Postoperative spondylodiscitis due to Kytococcus schroeteri in a diabetic woman.

Kytococcus schroeteri, a Gram-positive coccus, is usually regarded as part of the human skin flora. It has been described in prosthetic valve endocarditis but never as being involved in osteoarticular infections. We report here the first case of a spondylodiscitis due to K. schroeteri identified by 16S rRNA gene sequencing.

Evaluation of a bedside immunotest to predict individual anti-tetanus seroprotection: a prospective concordance study of 1018 adults in an emergency department.

BACKGROUND: Unscheduled tetanus prophylaxis (UTP) used in the emergency room (ER) in patients with wounds who are unaware of their vaccination history is erroneous in 40% of cases. Evaluation of bedside tetanus immunity with the Tétanos Quick Stick (TQS) test may improve UTP. OBJECTIVES: To show that (1) a positive TQS result reflects immunity to tetanus; and (2) TQS is reproducible by ER workers. METHODS: In a prospective concordance study, immunity to tetanus of patients with wounds was assessed by two techniques: (1) TQS at the bedside, which detects specific tetanus antitoxins at concentrations > or =0.2 IU/ml in whole blood or > or =0.1 IU/ml in serum; (2) ELISA in the laboratory (threshold >0.1 IU/ml). The study comprised three groups: (A) healthcare personnel self-tested with the two techniques to determine the effect of training; (B) selected patients with wounds were double-tested with TQS by two healthcare providers whose readings were compared to test reproducibility; and (C) all patients with wounds aged > or =15 years were consecutively included. RESULTS: Of 1018 individuals included, 60 were in group A, 50 were in group B and 908 were in group C. 403 patients who were not included were similar to those included for age, vaccination history and types of wounds. The reproducibility of the test was 98%. TQS sensitivity was 83.0%, specificity 97.5%, positive predictive value 99.6% and negative predictive value 42.9%. CONCLUSIONS: TQS reliably predicts tetanus immunity and is reproducible by healthcare providers. Although it may not accurately discriminate between patients with ongoing and declining immunity, it is currently the most sensitive and specific tool for guiding tetanus prophylaxis and should be included in current guidelines on UTP.

Appearance of aac(6')-Ib-cr gene among extended-spectrum beta-lactamase-producing Enterobacteriaceae in a French hospital.

OBJECTIVES: The aac(6')-Ib gene encodes many variants of an aminoglycoside-acetyltransferase enzyme that is responsible for amikacin resistance. Recently, a new variant aac(6')-Ib-cr capable of modifying aminoglycosides and fluoroquinolones has been described. The aim of our study was to observe the appearance and the location of the aac(6')-Ib gene in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae strains. METHODS: Sixty-six and nine non-clonal ESBL-producing Enterobacteriaceae strains were isolated, respectively, for one 3-year period from 1999 to 2001 and one 2-month period in 2005 in a French Hospital (Paris, France). RESULTS: Among these isolates, 35 of them carried the aac(6')-Ib gene. Fourteen out of the aac(6')-Ib genes of the period 1 and two of the period 2 were genes cassette located within class 1 integrons, whereas 16 and 3, respectively, were outside integrons. One of these encoded an aminoglycoside-acetyltransferase enzyme leading to an acetyltransferase that confers resistance to all aminoglycosides. The new -cr variant of aac(6')-Ib was detected in three Escherichia coli isolates in 2005 always associated with CTX-M-15 enzyme. CONCLUSIONS: The aac(6')-Ib-cr gene, responsible for antibiotic resistance to two very different drugs, is emerging in ESBL-producing Enterobacteriaceae strains isolated in France especially in strains carrying the bla(CTx-M-15) gene.

Cervical necrotizing fasciitis: 8-years' experience of microbiology.

Cervical necrotizing fasciitis (CNF) is a life-threatening complication of pharyngeal or dental infections. The aim of this paper was to investigate whether dental or pharyngeal source result from different pathogen(s) in CNF and whether antibiotics, given before admission, influence the antimicrobial resistance of pathogens. In 152 CNF patients, Streptococcus milleri group and Prevotella species were the predominant isolates, frequently copathogens, mostly in dental CNF samples. Penicillin and clindamycin resistance were observed in 39% and 37% of cases, respectively, independently of any previous antibiotic therapy. Thus, a combined aerobe-anaerobe infection may have a synergistic effect, which allows the infection to spread in cervical tissues.

Improved diagnosis specificity in bone and joint infections using molecular techniques.

OBJECTIVES: The microbiological diagnosis of osteoarticular infections currently relies on microbiological cultures, to specifically target treatment. However, these conventional methods sometimes lack of sensitivity and of specificity to establish definitive diagnosis. This study was conducted to determine whether molecular method could improve bacterial bone and joint infection diagnosis. METHODS: We evaluated the performance of nucleic acid extraction with the semi-automated NucliSens miniMAG instrument coupled to 16S rDNA sequencing on 76 samples collected from 51 patients with suspected infections: prosthetic-joint infection (15), spondylodiscitis (7) acute septic arthritis (11) and 18 controls. No pre-treatment of the sample was done before nucleic acid extraction. Classification in infected group required an accumulation of arguments. RESULTS: Our molecular method identified a broad spectrum of pathogenic bacteria including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Salmonella enterica, Escherichia coli, Pseudomonas aeruginosa, and fastidious bacteria like Neisseria gonorrhoeae and Fusobacterium nucleatum. The overall PCR sensitivity was 73.3%: 53.8% for prosthetic-joint infections and 88.2% for infections without prostheses. The overall PCR specificity was 95.2%, whereas culture specificity was only 85.7%. CONCLUSIONS: The instrument was simple to use and provided nucleic acids free of PCR inhibitors and free of contamination by foreign bacterial DNA. Our study highlights the need for still improved molecular diagnostic sensitivity.

[A novel colorimetric test to study the susceptibility of human cytomegalovirus DNA polymerase to foscarnet]. / Développement d'un test colorimétrique pour tester la sensibilité de l'ADN polymérase du cytomégalovirus humain au foscarnet.

We described a colorimetric method to determine the biochemical phenotype of wild-type and mutated cytomegalovirus (HCMV) DNA polymerases by measuring the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain. Mutations V715M and E756K, which are known to confer foscarnet-resistance, were used as controls. Mutation N495K and a combination of changes K415R and S291P, both observed in foscarnet-resistant isolates, were studied. The mutations were introduced by site-directed mutagenesis into wild-type gene UL54 cloned in an expression vector and then polymerases were synthesised by using a commercially available coupled transcription-translation system. The polymerase activity was measured with and without foscarnet. The activity of polymerases containing the V715M or E756K mutations was inhibited by foscarnet at concentrations 70- and 30-fold higher than that of wild-type polymerase, respectively. Change N495K and combination of K415R and S291P, induced a five- and ten-fold decrease in susceptibility to foscarnet, respectively. The results of this non-radioactive assay were consistent with those obtained with the conventional radioactive assay. Therefore, this novel phenotypic method could be useful for the characterisation of mutations that confer HCMV resistance to foscarnet.

Selection during cefepime treatment of a new cephalosporinase variant with extended-spectrum resistance to cefepime in an Enterobacter aerogenes clinical isolate.

Enterobacter aerogenes resistant to cefepime (MIC, 32 microg/ml) was isolated from a patient treated with cefepime for an infection caused by a strain of E. aerogenes overproducing its AmpC beta-lactamase (MIC of cefepime, 0.5 microg/ml). The AmpC beta-lactamase of the resistant strain had an L-293-P amino acid substitution and a high k(cat)/K(m) ratio for cefepime. Both of these modifications were necessary for resistance to cefepime.

Clostridium difficile brain empyema after prolonged intestinal carriage.

Clostridium difficile, the most common cause of antibiotic-associated diarrhea, is occasionally isolated from extraintestinal sites and is usually found as part of a polymicrobial flora. We report a case of brain empyema that occurred after the recurrent intestinal carriage of a nontoxigenic strain of C. difficile. Brain abscess cultures contained both toxigenic and nontoxigenic isolates. Pulsed-field gel electrophoresis showed that nontoxigenic isolates from the intestine and from the brain were identical.

Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate.

Enterobacter cloacae CHE, a clinical strain with overproduced cephalosporinase was found to be highly resistant to the new cephalosporins, cefepime and cefpirome (MICs> or =128 microg ml(-1)). The strain was isolated from a child previously treated with cefepime. The catalytic efficiency of the purified enzyme with the third-generation cephalosporins, cefepime and cefpirome, was 10 times higher than that with the E. cloacae P99 enzyme. This was mostly due to a decrease in K(m) for these beta-lactams. The clinical isolate produced large amounts of the cephalosporinase because introduction of the ampD gene decreased ampC expression and partially restored the wild-type phenotype. Indeed, MICs of cefepime and cefpirome remained 10 times higher than those for a stable derepressed clinical isolate (OUDhyp) transformed with an ampD gene. Sequencing of the ampC gene showed that 18 nucleotides had been deleted, corresponding to the six amino acids SKVALA (residues 289--294). According to the crystal structure of P99 beta-lactamase, this deletion was located in the H-10 helix. The ampR-ampC genes from the clinical isolates CHE and OUDhyp were cloned and expressed in Escherichia coli JM101. The MICs of cefpirome and cefepime of E. coli harboring ampC and ampR genes from CHE were 100--200 times higher than those of E. coli harboring ampC and ampR genes from OUDhyp. This suggests that the deletion, confirmed by sequencing of the ampC gene, is involved in resistance to cefepime and cefpirome. However, the high level of resistance to cefepime and cefpirome observed in the E. cloacae clinical isolate was due to a combination of hyperproduction of the AmpC beta-lactamase and structural modification of the enzyme. This is the first example of an AmpC variant conferring resistance to cefepime and cefpirome, isolated as a clinical strain.

A one-year prospective study (1994-1995) for a first evaluation of tuberculosis transmission in French prisons.

SETTING: Ten correctional facilities in Paris, including suburbs. OBJECTIVE: To prospectively determine the incidence of tuberculosis (TB) in prisons during a one-year period and to trace the transmission of tuberculosis by restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis strains from inmates. RESULTS: Of 93 cases of tuberculosis observed, 50 were culture-confirmed. The incidence of tuberculosis in correctional facilities was 215 cases per 100,000 inmates. A high turnover of inmates was observed. All patients were male, and a quarter had been homeless. Seventy-two per cent were diagnosed with pulmonary tuberculosis. Several severe cases of TB were observed, including three of tuberculous meningitis. No multidrug-resistant strains were noted. RFLP analysis (n = 24) revealed 22 distinct patterns which made up two clusters. Epidemiological investigation did not show direct tuberculosis transmission, which was, however, probable for one cluster. CONCLUSION: Independently of incarceration, prison inmates run a higher risk of developing active tuberculosis than the general population, which might be the main reason for the high incidence of tuberculosis observed in prisons. However, some cases of transmission may occur inside prisons.

Bacterial populations contaminating the upper gut in patients with small intestinal bacterial overgrowth syndrome.

OBJECTIVE: Small intestinal bacterial overgrowth syndrome (SIBOS) is characterized by an abnormally high bacterial population level in the upper gut, exceeding 10(5) organisms/ml (5 log colony-forming unit (CFU)/ml). To understand its origin and select an appropriate antibiotic treatment, we have analyzed the bacterial populations contaminating the upper gut in SIBOS patients. METHODS: Jejunal samples of 63 consecutive patients with diarrhea or malabsorption and conditions predisposing to SIBOS were cultured and antibiotic sensitivities determined. RESULTS: Concentrations of total, microaerophilic, and anaerobic bacteria were confirmed in 55 patients with SIBOS (mean +/- SE) 7.6 +/- 0.8, 7.4 +/- 0.9, and 6.1 +/- 0.7 log CFU/ml, respectively. Mean number of bacterial genera was 4.6 +/- 0.8. The main bacteria recovered were (mean +/- SE log CFU/ml) Streptococcus (71%; 6.4 +/- 0.8), Escherichia coli (69%; 7.2 +/- 0.9), Staphylococcus (25%; 6.2 +/- 0.6), Micrococcus (22%; 6.0 +/- 0.7), Klebsiella (20%; 7.1 +/- 0.8), Proteus (11%; 6.1 +/- 0.8) for microaerophilic bacteria, and Lactobacillus (75%; 6.1 +/- 1.1), Bacteroides (29%; 6.9 +/- 1.3), Clostridium (25%; 5.5 +/- 1.0), Veillonella (25%; 5.3 +/- 0.7), Fusobacterium (13%; 4.8 +/- 0.5), and Peptostreptococcus (13%; 6.1 +/- 0.7) for anaerobic bacteria. Amoxicillin-clavulanic acid and cefoxitin were efficient on >90% of strains. CONCLUSIONS: Contaminating flora isolated in SIBOS include commonly identified oropharyngeal and colonic flora, but these occur in SIBOS at different levels from those usually found in their original location. These data may hopefully serve as a starting point to further therapeutic controlled studies.

Helicobacter pylori alters exogenous antigen absorption and processing in a digestive tract epithelial cell line model.

To study the influence of Helicobacter pylori on epithelial barrier function, bacteria, bacterial sonicates, or broth culture supernatants were incubated for 24 h with HT29-19A intestinal cells grown as monolayers. Subsequently, the monolayers were mounted in Ussing chambers, and electrical resistance (R), fluxes of 22Na (JNa) and 14C-mannitol (JMan) (markers of the paracellular pathway), and fluxes of horseradish peroxidase (HRP) in total (J3H-HRP), intact (JHRPi), and degraded forms were measured. H. pylori did not induce any modification of the paracellular pathway (R = 148 +/- 10 versus 174 +/- 16 Omega. cm2; JNa = 4.16 +/- 0.44 versus 3.51 +/- 0.41 microEq/h. cm2; JMan = 0.081 +/- 0.01 versus 0.058 +/- 0.009 micromol/h. cm2), nor did it modify J3H-HRP (2,201 +/- 255 versus 2, 110 +/- 210 ng/h. cm2 for H. pylori-infected and control cells, respectively). However, in the presence of H. pylori, we observed a significant increase in JHRPi (520 +/- 146 versus 171 +/- 88 ng/h. cm2). This effect was not dependent of the cag status of the strain and was not reproduced by the sonicates or the culture supernatants. It was related to the presence of urease, since a urease-negative mutant of H. pylori did not induce this effect. Ammonia and bafilomycin A1, two agents known to increase the endolysosomal pH, reproduced the increase in JHRPi. In conclusion, H. pylori does not affect directly the integrity of intercellular junctions of epithelial cells in vitro, but it increases the passage of intact HRP, probably by inhibition of the intralysosomal degradation due to the release of ammonia. The increased transport of intact macromolecules may contribute to the induction and maintenance of gastric inflammation by H. pylori.

Qualitative immunoglobulin abnormalities in HIV-positive patients: long-term follow-up.

We studied the immunoglobulins of a cohort of 212 HIV-positive patients followed-up for 13 years. The qualitative study of immunoglobulins by immunoelectrophoresis and immunofixation distinguished three groups of patients: those with monoclonal immunoglobulins, those with minor abnormalities of immunoglobulins and those with polyclonal immunoglobulins. We characterized these groups according to age, sex, immunoglobulin isotypes, and survival curves. The results show that this population of immunoglobulinopathies has distinctive characteristics. In particular, the presence of immunoglobulin abnormalities has no significant prognostic value.

Failure of ganciclovir treatment associated with selection of a ganciclovir-resistant cytomegalovirus strain in a lung transplant recipient.

We report the case of a lung transplant recipient with progressive cytomegalovirus (CMV) disease due to a resistant CMV strain emerging under ganciclovir (GCV) therapy. A discriminative polymerase chain reaction (PCR) assay, designed to detect the resistance-related V460 mutation within the viral enzyme UL97, revealed the presence of a mutated strain in a heterogeneous isolate 51 days after transplantation. The conventional antiviral susceptibility assay had failed to demonstrate resistance to GCV. Under prolonged GCV therapy, the mutated strain dominated the wild-type strain, as shown by the PCR assay. This domination led to laboratory resistance, associated with recurrent fever and progressively severe retinitis. As this discriminative PCR assay was shown to be effective in detecting mutated strains that constitute a minority in the virus load, it should allow better management of patients with CMV disease.

[Resistance of cytomegalovirus to ganciclovir: rapid detection of the mutations 460 of the UL97 phosphotransferase]. / Résistance du cytomégalovirus au ganciclovir: détection rapide des mutations 460 de la phosphotransférase UL97.

The substitution of methionine by either isoleucine or valine at residue 460 in the UL97 phosphotransferase has been shown to be responsible for resistance to ganciclovir (GCV) in 30% of resistant cytomegalovirus (CMV) isolates [4]. These substitutions require one nucleotide change in the gene (G- > T 1 380 and A- > G 1378 respectively). The aim of this study was to develop a discriminative PCR assay for rapid detection of these DNA changes. A PCR assay was duplicated in parallel for each mutation; to detect G- > T 1380 each reaction mixture contained primer VSUL14 and either primer LNW to distinguish wild type residues or LNM to distinguish mutant residues, and for A- > G 1378 primers were VSUL8 and either MCMW to detect wild type sequences or MCMM to detect mutated residues. For optimal discrimination, primers MCMW and MCMM were designed with a mismatch at position 3'-1. The reference strains AD169, Davis and Towne, a laboratory GCV-resistant mutant RCL1.7, and 33 CMV isolates (10 resistant, 2 indetermined and 21 sensitive) were tested by PCR. AD169, Davis and Towne, and 30 isolates were amplified only with non modified primers, and the absence of 460 mutations was confirmed by sequencing. Two isolates P1 and P2, from a transplanted patient were amplified with both MCMM and MCMW: sequencing analysis shown the presence of a mixture of strain, one of them harbouring A- > G 1378 mutation. One resistant strain was amplified neither with MCMM nor with MCMW: a C- > T silent mutation at nt 1368 was present. As sequencing analysis confirmed PCR results, discriminative PCR enables isolates to be rapidly assessed for the presence or absence of 460 mutations. Moreover, it can distinguish Met to Val from Met to Ile mutations, and allows the analysis of mixtures of sensitive ad resistant strains.

Value of a new rapid non-radioactive sequencing method for analysis of the cytomegalovirus UL97 gene in ganciclovir-resistant strains.

Various DNA changes located within a restricted region of the UL97 open reading frame were shown to be associated with the resistance of cytomegalovirus strains to ganciclovir (GCV). In order to analyse this UL97 region in sensitive and GCV-resistant strains, a non-radioactive sequencing assay (Promega, Madison, WI, USA) which combines the dideoxy visualisation by silver-staining of the gel was used. Using this assay, polymerase chain reaction products from results were obtained within 1 day. Point mutations modifying the amino acid sequence of the putative UL97 catalytic site were detected in three isolates. These led to an alanine to valine substitution in residue 594 in one strain with reduced GCV sensitivity, and to a cysteine to glycine substitution in residue 592 in two GCV-resistant isolates. These mutations were different from the DNA changes previously mapped in GCV-resistant laboratory or field strains. No amino acid substitution in the UL97 catalytic site was found in GCV-sensitive isolates. Transfer marker experiments are in progress in order to test the significance of these DNA changes for GCV resistance. This rapid non-radioactive sequencing protocol could be a useful tool for analysing the UL97 region encoding the putative UL97 catalytic site of clinical isolates.

Prevalence of HIV infections among patients attending a Parisian anonymous testing center between 1988 and 1993.

The prevalence of HIV infection was assessed among 15,611 consecutive patients attending a Parisian anonymous testing center from April 1988 to June 1993. Sera (17,910) were tested for the presence of anti-HIV antibodies using two different enzyme-linked immunosorbent assays. Seropositivity was verified by Western blotting. The sera were also assayed for HIV antigenemia detection in 2,493 cases. Six hundred and seventy-seven patients were found to be anti-HIV antibody positive: among them 666 were infected by HIV-1 and only 11 by HIV-2. Antigenemia was detected in 108 samples (4.3%). In all cases but 5, antigenemia was associated with the presence of specific antibodies. Risk factors for HIV infection could be determined for 5,735 patients. The HIV prevalence rates were 5.2% in 1988-89, 4.9% in 1990, 3.4% in 1991, 2.8% in 1992 and 1.8% for the 6 first months of 1993 (p < 0.01). Only one patient was coinfected with HTLV-1. This study shows a trend of decreasing seropositivity rates among the patients attending the anonymous testing center since 1990. By contrast, the percentage of seropositive patients with antigenemia was stable between 1988 and 1993.