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In this study, the binding of F components of the staphylococcal bicomponent leukotoxins Panton-Valentine leucocidin (LukF-PV) and gamma-hemolysin (HlgB) on polymorphonuclear neutrophils (PMNs), monocytes, and lymphocytes was determined using labeled mutants and flow cytometry. Leukotoxin activity was evaluated by measuring Ca 2+ entry or pore formation using spectrofluorometry or flow cytometry. Although HlgB had no affinity for cells in the absence of an S component, LukF-PV had high affinity for PMNs (dissociation constant [ K d ], 6.2 ± 1.9 nM; n = 8), monocytes ( K d , 2.8 ± 0.8 nM; n = 7), and lymphocytes ( K d , 1.2 ± 0.2 nM; n = 7). Specific binding of HlgB was observed only after addition of LukS-PV on PMNs ( K d , 1.1 ± 0.2 nM; n = 4) and monocytes ( K d , 0.84 ± 0.31 nM; n = 4) or after addition of HlgC on PMNs, monocytes, and lymphocytes. Addition of LukS-PV or HlgC induced a second specific binding of LukF-PV on PMNs. HlgB and LukD competed only with LukF-PV molecules bound after addition of LukS-PV. LukF-PV and LukD competed with HlgB in the presence of LukS-PV on PMNs and monocytes. Use of antibodies and comparisons between binding and activity time courses showed that the LukF-PV molecules that bound to target cells before addition of LukS-PV were the only LukF-PV molecules responsible for Ca 2+ entry and pore formation. In contrast, the active HlgB molecules were the HlgB molecules bound after addition of LukS-PV. In conclusion, LukF-PV must be linked to LukS-PV and to a binding site of the membrane to have toxin activity.

Resume Introduction Les infections communautaires a Staphylococcus aureus resistant a la meticilline (SARM) sont un probleme emergent. Ces derniers sont parfois secreteurs de leucocidine de Panton-Valentine potentiellement responsable de pneumopathies necrosantes severes. Nous avons determine la prevalence des SARM et, parmi ces derniers, de ceux producteurs de leucocidine de Panton-Valentine, chez les consultants externes d’un service de dermatologie afin d’adapter, eventuellement, nos pratiques therapeutiques. Malades et methodes Il s’agissait d’une etude prospective incluant les sujets ayant consulte du 1 er mars 2005 au 31 decembre 2006 et chez lesquels un prelevement bacteriologique cutaneomuqueux a ete realise. Les principaux facteurs de risque d’acquisition de SARM etudies etaient les antecedents de consultations regulieres en milieu hospitalier, d’hospitalisation ou d’antibiotherapie dans les trois derniers mois et la vie en collectivite. D’autres facteurs de risque ont ete recueillis de maniere plus aleatoire : antecedent de voyage a l’etranger recent, de lesion similaire, facteur d’immunodepression, toxicomanie ou diabete. Resultats Deux cent trente-cinq prelevements ont ete realises chez 122 malades, 143 sur les lesions et 92 dans les narines. Un S. aureus a ete isole chez 68 de ces malades (56 %). Douze malades etaient porteurs de SARM (17,6 %) : il s’agissait de sept malades « ambulants » (10,3 %) et de cinq malades « consultants reguliers » (7,3 %). Les taux de resistance des SARM aux antibiotiques etaient de 64 % pour l’ofloxacine, 36 % pour l’amikacine et l’erythromycine, 27 % pour l’acide fusidique, 9,1 % pour l’association sulfamethoxasole–trimethoprime et 0 % pour la pristinamycine. Dans notre etude, seule la vie en collectivite etait un facteur de risque significatif d’etre confronte a un SARM ( p = 0,045). Quatre souches de SARM sur les 11 testees etaient secretrices de leucocidine de Panton-Valentine. Conclusion Les dermatologues sont desormais souvent confrontes a des infections cutanees a SARM. Les prelevements bacteriologiques devraient etre systematiques et les antibiotherapies probabilistes dirigees contre les SARM dans les infections severes.

Resume Objectif Streptococcus agalactiae (streptocoque du groupe B) est une cause importante d'infection invasive chez les adultes en dehors de la grossesse, touchant plus particulierement les personnes âgees ayant des facteurs favorisants sous-jacents. Nous avons cherche a decrire ces facteurs favorisants et les caracteristiques cliniques de ces infections dans notre hopital. Methode Soixante-quatre dossiers d'infections invasives a S. agalactiae de patients hospitalises entre janvier 1997 et janvier 2006 ont ete analyses retrospectivement. Resultats La moyenne d'âge des patients etait de 59 ± 17 ans. Le sex-ratio etait de 1,06. Au moins un antecedent etait trouve pour 90,6 % des patients. Le diabete (43,7 %), une arterite des membres inferieurs (34,4 %), une cardiopathie ischemique (20,3 %), une neoplasie (20,3 %), representaient les principaux antecedents. L'index moyen de comorbidite de Charlson etait de 2,5 ± 2. Les manifestations cliniques comprenaient : les infections urinaires hautes (32,8 %), les infections de la peau et des tissus mous (25 %), les osteoarthrites (21,9 %). Une bacteriemie survenait dans 31,2 % des cas, sans point de depart pour deux patients. Nous rapportons deux cas d'endocardite, un cas de meningite et quatre deces survenus dans le premier mois. Conclusion Malgre le caractere retrospectif de notre etude, nous avons pu decrire avec precision l'ensemble du spectre clinique des infections invasives a S. agalactiae. Nous confirmons l'importance du role du terrain sous-jacent dans l'emergence de cette pathologie et la gravite des infections.

Les infections de la periode perinatale sont contractees soit pendant la grossesse, soit pendant la periode de l’accouchement, soit parfois, plus de trois jours apres l’accouchement. Chez la mere, ces infections peuvent souvent etre evitees: Sur la base de donnees anamnestiques ou cliniques, une surveillance complementaire (soit plus frequente, soit vis-a-vis d’autres bacteries) peut etre realisee. Chez l’enfant, le probleme qui se pose est celui du diagnostic precoce de l’infection neonatale et de son traitement immediat. Le laboratoire de bacteriologie joue un role essentiel en effectuant l’analyse d’echantillons obtenus dans l’heure qui suit la naissance. Le liquide d’aspiration gastrique est un prelevement essentiel, car il est le reflet de la situation prevalantin utero dans le liquide amniotique. L’examen microscopique de ce liquide permet d’orienter rapidement le diagnostic d’infection neonatale. Ce prelevement est complete par un ou deux autres prelevements periorificiels. En dehors des bacteries associees a des infections sexuellement transmises, les bacteries a rechercher sont essentiellementStreptococcus agalactiae (groupe B),Listeria monocytogenes, Haemophilus influenzae etEscherichia coli. Le role pathogene d’Ureaplasma urealyticum est suspecte en cas de dysplasie broncho-pulmonaire chez les nouveau-nes hypotrophes.

Resume L'examen cyto-bacteriologique des urines est l'une des analyses microbiologiques les plus demandees. De nombreux parametres pre-analytiques (prelevement, transport) et analytiques (examen microscopique, tests rapides de depistage, culture quantitative) interviennent pour le diagnostic bacteriologique de l'infection urinaire.

Resume Introduction La borreliose europeenne, connue du public sous le nom de maladie de Lyme par analogie avec la forme americaine dont elle differe par de nombreux points, est endemique en Alsace, posant un probleme de sante publique. Le niveau de connaissance et de prevention de la population vis-a-vis de cette maladie n’a jamais ete evalue en France. Materiel et methodes Une enquete a ete realisee dans tous les centres d’examens de sante de la Securite Sociale d’Alsace a l’aide d’un auto-questionnaire. Les donnees recueillies portaient sur les caracteristiques socio-demographiques, les connaissances sur la maladie, les antecedents de piqures de tiques, la crainte suscitee par cette maladie, la frequence des sejours en foret, les habitudes de prevention lors de sorties en foret et l’attitude adoptee en cas de piqure de tique. Resultats Sur les 600 sujets inclus, 99 (16,5 p. 100) avaient ete piques une ou plusieurs fois par des tiques depuis un an. L’existence de la maladie de Lyme etait connue par 74 p. 100 des consultants, 63 p. 100 disaient en eprouver une crainte et 43 p. 100 savaient que sa premiere manifestation est une rougeur qui s’etend sur la peau. Vingt-sept pour cent s’habillaient d’un pantalon et de manches longues lors de sejours en foret et 19 p. 100 s’inspectaient le corps entier au retour. Les sujets les moins informes etaient egalement ceux qui se premunissaient le moins contre les piqures de tique. Il s’agissait des moins de 30 ans, des moins diplomes et des citadins. Les sujets se rendant frequemment en foret pour leurs loisirs et ceux ayant des antecedents de piqures etaient les mieux informes et avaient de meilleures habitudes de prevention. La crainte suscitee par la maladie etait associee a de meilleures connaissances et a un comportement mieux adapte. Discussion Cette etude montre qu’une proportion elevee de la population alsacienne est exposee aux piqures de tiques. Les borrelioses par piqure de tiques sont assez bien connues mais leur prevention demeure insuffisante. Une meilleure information est associee a une meilleure prevention. Les campagnes d’information et d’education de la population generale pourraient viser particulierement les jeunes, les citadins et les moins diplomes. Des messages specifiques de prevention pourraient egalement etre delivres aux sujets les plus exposes, c’est a dire ceux piques par des tiques ou se rendant souvent en foret, sur leurs lieux de consultation ou de loisirs.

The kinetics of infection and the pathogenic effects on the reproductive function of laboratory mice infected withBartonella birtlesiirecovered from anApodemusspecies are described.B. birtlesiiinfection, as determined by bacteremia, occurred in BALB/c mice inoculated intravenously. Inoculation with a low-dose inoculum (1.5 × 103CFU) induced bacteremia in only 75% of the mice compared to all of the mice inoculated with higher doses (≥1.5 × 104). Mice became bacteremic for at least 5 weeks (range, 5 to 8 weeks) with a peak ranging from 2 × 103to 105CFU/ml of blood. The bacteremia level was significantly higher in virgin females than in males but the duration of bacteremia was similar. In mice infected before pregnancy (n= 20), fetal loss was evaluated by enumerating resorption and fetal death on day 18 of gestation. The fetal death and resorption percentage of infected mice was 36.3% versus 14.5% for controls (P< 0.0001). Fetal suffering was evaluated by weighing viable fetuses. The weight of viable fetuses was significantly lower for infected mice than for uninfected mice (P< 0.0002). Transplacental transmission ofBartonellawas demonstrated since 76% of the fetal resorptions tested was culture positive forB. birtlesii. The histopathological analysis of the placentas of infected mice showed vascular lesions in the maternal placenta, which could explain the reproductive disorders observed. BALB/c mice appeared to be a useful model for studyingBartonellainfection. This study provides the first evidence of reproductive disorders in mice experimentally infected with aBartonellastrain originating from a wild rodent.

Since very little is known about the clinical expression of Lyme borreliosis in Western Europe, a 3-year prospective study was conducted that included all patients seen for suspected Lyme borreliosis at the Strasbourg University Hospital in northeastern France. The diagnosis of Lyme borreliosis was made on the basis of the presence of erythema migrans or on the basis of another suggestive clinical manifestation and laboratory confirmation. A total of 132 patients, 70 women and 62 men, mean age 54 years, had Lyme borreliosis according to these criteria. Within this study group, 77% of the patients were regularly exposed to tick bites and 64% could remember one. Erythema migrans, the most frequent clinical manifestation, occurred in 60% of the patients and was the only sign of Lyme borreliosis in 40%. Lymphocytoma and acrodermatitis chronica atrophicans were rare (1 and 3 patients, respectively). Nervous system involvement (mainly radiculoneuropathy), the second most common clinical manifestation, was found in 40% of the patients and was the only sign of Lyme borreliosis in 22%. Musculoskeletal involvement was present in 26% of the patients and was an isolated finding in 14%. During the study period, no patient was diagnosed with Lyme carditis. There was serological evidence of Lyme borreliosis in 75% of the cases and direct evidence of borrelial infection in 10 (7.5%). The results show that the clinical expression of Lyme borreliosis in northeastern France is similar to that in other European countries but different from that in North America.

The expanding genus Bartonella includes zoonotic and human-specific pathogens that can cause a wide range of clinical manifestations. A productive infection allowing bacterial transmission by blood-sucking arthropods is marked by an intraerythrocytic bacteremia that occurs exclusively in specific human or animal reservoir hosts. Incidental human infection by animal-adapted bartonellae can cause disease without evidence for erythrocyte parasitism. A better understanding of the intraerythrocytic lifestyle of bartonellae may permit the design of strategies to control the reservoir and transmittable stages of these emerging pathogens. We have dissected the process of Bartonella erythrocyte parasitism in experimentally infected animals using a novel approach for tracking blood infections based on flow cytometric quantification of green fluorescent protein–expressing bacteria during their interaction with in vivo–biotinylated erythrocytes. Bacteremia onset occurs several days after inoculation by a synchronous wave of bacterial invasion into mature erythrocytes. Intracellular bacteria replicate until reaching a stagnant number, which is sustained for the remaining life span of the infected erythrocyte. The initial wave of erythrocyte infection is followed by reinfection waves occurring at intervals of several days. Our findings unravel a unique bacterial persistence strategy adapted to a nonhemolytic intracellular colonization of erythrocytes that preserves the pathogen for efficient transmission by blood-sucking arthropods.

Blood samples were collected between February and June 1996 from a convenience sample of 436 domestic French cats living in Paris and its environs and were tested for Bartonella bacteremia and seropositivity. Seventy-two cats (16.5%) were Bartonella bacteremic, of which 36 cats (50%) were infected with Bartonella henselae type II (B.h. II) only, 15 cats (21%) were infected with Bartonella clarridgeiae (B.c.) only, and 11 cats (15%) were infected with B. henselae type I (B.h. I) only. Eight cats (11%) were co-infected with B. henselae and B. clarridgeiae (B.h. II/B.c.: five cats; B.h. I/B.c.: three cats). Two cats (2.8%) were concurrently bacteremic with B. henselae types I and II. Risk factors associated with bacteremia included ownership for

Staphylococcal Panton-Valentine leukocidin (PVL) is an important virulence factor, which causes leukocytolysis and tissue necrosis. Our previous report on the existence of the PVL genes (lukS-PV and lukF-PV) on the genome of prophage phiPVL in the Staphylococcus aureus strain V8 suggested the horizontal transmission of PVL genes by temperate bacteriophage among S. aureus (Kaneko, et al., 1998. Gene 215, 57-67). Here, we demonstrated the phage conversion of S. aureus leading to the production of PVL by discovery of a novel PVL-carrying phage, phiSLT (Staphylococcal Leukocytolytic Toxin) from a clinical isolate of S. aureus. phiSLT was able to lysogenize several clinical isolates of PVL-negative S. aureus strains as well as strain RN4220 at the conserved 29-bp sequence (attB) and all the lysogenized S. aureus strains had the ability to produce PVL. phiSLT had an elongated head of about 100x50 nm and a flexible tail of 400 nm long, that was quite different from phiPVL which had an isometric hexagonal head of about 60 nm diameter. The linear double-stranded phiSLT genome comprised 42,942 bp with 29-bp attachment core sequences and contained 62 open reading frames. Only 6.4 kbp region containing lysis cassette, PVL genes, attP, integrase, and orf204 of phiSLT was identical to that of phiPVL, while other regions were different from those of phiPVL. Thus, it can be concluded that PVL genes are carried by different temperate phages, which have the same attachment site.

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes ( Canis latrans ) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp. berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii . PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp. berkhoffii . Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp. berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31 Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case ( B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with Sma I restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, three Bartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii , especially to identify potential vectors, and to determine how humans become infected.

Three strains isolated from Apodemus spp. were similar to Bartonella species on the basis of phenotypic characteristics. Futhermore, genotypic analysis based on sequence analysis of the 16S rRNA and gltA genes and on DNA-DNA hybridization showed that the three isolates represented a distinct and new species of Bartonella. The name Bartonella birtlesii is proposed for the new species. The type strain of B. birtlesii sp. nov. is IBS 325T (= CIP 106294T = CCUG 44360T).

Since Lyme arthritis was first described in the United States, it has now been reported in many countries of Europe. However, very few strains of the causative bacterium, Borrelia burgdorferi , have been isolated from synovial samples. For this reason, different molecular direct typing methods were developed recently to assess which species could be involved in Lyme arthritis in Europe. We developed a simple oligonucleotide typing method with PCR fragments from the flagellin gene of B. burgdorferi sensu lato, which is able to differentiate seven different Borrelia species. Among 10 consecutive PCR-positive patients with Lyme arthritis from the northeastern France, two species were identified in synovial samples: B. burgdorferi sensu stricto in 9 cases and B. garinii in 1 case. Conversely, all B. burgdorferi sensu lato species detected in 10 consecutive PCR-positive biopsies from a second set of patients with erythema migrans from the same geographical area were identified as either B. afzelii or B. garinii ( P < 0.001). These results indicate that B. burgdorferi sensu stricto is the principal but not the only Borrelia species involved in Lyme arthritis in northeastern France.

Resume La realisation d'un examen de bacteriologie utile depend etroitement de la qualite de la prescription et des conditions techniques de recueil et de transport de l'echantillon biologique. Des l'etape de prescription il faut savoir si l'analyse demandee est a visee diagnostique ou epidemiologique. Pour une analyse a but diagnostique, les echantillons utiles sont habituellement ceux obtenus de maniere invasive, ainsi que tous ceux pour lesquels on recherche, eventuellement par ecouvillonnage, des bacteries nommement designees. Pour une analyse a but epidemiologique, les echantillons utiles sont ceux provenant du revetement superficiel ou d'aspirations tracheales par exemple, a condition que les bacteries a rechercher soient bien specifiees et qu'une methodologie stricte soit utilisee. La distinction entre ces deux grands types d'echantillons biologiques conditionne non seulement le mode de realisation du prelevement, mais aussi la technique d'analyse microbiologique a utiliser et l'interpretation des resultats. La realisation technique du prelevement et son transport sont souvent complexes et necessitent de se referer a un guide technique detaille, edite par le laboratoire d'analyse. Un certain nombre de prelevements souvent adresses au laboratoire de microbiologie apparaissent comme inutiles. Il est important de tenter d'obtenir des echantillons biologiques de qualite grâce a une collaboration clinico-biologique permanente. De plus, des renseignements administratifs et cliniques ainsi que des donnees sur les circonstances presidant au prelevement sont absolument necessaires pour realiser une analyse bacteriologique de qualite. Car de ces renseignements depend etroitement la strategie d'analyse utilisee par le bacteriologiste. La gestion des non-conformites, encore trop frequentes, est un outil qui doit etre utilise pour ameliorer la qualite des echantillons biologiques avec l'aide des cliniciens. L'etape de prescription, celle du prelevement et celle du transport representent donc trois points critiques qui conditionnent de facon essentielle la qualite de l'analyse bacteriologique.

Because of its high diagnostic sensitivity in detection of unknown primary tumors, F-18 FDG PET/CT was used in the exploration of a 51-year-old woman presenting with multiple lymphadenopathy of the hepatic hilum and several hepatic and splenic hypodensities on radiologic examinations for which malignancy was suspected. Despite the morphofunctional malignant appearance, both histologic examination and PCR analysis revealed Bartonella henselae infection. Moreover, repeat F-18 FDG PET showed disease regression during focused antibiotic therapy.

Two Bartonella strains from blood of two wild rats (Rattus norvegicus) living in a rural environment were isolated. These strains were distinct from all previously known Bartonella species based on phenotypic and genotypic characteristics. This new species is distinguished by its trypsin-like activity, the absence of the ability to hydrolyse proline and tributyrin, its 16S rRNA and citrate synthase gene sequences and by whole-DNA hybridization data. This new species, for which the name Bartonella tribocorum sp. nov. is proposed, seems to be genetically related to Bartonella elizabethae, an agent isolated in a case of human endocarditis. The type strain of Bartonella tribocorum sp. nov. is IBS 506T (CIP 105476T).

To evaluate the prevalence of sulfate-reducing bacteria in septic processes, we searched for these bacteria by culture in 100 consecutive abdominal and pleural pus specimens. Twelve isolates were obtained from abdominal samples and were identified by a multiplex PCR as Desulfovibrio piger (formerly Desulfomonas pigra ) (seven strains), Desulfovibrio fairfieldensis (four strains), and Desulfovibrio desulfuricans (one strain).

The genus Gordona was proposed by Tsukamura in 1971 (M. Tsukamura, J. Gen. Microbiol. 68:15-24,1971) and was revived in 1988 by Stackebrandt et al. (E. Stackebrandt, J. Smida, and M. D. Collins, J. Gen. Appl. Microbiol. 34:341-348, 1988) to accommodate Rhodococcus species containing mycolic acids with 48 to 66 carbon atoms and MK-9(H2) as the predominant menaquinone type. On the basis of mycolic acid composition as determined by high-performance liquid chromatography, DNA-DNA similarity data (S1 nuclease procedure), and the rRNA gene restriction patterns of the type strains, we propose that Rhodococcus chubuensis Tsukamura 1982 is a subjective synonym of Gordona sputi (Tsukamura 1978) Stackebrandt 1989 comb. nov. We found that R. chubuensis type strain ATCC 33609, and G. sputi type strain ATCC 29627 exhibit identical mycolic acid profiles, 97% DNA relatedness (difference in thermal denaturation midpoints, 1.0°C), and similar ribotypes after PvuII endonuclease digestion. Thus, as defined by the type strains and considering the priority of the name G. sputi, these two species should be regarded as a single species named Gordona sputi; an emended description of this taxon is given.

Pulsed-field gel electrophoresis (PFGE) after SmaI restriction of DNA from 239 methicillin-resistant Staphylococcus aureus isolates (from 142 patients) produced 26 different fingerprints. The deduced chromosome sizes ranged from 2,200 to 3,100 kb (+/- 100 kb). A total of 81 isolates taken from 65 patients were then typed by PFGE and ribotyping with ClaI, EcoRI, and HindIII. Ribotypes were less discriminating than PFGE. Ribotyping did not discriminate isolates from a given PFGE fingerprint into different subsets. PFGE may be a more effective epidemiological tool than ribotyping for the typing of methicillin-resistant S. aureus strains.

The BBL Crystal MRSA ID test (Becton Dickinson) was applied directly to blood culture vials containing clusters of gram-positive cocci. The sensitivity and specificity of the test were 84 and 100% and 54 and 100% for vials containing Staphylococcus aureus and coagulase-negative staphylococci, respectively. This test is a reliable method for direct detection of methicillin resistance in positive blood culture vials when S. aureus is identified in parallel by rapid identification procedures.

A one-step reverse transcriptase PCR (RT-PCR) method for detection of Borrelia burgdorferi mRNA in infected C3H mice is described. This simple procedure, less prone to nucleic acid cross-contamination than the standard method, was found to be 10-fold more sensitive than a classical two-step RT-PCR assay. By using one-step RT-PCR, flagellin mRNAs were detected in synovial and heart tissues from all seven infected mice tested.

This international collaborative study evaluates a new system (ATB 32 Staph) for the identification of staphylococci taking into account the new novobiocin-sensitive and -resistant species reported. This study involved eight laboratories and 792 strains were tested. The reproducibility obtained for the cumulative results of the inter- and intea-laboratory tests was more than 90%. For 713 strains relevant of a species 95.5% were correctly identified by the system. Eight strains (1.2%) were misidentified and 24 strains (3.3%) were not identified. For 79 strains initially considered as not-classified, 62% were identified at the species level by the new system. The newer ATB 32 Staph gallery is a performant and useful method for routine identification of the currently described staphylococci species from clinical and animal origin.

The automated MagNA Pure DNA extraction method for Chlamydia trachomatis was compared with the manual Cobas Amplicor protocol using 100 µL of input sample volume from 964 specimens. Agreement between protocols was 96.1%. The automated extraction method had a sensitivity of 99% and a specificity of 100%. Amplification inhibition observed after manual preparation of samples (3.8%) was not apparent following automated extraction. Using 200 µL of sample in the automated extraction process lowered the detection limit without raising the inhibition rate. Furthermore, the automated extraction method halved the hands-on time required for the procedure.

The growth of 29 human strains from the three main pathogenic species of Borrelia burgdorferi sensu lato on a solid BSK-based medium was compared in two culture atmospheres: 3% CO2 air and anaerobiosis. All strains grew under anaerobic conditions, whereas only 13 strains were able to grow in aerobiosis with 3% CO2 ( P 0.001 ). In the latter condition, 75% of the B. burgdorferi sensu stricto strains grew versus 33% of the B. garinii and B. afzelii strains. These data suggest that, especially for B. garinii and B. afzelii species, anaerobic conditions enhance growth yield and speed of low-passage Borrelia strains.

Cat scratch disease (CSD) is mostly due to Bartonella henselae after inoculation of the organism through a skin injury. Since the causative bacteria cannot be easily cultured from human lymph node samples, the diagnosis usually relies on epidemiological, clinical, histological, and serological criteria (classical criteria). A study was performed to determine the diagnostic value of PCR analysis for the detection of B. henselae for the diagnosis of CSD and its place in the diagnostic strategy alongside the classical criteria. Over a 7-year period, lymph node biopsy specimens or cytopunctures from 70 patients were systematically tested by PCR for the presence of B. henselae DNA ( htrA gene) in the Bacteriology Laboratory of the Hôpitaux Universitaires de Strasbourg. Serological testing by an immunofluorescence assay for B. henselae antibodies was also performed for each patient, and clinical, epidemiological, and histological data were collected. The patients were then divided into two groups according to the number of positive diagnostic criteria for CSD: 29 patients with definite CSD (two or more classical criteria) and 15 patients with possible CSD (less than two classical criteria). The remaining 26 patients for whom another diagnosis was retained were used as a control group. Among all criteria, PCR analysis had the best specificity (100%). The PCR assay for B. henselae was positive for 22 (76%; 95% confidence interval [CI 95 ], 56.5 to 89.7%) of the 29 definite CSD patients and 3 (20%; CI 95 , 4.3 to 48.1%) of the 15 possible CSD patients. We then studied combinations of diagnostic criteria, including B. henselae PCR analysis. The best diagnostic performance was observed if at least two criteria were present among serologic, epidemiologic, histological, and molecular criteria.

Two strains of bacteria isolated from the blood of French domestic cows were found to be similar to Bartonella species on the basis of phenotypic characteristics. Genotypic analysis based on sequence comparison of the 16S rRNA and citrate synthase (gltA) genes and on DNA–DNA hybridization showed that the two isolates represent a distinct and new species of Bartonella. Moreover, the phylogenetic analysis inferred from comparison of 16S rRNA and gltA sequences demonstrated that the new Bartonella species is related to other ruminant-derived Bartonella species. The name Bartonella chomelii is proposed for the new species. The type strain of Bartonella chomelii sp. nov. is A828T (=CIP 107869T=CCUG47497T).

Referred to by: Erratum to “Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S–23S intergenic spacer region” [Vet. Microbiol. 98 (2004) 63–69] Veterinary Microbiology, Volume 100, Issues 1-2, 20 May 2004, Pages 139-140, R Maillard, M Vayssier-Taussat, C Bouillin, C Gandoin, L Halos, B Chomel, Y Piémont, H.-J Boulouis; International audience; Of the 20 species or subspecies of Bartonella currently known, 7 cause various diseases in humans with many being zoonotic. However, some Bartonella species appear only to cause asymptomatic bacteraemia in their hosts. In ruminants, three Bartonella species (B. bovis, B. capreoli and B. schoenbuchensis) have recently been described. However, limited or no information has yet been published concerning their mode of transmission and their possible pathogenicity for domestic cattle. The phylogenetic relationship of these species with other bacteria of the Bartonella genus has only been recently investigated. It is therefore necessary to develop appropriate tools that will easily allow identification of these ruminant strains for epidemiological and clinical studies. A single-step PCR assay, based on the amplification of a fragment of the 16S-23S rRNA intergenic spacer (ITS), was evaluated for identification of Bartonella isolated from domestic cattle and from free-ranging or captive cervids. For each Bartonella species tested, the PCR assay led to a product that was unique either for its length or its sequence. All ruminant isolates tested could be easily differentiated among themselves and from the other Bartonella species. Furthermore, sequence analysis of the PCR products revealed a close relationship between all ruminant Bartonella strains. Therefore, ITS PCR testing appears to be a convenient tool for a quick diagnosis of ruminant Bartonella species.

In this report, we review two cases of brain infection due to Dialister pneumosintes in previously healthy patients. The bacterium was isolated from the first patient by blood culture and directly from a brain abscess in the second patient. In both cases, the infection was suspected to be of nasopharyngeal or dental origin. The patients had favorable outcomes following surgical debridement and antibiotic treatment. After in vitro amplification and partial sequencing of the 16S rRNA gene, two strains were classified as D. pneumosintes . However, traditional biochemical tests were not sufficient to identify the bacteria. In addition to causing periodontal and opportunistic infections, D. pneumosintes, contained in mixed flora, may behave as a clinically important pathogen, especially in the brain. In addition to phenotypic characterization, 16S rRNA partial sequencing was used to identify D. pneumosintes definitively.

Two novel species of Bartonella isolated from European ruminants are described. Bartonella capreoli sp. nov. was isolated from the blood of roe-deer (Capreolus capreolus) captured in Chize, France. The type strain is IBS 193T (= CIP 106691T = CCUG 43827T). It is distinct from another European ruminant isolate that originated from a cow from a French herd of 430 dairy cattle. The latter isolate belongs to a novel species named Bartonella bovis Bermond et al. sp. nov. The type strain is strain 91-4T (= CIP 106692T = CCUG 43828T). The two bacteria appeared as small, fastidious, aerobic, oxidase-negative, gram-negative rods. Their biochemical properties were similar to those of members of the genus Bartonella. The sequences of the 16S rRNA and citrate synthase genes obtained from the two type strains were highly related to sequences of the different Bartonella species. Hybridization values when testing type strains of recognized Bartonella species, obtained with the nuclease/trichloroacetic acid method, support the creation of two novel species.

Between 1986 and 1998, eight cases of community-acquired pneumonia due to Staphylococcus aureus strains carrying the gene for the Panton-Valentine leukocidin (PVL) were recorded in France, six of which were fatal. We aimed to assess the clinical features of these eight cases, and those of other cases identified prospectively, and to compare them with the characteristics of patients with pneumonia caused by PVL-negative strains.We compared eight retrospective and eight prospective cases of PVL-positive S aureus pneumonia with 36 cases of PVL-negative S aureus pneumonia. For all patients, we recorded age, length of hospital stay, risk factors for infection, signs and symptoms, laboratory findings, antibiotic treatment, and serial radiological findings.Median age was 14.8 years (IQR 5.4-24.0) for the PVL-positive patients and 70.1 years (59.2-81.4) for the others (p=0.001). Influenza-like illness had occurred during the 2 days before admission in 12 of the 16 PVL-positive patients, but in only three of 33 PVL-negative patients (p0.001). PVL-positive infections were more often marked by: temperature greater than 39 degrees C (p=0.01), heart rate above 140 beats per min (p=0.02), haemoptysis (p=0.005), onset of pleural effusion during hospital stay (p=0.004), and leucopenia (p=0.001). The survival rate 48 h after admission was 63% for the PVL-positive patients and 94% for PVL-negative individuals (p=0.007). Histopathological examination of lungs at necropsy from three cases of necrotising pneumonia associated with PVL-positive S aureus showed extensive necrotic ulcerations of the tracheal and bronchial mucosa and massive haemorrhagic necrosis of interalveolar septa.PVL-producing S aureus strains cause rapidly progressive, haemorrhagic, necrotising pneumonia, mainly in otherwise healthy children and young adults. The pneumonia is often preceded by influenza-like symptoms and has a high lethality rate.

Cat scratch disease (CSD) is a frequent clinical outcome of Bartonella henselae infection in humans. Recently, two case reports indicated Bartonella clarridgeiae as an additional causative agent of CSD. Both pathogens have been isolated from domestic cats, which are considered to be their natural reservoir. B. clarridgeiae and B. henselae can be distinguished phenotypically by the presence or absence of flagella, respectively. Separation of the protein content of purified flagella of B. clarridgeiae by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the flagellar filament is mainly composed of a polypeptide with a mass of 41 kDa. N-terminal sequencing of 20 amino acids of this protein revealed a perfect match to the N-terminal sequence of flagellin (FlaA) as deduced from the sequence of the flaA gene cloned from B. clarridgeiae . The flagellin of B. clarridgeiae is closely related to flagellins of Bartonella bacilliformis and several Bartonella -related bacteria. Since flagellar proteins are often immunodominant antigens, we investigated whether antibodies specific for the FlaA protein of B. clarridgeiae are found in patients with CSD or lymphadenopathy. Immunoblotting with 724 sera of patients suffering from lymphadenopathy and 100 healthy controls indicated specific FlaA antibodies in 3.9% of the patients' sera but in none of the controls. B. clarridgeiae FlaA is thus antigenic and expressed in vivo, providing a valuable tool for serological testing. Our results further indicate that B. clarridgeiae might be a possible etiologic agent of CSD or lymphadenopathy. However, it remains to be clarified whether antibodies to the FlaA protein of B. clarridgeiae are a useful indicator of acute infection.

Panton-Valentine leukocidin (PVL) is a cytotoxin that causes leukocyte destruction and tissue necrosis. It is produced by fewer than 5% of Staphylococcus aureus strains. A collection of 172 S. aureus strains were screened for PVL genes by polymerase chain reaction amplification. PVL genes were detected in 93% of strains associated with furunculosis and in 85% of those associated with severe necrotic hemorrhagic pneumonia (all community-acquired). They were detected in 55% of cellulitis strains, 50% of cutaneous abscess strains, 23% of osteomyelitis strains, and 13% of finger-pulp-infection strains. PVL genes were not detected in strains responsible for other infections, such as infective endocarditis, mediastinitis, hospital-acquired pneumonia, urinary tract infection, and enterocolitis, or in those associated with toxic-shock syndrome. It thus appears that PVL is mainly associated with necrotic lesions involving the skin or mucosa.

Bartonella species are considered as emerging human pathogens, with at least six different species pathogenic or possibly pathogenic for humans. However, little is known about Bartonella distribution, species polymorphism and pathogenicity in mammalian species. The objective of this work was to determine the presence, the frequency and the distribution of Bartonella species in wild rabbits (Oryctolagus cuniculus) caught in warrens in Alsace, France. Humans may come into contact with wild rabbits when hunting, especially when they are picked up with bare hands and at time of evisceration. Of 30 blood samples collected and cultured from wild rabbits, nine (30%) were positive for organisms morphologically similar to Bartonella spp. The bacteria appeared as small, fastidious, aerobic, oxidase-negative. Gram-negative rods which could be localized within erythrocytes. Their biochemical properties were similar to those of the genus Bartonella. The sequence of the 16S rRNA gene obtained from the rabbit isolates was highly related to the sequences of the different Bartonella species (97·8--99·3% similarity). The high DNA hybridization rate (81–90% similarity) between the three strains isolated from rabbit blood confirmed that they belong to the same bacterial species. Hybridization values, obtained with the nuclease-TCA method, when testing type strains of recognized Bartonella species (9--14% similarity), support the creation of a new species for the rabbit isolates. The name Bartonella alsatica is proposed for these strains isolated from the blood of wild rabbits. The type strain is IBS 382T(= CIP 105477T).

We collected 3,397 consecutive isolates of coagulase-positive staphylococci from various specimens of hospitalized patients. All were retrospectively classified as Staphylococcus aureus, except two which were identified as S. intermedius: one isolated from the nasal flora of a healthy carrier and the other isolated from pleural fluid, probably as a sample contaminant.

The aim of the present work was to determine by blood culture the prevalence of blood infection with Bartonella species in a well-defined, European, urban stray cat population. Therefore, 94 stray cats were trapped from 10 cat colonies. Blood samples of these cats were cultured on both blood agar and liquid medium in order to raise the likelihood of bacterial detection. Fifty blood samples (53%) gave a positive culture result for Bartonella species. Isolate identification was performed by sequencing the first 430 bases of the 16S ribosomal DNA. Three types of sequences were thus obtained. The first type (17 isolates; 34%) was identical to that of B. henselae Houston-1 and the corresponding strains were referred as B. henselae type I. The second sequence type (18 isolates; 36%) was identical to that initially described as "BA-TF," and the corresponding strains were referred to as B. henselae type II. The third sequence type (15 isolates; 30%) was identical to that of the Bartonella clarridgeiae type strain (ATCC 51734). Our study points out the major role of stray cats as a reservoir of Bartonella spp. which can be transmitted to pet cats and, consequently, to humans. The study also highlights the high prevalence of B. clarridgeiae (16%) in the blood of stray cats.

Objective. To compare the detection rates of chromosomal flagellin gene from Borrelia burgdorfen in synovial tissue (ST) and synovial fluid (SF) using polymerase chain reaction (PCR) techniques. Methods. B burgdorferi DNA was sought in SF and ST from 12 consecutive patients with Lyme arthritis and from 29 patients with noninfectious diseases (controls). Results. No DNA amplification was observed in samples obtained from the 29 control patients, whereas B burgdorferi DNA was detected in all ST and/or SF samples from the 12 patients with Lyme arthritis. Results from 1 ST sample were not interpretable because of PCR inhibitors. Among the 11 remaining patients, 10 had positive ST samples, whereas only 4 had positive SF samples (P < 0.05). Conclusion. These data suggest that detection of chromosomal B burgdorferi DNA may be more efficient in ST than SF.

Serum samples from 35 cat scratch disease (CSD) patients, 180 control patients (123 without lymph node enlargement and 57 with lymph node enlargement not evoking CSD), and 102 nonpatient subjects (35 with cat contact and 67 without cat contact) were tested by semiquantitative indirect immunofluorescence assay for the presence of antibodies directed to Afipia felis (ATCC 53690T) or Bartonella henselae (ATCC 49882T). The CSD group had statistically higher antibody titers against B. henselae than the control groups (P < 10(-5)), whereas no difference in A. felis antibody titers was evidenced among all groups tested. Among the 317 serum samples studied, the three with high A. felis antibody titers ( > or = 64) also had high antibody titers against other alpha-2 proteobacteria. The value of the indirect immunofluorescence assay with B. henselae antigen for the diagnosis of CSD was as follows: for a cutoff of 32, sensitivity was 0.80, specificity was 0.85, and the likelihood ratio was 5.1; for a cutoff of 64, the likelihood ratio was 12.1. In summary, in France, CSD is associated with high antibody titers against B. henselae, as previously described in the United States. However, the causes for B. henselae seronegativity in CSD patients and those for high antibody titers outside the typical nosological frame of CSD still have to be identified.

A new, well-documented case of Mycobacterium shimoidei infection is reported. This bacterium was isolated from a patient with preexisting pulmonary lesions who developed a tuberculosis-like cavity in the lung. Isolation of fewer than ten strains, not epidemiologically related, has been reported worldwide; all originated from human pulmonary samples. Identification of the strain described here was first accomplished by 16S rDNA direct sequencing after in vitro amplification. This method allows rapid identification of unusual bacteria. The identification was confirmed by specific phenotypical tests and by chromatographic methods.

a UMR BIPAR, Ecole Nationale Veterinaire d’Alfort, 7 Avenue du General de Gaulle, 94700 Maisons-Alfort, France b Unite de Pathologie du Betail, Ecole Nationale Veterinaire d’Alfort, 7 Avenue du General de Gaulle, 94700 Maisons-Alfort, France c Depailment of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA d Institute de Bacteriologie de la Faculte de Medecine, Universite Louis Pasteur, Hopitaux Universitaires de Strasbourg, 3 rue Koberle, 67000 Strasbourg, France

By using Taq polymerase, DNA amplification of a specific fragment of the macrophage infectivity potentiator (mip) gene from Legionella pneumophila was used to detect Legionella spp. in bronchoalveolar lavage (BAL) fluid specimens. We were able to detect DNAs from all 30 L. pneumophila strains tested (serogroups 1 to 14), L. micdadei, and L. bozemanii serogroup 1. DNA from bacteria of other species tested and DNA from human leukocytes were not amplified by this procedure. After optimization of the conditions for DNA extraction from BAL fluid, a 2-ml sample of BAL fluid seeded with 25 CFU/ml tested positive after DNA amplification. A total of 68 frozen BAL fluid specimens sent to the laboratory because of suspected legionellosis were tested in a retrospective study. The eight culture-positive samples were all positive after specific DNA amplification. Among 60 culture-negative samples, 7 were positive after amplification. Of these seven samples, four were from patients who had presented a typical clinical history of legionellosis; the samples had antibody titer increases of 2 dilutions. For the three remaining samples, serological diagnosis of legionellosis in the patients from whom the samples were obtained could not be documented, and although the causative agent of these pulmonary infections was not determined, the clinical features of the patients were in accordance with legionellosis.

The quantum yields of intrinsic fluorescence of staphylococcal exfoliative toxins ETA and ETB were determined after excitation at 295 nm and 275 nm respectively. The variations in the intrinsic fluorescence and degree of fluorescence polarization showed conformational modifications of ETA around 57–59°C and of ETB around 52–54°C. Above these transition temperatures, ETB precipitated out and its biological activity was lost, whereas ETA showed almost no precipitation and no change in its biological activity, despite the structural changes in the molecule.

The genus Bartonella comprises two human-specific pathogens and a growing number of zoonotic or animal-specific species. Domesticated as well as wild mammals can serve as reservoir hosts for the zoonotic agents and transmission to humans may occur by blood sucking arthropods or by direct blood to blood contact. Humans may come into intimate contact with free-ranging mammals during hunting, especially during evisceration with bare hands, when accidental blood to blood contact frequently occurs. The objective of this work was to determine the presence and the polymorphism of Bartonella strains in wild roe deer (Capreolus capreolus) as the most widely spread game in Western Europe. We report the isolation of four Bartonella strains from the blood of five roe deer. These strains carry polar flagella similar to Bartonella bacilliformis and Bartonella clarridgeiae. Based on their phenotypic and genotypic characteristics, three of the four roe deer isolates were different and they were all distinct from previously described Bartonella species. They can be distinguished from each other and from other Bartonella species by their protein profile, ERIC-PCR pattern, 16S rRNA and citrate synthase (gitA) gene sequences, as well as by whole DNA-DNA hybridization. In spite of their considerable heterogeneity, all four strains fulfil the criteria for belonging to a single new species. The name Bartonella schoenbuchii is proposed for this new species. The type strain R1T of Bartonella schoenbuchii has been deposited in the National Collection of Type Cultures as NCTC 13165T and the Deutsche Sammlung von Mikroorganismen und Zellkulturen as DSM 13525T.