Inactivation of Staphylococcus aureus and Salmonella enteritidis in tryptic soy broth and caviar samples by high pressure processing

We studied the action of high pressure processing on the inactivation of two foodborne pathogens, Staphylococcus aureus ATCC 6538 and Salmonella enteritidis ATCC 13076, suspended in a culture medium and inoculated into caviar samples. The baroresistance of the two pathogens in a tryptic soy broth suspension at a concentration of 10(8)-10(9) colony-forming units/ml was tested for continuous and cycled pressurization in the 150- to 550-MPa range and for 15-min treatments at room temperature. The increase of cycle number permitted the reduction of the pressure level able to totally inactivate both microorganisms in the tryptic soy broth suspension, whereas the effect of different procedure times on complete inactivation of the microorganisms inoculated into caviar was similar.

Detecção rápida de Salmonella Enteritidis em alimentos por ensaio imunoenzimático ELISA

O método convencional de detecção de Salmonella spp., além de trabalhoso, consome longo tempo, necessitando-se normalmente de 4 a 5 dias para a confirmação da presença dessa bactéria no alimento. Portanto, o emprego de métodos rápidos e simples é importante para o diagnóstico laboratorial de toxinfecção alimentar e para o controle de qualidade. O objetivo principal deste trabalho foi a padronização de ensaio imunoenzimático-ELISA para detecção de Salmonella Enteritidis em alimentos. O ensaio utilizou anticorpos policlonais para flagelina produzidos em coelho. O anti-soro apresentou pouca reação cruzada com os sorotipos de Salmonella e as diferentes espécies de enterobactérias testadas. A sensibilidade do ensaio foi de 10(4) células/mL, quando testado em cultivo puro. O conjugado peroxidase manteve-se estável durante dois meses a 4ºC e o seu uso deve ser exclusivamente durante este tempo. O ensaio padronizado apresentou simplicidade e rapidez, com sensibilidade de 1 célula/25g de maionese de batata e cenoura, após enriquecimento em água peptonada tamponada durante 24 horas a 37°C, sem necessidade de enriquecimento seletivo.

Cinética de crescimento de Salmonella Enteritidis envolvida em surtos alimentares no RS: uma comparação com linhagens de outros sorovares

No período de 1999 a 2002, uma linhagem geneticamente caracterizada de Salmonella Enteritidis esteve envolvida em mais de 90% das salmoneloses ocorridas no RS. Este trabalho teve por objetivo avaliar a cinética de crescimento dessa linhagem, comparando-a com o crescimento de uma linhagem de S. Typhimurium e de S. Bredeney não envolvida em surtos alimentares. Para tanto, cada linhagem foi semeada separadamente em caldo nutriente (CN) e em salada de batata com maionese caseira (SMC), os quais foram mantidos a 30 °C e 9,5 °C. Os resultados obtidos em laboratório foram comparados com os resultados modelados pelo Pathogen Modelling Program, USDA. Em CN, a cinética de crescimento a 30 °C foi semelhante para todas as linhagens, as quais atingiram aproximadamente 8 log UFC.mL-1. Em SMC, a 30 °C, a linhagem de S. Enteritidis apresentou maior quantidade de células nas primeiras 6 horas de crescimento, sendo que somente depois de 12 horas todos os microrganismos testados atingiram quantidades semelhantes de células, ou seja, aproximadamente 6 log UFC g-1. Em CN e em SMC, na temperatura de 9,5 °C, não foi detectado crescimento de nenhuma das linhagens testadas de Salmonella durante as primeiras 24 horas, sugerindo que essa temperatura foi suficiente para controlar a multiplicação desses microrganismos. A modelagem bacteriana confirmou a maioria dos resultados obtidos.

Effectiveness of cleaning and sanitizing procedures in controlling the adherence of Pseudomonas fluorescens, Salmonella Enteritidis, and Staphylococcus aureus to domestic kitchen surfaces

The effectiveness of cleaning and sanitizing procedures in controlling Staphylococcus aureus, Salmonella Enteritidis, and Pseudomonasfluorescens adhered to granite and stainless steel was evaluated. There was no significant difference (p > 0.05) in the adherence of pure cultures of these microorganisms to stainless steel. The numbers of P. fluorescens and S. Enteritidis adhered to granite were greater (p < 0.05) than the numbers of S. aureus. Additionally, the adherence of P. fluorescens was similar to the adherence of S. Enteritidis on granite surface. In a mixed culture with P. fluorescens, S aureus adhered less (p < 0.05) to stainless steel surfaces (1.31 log CFU.cm-2) than when in a pure culture (6.10 log CFU.cm-2). These results suggest that P. fluorescens inhibited the adherence of S. aureus. However, this inhibition was not observed in the adherence process for granite. There was a significant difference (p < 0.05) between the number of adhered cells before and after pre-washing for S. aureus on stainless steel and granite surfaces, and after washing with detergent for all microorganisms and surfaces. The efficiency of the cleaning plus sanitizing procedures was not significantly different (p > 0.05) between the surfaces. However, a significant difference was observed (p < 0.05) between the sanitizer solutions. Sodium hypochlorite and peracetic acid were more bactericidal (p < 0.05) than a quaternary ammonium compound. With regard to microorganisms, S. aureus was the least resistant to the sanitizers. These results show the importance of good cleaning and sanitization procedures to prevent bacterial adherence and biofilm formation.

Óleo essencial de orégano: interferência da composição química na atividade frente a Salmonella Enteritidis

Este trabalho avaliou a interferência da origem e do teor de compostos fenólicos de óleo essencial de orégano (OEO) de cinco marcas comerciais provenientes de diferentes regiões do mundo, na atividade inibitória frente à Salmonella Enteritidis (SE). A composição de cada OEO foi determinada por cromatografia gasosa acoplada ao espectrômetro de massas (CG-IE-MS). A atividade inibitória frente SE in vitro foi avaliada pela técnica de difusão em poços, empregando-se soluções alcoólicas a 0,1; 0,2; 0,5; 1,0; ou 2,0%, observando-se a formação de halo de inibição após incubação a 30 °C por 24 horas. Como resultado da análise por CG-IE-MS, foram identificados dezessete componentes voláteis. Todos OEO analisados neste trabalho possuíam carvacrol como componente principal e apresentaram atividade antimicrobiana frente a SE nas cinco concentrações testadas. A ação inibitória das cinco marcas comerciais avaliadas não apresentou diferença estatística significativa (p > 0,05), porém o OEO proveniente da região do Mediterrâneo com p-cimeno e γ-terpineno, além de carvacrol, apresentou maiores halos de inibição de SE que os demais OEO. Concluiu-se que a multiplicação de Salmonella Enteritidis in vitro pode ser inibida por OEO cuja ação antimicrobiana independe da região produtora de orégano. No entanto, óleos essenciais que possuem p-cimeno e γ-terpineno, além de carvacrol, podem ter o seu efeito antimicrobiano potencializado.

Étude de l’efficacité de la vaccination à Salmonella Enteritidis chez la poule pondeuse et de la protection contre l’infection

Les infections à Salmonella Enteritidis chez les humains sont associées à la consommation d’œufs ou d’ovoproduits contaminés. La vaccination est un outil utilisé pour diminuer les risques d’infection à SE chez la volaille, mais avec des résultats variables. Au Canada deux bactérines, MBL SE4C et Layermune, sont couramment utilisées pour lutter contre SE. Cependant, leur efficacité n’a pas été complètement déterminée chez les poules pondeuses plus âgées. Par ailleurs, la capacité de ces vaccins à prévenir la transmission verticale et horizontale n’a pas encore été étudiée. L’objectif principal de cette étude était d’évaluer l’effet des deux bactérines sur la réponse immunitaire chez les poules pondeuses, de vérifier la protection conférée par ces vaccins contre l’infection expérimentale à SE, et d’identifier des protéines immunogènes afin de développer un vaccin sous-unitaire. Les oiseaux ont été vaccinés avec deux protocoles d’immunisation en cours d’élevage (soit à 12 et 18, ou à 16 semaines d’âge). Le groupe contrôle a été injecté avec la solution saline. Les oiseaux ont été inoculés per os avec 2 x 109 CFU de la souche SE lysotype 4 à 55 ou à 65 semaines d’âge. Les anticorps (IgG et IgA) ont été mesurés à différents temps avec un ELISA maison en utilisant l’antigène entier de SE. La phagocytose, flambée oxydative, les populations des splénocytes B et T ont été analysées en utilisant la cytométrie en flux. Les signes cliniques, l’excrétion fécale, la contamination des jaunes d’œufs et l’invasion des salmonelles dans les organes ont été étudiés pour évaluer l’efficacité de protection. La transmission horizontale a aussi été étudiée en évaluant l’infection à SE chez les oiseaux mis en contact avec les oiseaux inoculés. Les protéines immunogènes ont été identifiées par SDS-PAGE et Western blot à l’aide d’antisérums prélevés suite à la vaccination et/ou à l’infection expérimentale/naturelle, puis caractérisées par la spectrométrie de masse. Le protocole de vaccination avec deux immunisations a généré un niveau élevé de séroconversion à partir de 3 jusqu’à 32-34 semaines post-vaccination par rapport à celui avec une seule immunisation (p < 0.02), mais il n’y avait plus de différence entre les groupes à 54 et 64 semaines d’âge. Il n’y a pas eu de corrélation entre les niveaux d’IgG et les taux d’isolement des salmonelles dans les organes et des jaunes d’œuf. La production des IgA n’a été observée que chez les oiseaux vaccinés avec 2 injections de MBL SE4C (p ≤ 0.04). Après l’infection expérimentale, la production des IgA a été significativement plus élevée aux jours 1 et 7 p.i dans l’oviducte des oiseaux vaccinés (sauf pour le groupe vacciné avec 2 injections de Layermune) par comparaison avec le groupe contrôle (p ≤ 0.03). Seule la bactérine MBL SE4C a eu un effet protecteur contre la contamination des jaunes d’œuf chez les oiseaux infectés. Ce vaccin réduit partiellement en utilisant deux immunisations, le taux d’excrétion fécale des salmonelles chez les oiseaux inoculés et les oiseaux horizontalement infectés (p ≤ 0.02). Cinq des protéines identifiées par la spectrométrie de masse sont considérées comme des protéines potentiellement candidates pour une étude plus approfondie de leur immonogénicité: Lipoamide dehydrogenase, Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase), Elongation factor Tu (EF-Tu), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) et DNA protection during starvation protein. En général, les bactérines ont induit une immunité humorale (IgG et IgA) chez les poules pondeuses. Cette réponse immunitaire a protégé partiellement les oiseaux quant à l’élimination des salmonelles, la contamination des jaunes d’œuf, ainsi que la transmission horizontale. Dans cette étude, la bactérine MBL SE4C (avec deux immunisations) s’est montrée plus efficace pour protéger les oiseaux que la bactérine Layermune. Nos résultats apportent des informations objectives et complémentaires sur le potentiel de deux bactérines pour lutter contre SE chez les poules pondeuses. Étant donné la protection partielle obtenue en utilisant ces vaccins, l’identification des antigènes immunogènes a permis de sélectionner des protéines spécifiques pour l’élaboration éventuelle d’un vaccin plus efficace contre SE chez les volailles.

Production and immunogenicity of selected proteins of Salmonella Enteritidis

Au cours des dernières années, Salmonella Enteritidis est devenus les sérotypes les plus souvent isolés chez les patients canadiens, les cas étant liés à la consommation de viande de poulet et d’œufs crus. Les vaccins tués commercialement disponibles pour la volaille, stimulent mal l'immunité mucosale, tandis que l'utilisation de vaccins vivants reste controversée. Par conséquent, un vaccin sous-unitaire par voie orale peut être une solution. Cinq protéines bactériennes ont été choisies comme candidates potentielles et identifiées, soit Glyceraldehyde-3-phosphate dehydrogenase, Enolase, Lipoamide dehydrogenase, DNA protection during starvation protein et Elongation factor-Tu. Notre objectif a été de produire et de purifier ces protéines et de démontrer leur immunogénicité. Les gènes des protéines ont été amplifiés et clonés dans le vecteur pQE-30 pour expression dans Escherichia coli M15. La purification a été effectuée par FPLC. Des poules pondeuses SPF ont été séparées en 6 groupes et injectées par voie intramusculaire à different âges avec une des 5 protéines, ou le PBS chez le groupe témoin. Les œufs ont été ramassés pendant l'expérience et du sang a été prélevé à 36 semaines d'âge. Les anticorps IgY ont été extraits à partir du jaune d'oeuf et du sérum, et les IgA à partir du blanc d'oeuf. Des immunodots, westernblots et ELISA ont évalué l'immunogénicité des protéines et les niveaux d'anticorps induits . Nous avons constaté que ces cinq protéines pourraient stimuler la production d'anticorps spécifiques in vivo. GAPDH, Enolase et DPS ont induit des titres d'anticorps plus élevés que LpdA et EF-Tu.

Prevalence and antimicrobial resistance of Salmonella enteritidis and other salmonellas in the eggs and egg-storing trays from retails markets of Coimbatore, South India

A study was conducted to determine the incidence of Salmonella enterica serovar Enteritidis and other Salmonella serovars on eggshell, egg contents and on egg-storing trays. A total of 492 eggs and 82 egg-storing trays were examined over a period of 1 year from different retail outlets of a residential area of Coimbatore city, South India. Salmonella contamination was recorded in 38 of 492 (7.7%) eggs out of which 29 was in eggshell (5.9%) and 9 in egg contents (1.8%). Around 7.5% of the egg-storing trays were also found to be contaminated with Salmonella. Serotyping of the Salmonella strains showed that 89.7% of the strains from eggshell, 100% of the strains from egg contents and 71.4% of the strains from egg-storing trays were Salmonella Enteritidis. Other serovarvars encountered were S. Cerro, S. Molade and S. Mbandaka from eggshell and S. Cerro from egg-storing trays. Seasonal variations in the prevalence pattern were identified with, a higher prevalence during monsoon months followed by post-monsoon and premonsoon. Further examination of the Salmonella strains was carried out by testing their antimicrobial sensitivity against 10 commonly used antimicrobials. Results revealed high prevalence of multiple antimicrobial resistance among these strains suggesting possible prior selection by use of antimicrobials in egg production

Salmonella cross contamination in retail chicken outlets and the efficacy of spice extracts on salmonella enteritidis growth inhibhition on various surfaces

The source of samonella cross contamination in 15 retail chicken outlets in aresidual area in coimbatore city ,sourthern India was studied. Chopping boards and the butchers hands were predominant followed by knives and the weighing balance tray. Serotyping of the salmonella strains revealed that all strains were salmonella enteritis, except one which was found to be salmonella cerro.The anti bacterial activity of commonly used spices were evaluated.

Prevalence and antimicrobial resistance of Salmonella enteritidis and other salmonellas in the eggs and egg-storing trays from retails markets of Coimbatore, South India

A study was conducted to determine the incidence of Salmonella enterica serovar Enteritidis and other Salmonella serovars on eggshell, egg contents and on egg-storing trays. A total of 492 eggs and 82 egg-storing trays were examined over a period of 1 year from different retail outlets of a residential area of Coimbatore city, South India. Salmonella contamination was recorded in 38 of 492 (7.7%) eggs out of which 29 was in eggshell (5.9%) and 9 in egg contents (1.8%). Around 7.5% of the egg-storing trays were also found to be contaminated with Salmonella. Serotyping of the Salmonella strains showed that 89.7% of the strains from eggshell, 100% of the strains from egg contents and 71.4% of the strains from egg-storing trays were Salmonella Enteritidis. Other serovarvars encountered were S. Cerro, S. Molade and S. Mbandaka from eggshell and S. Cerro from egg-storing trays. Seasonal variations in the prevalence pattern were identified with, a higher prevalence during monsoon months followed by post-monsoon and premonsoon. Further examination of the Salmonella strains was carried out by testing their antimicrobial sensitivity against 10 commonly used antimicrobials. Results revealed high prevalence of multiple antimicrobial resistance among these strains suggesting possible prior selection by use of antimicrobials in egg production

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized L-histidine, L-glutamine, L-proline, L-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

Studies into the role of the SEF14 fimbrial antigen in the pathogenesis of Salmonella enteritidis

To investigate the role of the SEF14 fimbrial antigen in pathogenesis, a single defined sefA (SEF14(-)) inactivated mutant of Salmonella enteritidis strain LA5 was constructed and tested in a number of biological assay systems. There was no significant difference between the wild-type strain and the isogenic SEF14(-) mutant in their abilities to adhere to and invade HEp-2 epithelial cells or their survival in mouse peritoneal macrophages, whereas the SEF14(-) mutant was ingested more rapidly by isolated human PMN. Both the strains colonized the intestine, invaded and spread systemically in 1 day-old chicks, laying hens and BALB/c mice equally well. A significantly greater number of chicks excreted the wildtype SEF14(+) strain during the first week following infection as compared to those infected with the SEF14(-) mutant. However, similar numbers of chicks excreted the two strains between 2 and 7 weeks after infection. These results indicate that possession of SEF14 fimbriae alone do not appear to play a significant role in the pathogenesis of S. enteritidis although its contribution to virulence may be dependent on the host species infected. (C) 1996 Academic Press Limited

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.

SEF17 fimbriae are essential for the convoluted colonial morphology of Salmonella enteritidis

Salmonella enteritidis isolated from poultry infections generated a convoluted colonial morphology after 48 h growth on colonisation factor antigen (CFA) agar at 25 degrees C. A mutant S. enteritidis defective for the elaboration of the SEF17 fimbrial antigen, in which the agf gene cluster was inactivated by insertion of an ampicillin resistance gene cassette, and other wild-type S. enteritidis transduced to this genotype failed to produce convoluted colonies. However, growth of SEF17(-) mutans at 25 degrees C on CFA agar supplemented with 0.001% Congo red resulted in partial recovery of the phenotype. Immunoelectron microscopy demonstrated that copious amounts of the SEF17 fimbrial antigen were present in the extracellular matrix of convoluted colonies of wild-type virulent S. enteritidis isolates. Bacteria were often hyperflagellated also. Immunoelectron microscopy of SEF17(-) mutants grown on CFA agar+0.001% Congo red demonstrated the elaboration of an as yet undefined fimbrial structure. Isolates of S. enteritidis which were described previously as avirulent and sensitive to environmental stress failed to express SEF17 or produce convoluted colonies. These data indicate an essential role for SEF17, and possibly for another fimbria and flagella, in the generation of the convoluted colonial phenotype. The relationship between virulence and colonial phenotype is discussed.

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (> 75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.