Detección y expresión de superantígenos y de resistencia antimicrobiana en aislamientos obtenidos de mujeres portadoras de Staphylococcus aureus que cuidan y alimentan niños / Detection and expression of superantigens and resistance in isolates from women carriers of Staphylococcus aureus who take care of and feed children

Resumen Introducción. Staphylococcus aureus coloniza mucosas y piel, y causa graves infecciones en el hombre y los animales. Es importante establecer el estatus de portadoras de cepas enterotoxigénicas de este microorganismo en manipuladoras de alimentos, con el fin de prevenir intoxicaciones alimentarias. Objetivo. Establecer las correlaciones entre los genes de enterotoxinas clásicas, el gen tsst-1, la producción de toxinas en cultivo y la resistencia antimicrobiana en aislamientos de S. aureus provenientes de manipuladoras de alimentos que cuidan niños en sus comunidades. Materiales y métodos. Se cultivaron muestras de las fosas nasales y las yemas de los dedos de las manos, y se identificó S. aureus empleando las pruebas de rutina y métodos automatizados. La extracción de ADN se hizo mediante el método de bromuro de cetil-trimetil-amonio (Cetyl-Trimethyl- Ammonium Bromide, CTAB) modificado. Para la detección de superantígenos se emplearon pruebas de reacción en cadena de la polimerasa (PCR) simple y múltiple, y para la de toxinas, estuches comerciales. Resultados. Se encontró que el 22,0 % de los aislamientos correspondía a portadoras de S. aureus: 17,0 % en los aislamientos de fosas nasales; 5,0 % en los de las manos y 6,7 % simultáneamente en los dos sitios. La prevalencia de superantígenos fue de 73,7 %. El genotipo más frecuente fue el sea-tsst-1, con 10,0 %. La resistencia a un solo antibiótico fue de 74,7 % y, a cuatro antibióticos, de 3,2 %; de los aislamientos, el 93,7 % correspondía a cepas productoras de betalactamasas. La detección de genes clásicos y de tsst-1 mediante PCR fue de 48,4 % y la de toxinas en el sobrenadante, de 42,1 %, con una correlación de 95,7 %. Las mayores correlaciones se establecieron entre las toxinas TSST-1 (22/22) y SEA (17/18). La correlación del gen tsst-1 con la proteína y la resistencia fue de 100 %. Todos los aislamientos con el genotipo sea-tsst-1 t fueron resistentes y productores de las toxinas. Conclusión. La tasa de aislamientos de S. aureus toxigénicos y resistentes obtenidos de mujeres que cuidan y preparan alimentos para niños fue de más de 70 %, lo que demostró su gran virulencia y la consecuente necesidad de aplicar estrictamente las normas higiénicas y sanitarias vigentes para evitar el riesgo de intoxicación alimentaria.

Abstract Introduction: Staphylococcus aureus colonizes mucous membranes and skin causing severe infections in humans and animals. It is important to determine carrier status of enterotoxigenic strains of this microorganism in food handlers to prevent food poisoning. Objective: To establish the correlations among classic enterotoxigenic genes, tsst-1 gene, the production of toxins in cultures and antimicrobial resistance in S. aureus isolates from women who handle the food, feed and take care of children in their communities. Materials and methods: Nasal swab and finger samples were cultured and S. aureus was identified using routine methods and automated systems. DNA extraction was done by the CTAB modified method, and superantigen detection by simple and multiplex PCR, while toxins were detected using commercial kits. Results: We found that 22.0% of subjects were S. aureus carriers: 17.0% corresponded to nose samples, 5.0% to hands and 6.7% to both nose and hands. The prevalence of superantigens was 73.7%. The most frequent genotype was sea-tsst-1 with 10%. Resistance to one antibiotic was 74.7%, and to four antibiotics, 3.2%; 93.7% of the isolates were betalactamase-positive. Classical genes and tsst-1 gene were detected by PCR in 48.4% of samples and toxins in supernatant were detected in 42.1% of them with 95.7% of correlation.The highest correlations were established for TSST-1 and SEA with 100% and 94.4%, respectively. The correlation of tsst-1 gene with toxin production and resistance was 100%. All isolates with genotype sea-tsst-1 were toxin-positive and resistant. Conclusion: The rate of toxigenic and resistant S. aureus isolates from women in charge of feeding and taking care of children was higher than 70%, which demonstrates its high virulence. This requires the strict application of hygienic and sanitary regulations in order to avoid the risk of food poisoning.

Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.

Clinical features of elderly chronic urticaria

BACKGROUND/AIMS: Chronic urticaria (CU) is defined as itchy wheals lasting 6 weeks or more. As the aged population increases worldwide, it is essential to identify the specific features of this disease in the elderly population. METHODS: We investigated the prevalence and clinical features of CU in elderly patients. Medical records of 837 CU patients from the outpatient Allergy Clinic of Ajou University Hospital, Korea were analyzed retrospectively. Patients with chronic spontaneous urticaria according to the EAACI/GA2LEN/EDF/WAO guidelines were included. Patients older than 60 years were defined as elderly. RESULTS: Of the 837 patients, 37 (4.5%) were elderly. In elderly versus nonelderly CU patients, the prevalence of atopic dermatitis (AD) was significantly higher (37.8% vs. 21.7%, respectively; p = 0.022), while that of aspirin intolerance was lower (18.9% vs. 43.6%, respectively; p = 0.003) in terms of comorbid conditions. The prevalences of serum specific immunoglobulin E antibodies to staphylococcal enterotoxin A and staphylococcal enterotoxin B were considerably higher in elderly CU patients with AD than in those without AD (37.5% vs. 0%, respectively). CONCLUSIONS: Elderly patients with CU had a higher prevalence of AD. Therefore, there is a need to recognize the existence of AD in elderly CU patients.

Distinct cytokine profiles of circulating mononuclear cells stimulated with Staphylococcus aureus enterotoxin A in vitro during early and late episodes of chronic osteomyelitis

We investigated the cytokine profile of peripheral mononuclear cells from chronic osteomyelitis (OST) patients following in vitro stimulation with staphylococcal enterotoxin A (SEA). We demonstrate that stimulation with SEA induced prominent lymphocyte proliferation and high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 secretion in both OST and non-infected individuals (NI). Even though stimulation with SEA had no impact on IL-6 production in either patient group, the baseline level of IL-6 production by cells from OST patients was always significantly less than that produced by cells from NI. After classifying the osteomyelitic episodes based on the time after the last reactivation event as "early" (1-4 months) or "late" osteomyelitis (5-12 months), we found that increased levels of TNF-α and IL-4 in combination with decreased levels of IL-6 were observed in the early episodes. By contrast, increased levels of IL-10, IL-2 and IL-6 were hallmarks of late episodes. Our data demonstrate that early osteomyelitic episodes are accompanied by an increased frequency of "high producers" of TNF-α and IL-4, whereas late events are characterised by increased frequencies of "high producers" of IL-10, IL-6 and IL-2. These findings demonstrate the distinct cytokine profiles in chronic osteomyelitis, with a distinct regulation of IL-6 production during early and late episodes.

Specific IgE against staphylococcal enterotoxin [superantigens] in allergic rhinitis: an independent risk factor for severe bronchial asthma

The study aimed to investigate whether IgE to Staphylococcus aureus enterotoxins might be relevant to disease severity in adult asthmatic patients. Specific IgE antibody concentrations in serum against enterotoxins, grass pollen [GP], and house dust mite [HDM] allergens and total IgE levels were measured in 69 adult control subjects, 152 patients with non-severe asthma, and 166 patients with severe asthma. Severe asthma was defined as inadequately controlled disease despite high-dose inhaled corticosteroids plus at least 2 other controller therapies, including oral steroids. Statistical analysis demonstrated Enterotoxin IgE positivity which was significantly greater in patients with severe asthma [59.67%] than in healthy control subjects [13% P< .001]. Twenty-one percent of patients with severe asthma showing positive enterotoxin IgE were considered non atopic. Also statistical analyses demonstrated significantly increased risks for enterotoxin IgE-positive subjects to have severe asthma [95%] versus enterotoxin IgE-negative subjects. The presence of GP or house dust IgE antibodies was not associated with either significantly increased risk for asthma or severity. Oral steroid use and hospitalizations were significantly increased in patients with positive enterotoxin IgE and non-atopic asthma. GP IgE was associated with a higher FEV1 percent predicted value and enterotoxin IgE was associated with a lower FEV1 percent predicted value. Staphylococcal enterotoxin IgE antibodies, but not IgE against inhalant allergens, are risk factors for asthma severity. We hypothesize that the presence of enterotoxin IgE in serum indicates the involvement of Staphylococcal superantigens in the pathophysiology of patients with severe bronchial asthma

Helicobacter pylori vaccine: mucosal adjuvant & delivery systems.

Vaccination, especially mucosal vaccination, is considered to be effective in the management of Helicobacter pylori infections. However, most antigens alone cannot induce immune responses when administered mucosally and need to be co-administered with adjuvants or delivery systems. The current research on the mucosal adjuvant and delivery systems of vaccine against H. pylori, including advantages and disadvantages, mechanisms and applications is discussed in this review. Mutants of cholera toxin (CT) and the heat labile enterotoxin of Escherichia coli (LT), CpG oligodeoxynucleotides, biocompatible and biodegradable polymers, and live attenuated bacterial vectors may be promising adjuvant and delivery systems for H. pylori vaccine.

Production and Characterization of Anti-Staphylococcal Toxic Shock Syndrome Toxin-1 Monoclonal Antibody / 대한진단검사의학회지

BACKGROUND: Recently the association between the virulence factors of Staphylococcus aureus and the outcome of the patients infected with the organism appears to be the subject of active investigation. Toxic shock syndrome toxin-1 (TSST-1) is thought to be a clinically more significant virulence factor than other staphylococcal toxins. We attempted to produce and characterize monoclonal antibodies to staphylococcal TSST-1. METHODS: An important epitope of TSST-1, amino acids 1-15 region, was synthesized into a peptide antigen, and Balb/c mice were immunized by intraperitoneal injection of the synthetic antigen. Hybridomas were produced by fusing immunized murine splenocytes with immortal myeloma cells. Hybridomas were cloned through a limiting dilution method. Stable cultured hybridoma was injected into the peritoneal cavity of Balb/c mice, and peritoneal fluid containing the monoclonal antibody was produced. RESULTS: One IgG2b type monoclonal antibody and two IgM type monoclonal antibodies were obtained. The IgG2b type monoclonal antibody was able to detect 5 microgram of TSST-1 with Western blot analysis and showed a strong reactivity to TSST-1 with ELISA. CONCLUSIONS: Highly immunoreactive anti-TSST-1 monoclonal antibody was produced by the use of synthesized peptide antigen. Diagnostic and protective capacity of this monoclonal antibody should be evaluated in the future.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes

Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.

Low cost production and purification of polyclonal antibodies to staphylococcal enterotoxin A

An immunization scheme for production of antiserum to staphylococcal enterotoxin A (SEA) is proposed. The reference method of Robbins and Bergdoll was modified to reduce the number of doses and the amount of toxin used per animal. The best immunization scheme used injections in days 0,8,24,59,62 and 67. The amount of toxin at each injection was 5,6,20,50,100 and 200ug, respectively. The total amount of toxin was 381ug, which corresponded to a reduction of 107ug in the amount of toxin for each animal when compared to the reference method. The average antiserum titer using the Optimum Sensitivity Plate - OSP was 1:60 and using ELISA the titer was 1:100.000. The lack of cross-reactivity with other staphylococcal enterotoxins indicated high specificity of the antibody to SEA. The proposed immunization scheme was adequate to produce specific SEA antisera, with high titers and the aditional advantage of reducing the amount of purified SEA required for immunization.

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.

Epítopos continuos y comunes presentes en las fimbrinas de Escherichia coli enterotoxigénica (ETEC) / Common continuos epitopes present in enterotoxigenic escherichia coli (ETEC) fimbriae

Escherichia coli enterotoxigénica (ETEC) es un agente causal de diarrea. ETEC cuenta con factores antigénicos de colonización (CFAs y PCFs), que son importantes en la virulencia. Los anticuerpos contra los CFAs son producidos al término de la infección por ETEC y han mostrado ser protectores contra la enfermedad. Sin embargo, los epítopos en CFA/I, los cuales inducen protección, no han sido caracterizados. El objetivo de este estudio es la detección de los epítopos continuos y comunes en CFA/I de ETEC, a nivel molecular, que conduzcan al desarrollo de métodos para la prevención de la enfermedad causada por ETEC. Se sintetizaron octapéptidos continuos unidos a fase sólida que comprendían a todo la secuencia de CFA/I por el método de Geysen para el escrutinio, con sueros de niños infectados por ETEC, con sueros de adultos de zona endémica y no endémica con el anticuerpo monoclonal CAF/I 1:6 (Mab CFA/I 1:6) y con los sueros hiperinmunes contra CFAs y PCFs diferentes a CFA/I. Los sueros de los niños, de los adultos de zona endémica y los sueros hiperinmunes reconocieron tres epítopos continuos y comunes en CFA/I. El Mab CFA/I 1:6 no reconoció nungún epítopo continuo

Diffuse adherence, ST-I enterotoxin and CFA/IV colonization factor are encoded by the same plasmid in the Escherichia coli O29: H21 strai

Escherichia coli O29:H21 is a human enterotoxigenic serotype that produces heat-stable (ST-I) enterotoxin, adheres diffusely to HeLa cells, and presents colonization factor antigen IV (CFA/IV) composed of CS5CS6 surface antigens. In one strain studied the genes for diffuse adherence and CFA/IV (CS5CS6) production were found to be present in the same plasmid encoding ST-I. The virulence plasmid (Ent) presented two unrelated basic replicons homologous to repFIC and repW. Gene(s) encoding diffuse adherence did not share homology with the probe for F1845 fimbrial adhesin which is responsible for this phenotype in other E. coli strains. Ent plasmid containing genes for diffuse adherence have not been described previously.

Construction of a hibrid plasmid coding for adherence antigen k88AB as well as the B subunit of Escherichia coli thermo-labile enterotoxin and mouse immune response to the encoded antigens

Um plasmídio híbrido (pUB3744) codificando para o antígeno de aderência K88ab e para a subunidade B da enterotoxina termo-lábil (LT-B) de Escherichia coli foi construído pela ligaçäo dos plasmídios pFM205 (K88ab) e pUB1844(LT-B). Estes 3 plasmídios foram subsequentemente transferidos para a amostra de E.coli O45:K, isolada de suíno, obtendo-se variantes K88ab, LT-B e K88ab/LT-B. Estas variantes foram inoculadas inoculadas por via oral em grupos de camundongos durante cinco dias consecutivos. A resposta de anticorpos isotipo-específica para K88ab, LT-B e para antígeno de bactérias sonicadas foi medida no soro e em raspados da mucosa intestinal cinco dias após a última inoculaçäo. A excreçäo da bactéria foi avaliada cultivando-se amostras de fezes. A bactéria LT-B foi eliminada por mais tempo pelos camundongos e induziu uma resposta imune local para LT-B (IgA, IgG e IgM). Näo foram detectados anticorpos para K88ab nos camundongos inoculados com a bactéria produzindo somente K88ab, mas o grupo de camundongos inoculados com as variantes 045:K apresentaram um aumento de anticorpos séricos para o antígeno de bactérias sonicadas e os níveis foram mais elevados no grupo inoculado com a variante K88ab/LT-B

Widespread emergence of Vibrio cholerae 0139 in India.

The National Institute of Communicable Diseases (NICD) has been monitoring the incidence of laboratory confirmed cases of cholera in Delhi in collaboration with Infectious Diseases Hospital (IDH) since 1965. Cholera and cholera-like cases from all hospitals in Delhi are admitted in IDH and the rectal swabs of all such cases are processed for isolation of Vibrio cholerae at NICD laboratory. Since April 1993, there has been isolation of Vibrio cholerae serotype 0139, in increasing numbers (831 out of 2,830, 29.2%) The isolates have been characterized and enterotoxin studies carried out. As a referral laboratory NICD has also confirmed the causative role of Vibrio cholerae 0139 in diarrhea outbreaks from various parts of the country. The implications of establishment of this newer serotype of Vibrio cholerae, as a potential epidemic strain are discussed.

Humoral immune response anti K99 pilus from enterotoxigenic Escherichia coli in experimentally inoculated calves

The bovine model is extremely interesting to study several basic aspects of mucosal local immunity. Many reports have shown that, in young calves, the infectivity of enterotoxigenic Escherichia coli may be inhibited by passively administered antibodies anti K99 pilus. We have measured, by immunoradiometric assays, the IgG response anti K99 pilus in the serum of calves, deprived of colostrum and orally inoculated with enteropathogenic K99+ E. coli. Although variable levels of IgG anti K99 pilus were detected, their protective value could not be ascertained in vivo due to the acute development of the infection. In an effort to correlate the presence of serum antibodies anti K99 pilus with their protective capacity, an ex-vivo assay to monitor the interaction of radiolabeled K99 pilus with the bovine mucosa was standardized. Paradoxically, although K99 pilus, purified by standard procedures, was recognized by polyclonal rabbit and calf antisera, its interaction with the bovine intestinal mucosa, quantitated in the ex-vivo system, was not inhibited by these reagents, indicating that the antibodies did not effectively block those K99 pilus domains involved in the interaction with mucosal receptors

Purification & antigenicity of Salmonella newport enterotoxin.

The enterotoxic moiety present in cell free culture supernatant (CFCS) of S. newport strains (MP/120 and BM/704) was purified and antigenically characterised. Purification was achieved to homogeneity by salt precipitation, dialysis and successive gel filtration through Sephadex G-100 and G-200 columns with the help of FPLC set. It was non-dialysable and purified enterotoxin yielded a single protein band in PAGE. It appeared to be of high molecular weight (100 KDa) and highly immunogenic in the rabbit. Antigenically, it was not related to cholera toxin, Shiga toxin or heat-labile enterotoxin of Escherichia coli.