MINIstock: Model for INsect Inclusion in sustainable agriculture: USDA-ARS's research approach to advancing insect meal development and inclusion in animal diets.

Animal agriculture is under pressure to increase efficiency, sustainability, and innovation to meet the demands of a rising global population while decreasing adverse environmental effects. Feed cost and availability are 2 of the biggest hurdles to sustainable production. Current diets depend on sources of grain and animal byproduct protein for essential amino acids which have limited sustainability. Insects have arisen as an attractive, sustainable alternative protein source for animal diets due to their favorable nutrient composition, low space and water requirements, and natural role in animal diets. Additionally, insects are capable of bioremediating waste streams including agricultural and food waste, manure, and plastics helping to increase their sustainability. The insect rearing industry has grown rapidly in recent years and shows great economic potential. However, state-of-the-art research is urgently needed to overcome barriers to adoption in commercial animal diets such as regulatory restrictions, production scale issues, and food safety concerns. To address this need, the USDA Agricultural Research Service "MINIstoc: Model for INsect Inclusion" project was created to bring together diverse scientists from across the world to synergistically advance insect meal production and inclusion in animal diets. Here, we provide a short review of insects as feed while describing the MINIstock project which serves as the inspiration for the Journal of Economic Entomology Special Collection "Insects as feed: sustainable solutions for food waste and animal production practices."

Bacterial proximity effects on the transfer of antibiotic resistance genes within the alimentary tract of yellow mealworm larvae.

The arthropod intestinal tract and other anatomical parts naturally carry microorganisms. Some of which are pathogens, secrete toxins, or carry transferable antibiotic-resistance genes. The risks associated with the production and consumption of edible arthropods are dependent on indigenous microbes, as well as microbes introduced during the processes of rearing. This mass arthropod production puts individual arthropods in close proximity, which increases the possibility of their exposure to antibiotic-resistant bacteria carried by bacteria from fellow insects, industry workers, or rearing hardware and substrates. The purpose of this study was to determine if the alimentary tract of the yellow mealworm provided an environment permitting horizontal gene transfer between bacteria. The effect of the concentration of bacterial exposure was also assessed. Antibiotic resistance gene transfer between marker Salmonella Lignières (Enterobacterales: Enterobacteriaceae) and Escherichia coli (Migula) (Enterobacterales: Enterobacteriaceae) introduced into the larval gut demonstrated that the nutrient-rich environment of the yellow mealworm gut provided favorable conditions for the transfer of antibiotic resistance genes. Conjugation frequencies were similar across inoculum concentrations; however, transconjugant production correlated positively to increased exposure concentration. The lowest concentration of bacterial exposure required enrichment to detect and thus may have been approaching a threshold level for the 2 bacteria to colocate within the expanse of the larval gut. While many factors can affect this transfer, the simple factor of the proximity of donor and recipient bacteria, as defined by the concentration of bacteria within the volume of the insect gut, likely primarily contributed to the efficiency of antibiotic gene transfer.

Campylobacter jejuni Response When Inoculated in Bovine In Vitro Fecal Microbial Consortia Incubations in the Presence of Metabolic Inhibitors.

Infection with the foodborne pathogen Campylobacter is the leading bacterial cause of human foodborne illness in the United States. The objectives of this experiment were to test the hypothesis that mixed microbial populations from the bovine rumen may be better at excluding Campylobacter than populations from freshly voided feces and to explore potential reasons as to why the rumen may be a less favorable environment for Campylobacter than feces. In an initial experiment, C. jejuni cultures inoculated without or with freshly collected bovine rumen fluid, bovine feces or their combination were cultured micro-aerobically for 48 h. Results revealed that C. jejuni grew at similar growth rates during the first 6 h of incubation regardless of whether inoculated with the rumen or fecal contents, with rates ranging from 0.178 to 0.222 h-1. However, C. jejuni counts (log10 colony-forming units/mL) at the end of the 48 h incubation were lowest in cultures inoculated with rumen fluid (5.73 log10 CFUs/mL), intermediate in cultures inoculated with feces or both feces and rumen fluid (7.16 and 6.36 log10 CFUs/mL) and highest in pure culture controls that had not been inoculated with the rumen or fecal contents (8.32 log10 CFUs/mL). In follow-up experiments intended to examine the potential effects of hydrogen and hydrogen-consuming methanogens on C. jejuni, freshly collected bovine feces, suspended in anaerobic buffer, were incubated anaerobically under either a 100% carbon dioxide or 50:50 carbon dioxide/hydrogen gas mix. While C. jejuni viability decreased <1 log10 CFUs/mL during incubation of the fecal suspensions, this did not differ whether under low or high hydrogen accumulations or whether the suspensions were treated without or with the mechanistically distinct methanogen inhibitors, 5 mM nitrate, 0.05 mM 2-bromosulfonate or 0.001 mM monensin. These results suggest that little if any competition between C. jejuni and hydrogen-consuming methanogens exists in the bovine intestine based on fecal incubations.

Chlortetracycline Concentration Impact on Salmonella Typhimurium Sustainability in the Presence of Porcine Gastrointestinal Tract Bacteria Maintained in Continuous Culture.

Concern exists that the continued use of antibiotics in animal feeds may lead to an increased prevalence of resistant bacteria within the host animal's gastrointestinal tract. To evaluate the effect of chlortetracycline on the persistence of Salmonella enterica serotype Typhimurium within a diverse population of porcine cecal bacteria, we cultured a mixed population of cecal bacteria without or with added chlortetracycline. When grown at a 24 h vessel turnover rate, chlortetracycline-susceptible S. Typhimurium exhibited more than 2.5 times faster (p < 0.05) disappearance rates than theoretically expected (0.301 log10 colony-forming unit/mL per day) but did not differ whether treated or not with 55 mg of chlortetracycline/L. Chlortetracycline-resistant S. Typhimurium was not recovered from any of these cultures. When the mixed cultures were inoculated with a chlortetracycline-resistant S. Typhimurium, rates of disappearance were nearly two times slower (p < 0.05) than those observed earlier with chlortetracycline-susceptible S. Typhimurium, and cultures persisted at >2 log10 colony-forming units/mL for up to 14 days of treatment with 110 mg of chlortetracycline/L. Under the conditions of this study, chlortetracycline-resistant S. Typhimurium was competitively enabled to persist longer within the mixed populations of porcine gut bacteria than chlortetracycline-susceptible S. Typhimurium, regardless of the presence or absence of added chlortetracycline.

Behavioral interplay between mosquito and mycolactone produced by Mycobacterium ulcerans and bacterial gene expression induced by mosquito proximity.

Mycolactone is a cytotoxic lipid metabolite produced by Mycobacterium ulcerans, the environmental pathogen responsible for Buruli ulcer, a neglected tropical disease. Mycobacterium ulcerans is prevalent in West Africa, particularly found in lentic environments, where mosquitoes also occur. Researchers hypothesize mosquitoes could serve as a transmission mechanism resulting in infection by M. ulcerans when mosquitoes pierce skin contaminated with M. ulcerans. The interplay between the pathogen, mycolactone, and mosquito is only just beginning to be explored. A triple-choice assay was conducted to determine the host-seeking preference of Aedes aegypti between M. ulcerans wildtype (MU, mycolactone active) and mutant (MUlac-, mycolactone inactive). Both qualitative and quantitative differences in volatile organic compounds' (VOCs) profiles of MU and MUlac- were determined by GC-MS. Additionally, we evaluated the interplay between Ae. aegypti proximity and M. ulcerans mRNA expression. The results showed that mosquito attraction was significantly greater (126.0%) to an artificial host treated with MU than MUlac-. We found that MU and MUlac produced differential profiles of VOCs associated with a wide range of biological importance from quorum sensing (QS) to human odor components. RT-qPCR assays showed that mycolactone upregulation was 24-fold greater for MU exposed to Ae. aegypti in direct proximity. Transcriptome data indicated significant induction of ten chromosomal genes of MU involved in stress responses and membrane protein, compared to MUlac- when directly having access to or in near mosquito proximity. Our study provides evidence of possible interkingdom interactions between unicellular and multicellular species that MU present on human skin is capable of interreacting with unrelated species (i.e., mosquitoes), altering its gene expression when mosquitoes are in direct contact or proximity, potentially impacting the production of its VOCs, and consequently leading to the stronger attraction of mosquitoes toward human hosts. This study elucidates interkingdom interactions between viable M. ulcerans bacteria and Ae. aegypti mosquitoes, which rarely have been explored in the past. Our finding opens new doors for future research in terms of disease ecology, prevalence, and pathogen dispersal outside of the M. ulcerans system.

Quorum sensing gene regulation in Staphylococcus epidermidis reduces the attraction of Aedes aegypti (L.) (Diptera: Culicidae).

Introduction: Identifying mechanisms regulating mosquito attraction to hosts is key to suppressing pathogen transmission. Historically, the ecology of the host microbial community and its influence on mosquito attraction, specifically, whether bacterial communication through quorum sensing (QS) modulates VOC production that affects mosquito behavior have not been extensively considered. Methods: Behavioral choice assays were applied along with volatile collection, followed by GC-MS and RNA transcriptome analyses of bacteria with and without a quorum-sensing inhibitor, furanone C-30. Results: Utilizing the quorum-sensing inhibitor on a skin-inhabiting bacterium, Staphylococcus epidermidis, we disrupted its interkingdom communication with adult Aedes aegypti and mitigated their attraction to a blood-meal by 55.1%. Discussion: One potential mechanism suppressing mosquito attraction could be the reduction (31.6% in our study) of bacterial volatiles and their associated concentrations by shifting S. epidermidis metabolic (12 of 29 up regulated genes) and stress (5 of 36 down regulated genes) responses. Manipulating the quorum-sensing pathways could serve as a mechanism to reduce mosquito attraction to a host. Such manipulations could be developed into novel control methods for pathogen-transmitting mosquitoes and other arthropods.

The bacterial and archaeal communities of flies, manure, lagoons, and troughs at a working dairy.

Background: Fundamental investigations into the location, load, and persistence of microbes, whether beneficial or detrimental, are scarce. Many questions about the retention and survival of microbes on various surfaces, as well as the load necessary for spread, exist. To answer these questions, we must know more about where to find various microbes and in what concentrations, the composition of the microbial communities, and the extent of dissemination between various elements. This study investigated the diversity, composition, and relative abundance of the communities associated with manure, lagoons, troughs, house flies, and stable flies present at a dairy, implementing two different free-stall management systems: flow-through and cross-vent. Shotgun metagenomics at the community level was used to compare the microbiomes within the dairy, allowing confident interpretation at the species level. Results: The results showed that there were significant difference in microbial composition between not only each of the dairy elements but also management styles. The primary exceptions were the microbiomes of the house fly and the stable fly. Their compositions heavily overlapped with one another, but interestingly, not with the other components sampled. Additionally, both species of flies carried more pathogens than the other elements of the dairy, indicating that they may not share these organisms with the other components, or that the environments offered by the other components are unsatisfactory for the survival of some pathogens.. Conclusion: The lack of overlapping pathogen profiles suggests a lack of transfer from flies to other dairy elements. Dairy health data, showing a low incidence of disease, suggests minimal sharing of bacteria by the flies at a level required for infection, given the health program of this dairy. While flies did carry a multitude of pathogenic bacteria, the mere presence of the bacteria associated with the flies did not necessarily translate into high risk leading to morbidity and mortality at this dairy. Thus, using flies as the sole sentinel of dairy health may not be appropriate for all bacterial pathogens or dairies.

Assessment of Potential Anti-Methanogenic and Antimicrobial Activity of Ethyl Nitroacetate, α-Lipoic Acid, Taurine and L-Cysteinesulfinic Acid In Vitro.

Livestock producers need new technologies to maintain the optimal health and well-being of their animals while minimizing the risks of propagating and disseminating pathogenic and antimicrobial-resistant bacteria to humans or other animals. Where possible, these interventions should contribute to the efficiency and profitability of animal production to avoid passing costs on to consumers. In this study, we examined the potential of nitroethane, 3-nitro-1-propionate, ethyl nitroacetate, taurine and L-cysteinesulfinic acid to modulate rumen methane production, a digestive inefficiency that results in the loss of up to 12% of the host's dietary energy intake and a major contributor of methane as a greenhouse gas to the atmosphere. The potential for these compounds to inhibit the foodborne pathogens, Escherichia coli O157:H7 and Salmonella Typhimurium DT104, was also tested. The results from the present study revealed that anaerobically grown O157:H7 and DT104 treated with the methanogenic inhibitor, ethyl nitroacetate, at concentrations of 3 and 9 mM had decreased (p < 0.05) mean specific growth rates of O157:H7 (by 22 to 36%) and of DT104 (by 16 to 26%) when compared to controls (0.823 and 0.886 h-1, respectively). The growth rates of O157:H7 and DT104 were decreased (p < 0.05) from controls by 31 to 73% and by 41 to 78% by α-lipoic acid, which we also found to inhibit in vitro rumen methanogenesis up to 66% (p < 0.05). Ethyl nitroacetate was mainly bacteriostatic, whereas 9 mM α-lipoic acid decreased (p < 0.05) maximal optical densities (measured at 600 nm) of O157:H7 and DT104 by 25 and 42% compared to controls (0.448 and 0.451, respectively). In the present study, the other oxidized nitro and organosulfur compounds were neither antimicrobial nor anti-methanogenic.

Inhibitory Effect of Select Nitrocompounds and Chlorate against Yersinia ruckeri and Yersinia aleksiciae In Vitro.

Yersinia ruckeri is an important fish pathogen causing enteric redmouth disease. Antibiotics have traditionally been used to control this pathogen, but concerns of antibiotic resistance have created a need for alternative interventions. Presently, chlorate and certain nitrocompounds were tested against Y. ruckeri as well as a related species within the genus, Y. aleksiciae, to assess the effects of these inhibitors. The results reveal that 9 mM chlorate had no inhibitory effect against Y. ruckeri, but inhibited growth rates and maximum optical densities of Y. aleksciciae by 20-25% from those of untreated controls (0.46 h-1 and 0.29 maximum optical density, respectively). The results further reveal that 2-nitropropanol and 2-nitroethanol (9 mM) eliminated the growth of both Y. ruckeri and Y. aleksiciae during anaerobic or aerobic culture. Nitroethane, ethyl nitroacetate and ethyl-2-nitropropionate (9 mM) were less inhibitory when tested similarly. Results from a mixed culture of Y. ruckeri with fish tank microbes and of Y. aleksiciae with porcine fecal microbes reveal that the anti-Yersinia activity of the tested nitrocompounds was bactericidal, with 2-nitropropanol and 2-nitroethanol being more potent than the other tested nitrocompounds. The anti-Yersinia activity observed with these tested compounds warrants further study to elucidate the mechanisms of action and strategies for their practical application.

Management Practices Affecting Lesser Mealworm Larvae (Alphitobius diaperinus) Associated Microbial Community in a Broiler House and After Relocating With the Litter Into Pastureland.

Lesser mealworms are often found infesting production houses used to raise broiler chickens. Previous studies have investigated pathogenic microorganisms associated with the larvae, but a more thorough study relating total microbiome changes due to management procedures and flock rotations was needed. Additionally, there is a question of what microbiota are transferred into the environment when the litter, in which larvae reside, is piled in pastureland for use as fertilizer and where interactions with the soil and other fauna can occur. This study chronicled, by the 16S rRNA sequencing, the bacterial community profile of larvae in a broiler grow-out house synchronizing to when birds were added to and removed from the house over 2.5 years. The profile was found to be relatively constant despite 11 flock rotations and management disruptions, specifically litter cleanout procedures and the addition of new birds or bedding. In contrast, once removed from the controlled broiler house environment and placed into open pastureland, the substantial microbial diversity brought with the larvae showed greater fluctuation in structure with environmental conditions, one of which was rainfall. Surprisingly, these larvae survived at least 19 weeks, so the potential for moving larval-associated microbes into the environment needs further assessment to minimize the risk of relocating foodborne pathogens and also to assess those bacteria-generating metabolites that have benefits to plant growth when using the litter as a fertilizer. The characterization of their microbiome is the first step to investigating the influences of their microbes on the manmade and environmental ecosystems.

Adult Alphitobius diaperinus Microbial Community during Broiler Production and in Spent Litter after Stockpiling.

The facilities used to raise broiler chickens are often infested with litter beetles (lesser mealworm, Alphitobius diaperinus). These beetles have been studied for their carriage of pathogenic microbes; however, a more comprehensive microbiome study on these arthropods is lacking. This study investigated their microbial community in a longitudinal study throughout 2.5 years of poultry production and after the spent litter, containing the mealworms, was piled in pastureland for use as fertilizer. The mean most abundant phyla harbored by the beetles in house were the Proteobacteria (39.8%), then Firmicutes (30.8%), Actinobacteria (21.1%), Tenericutes (5.1%), and Bacteroidetes (1.6%). The community showed a modest decrease in Firmicutes and increase in Proteobacteria over successive flock rotations. The beetles were relocated within the spent litter to pastureland, where they were found at least 19 weeks later. Over time in the pastureland, their microbial profile underwent a large decrease in the percent of Firmicutes (20.5%). The lesser mealworm showed an ability to survive long-term in the open environment within the spent litter, where their microbiome should be further assessed to both reduce the risk of transferring harmful bacteria, as well as to enhance their contribution when the litter is used as a fertilizer.

Disinfectant and Antimicrobial Susceptibility Studies of Staphylococcus aureus Strains and ST398-MRSA and ST5-MRSA Strains from Swine Mandibular Lymph Node Tissue, Commercial Pork Sausage Meat and Swine Feces.

Staphylococcus aureus (S. aureus) causes gastrointestinal illness worldwide. Disinfectants are used throughout the food chain for pathogenic bacteria control. We investigated S. aureus bioavailability in swine Mandibular lymph node tissue (MLT) and pork sausage meat (PSM), established susceptibility values for S. aureus to disinfectants, and determined the multilocus sequence type of MRSA strains. Antimicrobial and disinfectant susceptibility profiles were determined for 164 S. aureus strains isolated from swine feces (n = 63), MLT (n = 49) and PSM (n = 52). No antimicrobial resistance (AMR) was detected to daptomycin, nitrofurantoin, linezolid, and tigecycline, while high AMR prevalence was determined to erythromycin (50.6%), tylosin tartrate (42.7%), penicillin (72%), and tetracycline (68.9%). Methicillin-resistant S. aureus (MRSA) strains, ST398 (n = 6) and ST5 (n = 1), were found in the MLT and PSM, 4 MRSA in MLT and 3 MRSA strains in the PSM. About 17.5% of feces strains and 41.6% of MLT and PSM strains were resistant to chlorhexidine. All strains were susceptible to triclosan and benzalkonium chloride, with no cross-resistance between antimicrobials and disinfectants. Six MRSA strains had elevated susceptibilities to 18 disinfectants. The use of formaldehyde and tris(hydroxylmethyl)nitromethane in DC&R was not effective, which can add chemicals to the environment. Didecyldimethylammonium chloride and benzyldimethylhexadecylammonium chloride were equally effective disinfectants. ST398 and ST5 MRSA strains had elevated susceptibilities to 75% of the disinfectants tested. This study establishes susceptibility values for S. aureus strains from swine feces, mandibular lymph node tissue, and commercial pork sausage against 24 disinfectants. Since it was demonstrated that S. aureus and MRSA strains can be found deep within swine lymph node tissue, it may be beneficial for the consumer if raw swine lymph node tissue is not used in uncooked food products and pork sausage.

Antagonistic Effects of Lipids Against the Anti-Escherichia coli and Anti-Salmonella Activity of Thymol and Thymol-ß-d-Glucopyranoside in Porcine Gut and Fecal Cultures In Vitro.

Strategies are sought to reduce the carriage and dissemination of zoonotic pathogens and antimicrobial-resistant microbes within food-producing animals and their production environment. Thymol (an essential oil) is a potent bactericide in vitro but in vivo efficacy has been inconsistent, largely due to its lipophilicity and absorption, which limits its passage and subsequent availability in the distal gastrointestinal tract. Conjugation of thymol to glucose to form thymol-ß-d-glucopyranoside can decrease its absorption, but in vivo passage of effective concentrations to the lower gut remains suboptimal. Considering that contemporary swine diets often contain 5% or more added fat (to increase caloric density and reduce dustiness), we hypothesized that there may be sufficient residual fat in the distal intestinal tract to sequester free or conjugated thymol, thereby limiting the availability and subsequent effectiveness of this biocide. In support of this hypothesis, the anti-Salmonella Typhimurium effects of 6 mM free or conjugated thymol, expressed as log10-fold reductions of colony-forming units (CFU) ml-1, were diminished 90 and 58%, respectively, following 24-h in vitro anaerobic fecal incubation (at 39°C) with 3% added vegetable oil compared to reductions achieved during culture without added oil (6.1 log10 CFU ml-1). The antagonistic effect of vegetable oil and the bactericidal effect of free and conjugated thymol against Escherichia coli K88 tested similarly were diminished 86 and 84%, respectively, compared to reductions achieved in cultures incubated without added vegetable oil (5.7 log10 CFU ml-1). Inclusion of taurine (8 mg/ml), bile acids (0.6 mg/ml), or emulsifiers such as polyoxyethylene-40 stearate (0.2%), Tween 20, or Tween 80 (each at 1%) in the in vitro incubations had little effect on vegetable oil-caused inhibition of free or conjugated thymol. Based on these results, it seems reasonable to suspect that undigested lipid in the distal gut may limit the effectiveness of free or conjugated thymol. Accordingly, additional research is warranted to learn how to overcome obstacles diminishing bactericidal activity of free and conjugated thymol in the lower gastrointestinal tract of food-producing animals.

Black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), and house fly, Musca domestica L. (Diptera: Muscidae), larvae reduce livestock manure and possibly associated nutrients: An assessment at two scales.

The industrial production of insects for waste management or as a protein source is becoming vital to our society. Large volumes of manure are produced by concentrated animal facilities around the globe that must be managed, utilized, and disposed of properly. Flies offer a partial solution with their abilities to reduce these wastes and heavy metal pollutants. Meat and crop proteins are being supplemented by insect proteins for many feeds across the globe, yet science-based studies behind the mass-rearing of insects are still in their infancy. In the current study, the percent change in the composition of nutrients, heavy metals, and fiber, in dairy, poultry, and swine manure degraded by either black soldier fly (BSF) or house fly (HF) larvae was explored. Pre-digested and post-digested manure samples were collected from four independent studies that differed in production scale (number of larvae and feeding regimen): 1) BSF small-scale (100 larvae fed incrementally), 2) HF small-scale (100 larvae fed incrementally), 3) BSF large-scale (10,000 larvae fed a single time), and 4) HF large-scale (4,000 larvae fed a single time). Results indicate that nitrogen is a key nutrient impacted by larval digestion of manure by both species, regardless of scale. However, scale significantly impacted reductions of other nutrients, as did the type of manure in which the insects were reared. Ultimately, this study demonstrated that manure type and rearing scale impact the ability of BSF and HF larvae to reduce nutrients and heavy metals in manure, and thus insect management procedures need to be congruent with production emphases of the insects for waste management or protein products. Failure to take scale into consideration could lead to inaccurate assumptions related to industrialized efforts on this topic.

Poultry litter and the environment: Microbial profile of litter during successive flock rotations and after spreading on pastureland.

With the increasing demand for broiler meat, a thorough evaluation of the microbial community within the broiler houses and sites where litter is deposited is critical to animal and environmental wellbeing. However not much is known in this arena, so our work evaluates the litter bacterial microbial community within a house over a 2.5 year period through 11 flock rotations, a partial and a total cleanout, and the subsequent deposition of the litter as fertilizer on pastureland. The effects of both time and management practices correlated with alterations of the litter microbial community. The cleanout practices and introduction of new bedding had minimal influence on the house microbial community once it was established, which generally showed a consistent increase in the proportion of Actinobacteria and a decrease in Firmicutes over the 11 flock rotations. Analysis of the bacterial profile at the genus level gave increased resolution, revealing changes during the first and second flock rotation and after the total cleanout. The disturbance of the partial cleanout seemed to be buffered by the supporting conditions within the house while the total cleanout showed a small, but significant influence. The pastureland deposition of litter, however, was affected by time and abiotic factors that changed the litter microbial community structure weekly. The stockpiled litter had an increase in the phyla Actinobacteria and the class Bacilli that commonly have microbes utilizing nitrogen and decaying materials, in comparison to Native soil. Further, the soil beneath where the litter was stored for 20 weeks, lost diversity, indicating a possible effect of the litter stockpiling on environmental quality at that site. How management practices affect the composition of the microbial community within the litter of the broiler house is of interest in terms of bird health and environmentally for future utilization of spent litter.

Disinfectant and antimicrobial susceptibility studies of the foodborne pathogen Campylobacter jejuni isolated from the litter of broiler chicken houses.

Foodborne illness is an ongoing problem worldwide and is caused by bacteria that invade the food chain from the farm, slaughter house, restaurant or grocery, or in the home and can be controlled by strategies using biocides (antiseptics and disinfectants). Susceptibility profiles were determined for 96 Campylobacter jejuni strains obtained in 2011-2012 from broiler chicken house environments to antimicrobials and disinfectants as per the methods of the Clinical and Laboratory Standards Institute and TREK Diagnostics using CAMPY AST Campylobacter plates. Low prevalence of antimicrobial resistance was observed in C. jejuni strains to tetracycline (TET; 21.9%), ciprofloxacin (CIP; 13.5%), and nalidixic acid (NAL; 12.5%). The resistance profiles had a maximum of 3 antimicrobials, CIP-NAL-TET, with TET being the main profile observed. No cross-resistance was observed between antimicrobials and disinfectants. The C. jejuni strains (99%) were resistant to triclosan, 32% were resistant to chlorhexidine, and they all were susceptible to benzalkonium chloride. The strains had low-level minimum inhibitory concentrations (MICs) to the disinfectants P-128, Food Service Sanitizer, F-25 Sanitizer, Final Step 512 Sanitizer, OdoBan, dioctyldimethylammmonium chloride, didecyldimethylammonium chloride (C10AC), benzyldimethyldodecylammonium chloride (C12BAC), and benzyldimethyltetradecylammonium chloride (C14BAC). Intermediate MICs against DC&R, cetylpyridinium bromide hydrate, hexadecylpyridinium chloride, ethylhexadecyldimethylammonium bromide, and hexadecyltrimethylammonium bromide with elevated intermediate MICs against Tek-Trol, benzyldimethylhexadecylammonium chloride, tris(hydroxylmethyl)nitromethane (THN), and formaldehyde. The highest MIC were obtained for povidone-iodine. The components THN and the benzylammonium chlorides C12BAC and C14BAC were responsible for the inhibition by DC&R. The components C10AC and C12BAC may act synergistically causing inhibition of C. jejuni by the disinfectant P-128. The formaldehyde component in DC&R was not effective against C. jejuni compared with the ammonium chloride components. Its use in disinfectants may result in additional unnecessary chemicals in the environment. Didecyldimethylammonium chloride is the most effective ammonium chloride component against C. jejuni.

Reduced Environmental Microbial Diversity on the Cuticle and in the Galleries of a Subterranean Termite Compared to Surrounding Soil.

Termites are intimately tied to the microbial world, as they utilize their gut microbiome for the conversion of plant cellulose into necessary nutrients. Subterranean termites must also protect themselves from the vast diversity of harmful microbes found in soil. However, not all soil microbes are harmful, such as Streptomyces and methanotrophic bacteria that some species of termites harbor in complex nest structures made of fecal material. The eastern subterranean termite, Reticulitermes flavipes, has a simple nest structure consisting of fecal lined galleries. We tested the hypothesis that R. flavipes maintains a select microbial community in its nests to limit the penetration of harmful soilborne pathogens and favor the growth of beneficial microbes. Using Illumina sequencing, we characterized the bacterial and fungal communities in the surrounding soil, in the nest galleries, and on the cuticle of workers. We found that the galleries provide a more beneficial microbial community than the surrounding soil. Bacterial and fungal diversity was highest in the soil, lower in the galleries, and least on the cuticle. Bacterial communities clustered together according to the substrate from which they were sampled, but this clustering was less clear in fungal communities. Most of the identified bacterial and fungal taxa were unique to one substrate, but the soil and gallery communities had very similar phylum-level taxonomic profiles. Notably, the galleries of R. flavipes also harbored both the potentially beneficial Streptomyces and the methanotrophic Methylacidiphilales, indicating that these microbial associations in fecal material pre-date the emergence of complex fecal nest structures. Surprisingly, several pathogenic groups were relatively abundant in the galleries and on the cuticle, suggesting that pathogens may accumulate within termite nests over time while putatively remaining at enzootic level during the lifetime of the colony.

Inhibition of Salmonella Binding to Porcine Intestinal Cells by a Wood-Derived Prebiotic.

Numerous Salmonella enterica serovars can cause disease and contamination of animal-produced foods. Oligosaccharide-rich products capable of blocking pathogen adherence to intestinal mucosa are attractive alternatives to antibiotics as these have potential to prevent enteric infections. Presently, a wood-derived prebiotic composed mainly of glucose-galactose-mannose-xylose oligomers was found to inhibit mannose-sensitive binding of select Salmonella Typhimurium and Escherichia coli strains when reacted with Saccharomyces boulardii. Tests for the ability of the prebiotic to prevent binding of a green fluorescent protein (GFP)-labeled S. Typhimurium to intestinal porcine epithelial cells (IPEC-J2) cultured in vitro revealed that prebiotic-exposed GFP-labeled S. Typhimurium bound > 30% fewer individual IPEC-J2 cells than did GFP-labeled S. Typhimurium having no prebiotic exposure. Quantitatively, 90% fewer prebiotic-exposed GFP-labeled S. Typhimurium cells were bound per individual IPEC-J2 cell compared to non-prebiotic exposed GFP-labeled S. Typhimurium. Comparison of invasiveness of S. Typhimurium DT104 against IPEC-J2 cells revealed greater than a 90% decrease in intracellular recovery of prebiotic-exposed S. Typhimurium DT104 compared to non-exposed controls (averaging 4.4 ± 0.2 log10 CFU/well). These results suggest compounds within the wood-derived prebiotic bound to E. coli and S. Typhimurium-produced adhesions and in the case of S. Typhimurium, this adhesion-binding activity inhibited the binding and invasion of IPEC-J2 cells.

How Management Practices Within a Poultry House During Successive Flock Rotations Change the Structure of the Soil Microbiome.

The microbiome within a poultry production house influences the attainment of physiologically strong birds and thus food safety and public health. Yet little is known about the microbial communities within the house and the effects on the soil microbes onto which the houses are placed; nor the effects of management practices on their equilibrium. This study looked at the soil bacterial microbiome before a broiler house was constructed, then through 11 flock rotations (2.5 years) that included a partial clean-out and a total clean-out within the management regimen. Major shifts were observed, occurring at the taxonomic class level, related to the introduction of bedding and birds on top of the soil. The partial clean-out of litter did not change the soil bacterial community in any substantial way, only prompting a temporary increase in some genera; however, the total litter clean-out caused a major increase in a cohort of Actinobacteria. The underlying soil contained bacteria beneficial for poultry metabolism, such as Lactobacillus, Faecalibacterium, Bacteriodes, and Ruminococcus. Additionally, management practices affected the class structure of the soil bacterial community beneath the poultry house. The scheduling of these practices should be leveraged to exploit maintenance of beneficial bacteria that maximize microbiome contributions to bird production processes, while minimizing possible antibiotic-resistant bacteria and environmental effects.

Inhibition and Interactions of Campylobacter jejuni from Broiler Chicken Houses with Organic Acids.

Campylobacter jejuni is a bacterium that causes major diarrheal disease worldwide and is also one of the top five foodborne pathogens encountered in the United States. Poultry is a major source of C. jejuni, and a high-risk factor for contracting campylobacteriosis. Organic acids are used in the United States during food animal processing for removal of bacterial contamination from animal carcasses. Six organic acids were evaluated in inhibition studies of 96 C. jejuni strains obtained from shoe covers used in broiler chicken houses at different poultry farms in several states by determining the susceptibilities of the C. jejuni strains, along with the pH values at the molar minimum inhibitory concentrations (MICMs). The undissociated and dissociated organic acid concentrations were calculated at the MICMs with the Henderson-Hasselbalch equation. The results for the 96 C. jejuni strains were treated similarly for each different organic acid. Campylobacter jejuni inhibition did correlate with the dissociated organic acids, but did not correlate with pH or with the undissociated organic acids. When the concentrations of dissociated organic acids decreased, the C. jejuni strains were not disinfected. A carcass wash using organic acids should have the concentration of dissociated acid species carefully controlled. It is suggested to maintain a dissociated acid concentration for propionic, l-lactic, formic, citric, butyric, and acetic acids at 24, 40, 36, 21, 23, and 25 mM, respectively, and at these dissociated organic acid levels an acid wash would be expected to remove or inhibit 97% or more of the C. jejuni bacteria studied here. However, studies must be undertaken to confirm that the suggested concentrations of dissociated organic acids are adequate to remove C. jejuni bacteria in the field vs. the laboratory. Due to propionate, l-lactate, formate, butyrate, and acetate being utilized by C. jejuni, these organic acids may not be appropriate for use as a carcass wash to remove C. jejuni surface contamination. Of all tested organic acids, dissociated citric acid was the most efficient at inhibiting C. jejuni.