Proficiency program for real-time PCR diagnosis of Bordetella pertussis infections in French hospital laboratories and at the French National Reference Center for Whooping Cough and other Bordetelloses.

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 IS481 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/microl (0.2 to 2 CFU/microl). 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.

Invasive group B streptococcal infections in infants, France.

Clinical features and molecular characterization of 109 group B streptococci causing neonatal invasive infections were determined over an 18-month period in France. Sixty-four percent of the strains were from late-onset infections, and 75% were capsular type III. The hypervirulent clone ST-17 was recovered in 80% of meningitis cases.

Performance of chromID ESBL, a chromogenic medium for detection of Enterobacteriaceae producing extended-spectrum beta-lactamases.

The chromogenic agar medium chromID ESBL (bioMérieux) was compared with BLSE agar medium (AES) for selective isolation and presumptive identification of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae from clinical samples. A total of 765 samples (468 rectal swabs, 255 urine samples and 42 pulmonary aspirations) obtained from 547 patients was processed. All bacterial strains isolated on either medium were further characterized using biochemical tests, and ESBL producers were confirmed by synergy testing. Genetic characterization of ESBL genes was determined by PCR. A total of 33 ESBL-producing Enterobacteriaceae strains [Escherichia coli (n=16), Klebsiella pneumoniae (n=8), Enterobacter spp. (n=3), Citrobacter spp. (n=5) and Proteus mirabilis (n=1)] was recovered. The sensitivity after 24 h incubation was 88 % for chromID ESBL and 85 % for BLSE agar. At 48 h, the sensitivity of chromID ESBL increased to 94 % and was higher than that obtained with BLSE agar. The positive predictive value at 24 h for chromID ESBL was 38.7 % [95 % confidence interval (95 % CI) 28.3 -50.2 %)], which was significantly higher than that for BLSE agar [15.4 %, 95 % CI 10.1 -21.5 %]. On both media, false-positive results were mostly due to Pseudomonas aeruginosa and to Enterobacteriaceae overproducing chromosomal cephalosporinase (Enterobacter spp.) or a chromosomal penicillinase (Klebsiella oxytoca). This study showed that chromID ESBL, a ready-to-use chromogenic selective medium, is sensitive and specific for rapid, presumptive identification of ESBL-producing Enterobacteriaceae. Its chromogenic properties and its selectivity are particularly useful in specimens containing resident associated flora.

Comparative evaluation of Strepto B ID chromogenic medium and Granada media for the detection of Group B streptococcus from vaginal samples of pregnant women.

Two types of selective media, the chromogenic medium Strepto B ID and two non-chromogenic media Strepto B agar and the Granada medium, were tested and compared to blood agar plates (BAP) for screening of Group B streptococcus vaginal colonization in pregnant women. All tested media were comparable in terms of sensitivity however, their use in routine laboratories may markedly facilitate the rapid detection of GBS in vaginal samples.

Pertussis and respiratory syncytial virus infections.

During the winter 2005-2006, all infants <4 months of age admitted for bronchiolitis or acute respiratory tract infection in a tertiary care pediatric hospital in Paris were tested for respiratory syncytial virus (RSV) and pertussis with real-time polymerase-chain reaction (RT-PCR). A positive pertussis-PCR was found in 14/90 (16%) infants infected with RSV and in 5/30 negative for RSV. Similar clinical symptoms were found in all RSV-positive infants with or without pertussis co-infection. Most infants (73%) were not vaccinated against pertussis, and the other children had received one or two injections. In conclusion, pertussis-RSV co-infection is common in young infants, and pertussis-PCR should be used, whenever available.

Rapid detection of the "highly virulent" group B Streptococcus ST-17 clone.

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.

Comparative evaluation of 5 different selective media for Group B Streptococcus screening in pregnant women.

We compared the performances and the cost-effectiveness of 5 selective media for Group B Streptococcus (GBS) screening in vaginal samples from pregnant women. The usefulness of these media is unquestionable for GBS screening; the choice will depend largely on the laboratory organization.

Association between Staphylococcus aureus alone or combined with Pseudomonas aeruginosa and the clinical condition of patients with cystic fibrosis.

BACKGROUND: The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in cystic fibrosis (CF) patients has increased and MRSA seems to be associated with a poorer prognosis. The aim of this study was to assess the prevalence and clinical consequences of MRSA and methicillin-susceptible Staphylococcus aureus (MSSA), associated or not associated with Pseudomonas aeruginosa (PA). METHODS: In a retrospective study on 419 sputum producer patients (293 adults and 126 children >7 years of age), we recorded patient characteristics, lung function, nutritional status, i.v. antibiotics and hospitalisations, the presence of SA and/or PA and FEV1 decline over 2 years. RESULTS: SA was found in 72% of the patients: MSSA in 68.2% of children and 48.8% of adults; MRSA in 17.5% of children and 17.8% of adults. Sixty percent of MRSA patients and 60.4% of MSSA patients also harboured PA. The rate of deterioration of clinical status of the various groups, as assessed from respiratory function, i.v. antibiotic courses and hospitalisations, increased in the order: no SA/no PA, MSSA alone, MRSA alone, MSSA/PA, MRSA/PA, and PA alone. Nutritional status did not differ between groups. Results were roughly similar for children and adults. The yearly FEV1 decline was significantly higher only for MRSA/PA patients (p=0.03) compared to no SA/no PA patients. CONCLUSION: Clinical condition of CF patients with MSSA only or MRSA only appeared similar, whereas MRSA/PA patients had more severe respiratory function than MSSA/PA patients. In CF patients, MRSA might be more deleterious than MSSA only when associated with PA.

Methicillin-resistant Staphylococcus aureus expressing low-level methicillin resistance may not be detected by the VITEK2® system.

Low-level methicillin-resistant Staphylococcus aureus may be difficult to detect with the VITEK® 2 system (VK2). Here, we suggest that S. aureus exhibiting VK2-oxacillin MIC of 1 or 2 mg/L and a negative cefoxitin screen should be tested for the presence of mecA or its gene product.

[PCR testing for Bordetella pertussis in household contacts as a diagnostic tool for atypical whooping cough in unvaccinated young infants]. / La recherche de Bordetella pertussis par PCR chez les parents permet de diagnostiquer la coqueluche atypique chez les jeunes nourrissons non vaccinés.

INTRODUCTION: False-negative findings of polymerase chain reaction (PCR) for genuine pertussis as well as the numerous atypical forms of whooping cough make it difficult to diagnose this disease in young babies. METHODS: For two years, real-time PCR was performed to test for Bordetella pertussis in 86 infants younger than 6 months hospitalized for apnea or paroxysmal and/or vomiting cough and in 205 of their household contacts, whether or not they coughed. RESULTS: Group 1 included 30 infants for whom PCR detected B. pertussis (25 of whom were also RSV+). PCR was also positive for at least one household contact in 25/30 families. This group included 16 babies with apnea and 12 who developed a whooping cough during follow-up. Group 2 comprised 12 infants whose PCR was negative while at least one household contact had positive results. Five of these infants had severe apnea and 6 developed a whooping cough. Group 3 included 44 infants (28 RSV +) for whom PCR was negative in the index case and in the household contacts: none developed a whooping cough during follow-up. Only 3 of the 54 positive household contacts had a paroxysmal cough or a typical whooping cough and 12 had no cough at all. CONCLUSION: Positive PCR in a household contact, symptomatic or not, is helpful for the diagnosis of atypical whooping cough in young infants.

Ertapenem resistance of Escherichia coli.

An ertapenem-resistant Escherichia coli isolate was recovered from peritoneal fluid in a patient who had been treated with imipenem/cilastatin for 10 days. Ertapenem resistance may be explained by a defect in the outer membrane protein and production of extended-spectrum beta-lactamase CTX-M-2.

Comparative evaluation of VITEK 2 for antimicrobial susceptibility testing of group B Streptococcus.

OBJECTIVES: Intrapartum antibiotic prophylaxis is recommended to prevent neonatal group B streptococcal (GBS) disease in colonized women, and penicillin or aminopenicillin constitute the first-line antibiotics. Most isolates are resistant to tetracycline, and resistance to macrolide-lincosamide-streptogramin (MLS) antibiotics is increasing. Therefore, laboratory testing for MLS resistance in GBS is now recommended for penicillin-allergic patients. The aim of this study was to compare the antimicrobial susceptibility of GBS as determined by the VITEK 2 system (bioMérieux, Marcy l'Etoile, France), agar diffusion methods and PCR-genotypic detection of resistance genes. METHODS: One hundred and ten unrelated selected GBS clinical isolates were studied. The antibiotics tested (VITEK 2 and agar diffusion method) were benzylpenicillin, ampicillin, erythromycin, clindamycin, co-trimoxazole, tetracycline, kanamycin, streptomycin and vancomycin. A standardized double-disc (DD) diffusion test was performed for MLS antibiotics. Genotypic characterization of tetracycline, MLS and aminoglycoside resistance genes was performed by PCR. RESULTS: All strains were susceptible to benzylpenicillin, ampicillin and vancomycin [category agreement (CA) between VITEK 2 and the diffusion method was 100%]. Ninety-five (86%) strains were resistant to tetracycline (CA was 98.9%). Eighty-one strains (73.6%) harboured an MLS resistance phenotype; 50 (61.8%) an MLS(B)-constitutive phenotype, 25 (30.8%) an MLS(B)-inducible phenotype and 6 (7.4%) an M phenotype. The agreement between data of VITEK 2 and the DD diffusion test for the detection of MLS(B)-constitutive, MLS(B)-inducible and M phenotype isolates was 76%, 36% and 100%, respectively. Almost all discrepancies were due to failure to detect erythromycin resistance by VITEK 2. CONCLUSIONS: VITEK 2 allows accurate determination of GBS susceptibility for the majority of antibiotics, but has to be improved for erythromycin. Thus, the DD diffusion test remains the most simple and reliable method for macrolide resistance detection among this streptococcal species.

Multiplex PCR assay for rapid and accurate capsular typing of group B streptococci.

We developed a simple, specific, and sensitive two-multiplex-PCR assay that enabled the detection of all known group B streptococcal (GBS) capsular polysaccharides. This test is well adapted for GBS capsular polysaccharide typing in large-scale epidemiological studies.

A hypermutator phenotype attenuates the virulence of Listeria monocytogenes in a mouse model.

The integrity of the genetic material of bacteria is guaranteed by a set of distinct repair mechanisms. The participation of these repair systems in bacterial pathogenicity has been addressed only recently. Here, we study for the first time the participation in virulence of the MutSL mismatch repair system of Listeria monocytogenes. The mutS and mutL genes, which are contiguous in the L. monocytogenes chromosome, were identified after in silico analysis. The deduced MutS shares 62% identity with MutS of Bacillus subtilis and 50% identity with HexA, its homologue in Streptococcus pneumoniae; MutL shares 59% identity with MutL of B. subtilis and 47% identity with HexB of S. pneumoniae. Functional analysis of the mutSL locus was studied by constructing a double knock-out mutant. We showed that the deletion DeltamutSL induces: (i) a 100- to 1000-fold increase in the spontaneous mutation rate; and (ii) a 10- to 15-fold increase in the frequency of transduction, thus demonstrating the role of mutSL of L. monocytogenes in both mismatch repair and homologous recombination. We found that the deletion DeltamutSL moderately affected bacterial virulence, with a 1-log increase in the lethal dose 50% (LD50) in the mouse. Strikingly, repeated passages of the mutant strain in mice reduced virulence further. Competition assays between wild-type and mutant strains showed that the deletion DeltamutSL reduced the capacity of L. monocytogenes to survive and multiply in mice. These results thus demonstrate that, for the intracellular pathogen L. monocytogenes, a hypermutator phenotype is more deleterious than profitable to its virulence.

Identification of LpeA, a PsaA-like membrane protein that promotes cell entry by Listeria monocytogenes.

The intracellular life of Listeria monocytogenes starts by a complex process of entry involving several bacterial ligands and eukaryotic receptors. In this work, we identified in silico from the sequence of the genome of L. monocytogenes a previously unknown gene designated lpeA (for lipoprotein promoting entry) encoding a 35-kDa protein homologous to PsaA, a lipoprotein belonging to the LraI family and implicated in the cell adherence of Streptococcus pneumoniae and related species. By constructing a mutant of L. monocytogenes in which lpeA is deleted (lpeA mutant), we show that the PsaA-like protein LpeA is not involved in bacterial adherence but is required for entry of L. monocytogenes in eukaryotic cells. In contrast to wild-type bacteria, mutant bacteria failed to invade the epithelial Caco-2 and hepatocyte TIB73 cell lines, as confirmed by confocal microscopy. The mutant bacteria rapidly penetrated in mouse bone marrow-derived macrophages. Surprisingly, lpeA mutant bacteria survive better in macrophages than do wild-type bacteria. This was correlated with a weak exacerbation of virulence of the lpeA mutant in the mouse. LpeA is therefore a novel invasin favoring the entry of L. monocytogenes into nonprofessional phagocytes but not its invasion of macrophages. This is the first report of a lipoprotein promoting cell invasion of an intracellular pathogen.

The sortase SrtA of Listeria monocytogenes is involved in processing of internalin and in virulence.

Listeria monocytogenes is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in Staphylococcus aureus that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of L. monocytogenes a gene, designated srtA, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the srtA mutation on cell wall anchoring of this LPXTG protein in L. monocytogenes. We show that the srtA mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that srtA of L. monocytogenes acts as a sortase and plays a role in the pathogenicity.

Differential roles of multiple signal peptidases in the virulence of Listeria monocytogenes.

Most bacteria contain one type I signal peptidase (Spase I) for cleavage of signal peptides from exported and secreted proteins. Here, we identified a locus encoding three contiguous Spase I genes in the genome of Listeria monocytogenes. The deduced Sip proteins (denoted SipX, SipY and SipZ) are significantly similar to SipS and SipT, the major SPase I proteins of Bacillus subtilis (38% to 44% peptidic identity). We studied the role of these multiple signal peptidases in bacterial pathogenicity by constructing a series of single- and double-chromosomal knock-out mutants. Inactivation of sipX did not affect intracellular multiplication of L. monocytogenes but significantly reduced bacterial virulence (approximately 100-fold). Inactivation of sipZ impaired the secretion of phospholipase C (PC-PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD(50) of 10(8.3)), inactivation of sipY had no detectable effects. Most importantly, a mutant expressing only SipX was impaired in intracellular survival and strongly attenuated in the mouse (LD(50) of 10(7.2)), whereas, a mutant expressing only SipZ behaved like wild-type EGD in all the assays performed. The data establish that SipX and SipZ perform distinct functions in bacterial pathogenicity and that SipZ is the major Spase I of L. monocytogenes. This work constitutes the first report on the differential role of multiple Spases I in a pathogenic bacterium and suggests a possible post-translational control mechanism of virulence factors expression.

Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape of Listeria monocytogenes.

Lipoproteins of Gram-positive bacteria are involved in a broad range of functions such as substrate binding and transport, antibiotic resistance, cell signaling, or protein export and folding. Lipoproteins are also known to initiate both innate and adaptative immune responses. However, their role in the pathogenicity of intracellular microorganisms is yet poorly understood. In Listeria monocytogenes, a Gram-positive facultative intracellular human pathogen, surface proteins have important roles in the interactions of the microorganism with the host cells. Among the putative surface proteins of L. monocytogenes, lipoproteins constitute the largest family. Here, we addressed the role of the signal peptidase (SPase II), responsible for the maturation of lipoproteins in listerial pathogenesis. We identified a gene, lsp, encoding a SPase II in the genome of L. monocytogenes and constructed a deltalsp chromosomal deletion mutant. The mutant strain fails to process several lipoproteins demonstrating that lsp encodes a genuine SPase II. This defect is accompanied by a reduced efficiency of phagosomal escape during infection of eucaryotic cells, and leads to an attenuated virulence. We show that lsp gene expression is strongly induced when bacteria are still entrapped inside phagosomes of infected macrophages. The data presented establish, thus, that maturation of lipoproteins is critical for efficient phagosomal escape of L. monocytogenes, a process temporally controlled by the regulation of Lsp production in infected cells.