Prevalence and characterization of Salmonella enterica serovar Weltevreden from imported seafood

During 2001-2005, 210 Salmonella enterica strains were isolated from seafood samples imported into US. Strains of S. enterica serovar Weltevreden were the most predominantly found among the 64 different serovars isolated. A total of 37 Salmonella Weltevreden isolates were characterized by pulsed-field gel electrophoresis (PFGE), plasmid profiles and antibiotic susceptibility to assess genetic diversity. Our results showed a low frequency of antibiotic resistance; 35 of the 37 isolates were sensitive to ampicillin, tetracycline, chloramphenicol, gentamicin, sulfisoxazole, streptomycin and kanamycin. Only two isolates, from samples originating in the Philippines and India, showed resistance to ampicillin and tetracycline and to streptomycin, sulfisoxazole and tetracycline, respectively. Of the 37 isolates, two isolates did not carry any plasmid and 35 isolates harbored several small and mega-plasmids. These isolates were differentiated into 10 distinct types based on plasmid profiles. Four different PFGE clusters were obtained with a genetic similarity of 66-76%. Four groups of isolates (formed by two or three isolates each) showed 100% similarity in the PFGE profiles. One of these groups included strains isolated in Vietnam in 2003, 2004 and 2005 from fish and shrimp. The other groups included strains isolated in Vietnam, Indonesia and Thailand in 2000, 2004 and 2005 from snail, shrimp and fish. Our findings show genetic diversity and temporal persistence of S. enterica serovar Weltevreden in recently monitored seafood imports.     

Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars

Salmonella enterica serovar Typhimurium is a major foodborne pathogen throughout the world. Until now, the specific target genes for the detection and identification of serovar Typhimurium have not been developed. To determine the specific probes for serovar Typhimurium, the genes of serovar Typhimurium LT2 that were expected to be unique were selected with the BLAST (Basic Local Alignment Search Tool) program within GenBank. The selected genes were compared with 11 genomic sequences of various Salmonella serovars by BLAST. Of these selected genes, 10 were expected to be specific to serovar Typhimurium and were not related to virulence factor genes of Salmonella pathogenicity island or to genes of the O and H antigens of Salmonella. Primers for the 10 selected genes were constructed, and PCRs were evaluated with various genomic DNAs of Salmonella and non-Salmonella strains for the specific identification of Salmonella serovar Typhimurium. Among all the primer sets for the 10 genes, STM4497 showed the highest degree of specificity to serovar Typhimurium. In this study, a specific primer set for Salmonella serovar Typhimurium was developed on the basis of the comparison of genomic sequences between Salmonella serovars and was validated with PCR. This method of comparative genomics to select target genes or sequences can be applied to the specific detection of microorganisms.     

Efficacy of egg cleaning compounds on eggshells contaminated with Salmonella enterica serovar Enteritidis

Salmonella Enteritidis infections of egg contents can be related to external contamination of the shell. In this study, the efficacy of three commercial cleaning and/or sanitizing compounds (sodium carbonate, sodium hypochlorite, and potassium hydroxide) was evaluated for bactericidal activity at pH values of 10, 11, and 12 against various concentrations (10(2), 10(4), or 10(6) CFU/ml) of Salmonella Enteritidis inoculated onto the eggshell surface. Efficacy of these chemical agents was also assessed against Salmonella Enteritidis in aqueous suspension. Our results indicated that none of the chemicals applied at the recommended manufacturer's concentrations (sodium carbonate, 36 ppm; other treatments, 200 ppm) could eliminate Salmonella Enteritidis from eggshells artificially contaminated with the highest bacterial concentrations (10(4) or 10(6) CFU/ml). Higher concentrations of each product, at least 5 to 20 times greater than recommended doses, were needed to destroy the bacteria on egg surfaces. However, at or slightly above the manufacturer's recommended use concentrations, all three formulations were effective against Salmonella Enteritidis in aqueous suspension (10(8) CFU/ml) or on eggshells contaminated with 10(2) CFU/ml. For both shell and suspension assays, inactivation of Salmonella Enteritidis occurred at lower concentrations at pH 12 than at pH 11 and 10. Contact time between chemicals and Salmonella apparently influenced bacterial inactivation. Extended contact times (2 to 10 min) reduced minimum chemical concentrations necessary to inactivate the bacteria. However, neither pH nor contact time influenced Salmonella Enteritidis inactivation when the initial bacterial numbers on eggshells were high.     

Fermented whey dairy beverage offers protection against Salmonella enterica ssp. enterica serovar Typhimurium infection in mice

Fermented whey dairy beverages are dairy products obtained by fermentation from a mixture of milk and whey. These beverages have important health benefits, which could be improved with the addition of probiotic cultures. This study assessed the protective effect of the cosupplementation of a probiotic culture (Lactobacillus casei 01) with a fermented whey dairy beverage against infection by Salmonella enterica ssp. enterica serovar Typhimurium in a murine model. Two fermented whey dairy beverages were prepared: conventional (FWB; starter culture) and probiotic (PFWB; starter and probiotic cultures). In the first set of experiments, Balb/C female mice were treated with FWB or PFWB, challenged with Salmonella Typhimurium, and analyzed for clinical signs, weight loss, and mortality for 20 d postinfection. In the second set of experiments, mice were treated with FWB or PFWB, challenged with Salmonella Typhimurium, and killed on d 10 postinfection. The liver, colon, and ileum were used for myeloperoxidase, eosinophil peroxidase, and histological analysis and translocation to the liver. The contents from the small intestine were used for secretory IgA determination. The FWB treatment showed a better effect on animal survival (70%), translocation of the pathogen to the liver (2 out of 10), histopathology (fewer lesions), and inflammation than PFWB, which presented 50% animal survival, translocation in 5 out of 10 animals, and higher lesions. The control group presented 40% animal survival, translocation in 6 out of 10 animals, and severe lesions. Therefore, FWB was deemed to have a greater protective effect against Salmonella Typhimurium infection in the murine model compared with PFWB.     

Pulsed field,ribotype and integron analysis of multidrug resistant isolates of Salmonella enterica serovar Newport

Twenty-one isolates of Salmonella enterica serovar Newport were evaluated for their antimicrobial resistance, pulsed field gel electrophoresis (PFGE) profiles, ribotype profiles, and their integron profiles. Antimicrobial resistance profiles indicated that 20 of the 21 isolates were resistant to the following antibiotics: amoxicillin–clavulanic acid (AMOX/CA), ampicillin (AMPC), cefoxitin (CFOX), ceftiofur (TIO), cephalothin (CRIN), chloramphenicol (CHL), streptomycin (STR), tetracycline (TET), and sulfamethoxazole (SMX). Five isolates showed resistance to gentamycin (GEN) and kanamycin (KAN). Trimethoprim–sulfamethoxazole (SMX/TMP) resistance was observed in six isolates. Eight of the twenty one isolates showed intermediate resistance to ceftriaxone (CTRX), with one isolate exhibiting complete resistance. PFGE clearly resolved the Salmonella Newport isolates into nine distinct clusters, and a good congruence was observed between PFGE and antibiotic resistance patterns. Automated riboprinting clearly distinguished between antibiotic resistant and sensitive strains of Salmonella Newport, and resolved the isolates into two ribogroups. One group consisted of the multidrug resistant isolates, and the other grouping contained the sensitive isolate. Three different integrons (1.0, 1.2, and 1.8 kb) were observed in many of the isolates, and several isolates contained more than one integron. Restriction fragment length polymorphisms (RFLP) indicated that integrons of the same size were indistinguishable.When integron analysis and ribotype analysis were used in conjunction, four subtypes of multidrug resistant Salmonella Newport isolates were clearly defined. These results demonstrate the possibility of utilizing automated ribotyping and integron analysis to rapidly subtype multidrug resistant Salmonella Newport isolates.     

Interaction of Salmonella enterica subspecies enterica serovar Typhimurium and mung bean (Phaseolus aureus) plants

The effect of Salmonella enterica subspecies enterica serovar Typhimurium, a zoonotic serovar, on mung bean (Phaseolus aureus) cultivar Pant Mung-3 plants was studied. Inoculation of mung bean seeds with Salmonella Typhimurium (7.2 x 10(5) CFU/ml) reduced sprouting rate (P < 0.07). This effect was more pronounced at higher levels of contamination. In the soil inoculated with Salmonella Typhimurium (7.2 x 10(6) CFU/g), germination was retarded and the number of defective sprouts was also significantly higher (P < 0.002). Salmonella Typhimurium grew inside germinating seeds and plant tissues and persisted in seedlings, adult plants, and harvested seedlings dried and stored at room temperature (30 degrees C) up to 45 days. Phaseolus aureus plants grown in sterile soil was resistant to Salmonella Typhimurium infection at 15 days of age and cleared Salmonella from all the aerial parts within 3 h of infection. However, Salmonella Typhimurium could be reisolated from the basal area of the stem and from soil even after 45 days of exposure to the pathogen.     

Survival characteristics of Salmonella enterica serovar Newport in the dairy farm environment

Multi-drug resistant (MDR) Salmonella enterica serovar Newport (S. Newport) has established a reservoir in dairy cattle. Infected herds suffer significant mortality in both adult and young animals, posing a considerable economic loss to producers. Land application of manure from infected animals may further spread the pathogen into the agroecosystem, causing public health concerns. Previous work by our group demonstrated that the organism persisted in manure and manured soil for 6 to 10 mo under laboratory conditions. In the present study, we determined the survival characteristics of MDR S. Newport in a dairy lagoon, compost pile, and soil of a grass field under natural conditions using environmental sentinel chambers with an initial concentration of S. Newport around 7 log₁₀ per gram. In the static compost pile at 64°C, S. Newport was eliminated within 18 h. In the dairy effluent lagoon, the pathogen survived for >137 d, whereas in the field soil, the organisms persisted for over 276 d. The survival of MDR S. Newport in both the lagoon and field soil followed a pattern of (1) an increase or plateau for a few days, (2) log-linear decline for 6 to 13 wk, and (3) a long tailing phase at low and variable concentration for 4 to 9 mo. Log reduction times (days required for 90% decrease in concentration) based on the log-linear decline phase were 7 d in the lagoon and 14 to 20 d in the soil. Conditions leading to faster inactivation during the initial phase do not necessarily translate into a quicker elimination of the pathogen. Regression models of the log-linear phase may be inaccurate for estimating complete pathogen elimination.     

Antimicrobial resistance of Salmonella enterica serovar Typhimurium isolated from cattle in Japan

The antimicrobial susceptibility of 144 Salmonella enterica serovar Typhimurium isolates collected from all over Japan between 1973 and 1998 were investigated. All the isolates exhibited resistance to four or more antimicrobials and 22 resistance patterns were observed. Isolates showing resistance patterns to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfonamides (Su) and tetracycline (T), which are typical resistance patterns for S. Typhimurium DT104 (DT104), were predominant. Thirty-six of the 68 isolates that exhibited resistance to five or more antimicrobials (ACSSuT+) were identified as DT104 by phage typing. Another 103 S. Typhimurium strains gathered from cattle between 1977 and 1999 in a limited area of Japan were analyzed for molecular epidemiological studies. Results using fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis suggest that clonal exchange of S. Typhimurium among cattle in Japan has occurred since 1992, and that contemporary strains show a remarkable degree of homogeneity with DT104 at a molecular level. The clonal replacement by DT104 affected the antimicrobial resistance pattern of S. Typhimurium from cattle in Japan.     

High prevalence of Salmonella enterica subsp. diarizonae in tonsils of sheep at slaughter

The presence of Salmonella was studied in tonsils and feces of sheep and goats at slaughter using PCR and culturing. The isolates were further characterized using PFGE to get more information about the genetic diversity of Salmonella strains circulating among sheep and goats. Antimicrobial resistance was studied because resistance to multiple antimicrobial agents among Salmonella is increasing. The prevalence of Salmonella was 43% and 2% in the tonsils of sheep and goat, respectively. Salmonella was not detected in the feces of adult animals and only sporadically in the feces of juveniles (2%). S. enterica subsp. diarizonae 61:k:1,5,(7) was isolated from 20% of the sheep tonsils and 1% of the goat tonsils. In total, 9 genotypes were obtained with PFGE using SpeI, XbaI, NotI and XhoI restriction enzymes; however, one genotype was predominant. All strains were sensitive to most (13/16) of the antimicrobials. Resistance to sulfamethoxazole was high (95%). Three (15%) strains, which were isolated from lambs, were also resistant to colistin. No correlation between the antimicrobial resistance pattern and the genotype was noticed. These results demonstrate that slaughtered sheep are an important reservoir for S. enterica subsp. diarizonae 61:k:1,5,(7) carrying this pathogen frequently in the tonsils. Future studies are needed to elucidate the significance of the tonsils in the contamination of sheep carcasses and meat with Salmonella.     

Viability of Salmonella enterica subsp. enterica during the preparation and cold storage of Egyptian soft cheeses and ice-cream

Changes occurring in the viability of Salmonella enterica subsp. enterica during the preparation and cold storage of Domiati cheese, Kariesh cheese and ice-cream were examined. A significant decrease in numbers was observed after whey drainage during the manufacture of Domiati cheese, but Salmonella remained viable for 13 weeks in cheeses prepared from milks with between 60 and 100 g/L NaCl; the viability declined in Domiati cheese made from highly salted milk during the later stages of storage. The method of coagulation used in the preparation of Kariesh cheese affected the survival time of the pathogen, and it varied from 2 to 3 weeks in cheeses made with a slow-acid coagulation method to 4–5 weeks for an acid-rennet coagulation method. This difference was attributed to the higher salt-in-moisture levels and lower pH values of Kariesh cheese prepared by the slow-acid coagulation method. A slight decrease in the numbers of Salmonella resulted from ageing ice-cream mix for 24 h at 0°C, but a greater reduction was evident after one day of frozen storage at −20°C. The pathogen survived further frozen storage for four months without any substantial change in numbers.     

Sequencing of plasmids from a multi-antimicrobial resistant Salmonella enterica serovar Dublin strain

Salmonella enterica serovar Dublin (S. Dublin) is a host-adapted serotype whose primary host is cattle, which can serve as a potential reservoir for human infections. S. Dublin remains one of the leading causes of severe invasive infections and deaths associated with salmonellosis. Because of their propensity to cause severe infection, antimicrobial therapy is often required, thus antimicrobial resistance is an important concern. Plasmids play a key role in facilitating drug resistance in these pathogens. This study reports the results of DNA sequencing and sequence analysis of plasmids from a highly multidrug resistant strain (resistant to 11/15 drugs tested) of S. Dublin that originated from cattle. The strain was found to contain four plasmids of approximately 8, 77, 89, and 174 kb. The 174 kb plasmid is an incompatibility group (Inc) A/C plasmid containing genes associated with resistance to at least 9 different antimicrobials, as well as disinfectants and metals. The 88.5 kb plasmid is an IncFIB plasmid containing genes associated with resistance to at least 3 antimicrobial agents and mercurial compounds. The 77 kb plasmid is a S. Dublin virulence plasmid containing multiple virulence-associated genes and the 7.9 kb plasmid encodes mobilization and replication genes. Overall, sequencing identified multiple plasmids containing antimicrobial resistance and virulence genes. The resistance genes identified correlated to the observed resistance phenotype, further indicating the importance of plasmids in antimicrobial resistance in many Salmonella.     

Standardized laboratory-scale preparation of mayonnaise containing low levels of Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis PT4 and PT6 are associated with food poisoning outbreaks and are often found in food only in low concentrations. In this study a reproducible laboratory-scale procedure for preparation of mayonnaise is presented. The mayonnaise that simulates a naturally low-level contaminated product can be used for validation of new methods and is also suitable to study the behavior of low numbers of food pathogenic spoilage microorganisms in a food environment. During processing, liquid egg was artificially contaminated with low levels of S. enterica serovar Enteritidis that resulted in levels of 1 to 3 log10 CFU/g in the final mayonnaise. Cells of S. enterica serovar Enteritidis had increased stability in the mayonnaise when they were subjected to low pH in two stages, first to pH 5.8 and afterward to pH 4.5 before addition to the mayonnaise. The pH of the mayonnaise was between 4.2 to 4.5 and remained stable over the storage period. Low-level S. enterica serovar Enteritidis remained stable in artificially contaminated mayonnaise for 4 weeks at 4 degrees C.     

Quantification of low and high levels of Salmonella enterica serovar Typhimurium on leaves

Precise and rapid quantification of low levels of pathogens associated with fresh produce may be particularly challenging and yet evermore necessary to guarantee microbial safety of the produce and to carry out research on the subject of persistence of the pathogens. Here, microbiological and molecular based methods were examined for their ability to precisely quantify different amounts of Salmonella enterica serovar Typhimurium artificially inoculated on parsley leaves. Recovery of S. Typhimurium from parsley by mechanical detachment using stomacher, mortar and pestle, vortex, sonicator or homogenizer followed by plating resulted in underestimation with less than 1% recovery when leaves were inoculated with 3.5–6.5 log CFU/g. Lower levels were undetectable by most assayed methods, and only recovery with mortar and pestle or adding of enrichment step resulted in partial detection of 300 CFU/g. Implementation of PCR based methods with/without pre extraction of the DNA from the contaminated leaves resulted in more accurate values of the pathogen (about 20% of the initial inocula) and as low as 300 CFU/g were detected even without an enrichment step. These methods can be applied to study transfer of Salmonlla from contaminated water or soil to plants using low and more reasonable levels of contamination.     

Eggshell penetration of various types of hens' eggs by Salmonella enterica serovar Enteritidis

Egg weight, shell thickness, number of pores, cuticle deposition, eggshell strength (dynamic stiffness and damping ratio), and the ability of Salmonella enterica serovar Enteritidis (SE) to penetrate the eggshell were determined. Penetration was assessed by filling the eggs with a selective medium that allowed viewing of Salmonella growth on the inside of the shell and membrane complex. After inoculation of each shell with on average 2.71 log CFU, the eggs were stored for up to 14 days at 20 degrees C and 60% relative humidity. Commercially available eggs were used. At 14 days of storage, only 6.0% of the eggs from free-range hens and 16.0% of the generic (i.e., eggs from hens in conventional battery cages that were given standard feed) white eggs were penetrated. The generic brown, organic, and omega-3-enriched eggs were penetrated at a frequency of 30 to 34%. In a second experiment it was shown that the layer strains of the hen (ISA-Brown Warren versus Bovans Goldline), which were kept in furnished cages, did not affect eggshell penetration by SE. For Bovans Goldline hens, the housing system (furnished cage versus aviary) did not affect penetration, while a trend was visible toward a higher fraction of penetrated eggshells when hens were fed corncob mix rather than standard feed. Eggshell penetration was observed more frequently in the absence of cuticle spots and for eggs having lower dynamic stiffness values. Shell contamination at the end of storage was highly correlated with SE penetration.     

鲜切生菜清洗过程中肠炎沙门氏菌转移模型的建立

研究了肠炎沙门氏菌(Salmonella enterica serovar Enteritidis,SE)在鲜切生菜清洗过程中的转移并建立转移模型,选择NaClO及复合抑菌剂(250IU/mL nisin+0.15%柠檬酸+0.05%双乙酸钠,nisin-CA-SD)为清洗剂,对比二者在控制SE转移能力上的差异,考察幂函数与指数函数2种模型在清洗环境中的适用性。研究结果表明,随着清洗次数的增加,SE从接种生菜转移到未接种生菜上的数量逐渐减少;NaClO及复合抑菌剂均不能完全抑制SE在清洗过程中的转移,但复合抑菌剂更有助于抑制SE的交叉污染。清洗剂多次利用时,幂函数模型能更好地描述及预测SE的转移情况。     

Influence of eggshell condensation on eggshell penetration and whole egg contamination with Salmonella enterica serovar enteritidis

Shells of agar-filled and whole eggs were inoculated with 10(3) to 10(4) CFU of Salmonella enterica serovar Enteritidis per eggshell. The agar-filled eggs were used to study bacterial eggshell penetration, and the whole egg results were used to characterize contamination of the egg contents. In each group, half of the eggs were stored for 21 days at 20 degrees C and 60% relative humidity (RH), and the other half was stored for 24 h at 6 degrees C and then for 20 days at 20 degrees C. The latter conditions resulted in condensation on the eggshell for 30 min from the moment the eggs were placed in the 20 degrees C chamber. Taking into account the ages at which hens were studied (39, 53, and 67 weeks), an average of 62% of the eggshells with condensate were penetrated compared with 43% for the control group; this difference was significant (P < 0.01). No significant difference in whole egg contamination was found; 18% of the control eggs were contaminated compared with 22% of the condensate eggs. Whole egg contamination was significantly higher for eggs from the hens at an older age (67 weeks). This difference probably was not due to a higher penetration potential because differences were not observed for the corresponding agar-filled eggs. Condensation on the eggshell seemed to encourage bacterial penetration of the eggshell but had a smaller impact on whole egg contamination.     

Recovery of Salmonella enterica serovars Typhimurium and Tennessee in peanut butter after electron beam exposure

The effect of electron beam (e-beam) radiation on the recovery of Salmonella serotypes Tennessee (ATCC 10722) and Typhimurium (ATCC 14028) in creamy peanut butter over a 14-d storage period at 22 °C was studied. Each Salmonella type was independently inoculated into peanut butter and subjected to e-beam doses that ranged from 0 to 3.1 kGy, confirmed by film dosimetry. After 2-, 4-, 6-, 8-, and 14-d of storage, microbial analyses were conducted. Survivors were recovered on growth and selective media using standard spread-plating methods. Microbial counts (CFU/g) were log-converted and differences were determined by ANOVA and Tukey's Honestly Significant Differences test. When samples were not e-beam-treated, there were no significant changes (P > 0.05) in microbial numbers over time. In e-beamed samples, microbial numbers decreased over time; however, reductions were not always significant. Initial recovery rates (R-rates) 2 d after e-beam treatment were significantly different for the 2 strains of Salmonella and between recovery media (P < 0.05); however, these differences did not persist for the remainder of the storage period (P > 0.05) indicating that injured cells were not able to survive in the high-fat, low-water activity peanut butter environment. R-rates for both strains of Salmonella were maintained until day 14 when there were significant reductions in Salmonella Typhimurium (P < 0.05). These results indicate that Salmonella Tennessee and Salmonella Typhimurium will survive in peanut butter when exposed to nonlethal doses of e-beam irradiation. PRACTICAL APPLICATION: Electron beam (e-beam) irradiation is an alternative to thermal processing; this technique inactivates microorganisms and insects that might be present in a food by generating radiation by accelerated electrons that inactivate organisms directly because of interaction with cell components and indirectly by producing free radicals that disrupt integrity of the cell membrane. E-beam radiation will reduce the number of probable microbiological hazards that could be present while the food remains generally unaffected in texture, taste, and nutritional value. A recent study showed e-beam irradiation to be effective at reducing both Salmonella Tennessee and Typhimurium in peanut butter by one log after exposure to less than 1 kGy, highlighting the need to explore this process further.     

Heat tolerance of Salmonella enterica serovars Agona, Enteritidis, and Typhimurium in peanut butter

Recent large foodborne outbreaks caused by Salmonella enterica serovars have been associated with consumption of foods with high fat content and reduced water activity, even though their ingredients usually undergo pasteurization. The present study was focused on the heat tolerance of Salmonella enterica serovars Agona, Enteritidis, and Typhimurium in peanut butter. The Salmonella serovars in the peanut butter were resistant to heat, and even at a temperature as high as 90 degrees C only 3.2-log reduction in CFU was observed. The obtained thermal inactivation curves were upwardly concave, indicating rapid death at the beginning (10 min) followed by lower death rates and an asymptotic tail. The curves fitted the nonlinear Weibull model with beta parameters < 1, indicating that the remaining cells have a lower probability of dying. beta at 70 degrees C (0.40 +/- 0.04) was significantly lower than beta at 80 degrees C (0.73 +/- 0.19) and 90 degrees C (0.69 +/- 0.17). Very little decrease in the viable population (less than 2-log decrease) was noted in cultures that were exposed to a second thermal treatment. Peanut butter is a highly concentrated colloidal suspension of lipid and water in a peanut meal phase. We hypothesized that differences in the local environments of the bacteria, with respect to fat content or water activity, explained the observed distribution and high portion of surviving cells (0.1%, independent of the initial cell number). These results demonstrate that thermal treatments are inadequate to consistently destroy Salmonella in highly contaminated peanut butter and that the pasteurization process cannot be improved significantly by longer treatment or higher temperatures.     

Genetic characterization of antimicrobial resistance in Salmonella enterica serovars isolated from dairy cattle in Wisconsin

Salmonella enterica is a pathogen of humans and animals, and is one of the most frequent causes of bacterial foodborne illness worldwide. People consuming contaminated foods or working with infected livestock have the potential to become infected with Salmonella and may require antimicrobial therapy. Antimicrobial resistance in Salmonella has become a problem worldwide due in part to the inappropriate use of antimicrobial agents in human and veterinary medicine. In this study, forty-five Salmonella isolates from diagnostic fecal samples of Wisconsin dairy cattle were serotyped and characterized by antimicrobial susceptibility testing using agar disk diffusion, antimicrobial resistance gene detection by PCR, plasmid analysis and conjugation studies. The predominant serovars detected were Kentucky, Newport, Typhimurium, Cerro, Dublin and Montevideo. Over half (51%) of all isolates were resistant to at least one antimicrobial agent, and 29% were resistant to 8–10 drugs. The most commonly observed resistance phenotypes were to streptomycin (44%), tetracycline (42%), sulfisoxazole (40%), chloramphenicol (35%), ampicillin (33%), and cefoxitin (33%). When resistance was detected phenotypically, a corresponding resistance gene was detected 86.2% of the time. Plasmids ranging in size from < 8 to 165 kb were detected in 45% of the isolates. A greater understanding of the factors associated with antimicrobial resistance in Salmonella should provide insights into the factors that contribute to the development of resistant pathogens during dairy production, which in turn can lead to strategies to minimize the spread of antimicrobial resistant Salmonella in the food supply.     

Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis

Eight Lactobacillus kefir strains isolated from different kefir grains were tested for their ability to antagonize Salmonella enterica serovar Enteritidis (Salmonella enteritidis) interaction with epithelial cells. L. kefir surface properties such as autoaggregation and coaggregation with Salmonella and adhesion to Caco-2/TC-7 cells were evaluated. L. kefir strains showed significantly different adhesion capacities, six strains were able to autoaggregate and four strains coaggregated with Salmonella. Coincubation of Salmonella with coaggregating L. kefir strains significantly decreased its capacity to adhere to and to invade Caco-2/TC-7 cells. This was not observed with non coaggregating L. kefir strains. Spent culture supernatants of L. kefir contain significant amounts of S-layer proteins. Salmonella pretreated with spent culture supernatants (pH 4.5-4.7) from all tested L. kefir strains showed a significant decrease in association and invasion to Caco-2/TC-7 cells. Artificially acidified MRS containing lactic acid to a final concentration and pH equivalent to lactobacilli spent culture supernatants did not show any protective action. Pretreatment of this pathogen with spent culture supernatants reduced microvilli disorganization produced by Salmonella. In addition, Salmonella pretreated with S-layer proteins extracted from coaggregating and non coaggregating L. kefir strains were unable to invade Caco-2/TC-7 cells. After treatment, L. kefir S-layer protein was detected associated with Salmonella, suggesting a protective role of this protein on association and invasion.