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Objectives: This study measured the impact of the first wave of COVID-19 pandemic (COVID-19) (March–April 2020) on the incidence of bloodstream infections (BSIs) at Assistance Publique – Hôpitaux de Paris (APHP), the largest multisite public healthcare institution in France. Methods: The number of patient admission blood cultures (BCs) collected, number of positive BCs, and antibiotic resistance and consumption were analysed retrospectively for the first quarter of 2020, and also for the first quarter of 2019 for comparison, in 25 APHP hospitals (ca. 14 000 beds). Results: Up to a fourth of patients admitted in March–April 2020 in these hospitals had COVID-19. The BSI rate per 100 admissions increased overall by 24% in March 2020 and 115% in April 2020, and separately for the major pathogens (Escherichia coli, Klebsiella pneumoniae, enterococci, Staphylococcus aureus, Pseudomonas aeruginosa, yeasts). A sharp increase in the rate of BSIs caused by microorganisms resistant to third-generation cephalosporins (3GC) was also observed in March–April 2020, particularly in K. pneumoniae, enterobacterial species naturally producing inducible AmpC (Enterobacter cloacae...), and P. aeruginosa. A concomitant increase in 3GC consumption occurred. Conclusions: The COVID-19 pandemic had a strong impact on hospital management and also unfavourable effects on severe infections, antimicrobial resistance, and laboratory work diagnostics.

International audience; Background: Culture conditions sometimes make it difficult to detect non-tuberculous mycobacteria (NTM), particularly Mycobacterium abscessus, an emerging cystic fibrosis (CF) pathogen. The diagnosis of NTM positive cases not detected by classical culture methods might benefit from the development of a serological assay.Methods: As part of a diagnostic accuracy study, a total of 173 sera CF-patients, including 33 patients with M. abscessus positive cultures, and 31 non-CF healthy controls (HC) were evaluated. Four M. abscessus antigens were used separately, comprising two surface extracts (Interphase (INP) and a TLR2 positive extract (TLR2eF)) and two recombinant proteins (rMAB_2545c and rMAB_0555 also known as the phospholipase C (rPLC)).Results: TLR2eF and rPLC were the most efficient antigens to discriminate NTM-culture positive CF-patients from NTM-culture negative CF-patients. The best clinical values were obtained for the detection of M. abscessus-culture positive CF-patients; with sensitivities for the TLR2eF and rPLC of 81.2% (95% CI:65.7-92.3%) and 87.9% (95% CI:71.9-95.6%) respectively, and specificities of 88.9% (95% CI:85.3-94.8%) and 84.8% (95% CI:80.6-91.5%) respectively. When considering as positive all sera, giving a positive response in at least one of the two tests, and, as negative, all sera negative for both tests, we obtained a sensitivity of 93.9% and a specificity of 80.7% for the detection of M. abscessus-culture positive CF-patients.Conclusion: High antibody titers against TLR2eF and rPLC were obtained in M. abscessus-culture positive CF-patients, allowing us to consider these serological markers as potential tools in the detection of CF-patients infected with M. abscessus.

BackgroundChronic lung infection of cystic fibrosis (CF) patients by Staphylococcus aureus is a well-established epidemiological fact. Indeed, S. aureus is the most commonly identified pathogen in the lungs of CF patients. Strikingly the molecular mechanisms underlying S. aureus persistency are not understood.MethodsWe selected pairs of sequential S. aureus isolates from 3 patients with CF and from one patient with non-CF chronic lung disease. We used a combination of genomic, proteomic and metabolomic approaches with functional assays for in-depth characterization of S. aureus long-term persistence.ResultsFor the first time, we show that late S. aureus isolates from CF patients have an increased ability for intracellular survival in CFBE-F508del cells compared to ancestral early isolates. Importantly, the increased ability to persist intracellularly was confirmed for S. aureus isolates within the own patient F508del epithelial cells. An increased ability to form biofilm was also demonstrated.Furthermore, we identified the underlying genetic modifications inducing altered protein expression profiles and notable metabolic changes. These modifications affect several metabolic pathways and virulence regulators that could constitute therapeutic targets.ConclusionsOur results strongly suggest that the intracellular environment might constitute an important niche of persistence and relapse necessitating adapted antibiotic treatments.SummaryS. aureus persists for years in the lungs of patients with cystic fibrosis despite antibiotic therapies. We demonstrate that S. aureus adaptation leads to increased intracellular persistence suggesting a key role for intracellular niche during S. aureus chronic lung infection.

UMR BFP - Equipe Mollicutes; International audience; We report a disseminated infection caused by Spiroplasma apis, a honeybee pathogen, in a patient in France who had X-linked agammaglobulinemia. Identification was challenging because initial bacterial cultures and direct examination by Gram staining were negative. Unexplained sepsis in patients with agammaglobulinemia warrants specific investigation to identify fastidious bacteria such as Spiroplasma spp

International audience; ABSTRACT Staphylococcus epidermidis is a pathogen emerging worldwide as a leading cause of health care-associated infections. A standardized high-resolution typing method to document transmission and dissemination of multidrug-resistant S. epidermidis strains is needed. Our aim was to provide a core genome multilocus sequence typing (cgMLST) scheme for S. epidermidis to improve the international surveillance of S. epidermidis . We defined a cgMLST scheme based on 699 core genes and used it to investigate the population structure of the species and the genetic relatedness of isolates recovered from infants hospitalized in several wards of a French hospital. Our results show the long-lasting endemic persistence of S. epidermidis clones within and across wards of hospitals and demonstrate the ability of our cgMLST approach to identify and track these clones. We made the scheme publicly available through the Institut Pasteur BIGSdb server ( http://bigsdb.pasteur.fr/epidermidis/ ). This tool should enable international harmonization of the epidemiological surveillance of multidrug-resistant S. epidermidis clones. By comparing gene distribution among infection and commensal isolates, we also confirmed the association of the mecA locus with infection isolates and of the fdh gene with commensal isolates. (This study has been registered at ClinicalTrials.gov under registration no. NCT03374371.)

Background Early bacterial infection is a major and severe complication after liver transplantation (LT). The rise of antimicrobial resistance, especially extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE), is a growing concern for these patients. This study aimed to assess the epidemiology of early bacterial infections in a pediatric population, including those caused by multidrug-resistant (MDR) pathogens, and to identify risk factors for infection. Methods We conducted a monocentric retrospective study including 142 consecutive LTs performed in 137 children between 2009 and 2017. Results Ninety-three bacterial infections occurred after 67 (47%) LTs. Among the 82 isolated pathogens, the most common was Klebsiella pneumoniae (n = 19, 23%). Independent risk factors for early bacterial infection were low weight [odds ratio (OR) = 0.96; 95% confidence interval (CI): 0.9-0.99; P = 0.03] and the presence of a prosthetic mesh (OR = 2.4; 95% CI: 1.1-5.4; P = 0.046). Sixty-one children (45%) carried MDR bacteria and 16 infections were caused by MDR pathogens, especially ESBL-producing K. pneumoniae (n = 12). ESBL-PE stool carriage was associated with ESBL-PE infection (OR = 4.5; 95% CI: 1.4-17.4; P = 0.02). Four children died from an infection, three due to ESBL-producing K. pneumoniae. Conclusions This study confirmed a shift toward a predominance of Gram-negative early bacterial infections after pediatric LT. The risk factors for infection were low weight and the presence of a prosthetic mesh. ESBL-PE stool carriage was associated with ESBL-PE infection. Adapted antimicrobial prophylaxis and personalized antibiotherapy are mandatory to reduce infection prevalence and mortality.

Background The population structure of extraintestinal pathogenic Escherichia coli evolves over time, notably due to the emergence of antibiotic-resistant clones such as ESBL-producing Enterobacteriaceae (ESBL-E). Objectives To analyse by WGS the genetic diversity of a large number of ESBL-E isolated from urinary tract infections in children from paediatric centres across France between 2014 and 2017 and collected by the National Observatory of febrile urinary tract infection (FUTI) caused by ESBL-E. Methods A total of 40 905 Enterobacteriaceae-positive urine cultures were identified. ESBL-E were found in 1983 samples (4.85%). WGS was performed on 251 ESBL-E causing FUTI. STs, core genome MLST (cgMLST), serotype, fimH allele, ESBL genes and presence of papGII key virulence factor were determined. Results E. coli and Klebsiella pneumoniae were found in 86.9% (218/251) and 11.2% (28/251) of cases, respectively. Several STs predominate among E. coli such as ST131, ST38, ST69, ST73, ST95, ST405, ST12 and ST1193, while no ST emerged in K. pneumoniae. E. coli ST131, ST38 and ST1193 increased during the study period, with a heterogeneity in papGII prevalence (64.5%, 35% and 20% respectively). Most isolates harboured the CTX-M type (97%) with a predominance of blaCTX-M-15. blaCTX-M-27, an emerging variant in E. coli, is found in various STs. cgMLST enabled discrimination of clusters within the main STs. Conclusions The predominance of ST131, and the emergence of other STs such as ST38 and ST1193 combined with ESBL genes deserves close epidemiological surveillance considering their high threat in infectious disease. cgMLST could be a discriminant complementary tool for the analyses.

International audience; Objectives: Achromobacter spp. are emerging pathogens in respiratory samples from cystic fibrosis patients. The current reference methods (nrdA-sequencing or multilocus sequence typing) can identify 18 species which are often misidentified by conventional techniques as A. xylosoxidans. A few studies have suggested that matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) provides accurate identification of the genus but not of species. The aims of this study were (a) to generate a database for MALDI-TOF/MS Bruker including the 18 species, (b) to evaluate the suitability of the database for routine laboratory identification, and (c) to compare its performance with that of the currently available Bruker default database.Methods: A total of 205 isolates belonging to the 18 species identified by nrdA sequencing were used to build a local database. Main spectra profiles (MSPs) were created according to Bruker's recommendations for each isolate with the Biotyper software. Performance of the default Bruker database and ours for routine use were compared by testing 167 strains (including 38 isolates used from MSP creation) belonging to the 18 species identified by nrdA sequencing directly from colonies cultivated on various media.Results: Our new database accurately identified 99.4% (166/167) of the isolates from the 18 species (score ≥2.0) versus only 50.9% (85/167) with the Bruker database. In the Bruker database 17.3% of the isolates (29/167) were incorrectly identified as another species despite a score of ≥2.0.Conclusions: The use of MALDI-TOF/MS in combination with a database developed with samples from 18 Achromobacter species provides rapid and accurate identification. This tool could be used to help future clinical studies.

International audience; We previously reported the distribution of Achromobacter spp. (species and Sequence Types (ST)) in our French Cystic Fibrosis (CF) centre. In the present study we collected 109 Achromobacter isolates (1/patient) from 9 other French CF Centres for species identification, antimicrobial susceptibility testings and Multilocus-Sequence-Typing (MLST) analysis. Ten species were detected, A. xylosoxidans being the most predominant one (73.4% of the isolates). Piperacillin-tazobactam, ceftazidime, imipenem, meropenem and ciprofioxacin were respectively active against 88, 70, 79, 72 and 23% of the isolates. Among the 79 A. xylosoxidans isolates, 46 STs were detected. Interestingly, ST 137, recovered in 4 centres (5 patients), was previously detected in our centre (2 patients). The strains from the 7 patients belonged to the same pulsotype (pulsed-field-gel-electrophoresis analysis) and harboured acquired resistance to meropenem, ceftazidime, ciprofioxacin, and except for 2 isolates, to imipenem and piperacillin-tazobactam. This is the first description in France of a circulating multiresistant A. xylosoxidans strain.

Lyme neuroborreliosis is a bacterial infection caused by the dissemination and proliferation of a Borrelia species in the central nervous system. Neuroborreliosis occurs after transmission of the pathogen from an infected tick to a human host during a tick bite. We report nine cases of pediatric neuroborreliosis collected by the National Observatory of Pediatric Bacterial Meningitis in France between 2001 and 2012. The nine children, aged 4-13 years, were identified in northern and eastern France and had the following clinical features: meningeal irritation alone or with facial palsy, or isolated facial palsy. All cases showed anti-Borrelia antibodies in cerebrospinal fluid or serum, or with a positive Borrelia PCR in the CSF. The outcome was favorable in all cases after a 2- to 3-week course of third-generation cephalosporin. On the basis of these nine pediatric cases, this study provides an update on the epidemiology, pathophysiology, diagnostic strategy, and treatment of neuroborreliosis, with insight into the specific features of pediatric neuroborreliosis and the difficulties encountered in the diagnosis of this infection.

Three cases of Bacillus cereus infection or colonization occurred in the same region in France, and milk from the milk bank was suspected as a possible common source of contamination. All Batches delivered to the three cases complied with the requirements of the bacteriological reference method recommended by good practices guidelines. Still, a retrospective analysis with a more sensitive method showed one batch to contain B. cereus, however straincomparison revealed no epidemiological link betweenisolates from patients and those from the milk. Consequently, in accordance with the precautionary principle, we developed a new sensitive method for the screening of pasteurized milk for pathogenic bacteria. From January 1 to August 31, 2017, 2526 samples of pasteurized milk were prospectively included in the study. We showed that a 20 mL sample of pasteurized milk incubated for 18 h at 37 °C under aerobic conditions was favoring the detection of B. Cereus. The nonconformity rate was 6.3% for the reference method and 12.6% for the improved method (p

Until now, the identification of micro-organisms has been based on the cultural and biochemical characteristics of bacterial and fungal species. Recently, Mass Spectrometry type Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS) was developed in clinical microbiology laboratories. This new technology allows identification of micro-organisms directly from colonies of bacteria and fungi within few minutes. In addition, it can be used to identify germs directly from positive blood culture bottles or directly from urine samples. Other ways are being explored to expand the use of MALDI-TOF in clinical microbiology laboratories. Indeed, some studies propose to detect bacterial antibiotic resistance while others compare strains within species for faster strain typing. The main objective of this review is to update data from the recent literature for different applications of MALDI-TOF technique in microbiological diagnostic routine.

International audience; Helicobacter bilis is a commensal bacterium causing chronic hepatitis and colitis in mice. In humans, enterohepatic Helicobacter spp. are associated with chronic hepatobiliary diseases. Purpose : We aimed at understanding the microbial etiology in a patient with X-linked agammaglobulinemia presenting with suppurative cholangitis. Methods : 16S rDNA PCR directly performed on a liver biopsy retrieved DNA of H. bilis. Results : Clinical outcome resulted in the normalization of clinical and biological parameters under antibiotic treatment by a combination of ceftriaxone, metronidazole, and doxycyclin followed by a 2-week treatment with moxifloxacin and a 2-month treatment with azithromycin. Conclusion : In conclusion, these data suggest a specific clinical and microbiological approach in patients with humoral deficiency in order to detect H. bilis hepatobiliary diseases.

Effective antibiotic therapy is crucial for the outcome of septic patients and requires early diagnosis of β-lactam resistance in cases of Gram-negative bacteremia. Here, we report high sensitivity of the β-LACTA™ test in rapid detection of extended-spectrum β-lactamase–producing Enterobacteriaceae in blood cultures positive for Gram-negative bacilli.

Bordetella bronchiseptica is an opportunistic bacteria infecting the respiratory tract of patients with cystic fibrosis. We present a case of B. bronchiseptica chronic pulmonary infection and documentation of some phenotypic attributes of the clinical isolates allowing the microorganism to induce progressive respiratory degradation and chronic sputum colonization. We recommend implementing adequate treatment aiming eradication from the first isolation of this bacterium. We advise for practices that minimize opportunities for zoonotic transmission of B. bronchiseptica from family pets.

Pyomyositis is a primary purulent infection of skeletal muscles mostly described in tropical areas. Staphylococcus aureus is by far the main causative microorganisms. In temperate areas, pyomyositis more frequently affects male adults with immunodeficiency. Gram-negative bacilli pyomyositis are uncommon and occur mainly in this context of immunodeficiency. Only one case of Enterobacter cloacae pyomyositis has been reported so far. Chronic Granulomatous Disease (CGD) is a rare inherited immunodeficiency characterized by a loss of NADPH oxidase activity in phagocytic cells that favors infections due to catalase-positive microorganisms. Pyomyositis has only been described once as a complication of CGD. We report here a case of Enterobacter cloacae-associated pyomyositis of the left thigh in a patient with CGD, and review the literature on Gram-negative pyomyositis.

Limited data is available on pediatric community-onset infections with extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE), but such infections may affect both the efficacy of empiric antibiotic therapy and the rational use of antibiotics. We retrospectively analyzed data from 2007 to 2012 for all children ≤16 years old with a positive ESBL-PE strain from usually sterile sites within 48 hours of admission in a tertiary hospital in France. We analyzed healthcare- and community-associated infections among community-onset infections. In total, 3612 Enterobacteriaceae isolates were collected; the prevalence of ESBL-PE infection increased over the study period, from 2.4% to 5.1% (P

All organisms usually isolated in our laboratory are now routinely identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the Andromas software. The aim of this study was to describe the use of this strategy in a routine clinical microbiology laboratory. The microorganisms identified included bacteria, mycobacteria, yeasts and Aspergillus spp. isolated on solid media or extracted directly from blood cultures. MALDI-TOF MS was performed on 2665 bacteria isolated on solid media, corresponding to all bacteria isolated during this period except Escherichia coli grown on chromogenic media. All acquisitions were performed without extraction. After a single acquisition, 93.1% of bacteria grown on solid media were correctly identified. When the first acquisition was not contributory, a second acquisition was performed either the same day or the next day. After two acquisitions, the rate of bacteria identified increased to 99.2%. The failures reported on 21 strains were due to an unknown profile attributed to new species (9) or an insufficient quality of the spectrum (12). MALDI-TOF MS has been applied to 162 positive blood cultures. The identification rate was 91.4%. All mycobacteria isolated during this period (22) were correctly identified by MALDI-TOF MS without any extraction. For 96.3% and 92.2% of yeasts and Aspergillus spp., respectively, the identification was obtained with a single acquisition. After a second acquisition, the overall identification rate was 98.8% for yeasts (160/162) and 98.4% (63/64) for Aspergillus spp. In conclusion, the MALDI-TOF MS strategy used in this work allows a rapid and efficient identification of all microorganisms isolated routinely.

Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae , 13 C. pseudotuberculosis , 19 C. ulcerans , and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia , 75 Actinomyces , 18 Actinobaculum , 11 Propionibacterium acnes , 18 Propionibacterium avidum , 30 Lactobacillus , 21 Bacillus , 2 Rhodococcus equi , 2 Erysipelothrix rhusiopathiae , and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non- Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

There is a causal association between Helicobacter pylori (Hp) gastric infection and the development of gastric MALT lymphoma. In contrast, the link between Hp gastric infection and the development of extragastric lymphoma has not been thoroughly investigated. We, therefore, studied the prevalence of gastric Hp infection at initial diagnosis of ophthalmologic and nonophthalmologic extragastric lymphoma patients. Three cohorts of patients were studied: a first one of 83 patients with OAL, a second one of 101 patients with extraophthalmologic extragastric lymphoma, and a third one of 156 control individuals (control) without malignant lymphoma. Gastric Hp infection was investigated by histopathological analysis and Hp-specific PCR assay on gastric biopsy tissue samples. We found gastric Hp infection in 37 OAL patients (45%), in 25 extraophthalmologic extragastric lymphoma cases (25%), and in 18 controls individuals (12%) (P < 0.0001 OAL/C and P < 0.01 OAL/extra-OAL cases). Gastritis was found in 51% and 9% of Hp-positive and Hp-negative lymphoma patients, respectively (P < 10−4). Gastric Hp infection only correlated with MALT/LPL lymphoma (P = 0.03). There is a significant association between gastric Hp infection and MALT/LPL OAL. This suggests a novel mechanism of indirect infection-associated lymphomagenesis whereby chronic local antigen stimulation would lead to the emergence of ectopic B-cell lymphoma. © 2010 Wiley-Liss, Inc. Am. J. Hematol.

A study was performed to compare matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), linked to a recently engineered microbial identification database, and two rapid identification (ID) automated systems, BD Phoenix (Becton Dickinson Diagnostic Systems, France) and VITEK-2 (bioMérieux, Marcy L'Etoile, France), for the ID of coagulase-negative staphylococci (CoNS). Two hundred and thirty-four clinical isolates of CoNS representing 20 species were analyzed. All CoNS isolates were characterized by sodA gene sequencing, allowing interpretation of the ID results obtained using the respective database of each apparatus. Overall correct ID results were obtained in 93.2%, 75.6% and 75.2% of the cases with the MALDI-TOF-MS, Phoenix and VITEK-2 systems, respectively. Mis-ID and absence of results occurred in 1.7% and 5.1% of the cases with MALDI-TOF-MS, in 23.1% and 1.3% with the Phoenix, and in 13.7% and 0.9% with the VITEK-2 systems, respectively. In addition, with the latter automate, 10.3% of the IDs were proposed with remote possibility. When excluding the CoNS species not included in the databases of at least one of the three systems, the final percentage of correct results, Mis-ID and absence of ID were 97.4%, 1.3% and 1.3% with MALDI-TOF-MS, 79%, 21% and 0% with the Phoenix, and 78.6%, 10.3% and 0.9% with the VITEK-2 system, respectively. The present study demonstrates the robustness and high sensitivity of our microbial identification database used with MALDI-TOF-MS technology. This approach represents a powerful tool for the fast ID of clinical CoNS isolates.

With the support of a ministerial program for innovative and expensive technologies, dedicated to the economic evaluation of laboratory diagnosis of pertussis by real-time PCR, external quality assessment for real-time IS 481 PCR was carried out. Coordinated by the National Centre of Reference of Pertussis and other Bordetelloses (NCR), this study aimed to harmonize and to assess the performances of eight participating microbiology hospital laboratories throughout the French territory. Between January 2006 and February 2007, 10 proficiency panels were sent by the NCR (ascending proficiency program), representing a total of 49 samples and including eight panels to analyze and evaluate the global sensitivity and specificity of real-time PCR, one to assess the limit of detection, and one to evaluate nucleic acid extraction methods. As part of the descending proficiency program, extracted DNA from clinical samples was sent by the eight participating laboratories in different panels and analyzed by the NCR. In the ascending proficiency analysis, the sensitivity and specificity of the real-time PCR methods were 92.2% and 94.3%, respectively. The limit of detection of the different methods ranged between 0.1 and 1 fg/μl (0.2 to 2 CFU/μl). The nucleic acid extraction methods showed similar performances. During the descending proficiency analysis, performed with 126 samples, the result of the NCR for 15 samples (11.9%) was discordant with the result obtained by the source laboratory. Despite several initial differences, harmonization was easy and performances were homogeneous. However, the risk of false-positive results remains quite high, and we strongly recommend establishment of uniform quality control procedures performed regularly.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) of intact bacteria yields a reproducible spectrum depending upon growth conditions, strain, or species. Using whole viable bacteria we describe here the application of MALDI-TOF-MS to the identification of coagulase-negative staphylococci (CoNS). Our aim was, once a bacterium has been recognized as Micrococcaceae , to identify peaks in the spectrum that can be used to identify the species or subspecies. MALDI-TOF-MS was performed using bacteria obtained from one isolated colony. One reference strain for each of the 23 clinically relevant species or subspecies of Micrococcaceae was selected. For each reference strain, the MALDI-TOF-MS profile of 10 colonies obtained from 10 different passages was analyzed. For each strain, only peaks that were conserved in the spectra of all 10 isolated colonies and with a relative intensity above 0.1 were retained, thus leading to a set of 3 to 14 selected peaks per strain. The MALDI-TOF-MS profile of 196 tested strains was then compared with that of the set of selected peaks of each of the 23 reference strains. In all cases the best hit was with the set of peaks of the reference strain belonging to the same species as that of the tested strain, thus demonstrating that the 23 sets of selected peaks can be used as a database for the rapid species identification of CoNS. Similar results were obtained using four different growth conditions. Extending this strategy to other groups of relevant pathogenic bacteria will allow rapid bacterial identification.

Objectives: To compare cefoxitin and/or moxalactam 30 μg disc diffusion (DD) methods to detect methicillin resistance in coagulase-negative staphylococci (CoNS) using both high- and low-density (HD/LD) inoculum techniques. Methods: A challenge set of 192 CoNS was tested. DD test results were compared with PBP2a detection. Results: With the LD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 94.4%/100% and 100%/92.4%, respectively, using the DD breakpoints of the Comite de l'Antibiogramme de la Societe Francaise de Microbiologie. With the HD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 93.7%/100% and 100%/96.9%, using the cefoxitin DD breakpoints of the CLSI and a resistant/susceptible breakpoint of

Aim: To study the bacterial aetiology of acute osteoarticular infections in children and to analyse the efficiency of culture methods. Methods: Bacteriological data of 407 cases of clinically suspected osteoarticular infections affecting 406 children hospitalized in an orthopaedic surgery department between 1999 and 2002 were retrospectively reviewed. Results: Bacterial cultures from clinical specimens were positive in 74 cases (18%): 38 cases of septic arthritis and 36 cases of bone infections (osteitis, osteomyelitis or osteoarthritis). The use of liquid medium bottles to grow bacteria from articular fluids increased the rate of positive cultures compared to the use of standard solid media (p=0.0001). The most commonly recovered pathogen was Staphylococcus aureus (44%) followed by Kingella kingae (14%), Streptococcus pyogenes (10%) and Streptococcus pneumoniae (10%). K. kingae was most frequently isolated among children under 36 mo of age (p=0.0003), whereas S. aureus was most frequently isolated among children over 36 mo (p=0.0015). Conclusion: By improving our culture method, we observed a recrudescence of isolation of K. kingae, but S. aureus remains the main pathogen isolated from osteoarticular infections in children. This finding is useful for the adaptation of a probabilistic antibiotic treatment of these infections.

The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria (Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.

The diagnosis and prognosis of lung infections due to the emerging nontuberculous mycobacterium (NTM) Mycobacterium abscessus are difficult to establish in children with cystic fibrosis.We evaluated the usefulness of an enzyme-linked immunosorbent assay for detecting serum IgG antibodies against the ubiquitous mycobacterial antigen A60.A total of 186 patients with cystic fibrosis (mean age+/-SD, 12.0+/-5.0 years) were studied, including 15 M. abscessus-positive patients who fulfilled American Thoracic Society (ATS) criteria for NTM infection (M. abscessus-infected patients), 7 M. abscessus-positive patients who did not fulfill ATS criteria, 20 patients positive for various other NTM who did not fulfill ATS criteria, and 144 NTM-negative control patients; mean IgG titers (+/-SD) for these 4 groups were 718+/-342 U, 193+/-113 U, 129+/-49 U, and 121+/-53 U, respectively (M. abscessus-infected patients vs. each of the other groups, P.005). The A60 IgG test was both sensitive (approximately 87%) and specific (approximately 95%) if adapted cutoff values were used (150 U and 250 U for patients agedor=10 years and patients aged10 years, respectively) and correlated well with results of acid-fast bacilli smears.Measurement of anti-A60 IgG may be useful for both the diagnosis and assessment of activity of M. abscessus lung infection in persons with cystic fibrosis.

We prospectively studied 298 patients with cystic fibrosis (mean age 11.3 years; range 2 months to 32 years; sex ratio, 0.47) for nontuberculous mycobacteria in respiratory samples from January 1, 1996, to December 31, 1999. Mycobacterium abscessus was by far the most prevalent nontuberculous mycobacterium: 15 patients (6 male, 9 female; mean age 11.9 years; range 2.5–22 years) had at least one positive sample for this microorganism (versus 6 patients positive for M. avium complex), including 10 with >3 positive samples (versus 3 patients for M. avium complex). The M. abscessus isolates from 14 patients were typed by pulsed-field gel electrophoresis: each of the 14 patients harbored a unique strain, ruling out a common environmental reservoir or person-to-person transmission. Water samples collected in the cystic fibrosis center were negative for M. abscessus. This major mycobacterial pathogen in children and teenagers with cystic fibrosis does not appear to be acquired nosocomially.

Resume But de l’etude. – Evaluer la prevalence et les caracteristiques des souches atypiques de bacilles a Gram negatif non fermentants isolees au cours de la mucoviscidose, en determinant l’impact clinique et epidemiologique de leur identification. Materiel et methodes. – Le sequencage partiel du gene codant pour l’ARN ribosomal 16S (ADNr 16S) a ete utilise pour l’identification prospective des souches atypiques isolees chez les patients suivis a l’hopital Necker–Enfants Malades. Resultats. – Au cours de l’annee 1999, 1093 isolats de bacilles a Gram negatif non fermentants ont ete retrouves dans 702 expectorations provenant de 148 patients. Parmi ces isolats, 46 (27 patients) n’ont pas ete identifies de maniere satisfaisante par les methodes phenotypiques. L’analyse genotypique a permis d’identifier Pseudomonas aeruginosa (19 isolats, 12 patients), Achromobacter xylosoxidans (10 isolats, 8 patients), Stenotrophomonas maltophilia (9 isolats, 9 patients), Burkholderia cepacia genomovar I/III (3 isolats, 3 patients), Burkholderia vietnamiensis (1 isolat), Burkholderia gladioli (1 isolat) et Ralstonia mannitolilytica (3 isolats, 2 patients). Quinze isolats (33 %) etaient resistants a tous les antibiotiques testes en routine. Seize isolats (37 %) resistants a la colistine ont ete retrouves sur le milieu selectif cepacia : 2 P. aeruginosa, 3 A. xylosoxidans, 3 S. maltophilia et les 8 especes de Burkholderia - Ralstonia. La galerie API 20NE n’a donne aucune identification pour 35 isolats et une identification erronee pour 11 isolats, dont 2 P. aeruginosa, 2 A. xylosoxidans et 1 S. maltophilia identifies a tort B. cepacia. Les mesures d’isolement et/ou le traitement antibiotique ont ete nettement ameliores apres le resultat de l’identification par sequencage dans 3 cas. Conclusion. – Cette etude confirme l’insuffisance des methodes phenotypiques pour l’identification precise des bacilles a Gram negatif non fermentants retrouves au cours de la mucoviscidose. L’utilisation en routine d’une methode genotypique d’identification telle que le sequencage de l’ADNr 16S peut ameliorer la connaissance de l’epidemiologie de certaines especes bacteriennes et identifier eventuellement l’emergence de nouvelles especes au cours de la mucoviscidose.

Mycobacterium abscessus is a rapidly growing mycobacterium that causes respiratory tract infections in predisposed patients, such as those with cystic fibrosis and nosocomial skin and soft tissue infections. In order to investigate the clonal relationships between the strains causing epidemic episodes, we evaluated the discriminatory power of the semiautomated DiversiLab (DL) repetitive extragenic palindromic sequence PCR (REP-PCR) test for M. abscessus genotyping. Since M. abscessus was shown to be composed of subspecies ( M. abscessus subsp. massiliense , M. abscessus subsp. bolletii , and M. abscessus subsp. abscessus ), we also evaluated the ability of this technique to differentiate subspecies. The technique was applied to two collections of clinical isolates, (i) 83 M. abscessus original isolates (43 M. abscessus subsp. abscessus , 12 M. abscessus subsp. bolletii , and 28 M. abscessus subsp. massiliense ) from infected patients and (ii) 35 repeated isolates obtained over 1 year from four cystic fibrosis patients. The DL REP-PCR test was standardized for DNA extraction, DNA amplification, and electrophoresis pattern comparisons. Among the isolates from distinct patients, 53/83 (62%) isolates showed a specific pattern, and 30 were distributed in 11 clusters and 6 patterns, with 2 to 4 isolates per pattern. The clusters and patterns did not fully correlate with multilocus sequence typing (MLST) analysis results. This revealed a high genomic diversity between patients, with a discriminatory power of 98% (Simpson's diversity index). However, since some isolates shared identical patterns, this raises the question of whether it is due to transmission between patients or a common reservoir. Multiple isolates from the same patient showed identical patterns, except for one patient infected by two strains. Between the M. abscessus subspecies, the indexes were M. abscessus infections.

Resume L'isolement d'une souche de Bacillus cereus dans un liquide cephalorachidien est le plus souvent du a une contamination du prelevement ou de sa mise en culture. Il a cependant ete decrit quelques cas, dont six en pediatrie, de meningites a ce germe. Observation Nous presentons le cas d'un nourrisson âge de 2,5 mois ayant developpe une meningite a B cereus , 3 jours apres son entree a l'hopital pour un bilan metabolique dans le cadre du suivi d'un syndrome de Reye. L'identification du germe a ete confirmee par sequencage de l'ARN 16S. Le traitement antibiotique precoce par l'association imipeneme-amikacine et corticoides a permis une evolution favorable de l'infection. Aucun facteur de risque evident n'a pu etre montre; notamment, le bilan immunitaire effectue etait normal et il n'y a pas eu de manœuvre invasive profonde. Conclusion Bien que souvent en rapport avec une contamination, l'isolement repete d'une souche de B cereus doit faire evoquer la possibilite d'une reelle infection a ce germe.

An outbreak of 14 cases of urinary tract infections by Pseudomonas aeruginosa, including six symptomatic infections, occurred from September to November 1994 in a paediatric surgical unit. During the outbreak, urine samples from patients and multiple samples from the environment of patients were tested for the presence of P. aeruginosa. Bacterial isolates were studied by pulsed-field gel electrophoresis. Genotypic analysis showed that most of the isolates from children were different. Multiple P. aeruginosa isolates were also found in the tap water, as the only putative source of contamination. Two of these isolates were identified in two infected patients, indicating possible direct contamination of patients via tap water and this was related to the distal colonization of faucets. Bacteria were eradicated from tap water by replacement of taps. The cluster of cases of P. aeruginosa urinary infection was, therefore, related to multiple contaminations through tap water. These results illustrate an unexpected risk of nosocomial infection and emphasizes the importance of checking tap water to prevent bacterial contamination through handwashing in contaminated water.

During an open clinical trial in an area where streptococcal infections are hyperendemic, we studied the genetic polymorphism of Streptococcus pyogenes isolates collected from patients and from healthy carriers living in close contact with them. The clonal diversity of isolates was analyzed by pulsed-field gel electrophoresis with three restriction enzymes (SmaI, ApaI, and SacII). The pharynx of each patient and healthy carrier was colonized by a single clone, suggesting the clonal nature of streptococcal colonization in individuals. Among 52 isolates obtained from patients with acute pharyngotonsillitis, we found 14 genetically unrelated clones, showing the genetic diversity of S. pyogenes. However, two clones belonging to the M1 and M12 serotypes represented about 70% of isolates in carriers and patients. Pharyngeal colonization in cured patients was monitored for 3 to 4 months. After the initial elimination of S. pyogenes following antibiotic therapy, the rate of recolonization was high by day 30 (about 20%) and was also at that level between days 90 and 120; this was similar to the carriage rate in family contacts. Thus, cured patients can be recontaminated by unrelated clones, suggesting that colonization of healthy carriers might be a potential source of spread and redistribution of S. pyogenes isolates.

The epidemiology of acute paediatric osteoarticular infections (OAI) has recently evolved, mainly due to the improvement of microbiological diagnosis. We conducted a prospective study to analyse the recent epidemiology and the clinical evolution of paediatric OAI in order to validate the adequacy of our probabilistic first-line antibiotic treatment (intraveinous cefamandole + gentamicin). All children suspected of community acquired OAI were included and followed-up for 3 years. The etiologic diagnosis was based on blood cultures, joint aspirations and bone punctures. All osteoarticular (OA) samples were systematically inoculated into blood culture bottles. Real-time universal 16S rRNA and PCR targeted on Staphylococcus aureus, Kingella kingae, Streptococcus pneumoniae and Streptococcus pyogenes were performed twice a week. From 17 March 2007 to 26 February 2009, 98 septic arthritis, 70 osteomyelitis, 23 osteoarthritis and six spondylodiscitis were analysed. A portal of entry was suspected in 44% of cases, including 55% of otorhinolaryngological infections. C reactive protein was the most sensitive inflammatory marker. PCR increased by 54% the performance of bacteriological diagnosis. Among the patients completely investigated (blood culture and OAI samples), there were 63% documented OAI. The main pathogens found were K. kingae (52%), S. aureus (28%), S. pyogenes (7%), S. pneumoniae (3%) and Streptococcus agalactiae (2%). All isolated bacteria were sensitive to the probabilist treatment and outcome was favorable. PCR has significantly improved the performance and the delay of IOA diagnosis in children, for which K. kingae turned out to be the first causative agent. The probabilistic treatment was active against the main bacteria responsible for paediatric OAI.

Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640-12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.

We report on six cases of Pasteurella multocida (P. multocida) meningitis occurring between 2001 and 2011 by a French nationwide active surveillance network of paediatric bacterial meningitis (ACTIV/GPIP). The cases accounted for 0.15 % of the paediatric meningitis cases reported between 2001 and 2011 in France, all in infants

We studied the clonality of clinical isolates of Streptococcus pneumoniae 23F, the serotype most often associated with penicillin resistance in France. Clinical isolates obtained between November 1992 and April 1993 from nasopharyngeal samples from children with acute otitis media from different regions of the country were analyzed. The genetic polymorphism of penicillin-susceptible and -resistant 23F isolates (MIC, 2 mg/liter) was studied by pulsed-field gel electrophoresis. The resistant isolates were closely related, whereas the susceptible isolates were genetically heterogeneous. PCR amplification and restriction of the genes encoding penicillin-binding proteins (PBPs) 1A, 2B, and 2X also showed that the 24 resistant isolates had similar patterns which were very different from those of the susceptible isolates. All resistant isolates gave the same PBP pattern, with low affinities of PBPs for penicillin. Our results indicate that, in contrast to penicillin-susceptible 23F isolates, the penicillin-resistant 23F isolates have a single clonal origin, suggesting the rapid clonal spread of a resistant epidemic strain throughout the country.

A case of persistent bloodstream infection with Kocuria rhizophila related to a damaged central venous catheter in a 3-year-old girl with Hirschsprung's disease is reported. The strain was identified as K. rhizophila by 16S rRNA gene sequencing and matrix-assisted laser desorption ionization–time of flight mass spectrometry. Arbitrarily primed PCR analysis showed a clonal strain. The repeated septic episodes were resolved with the catheter repair.

Kingella kingae is an emerging osteoarticular pathogen in young children. Its isolation by traditional culture methods remains difficult, underscoring the need to implement other diagnostic methods for its detection and identification, such as nucleic acid amplification tests. Although the genome of this bacterium has not yet been sequenced, a toxin named RTX has been identified. The goal of this study was to develop sensitive, specific, and rapid molecular methods based on the rtxA toxin gene sequence to diagnose this infection. Two real-time PCR assays (SYBR green and TaqMan chemistries) targeting this gene are reported. Sensitivity and specificity were first evaluated successfully with 67 strains: 31 Kingella kingae isolates and 36 strains from other bacterial species. Then, 52 clinical specimens positive or negative by culture and/or PCR (16S rRNA and cpn60 genes) were tested with these assays. A nested PCR assay with subsequent sequencing was also developed to confirm the presence of Kingella kingae isolates in these clinical specimens. The results obtained demonstrate that these assays are accurate for the diagnosis of Kingella kingae infection.

Establishing the diagnosis of pulmonary infection in patients with hematological malignancies (HM) is a major challenge. Molecular diagnostic tools and antigen assays may improve diagnosis rates, although their performance has not been specifically evaluated in patients with HM. PCR is useful for circumventing the difficulties encountered in culturing atypical organisms (i.e., Mycoplasma pneumoniae and Chlamydophila pneumoniae). Similarly, the rapid PCR diagnosis of Legionella pneumophila in lung samples is a helpful complement to antigen detection in urine and may improve the detection of Legionella species other than L. pneumophila serogroup 1. Mycobacterium tuberculosis PCR can significantly shorten the time to diagnosis when direct detection is positive. PCR identification of pyogenic bacteria, particularly Streptococcus pneumoniae, can overcome the limited sensitivity of culture and antigen detection in urine, although standardized quantification is crucial to differentiate infection from contamination by the nasopharyngeal tract, and a thorough evaluation is therefore required. Universal PCR is valuable for detecting nontargeted bacteria in culture-negative samples, but has been evaluated only with pleural fluid. Finally, multiplex PCRs, by screening a large panel of microorganisms, are very promising techniques for one-step identification of viruses and atypical bacteria responsible for pulmonary infections. Urinary antigen detection is available only for L. pneumophila serogroup 1 and S. pneumoniae. Some studies obtained good results with S. pneumoniae antigen detection in pleural fluid and bronchoalveolar lavage fluid.

Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube [MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species.

Delays in the identification of microorganisms are a barrier to the establishment of adequate empirical antibiotic therapy of bacteremia. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) allows the identification of microorganisms directly from colonies within minutes. In this study, we have adapted and tested this technology for use with blood culture broths, thus allowing identification in less than 30 min once the blood culture is detected as positive. Our method is based on the selective recovery of bacteria by adding a detergent that solubilizes blood cells but not microbial membranes. Microorganisms are then extracted by centrifugation and analyzed by MALDI-TOF-MS. This strategy was first tested by inoculating various bacterial and fungal species into negative blood culture bottles. We then tested positive patient blood or fluid samples grown in blood culture bottles, and the results obtained by MALDI-TOF-MS were compared with those obtained using conventional strategies. Three hundred twelve spiked bottles and 434 positive cultures from patients were analyzed. Among monomicrobial fluids, MALDI-TOF-MS allowed a reliable identification at the species, group, and genus/family level in 91%, 5%, and 2% of cases, respectively, in 20 min. In only 2% of these samples, MALDI-TOF MS did not yield any result. When blood cultures were multibacterial, identification was improved by using specific databases based on the Gram staining results. MALDI-TOF-MS is currently the fastest technique to accurately identify microorganisms grown in positive blood culture broths.

BackgroundDevelopment of T-cells based-Interferon gamma (IFNgamma) assays has offered new possibilities for the diagnosis of latent tuberculosis infection (LTBI) and active disease in adults. Few studies have been performed in children, none in France. With reference to the published data on childhood TB epidemiology in the Paris and Ile de France Region, we considered it important to evaluate the performance of IGRA (QuantiFERON TB Gold In Tube(R), QF-TB-IT) in the diagnosis and the follow-up through treatment of LTBI and active TB in a cohort of French children.Methodology/principal findings131 children were recruited during a prospective and multicentre study (October 2005 and May 2007; Ethical Committee St Louis Hospital, Paris, study number 2005/32). Children were sampled at day 0, 10, 30, 60 (except Healthy Contacts, HC) and 90 for LTBI and HC, and a further day 120, and day 180 for active TB children. Median age was 7.4 years, with 91% of the children BCG vaccinated. LTBI and active TB children undergoing therapy produced significant higher IFNgamma values after 10 days of treatment (p = 0.035). In addition, IFNgamma values were significantly lower at the end of treatment compared to IFNgamma values at day 0, although the number of positive patients was not significantly different between day 0 and end of treatment.Conclusions/ significanceBy following quantitative IFNgamma values in each enrolled child with LTBI or active TB and receiving treatment, we were able to detect an increase in the IFNgamma response at day 10 of treatment which might allow the confirmation of a diagnosis. In addition, a decline in IFNgamma values during treatment makes it possible for clinicians to monitor the effect of preventive or curative therapy.

The identification of nonfermenting gram-negative bacilli isolated from cystic fibrosis (CF) patients is usually achieved by using phenotype-based techniques and eventually molecular tools. These techniques remain time-consuming, expensive, and technically demanding. We used a method based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the identification of these bacteria. A set of reference strains belonging to 58 species of clinically relevant nonfermenting gram-negative bacilli was used. To identify peaks discriminating between these various species, the profile of 10 isolated colonies obtained from 10 different passages was analyzed for each referenced strain. Conserved peaks with a relative intensity greater than 0.1 were retained. The spectra of 559 clinical isolates were then compared to that of each of the 58 reference strains as follows: 400 Pseudomonas aeruginosa , 54 Achromobacter xylosoxidans , 32 Stenotrophomonas maltophilia , 52 Burkholderia cepacia complex (BCC), 1 Burkholderia gladioli , 14 Ralstonia mannitolilytica , 2 Ralstonia pickettii , 1 Bordetella hinzii , 1 Inquilinus limosus , 1 Cupriavidus respiraculi , and 1 Burkholderia thailandensis . Using this database, 549 strains were correctly identified. Nine BCC strains and one R. mannnitolilytica strain were identified as belonging to the appropriate genus but not the correct species. We subsequently engineered BCC- and Ralstonia -specific databases using additional reference strains. Using these databases, correct identification for these species increased from 83 to 98% and from 94 to 100% of cases, respectively. Altogether, these data demonstrate that, in CF patients, MALDI-TOF-MS is a powerful tool for rapid identification of nonfermenting gram-negative bacilli.

Confirmation of infectious endophthalmitis is a major challenge. Traditional microbial tools are frequently unsuccessful mostly due to very small fluid samples and prior use of antibiotics. Here we report the first case of Streptococcus pneumoniae endophthalmitis diagnosed by polymerase chain reaction. Molecular biology should be considered as a new tool for the diagnosis of such sequestered sites as the eye.

OBJECTIVE. The objective of this study was to determine the incidence of multidrug-resistant bacteria in hospitalized children METHODS. This multicenter study was conducted in 5 hospitals in the Paris area from 1999 to 2003. We recorded all isolations of multidrug-resistant bacteria from clinical samples that were obtained from hospitalized children. Strains that were isolated during systematic screening for carriers were excluded. RESULTS. The mean incidences were 0.9 per 1000 hospitalization-days for methicillin-resistant Staphylococcus aureus, 0.45 for extended-spectrum β-lactamase–producing Klebsiella pneumoniae, 0.32 for extended-spectrum β-lactamase–producing Enterobacteriaceae other than Klebsiella pneumoniae, 0.40 for Enterobacter species with derepressed cephalosporinase, and 0.01 for vancomycin-resistant Enterococcus. The incidences per 1000 hospitalization-days of methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase–producing Klebsiella pneumoniae, extended-spectrum β-lactamase–producing Enterobacteriaceae other than Klebsiella pneumoniae, and Enterobacter species with derepressed cephalosporinase decreased significantly from 1999 to 2003, whereas the incidence of vancomycin-resistant Enterococcus remained very low. The proportion of resistant strains within the species did not vary significantly for methicillin-resistant Staphylococcus aureus (11% to 9.6%), extended-spectrum β-lactamase–producing Enterobacteriaceae other than Klebsiella pneumoniae (1.1%), and vancomycin-resistant Enterococcus (0.03% to 0.023%). In contrast, the frequency of extended-spectrum β-lactamase–producing Klebsiella pneumoniae decreased from 31.6% to 7.4%, and that of Enterobacter species with derepressed cephalosporinase decreased from 38.8% to 18.5%. CONCLUSIONS. We report significant decreases in the incidence of methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase–producing Klebsiella pneumoniae, extended-spectrum β-lactamase–producing Enterobacteriaceae other than Klebsiella pneumoniae, and Enterobacter species with derepressed cephalosporinase in hospitalized children during a 5-year period.

The chlorhexidine method was compared to the N -acetyl- l -cysteine-NaOH-oxalic acid decontamination method currently recommended for the recovery of nontuberculous mycobacteria (NTM) from patients with cystic fibrosis. Sputum samples ( n = 827) treated with chlorhexidine yielded twice as many NTM-positive cultures as those treated by the reference method (54 [6.50%] versus 27 [3.25%]; P < 0.0001) despite a higher contamination rate (20% versus 14.2%; P = 0.0017).