In vitro antimicrobial properties of plant essential oils thymus vulgaris, cymbopogon citratus and laurus nobilis against five important foodborne pathogens

Several essential oils of condiment and medicinal plants possess proven antimicrobial activity and are of important interest for the food industry. Therefore, the Minimum Inhibitory Concentrations (MIC) of those oils should be determined for various bacteria. MIC varies according to the oil used, the major compounds, and the physiology of the bacterium under study. In the present study, the essential oils of the plants Thymus vulgaris (time), Cymbopogon citratus (lemongrass) and Laurus nobilis (bay) were chemically quantified, and the MIC was determined on the bacteria Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19117, Salmonella enterica Enteritidis S64, and Pseudomonas aeruginosa ATCC 27853. The essential oil of C. citratus demonstrated bacterial activity at all concentrations tested and against all of the bacteria tested. The majority of essential oil compounds were geranial and neral. The major constituent of T. vulgaris was 1.8-cineol and of L. nobilis was linalool, which presented lower antibacterial activity, followed by 1.8-cineol. The Gram-negative bacteria demonstrated higher resistance to the use of the essential oils tested in this study. E. coli was the least sensitive and was inhibited only by the oils of C. citratus and L. nobilis.

Salmonella Typhimurium, Infantis, Derby, and Enteritidis survival in pasty dulce de leche

Dulce de leche is a food obtained by concentration and heating of milk with the addition of sucrose. The common practice of opening the dulce de leche containers in retail markets can lead to food contamination by Salmonella. The objective of this study was to evaluate the survivability of Salmonellaenterica subsp. enterica serotypes Typhimurium, Enteritidis, Infantis and Derby in pasty dulce de leche. Aliquots of this sweet were experimentally contaminated with these microorganisms and later analyzed to evaluate microorganism viability after storage for 0, 1, 2, 3, 5, 10, and 20 days. Salmonella was recovered up to the 20th day. These results are a warning about the need to adopt proper sanitary-hygienic measures for handling and packaging this food aiming at food safety.

Caractérisation des souches de Salmonella Typhimurium responsables de septicémies chez le porc

Depuis quelques années, il y a émergence de souches de Salmonella enterica sérovar Typhimurium multirésistantes causant une septicémie et la mort chez le porc. Ceci constitue un problème majeur pour l’industrie porcine et possiblement pour la santé publique. L’objectif de ce projet était de comparer et de caractériser une souche capable de causer une septicémie chez le porc et une souche commensale, en observant l’interaction avec des cellules épithéliales, des macrophages humains et d’identifier des gènes exprimés par les souches septicémiques et les souches commensales. Tout d’abord, l’infection de cellules épithéliales permet d’observer l’adhérence et l’invasion des bactéries, pour ainsi mettre en évidence la capacité des souches à coloniser le tractus gastro-intestinal. La souche commensale possède un pouvoir d’adhésion supérieur à la souche septicémique. Par la suite, l’infection de macrophages permet de caractériser le niveau de phagocytose et de survie. L’importance de la survie dans les macrophages pourrait permettre de faire un lien avec la septicémie. Toutefois, aucune différence n’est observable dans les conditions qui ont été testé. Ensuite, la technique SCOTS (Selective Capture of Transcribed Sequences) est utilisée pour capturer des gènes uniques à la souche septicémique et un autre SCOTS est fait pour capturer les gènes spécifiques à la souche commensale. Finalement, les gènes sont clonés, leur spécificité face aux souches est analysé par dot blot et ils sont identifiés par séquençage suivient d’une analyses bioinformatiques. Les gènes identifiés par SCOTS, lors des captures pour la souche septicémique et la souche commensale, se trouvent à être des gènes communs aux Salmonella. Toutefois, la différence de pathologie causée par les deux souches, n’est peut-être pas l’acquisition de nouveaux gènes, mais plutôt une différence d’expression entre les deux souches.

Physicochemical and molecular characterization of bacteriophages ΦSP-1 and ΦSP-3, specific for pathogenic Salmonella and evaluation of their potential as biocontrol agent

This thesis entitled Physicochemical and molecular characterization of bacteriophages ΦSP-1and ΦSP-3, specific for pathogenic Salmonella and evaluation of their potential as biocontrol agent . Salmonella were screened using standard methodologies from various environmental samples including chicken caecum. Salmonella strains, which were previously isolated and stocked in the lab, were also included in this study as host, for screening Salmonella specific lytic phages. The Salmonella strain in this study designated as S49 which helped in phage propagation by acting as host bacteria was identified as Salmonella enterica subsp. enterica by 16S rRNA gene analysis and serotyping . A total of three Salmonella specific phage named as ΦSP-1, ΦSP-2 and ΦSP-3 were isolated from chicken intestine samples via an enrichment protocol employing the double agar overlay method. ΦSP-1 and ΦSP-3 showing consistent lytic nature were selected for further study and were purified by repeated plating after picking of single isolated plaques from the lawns of Salmonella S49 plates. Both the phages produced small, clear plaques indicating their lytic nature. ΦSP-1 and ΦSP-3 were concentrated employing PEG-NaCl precipitation method before further characterization. The focus of present study was to isolate, characterize and verify the efficacy of lytic bacteriophages against the robust pathogen Salmonella, capable of surviving under various hostile conditions. Two phages, ΦSP-1 and ΦSP-3, belonging to two families, Podovoridae and Siphoviridae were isolated.

Influence of a Salt Water Regulator on the Survival Response of Salmonella Paratyphi in Vembanadu Lake: India

Contamination of environmental water by pathogenic microorganisms and subsequent infections originated from such sources during different contact and non- contact recreational activities are a major public health problem worldwide particularly in developing countries. The main pathogen frequently associated with enteric infection in developing countries are Salmonella enterica serovar typhi and paratyphi. Although the natural habitat of Salmonella is the gastrointestinal tract of animals, it find its way into natural water through faecal contamination and are frequently identified from various aquatic environments (Baudart et al., 2000; Dionisio et al., 2000; Martinez -Urtaza et al., 2004., Abhirosh et al., 2008). Typhoid fever caused by S. enterica serotype typhi and paratyphi are a common infectious disease occurring in all the parts of the world with its highest endemicity in certain parts of Asia, Africa, Latin America and in the Indian subcontinent with an estimated incidence of 33 million cases each year with significant morbidity and mortality (Threlfall, 2002). In most cases the disease is transmitted by polluted water (Girard et al., 2006) because of the poor hygienic conditions, inadequate clean water supplies and sewage treatment facilities. However in developed countries the disease is mainly associated with food (Bell et al., 2002) especially shellfish (Heinitz et al., 2000

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

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

Estudio de las comunidades microbianas de embutidos fermentados ligeramente acidificados mediante técnicas moleculares. Estandarización, seguridad y mejora tecnológica.

Los embutidos fermentados ligeramente acidificados son un grupo de productos tradicionales mediterráneos, caracterizados por un pH superior a 5,3. Para un control eficiente de la seguridad microbiológica de los embutidos se necesitan técnicas rápidas para la identificación y recuento de los microorganismos patógenos a estudiar. En el presente trabajo, se desarrolló una técnica para la enumeración de L. monocytogenes que combinó el método del número más probable y la identificación mediante PCR específica. Para la detección de Salmonella spp. y L. monocytogenes se desarrolló un sistema de PCR-multiplex que permitió la identificación de ambos patógenos de forma simultánea en una sola reacción. El estudio de la calidad microbiológica de los embutidos fermentados ligeramente acidificados se completó con la caracterización de las comunidades microbianas más importantes en estos productos. Se identificaron a nivel de especie los aislados de bacterias del ácido láctico (BAL), de enterococos y de cocos gram-positivos catalasa-positivos (CGC+). Posteriormente se realizó una tipificación molecular de los mismos mediante RAPD y análisis del perfil plasmídico y se estudiaron las principales características de interés higiénico-sanitario y tecnológico de las cepas. Mediante PCR se identificó Lactobacillus sakei como la especie predominante (74%), seguida por Lactobacillus curvatus (21,2%). La actividad aminoácido-descarboxilasa se asoció a la especie L. curvatus (el 66% de los aislados presentaron esta actividad). La identificación de los enterococos se realizó mediante PCR-multiplex y por secuenciación del gen sodA. Enterococcus faecium fue la especie de enterococos predominante (51,9%) seguida por Enterococcus faecalis (14,2%). Todas las cepas de E. faecalis presentaron genes asociados a factores de virulencia. E. faecalis presentó mayor resistencia a antibióticos que el resto de las especies de enterococos estudiadas. Tan sólo una cepa de E. faecium presentó el genotipo vanA (que confiere resistencia de alto nivel a la vancomicina). La identificación de los aislados de CGC+ (mediante PCR específica y amplificación de la región intergénica 16S-23S ARNr) demostró que Staphylococcus xylosus es la especie predominante en los embutidos fermentados ligeramente acidificados (80,8%). La amina biógena más común en los CGC+ fue la feniletilamina, producida por un 10,8% de aislados. Un pequeño porcentaje de aislados fueron mecA+ (4,6%), presentando además resistencia a múltiples antibióticos. El potencial enterotoxigénico de las cepas de CGC+ fue muy reducido (3,3% de los aislados), detectándose únicamente el gen entC. El estudio pormenorizado de las comunidades bacterianas de interés permitió la selección de 2 cepas de L. sakei y 2 cepas de S. xylosus con características tecnológicas e higiénico-sanitarias óptimas. Para evaluar su efectividad como cultivos iniciadores se elaboraron dos tipos de embutidos ligeramente ácidos, chorizo y fuet, inoculados con microorganismos patógenos (Salmonella spp., L. monocytogenes y S. aureus). El uso de cultivos iniciadores permitió el control de L. monocytogenes, Enterobacteriaceae y Enterococcus así como del contenido en aminas biógenas. Los recuentos de Salmonella spp. disminuyeron de forma significante durante la maduración de los embutidos, independientemente del uso de cultivos iniciadores. El uso del tratamiento de alta presión (400 MPa) en los embutidos madurados consiguió la ausencia de Salmonella spp. en los lotes tratados.

Osmotic upshift transiently inhibits uptake via ABC transporters in gram-negative bacteria

ATP-binding cassette transporters from several rhizobia and Salmonella enterica serovar Typhimurium, but not secondarily coupled systems, were inhibited by high concentrations (100 to 500 mM) of various osmolytes, an effect reversed by the removal of the osmolyte. ABC systems were also inactivated in isolated pea bacteroids, probably due to the obligatory use of high-osmolarity isolation media. Measurement of nutrient cycling in isolated pea bacteroids is impeded by this effect.

A chimeric N-terminal Escherichia coli C-terminal Rhodobacter sphaeroides FliG rotor protein supports bidirectional E coli flagellar rotation and chemotaxis

Flagellate bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium typically express 5 to 12 flagellar filaments over their cell surface that rotate in clockwise (CW) and counterclockwise directions. These bacteria modulate their swimming direction towards favorable environments by biasing the direction of flagellar rotation in response to various stimuli. In contrast, Rhodobacter sphaeroides expresses a single subpolar flagellum that rotates only CW and responds tactically by a series of biased stops and starts. Rotor protein FliG transiently links the MotAB stators to the rotor, to power rotation and also has an essential function in flagellar export. In this study, we sought to determine whether the FliG protein confers directionality on flagellar motors by testing the functional properties of R. sphaeroides FliG and a chimeric FliG protein, EcRsFliG (N-terminal and central domains of E. coli FliG fused to an R. sphaeroides FliG C terminus), in an E. coli FliG null background. The EcRsFliG chimera supported flagellar synthesis and bidirectional rotation; bacteria swam and tumbled in a manner qualitatively similar to that of the wild type and showed chemotaxis to amino acids. Thus, the FliG C terminus alone does not confer the unidirectional stop-start character of the R. sphaeroides flagellar motor, and its conformation continues to support tactic, switch-protein interactions in a bidirectional motor, despite its evolutionary history in a bacterium with a unidirectional motor.

A novel galactooligosaccharide mixture increases the bifidobacterial population numbers in a continuous in vitro fermentation system and in the proximal colonic contents of pigs in vivo

Prebiotics are nondigestible food ingredients that encourage proliferation of selected groups of the colonic microflora, thereby altering the composition toward a more beneficial community. In the present study, the prebiotic potential of a novel galactooligosaccharide (GOS) mixture, produced by the activity of galactosyltransferases from Bifidobacterium bifidum 41171 on lactose, was assessed in vitro and in a parallel continuous randomized pig trial. In situ fluorescent hybridization with 16S rRNA-targeted probes was used to investigate changes in total bacteria, bifidobacteria, lactobacilli, bacteroides, and Clostridium histolyticum group in response to supplementing the novel GOS mixture. In a 3-stage continuous culture system, the bifidobacterial numbers for the first 2 vessels, which represented the proximal and traverse colon, increased (P < 0.05) after the addition of the oligosaccharide mixture. In addition, the oligosaccharide mixture strongly inhibited the attachment of enterohepatic Escherichia coli (P < 0.01) and Salmonella enterica serotype Typhimurium (P < 0.01) to HT29 cells. Addition of the novel mixture at 4% (wt:wt) to a commercial diet increased the density of bificlobacteria (P < 0.001) and the acetate concentration (P < 0.001), and decreased the pH (P < 0.001) compared with the control diet and the control diet supplemented with inulin, suggesting a great prebiotic potential for the novel oligosaccharide mixture. J. Nutr. 135: 1726-1731, 2005.

Modulation of anti-pathogenic activity in canine-derived Lactobacillus species by carbohydrate growth substrate

Aims: To investigate the effect of various carbon sources on the production of extracellular antagonistic compounds against two Escherichia coli strains and Salmonella enterica serotype Typhimurium by three canine-derived lactobacilli strains. Methods and Materials: Cell-free preparations, pH neutralized, were used in antibiotic disc experiments as an initial screening. The bacteria/carbohydrate combinations that showed inhibition of the growth of those pathogens, were further investigated in batch co-culture experiments. The cell-free supernatants of the cultures, that decreased the population number of the pathogens in the co-culture experiments to log CFU ml(-1) less than or equal to 4, were tested for inhibition of the pathogens in pure cultures at neutral and acidic pH. Conclusions: The results showed that the substrate seems to affect the production of antimicrobial compounds and this effect could not just be ascribed to the ability of the bacteria to grow in the various carbon sources. L. mucosae, L. acidophilus and L. reuteri, when grown in sugar mixtures consisting of alpha-glucosides (Degree of Polymerization (DP) 1-4) could produce antimicrobial compounds active against all three pathogens in vitro. This effect could not be attributed to a single ingredient of those sugar mixtures and was synergistic. This inhibition had a dose-response characteristic and was more active at acidic pH. Significance and Impact of the Study: Knowledge of the effect that the carbon source has on the production of antimicrobial compounds by gut-associated lactobacilli allows the rational design of prebiotic/probiotic combinations to combat gastrointestinal pathogens.

Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media

Aims: To make a preliminary assessment of the incidence of Salmonella in Egyptian dairy products, and to investigate the effectiveness of various protocols for the detection of the pathogen in these products. Methods and Results: Samples of milk and related dairy products were randomly collected from local markets and examined for the presence of Salmonella. While most samples were free of the organism, isolates of Salmonella enterica subsp. enterica serovar Typhimurium PT 8 could be recovered from 'matared' cream specimens. These isolates were susceptible to antibiotics usually used to challenge infections caused by Salmonella. A combination of buffered peptone water, Muller-Kauffman tetrathionate broth, and brilliant green phenol red agar gave the best results for the detection of the pathogen. Selenite-cystine broth and Hektoen enteric agar were ineffective as an enrichment and a plating medium, respectively, in the isolation of Salmonella. A modified identification strategy that reduces the burden of serological testing of presumptive isolates is proposed. Conclusions, Significance and Impact of the Study: 'Matared' cream could be a vehicle for transmitting Salmonella. Using the above combination of media, beside the suggested modified confirmatory procedure, should increase the effectiveness and ease of the detection of Salmonella in milk and dairy products.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain

To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain. Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates. Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1. Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.