High voltage atmospheric cold plasma inactivation of Listeria monocytogenes in fresh Queso Fresco cheese.

Listeria (L.) monocytogenes is a significant pathogen found in ready-to-eat meat and dairy products. Soft cheeses, such as Queso Fresco cheese (QFC), are particularly sensitive to Listeria contamination, and occasionally serve as a source of food-borne illness outbreaks. In the present study, clinical and cheese isolates of L. monocytogenes were assayed for phenotypic characteristics following sub-lethal high voltage atmospheric cold plasma (HVACP) treatment. Reductions in biofilm formation, swimming motility, and growth dynamics were observed following HVACP treatment. Microbial enumeration of 1-, 10-, and 100-g fresh QFC following 0, 1, 2, or 3 min of HVACP demonstrated significant reductions in L. monocytogenes after 1 min (P-value <0.05), with increasing efficacy with prolonged exposure. A mass-dependent effect was observed between treatments of 1-, 10-, and 100-g QFC in regard to treatment efficacy. This result indicates that greater L. monocytogenes reduction on a larger QFC mass requires greater exposure of the L. monocytogenes to the reactive gas species. Optical absorption spectroscopy confirmed a reduction in reactive gas species for each log increase in QFC mass, however, an equivalent volume of inert foam resulted in increased reactive gas generation compared to QFC. In conclusion, we demonstrate both the application and limitations of HVACP treatments of QFC in the currently defined experimental parameters.

High voltage atmospheric cold plasma treatment inactivates Aspergillus flavus spores and deoxynivalenol toxin.

Fungal contamination is a concern for the food industry. Fungal spores resist food sterilization treatments and produce mycotoxins that are toxic for animals and humans. Technologies that deactivate spores and toxins without impacting food quality are desirable. This study demonstrates the efficiency of a high voltage atmospheric cold plasma (HVACP) technology using air to generate reactive oxygen (ROS) and nitrogen (RNS) species for the degradation of Aspergillus flavus cultures and the deoxynivalenol (DON) mycotoxin. Optical emission and absorption spectroscopy demonstrate ionization of hydroxyl groups, atomic oxygen and nitrogen, and confirm production of ROS and RNS, e.g. O3, NO2, NO3, N2O4, and N2O5. Fungal cultures show a depletion in pigmentation and an ~50% spore inactivation after 1-min treatments. Treated spores show surface ablation and membrane degradation by scanning electron microscopy. Twenty-minute direct HVACP treatments of 100 µg of DON in one mL aqueous suspensions resulted in a greater than 99% reduction in DON structure and rescued over 80% of Caco-2 cell viability; however, the same treatment on 100 µg of powdered DON toxin only showed a 33% reduction in DON and only rescued 15% of cell viability. In summary, HVACP air treatment can inactivate both fungal spores and toxins in minutes.

Short-chain fatty acids inhibit bacterial plasmid transfer through conjugation in vitro and in ex vivo chicken tissue explants.

The animal gut acts as a potent reservoir for spreading and maintaining conjugative plasmids that confer antimicrobial resistance (AMR), fitness, and virulence attributes. Interventions that inhibit the continued emergence and expansion of AMR and virulent strains in agricultural and clinical environments are greatly desired. This study aims to determine the presence and efficacy of short-chain fatty acids (SCFA) inhibitory effects on the conjugal transfer of AMR plasmids. In vitro broth conjugations were conducted between donor Escherichia coli strains carrying AMP plasmids and the plasmid-less Escherichia coli HS-4 recipient strain. Conjugations were supplemented with ddH2O or SCFAs at 1, 0.1, 0.01, or 0.001 molar final concentration. The addition of SCFAs completely inhibited plasmid transfer at 1 and 0.1 molar and significantly (p < 0.05) reduced transfer at 0.01 molar, regardless of SCFA tested. In explant models for the chicken ceca, either ddH2O or a final concentration of 0.025 M SCFAs were supplemented to the explants infected with donor and recipient E. coli. In every SCFA tested, significant decreases in transconjugant populations compared to ddH2O-treated control samples were observed with minimal effects on donor and recipient populations. Finally, significant reductions in transconjugants for plasmids of each incompatibility type (IncP1ε, IncFIß, and IncI1) tested were detected. This study demonstrates for the first time the broad inhibition ability of SCFAs on bacterial plasmid transfer and eliminates AMR with minimal effect on bacteria. Implementing interventions that increase the concentrations of SCFAs in the gut may be a viable method to reduce the risk, incidence, and rate of AMR emergence in agricultural and human environments.

Segmented filamentous bacteria-based treatment to elicit protection against Enterobacteriaceae in Layer chickens.

Introduction: Gut microbes like segmented filamentous bacteria (SFB) play a key role in gut maturation during early life, as demonstrated in humans and mice. Our previous study demonstrated oral inoculation of ileum-spores containing SFB to chickens after hatch increases early SFB gut colonization, which increases immune maturation and resistance to bacteria, like Salmonella, as tested in vitro; however, more studies are needed for treatment optimization and in vivo testing. The objectives of this study were to (1) test a treatment that includes both spores and filamentous SFB, (2) validate antimicrobial ability of the treatment in layer hens in vivo, and (3) elucidate its molecular mechanism. Methods: One-day-old specific pathogen-free layers (n = 12 per group) were orally treated with either PBS (CON) or SFB-based treatment (SFB). At 4 days post-inoculation (DPI), both CON and SFB groups were orally challenged with Salmonella Typhimurium. Total Enterobacteriaceae and Salmonella were examined by plating and enumeration in feces at 7,10 and 14 dpi; and in the ileum, cecum, and spleen at 16 dpi in euthanized birds. The presence and levels of SFB were determined from ilea scrapings via microscopy and qPCR, respectively. Relative gene expression of host-derived antimicrobial peptides and cytokines in the distal ileum was determined by RT-qPCR. Results: At 10 and 14 dpi, a significant decrease in total Enterobacteriaceae was observed in the feces of the SFB group. At necropsy, the level of SFB was significantly higher in the SFB group than in the CON group, while a significant decrease in total Enterobacteriaceae and Salmonella was observed in the ceca of the SFB group. RT-qPCR revealed increased expression of ß-defensin 14, and cytokines IL-10 and IFNγ. Discussion: The introduction of SFB at hatch as a prophylactic treatment may benefit commercial partners as well as consumers by reducing the incidence of Enterobacteriaceae in food animals. Reduction of these bacteria in animals would, in turn, increase animal health, productivity, and safety for consumers. Studies to optimize the treatment for poultry industry applications are ongoing in our lab.

Models for Gut-Mediated Horizontal Gene Transfer by Bacterial Plasmid Conjugation.

The emergence of new antimicrobial resistant and virulent bacterial strains may pose a threat to human and animal health. Bacterial plasmid conjugation is a significant contributor to rapid microbial evolutions that results in the emergence and spread of antimicrobial resistance (AR). The gut of animals is believed to be a potent reservoir for the spread of AR and virulence genes through the horizontal exchange of mobile genetic elements such as plasmids. The study of the plasmid transfer process in the complex gut environment is limited due to the confounding factors that affect colonization, persistence, and plasmid conjugation. Furthermore, study of plasmid transfer in the gut of humans is limited to observational studies, leading to the need to identify alternate models that provide insight into the factors regulating conjugation in the gut. This review discusses key studies on the current models for in silico, in vitro, and in vivo modeling of bacterial conjugation, and their ability to reflect the gut of animals. We particularly emphasize the use of computational and in vitro models that may approximate aspects of the gut, as well as animal models that represent in vivo conditions to a greater extent. Directions on future research studies in the field are provided.

Drosophila Model for Gut-Mediated Horizontal Transfer of Narrow- and Broad-Host-Range Plasmids.

Horizontal gene transfer (HGT) is a driving force of microbial evolution. The gut of animals acts as a potent reservoir for the lateral transfer of virulence, fitness, and antimicrobial resistance genes through plasmids. Reduced-complexity models for the examination of host-microbe interactions involved in plasmid transfer are greatly desired. Thus, this study identifies the use of Drosophila melanogaster as a model organism for the conjugation of plasmids of various incompatibility groups in the gut. Enterobacteriaceae conjugation pairs were identified in vitro and used for oral inoculation of the Drosophila gut. Flies were enumerated for the donor, recipient, and transconjugant populations. Each donor-recipient pair was observed to persist in fly guts for the duration of the experiment. Gut concentrations of the donors and recipients were significantly different between male and female flies, with females generally demonstrating increased concentrations. Furthermore, host genetics significantly altered the concentrations of donors and recipients. However, transconjugant concentrations were not affected by host sex or genetics and were detected only in the IncPε and IncI1 plasmid groups. This study demonstrates Drosophila melanogaster as a model for gut-mediated plasmid transfer. IMPORTANCE Microbial evolution in the gut of animals due to horizontal gene transfer (HGT) is of significant interest for microbial evolution as well as within the context of human and animal health. Microbial populations evolve within the host, and factors from the bacteria and host interact to regulate this evolution. However, little is currently known about how host and bacterial factors regulate plasmid-mediated HGT in the gut. This study demonstrates the use of Drosophila and the roles of sexual dimorphism as well as plasmid incompatibility groups in HGT in the gut.

Bacteria Broadly-Resistant to Last Resort Antibiotics Detected in Commercial Chicken Farms.

Resistance to last resort antibiotics in bacteria is an emerging threat to human and animal health. It is important to identify the source of these antimicrobial resistant (AMR) bacteria that are resistant to clinically important antibiotics and evaluate their potential transfer among bacteria. The objectives of this study were to (i) detect bacteria resistant to colistin, carbapenems, and ß-lactams in commercial poultry farms, (ii) characterize phylogenetic and virulence markers of E. coli isolates to potentiate virulence risk, and (iii) assess potential transfer of AMR from these isolates via conjugation. Ceca contents from laying hens from conventional cage (CC) and cage-free (CF) farms at three maturity stages were randomly sampled and screened for extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, carbapenem-resistant Acinetobacter (CRA), and colistin resistant Escherichia coli (CRE) using CHROMagar™ selective media. We found a wide-spread abundance of CRE in both CC and CF hens across all three maturity stages. Extraintestinal pathogenic Escherichia coli phylogenetic groups B2 and D, as well as plasmidic virulence markers iss and iutA, were widely associated with AMR E. coli isolates. ESBL-producing Enterobacteriaceae were uniquely detected in the early lay period of both CC and CF, while multidrug resistant (MDR) Acinetobacter were found in peak and late lay periods of both CC and CF. CRA was detected in CF hens only. blaCMY was detected in ESBL-producing E. coli in CC and CF and MDR Acinetobacter spp. in CC. Finally, the blaCMY was shown to be transferrable via an IncK/B plasmid in CC. The presence of MDR to the last-resort antibiotics that are transferable between bacteria in food-producing animals is alarming and warrants studies to develop strategies for their mitigation in the environment.

Mouse Genetic Background Affects Transfer of an Antibiotic Resistance Plasmid in the Gastrointestinal Tract.

Dissemination of antibiotic resistance (AR) genes, often on plasmids, leads to antibiotic-resistant bacterial infections, which is a major problem for animal and public health. Bacterial conjugation is the primary route of AR gene transfer in the mammalian gastrointestinal tract. Significant gaps in knowledge about which gastrointestinal communities and host factors promote plasmid transfer remain. Here, we used Salmonella enterica serovar Kentucky strain CVM29188 carrying plasmid pCVM29188_146 (harboring streptomycin and tetracycline resistance genes) to assess plasmid transfer to Escherichia coli under in vitro conditions and in various mouse strains with a conventional or defined microbiota. As an initial test, the transfer of pCVM29188_146 to the E. coli strains was confirmed in vitro Colonization resistance and, therefore, a lack of plasmid transfer were found in wild-type mice harboring a conventional microbiota. Thus, mice harboring the altered Schaedler flora (ASF), or ASF mice, were used to probe for host factors in the context of a defined microbiota. To assess the influence of inflammation on plasmid transfer, we compared interleukin-10 gene-deficient 129S6/SvEv ASF mice (proinflammatory environment) to wild-type 129S6/SvEv ASF mice and found no difference in transconjugant yields. In contrast, the mouse strain influenced plasmid transfer, as C3H/HeN ASF mice had significantly lower levels of transconjugants than 129S6/SvEv ASF mice. Although gastrointestinal members were identical between the ASF mouse strains, a few differences from C3H/HeN ASF mice were detected, with C3H/HeN ASF mice having significantly lower abundances of ASF members 356 (Clostridium sp.), 492 (Eubacterium plexicaudatum), and 502 (Clostridium sp.) than 129S6/SvEv ASF mice. Overall, we demonstrate that microbiota complexity and mouse genetic background influence in vivo plasmid transfer.IMPORTANCE Antibiotic resistance is a threat to public health. Many clinically relevant antibiotic resistance genes are carried on plasmids that can be transferred to other bacterial members in the gastrointestinal tract. The current study used a murine model to study the transfer of a large antibiotic resistance plasmid from a foodborne Salmonella strain to a gut commensal E. coli strain in the gastrointestinal tract. We found that different mouse genetic backgrounds and a different diversity of microbial communities influenced the level of Escherichia coli that acquired the plasmid in the gastrointestinal tract. This study suggests that the complexity of the microbial community and host genetics influence plasmid transfer from donor to recipient bacteria.