Regulation of Staphylococcus aureus type 5 and type 8 capsular polysaccharides by CO(2).

Staphylococcus aureus expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO(2). Here we show that CO(2) reduces CP5 expression at the transcriptional level and that CO(2) regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO(2) caused a significant decrease in CP8 expression in four S. aureus strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine S. aureus strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of cap5 and cap8. To test whether these sequence variations are responsible for the different responses to CO(2), promoter regions from selected strains were fused to the reporter gene xylE in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO(2) in all derivatives of strain Newman and was always positively regulated by CO(2) in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO(2) response. trans-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive S. aureus strains.

Preparation, immunogenicity, and protective efficacy, in a murine model, of a conjugate vaccine composed of the polysaccharide moiety of the lipopolysaccharide of Vibrio cholerae O139 bound to tetanus toxoid.

The epidemic and pandemic potential of Vibrio cholerae O139 is such that a vaccine against this newly emerged serogroup of V. cholerae is required. A conjugate made of the polysaccharide moiety (O-specific polysaccharide plus core) of the lipopolysaccharide (LPS) of V. cholerae O139 (pmLPS) was prepared by derivatization of the pmLPS with adipic acid dihydrazide and coupling to tetanus toxoid (TT) by carbodiimide-mediated condensation. The immunologic properties of the conjugate were tested using BALB/c mice injected subcutaneously three times at 2 weeks interval and then a fourth time 4 weeks later. Mice were bled 7 days after each injection and then once each month for the following 6 months. LPS and TT antibody levels were determined by enzyme-linked immunosorbent assay using immunoplates coated with either O139 LPS or TT. Both pmLPS and pmLPS-TT conjugate elicited low levels of immunoglobulin M (IgM), peaking 5 weeks after the first immunization. The conjugate elicited high levels of IgG antibodies, peaking 3 months after the first immunization and declining slowly during the following 5 months. TT alone, or as a component of conjugate, induced mostly IgG antibodies. Antibodies elicited by the conjugate recognized both capsular polysaccharide and LPS from V. cholerae O139 and were vibriocidal. They were also protective in the neonatal mouse model of cholera infection. The conjugation of the O139 pmLPS, therefore, enhanced its immunogenicity and conferred T-dependent properties to this polysaccharide.

Genetic analysis of the cap5 locus of Staphylococcus aureus.

Staphylococcus aureus expresses at least eight distinct serotypes of capsular polysaccharide (CP). Gene clusters involved in the expression of serotypes 1, 5 and 8 have been cloned and sequenced. In this report we describe the isolation and analysis of serotype 5 capsular polysaccharide-defective mutants. A naturally occurring cap mutation in the laboratory strains 8325-4 and RN4220 was mapped to the cap5E gene by genetic complementation. The cap5H-K genes were shown to be responsible for CP5 serotype specificity by transduction and complementation.

Regulation of Staphylococcus aureus capsular polysaccharide type 5: CO2 inhibition in vitro and in vivo.

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats, and in vitro using ELISA and IFA. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1%-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1%-5% of CP5-positive cells were detectable in pouch exudates. CF and pouch isolates, however, reexpressed CP5 (70%-90% of cells) when grown in vitro with air. Addition of > or = 1% CO2 to air or to O2/N2 gas mixtures reduced CP5 expression significantly (P < .001) in a dose-dependent manner (6%-1% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal that regulates CP5 expression.

[Regulation of Staphylococcus aureus capsular polysaccharide type 5: in vitro and in vivo inhibition by CO2]. / Regulation des Staphylococcus aureus-Kapselpolysaccharids vom Typ 5: Hemmung durch CO2 in vitro und in vivo.

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats and in vitro using ELISA and immunofluorescence. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1-5% CP5-positive cells were detectable in pouch exsudates. CF and pouch isolates, however, re-expressed CP5 (70-90% of cells) when grown in vitro with air. Addition of 1% CO2 or more to air or to O2/N2 gas mixtures reduced CP5 expression significantly (p < 0.001) in a dose-dependent manner (1-6% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal which regulates CP5 expression.

Respiratory activity is essential for post-exponential-phase production of type 5 capsular polysaccharide by Staphylococcus aureus.

Capsule formation is believed to have a significant role in bacterial virulence. To examine the possible involvement of capsular polysaccharide (CP) from Staphylococcus aureus in the pathological mechanisms associated with staphylococcal infections, we investigated the influence of respiratory activity on type 5 CP production by S. aureus grown in the presence of various concentrations of dissolved oxygen or nitrate. The effects of several metabolic inhibitors (arsenite, cyanide, azide, trimethylamine N-oxide, 2-heptyl-4-hydroxyquinoline N-oxide, and 2,4-dinitrophenol) were also tested. The metabolism of the bacteria was estimated by measuring their reductive capacity and by monitoring the pH and concentrations of fermentation products. Type 5 CP was always produced by S. aureus during the exponential phase of growth under all culture conditions tested. In contrast, post-exponential-phase CP production appeared to be strictly dependent on the respiratory activity. Since post-exponential-phase CP production contributes at least two-thirds of the total CP obtained, the influence of S. aureus respiration on CP production might be of some importance in the process of infection.

Involvement of the accessory gene regulator (agr) in expression of type 5 capsular polysaccharide by Staphylococcus aureus.

The effect of an agr mutation on expression of type 5 capsular polysaccharide (CP) by Staphylococcus aureus Newman was investigated in different complex and synthetic media. CP expression by the agr mutant was strongly reduced in certain media but slightly in others, indicating that CP synthesis is positively controlled by agr. CP expression occurred in the post-exponential growth phase in both wild-type and mutant strains, suggesting that other regulatory systems could act in conjunction with agr.

Production of type 5 capsular polysaccharide by Staphylococcus aureus grown in a semi-synthetic medium.

The concentration of the type 5 capsular polysaccharide (CP) antigen of Staphylococcus aureus can be measured directly in cultures or cell suspensions by a two-step inhibition enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies. CP was synthesized during growth on a variety of carbon substrates and its production was not affected by the nature of the carbon source. High levels of yeast extract inhibited CP formation. CP was synthesized in batch culture at the same rate during exponential growth as in the post-exponential phase. Post-exponential CP production contributed at least half the final amount of CP measured. This phenomenon was observed in different culture media, although the specific yield of polysaccharide varied from one medium to another. Post-exponential CP production was observed in the pH range 6-7, but not at pH 8. Post-exponential production was strictly dependent on oxygen availability and did not occur under anaerobic conditions.

Staphylococcus aureus growth and type 5 capsular polysaccharide production in synthetic media.

The production of type 5 capsular polysaccharide by Staphylococcus aureus in synthetic media was investigated. The influence of medium components on capsular polysaccharide synthesis appeared to relate to the presence or absence of the component rather than to concentration gradient. The production of type 5 capsular polysaccharide was linked to energy availability and energy source, but not to carbohydrate concentration or carbon/nitrogen ratio. Regulation of capsular polysaccharide production by S. aureus in response to medium changes would appear to differ from that typically displayed in other organisms that produce polysaccharides.

[Epidemiologic study of acute infantile gastroenteritis in New Caledonia]. / Etude épidémiologique des gastro-entérites infantiles aiguës en Nouvelle-Calédonie.

In a test area (suburbs of Nouméa), a survey on acute infantile gastro-enteritis showed an annual incidence of 2.2% and a hospitalization rate of 27.5%. The 0 to 23 months age group was the most exposed. The factors of severity were: a low age, a high frequency of liquid stool, vomits, fever and associated acute respiratory infection. The etiologic diagnosis was possible in 76% of cases: 49.5% enteropathogens (22% bacteria, 27.5% viruses), 26.5% non-intestinal infections. The asymptomatic carriers were 11.5% for bacteria and 27.4% for viruses. In regard to epidemiology, the housing hygiene, the potable water supply and the presence of suitable water closet were over 90%. On the contrary, the individual hygiene was neglected, especially the hand washing. The pathogens are transmitted by the dirty hands of asymptomatic carriers. Mass media campaigns and health education of parents and children are the recommended prophylactic measures.

[Regional characteristics of enterotoxigenic Escherichia coli serotypes isolated from children with gastroenteritis in the South Pacific (New Caledonia, Vanuatu Republic, Wallis and Futuna]. / Particularités régionales des sérotypes de Escherichia coli entérotoxinogènes isolés de gastro-entérites infantiles dans le Pacifique Sud (Nouvelle-Calédonie, République de Vanuatu, Wallis et Futuna).

One hundred and fifty enterotoxigenic strains of Escherichia coli (ETEC) isolated from infants with acute diarrhoeas in New Caledonia, Vanuatu and Wallis and Futuna were examined for serotypes. Twenty serotypes were identified, 5% of E. coli (7 strains) were rough and 8% (12 strains) were untypable. The serotypes 06, 025, and 078 are enterotoxigenic at 100%. The serotypes 027 and 073 classically enterotoxigenic did not produce any enterotoxin. This study opens up a possibility that the predominant enterotoxigenic serotypes in that region of the South Pacific are probably different from the serotypes proposed in the pool of sera to identify ETEC.

[Production of heat-labile Escherichia coli enterotoxin in a synthetic medium and its assay on a culture of Vero cells]. / Production de l'entérotoxine thermolabile de Escherichia coli sur milieu synthétique en vue de son dosage sur culture de cellules Vero.

A modification of the technique described by Evans for the production of Escherichia coli heat-labile enterotoxin was employed. Instead of Casamino yeast extract medium, which is toxic for Vero cells, Eagle's minimal essential medium (MEM) commonly used in cell cultures was substituted and the LT toxin produced in MEM was assayed on Vero cells.

[Method for the detection and determination of heat-labile Escherichia coli enterotoxin by an immunoenzyme technic on a new polystyrene support adapted for a test kit]. / Méthode de détection et de dosage de l'entérotoxine thermolabile de Escherichia coli par une technique immunoenzymatique sur un nouveau support de polystyrène adapté en trousse autonome.

An ELISA method on microtitration plates to detect and assay Escherichia coli heat-labile enterotoxin (LT) is described. This technique is rapid and simple to perform in any laboratory. It allows detection of the presence of LT with the naked eye within 10 h in a 12-h E. coli culture supernatant. The reaction is based on immunological cross-reaction between LT and the Vibrio cholerae toxin (CT). In place of traditional microtitration plates coated with ganglioside Gm1, we propose a new polystyrene support coated with purified anti-CT antibodies. This coated support has been conditioned in a kit to be used in laboratories in bush dispensaries of endemic areas. It was tested with 40 enterotoxigenic (LT+) strains isolated from stools of diarrhoeal children and with 14 LT- strains. All supernatants LT+ and LT- were found positive and negative, respectively, with the ELISA method and with the new polystyrene support. Field tests (in Wallis, Futuna et Vanuatu) with the new kit and standard method were satisfactory.

[Diarrhea due to enterotoxigenic Escherichia coli in New Caledonia. Epidemiology and bacteriologic aspects]. / Les diarrhées a escherichia coli entérotoxinogène (ECET) en Nouvelle-Calédonie. Etude épidémiologique et aspect bactériologique.

Three types of Escherichia coli play important roles in the etiology of acute diarrhoea. Depending on the pathogenicity mechanisms involved, the E. coli intestinal strains can be divided into enteropathogenic (EPEC), enteroinvasive (EIEC) and enterotoxigenic (ETEC) strains. We have studied ETEC in New-Caledonia. In one year 53 strains were isolated. ETEC are isolated frequently during the wet season in melanesian children with a law hygiene level. A correlation has been established between the fermentation of the rhamnose and the production of heat-stable toxin (ST). All the strains ST+ had the same antibiotype (8 resistances). The strains producing heat-labile toxin (LT) had various antibiotypes. Eleven O serotypes have been identified and the 078 is the most frequent. An another plan we have identified serotypes found in Australia and in the SE Asian.

[Assay of rabbit anti-thermolabile enterotoxin antibodies from Escherichia coli by an immunoenzyme technic]. / Dosage des anticorps de lapin anti-entérotoxine thermolabile de Escherichia coli par une technique immunoenzymatique.

The titration of rabbit anti-Escherichia coli heat-labile enterotoxin antibodies by an immunosorbent assay (ELISA) generally uses a GM1 coating step to lend specificity to the ELISA reaction at a time when the method is tempered by a lack of purified E. coli toxin. We have developed an adsorption method of the toxin to the polystyrene in order to simplify the technique. Three different immunosorbent preparations were tested to determine which of them yielded the most sensitive results.

Growth of Streptococcus pyogenes and streptolysin O production in complex and synthetic media.

A comparative study of the growth of a highly haemolytic group A streptococcal strain in Trypticase-yeast extract medium and in a chemically defined medium was undertaken. Appreciable growth was obtained in the latter medium with the release of significant amounts (120 haemolytic units) of streptolysin O. This indicates that toxinogenic factors present only in the peptones of complex media are not essential for biosynthesis and release of this cytolytic toxin, as is the case for many bacterial toxins. NADase was also released in the synthetic medium. Bacterial cell mass, growth rate, and streptolysin O production were threefold higher in the complex medium. The effects of various carbohydrates on streptolysin O production in complex medium are investigated. No repression of toxin formation by glucose was observed. The relationship between growth and toxinogenesis in streptococci and in other toxinogenic bacteria is discussed.