The Streptococcus agalactiae Exonuclease ExoVII Is Required for Resistance to Exogenous DNA-Damaging Agents.

Streptococcus agalactiae is a human pathogen responsible for severe invasive infections in newborns. In this bacterium, XseB, a part of the ExoVII exonuclease, was shown to be specifically more abundant in the hypervirulent ST-17 strains. In Escherichia coli, ExoVII is associated either with mismatch repair or with recombinational DNA repair and is redundant with other exonucleases. In this study, the biological role of S. agalactiae ExoVII was examined. The ΔexoVII mutant strain was subjected to different DNA-damaging agents, as well as a large set of mutants impaired either in the mismatch repair pathway or in processes of recombinational DNA repair. Our results clarified the role of this protein in Gram-positive bacteria as we showed that ExoVII is not significantly involved in mismatch repair but is involved in bacterial recovery after exposure to exogenous DNA-damaging agents such as ciprofloxacin, UV irradiation, or hydrogen peroxide. We found that ExoVII is more particularly important for resistance to ciprofloxacin, likely as part of the RecF DNA repair pathway. Depending on the tested agent, ExoVII appeared to be fully redundant or nonredundant with another exonuclease, RecJ. The importance of each exonuclease, ExoVII or RecJ, in the process of DNA repair is thus dependent on the considered DNA lesion. IMPORTANCE This study examined the role of the ExoVII exonuclease of Streptococcus agalactiae within the different DNA repair processes. Our results concluded that ExoVII is involved in bacterial recovery after exposure to different exogenous DNA-damaging agents but not in the mismatch repair pathway. We found that ExoVII is particularly important for resistance to ciprofloxacin, likely as part of the RecF DNA repair pathway.

Defining the Role of the Streptococcus agalactiae Sht-Family Proteins in Zinc Acquisition and Complement Evasion.

Streptococcus agalactiae is not only part of the human intestinal and urogenital microbiota but is also a leading cause of septicemia and meningitis in neonates. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including the transition metal ion zinc. The primary zinc acquisition system of the pathogen is the Adc/Lmb ABC permease, which is essential for viability in zinc-restricted environments. Here, we show that in addition to the AdcCB transporter and the three zinc-binding proteins, Lmb, AdcA, and AdcAII, S. agalactiae zinc homeostasis also involves two streptococcal histidine triad (Sht) proteins. Sht and ShtII are required for zinc uptake via the Lmb and AdcAII proteins with apparent overlapping functionality and specificity. Both Sht-family proteins possess five-histidine triad motifs with similar hierarchies of importance for Zn homeostasis. Independent of its contribution to zinc homeostasis, Sht has previously been reported to bind factor H leading to predictions of a contribution to complement evasion. Here, we investigated ShtII to ascertain whether it had similar properties. Analysis of recombinant Sht and ShtII reveals that both proteins have similar affinities for factor H binding. However, neither protein aided in resistance to complement in human blood. These findings challenge prior inferences regarding the in vivo role of the Sht proteins in resisting complement-mediated clearance.IMPORTANCE This study examined the role of the two streptococcal histidine triad (Sht) proteins of Streptococcus agalactiae in zinc homeostasis and complement resistance. We showed that Sht and ShtII facilitate zinc homeostasis in conjunction with the metal-binding proteins Lmb and AdcAII. Here, we show that the Sht-family proteins are functionally redundant with overlapping roles in zinc uptake. Further, this work reveals that although the Sht-family proteins bind to factor H in vitro this did not influence survival in human blood.

Detection of Bartonella in cat scratch disease using a single-step PCR assay kit.

PURPOSE: Bartonella is an increasingly isolated emerging pathogen that can cause severe illness in humans, including cat scratch disease (CSD). The bacteria are difficult to grow and thus many detection methods have been developed, especially molecular. We previously developed a PCR method targeting ribC to identify Bartonella sp. A manufactured kit (RealCycler BART, Progenie Molecular) was commercialised shortly thereafter for the detection of Bartonella infection, including Bartonella henselae. METHODOLOGY: We performed a comparison between this test and our in-house PCR assay on 73 lymphadenopathy samples sent to the laboratory for suspicion of CSD.Results/Key findings. Among the 28 positive samples for Bartonella, 21 were identified by the two PCR assays, and seven by the commercial kit only. CONCLUSION: The performance of this commercial kit suggests that it could be a suitable alternative to our in-house PCR assay, highlighting the importance of the molecular methods used to diagnose CSD.

[Prevalence of extended-spectrum beta-lactamases of the CTX-M type producing Escherichia coli and Klebsiella pneumoniae in Bretonneau hospitals (CHRU Tours)]. / Prévalence des souches de Escherichia coli et de Klebsiella pneumoniae productrices de beta-lactamases à spectre étendu de type CTX-M à l'hôpital Bretonneau (CHRU de Tours).

The CTX-M type extended spectrum beta-lactamases constitute a rapidly growing cluster of enzymes that have disseminated geographically. This study evaluates the prevalence of these enzymes in the university hospital in Tours, in 2007. Twenty-eight strains were studied: 21 Escherichia coli and seven Klebsiella pneumoniae. The gene bla(CTX-M) was detected by real-time PCR for 27 strains. The CTX-M-1 group, including CTX-M-15, was the most frequent CTX-M-type enzyme.

[PCR rDNA 16S used for the etiological diagnosis of blood culture negative endocarditis]. / PCR rDNA 16S dans le diagnostic étiologique des endocardites infectieuses à hémocultures négatives.

We report the case of a 55 year-old man presenting with a double aortic and mitral endocarditis for which resected valve culture was repeatedly negative. Specific PCR made on valves because of highly positive blood tests for Bartonella henselae remained negative. A molecular approach was made with 16S rDNA PCR, followed by sequencing. Bartonella quintana was identified as the etiology of endocarditis. B. quintana, "fastidious" bacteria, even if hard to identify in a laboratory, is often reported as a blood culture negative endocarditis (BCNE) agent. Molecular biology methods have strongly improved the diagnosis of BCNE. We propose a review of the literature focusing on the interest of broad-spectrum PCR on valve for the etiological diagnosis of BCNE.

A Listeria monocytogenes strain is still virulent despite nonfunctional major virulence genes.

The low-virulence Listeria monocytogenes strains have been previously assigned to 4 phenotypic groups. This study aimed to characterize the A23 strain, which exhibits a pulsed-field gel electrophoresis profile specific to low-virulence strains. This strain has the same causal mutations as the group III strains and a supplementary mutation in the mpl gene, leading to the absence of internalin A expression and the presence of inactive internalin B, phosphatidyl-inositol phospholipase C, and phosphatidylcholine phospholipase C. Despite these mutations in major virulence genes, the A23 strain formed plaques in cell monolayers and contaminated 100% of inoculated mice, suggesting that it evolved from group III strains by acquiring new virulence genes.

[Pediatric osteoarticular infections caused by Kingella kingae from 1995 to 2006 at CHRU de Tours]. / Infections ostéo-articulaires pédiatriques à Kingella kingae de 1995 à 2006 au CHRU de Tours.

Use of molecular biology shows that Kingella kingae is a pathogen frequently involved in osteoarticular infections in young children. This study describes the cases of osteoarticular infections due to K. kingae which happened from 1995 to 2006 in the CHRU of Tours. The description is based on clinical and biological features. A K. kingae specific polymerase chain reaction was performed in our laboratory in order to improve K. kingae osteoarticular infections diagnosis, and is detailed here.

Application of the French guidelines for preventing neonatal group B streptococcal disease in a university hospital.

This study evaluated the application of the French guidelines for prevention of neonatal group B streptococcus (GBS) infections. The prevalence of GBS vaginal carriage by pregnant women during the study period was 6%. Less than 50% of pregnant women testing positive for GBS were treated with at least two doses of antibiotics during labour, and most received only one dose or no antibiotics. In addition, several neonates were colonised or infected by GBS although their mothers were GBS-negative. These results are consistent with vaginal screening having a poor sensitivity, as suggested by the low prevalence of GBS carriage.

[Molecular identification of species within the Mycobacterium tuberculosis complex using regions of difference]. / Identification moléculaire des espèces de mycobactéries du groupe de la tuberculose par étude des régions de différence.

The differentiation within the Mycobacterium tuberculosis complex (MTBC) based on phenotypic methods is long and does not give an unambiguous result in every case whereas the advance in genetic knowledge leads to new views. Thus, regions of difference (RD), that seem to characterize the species of the MTBC, have been identified. Amplification methods, targeted on these zones, have been developed then. The study of four regions (RD1, RD5, RD9, RD10) has been done on 64 isolates formerly identified thanks to phenotypic methods. Genotypic results confirm phenotypic identifications except in one case. This strain initially identified as M. tuberculosis and isolated from a Gabonese patient, corresponds, according to genotypic identification, to M. africanum. Since phenotypic characterization of M. africanum is difficult, this method would allow to accurately determine the true prevalence of this specie. Moreover, the study of RD10 doesn't seem to be informative. The amplification of only two RD, RD1 and RD9 carries out the identification of all M. tuberculosis and M. bovis BCG isolates. M. bovis and M. africanum will be then identified thanks to RD5. Thus, this easy and rapid method of identification of the major species of MTBC seems to be appropriated for a routine use.

[Systematic screening tests for Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in urogenital specimens of infertile couples]. / Existe-t-il un bénéfice au dépistage systématique de Chlamydia trachomatis, Mycoplasma hominis et Ureaplasma urealyticum dans les prélèvements génito-urinaires réalisés au cours d'un bilan d'infertilité?

We conducted a prospective study on 100 couples consulting for infertility at the teaching Hospital of Tours, with the scope to determine if there is a benefit for systematic screening of Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum among genito-urinary specimen when exploring couples infertility. C. trachomatis was detected by PCR on sperm, endocervix and urine specimen. M. hominis and U. urealyticum were detected by culture on A7 agar medium and with minigaleries on sperm and endocervix specimen. Standard cultures were also performed on sperm, endocervix, vaginal and urine specimen. Only one specimen (sperm) was positive for C. trachomatis. Three percent of the specimen were positive for U. urealyticum (from which 2,5% of the sperm specimen). No specimen was positive for M. hominis. Our results show that screening of C. trachomatis, M. hominis and U. urealyticum is not systematically required for among check up of infertile couples, given the prevalence of chlamydiosis among the population studied. However, it would be interesting to perform it on a targeted population, according to anamnestic or clinical criteria. In addition, an important modification of vaginal flora was observed in 12% of cases, and 2 vaginosis were diagnosed; the putative consequences of this disequilibrium has to be further investigated.

Outbreak in France of Neisseria meningitidis B:15:P1.12 belonging to sequence type 1403.

This report describes a meningococcal outbreak in France caused by Neisseria meningitidis B:15:P1.12 of sequence type 1403, which affected eight young patients, between November 2000 and February 2002. Epidemiological typing confirmed that a single strain was responsible. Favourable outcome, sequelae or death resulted in similar proportions as in other cases of meningococcal disease in France during the same period, but purpura was observed in all eight cases. The patients were aged between 14 and 28 years, whereas the median age of patients affected by other meningococcal strains during this period in the same area was 60.4 years.

Spread of Stenotrophomonas maltophilia colonization in a pediatric intensive care unit detected by monitoring tracheal bacterial carriage and molecular typing.

In our pediatric intensive care unit in Tours (France), intubated and ventilated inpatients are systematically monitored for tracheal bacterial colonization twice a week. This led us to detect five patients colonized with Stenotrophomonas maltophilia over a 4-month period. Molecular typing of the isolates using random amplified polymorphism DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) confirmed that four of the five isolates were genetically related. The strict isolation of carriers and improvements in hygiene measures stopped the spread. This systematic strategy prevented pulmonary nosocomial infections or allowed their early detection. Moreover, it has made it possible to assess the efficiency of care practices continuously.

Pseudomonas aeruginosa and cystic fibrosis: correlation between exoenzyme production and patient's clinical state.

In this study, we investigated the correlation between the production by Pseudomonas aeruginosa isolates of four exoenzymes (protease, elastase, neuraminidase, and phospholipase C (PLC)) and the clinical state of cystic fibrosis (CF) patients. We studied 212 P. aeruginosa isolates from 22 CF patients chronically infected with this bacterium. Patients were classified into three clinical groups according to a modified Shwachman-Kulczycki-Khaw (SKK) scoring system. The production of enzymes by isolates from patients in the three populations was analyzed and compared using four statistical tests: chi-square, Mann-Whitney U, principal component analysis, and discriminant analysis. Isolates from patients with excellent or good clinical status (group I, SKK score >/=71) had higher elastase and neuraminidase activities than isolates from the other patients. In contrast, PLC activity, a common characteristic of CF isolates, was higher in isolates from patients with poor or weak clinical status (group III, SKK score

Identification of new phages to type Staphylococcus aureus strains and comparison with a genotypic method.

The aim of this study was to optimize the epidemiological monitoring of strains of Staphylococcus aureus, a major cause of hospital-acquired infections. From September to December 1998 47 S. aureus strains isolated from swabs taken from orthopaedic and trauma patients were in studied. Thirty-five isolates were sensitive to methicillin (MSSA) and 12 were methicillin-resistant (MRSA). Ten of the 47 isolates could not be phage-typed using the international set of typing phages: five of these isolates were MSSA and five were MRSA. These MRSA isolates, which were also not typeable by the phages currently recommended for phage-typing MRSA, were lysed by locally isolated experimental phages 584 and 1814. Phage 1814 lysed the gentamicin-resistant MRSA and phage 584 acted on the gentamicin-sensitive MRSA. Both new phages were inactive against the methicillin-sensitive isolates. Cloning of certain isolates was confirmed by macrorestriction genomic profiles obtained by pulsed-field gel electrophoresis analysis (PFGE). The results showed good discriminatory ability of antibiotic-resistance pattern phenotyping and phage-typing when the phages used were adapted to epidemic-associated MRSA strains.

Staphylococcus lugdunensis infections: high frequency of inguinal area carriage.

Following a change in surgical practice, we noted that the rate at which Staphylococcus lugdunensis was isolated from samples from the plastic surgery unit of our hospital increased considerably. We investigated the sources of these S. lugdunensis strains, and we found that in the case of drain colonization or surgical site infection, the strain was more likely to have come from the patient's skin bacteria when the pubic site had been shaved preoperatively. To test the hypothesis of pubic site colonization, we evaluated the prevalence of S. lugdunensis carriage among the cutaneous flora of the inguinal area. We found that 22% of 140 incoming patients carried S. lugdunensis in this area and that carriage at both inguinal folds was frequent (68% of carriers). A study of the genetic structure of the total population, including the clinical (n = 18) and the commensal (n = 53) strains, revealed that the diversity of the species was low and that the population was composed of two major groups that diverged at a distance of 35%. No particular characteristics made it possible to distinguish between clinical and commensal strains. Only isolates producing beta-lactamase were homogeneous; six of the eight beta-lactamase-positive strains displayed the same pulsed-field gel electrophoresis pattern.

Genetic relationship between Escherichia coli strains isolated from the intestinal flora and those responsible for infectious diseases among patients hospitalized in intensive care units.

The exact origin of strains of Escherichia coli responsible for infectious diseases in intensive care units (ICUs) remains partly unknown. Our aim was to determine the nature of the link between strains from the intestinal flora of hospital staff, strains from the intestinal flora of patients hospitalized in ICUs and strains isolated from ICU patients with invasive diseases. For this purpose, 77 strains of E. coli were genetically characterized by exploring their entire genomes by random amplified polymorphism of DNA (RAPD), and by determining their phylogenetic position in ECOR (E. coli reference) groups, the virulence factors harboured (pap, sfa, afa, hly, aer and cnf) and their ability to mutate. The strains isolated from the intestinal flora of hospital staff were found to constitute a genetically heterogeneous population compared with the strains isolated from ICU carriers, which were highly clustered. The latter strains harboured numerous virulence factors, and 80% belonged to the group ECOR B2. The strains isolated from infected patients harboured fewer virulence factors than those from the ICU carriers, and only half belonged to ECOR B2. Moreover, these strains were more genetically related to strains from hospital staff than to strains from ICU carriers. Thus, the exogenous origin of the E. coli strains is probably almost as important as translocation from intestinal flora in ICUs. Moreover, a strong mutator phenotype had a minor, or no, role in the rapid adaptation to modifications in the ecological environment.

Combined ribotyping and random multiprimer DNA analysis to probe the population structure of Listeria monocytogenes.

To improve our understanding of the genetic links between strains originating from food and strains responsible for human diseases, we studied the genetic diversity and population structure of 130 epidemiologically unrelated Listeria monocytogenes strains. Strains were isolated from different sources and ecosystems in which the bacterium is commonly found. We used rRNA gene restriction fragment length polymorphism analysis with two endonucleases and random multiprimer DNA analysis with seven oligonucleotide primers to study multiple genetic features of each strain. We used three clustering methods to identify genetic links between individual strains and to determine the precise genetic structure of the population. The combined results confirmed that L. monocytogenes strains can be divided into two major phylogenetic divisions. The method used allowed us to demonstrate that the genetic structure and diversity of the two phylogenetic divisions differ. Division I is the most homogeneous and can easily be divided into subgroups with dissimilarity distances of less than 0.30. Each of these subgroups mainly, or exclusively, contains a single serotype (1/2b, 4b, 3b, or 4a). The serotype 4a lineage appears to form a branch that is highly divergent from the phylogenetic group containing serotypes 1/2b, 4b, and 3b. Division II contains strains of serotypes 1/2a, 1/2c, and 3a. It exhibits more genetic diversity with no peculiar clustering. The fact that division II is more heterogeneous than division I suggests that division II evolved from a common ancestor earlier than division I. A significant association was found between division I and human strains, suggesting that strains from division I are better adapted to human hosts.

Evaluation of the international phage typing set and some experimental phages for typing of Listeria monocytogenes from poultry in Spain.

AIMS: The validity of the international phage set and 13 experimental phages for subtyping Listeria monocytogenes strains isolated from poultry in Spain was investigated. METHODS AND RESULTS: Ninety-six L. monocytogenes strains (52 from serogroup 1/2 and 44 from serogroup 4) were phage-typed using the international phage set, 10 experimental phages for typing serogroup 1/2 strains (seven isolated in France: 1313, 9425, 1807, 351, 881, 717 and 586-, and three from Denmark: 5775, 12682 and 6223-) and three experimental phages isolated in France for typing serogroup 4 strains (2425 A, 4286 and 197). Percentages of serogroup 1/2, serogroup 4 and total phage-typeable strains were 57.7%, 52.3% and 55.2%, respectively. Important differences in the behaviour of the phages tested were found. The typeability rate, the specificity index and the percentage of strong reactions were greater in the phages of international set than in the experimental phages. The number of phage typeable strains and the number of phage types (42) were not modified by the use of experimental phages. CONCLUSIONS: The phage set used was not effective for typing L. monocytogenes strains from poultry in Spain, because a low typeability rate was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest the importance of the availability of new phages specific to a geographical area in order to improve the typeability of the system.

Low sensitivity of Listeria monocytogenes to quaternary ammonium compounds.

Ninety-seven epidemiologically unrelated strains of Listeria monocytogenes were investigated for their sensitivities to quaternary ammonium compounds (benzalkonium chloride and cetrimide). The MICs for seven serogroup 1/2 strains were high. Three came from the environment and four came from food; none were isolated from human or animal samples. All 97 strains carried the mdrL gene, which encodes a multidrug efflux pump, and the orfA gene, a putative transcriptional repressor of mdrL. The absence of plasmids in four of the seven resistant strains and the conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid borne. Moreover, PCR amplification and Southern blot hybridization experiments failed to find genes phylogenetically related to the qacA and smr genes, encoding multidrug efflux systems previously described for the genus Staphylococcus. The high association between nontypeability by phages and the loss of sensitivity to quaternary ammonium compounds are suggestive of an intrinsic resistance due to modifications in the cell wall.