Paediatric epidemiology of Pasteurella multocida meningitis in France and review of the literature.

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 <4 months old. A review of the literature allowed us to gather information on 42 other cases of P. multocida meningitis in infants <1 year old reported since 1963. Among all 48 cases, 44 % were newborns. An animal source of the infection, including 39 household dogs and cats, was suspected or identified in 42 of 48 cases. A traumatic contact between the child and a pet occurred in 8 % of cases, and a vertical transmission from mother to child during birth in 10.4 %. Most of the time, the infection resulted from non-traumatic contact between the child and the pet, through licking or sniffing. The absence of host risk factors suggests that an immature immune system is responsible, given the young age of the children. Although complications, especially neurological lesions, were not rare (37.5 %), the long-term outcome was usually good. Four infants died of meningitis. This rare disease could be prevented by reducing contact between infants and household pets, and by performing simple hygiene measures before handling babies.

Evaluation of the Andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing Gram-positive bacilli.

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.

The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo.

The intracellular parasite Listeria monocytogenes is able to induce its internalization by cultured mammalian cells that are not normally phagocytic. This process requires the expression of the chromosomal locus inlAB. We studied the virulence of an inlAB mutant and of its parent in murine listeriosis. Irrespective of the route of inoculation, the inlAB mutant was severely attenuated for growth in the liver. The livers of mice inoculated with the inlAB mutant displayed much smaller infectious foci than the parent as early as 24 h after infection. Electron microscopy showed that these foci consisted of a few inflammatory cells, with few bacteria; bacteria were rarely found within hepatocytes. In contrast, foci in livers of mice inoculated with the parent consisted of islets of heavily infected hepatocytes that were infiltrated by numerous neutrophils; bacteria seemed intact within hepatocytes and damaged within neutrophils. A direct role of inlAB for the entry of L. monocytogenes into hepatocytes was confirmed in a cell infection system using the murine embryonic hepatocyte cell line TIB73. The inlAB mutant was approximately 20-fold less invasive in trans. The "invasion locus" inlAB contributes to protect L. monocytogenes from the host's innate defense mechanisms by promoting its entry into hepatocytes.

Non-H-2 restriction of expression of passively transferred delayed sensitivity.

The results of this study of allogeneic restriction of passively transferred delayed sensitivity to Listeria antigens serve to illustrate the complexity of in vivo models. They show that the H-2 restriction observed when delayed-type hypersensitivity was transferred between H-2-congenic strains was no more severe than the restriction observed when delayed-type hypersensitivity was transferred between parental and F1 mice and between different strains sharing the same H-2 haplotype. It is obvious that genes, in addition to those of the H-2 locus, can be responsible for allogeneic restriction in vivo.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Detection of circulating Aspergillus fumigatus DNA by real-time PCR assay of large serum volumes improves early diagnosis of invasive aspergillosis in high-risk adult patients under hematologic surveillance.

Detection of galactomannan antigen (GMA) in serum is the standard assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematological disorders. Detection of Aspergillus DNA in serum has been proposed, but its sensitivity is lower than that of GMA when small serum volumes (SSV) are used. In this study, we investigated whether extraction of DNA from large serum volumes (LSV) improves diagnostic yield. In a 13-month prospective study, we compared the performances of twice-weekly screening of serum for GMA by an enzyme immunoassay and weekly screening for Aspergillus fumigatus DNA by a real-time PCR (RT-PCR) assay of 1.0 ml (LSV) or 100 mul (SSV) of serum. We included 124 patients (138 treatment episodes), with 17 episodes of EORTC (European Organization for Research and Treatment of Cancer)/MSG (Mycoses Study Group)-documented IA. In all, 1,870 samples were screened for GMA. The sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV, respectively) of GMA for IA were 88.2%, 95.8%, 75%, and 98.3%, respectively. We screened 938 samples for Aspergillus DNA by using LSV; 404 of these samples were also tested with SSV. The Se, Sp, PPV, and NPV of RT-PCR were 100%, 96.7%, 81%, and 100%, respectively, with LSV and 76.5%, 96.7%, 81.3%, and 95.6%, respectively, with SSV. DNA detection gave a positive result when performed on LSV in two cases of IA where the GMA assay result remained negative. Furthermore, in four IA cases, DNA was detected earlier than GMA. The use of LSV for extraction improved the performance of the RT-PCR, which appears highly sensitive and specific for the early diagnosis of IA in high-risk patients with hematological disorders.

The threat of smallpox and bioterrorism.

Smallpox (variola) was a devastating disease with a high case-fatality rate. Although the disease was eradicated in 1977, the remaining stocks of smallpox virus constitute one of the most dangerous threats to humanity. The smallpox virus is highly specific for humans and non-pathogenic in animals. There is no antiviral treatment and a vaccine is active only if administered in the first four days post-exposure. Smallpox virus represents a potential biological weapon that could be used by terrorists, and the destruction of stocks raises political, social, scientific and ethical issues.

Evaluation of the quality of hospital food from the kitchen to the patient.

Food-borne pathogens can multiply if food is not maintained at an appropriate temperature and if there are delays between food preparation and distribution. The aim of this study was to evaluate the quality of meals during transport from the kitchens to the patients in three departments of a university hospital. Meals were transported inside insulated, cooled food carts. We analysed the delays at each step of the transport process, and measured the temperature inside the food cart and inside the meals. The total duration of the transport (mean=85.3 min; range 44-123 min) conformed to the official recommendations (<2 h at a temperature <10 degrees C before consumption). The internal temperature of 73.6% of the 30 food carts followed was below 10 degrees C. The internal temperature of the meals was below 10 degrees C in 91.7% of cases when the food cart was first opened, but in only 12% of cases by the time the last patient was served. No pathogens were isolated from any of the samples. However, 10% of meals, all of which were salads, had total viable counts of bacteria above the recommended limits. This study confirms that it is essential to control time and temperature to ensure food quality and safety in hospitals.

Listeria monocytogenes skin infection with cerebritis and haemophagocytosis syndrome in a bone marrow transplant recipient.

In this report, we describe unusual and unreported manifestations of Listeria monocytogenes infection in a bone marrow transplant recipient, including cutaneous infection with an hamophagocytosis syndrome and cerebritis. L. monocytogenes occurred despite a broad spectrum antibiotherapy. L. monocytogenes was isolated from a skin biopsy. Outcome was favorable with amoxicillin and gentamicin therapy. L. monocytogenes infection should be suspected in patients with cerebritis despite large spectrum antibiotherapy and this report underscores the usefulness of skin biopsies in febrile immunocompromised patients.

[Opportunistic infections caused by Shewanella, new emergent bacteria]. / Les infections à Shewanella, un pathogène opportuniste émergent.

Shewanella putrefaciens and Shewanella algae are Gram negative, nonfermentative and oxidative bacilli whose the main phenotypic feature is the production of hydrogen sulfide gas. Widespread in the environment, both S. putrefaciens and S. algae species are rare human bacteria although they are reported with increasing frequency as a cause of opportunistic infection in humans, such as skin and soft tissue infections and bacteremia. Chronic infections of the lower limbs and liver disease have been identified as risk factors for bloodstream infection, with a faster course and a poorer prognosis in the last case. S. algae appears to be more virulent than S. putrefaciens. Most human S. putrefaciens strains are isolated from bacterial flora, which puts to question its clinical significance. Molecular biology must be used for an adequate identification because S. algae can easily be mistaken for S. putrefaciens with usual tests.

The rfaD locus: a region of rearrangement in Vibrio cholerae O139.

We analyzed the rfaD locus of the novel epidemic Vibrio cholerae strain O139, a putative region of rearrangement. This region includes 4 orfs in the same orientation. Two orfs, rfaD(O139) and orf2(O139) were almost identical to those described in V. cholerae O1. In contrast, the two other orfs upstream from rfaD(O139), designated orfA(O139) and orfB(O139), were absent from V. cholerae O1, but present in environmental strains of V. cholerae O22, O141 and O155. These results suggest that a chromosomal rearrangement might have occurred in the vicinity of rfaD in V. cholerae O1, resulting in the emergence of V. cholerae O139. The putative source of exogenous DNA might have been V. cholerae O22, O141 and O155.

Molecular characterization and expression analysis of the superoxide dismutase gene from Streptococcus agalactiae.

We have cloned and sequenced a 3103-bp DNA fragment carrying the gene encoding the Mn-SOD from Streptococcus agalactiae NEM318 serotype III. This DNA fragment contained four orfs that have the same polarity of transcription. Orf1 was truncated by molecular cloning and the corresponding 228-aa-long polypeptide did not exhibit any significant homology with other cognate proteins. Orf2 encodes a protein of 345 aa that displays some similarity (29% identity) with the YqeN peptide of Bacillus subtilis, the function of which is unknown. Orf3 encodes the 202-aa-long Mn-SOD which was functionally expressed in Escherichia coli. Orf4 was also truncated by molecular cloning and encodes 99 aa of the N-terminal moiety of a protein that displays significant homology (40% f identity) with the antiterminator LicT of B. subtilis. Transcriptional analysis revealed that the sodA gene of S. agalactiae NEM318 was transcribed monocistronically from a promoter, the activity of which is neither regulated by pH, O2, nor CO2 concentrations of the culture medium. Analysis by high resolution agarose gel electrophoresis of the AluI DNA polymorphism of the sodA locus in wild-type strains of S. agalactiae belonging to serogroups I, II, or III revealed no detectable difference.

Clinical efficacy and tolerability of cefixime in the treatment of acute sinusitis.

In a noncomparative trial, 73 adults with acute sinusitis confirmed by x-ray received cefixime 400mg once daily for approximately 10 days. At the end of treatment, 60 patients (82%) were cured, 2 (2.7%) had improved and 7 (9.6%) had failed therapy; 4 patients were not evaluable. No relapses were observed at follow-up. Haemophilus influenzae, Streptococcus pneumoniae and Branhamella (Moraxella) catarrhalis were the main pretreatment pathogens, accounting for 65% of all bacterial isolates. Overall, 84% of pathogens were eradicated after treatment. Cefixime was well tolerated, moderate gastrointestinal disturbances being the most frequent adverse effects noted (3 of 4 patients with adverse effects). These results are comparable to those obtained with cefixime 400mg administered orally in 2 divided doses.

Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition.

A panel of 103 Listeria monocytogenes strains of different origins was examined for haemolysin and lecithinase production in brain-heart infusion (BHI). Three distinct phenotypes were observed. Phenotype 1 was characterized by low to undetectable levels of expression and was exhibited by almost all strains tested. Phenotype 2 expressed high levels of haemolysin and lecithinase and was displayed by five strains: one (P14-A) was a spontaneous mutant derived from a type 1 isolate (P14); the four others (EGD-A, NCTC 7973, SLCC 2373 and CLIP 545) were all laboratory strains kept under in vitro conditions for a long period. Phenotype 3 was intermediate and was exhibited by another laboratory strain (L028). We therefore concluded that phenotype 1 corresponded to the wild type, whereas phenotypes 2 and 3 represented mutant or variant phenotypes. Interestingly, wild-type strains were able to dramatically increase the expression of virulence factors when cultured in BHI treated with activated charcoal (BHIC), up to levels similar to those constitutively expressed by the hyperhaemolytic/lecithinase variants in BHI. Experiments with P14 and P14-A demonstrated that both charcoal and the hyperhaemolytic/lecithinase mutation exerted their effect by inducing (or derepressing) transcription of prfA, the pleiotropic transcriptional activator of the L. monocytogenes virulence regulon. Moreover, P14 and P14-A were equally virulent for mice despite the different levels of virulence factor expression in BHI. Taken together, these observations indicate that L. monocytogenes turns off virulence gene expression when growing in vitro in a rich medium, and suggest that the increased levels of virulence factors in the hyperhaemolytic/lecithinase mutants and in wild-type strains grown in BHIC might represent the levels of expression needed in vivo by L. monocytogenes for infecting host tissues.

Optimization of green fluorescent protein expression vectors for in vitro and in vivo detection of Listeria monocytogenes.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule for monitoring in vivo gene expression in eukaryotic and prokaryotic cells. We constructed a series of GFP vectors for in situ detection of the intracellular pathogen Listeria monocytogenes. The gfp-mutl gene, which encodes a red-shifted GFP, was transcriptionally fused to a strong L. monocytogenes promoter and inserted into various Escherichia coli-Listeria shuttle vectors: i) the integrative monocopy plasmid pAT113; ii) the low copy number plasmid pTCV-Exl; iii) the high copy number plasmid pAT18. Listeria cells harboring pNF6 and pNF7, constructed from pAT113 and pTCV-Exl, respectively, gave low fluorescence intensities, and were optically detected in cultured macrophages, but not in tissue sections. The fluorescence of Listeria with the pAT18 derivative pNF8 was about 40 times greater than that with pNF6 and 15 times greater than that with pNF7. Listeria cells harboring pNF8 were readily detected in both cultured macrophages and tissue sections. Constructed GFP vectors did not affect the virulence of L. monocytogenes in a murine model of infection.

Pathogens isolated during treatment failures in otitis.

OBJECTIVES: A prospective study in the Paris region to evaluate the clinical and bacteriologic epidemiology of acute otitis media in infants in whom oral antibiotic therapy resulted in clinical failure. METHODS: The study included 186 children with a mean age of 17.5 +/- 13.1 months. Two-thirds of them attended a day-care center and 40.8% had a history of recurrent otitis media. The most frequently prescribed prior antibiotics were amoxicillin-clavulanic acid (43% of cases), an oral third generation cephalosporin (22.6%), erythromycin-sulfisoxazole (11.8%) and a first generation cephalosporin (10.2%). The average duration of antibiotic therapy was 6.9 +/- 2.65 days. Specimens for bacterial cultures included 188 samples of middle ear fluid obtained by tympanocentesis and 37 collected from otorrhea fluid. RESULTS: One hundred forty-one samples (62.7%) from 126 children yielded 170 bacterial isolates. In 60 children (32.3%) the culture of the ear pus was sterile. Among the 170 bacterial isolates: 67 (39.4%) were Streptococcus pneumoniae (59 patients), of which 77.6% had reduced susceptibility to penicillin (PRSP with penicillin MIC > or = 0.125 mg/l); 61 (35.9%) were Haemophilus influenzae (56 patients) of which 49.2% were beta-lactamase producers; and 8 were Moraxella catarrhalis (8 patients), of which 87.5% were beta-lactamase producers. Thirty-six patients were infected by S. pneumoniae with penicillin MIC > or =1 mg/l. In our study attending day-care center (P = 0.04), temperature >38 degrees C with signs of otalgia (P = 0.02), age <2 years (P = 0.048) and prior antibiotic treatment with erythromycin-sulfisoxazole (P = 0.006) were independently predictive risk factors for patients infected with penicillin-resistant S. pneumoniae. Pneumococcal serogroups 23, 14 and 19 were predominant (25.4, 25.4 and 23.8%, respectively). Penicillin resistance was mainly associated with serogroups 23 and 14. CONCLUSIONS: Penicillin-resistant S. pneumoniae isolates are frequently responsible for therapeutic failure in cases of acute otitis media in the Paris region.

Evaluation of nasopharyngeal cultures for bacteriologic assessment of acute otitis media in children.

BACKGROUND: The recent emergence of penicillin-resistant Streptococcus pneumoniae, particularly in acute otitis media (AOM), has increased interest in the development of noninvasive procedures that might help to predict the bacterial etiology of this condition. We conducted an open multicenter study to evaluate the predictive value of the nasopharyngeal (NP) sampling in children with AOM by comparing the bacteriologic results of NP cultures with those of pus collected by myringotomy in the same patients. METHODS: The NP secretions and the pus obtained by myringotomy were collected concomitantly in 354 children younger than 6 years of age with clinical signs of AOM. The clinical usefulness of NP culture was determined by calculating its sensitivity and specificity, and especially its positive and negative predictive values for the three main pathogens responsible for AOM, Haemophilus influenzae, S. pneumoniae and Moraxella catarrhalis. RESULTS: A positive NP culture was found to have little predictive value for H. influenzae (52%), S. pneumoniae (43%) and M. catarrhalis (19%). In contrast the negative predictive value of NP cultures was much greater and was accompanied by negative middle ear fluid cultures in more than 95% of children, especially for S. pneumoniae. Furthermore the incidence of beta-lactamase-producing strains of H. influenzae at both sampling sites was similar (30 and 35%, respectively), as was the incidence of penicillin-resistant S. pneumoniae (50 and 54%). CONCLUSION: It appears that the correlation between results of NP and middle ear fluid cultures in children with AOM is too weak to allow NP culture to be recommended for the bacteriologic documentation of this disease. However, these results should not overshadow the considerable epidemiologic value of NP cultures, particularly with reference to the monitoring of pneumonococcal susceptibility in children. The collection of NP cultures should therefore be promoted for their collective epidemiologic value.