Genetic and redox determinants of nitric oxide cytotoxicity in a Salmonella typhimurium model.

Paradoxically, nitric oxide (NO) has been found to exhibit cytotoxic, antiproliferative, or cytoprotective activity under different conditions. We have utilized Salmonella mutants deficient in antioxidant defenses or peptide transport to gain insights into NO actions. Comparison of three NO donor compounds reveals distinct and independent cellular responses associated with specific redox forms of NO. The peroxynitrite (OONO-) generator 3-morpholinosydnonimine hydrochloride mediates oxygen-dependent Salmonella killing, whereas S-nitrosoglutathione (GSNO) causes oxygen-independent cytostasis, and the NO. donor diethylenetriamine-nitric oxide adduct has no antibacterial activity. GSNO has the greatest activity for stationary cells, a characteristic relevant to latent or intracellular pathogens. Moreover, the cytostatic activity of GSNO may best correlate with antiproliferative or antimicrobial effects of NO, which are unassociated with overt cell injury. dpp mutants defective in active dipeptide transport are resistant to GSNO, implicating heterolytic NO+ transfer rather than homolytic NO. release in the mechanism of cytostasis. This transport system may provide a specific pathway for GSNO-mediated signaling in biological systems. The redox state and associated carrier molecules are critical determinants of NO activity.

Vegetais minimamente processados prontos para o consumo: influência da etapa de desinfecção na inativação de Salmonella Typhimurium, na ocorrência da contaminação cruzada e na avaliação quantitativa de risco microbiológico em relação a este patógeno

Dados mundiais apontam haver uma associação entre o aumento do comércio de vegetais minimamente processados prontos para o consumo (VPC) e o aumento da ocorrência de surtos de enfermidades transmitidas por alimentos. Durante o processamento industrial de VPC, a desinfecção é a principal etapa de inativação de micro-organismos patogênicos presentes, mas nessa etapa também pode ocorrer contaminação cruzada, com transferência de contaminantes de produtos contaminados para não-contaminados. Neste trabalho, foram coletadas informações sobre as práticas empregadas na etapa de desinfecção em dez importantes indústrias produtoras de VPC no Estado de São Paulo, avaliando-se, em seguida, a influência dessas práticas na qualidade microbiológica dos produtos e na inativação de Salmonella Typhimurium, bem como na ocorrência de contaminação cruzada por este patógeno. Um modelo de avaliação quantitativa de risco microbiológico foi elaborado para estimar o impacto da contaminação cruzada durante a etapa de desinfecção no risco de infecção por Salmonella devido ao consumo de VPC. Observou-se que, em todas as indústrias visitadas, a desinfecção dos vegetais era feita com produtos à base de cloro em concentrações de 50 a 240 mg/L, que resultava em redução de até 1,2 log na carga microbiana dos vegetais que entravam na linha de processamento. Ao avaliar a influência das características da água de processamento (pH, temperatura, concentração de matéria orgânica e concentração de dicloroisocianurato de sódio) e do tempo de contato entre a água clorada e os vegetais na redução de Salmonella, observou-se que a concentração do produto à base de cloro foi o parâmetro que apresentou maior influência (p<0.05). Concentrações de dicloroisocianurato de sódio acima de 10 mg/L foram necessárias para controle da contaminação cruzada durante a etapa de lavagem. O modelo de avaliação de risco construído indicou quantitativamente haver uma relação entre a concentração de dicloroisocianurato de sódio na água de desinfecção e o risco de ocorrência de surtos causados por Salmonella em VPC. Cenários simulando uso de dicloroisocianurato de sódio em concentrações abaixo de 5 mg/L indicaram que mais de 96% dos casos preditos de infecção por Salmonella poderiam ser atribuídos à ocorrência de contaminação cruzada, enquanto que em cenários com concentrações acima de 50 mg/L, casos de infecção devidos à contaminação cruzada não foram preditos. Estes resultados mostram que o controle da qualidade da água e o monitoramento da concentração de sanitizante na etapa de desinfecção são essenciais para evitar a ocorrência de contaminação cruzada e garantir a produção de VPC seguros para o consumo.

Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid

Sterile immunity against malaria can be achieved by the induction of IFNgamma-producing CD8(+) T cells that target infected hepatocytes presenting epitopes of the circumsporozoite protein (CSP). In the present study we evaluate the protective efficacy of a heterologous prime/boost immunization protocol based on the delivery of the CD8(+) epitope of Plasmodium berghei CSP into the MHC class I presentation pathway, by either a type III secretion system of live recombinant Salmonella and/or by direct translocation of a recombinant Bordetella adenylate cyclase toxoid fusion (ACT-CSP) into the cytosol of professional antigen-presenting cells (APCs). A single intraperitoneal application of the recombinant ACT-CSP toxoid, as well as a single oral immunization with the Salmonella vaccine, induced a specific CD8(+) T cell response, which however conferred only a partial protection on mice against a subsequent sporozoite challenge. In contrast, a heterologous prime/boost vaccination with the live Salmonella followed by ACT-CSP led to a significant enhancement of the CSP-specific T cell response and induced complete protection in all vaccinated mice.

The diagnosis of Salmonella types

Includes bibliographical references

Proceedings of the 3rd International Symposium on the Epidemiology and Control of Salmonella in Pork : Renaissance Mayflower Hotel, Washington, D.C., August 5 to 7, 1999 /

Includes bibliographical references.

Isolation and identification of Salmonella [and] Shigella /

Tables.

Quantification and prevalence of Salmonella in beef cattle presenting at slaughter

Aims: A survey to determine the prevalence and numbers of Salmonella in beef cattle presented for slaughter at abattoirs across Australia was conducted between September 2002 and January 2003. Methods and Results: Automated immunomagnetic separation (AIMS) was used for detection and isolation of Salmonella enriched from cattle faeces. Salmonella were enumerated from positive samples using a combination of the Most Probable Number (MPN) technique and AIMS. A total of 310 faecal samples were tested, 155 were from lot-fed cattle and 155 from grass-fed cattle. Salmonella spp. were isolated from 21 (6.8%) of the cattle and the prevalence amongst grass-fed cattle (4.5%) was not significantly different to that found in lot-fed cattle (9%). Counts of Salmonella in positive faeces varied from

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome.

Long-term persistence of multi-drug-resistant Salmonella enterica serovar Newport in two dairy herds

Objective - To evaluate the association between maintaining joint hospital and maternity pens;and persistence of multi-drug-resistant (MDR) Salmonella enterica serovar Newport on 2 dairy farms. Design - Observational study. Sample Population - Feces and environmental samples from 2 dairy herds. Procedure - Herds were monitored for fecal shedding of S enterica Newport after outbreaks of clinical disease. Fecal and environmental samples were collected approximately monthly from pens housing sick cows and calving cows and from pens containing lactating cows. Cattle shedding the organism were tested serially on subsequent visits to determine carrier status. One farm was resampled after initiation of interventional procedures, including separation of hospital and maternity pens. Isolates were characterized via serotyping, determination of antimicrobial resistance phenotype, detection of the CMY-2 gene, and DNA fingerprinting. Results - The prevalence (32.4% and 33.3% on farms A and B, respectively) of isolating Salmonella from samples from joint hospital-maternity pens was significantly higher than the prevalence in samples from pens housing preparturient cows (0.8%, both farms) and postparturient cows on Farm B (8.8%). Multi-drug-resistant Salmonella Newport was isolated in high numbers from bedding material, feed refusals, lagoon slurry, and milk filters. One cow excreted the organism for 190 days. Interventional procedures yielded significant reductions in the prevalences of isolating the organism from fecal and environmental samples. Most isolates were of the C2 serogroup and were resistant to third-generation cephalosporins. Conclusions and Clinical Relevance - Management practices may be effective at reducing the persistence of MDR Salmonella spp in dairy herds, thus mitigating animal and public health risk.

Investigating the prevalence of Salmonella in dogs within the Midlands region of the United Kingdom

Background - The intimate relationship between dogs and their owners has the potential to increase the risk of human exposure to bacterial pathogens. Over the past 40 years, there have been several reports on transmission of salmonellae from dogs to humans. This study therefore aimed to determine the prevalence of Salmonella in the faeces of dogs from the Midlands region of the United Kingdom to assess exposure risk and potential for zoonotic transmission. Results - A total of 436 apparently healthy dogs without diarrhoea from households (n = 126), rescue centres (n = 96), boarding kennels (n = 43), retired greyhound kennels (n = 39) and a pet nutrition facility (n = 132) were investigated for Salmonella shedding. Faecal samples were processed by an enrichment culture based method. The faeces from one dog (0.23 %; 95 % confidence limit 0.006 %, 1.27 %) was positive for Salmonella. The species was S. enterica subspecies arizonae. Conclusion - This study showed that the prevalence of Salmonella from faeces from apparently healthy dogs from a variety of housing conditions is low; however, Salmonella shedding was still identified.

Exploring the biological basis for Salmonella persistence in food manufacturing environments

The persistence of Salmonella spp. in low moisture foods is a challenge for the food industry as despite control strategies already in place, notable outbreaks still occur. The aim of this study was to characterise isolates of Salmonella, known to be persistent in the food manufacturing environment, by comparing their microbiological characteristics with a panel of matched clinical and veterinary isolates. The gross morphology of the challenge panel was phenotypically characterised in terms of cellular size, shape and motility. In all the parameters measured, the factory isolates were indistinguishable from the human, clinical and veterinary strains. Further detailed metabolic profiling was undertaken using the biolog Microbial ID system. Multivariate analysis of the metabolic microarray revealed differences in metabolism of the factory isolate of S.Montevideo, based on its upregulated ability to utilise glucose and the sugar alcohol groups. The remainder of the serotype-matched isolates were metabolically indistinguishable. Temperature and humidity are known to influence bacterial survival and through environmental monitoring experimental parameters were defined. The results revealed Salmonella survival on stainless steel was affected by environmental temperatures that may be experienced in a food processing environment; with higher survival rates (D25=35.4) at temperatures at 25°C and lower humidity levels of 15% RH, however a rapid decline in cell count (D10=3.4) with lower temperatures of 10°C and higher humidity of 70% RH. Several resident factories strains survived in higher numbers on stainless steel (D25=29.69) compared to serotype matched clinical and veterinary isolates (D25=22.98). Factory isolates of Salmonella did not show an enhanced growth rate in comparison to serotype matched solates grown in Luria broth, Nutrient broth and M9 minimal media indicating that as an independent factor, growth was unlikely to be a major factor driving Salmonella persistence. Using a live / dead stain coupled with fluorescence microscopy revealed that when no longer culturable, isolates of S.Schwarzengrund entered into a viable nonculturable state. The biofilm forming capacity of the panel was characterised and revealed that all were able to form biofilms. None of the factory isolates showed an enhanced capability to form biofilms in comparison to serotype-matched isolates. In disinfection studies, planktonic cells were more susceptible to disinfectants than cells in biofilm and all the disinfectants tested were successful in reducing bacterial load. Contact time was one of the most important factors for reducing bacterial populations in a biofilm. The genomes of eight strains were sequenced. At the nucleotide and amino acid level the food factory isolates were similar to those of isolates from other environments; no major genomic rearrangements were observed, supporting the conclusions of the phenotypic and metabolic analysis. In conclusion, having investigated a variety of morphological, biochemical and genomic factors, it is unlikely that the persistence of Salmonella in the food manufacturing environment is attributable to a single phenotypic, metabolic or genomic factor. Whilst a combination of microbiological factors may be involved it is also possible that strain persistence in the factory environment is a consequence of failure to apply established hygiene management principles.