[MALDI-TOF mass spectrometry to identify clinical bacterial isolates: evaluation in a teaching hospital in Lille]. / Identification bactérienne par spectrométrie de masse de type MALDI-TOF: évaluation au CHU de Lille.

PURPOSE: The aim of our study was to evaluate the capacity of MALDI-TOF mass spectrometry to identify clinical bacterial isolates, as compared to the automated identification system Vitek 2 (bioMérieux) used routinely in a teaching hospital. METHODS: Three hundred and sixty-two strains representing 178 species from the laboratory collection were analysed by a Microflex spectrometer (Bruker Daltonics) and Vitek 2. Discrepancies between MALDI-TOF and Vitek 2 identifications were investigated by genetic identification (rrS, sodA, rpoB), considered as a reference. RESULTS: Among the 362 isolates, 264 (73%) were consistently identified by Vitek 2 and Microflex. Taking into account genetic identification, we found that 44 (44.9%) of the 98 remaining isolates were correctly identified by mass spectrometry but not by Vitek 2. Conversely, 33 isolates (33.7%) were correctly identified by Vitek 2, but not by Microflex. The genetic identification of the 21 remaining isolates (21,4%) did not match either Vitek 2 or Microflex results. CONCLUSION: The performances of MALDI-TOF mass spectrometry for bacterial identification correspond to those of a reference automated identification system.

Development of an animal model to study meshes used in genital prolapse surgery.

OBJECTIVE: The objective was to develop an animal model using bacterial inoculation to evaluate tissue integration and tolerance to meshes used in genital prolapse surgery. STUDY DESIGN: We placed three different meshes under the abdominal skin of 120 Wistar rats: a polypropylene monofilament non-coated mesh (Parietene), a polypropylene monofilament collagen-coated mesh (Ugytex) and a polyethylene terephthalate mesh (Mersuture). We performed bacterial inoculation just after implantation with 1 ml of 10(7) colonies forming unit (CFU) of Staphylococcus epidermidis or Escherichia coli. Rats were sacrificed 7, 14, 60, and 90 days after intervention. We used polarised light microscopy to analyse the collagen deposition and organisation. We quantified the inflammation cells. Bacterial analysis and quantification of the explanted meshes were performed. The exact Fisher's test and Kruskal-Wallis test were used for statistics. RESULTS: We did not find any significant difference between inoculated or non-inoculated meshes in terms of collagen deposition. The scarring process seemed stable at day 90. Tissue integration was best with the polypropylene meshes, which allowed the development of a well-organised, mature connective tissue. Inflammatory reaction was higher in inoculated meshes, but only at day 7. At day 90, we found a high number of macrophages and multinuclear cells around all the meshes. There was no significant difference between prostheses that had been inoculated and those that had not with regard to positive bacterial culture. Quantification of bacterial colonies decreased with time. CONCLUSION: In this animal model, we did not find any clinically related difference in infection and tissue integration between the meshes used in genital prolapse. Such experimental studies must be carried out whenever new prostheses become available before their use is validated in common practice.

[Spondylodiscitis due to Salmonella enteritica serotype Typhi]. / Spondylodiscite à Salmonella enteritica sérotype Typhi.

We reported a case of lombar spondylodiscitis caused by Salmonella enteritica serotype Typhi in an immunocompetent patient. Salmonella is a rare causative agent of spondylodiscitis. Early bacteriological diagnosis is essential to avoid longterm sequelae.

Automated reading of a microtitre plate: preliminary evaluation in antimicrobial susceptibility tests and Enterobacteriaceae identification.

An automated microELISA Reader was evaluated for its ability to read and interpret microtitre plates. A total of 309 microtitre plates were investigated by automated and visual methods. There was disagreement between the methods in one hundred and twelve (0.6%) wells. However agreements between the two methods for susceptibility tests and Enterobacteriaceae identification were respectively 98.8% and 89.3%.

A microcomputer system for clinical bacteriology: experience of 12 months' trial.

A data processing system using microcomputers was developed in a hospital bacteriology laboratory processing more than 60 000 specimens yearly. The purchase price of the hardware was frs 200 000 (17 500 pounds) and the software was written by the authors. The system has been running since May 1980 without general breakdown. The present configuration allows the processing of specimens, enquiries, scientific and administrative tasks but multiprogramming and cumulative reports are not possible.

Iron depletion and virulence in Staphylococcus aureus.

Staphylococcus aureus is able to grow in the presence of extremely low iron concentrations (0.04 microM). In iron-limiting conditions, this species develops alternative metabolic strategies such as highly efficient iron-uptake mechanisms which are only partially shared with S. epidermidis. Here we summarize the mechanisms induced by iron starvation in S. aureus in order to elucidate the virulence characteristics of this bacterium.

Influence of iron depletion on growth kinetics, siderophore production, and protein expression of Staphylococcus aureus.

Growth rates, siderophore secretion, and bacterial proteins of two clinical isolates of Staphylococcus aureus were studied over 72 h of growth in iron-supplemented and iron-restricted chemically defined media. Under iron restriction the growth rates were decreased to different extents depending on the strain. Production of siderophore was detected in the mid-exponential and stationary phases of growth. The expression of iron-regulated proteins of 81, 23, and 17 kDa was time-dependent, associated with the same stage of growth, and might be involved in siderophore efficiency.

Characterization of the Acinetobacter baumannii Fur regulator: cloning and sequencing of the fur homolog gene.

Growth kinetics, siderophore activity and iron-regulated bacterial proteins of Acinetobacter baumannii BM2580 were studied in iron-restricted and iron-supplemented chemically defined media. Iron-regulated outer membrane proteins of 75 kDa and 80 kDa were expressed under iron-restricted conditions. Cloning and sequencing of the complete iron-uptake regulatory (fur) gene from A. baumannii BM2580 is reported for the first time. This gene is preceded by a single autoregulated promoter whose -10 region overlaps the Fur binding site. The open reading frame identified encodes a polypeptide consisting of 145 amino acids. The fur gene is followed by a divergent open reading frame coding for the C-terminus of a putative PilU protein. Sequence analysis indicates that the Fur protein of A. baumannii was 63% identical to the Escherichia coli Fur protein.

In vitro bactericidal effect of a beta-lactam+aminoglycoside combination against multiresistant Pseudomonas aeruginosa and Acinetobacter baumannii.

Pseudomonas aeruginosa and Acinetobacter baumannii are frequently isolated in hospital outbreaks of nosocomial infections. In our hospital, among 1018 strains isolated one year in an intensive care unit, 84 strains (8.3%) of P. aeruginosa and 155 strains (15.2%) of A. baumannii were considered responsible for infections. The major problem related to these bacteria is their multiresistant characteristic which confers great difficulty in treating infections. We carried out a 24 h time-kill study to assess the bactericidal effect of three beta-lactams [imipenem (IPM), ticarcillin+clavulanic acid (TCC), piperacillin+tazobactam (PTB)] in combination with each other and with sulbactam (SUL) and amikacin (AKN) against 8 P. aeruginosa strains and 8 A. baumannii strains. The initial inoculum was 10(6) cfu/ml. Antibiotics were tested at clinically achievable concentrations: TCC (112 mg/l), PTB (100 mg/l), IPM (25 mg/l) and AKN (15 mg/l). The results showed: IMP + TCC + AKN = PTB + SUL + AKN = PTB + TCC + AKN > > IMP + SUL + AKN against P. aeruginosa; and PTB + SUL + AKN = PTB + TCC + AKN > IMP + SUL + AKN or IMP + TCC + AKN against A. baumannii. When infection due to these multiresistant strains was suspected, PTB + AKN combined with either TCC or SUL was bactericidal against both strains. These combinations appeared to be an alternative therapy in the treatment of undocumented nosocomial infections in intensive care units. These in vitro results are being evaluated in patients and seem to give good results for the moment.

Effect of vancomycin and teicoplanin alone or in combination with fusidic acid or amikacin against methicillin-resistant staphylococci.

The action of two glycopeptides (vancomycin and teicoplanin) alone or in combination with amikacin or fusidic acid has been studied against 10 non-repetitive strains of methicillin-resistant Staphylococcus. The plotted killing curves show that the combination of vancomycin or teicoplanin plus fusidic acid always exhibited an antagonist effect at 24 h for the 10 strains. The combination vancomycin or teicoplanin plus amikacin shows the same effect at 24 h as vancomycin or teicoplanin alone. Thus, the combination glycopeptide plus amikacin does not alter the activity of the glycopeptide but does increase the spectrum of activity of the combination versus Gram-negative nosocomial bacteria (Pseudomonas aeruginosa, Acinetobacter sp). The vancomycin or teicoplanin plus fusidic acid combination may be responsible for bacteriological failure against methicillin-resistant Staphylococcus.

Pharmacokinetics of dibekacin in children with cystic fibrosis.

Dosages and pharmacokinetics of dibekacin were studied in a group of 15 children with cystic fibrosis (CF) and a control group of 9 children. The mean dosage regimens of dibekacin were respectively equal to 2.19 mg/kg and 2.16 mg/kg in the CF and non-CF patients. The mean peak serum values for the CF and non-CF groups were respectively equal to 5.3 micrograms/ml. Comparison of pharmacokinetic parameters (i.e. half-life, distribution volume, total body clearance and area under the curve) showed no significant difference between the two groups (p greater than 0.05).

[Campylobacter fetus subsp fetus bacteremia in a female patient infected by the hepatitis C virus]. / Bactériémie à Campylobacter fetus subsp fetus chez une patiente infectée par le virus de l'hépatite C.

Campylobacter fetus subsp fetus was identified as an unusual etiologic agent of septicemia in an immuno-compromized patient VHC positive by utilizing a 16S rRNA molecular kit in our hospital's clinical laboratory. This method would appear as a performing approach to identify pathogens when discrepancies exist between phenotypical tests.

Amikacin assay: correlation between rapid bioassay, enzyme immuno-assay (EMIT) and fluoro-immuno-assay (FIA).

Three methods (rapid bioassay, enzyme immunoassay and fluoroimmunoassay) for the determination of amikacin level in serum are compared. The statistical analysis of the results show correlation coefficients equal to 0.967 (EMIT versus FIA), 0.970 (bioassay versus FIA) and 0.983 (bioassay versus EMIT). The three methods are acceptable for routine clinical use. The enzyme immunoassay adapted on a centrifugal analyzer appears to be a method of choice for laboratories with a large workload. However, the rapid bioassay described in this paper remains adequate for laboratories testing 5 to 10 sera per day.

Bacterial epidemiology of osteoarticular infections in a referent center: 10-year study.

INTRODUCTION: Management of osteoarticular infections combines surgical treatment with antibiotic therapy. For some teams the immediate postoperative regimen requires at least partly wide-spectrum probabilistic treatment while waiting for the microbiological results. This protocol exposes the patient to the selection of resistant bacteria and the hospital unit to a modification of its bacterial ecology. The objective of this study was to retrospectively describe the microbial epidemiology of the Traumatology and Orthopaedics Department of the Lille University Hospital over 10 years (2002-2011). MATERIALS AND METHODS: The bacterial species isolated in culture of osteoarticular samples were listed, after removing any duplicates. The antibiotics retained for follow-up were those used in treatment of these infections as well as those recognized as markers of resistance. For Gram-positive species, the antibiotics considered were methicillin, rifampicin, fluoroquinolones, glycopeptides, and linezolid; for the Gram-negative species, cefotaxime, cefepime, imipenem, and fluoroquinolones were considered. RESULTS: Of the 5006 strains isolated between 2002 and 2011, Gram-positive cocci accounted for more than 71%; Staphylococcus aureus 27%, and coagulase-negative staphylococci (CoNS) 54%. Contrary to S. aureus, resistance to methicillin, fluoroquinolones, and teicoplanin significantly increased in CoNS, reaching 44%, 34%, and 22%, respectively, of the strains in 2011. The proportion of streptococcal and enterococcal infections remained stable, a mean 7.4% and 5.3%, respectively, per year. Enterobacteria (12.5% of the isolates) were producers of extended-spectrum beta-lactamase in 7.8% of the cases. Pseudomonas aeruginosa was involved in 3.6% of the infections, and 12% of the strains remained resistant to ceftazidime. Propionibacterium acnes accounted for 5.8% of the bacteria isolated and showed few antibiotic resistance problems. DISCUSSION: Stability in the distribution and the susceptibility of different bacterial species was noted over this 10-year period. Although the evolution of S. aureus resistance was favourable, the resistance of CoNS specially to methicillin and glycopeptides increased. LEVEL OF EVIDENCE: Level IV. Retrospective cohort study.

Preliminary clinical study using a multiplex real-time PCR test for the detection of bacterial and fungal DNA directly in blood.

Early diagnosis of sepsis, rapid identification of the causative pathogen(s) and prompt initiation of appropriate antibiotic treatment have a combined impact on mortality due to sepsis. In this observational study, a new DNA-based system (LightCycler SeptiFast (LC-SF) test; Roche Diagnostics) allowing detection of 16 pathogens at the species level and four groups of pathogens at the genus level has been evaluated and compared with conventional blood cultures (BCs). One hundred BC and LC-SF results were obtained for 72 patients admitted to the intensive-care unit over a 6-month period for suspected sepsis. Microbiological data were compared with other biological parameters and with clinical data. The positivity rate of BCs for bacteraemia/fungaemia was 10%, whereas the LC-SF test allowed detection of DNA in 15% of cases. The LC-SF performance, based on its clinical relevance, was as follows: sensitivity, 78%; specificity, 99%; positive predictive value, 93%; and negative predictive value, 95%. Management was changed for four of eight (50%) of the patients because organisms were detected by the LC-SF test but not by BC. LC-SF results were obtained in 7-15 h, in contrast to the 24-72 h required for BC. According to the LC-SF results, initial therapy was inadequate in eight patients, and antibiotic treatment was changed. Our results suggest that the LC-SF test may be a valuable complementary tool in the management of patients with clinically suspected sepsis.

Bacterial flora of the low male genital tract in patients consulting for infertility.

The physiological aerobic bacterial flora of the low male genital tract was determined. This prospective study was performed on 600 semen specimens collected from 543 asymptomatic males consulting for infertility. Semen cultures were sterile in 28.8%, with a polymicrobial flora and/or absence or low titres of Ureaplasma urealyticum in 49.3%, and with one or two aerobic and facultative bacteria > or =1 x 10(3) CFU ml(-1) and/or U. urealyticum with titres > or =10(4) CCU ml(-1) (colour changing units) in 21.8%. In standard aerobic cultures, Gardnerella vaginalis was the most commonly isolated species (26.1%), followed by coagulase-negative staphylococci (15.7%) and Streptococcus anginosus (14.2%). Ureaplasma urealyticum was absent in 84.5% of semen samples, but when recovered, high (> or =10(4) CCU ml(-1)) and low titres (< or =10(3) CCU ml(-1)) were counted in 7.2% and 8.3% respectively. Of 48 patients, the follow-up of semen cultures showed marked variations in time. This study shows that (i) there was no relationship between the bacterial flora and the leucocytospermia; (ii) low titres of U. urealyticum in semen were not associated with a disturbance of the ecosystem; (iii) the critical threshold for U. urealyticum should be raised to > or =10(4) CFU ml(-1) and (iv) a positive semen culture should be repeated before any treatment.

Evaluation of an automated system for identification of Enterobacteriaceae and nonfermenting bacilli.

The performance of the Vitek 2 (bioMérieux, France), a new fully automated system allowing rapid identification of microorganisms and susceptibility testing, and the Vitek 2 ID-GNB card (bioMérieux) was evaluated using 502 clinical isolates and stock collection strains of gram-negative rods belonging to 70 taxa. The number of isolates correctly identified to species and genus levels was 430 (85.7%) and 485 (96.6%), respectively. Clinical isolates of both Enterobacteriaceae and non-Enterobacteriaceae were better identified at the species level (95.3% and 74%, respectively) than stock collection strains (86.4% and 52.2%, respectively). The Vitek 2 ID-GNB card provides after 3 h a highly acceptable level of accuracy for identification of Enterobacteriaceae and non-Enterobacteriaceae, including most atypical strains encountered in clinical situations.

Effects of host iron transport compounds on growth kinetics and outer-membrane protein expression of Bilophila wadsworthia.

Since the environmental iron concentration has emerged as an important attribute in the expression of bacterial virulence, the purpose of this study was to determine the effects of transferrin, lactoferrin, heme compounds, and inorganic iron sources (ferric and ferrous sulfate) on the growth of Bilophila wadsworthia and to study its outer membrane composition when grown under these different simulated in vivo conditions. Lactoferrin, transferrin, hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources. Bilophila wadsworthia was also capable of growing in the presence of ferrous and ferric sulfate. Profiles obtained by SDS-PAGE showed two iron-regulated outer membrane proteins (IROMPs) of 190 kDa and 88 kDa. The 190 kDa was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. These two major IROMPs were expressed in iron-restricted media supplemented with iron-bound organic sources and repressed by the addition of inorganic iron sources.