Draft whole-genome sequences of Bacillus strains of potential probiotic for poultry.

Bacillus species have been widely selected and used as probiotics for humans and animals. In this article, we reported draft whole-genome sequences of four Bacillus strains isolated from sourdough and chicken cecum and previously selected as potential probiotics for poultry. These genome sequences will provide a foundation for further characterization and understanding of their probiotic attributes.

Draft whole-genome sequences of competitive exclusion Ligilactobacillus salivarius strains for poultry.

Competitive exclusion (CE) bacteria have been used to control the colonization of chickens by major foodborne pathogens. In this article, we report draft whole-genome sequences of three Ligilactobacillus salivarius strains isolated from chicken gastrointestinal tracts and previously selected as CE for poultry. These genome sequences will provide a foundation for further characterization and understanding of their CE attributes.

Prevalence of Shiga Toxin-Producing Escherichia coli and Salmonella in Native Pecan Orchards as Influenced by Waiting Periods between Grazing and Harvest.

ABSTRACT: Animals (grazing, working, or intrusion) in produce production areas may present a potential contamination source of foodborne pathogens on produce. Cattle grazing on native pecan production orchards, a common practice in the southern United States, provides an opportunity to study the impact of grazing practice and waiting periods on contamination rates of foodborne pathogens of tree nuts. Therefore, the objective of this study was to determine the prevalence of Salmonella and Shiga toxin-producing Escherichia coli (STEC) in native pecan production orchards as influenced by waiting periods between grazing cattle and pecan harvest. Soil (10 g), cattle feces (10 g), and in-shell pecans (25 g) were sampled from five cattle-grazed orchards in areas with cattle removed 2 or 4 months before harvest and not removed. Five nongrazing orchards were sampled at harvest for comparison. Detection and isolation of the pathogens were performed by enrichment, selective isolation, and multiplex PCR. Statistical analyses were performed using contingency tables with Pearson's chi-square test. The prevalence of STEC (36%) and Salmonella (29%) in cattle-grazed orchards was significantly higher than in nongrazed orchards (13 and 7%, respectively). STEC prevalence in cattle-grazed orchards was higher (38%) in areas with cattle at harvest than in fenced areas where cattle were removed 2 (29%) and 4 (27%) months before harvest. Salmonella prevalence was similar in areas without fencing (31%) and areas with cattle removed at 2 (22%) and 4 (30%) months before harvest. However, there were no significant differences (P > 0.05) in contamination rates between waiting periods for either pathogen, suggesting a limited impact of waiting periods on reducing the risk of contamination.

Isolation and characterization of Bacillus spp. strains as potential probiotics for poultry.

Probiotics have become one of the potential solutions to global restriction on antibiotic use in food animal production. Bacillus species have been attractive probiotics partially due to their long-term stability during storage. In this study, 200 endospore-forming bacteria isolates were recovered from sourdough and the gastrointestinal tract (GIT) of young broiler chicks. Based on the production of a series of exoenzymes and survivability under stress conditions similar to those in the poultry GIT, 42 isolates were selected and identified by 16S rRNA gene sequencing. Seven strains with a profile of high enzymatic activities were further evaluated for sporulation efficiency, biofilm formation, compatibility among themselves (Bacillus spp.), and antagonistic effects against three bacteria pathogenic to poultry and humans: Enterococcus cecorum, Salmonella enterica, and Shiga-toxin-producing Escherichia coli. The strains from sourdough were identified as Bacillus amyloliquefaciens whereas the ones from the chicks' GIT were Bacillus subtilis. These strains demonstrated remarkable potential as probiotics for poultry.

Investigation of the Roles of Plasma Species Generated by Surface Dielectric Barrier Discharge.

As an emerging sterilization technology, cold atmospheric plasma offers a dry, non-thermal, rapid process that is minimally damaging to a majority of substrates. However, the mechanisms by which plasma interacts with living cells are poorly understood and the plasma generation apparatuses are complex and resource-intensive. In this study, the roles of reactive oxygen species (ROS), nitric oxide (NO), and charged particles (ions) produced by surface dielectric barrier discharge (SDBD) plasma on prokaryotic (Listeria monocytogenes (Gram-positive)) and eukaryotic (human umbilical vein endothelial cells (HUVEC)) cellular function were evaluated. HUVEC and bacterial oxidative stress responses, the accumulation of nitrite in aqueous media, air ion density, and bacterial inactivation at various distances from SDBD actuators were measured. SDBD actuator designs were also varied in terms of electrode number and length to evaluate the cellular effects of plasma volume and power distribution. NO and ions were found to contribute minimally to the observed cellular effects, whereas ROS were found to cause rapid bacterial inactivation, induce eukaryotic and prokaryotic oxidative stress, and result in rapid oxidation of bovine muscle tissue. The results of this study underscore the dominance of ROS as the major plasma generated species responsible for cellular effects, with ions and RNS having a secondary, complimentary role.

Multiple-locus variable-number tandem repeat analysis for strain discrimination of non-O157 Shiga toxin-producing Escherichia coli.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of growing concern worldwide that have been associated with several recent multistate and multinational outbreaks of foodborne illness. Rapid and sensitive molecular-based bacterial strain discrimination methods are critical for timely outbreak identification and contaminated food source traceback. One such method, multiple-locus variable-number tandem repeat analysis (MLVA), is being used with increasing frequency in foodborne illness outbreak investigations to augment the current gold standard bacterial subtyping technique, pulsed-field gel electrophoresis (PFGE). The objective of this study was to develop a MLVA assay for intra- and inter-serogroup discrimination of six major non-O157 STEC serogroups-O26, O111, O103, O121, O45, and O145-and perform a preliminary internal validation of the method on a limited number of clinical isolates. The resultant MLVA scheme consists of ten variable number tandem repeat (VNTR) loci amplified in three multiplex PCR reactions. Sixty-five unique MLVA types were obtained among 84 clinical non-O157 STEC strains comprised of geographically diverse sporadic and outbreak related isolates. Compared to PFGE, the developed MLVA scheme allowed similar discrimination among serogroups O26, O111, O103, and O121 but not among O145 and O45. To more fully compare the discriminatory power of this preliminary MLVA method to PFGE and to determine its epidemiological congruence, a thorough internal and external validation needs to be performed on a carefully selected large panel of strains, including multiple isolates from single outbreaks.

Surface survival and internalization of salmonella through natural cracks on developing cantaloupe fruits, alone or in the presence of the melon wilt pathogen Erwinia tracheiphila.

Outbreaks of foodborne illness attributed to the consumption of Salmonella-tainted cantaloupe have occurred repeatedly, but understanding of the ecology of Salmonella on cantaloupe fruit surfaces is limited. We investigated the interactions between Salmonella enterica Poona, the plant pathogenic bacterium Erwinia tracheiphila, and cantaloupe fruit. Fruit surfaces were inoculated at the natural cracking stage by spreading S. enterica and E. tracheiphila, 20 µl at 107 cfu/ml, independently or together, over a 2×2 cm rind area containing a crack. Microbial and microscopic analyses were performed at 0, 9 and 24 days post inoculation (DPI). Even at 24 DPI (fruit maturity) S. enterica was detected on 14% and 40% of the fruit inoculated with S. enterica alone and the two-pathogen mixture, respectively. However, the population of S. enterica declined gradually after initial inoculation. E. tracheiphila, inoculated alone or together with Salmonella, caused watersoaked lesions on cantaloupe fruit; but we could not conclude in this study that S. enterica survival on the fruit surface was enhanced by the presence of those lesions. Of fruit inoculated with E. tracheiphila alone and sampled at 24 DPI, 61% had watersoaked lesions on the surface. In nearly half of those symptomatic fruits the watersoaking extended into the sub-rind mesocarp, and E. tracheiphila was recovered from that tissue in 50% of the symptomatic fruit. In this work, E. tracheiphila internalized through natural cracks on developing fruits. S. enterica was never detected in the fruit interior (ca. 2-3 mm below rind surface) under the limited conditions of our experiments, but the possibility that it, or other human pathogens that contaminate fresh produce, might also do so should be investigated under a wider range of conditions and produce types.

Survival and growth of foodborne pathogens in pesticide solutions routinely used in leafy green vegetables and tomato production.

BACKGROUND: The consumption of fresh produce has increased tremendously in the past few years as have outbreaks of foodborne illnesses associated with these commodities. Pesticides routinely used in crop production could influence the outcomes of foodborne pathogen contamination of fresh produce. Experiments were performed to determine the effects of pesticides on the survival and growth characteristics of Escherichia coli O157:H7 and Salmonella spp. Eight commercial fungicides and insecticides commonly used for disease and insect pest control on leafy green vegetables and tomatoes were evaluated. RESULTS: Among the pesticides tested, copper hydroxide, acetamiprid, cypermethrin and permethrin were found to be significantly (P < 0.05) inhibitory to pathogens while no effect was observed for chlorothalonil, flonicamid and methoxyfenozide. At the highest concentration tested (2.66%), azoxystrobin had a significant (P < 0.05) stimulatory effect on the growth of E. coli O157:H7 after 24 h incubation. The results indicated that some pesticides can stimulate the growth of human pathogens if contaminated water is used in their preparation, whereas others were likely to inhibit or reduce pathogen populations. CONCLUSION: This information is helpful in mitigating the risk of microbial contamination in fresh produce, which is critical to public health and safety.

Primers with 5' flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7.

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.