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.

[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.

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.

[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.

[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.

Multicenter evaluation of an automated system using selected bacteria that harbor challenging and clinically relevant mechanisms of resistance to antibiotics.

A multicenter study was carried out to evaluate the performance of a new commercial automated system in comparison with that of the reference agar dilution method. Ten clinical microbiology laboratories tested a collection of 61 strains of gram-negative bacilli (49 Enterobacteriaceae and 12 Pseudomonas aeruginosa), and 6 other laboratories tested a collection of 55 strains of gram-positive cocci (10 enterococci and 45 staphylococci) against 10-20 antimicrobial agents. The strains were selected on the basis that they harbored challenging and characterized mechanisms of resistance. In comparison with the agar reference method, the automated system gave an overall essential agreement (+/-1 dilution) of 94.5%, 93.5%, and 97% for the gram-negative bacilli, enterococci, and staphylococci, respectively. According to the interpretive standards of the National Committee for Clinical Laboratory Standards, the category agreement ranged from 96 to 96.4% for the three sets of organisms. The accuracy of the automated system, as determined by the kappa test, ranged from 0.80 to 0.88, reflecting an almost perfect agreement with the reference technique. Very major, major, and minor errors obtained with the automated system were 0.3%, 2.9%, and 6.6% for gram-negative bacilli, 3.4%, 0%, and 5% for enterococci, and 1%, 1.6%, and 2.7% for staphylococci, respectively. The high rate of very major errors in enterococci was mostly due to a single strain of multidrug-resistant Enterococcus faecium, which was found susceptible to several antibiotics in a majority of participant laboratories. The use of a heavy inoculum and of a broth test medium by the automated system might account for a better expression of certain resistance mechanisms, including beta-lactamases, as compared to the agar dilution reference method. The interlaboratory reproducibility was acceptable, as shown by the narrow dispersion of MICs and by the results of quality control.

[Bacterial epidemiological study of acute otitis media in children observed at home in the Nord Pas-de-Calais region]. / Etude de l'épidémiologie bactérienne de l'otite moyenne aiguë de l'enfant observée en ville dans la région Nord Pas-de-Calais.

BACKGROUND: The aim of this work is the study of the bacteriologic epidemiology of acute otitis media in infants observed at home in Nord Pas-de-Calais area, and the analysis of bacteria associated to recurrent otitis and clinical failure. OBSERVATIONS: A total of 295 specimens of ear pus specimens were collected from children (mean age: 18 months; average: one month-12 years). Pneumococcus strains were isolated from 52% of samples and 80% of these showed resistance to penicillin. H. influenzae was found in 35% of specimens and the half produced a beta lactamase. Pneumococcus is the predominant pathogen isolated in prolonged otitis media, while H. influenzae is preferentially found during recurrent otitis media. The main bacteriologic cause of failure traitement was penicillin-intermediate or -resistant pneumococci. The therapy administered 48 to 72 hours before collection of ear pus sample in therapeutic failure was ineffective (oral cephalosporins or macrolides), or administered to low dosage (50 mg/kg/j). CONCLUSION: Our results demonstrate, in opposition to other studies, Streptococcus pneumoniae as the most frequent pathogen in acute otitis media. They also show the excellent correlation between antibiotic therapy and clinical failures.

Molecular identification of Pasteurella dagmatis peritonitis in a patient undergoing peritoneal dialysis.

Pasteurella dagmatis was identified as the etiologic agent of peritonitis in a continuous ambulatory peritoneal dialysis patient by utilizing a molecular kit in our hospital's clinical laboratory. This method would appear a useful approach to identify a species of Pasteurella not included in the existing database of commercial identification kits when discrepancies exist between phenotypic tests.

Peritonitis due to Staphylococcus sciuri in a patient on continuous ambulatory peritoneal dialysis.

Among the coagulase-negative staphylococci, Staphylococcus sciuri has rarely been described as the aetiology of continuous ambulatory peritoneal dialysis (CAPD) peritonitis. It has been reported in 1 case of endocarditis and has been isolated from peritoneal dialysis fluid in 2 patients. The case reported here describes CAPD peritonitis due to S. sciuri shortly after a previous episode due to S. aureus, showing the necessity to identify coagulase-negative staphylococci to find new species that cause CAPD peritonitis.

[Contribution of direct bacteriologic examinations to the diagnosis of early materno-fetal bacterial infection: the Lille experience]. / Apport des examens directs bactériologiques au diagnostic de l'infection bactérienne materno-foetale précoce: expérience lilloise.

A prospective study was conducted in 3056 live-born infants delivered at the Jeanneade-Flandre maternity hospital of the Lille Teaching Hospital between January and August 1997. Clinical, laboratory test, and microbiological test findings were compared. A cohort of 1003 infants who remained in the maternity ward but were considered at increased risk of maternofetal infection (MFI) based on history and/or obstetrical criteria and/or neonatal criteria underwent routine collection of specimens including gastric fluid, auricular and anal swabs, amniotic fluid, and placental fragments. Microscopic examination of gastric fluid smears, the first result available to the clinician, was found to have 27.5% sensitivity (983 samples). Positive predictive value (PPV) was only 17.8% because of a high rate of colonization (16.8%), defined as absence of clinical symptoms and three peripheral specimens positive for the same organism. However, negative predictive value (NPV) was as high as 99.8% as a result of high sensitivity (97.8%) in the infected neonates. The gastric fluid smear was positive in 30% and 35% of neonates born to mothers with hyperpyrexia during early and late labor, respectively, and in 42% of neonates born to mothers with a history of group B streptococcus carriage during the pregnancy. Forty-two per cent of neonates with a history of fetal tachycardia had a positive gastric fluid smear. Diagnostic criteria for infection were three peripheral specimens positive for the same organism, C-reactive protein elevation, and/or one or more clinical signs suggestive of infection, and/or a positive central specimen (blood, CSF). The infection rate in infants who remained in the maternity ward was 1.6%. The most common causative organisms were group B streptococci. These findings illustrate the useful contribution of gastric fluid smears to the early diagnosis of MFI and confirm the predominant role of group B streptococci.

[Determination of the nosocomial origin of vancomycin-resistance strains of Enterococcus isolated from stool of patients in a hematology department]. / Recherche de l'origine nosocomiale des souches d'entérocoques vancomycine-résistantes isolées des selles de patients du service des maladies du sang.

Patients severely neutropenic, when hospitalized, occasionally receive selective digestive decontamination, and the risk of vancomycin-resistant strain selection is a drawback since glycopeptide resistance is often associated with betalactam and aminoglycosid resistance. Bacterial translocation can lead to multiresistant bacterial sepsis. Eighteen Enterococcus faecium strains were collected from patients hospitalized in the leukemia unit of the Universitary Hospital of Lille (CHRU, Pr Bauters) between October 1992 and July 1997 and were studied. Nosocomial acquisition or endogenous origin were investigated to choose well-adapted prevention. All the vancomycin-resistant strains were shown by Polymerase Chain Reaction having the van A gene. The clonality of these strains was investigated by Pulsed-Field-Gel-Electrophoresis after Sma I restriction. Pulsotype analysis showed variable homology (52%-100%). Our results do not show evidence of patient-to-patient E. faecium transmission and suggest vancomycin-resistant strains were independently selected by antibiotic therapy from individual fecal flora. Except when epidemic events or happen, this strain isolation is more related to antibiotic prescription than misuse of isolation techniques.

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.

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.

Siderophore production by Staphylococcus aureus and identification of iron-regulated proteins.

Siderophore activity of Staphylococcus aureus was detected in an iron-restricted chemically defined medium. The molecular mass of this siderophore, called aureochelin, was 577 Da. Surface-associated proteins of 120, 88, 57, 35, and 33 kDa were mainly expressed under iron restriction conditions. Results showed a relationship between siderophore production and the existence of the 120- and 88-kDa proteins. Western blotting of surface-associated proteins revealed that these proteins were recognized both by patients sera and polyclonal rabbit serum.

The binding of surface proteins from Staphylococcus aureus to human bronchial mucins.

Colonization by Staphylococcus aureus is frequently observed in obstructive lung diseases, particularly in cystic fibrosis. It has been shown that the bacteria bind to mucins, the main constituent of bronchial secretions. The binding mechanism, however, remains unclear. We have investigated the interactions of two strains of S. aureus, one mucoid and one nonmucoid, with human bronchial mucins. Using a solution phase assay, the binding capacity of the two strains to radiolabelled bronchial mucins was assessed. The bacterial constituents were released by lysostaphin lysis and the surface components of the nonmucoid strain were extracted with the use of a detergent (3-([3-cholamidopropyl] dimethylammonio)-1-propane sulphonate (CHAPS)). All were analysed for mucin-binding using an overlay assay. The amount of mucins bound to the nonmucoid strain was threefold greater than that of the mucoid strain. In the lysostaphin extract from the mucoid strain, only a 57 kDa protein faintly bound 125I-labelled mucins, whereas three mucin-binding proteins (52, 57 and 71 kDa) were identified from the nonmucoid strain. Two surface proteins, one major at 60 kDa and one minor at 71 kDa, bound radiolabelled bronchial mucins and their binding was almost completely inhibited by ovine submaxillary mucin. These results indicate: 1) differences in the mucin-binding capacity from one strain of S. aureus to another; and 2) the presence of external and internal adhesins binding to human respiratory mucins in the nonmucoid strain.