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.

Large scale isolation of human blood monocytes by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation for adoptive cellular immunotherapy in cancer patients.

The increasing interest in mononuclear phagocytes for adoptive cellular immunotherapy (ACI) trials in cancer patients led us to define a procedural approach to harvest reproducibly highly purified single-cell suspensions of large numbers of functional human circulating blood monocytes (Mo). A semiclosed counterflow centrifugal elutriation (CCE) system has been developed, using a new large capacity Beckman JE 5.0 rotor with one interchangeable 40 ml or 5 ml separation chamber, to purify Mo from mononuclear cell (MNC) concentrates of healthy donors and cancer patients obtained by continuous flow centrifugation leukapheresis (CFCL). This method does not require a Ficoll density gradient centrifugation step. A total of 115 leukapheresis procedures were carried out in 35 patients and in 30 healthy donors by either Cobe 2997 or Cobe Spectra, with a similar efficiency in MNC apheresis. The average yield per leukapheresis procedure was 5.6 x 10(9) MNC of purity 90-100% (25-45% Mo, 40-65% lymphocytes). The average yields per elutriation procedure (R/O fraction) were 1.1 x 10(9) cells (purity 93% Mo) using the 5 ml separation chamber, and 1.5 x 10(9) cells (purity 91%) using the 40 ml separation chamber, with a respective recovery of 82 +/- 7% and 78 +/- 8% Mo. In vitro analysis of the viability and function of the purified Mo shows that neither morphological integrity nor physiological activity was compromised by this two-step isolation procedure, which additionally provides highly purified human Mo suspensions, in a quantity suitable for ACl of cancer patients.

Improved purification of leukocidin from Staphylococcus aureus and toxin distribution among hospital strains.

For purification of F and S components of the Panton-Valentine leukocidin, an easy three-step method using fast protein liquid chromatography was developed to replace the time-consuming purification procedures previously published. This technique enabled the recovery of 13 and 17 mg of purified F and S, respectively, per litre of culture supernatant. Affinity-purified neutralizing polyclonal antibodies were obtained against each individual component. One hundred and thirty-nine Staphylococcus aureus strains isolated from various clinical samples of hospitalized patients were screened by immunoprecipitation for Panton-Valentine leukocidin (PVL) production. Only 8 strains produced PVL; all originated from injured superficial soft tissues. Contrary to widespread opinion, the 8 PVL-producing strains were never associated with severe infection.

A structurally novel staphylococcal protein A from the V8 strain.

Protein A from the Staphylococcus aureus strain V8 has a molecular mass about 8000 Da less than that of known proteins A. The corresponding gene was cloned and expressed in Escherichia coli. Sequence analysis of this structurally new protein A revealed that it lacked an IgG-binding domain (58 amino acids), and that it also lacked two octapeptide repetitions located in the membrane/wall attaching region. Contrary to what had been proposed previously, the first translated amino acid is probably not a leucine, but very likely a methionine located 12 residues upstream.

Epidemiological data on Staphylococcus aureus strains producing synergohymenotropic toxins.

DNA hybridisation of 309 consecutive Staphylococcus aureus clinical isolates with oligonucleotide probes specific for genes encoding Panton-Valentine leucocidin (luk-PV) and gamma-haemolysin (hlg) revealed that 99% of randomly selected strains carried the hlg locus whereas only 2% harboured the luk-PV as well as the hlg loci. Only 1% of the strains did not possess either gene. In a clinical prospective study of independent S. aureus strains, 58 Panton-Valentine leucocidin (PVL)-producing isolates were shown to be responsible for primary skin infections, mainly furuncles (86%). Phage susceptibility patterns and pulsed field gel electrophoresis (PFGE) profiles of DNA were shown to be polymorphic epidemiological markers of PVL-producing strains. In eight patients with recurrent furuncles, the PVL-producing strains isolated either from furuncles or from the anterior nares were considered to be identical in each based upon phage sensitivity profiles or PFGE patterns.

Pulsed field gel electrophoresis as a new epidemiological tool for monitoring methicillin-resistant Staphylococcus aureus in an intensive care unit.

Pulsed field gel electrophoresis (PFGE) of bacterial DNA was used in a 1-month epidemiological study of methicillin-resistant Staphylococcus aureus (MRSA) in a 15-bed Intensive Care Unit (ICU). Patient and hospital staff carriage as well as distribution of MRSA in the ICU environment were investigated, and a total of 3802 samples produced 175 isolates. The stability and the reproducibility of the PFGE method were satisfactory. Moreover, the plasmid content of the strains so far examined had no influence on the PFGE profiles of the MRSA strains. The polymorphic profiles observed also account for the use of this method as an epidemiological tool for investigating MRSA. Among 30 patients who stayed more than 4 days in the unit, PFGE analysis showed 11 episodes of colonization in nine patients, whereas lysotyping and plasmid DNA analysis demonstrated only eight and seven such episodes in the same patients, respectively. The combination of PFGE with lysotyping and plasmid analysis may provide a greater discriminatory capacity between MRSA isolates.

Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S-23S intergenic spacer region.

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.

Epidemiology of Bartonella infection in domestic cats in France.

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 <6months (prevalence ratio (PR)=1.80; 95% confidence interval (CI)=1.13-2.85), adoption from the pound or found as a stray (PR=1.67, 95% CI=1.05-2.65), and cohabitation with one or more cats (PR=1.60, 95% CI=1.01-2.53). Bartonella antibodies to either B. henselae or B. clarridgeiae were detected in 179 cats (41.1%). Risk factors associated with seroposivity paralleled those for bacteremia, except for lack of association with time of ownership. Prevalence ratios of bacteremic or seropositive cats increased with the number of cats per household (p=0.02). The lack of antibodies to B. henselae or B. clarridgeiae was highly predictive of the absence of bacteremia (predictive value of a negative test=97.3%). Multiple logistic regression analysis indicated that bacteremia, after adjustment for age and flea infestation, and positive serology, after adjustment for age, were associated with origin of adoption and number of cats in the household. Flea infestation was associated with positive serology.

Action of staphylococcal exfoliative toxins on epidermal cell cultures and organotypic skin.

In the staphylococcal scalded skin syndrome, spontaneous intraepithelial cleavages are due to the exfoliative toxins A or B (ETA or ETB). Until now, these toxins have been studied either on epidermis or on organotypic skin cultures. In the present study, we compare the effects of these toxins on human keratinocyte cell cultures to those on human and mouse organotypic skin cultures. With concentrations of ETA or ETB of 1 mg/ml for 3 hours, spontaneous intraepithelial cleavages were noted in both cell and organotypic cultures. Keratinocyte cell cultures were as sensitive as organotypic skin cultures to these toxins. Since keratohyaline granules may represent a possible binding site for ETA or ETB, we tried to correlate the expression of keratohyaline granules with the appearance of intraepithelial clefts due to the toxins. However, when cultured in liquid medium, epithelia were not differentiated enough to allow the detection of the binding site of ETA-ETB.

A randomized study of on-line plasma perfusion over protein A-sepharose and 5-fluorouracil chemotherapy in patients with metastatic colorectal carcinoma.

To evaluate the safety of on-line plasma perfusion over protein-A sepharose and the therapeutic advantage of combining plasma perfusion (PP) over protein-A sepharose with 5-fluorouracil (5-FU) chemotherapy in patients with metastatic colorectal carcinoma (MCRC), thirty patients were randomized after surgery of primary CRC to receive a combination of 5-FU and PP over protein-A sepharose (group A), or a combination of 5-FU and PP over sepharose (group B), or 5-FU alone (group C). Bi-weekly on-line PP over 200 ml protein-A sepharose gel (group A) or 200 ml sepharose gel (group B) were performed with a Cobe 2997 blood cell separator for a maximum of 19 treatments per patient. 5-FU was given at 1000 mg/m2/d on days 1-5 of a 4-weekly cycle until progression. PP was well tolerated and no severe or life-threatening toxicity was observed. Mild clinical side-effects consisted of fever and chills (36% in group A, 23% in group B). The most common biological effects of PP over protein-A sepharose were significant drops in IgG (66% of pre-PP values), CH50 and C3 (73% of pre-PP values) and a significant generation of C3a and C5a anaphylatoxins. Tumor response rates were 40% for group A, 0% for group B and 20% for group C. The median survival times tended to be longer in group A (17 months) than in group B (10 months) and in group C (9 months). This is the first randomized trial showing some therapeutic advantage in combining PP over protein-A sepharose with conventional chemotherapy in MCRC.

[Experimental models for studying the effects induced by staphylococcal toxins A and B on human keratinocytes in culture]. / Modelli sperimentali per lo studio degli effetti indotti dalla tossina staphylococcica A. e B. su cheratinociti umani in coltura.

The toxic effects of the two serotypes of staphylococcal exotoxin: exfoliatin A (ETA) and B (ETB) on two experimental models: organotypic cultures of the skin and cellular cultures of the epidermis reconstructed "in vitro" have been studied. The results show that, in both cases, purified ETB (Fig. 4a, 6-6a, b, c) reproduces the characteristics of Lyell's Staphylococcal Syndrome that is the intraepidermal cleavage either between the granulosa and the spinous layers or at the granulosa layer of the epidermis. As the images of the LM and EM demonstrate, purified ETA behaves differently in the two experimental models; in fact, it produces the same effect as purified toxin B on the organotypic cultures (Fig. 3a, b), whereas it causes no alterations in the epithelial cultures reproduced "in vitro" (Fig. 5a, b).