nasal swabs and umbilical swabs, respectively. S. aureus isolates were polymerase chain reaction (PCR) screened for etA gene and genotyped by pulsed-field gel electrophoresis (PFGE). Two clusters of eight and five cases were identified. Receiving more than one early umbilical care procedure by the same ancillary nurse was the only risk factor identified in the case-control study (odds ratio=15, 95% confidence intervals 2-328). The ancillary nurse suffered from chronic dermatitis on her hands that favoured S. aureus carriage. Exfoliative-toxin-A-producing strains, as evidenced by PCR and indistinguishable by PFGE, were isolated from all but one of the SSSS cases, from four asymptomatic neonates, from two staff members and from the ancillary nurse's hands. Removal of the ancillary nurse from duty, infection control measures (isolation precautions, chlorhexidine handwashing and barrier protections), and treatment of the carriers (nasal mupirocin and chlorhexidine showers) led to control of the epidemic. In conclusion, this study emphasizes the need for tight surveillance of chronic dermatitis in healthcare workers.

Interaction between roxithromycin and cyclosporin in heart transplant patients.

Cyclosporin is an immunosuppressive agent commonly used in transplant patients. It is actively metabolised by the cytochrome P450 system and interactions with drugs metabolised by the same system are predictable. This is particularly relevant since cyclosporin has a low therapeutic index and its renal toxicity is concentration-related. Roxithromycin, a new, well-tolerated macrolide with a weak interactive profile, uses the same isoenzyme of the P450 system as cyclosporin. To evaluate its interaction potential in clinical practice, 8 heart transplant recipients treated with cyclosporin for at least 1 month received roxithromycin for 11 days (150 mg twice daily). Bi-weekly controls of plasma cyclosporin concentrations and creatinine levels were carried out before, during and after roxithromycin treatment. A slight nonsignificant rise in cyclosporin concentrations was observed, but creatinine levels remained stable during roxithromycin treatment. Values of cyclosporin concentrations diminished after withdrawal of roxithromycin. Cyclosporin dosage adjustment was not necessary. There was a minor pharmacokinetic interaction, which can be considered safe for the usual therapeutic dosage of roxithromycin used.

[Critical analysis of drug interactions with cyclosporine. Evaluation of interactions with cyclosporine]. / Analyse critique des interactions médicamenteuses de la ciclosporine. Evaluation des interactions de la ciclosporine.

Cyclosporine A is a potent immunosuppressive agent, widely used in organ transplantation, in bone marrow transplantation and in the treatment of some autoimmune diseases. Changes of its absorption, a metabolism mainly processed by the liver and a concentration-related nephrotoxicity lead to the need of a careful drug monitoring, allowing to obtain blood levels that must be low and nevertheless sufficiently efficient. Cyclosporin A may additionally yield some numerous drug interactions. Those with potentially serious issue must be mandatory avoided and distinguished from those less severe that only have to be followed up. The strategy differs according to the nature of the interaction (i.e. pharmacokinetic/pharmacodynamic): the posology will have either to be adjusted or the risk/benefit ratio will have to be taken into account to decide any change in the dosage regimen.

Plasmid-mediated and inducible cephalosporinase DHA-2 from Klebsiella pneumoniae.

A Klebsiella pneumoniae strain resistant to cefoxitin and oxyimino-cephalosporins was cultured from a child hospitalized in Paris, France, in 1992. This isolate harboured a beta-lactamase gene located on an approximately 200 kb non-self-transferable plasmid. The beta-lactamase identified, DHA-2, shared 99% amino acid identity with the AmpC enzyme of Morganella morganii. DHA-2 was a point-mutant derivative of DHA-1 identified previously in a Salmonella enteritidis isolate. DHA-2 expression was inducible due to an ampR regulatory gene. This is the first report of an inducible and plasmid-located cephalosporinase from K. pneumoniae.

Immunization with live aroA recombinant Salmonella typhimurium producing invasin inhibits intestinal translocation of Yersinia pseudotuberculosis.

The Yersinia pseudotuberculosis inv gene encodes invasin, a 103-kDa outer membrane protein that allows bacteria to enter mammalian cells. The gene was subcloned into the attenuated aroA mutant of Salmonella typhimurium SL3261. Invasin was produced by the recombinant Salmonella strain and increased the ability of microorganisms to translocate from the intestinal lumen to the mesenteric lymph nodes. Specific antibodies for invasin were detected in sera and intestinal secretions of mice following oral immunization with the live Inv+ Salmonella strain. The immunization strongly inhibited intestinal translocation of Y. pseudotuberculosis when this pathogen was inoculated to mice but failed to prevent Yersinia dissemination from the gut lymphoid tissue.

SHV-type extended-spectrum beta-lactamase in a Shigella flexneri clinical isolate.

A Shigella flexneri isolate resistant to oxyimino-cephalosporins was recovered from a stool sample of a 16 month-old Algerian child hospitalized in Paris, France. This isolate harboured an SHV-2 beta-lactamase gene located on a c. 80 kb self-transferable plasmid. This is the first report of an Ambler class A extended-spectrum beta-lactamase from Shigella spp.

Lack of antibody response to invasin in humans with yersiniosis.

The Yersinia pseudotuberculosis inv gene encodes invasin, a 103-kDa outer membrane protein allowing bacteria to penetrate mammalian cells. This protein is produced in vitro at below 30 degrees C. In this work, we studied the antibody response against invasin in humans suffering from yersiniosis and in mice orally infected with a virulent strain of Y. pseudotuberculosis. Infection with enteropathogenic Yersinia strains did not induce either a systemic or a gut antibody response to invasin. Our results suggest that the inv gene is not expressed in the gut at 37 degrees C and, therefore, that invasin is not present to the immune system when microorganisms multiply in the host tissues.

Evaluation of CHROMagar Staph. aureus, a new chromogenic medium, for isolation and presumptive identification of Staphylococcus aureus from human clinical specimens.

CHROMagar Staph. aureus (CSA) is a new chromogenic medium for presumptive identification of Staphylococcus aureus as mauve colonies after 24 h of incubation. We conducted a preliminary study with 100 S. aureus and 45 coagulase-negative Staphylococcus (CoNS) stock isolates plated on CSA. All S. aureus isolates yielded mauve colonies after 24 h of incubation at 37 degrees C, while CoNS isolates grew as blue, white, or beige colonies. Culture on CSA was then prospectively compared to a conventional laboratory method, i.e. , culture on 5% horse blood agar (HBA), catalase test, and latex agglutination test (HBA-catalase-latex), for isolation and presumptive identification of S. aureus from 2,000 consecutive clinical samples. Among the 310 S. aureus isolates recovered by at least one of the two methods, 296 grew as typical mauve colonies on CSA, while only 254 yielded catalase-positive, latex-positive colonies on HBA. The sensitivity of CSA was significantly higher than that of the conventional method (95.5 and 81.9%, respectively; P < 0.001) and allowed the recovery of important clinical isolates that were undetected on blood agar. The specificities of the two methods were not significantly different, although that of CSA was slightly higher (99.4% versus 98.9% for HBA-catalase-latex; P = 0. 08). On the basis of its excellent sensitivity and specificity, ease of identification of positive colonies, and absence of complementary testing, CSA can be recommended as a routine plating medium for presumptive identification of S. aureus in clinical specimens.

Invasin production by Yersinia pestis is abolished by insertion of an IS200-like element within the inv gene.

The two enteropathogens Yersinia pseudotuberculosis and Yersinia enterocolitica penetrate eukaryotic cells in vitro through invasin, a surface-exposed protein. In contrast, Yersinia pestis, the causative agent of plague, is unable to enter mammalian cell lines, although the inv gene is present on its chromosome. Although 99.3% identical to the inv gene of Y. pseudotuberculosis, the Y. pestis inv gene was disrupted in its central region by a 708-bp IS200-like element. Multiple copies of this insertion sequence element were found within the genome of the plague bacillus.

Biochemical-genetic characterization of the chromosomally encoded extended-spectrum class A beta-lactamase from Rahnella aquatilis.

From whole-cell DNA of a clinical isolate of the enterobacterial species Rahnella aquatilis, a beta-lactamase gene was cloned that encoded a chromosomally encoded Ambler class A enzyme, RAHN-1. RAHN-1, with a pI of 7.2, shares 76, 73, and 71% amino acid identity with the extended-spectrum beta-lactamase of chromosomal origin from Serratia fonticola and with the plasmid-mediated beta-lactamases CTX-M-2 and CTX-M-1, respectively. The hydrolysis spectrum of the clavulanic acid-inhibited RAHN-1 was expanded to cephalosporins such as cefuroxime, cefotaxime, and ceftriaxone, but not to ceftazidime. Its expression was not inducible.