VISLISI trial, a prospective clinical study allowing identification of a new metalloprotease and putative virulence factor from Staphylococcus lugdunensis.

OBJECTIVE: Staphylococcus lugdunensis is a coagulase-negative staphylococcus that displays an unusually high virulence rate close to that of Staphylococcus aureus. It also shares phenotypic properties with S. aureus and several studies found putative virulence factors. The objective of the study was to describe the clinical manifestations of S. lugdunensis infections and investigate putative virulence factors. METHOD: We conducted a prospective study from November 2013 to March 2016 at the University Hospital of Strasbourg. Putative virulence factors were investigated by clumping factor detection, screening for proteolytic activity, and sequence analysis using tandem nano-liquid chromatography-mass spectrometry. RESULTS: In total, 347 positive samples for S. lugdunensis were collected, of which 129 (37.2%) were from confirmed cases of S. lugdunensis infection. Eighty-one of these 129 patients were included in the study. Bone and prosthetic joints (PJI) were the most frequent sites of infection (n=28; 34.6%) followed by skin and soft tissues (n=23; 28.4%). We identified and purified a novel protease secreted by 50 samples (61.7%), most frequently associated with samples from deep infections and PJI (pr 0.97 and pr 0.91, respectively). Protease peptide sequencing by nano-liquid chromatography-mass spectrometry revealed a novel protease bearing 62.42% identity with ShpI, a metalloprotease secreted by Staphylococcus hyicus. CONCLUSION: This study confirms the pathogenicity of S. lugdunensis, particularly in bone and PJI. We also identified a novel metalloprotease called lugdulysin that may contribute to virulence.

[Comparative study of Vitek-2™ AIX versus Vitek-2™ PC: phenotypic resistance of enterobacteriaceae to ß-lactams]. / Comparaison du Vitek-2™ AIX versus Vitek-2™ PC par l'étude des phénotypes de résistance des entérobactéries aux ß-lactamines.

AIM OF THE STUDY: Vitek-2™ AIX versus Vitek-2™ PC have different rules for phenotypic interpretation. The aim of this study is to ensure that the raw results determined by these two versions of Vitek-2™ allow biologists to conclude to the same resistance phenotype, but also to evaluate their own phenotypic interpretation system (advanced expert system). MATERIAL AND METHODS: A total of 251 strains of Enterobacteriaceae of different groups and phenotypes was tested. Each strain was studied simultaneously on both types of Vitek-2™ from the same calibrated inoculum. We then compared their resistance phenotype to beta-lactams. RESULTS: For strains not producing ESBL or CHN, the biologist concluded in 99.3% of cases to the same resistance phenotype by interpreting the raw results of Vitek-2™ AIX versus PC. The phenotypic interpretation of biologist is different from the Vitek-2™ in respectively 40% versus 43% of cases for AIX and PC versions. For multi-resistant strains, the biologist concluded in 100% of cases to the same resistance phenotype by interpreting the raw results of Vitek-2™ AIX versus PC. In 51.5% of cases the biologist use the disk diffusion method (DD). The results of this technique put forward 29% discrepancy with the two types of Vitek-2™. Finally, when Vitek-2™ claims the presence of an ESBL alone, this result is routinely confirmed by DD. CONCLUSION: The switch from Vitek-2™ AIX to Vitek-2™ PC does not alter the results of the phenotypic interpretation of biologist.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now widely used for marker/multi-biomarker detection in medical diagnosis. We tested a new protocol for bacterial identification from blood culture broths in hospital routine by using collection tubes with separator gels on 503 included samples examined over 3 months, where 1.5 mL was injected by a syringe into BD Vacutainer tubes from BACTEC-positive bottles, before processing for bacterial protein extraction. Samples were loaded in duplicate onto the MALDI MS target, allowing a series of 12 samples to be processed in duplicate within 80 min by using Biflex III and BioTyper 2.0 software (Bruker). Including polymicrobial samples, 193 of 213 of Gram-negative bacteria (91.08%) and 284 of 319 of Gram-positive bacteria (89.02%) were correctly identified at the species level. Enterobacteriaceae constituted 35.15% of all species found, Staphylococaceae 37.96%, Streptococaceae and Enterococaceae 20.85%, Pseudomonadaceae 1.69%, and anaerobes 2.44%. In most of the polymicrobial samples, one of the species present was identified (80.9%). Seven isolates remained misidentified as Streptococcus pneumoniae, all belonging to Streptococcus mitis. Staphylococcus aureus was identified better when grown on anaero-aerobic medium, and MALDI BioTyper identification scores as low as 1.4 were pertinent, provided that four successive proposals of the same species were given. This new protocol correlates with conventional microbiology procedures by up to 90%, and by >95% for only monomicrobial samples, and provides a decreased turn-around time for identification of bacteria isolated from blood cultures, making this technology suitable also for blood cultures, with less delay and cost decreases in bacterial diagnostics, and favouring better care of patients.

Virulence factors produced by strains of Staphylococcus aureus isolated from urinary tract infections.

Staphylococcus aureus infections are widely prevalent in West Africa and are often associated with urinary tract infections (UTIs). Virulence factors from S. aureus have rarely been described for such infections. The purpose of the current study was to determine the prevalence of toxins and adhesion factors obtained from S. aureus isolated from presumed primary UTIs at the Cotonou University Hospital (CUH) in Benin as compared with the Strasbourg University Hospital (SUH) in France. Both ambulatory and hospitalised patients were included in the study. Sixty-five independent strains of S. aureus from CUH and 35 strains from SUH were obtained over a four-month period. Virulence factors were characterised by immunodetection or multiplex polymerase chain reaction, and meticillin susceptibility was recorded. Approximately 50% of all isolates produced at least one enterotoxin. No isolate from SUH produced Panton-Valentine leucocidin (PVL), whereas 21.5% of the S. aureus isolates from CUH produced PVL (P<0.01). Six of 14 (43%) PVL-positive isolates were meticillin-resistant. At SUH, the incidence of MRSA (57%) was significantly higher (P<0.01) than at CUH (14%). Genes encoding clumping factor B, and elastin and laminin binding proteins were detected in almost all isolates (80%), irrespective of the geographical origin. The results for elastin binding protein differed significantly from published data regarding isolates from other clinical origins. Staphylococcal toxins and adhesion factors may be important in the physiopathology of UTI.

[Invasive Streptococcus agalactiae infections in non-pregnant adults]. / Les infections invasives à Streptococcus agalactiae chez l'adulte (femme enceinte exclue).

OBJECTIVES: Streptococcus agalactiae (Group B streptococcus) is a major cause of invasive diseases in non-pregnant adults, particularly in the elderly and those with underlying conditions. We describe these conditions and clinical characteristics of patients followed in our teaching hospital. METHODS: We retrospectively reviewed clinical records of 64 patients with S. agalactiae-related invasive infection, hospitalized between January 1997 and January 2006. RESULTS: The mean age of patients was 59 (+/-17 years). The H:F sex ratio was 1.06. At least one underlying condition was found in 90.6%. Diabetes mellitus (43.7%), peripheral vascular disease (34.4%), myocardial ischemia (20.3%) and malignant neoplasms (20.3%) were among the most frequent conditions. The mean index of comorbidity (Charlson) was 2.5 (+/-2). Common clinical manifestations included infection of the urinary tract (32.8%), skin and soft-tissue (25%), and osteoarthritis (21.9%). Bacteremia occurred in 31.2% with no identified source in 2 patients. During the first month, 2 cases of endocarditis, 1 case of meningitis, and 4 deaths occurred. CONCLUSION: We confirm the importance of underlying diseases in the emergence of S. agalactiae infections.

[A young woman with granulomatous mastitis: a corynebacteria may be involved in the pathogenesis of these disease]. / Mastite granulomateuse récidivante chez une jeune femme: rôle potentiel de "corynebacterium kroppenstedtii".

INTRODUCTION: The granulomatous mastitis is an inflammatory pseudotumor of the breast of which evolution benign but likely to generate important morphological after-effects among young women. This anatomoclinic entity of dubious etiology until these last years poses a problem of differential diagnosis with other etiologies of granulomatosis and especially with inflammatory carcinoma of the breast. The infectious theory is actually based on solid arguments and mainly explains the physiopathology of this affection. INTERPRETATION: A 26 years old young woman developed an inflammatory tumor of the left breast of which the catch of load by surgery and an antibiotherapy had shown trailing local continuations and of the esthetic after-effects. One year later, a very inflammatory repetition on the level of the right breast was dealt with in a different way: by steroids and immunomodulating drugs associated with iterative punctures with the purulent collections, the objective being to be less dilapidating that left side. The initial answer was rather favorable and encouraging but the purulent reappearance bulky granulomas with sinus way made reconsider the therapeutic attitude and antibiotics were undertaken after description of a lipophilic corynebactery in the material of puncture (Corynebacteria kroppenstedtii). The effectiveness of the amoxicilline introduced on the data of the antibiogram was undeniable. CONCLUSION: This observation illustrates the therapeutic and diagnostic difficulties of an exceptional affection. Potentially accessible to antibiotics it generally requires a joint surgical assumption of responsibility, at the same time to ensure the histological diagnosis but also with a therapeutic aim. The interest of steroids and the immunomodulation by methotrexate is debatable, these treatments cannot however be conceived without antibiotherapy and sometimes surgery.

[Actinomycosis of the buttock]. / Actinomycose fessière.

INTRODUCTION: Cutaneous actinomycosis of the buttocks is a rare granulomatous bacterial infection that usually starts in the perianal area. We present an exceptional case in the form of a pseudo-tumor. CASE REPORT: A 69 year-old woman, in general good health, developed an indurate mass on the supra-external quadrant of the right buttock. The tumor was centered by an ulcerated nodule with a diameter of around 10 centimeters. Imaging showed invasion of the soft tissue of the skin in the internal psoas muscle, the adipose tonality of which was compatible with a liposarcoma. The skin biopsy revealed characteristic bacterial grain in the center of a cholesterol granuloma. Subsequent culture in aerobic milieu identified Actinomyces gerencseriae. Cure was obtained following complete exeresis of the fibrous tissue and 8 months of antibiotic amoxicillin-clavulanic acid therapy. DISCUSSION: Other than the most unusual clinical aspect, the originality of this case of actinomycosis of the buttocks is based on its potential appendix origin, 4 years after acute appendicitis, with slow posterior fistulation. Other cases of actinomycosis of appendix origin have been reported and its delayed onset following the intervention has been documented. The pseudo-sarcomatous aspect was responsible for diagnostic wandering. The histological image and, subsequently, the results of the bacteriological culture confirmed the diagnosis.

Corynebacterium freneyi sp. nov., alpha-glucosidase-positive strains related to Corynebacterium xerosis.

Three coryneform strains from clinical specimens were studied. They belonged to the genus Corynebacterium, since they had type IV cell walls containing corynemycolic acids. They had phenotypic characteristics that included alpha-glucosidase, pyrazinamidase and alkaline phosphatase activities and fermentation of glucose, ribose, maltose and sucrose. These are the characteristics of Corynebacterium xerosis. Since this species is very rare in human pathology, the strains were studied in more detail by comparing the 16S-23S intergenic spacers, rDNA sequences and levels of DNA similarity of these three strains and those of the reference strains C. xerosis ATCC 373T and Corynebacterium amycolatum CIP 103452T. According to DNA-DNA hybridization data, the three novel strains are members of the same species (level of DNA similarity >72%). Phylogenetic analysis revealed that these strains are closely related to C. xerosis and C. amycolatum, but DNA-relatedness experiments showed clearly that they constitute a distinct new species, with levels of DNA relatedness of less than 23% to C. xerosis ATCC 373T and less than 5% to C. amycolatum CIP 103452T. Two other alpha-glucosidase-positive strains presenting the same biochemical characteristics were included in the study and proved to be C. amycolatum. This new species can be differentiated from C. xerosis and C. amycolatum strains by carbon source utilization, intergenic spacer region length profiles and some biochemical characteristics such as glucose fermentation at 42 degrees C and growth at 20 degrees C. The name Corynebacterium freneyi sp. nov. is proposed with the type strain ISPB 6695110T (= CIP 106767T = DSM 44506T).

Identification of Corynebacterium glucuronolyticum strains from the urogenital tract of humans and pigs.

Bacterial strains isolated from the genital tracts of humans (predominantly males), semen of boars, and uterine and vaginal secretions of sows were identified as Corynebacterium glucuronolyticum and were compared with the type strains of the recently proposed species Corynebacterium glucuronolyticum and Corynebacterium seminale. The two type strains as well as the clinical strains were shown by DNA-DNA hybridization and sequencing of the 16S rRNA gene to be related at the species level. All strains were classified as C. glucuronolyticum, because this name has nomenclatural priority over C. seminale.

Catalase-negative Actinomyces neuii subsp. neuii isolated from an infected mammary prosthesis.

In this case report a catalase-negative strain of Actinomyces neuii subsp. neuii is described as the possible causative agent of an infected mammary prosthesis. DNA hybridization studies and 16S rRNA analysis confirmed that the strain belongs to the species Actinomyces neuii subsp. neuii. Since this strain is the first A. neuii subsp. neuii strain reported to be catalase negative, the catalase reaction should no longer be considered a key reaction for the diagnosis of this species but must be interpreted in conjunction with other characteristics.

Bacteremia and endocarditis caused by a Gordonia species in a patient with a central venous catheter.

We report the first case of endocarditis caused by a Gordonia species genetically related to G. sputi but exhibiting some atypical biochemical features in a 31-year-old woman with a central venous catheter. This unusual pathogen may be a new cause of opportunistic infections in patients with severe underlying diseases.

Identification of nonlipophilic corynebacteria isolated from dairy cows with mastitis.

Nonlipophilic corynebacteria associated with clinical and subclinical mastitis in dairy cows were found to belong to four species: Corynebacterium amycolatum, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, and Corynebacterium minutissimum. These species may easily be confused. However, clear-cut differences between C. ulcerans and C. pseudotuberculosis were found in their acid production from maltotriose and ethylene glycol, susceptibility to vibriostatic agent O129, and alkaline phosphatase. Absence of growth at 20 degrees C and lack of alpha-glucosidase and 4MU-alpha-D-glycoside hydrolysis activity differentiated C. amycolatum from C. pseudotuberculosis and C. ulcerans. The mastitis C. pseudotuberculosis strains differed from the biovar equi and ovis reference strains and from caprine field strains in their colony morphologies and in their reduced inhibitory activity on staphylococcal beta-hemolysin. C. amycolatum was the most frequently isolated nonlipophilic corynebacterium.

Bartonella alsatica sp. nov., a new Bartonella species isolated from the blood of wild rabbits.

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).

Corynebacterium confusum sp. nov., isolated from human clinical specimens.

Three strains of a previously unknown coryneform bacterium were isolated from two patients with foot infections and from a blood culture of a third patient. The three non-lipophilic strains exhibited very slow fermentative acid production from glucose but not from maltose or sucrose, nitrate reductase activity, no tyrosinase activity and the presence of small amounts of tuberculostearic acid as the most significant phenotypic features. Differentiation of these strains from all other presently defined coryneform bacteria was readily achieved. Chemotaxonomic investigations revealed that the three strains unambiguously belonged to the genus Corynebacterium. Comparative 16S rRNA gene sequence analysis demonstrated that the isolates were almost identical and represented a new subline within the genus Corynebacterium, for which the designation Corynebacterium confusum sp. nov. is proposed. The type strain of Corynebacterium confusum is CCUG 38267T.

Bartonella tribocorum sp. nov., a new Bartonella species isolated from the blood of wild rats.

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).

Differentiation of Corynebacterium amycolatum, C. minutissimum, and C. striatum by carbon substrate assimilation tests.

We tested the carbon substrate assimilation patterns of 40 Corynebacterium amycolatum strains, 19 C. minutissimum strains, 50 C. striatum strains, and 1 C. xerosis strain with the Biotype 100 system (bioMérieux, Marcy-l'Etoile, France). Twelve carbon substrates of 99 allowed discrimination among the species tested. Additionally, assimilation of 3 of these 12 carbon substrates (maltose, N-acetyl-D-glucosamine, and phenylacetate) was tested with the API 20 NE identification system (bioMérieux). Since concordant results were observed with the two systems for these three carbon substrates, either identification system can be used as a supplementary tool to achieve phenotypic differential identification of C. amycolatum, C. minutissimum, and C. striatum in the clinical microbiology laboratory.