Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods.

[Contamination of human milk with aerobic flora: Evaluation of losses for a human milk bank]. / Contamination du lait maternel par une flore aérobie : évaluation des pertes pour un lactarium.

INTRODUCTION: In France, human milk banks pasteurize milk for the mother's own hospitalized baby (personalized milk) and for donation. There is specific legislation regulating the activity of human milk banks with bacterial screening of donor milk before and after pasteurization. Milk should be tested for Staphylococcus aureus and total aerobic flora. Any sample of milk positive for aerobic flora and/or S. aureus before and/or after pasteurization should be discarded. The real pathogenicity of the total aerobic flora is actually debated as well as the usefulness of systematic postpasteurization screening. The aim of this study was to quantify milk losses related to prepasteurization contamination by total aerobic flora in a regional milk bank, to identify losses due to contamination with S. aureus or aerobic flora, and to analyze differences between centers. METHODS: This was a prospective observational study conducted in the regional human milk bank of the Nord-Pas-de-Calais area in France. Data were collected from six major centers providing 80% of the milk collected between June 2011 and June 2012. Variables were the volumes of personalized milk collected by each center, volumes of contaminated milk, and the type of bacteria identified. RESULTS: During the study period, the regional human milk bank treated 4715 L (liters) of personalized milk and 508 L (10.8%) were discarded due to bacteriological screening. Among these 508 L, 43% were discarded because of a prepasteurization contamination with aerobic flora, 55% because of a prepasteurization contamination with S. aureus, and 2% because of other pathogenic bacteria. Postpasteurization tests were positive in 25 samples (0.5%). Only five of these 25 samples were positive before pasteurization and in all cases with S. aureus. A total of 218 L were destroyed because of prepasteurization contamination with total aerobic flora, while the postpasteurization culture was sterile. There was a great difference between centers in the percentage of discarded milk and the type of contamination. The percentage of discarded milk varied from 4 to 16% (P<0.001) and the percentage of prepasteurization positive samples with aerobic flora from 0 to 70% (P<0.001). Costing 80 €/L in France, this represented an economic loss of €17,440. CONCLUSION: A significant volume of milk is discarded because of contamination with total aerobic flora found only in prepasteurization tests. Reassessment of the French regulations with regard to microbiological safety could save human milk to cover the needs of a larger group of preterm babies.

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.

Assessment of the bio-preparedness and of the training of the French hospital laboratories in the event of biological threat.

A national laboratory network 'Biotox-Piratox' was created in 2003 in France with the purpose of detecting, confirming and reporting potential biological and chemical threat agents. This network is divided into three levels: Level 1 is dedicated to the evaluation of risks (biological, chemical, radiological), to sampling and packing. Level 2 consists of university and military hospitals, who deal with biological specimens, and of environmental and veterinary laboratories, who deal with environmental and animal samples. Level 3 comprises national reference laboratories and the Jean Mérieux biosafety level (BSL)-4 laboratory in Lyon. This report presents the results of four bio-preparedness exercises to check critical points in the processing of samples. These exercises took place in 2007, 2009, 2010 and 2011. Each of them consisted of two parts. The first part was the identification of an unknown bacterial strain and its susceptibility to antibiotics used as a default in case of a bioterrorist event. The second part was the detection of Class III microorganisms, mainly by molecular techniques. The main lesson learnt in these exercises was that the key to successful detection of biological agents in case of a biological threat was standardisation and validation of the methods implemented by all the laboratories belonging to the network.

[Bacteriospermia in Assisted Reproductive Techniques: effects of bacteria on spermatozoa and seminal plasma, diagnosis and treatment]. / Les bactériospermies en AMP: comment réaliser et interpréter une spermoculture? Qui traiter? Pourquoi? Comment?

The presence of bacteria in semen could induce impairment of sperm morphology, alteration of sperm function and mechanical or functional obstruction of the seminal tract. The term of bacteriospermia does not signify infection. Bacteriospermia and male accessory gland infection (MAGI) have indeed to be distinguished. They may lead both to male infertility but their diagnosis and treatment options differ. This review summarizes effects of bacteria and leucocytospermia on sperm parameters and functions. Then, indications, benefits and risks of treatment of bacteriospermia and MAGI, in assisted reproductive techniques (ART) will be discussed. For bacteria commonly observed in semen, this review aims at defining some thresholds above which a treatment is required. These thresholds were established according to literature, according to French microbiology society and in function of our usual practice. This review should help practitioners of reproductive medicine to take care of bacteriospermia in semen.

[Detection of extended-spectrum ß-lactamase-producing Enterobacteriaceae in rectal swabs with the Mastdiscs™ ID AmpC ßLSE detection set]. / Dépistage du portage digestif des entérobactéries productrices de ß-lactamase à spectre étendu par ensemencement direct des écouvillons rectaux à l'aide de la méthode Mastdiscs™ ID AmpC ßLSE.

OBJECTIVES: The double-disk synergy test was compared to the Mastdiscs™ ID AmpC and ESßL method for detection of ESßL production in rectal swab. METHODS: Two hundred and forty-nine rectal swabs were directly inoculated onto Mueller-Hinton plates and analyzed according to both methods. RESULTS: A total of 41 (16%) and 208 (84%) were positive and negative for ESßL, respectively. Twelve (29%) and 20 (49%) of the 41 rectal swabs positive for ESßL were detected after 24h of incubation with the double-disk synergy test and the Mastdiscs™ method, respectively (P=0.013). One hundred fifty-eight (76%) et 183 (88%) of the 208 rectal swabs were detected negative for ESßL after 24h of incubation with the double-disk synergy test and the Mastdiscs™ method, respectively (P<0.001). Finally, 79 (32%) and 46 (18%) rectal swabs respectively inoculated according to the double-disk synergy test and the Mastdiscs™ method were inconclusive after 24h of incubation. The better performance of the Mastdiscs™ method was due to an easier detection of cephalosporinase producing bacteria. CONCLUSIONS: The Mastdiscs™ method is a simple phenotypic method that detects more easily ESßL and non-ESßL producing bacteria in rectal swab.

Risk of acquiring multidrug-resistant Gram-negative bacilli from prior room occupants in the intensive care unit.

The objective of this prospective cohort study was to determine whether admission to an intensive care unit (ICU) room previously occupied by a patient with multidrug-resistant (MDR) Gram-negative bacilli (GNB) increases the risk of acquiring these bacteria by subsequent patients. All patients hospitalized for >48 h were eligible. Patients with MDR GNB at ICU admission were excluded. The MDR GNB were defined as MDR Pseudomonas aeruginosa, Acinetobacter baumannii and extended spectrum ß-lactamase (ESBL) -producing GNB. All patients were hospitalized in single rooms. Cleaning of ICU rooms between two patients was performed using quaternary ammonium disinfectant. Risk factors for MDR P. aeruginosa, A. baumannii and ESBL-producing GNB were determined using univariate and multivariate analysis. Five hundred and eleven consecutive patients were included; ICU-acquired MDR P. aeruginosa was diagnosed in 82 (16%) patients, A. baumannii in 57 (11%) patients, and ESBL-producing GNB in 50 (9%) patients. Independent risk factors for ICU-acquired MDR P. aeruginosa were prior occupant with MDR P. aeruginosa (OR 2.3, 95% CI 1.2-4.3, p 0.012), surgery (OR 1.9, 95% CI 1.1-3.6, p 0.024), and prior piperacillin/tazobactam use (OR 1.2, 95% CI 1.1-1.3, p 0.040). Independent risk factors for ICU-acquired A. baumannii were prior occupant with A. baumannii (OR 4.2, 95% CI 2-8.8, p <0.001), and mechanical ventilation (OR 9.3, 95% CI 1.1-83, p 0.045). Independent risk factors for ICU-acquired ESBL-producing GNB were tracheostomy (OR 2.6, 95% CI 1.1-6.5, p 0.049), and sedation (OR 6.6, 95% CI 1.1-40, p 0.041). We conclude that admission to an ICU room previously occupied by a patient with MDR P. aeruginosa or A. baumannii is an independent risk factor for acquisition of these bacteria by subsequent room occupants. This relationship was not identified for ESBL-producing GNB.

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.

Quorum-sensing activity and related virulence factor expression in clinically pathogenic isolates of Pseudomonas aeruginosa.

Respiratory isolates of Pseudomonas aeruginosa were collected from 58 critically-ill patients with ventilator-associated pneumonia. Expression of elastase and pyocyanin was assessed semi-quantitatively, while quorum-sensing activity was assessed by quantifying the levels of the autoinducers N-3-oxododecanoyl-L-homoserine lactone (C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL). Correlations were sought between quorum-sensing activity and the expression of these two virulence factors, and all results were compared to those obtained with the laboratory reference strains PA103, a strain defective in quorum-sensing, and PAO1, a functional quorum-sensing strain. More than two-thirds of clinically pathogenic isolates had increased levels of elastase and/or pyocyanin, and high quorum-sensing activity, as assessed by autoinducer levels. However, a strong correlation between quorum-sensing activity and virulence factor production was revealed only for elastase and not for pyocyanin (C12-HSL/elastase, r = 0.7, p 2 x 10(-9); C4-HSL/elastase, r = 0.7, p 2 x 10(-9)). These data suggest that the pathogenicity of P. aeruginosa isolates from critically-ill patients with ventilator-associated pneumonia is caused, at least in part, by an increase in elastase production regulated by quorum-sensing, while increased pyocyanin production in these isolates may be regulated predominantly by mechanisms other than quorum-sensing.

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.

[Self-collected vaginal swabs to diagnosis bacterial vaginosis during pregnancy: A pilot study]. / Autoprélèvement vaginal à la recherche d'une vaginose bactérienne pendant la grossesse: étude pilote.

OBJECTIVE: To study the feasibility of a screening for bacterial vaginosis by a self-collected vaginal swab during pregnancy. To measure bacterial vaginosis prevalence in a non-representative sample of women. PATIENTS AND METHODS: A self-collected swab was suggested to 398 women who consulted between 15 and 33 weeks of gestation in three different centres. Gram stain evaluation using Nugent criteria was used for the diagnosis of bacterial vaginosis. RESULTS: Three hundred and forty-one women agreed to take part in the study (86%). The quality of the swabs was satisfactory in 93% of the cases. Concerning the 15 non-interpretable slides, the cellular and bacterial density was too poor, owing to a poor quality or a low vaginal flora. Thirty-one women (9%) had a bacterial vaginosis--Nugent score included between 7 and 10--and this frequency did not vary according to the centre. Thirty-five women (10%) had an intermediate flora--score between 4 and 6--and this result varied from 2 to 12% depending on the centre, but the difference was not significant. DISCUSSION AND CONCLUSION: Self-collected swabs to detect bacterial vaginosis are well accepted by most of pregnant women, and the quality of the swabs seems to be satisfactory. In case vaginal flora is intermediate--between 4 and 6--the interpretation of the slides could be difficult.

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.

Local symptoms at the site of pacemaker implantation indicate latent systemic infection.

BACKGROUND: To determine whether local complications at the site of pacemaker implantation indicate infection of the intravascular part of the lead as well as of the pacemaker pocket. METHODS: 105 patients admitted for local inflammatory findings, impending pacemaker or lead exteriorisation, frank pacemaker or lead exteriorisation, or overt infection were studied prospectively. After systematic lead extraction, the initial clinical presentation was related to the results of lead cultures. RESULTS: Regardless of the initial presentation, the intravascular parts of the leads gave positive cultures in 79.3% of patients. Additionally, 91.6% of the cultures of the extravascular lead segments were positive, in contrast to 38.1% positivity for wound swab cultures. No clinical observations or laboratory investigations permitted identification of patients with negative lead cultures. In a subgroup of 50 patients with manifestations strictly limited to the pacemaker implantation site, cultures of intravascular lead segments were positive in 72%. Infection recurred in 4/8 patients without complete lead body extraction (50%) v 1/97 patients (1.0%) whose leads were totally extracted (p < 0.001). CONCLUSIONS: Local complications at the site of pacemaker implantation are usually associated with infection of the intravascular part of the leads, with a risk of progressing to systemic infection. Such local symptoms should prompt the extraction of leads even in the absence of other infectious manifestations.

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.