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).

Mycobacterium avium-intracellulare contamination of mammalian cell cultures.

Mycobacterium avium contamination has been described as a putative contaminant of nonphagocytic mammalian cells. Screening of numerous cultured nonphagocytic mammalian cell lines revealed the presence of intracellular bacteria that were identified as M. avium-intracellulare. An extensive and critical analysis of the origin of infection, of cure protocols, and of biological manifestations in M. avium-infected cells is presented. As no tremendous visible alteration of turbidity or pH of cell culture media, and no morphological change occurred in most M. avium-infected cell cultures, detection of an infection by these bacteria is rather difficult. Recommendations are given for treatment of irreplaceable cultures and prevention of mycobacterial contamination in a tissue culture facility.

[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.

Comparison of microbiological results of needle puncture vs. superficial swab in infected diabetic foot ulcer with osteomyelitis.

AIM: To study prospectively two methods for the bacteriological diagnosis of osteomyelitis related to diabetic foot ulcer: needle puncture performed across normal skin surrounding the foot ulcer and superficial swabbing of the ulcer. PATIENTS AND METHODS: Diabetic patients with a foot ulcer complicated by bone or joint infection, as detected by X-ray imaging, were included in the study. Ulcer swabbing and needle puncture were performed in each patient. To reach the tissue nearest the bone surface, needle puncture was guided by X-ray imaging and the drop of fluid obtained by aspiration was used for both aerobic and anaerobic bacterial culture. RESULTS: Twenty-one diabetic patients were included. The mean number of microorganisms isolated by needle puncture was significantly lower compared with that obtained by superficial swabbing: 1.09 vs. 2.04 (P < 0.02). Three bacterial species were isolated by needle puncture only in one patient while three or more bacterial isolates were obtained by superficial swabbing in six patients. No bacterial isolate was detected in five patients by needle puncture and in two patients by superficial swabbing. Staphylococcus aureus accounted for 70% of cases (seven patients) when a single bacterial species was obtained by needle puncture. After needle puncture, no wound complication or infection was observed. CONCLUSION: Culture of samples obtained by needle puncture revealed one or two bacterial isolates in two-thirds of diabetic patients with osteomyelitis following foot ulcer. Given the lack of complications, this invasive diagnostic technique should be considered for deep direct sampling in diabetic patients with osteomyelitis related to foot ulcer when surgical debridement is contraindicated or delayed.

Peripartum bacteremias due to Leptotrichia amnionii and Sneathia sanguinegens, rare causes of fever during and after delivery.

We report three cases of delivery and postpartum bacteremia due to unusual anaerobic bacteria in healthy young women. Leptotrichia amnionii bacteremia occurred during delivery in two mothers and was associated with fetal distress during labor. Conversely, Sneathia sanguinegens bacteremia occurred postpartum, 2 days after delivery, without consequence for the neonate.

[Knowledge and prevention of tick-bite borreliosis: survey of the population in Alsace, an endemic area]. / Connaissance et prévention des borrélioses par piqûres de tiques. Enquête dans la population d'une région endémique européenne, l'Alsace.

INTRODUCTION: European borreliosis, known to the general public as 'Lyme disease' in analogy with the American form, which it differs from in many points, is endemic in the area of Alsace and is a public health problem. The level of knowledge and prevention of the population with regard to the disease has never been assessed in France. MATERIAL AND METHODS: A survey was conducted in all the national health examination centers in Alsace using a self-applied questionnaire. The data collected concerned the socio-demographical characteristics, knowledge on the disease, history of tick bites, fear the disease creates, frequency of visits to the forest, prevention habits when visiting the forest and the attitude adopted in the case of a tick bite. RESULTS: Out of the 600 persons included, 99 (16.5 p.cent) had been bitten by ticks once or more over the past year. The existence of Lyme's disease was known to 74 p.cent of the consultants, 63 p.cent claimed they were worried by the disease and 43 p.cent knew that the first manifestation is redness spreading over the skin. Twenty-seven percent wore trousers and long sleeves when visiting the forest and 19 p.cent inspected their body carefully on their return. The persons least well informed were also those who did little to protect themselves against tick bites. They often were under 30 years old, lived in urban settings and had few diplomas. Those who had frequent spare time in forest and those who had a history of tick bites were the best informed and protected themselves better. The fear of the disease was associated with better knowledge and more appropriate behaviour. DISCUSSION: This study shows that a large percent of the population in Alsace is exposed to tick bites. Tick bite borreliosis is relatively well known, but protection remains insufficient. Better knowledge of the disease is related to better prevention. Information and teaching campaigns for the general public could specifically target the young people, urban dwellers and those with few diplomas. Specific messages of prevention could be delivered to the most exposed at-risk subjects (i.e. those bitten by ticks or having leisure in forest) at the places of their leisure or medical consultations.

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.

Dialister pneumosintes associated with human brain abscesses.

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.

Kinetics of Bartonella birtlesii infection in experimentally infected mice and pathogenic effect on reproductive functions.

The kinetics of infection and the pathogenic effects on the reproductive function of laboratory mice infected with Bartonella birtlesii recovered from an Apodemus species are described. B. birtlesii infection, as determined by bacteremia, occurred in BALB/c mice inoculated intravenously. Inoculation with a low-dose inoculum (1.5 x 10(3) CFU) induced bacteremia in only 75% of the mice compared to all of the mice inoculated with higher doses (> or =1.5 x 10(4)). Mice became bacteremic for at least 5 weeks (range, 5 to 8 weeks) with a peak ranging from 2 x 10(3) to 10(5) CFU/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 of Bartonella was demonstrated since 76% of the fetal resorptions tested was culture positive for B. 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 studying Bartonella infection. This study provides the first evidence of reproductive disorders in mice experimentally infected with a Bartonella strain originating from a wild rodent.

Bartonella schoenbuchii sp. nov., isolated from the blood of wild roe deer.

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.

Disease expression of Lyme borreliosis in northeastern France.

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.

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.

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.

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.