Evaluation of a new chromogenic medium, chromID Vibrio, for the isolation and presumptive identification of Vibrio cholerae and Vibrio parahaemolyticus from human clinical specimens.

The aim of this study was to evaluate the performance of the chromID Vibrio medium for the detection of Vibrio cholerae and V. parahaemolyticus in stool and swab specimens in comparison with thiosulfate citrate bile salts sucrose (TCBS) medium. A total of 96 samples including 30 fresh stool, 32 stool, and 34 swab specimens originating from routine laboratories were tested. All samples were seeded on both media, the TCBS medium and the chromID Vibrio, directly and after an enrichment step on alkaline peptone water. Of the 96 samples studied, 34 were positive for V. cholerae and 30 were positive for V. parahaemolyticus. The sensitivity for the isolation of V. cholerae in fresh stool specimens was identical for both media, 78.5% and 100% before and after enrichment, respectively. However, positive test with chromID Vibrio concluded immediately to the presence of V. cholerae. In the case of artificial contaminations, the sensitivity of chromID Vibrio was more important than TCBS after enrichment for V. cholerae and for V. parahaemolyticus before and after enrichment. In fresh stool specimens, the specificity of chromID Vibrio for screening V. cholerae was significantly higher than TCBS (100% and 100% compared to 50% and 50% before and after enrichment, respectively) and was important for V. parahaemolyticus (100% chromID Vibrio; 93.33% TCBS).

Invasion and persistent intracellular colonization of erythrocytes. A unique parasitic strategy of the emerging pathogen Bartonella.

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.

[Community-acquired methicillin-resistant Staphylococcus aureus in a dermatology outpatient clinic]. / Les souches de Staphylococcus aureus méticilline-résistant communautaires en consultation externe hospitalière de dermatologie.

BACKGROUND: Community-acquired cutaneous infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing concern. These bacteria may produce Panton-Valentine leucocidin potentially leading to necrotizing pneumonia. We studied the prevalence of MRSA and Panton-Valentine leucocidin in dermatology clinic outpatients in order to adapt therapy where possible. PATIENTS AND METHODS: This was a prospective study including all patients seen at a dermatology outpatient clinic between 1st March 2005 and 31st December 2006 and presenting mucocutaneous bacteriological samples. The main MRSA risk factors studied were frequent hospital consultations, hospitalization, antibiotic therapy within the last three months and community life. The following risk factors were also analysed, although less routinely: substance abuse, immunosuppression, diabetes mellitus, recent travel abroad and a history of similar lesions. RESULTS: One hundred and twenty-two patients were included in the study and 235 samples (143 lesion samples and 92 nasal swabs) were carried out and S. aureus was isolated in 68 patients (56%). Twelve patients had MRSA (17.6%); seven of these were normal outpatients but five attended frequent hospital consultations (7.3%). MRSA resistance rates were as follows: 64% to ofloxacin, 36% to amikacin and erythromycin, 27% to fusidic acid, 9.1% to sulfamethoxazole-trimethoprim and 0% to pristinamycin. Community life was the only significant risk factor for MRSA in this study (p=0.045). Four of the 11 MRSA strains tested produced Panton-Valentine leucocidin. CONCLUSION: Dermatologists are increasingly faced with cutaneous infections caused by community-acquired MRSA. Bacterial samples should be taken routinely and probabilistic antibiotic therapy for MRSA instituted in severe infections.

[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.

[Bacterial cultures and empirical antimicrobial therapy for community-acquired secondary peritonitis]. / Prélèvements microbiologiques et traitements antibiotiques probabilistes des péritonites secondaires communautaires.

Surgeons and anesthetists are frequently confronted with community-acquired secondary peritonitis. We summarize literature results and consensus conferences concerning the types of bacteriologic sampling and cultures and the empiric choice of an antibiotic regimen based on the probable pathogens encountered in community-acquired secondary peritonitis. These studies leave some doubt as to the necessity for routine blood cultures and the need for anaerobic cultures of peritoneal fluid. No one disputes the need for broad spectrum antibiotic therapy, but there is no consensus regarding one, two, or three drug antibiotic regimens or whether an aminoglycoside is an essential part of the recipe. Duration of antibiotic therapy is still a subject of controversy with recommendations varying from 24 hours to 10 days. The need for antibiotics with activity against enterococcus and the need for systematic antifungal therapy when fungal growth is noted in the peritoneal fluid remain undefined. These uncertainties underline the need for treating physicians within each establishment to elaborate a written consensus of antibiotic therapy.

Detection of Chlamydia trachomatis DNA using MagNA Pure DNA extraction and Cobas Amplicor CT/NG amplification.

The automated MagNA Pure DNA extraction method for Chlamydia trachomatis was compared with the manual Cobas Amplicor protocol using 100 microL 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 microL 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 structure of Staphylococcus aureus epidermolytic toxin A, an atypic serine protease, at 1.7 A resolution.

BACKGROUND: Staphylococcal epidermolytic toxins A and B (ETA and ETB) are responsible for the staphylococcal scalded skin syndrome of newborn and young infants; this condition can appear just a few hours after birth. These toxins cause the disorganization and disruption of the region between the stratum spinosum and the stratum granulosum--two of the three cellular layers constituting the epidermis. The physiological substrate of ETA is not known and, consequently, its mode of action in vivo remains an unanswered question. Determination of the structure of ETA and its comparison with other serine proteases may reveal insights into ETA's catalytic mechanism. RESULTS: The crystal structure of staphylococcal ETA has been determined by multiple isomorphous replacement and refined at 1.7 A resolution with a crystallographic R factor of 0.184. The structure of ETA reveals it to be a new and unique member of the trypsin-like serine protease family. In contrast to other serine protease folds, ETA can be characterized by ETA-specific surface loops, a lack of cysteine bridges, an oxyanion hole which is not preformed, an S1 specific pocket designed for a negatively charged amino acid and an ETA-specific specific N-terminal helix which is shown to be crucial for substrate hydrolysis. CONCLUSIONS: Despite very low sequence homology between ETA and other trypsin-like serine proteases, the ETA crystal structure, together with biochemical data and site-directed mutagenesis studies, strongly confirms the classification of ETA in the Glu-endopeptidase family. Direct links can be made between the protease architecture of ETA and its biological activity.

Preliminary crystallographic data for exfoliative toxin B from Staphylococcus aureus.

The serotype B of exfoliative toxin, isolated from Staphylococcus aureus, strain TC 142, has been crystallized. The monoclinic crystals belong to space group P21, with a = 55.9 A, b = 107.9 A, c = 42.8 A, and beta = 90.9 degrees. The asymmetric unit contains two molecules of molecular weight 30,000.

Phage conversion of Panton-Valentine leukocidin in Staphylococcus aureus: molecular analysis of a PVL-converting phage, phiSLT.

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.

Characterisation of a synergohymenotropic toxin produced by Staphylococcus intermedius.

Staphylococcal synergohymenotropic (SHT) toxins damage membranes of host defence cells and erythrocytes by the synergy of two secreted and non-associated proteins: class S and class F components. Whereas Panton-Valentine leucocidin (PVL), gamma-hemolysin and Luk-M from Staphylococcus aureus are members of this toxin family, a new bi-component toxin (LukS-I + LukF-I) from Staphylococcus intermedius, a pathogen for small animals, was characterised and sequenced. It is encoded as a luk-I operon by two cotranscribed genes, like PVL, LukS-I + LukF-I shares a strong leukotoxicity of various PMNs, but only slight haemolytic properties on rabbit erythrocytes. When intradermally injected into rabbit skin, a 100 ng dose caused acute inflammatory reaction leading to tissue necrosis. The new SHT seemed to be largely distributed among various Staphylococcus intermedius strains.