From April 1996 to July 2004, an outbreak of metallo-beta-lactamase-positive (MBL) Pseudomonas aeruginosa occurred in the haematology ward at Nantes University Hospital in France. Fifty-nine patients were carriers of VIM-2-positive strains of whom 14 were infected (mostly urinary tract infections and pneumonia). Pulsed-field gel electrophoresis identified related isolates demonstrating resistance to all beta-lactams, aminoglycosides, fluoroquinolones, fosfomycin, rifampicin but not colistin. The bla(VIM-2) gene responsible for VIM-2 MBL was not plasmid-encoded but part of a novel type of class 1 integron. VIM-2-positive strains were mostly from urine samples and clinical data suggest that in the absence of therapeutic guidelines, piperacillin-tazobactam or aztreonam may be a reliable choice for treating infections with MBL-producing strains.

Objective: To develop an effective outbreak-control strategy by identifying the source and modes of transmission of Staphylococcus capitis in a 60-bed neonatal intensive care unit ( NICU).Study Design: We conducted a study among neonates hospitalized during the outbreak ( June 2000 through November 2003). All cases of S. capitis colonization or infection detected by clinical samples during the outbreak were included. The molecular analysis of the isolated was assessed by pulsed-field electrophoresis. We reported the description of the outbreak and the measures taken during this investigation.Result: Thirty-three patients were colonized or infected by S. capitis. Mean gestational age was 28.5 +/- 4.4 weeks of gestation, mean birth weight was 1068 +/- 637.3 g and the mean length of hospital stay was 77.9 +/- 35.9 days. We observed that positive S. capitis cultures were over-represented in six beds of the NICU. Because S. capitis is known to thrive in lipid media, we cultured samples from the almond oil bottles assigned to these beds. S. capitis strain recovered from one of the almond oil sample was genetically identical to the strain recovered from the cases.Conclusion: Almond oil is an unusual reservoir infection. Control policy allowed prompt institution of measures that were successful in ending the outbreak.

Polyclonal Escherichia coli strains were isolated in a transplanted patient who experienced successive septic shocks. Fluoroquinolone susceptible and resistant strains were corresponding to different PFGE fragment profiles. The gyrA S83L mutation was associated with a reduction in biological fitness. Resistant strain was selected by a long-term single use of ofloxacin. (c) 2006 Elsevier Masson SAS. All rights reserved.

The prospective cohort study presented here assessed the risk factors associated with Pseudomonas aeruginosa gastrointestinal colonization (PAGIC) in 933 patients hospitalized in five different wards in a French university hospital. A total of 195 patients were colonized. By logistic regression, hospitalization in an intensive care unit and length of hospital stay were independent risk factors. A significant association was observed between fluoroquinolone use and PAGIC caused by an ofloxacin-resistant strain (p<0.0001), imipenem use and PAGIC caused by an imipenem-resistant strain (p<0.0002) and ceftazidime use and PAGIC caused by a ceftazidime-resistant strain (p<0.02). The ecological impact of antibiotic use is of great clinical relevance and clinicians should consider antimicrobial resistance in order to limit the development and dissemination of resistant microorganisms.

This study assessed the incidence of gastrointestinal colonisation by resistant Enterobacteriaceae among hospitalised patients, and identified risk-factors for ceftazidime and ofloxacin resistance. A prospective cohort study was performed in five wards in a French teaching hospital during a 2-year period. Patients hospitalised for > 48 h were enrolled between 17 April 2000 and 30 April 2002. A rectal swab was taken at admission, then once-weekly and/or on the day of discharge. In total, 933 patients were investigated and 585 amoxycillin-resistant isolates were obtained. Resistance rates for ceftazidime and ofloxacin were 9.4% and 4.8%, respectively. Multivariate analysis indicated that previous hospitalisation (p < 0.004) and exposure to amoxycillin-clavulanate (p < 0.003) and ceftriaxone (p < 0.002) were associated significantly with ceftazidime resistance. Hospitalisation in the urology ward (p < 0.02) and previous exposure to fluoroquinolones (p < 0.01) were the two independent risk-factors associated with ofloxacin resistance. The results of the study confirmed that antibiotic use selected resistant Enterobacteriaceae from the gut flora. Resistance was observed mostly in patients with previous antibiotic exposure and previous hospitalisation in wards with a high antibiotic selection pressure.

The clinical significance of coagulase-negative staphylococci isolated from blood culture is typically assessed on the basis of a combination of clinical and microbiological criteria. However, these criteria are difficult to apply to haematology patients who are highly immunosuppressed and from whom blood cultures are obtained most frequently through a central venous catheter. This study analysed 112 episodes of Staphylococcus epidermidis bacteraemia that occurred in 79 bone marrow transplant recipients. In 73 (65%) episodes, only one blood culture set was positive for S. epidermidis, while 39 (35%) episodes grew S. epidermidis from multiple blood cultures. Nine patients had two or more episodes of bacteraemia with the same strain, as determined by pulsed-field gel electrophoresis (PFGE). The PFGE method also showed that 34 (31%) isolates belonged to seven clusters, indicating the persistence of certain clones in the environment. Of the 109 isolates analysed, 59 (54%) produced biofilm and 91 (83.5%) carried the ica operon. Isolates that produced biofilm were observed to colonise central venous catheters faster than non-biofilm-producing isolates (18 vs. 37 days; p 0.03). No clinical features were associated with carriage of the ica operon, but the ica operon was carried more frequently by the isolates that formed clusters.

To understand the regulation of the MexAB OprM efflux system in a clinical strain of Pseudomonas aeruginosa presenting a decreased susceptibility to ticarcillin and aztreonam, the mexR repressor gene was amplified by polymerase chain reaction (PCR) and was shown to be disrupted by an insertion sequence of more than 2 kb, with characteristic direct and inverted repeat sequences. Sequencing revealed a 2131-bp IS21 insertion sequence. A reverse transcription PCR method was used to quantify mexA transcripts and showed an increased transcription rate of mexA in this strain, compared with a PAO1 control strain. The nalB phenotype in P. aeruginosa may be due to point mutations, but also to the presence of an insertion sequence in the mexR regulator gene.

In Escherichia coli, beta-lactam resistance usually depends on beta-lactamase production. AmpC chromosomal cephalosporinase hyperproduction is generally due to mutations in the ampC gene promoter. In order to study ampC expression in E coli clinical strains, we have compared two methods: conventional and real-time reverse transcription-polymerase chain reaction (RT-PCR). With both methods, ampC mRNA was found to be greatly increased in strains presenting -42 or -32 mutations in the ampC promoter, and moderately increased when a -11 mutation was present in the Pribnow box. Real-time RT-PCR represents a powerful tool combining amplification, fluorescent detection and analysis. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

An environmental investigation and a cohort study were carried out to analyse an outbreak of infection caused by a serotype O10 Pseudomonas aeruginosa in a neonatal intensive care unit. Thirty one cases of infection were recorded, including four lethal ones. The outbreak was stopped by eradicating the environmental sources: a contaminated milk bank pasteuriser and bottle warmer.

OBJECTIVE:To compare the genetic environments of ampC genes in different Acinetobacter baumannii isolates showing different levels of beta-lactam resistance. METHODS:The patterns of beta-lactam resistance and beta-lactamase production were investigated for 42 A. baumannii clinical strains. The MICs of various beta-lactams were determined in the presence or absence of the class C cephalosporinase inhibitor, cloxacillin (500 mg/L). The ampC gene and its 5' adjacent sequence were analysed by PCR and DNA sequencing. An RT-PCR method was developed to evaluate ampC transcript levels. RESULTS:Strains fell into three resistance groups: first, strains with a ceftazidime MIC < or =8 mg/L (20 strains, 47.6%); secondly, strains with a ceftazidime MIC 32 mg/L, which was reduced four-fold in the presence of cloxacillin (eight strains, 19%); and thirdly, strains with a ceftazidime MIC > or =256 mg/L, which did not decrease in the presence of cloxacillin (14 strains, 33.4%). In all of the resistant isolates (groups II and III), but not in any of the ceftazidime-susceptible isolates (group I), a 1180 bp insert showing all the characteristics of an insertion sequence was detected upstream from the ampC gene. Isolates having this insert overexpress ampC, according to RT-PCR experiments. CONCLUSION:Presence of an insertion sequence upstream of ampC in A. baumannii clinical isolates, possibly including a strong promoter, has the potential to cause over-expression of AmpC, resulting in high-level ceftazidime resistance.

Streptococcus salivarius meningitis are uncommon and often iatrogenic. We describe the case of a patient with community-acquired Streptococcus salivarius meningitis that revealed petrous, apex cholesteatoma. A 50-years old patient was hospitalized because of severe headache, fever (39 degreesC), vomiting, and confusion. Cerebrospinal fluid (CSF) showed 2044 cells/mm(3) with 91% neutrophils, Gram positive cocci, protein level > 2.5g/L, glucose level 2.5 mmol/L (glycemia 7.1 mmol/L). Two blood cultures and CSF culture were positive with Streptococcus salivarius. Evolution was good with amoxicillin treatment. The patient had rhinorrhea and chronic otitis. Cholesteatoma was revealed in a scanner, and was opered. Community-acquired Streptococcus salivarius meningitis are unfrequent, and can reveal ENT pathology like cholesteatoma. (C) 2002 Editions scientifiques et medicales Elsevier SAS.

ABSTRACT A mutation was discovered in the Pribnow box of the ampC promoter in a clinical Escherichia coli strain. This −11 C-to-T transition created a perfect homology with the −10 consensus sequence. The new promoter was cloned upstream of the cat gene of pKK232-8 and induced a sixfold increase in promoter strength.

We assessed the risk factors for acquisition of fluoroquinolone-resistant, gram-negative organisms in the gastrointestinal tract of hospitalized patients. We analyzed stool samples from 204 patients and recovered fluoroquinolone-resistant, gram-negative organisms from 63. Receipt of fluoroquinolone during the month preceding admission was the only risk factor identified, whereas female sex, duration of hospitalization, exposure to indwelling devices, admission from another hospital, and history of infection were risk factors for fecal colonization after day 4.

Escherichia coli usually produces only very small amounts of a constitutive AmpC beta-lactamase, but clinical strains overproducing this enzyme have been isolated. Three different ampC promoters of E. coli clinical strains were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene in the pKK232-8 reporter plasmid and their relative strengths were compared by two different methods. The strength of the promoters from AmpC hyperproducers was 70- to 120-fold higher than those from a low-level AmpC producer. One of the strong promoters, which differs from strain K12 at bases -88, -82, -42, -18, -1 and +58, was mutated to abolish the -42 mutation. This change resulted in a 43-fold decrease in CAT concentration. In another promoter, with eight different mutations at positions -88, -82, -32, -18, -1, +5, +24 and +58, the -32T-->A transversion, which created perfect homology with the -35 consensus sequence, was reverted; this led to a 13-fold decrease in CAT concentration. The -42 and -32 mutations play an important role in E. coli resistance to beta-lactams by increasing ampC transcription.

Escherichia coli strains showing an increased resistance to oxyiminocephalosporins without an extended spectrum β-lactamase production have been screened for mutations in their ampC β-lactamase gene promoter. Mutations were found by direct sequencing of seven promoters at positions −42, −32 (box −35), −18, −1, +5, +24 (attenuator), +31 (attenuator) and +58. By using rapid and simple methods, three of these mutations (−42, −32 and +24), which could enhance transcription, were searched in 37 resistant and 25 sensitive isolates. The −42 mutation was present in 33 of the 37 promoters from the resistant isolates. The −32 and +24 mutations were present only in three and two promoters, respectively, and they were combined in the most resistant strain of the study. The +24 mutation was detected in another strain associated with a 1-bp insertion between the −35 and −10 conserved sequences. None of these mutations was detected in the ampC β-lactamase from the sensitive isolates.

Background: Acinetobacter baumannii is an important opportunistic pathogen that is rapidly evolving toward multidrug resistance and is involved in various nosocomial infections that are often severe. It is difficult to prevent A. baumannii infection because A. baumannii is ubiquitous and the epidemiology of the infections it causes is complex.Objective: To study the epidemiology of A. baumannii infections and assess the relation between fluoroquinolone use and the persistence of multidrug-resistant clones.Design: Three case-control studies and a retrospective cohort study.Setting: A 20-bed medical and surgical intensive care unit.Patients: Acinetobacter baumannii was isolated from 45 patients in urine (31%), the lower respiratory tract (26.7%), wounds (17.8%), blood (11.1%), skin (6.7%), cerebrospinal fluid (4.4%), and sinus specimens (2.2%). One death was due to A. baumannii infection.Measurements: Antimicrobial resistance pattern and molecular typing were used to characterize isolates. The incidence of A. baumannii infection and the use of fluoroquinolones were calculated annually.Results: Initially, 28 patients developed A. baumannii infection. Eleven isolates had the same antimicrobial susceptibility profile, genotypic profile, or both (epidemic cases), and 17 were heterogeneous (endemic cases). A surgical procedure done in an emergency operating room was the main risk factor for epidemic cases, whereas previous receipt of a fluoroquinolone was the only risk factor for endemic cases. The opening of a new operating room combined with the restriction of fluoroquinolone use contributed to a transitory reduction in the incidence of infection. When a third epidemiologic study was done, previous receipt of a fluoroquinolone was again an independent risk factor and a parallel was seen between the amount of intravenous fluoroquinolones prescribed and the incidence of endemic infection.Conclusion: Epidemic infections coexisted with endemic infections favored by the selection pressure of intravenous fluoroquinolones.

Rahnella aquatilis was isolated from the blood cultures of two patients who were in different units of the same hospital. Both isolates were susceptible to aminoglycosides, fluoroquinolones, cotrimoxazole, piperacillin, third generation cephalosporins and amoxicillin-clavulanate, but resistant to amoxicillin, ticarcillin, and first generation cephalosporins. The synergistic activity of amoxicillin and clavulanic acid suggested the presence of aΒ-lactamase, confirmed by a positive nitrocefin test and by analytical isoelectric focusing. Pulsed-field gel electrophoresis and ribotyping with the pKK3535 probe showed that the isolates shared the same banding pattern. The results of an epidemiological study suggested that an in-house total parenteral nutrition solution might be the source of this unusual gram-negative rod.

Accompanying increased commercial applications and production of silver nanomaterials is an increased probability of human exposure, with inhalation a key route. Nanomaterials that deposit in the pulmonary alveolar region following inhalation will interact firstly with pulmonary surfactant before they interact with the alveolar epithelium. It is therefore critical to understand the effects of human pulmonary surfactant when evaluating the inhalation toxicity of silver nanoparticles. In this study, we evaluated the toxicity of AgNPs on human alveolar type-I-like epithelial (TT1) cells in the absence and presence of Curosurf® (a natural pulmonary surfactant substitute), hypothesising that the pulmonary surfactant would act to modify toxicity. We demonstrated that 20 nm citrate-capped AgNPs induce toxicity in human alveolar type I-like epithelial cells and, in agreement with our hypothesis, that pulmonary surfactant acts to mitigate this toxicity, possibly through reducing AgNP dissolution into cytotoxic Ag+ ions. For example, IL-6 and IL-8 release by TT1 cells significantly increased 10.7- and 35-fold, respectively (P < 0.01), 24 h after treatment with 25 μg/ml AgNPs. In contrast, following pre-incubation of AgNPs with Curosurf®, this effect was almost completely abolished. We further determined that the mechanism of this toxicity is likely associated with Ag+ ion release and lysosomal disruption, but not with increased reactive oxygen species generation. This study provides a critical understanding of the toxicity of AgNPs in target human alveolar type-I-like epithelial cells and the role of pulmonary surfactant in mitigating this toxicity. The observations reported have important implications for the manufacture and application of AgNPs, in particular for applications involving use of aerosolised AgNPs.