An optimized live bacterial delivery vehicle safely and efficaciously delivers bacterially transcribed therapeutic nucleic acids.

There is an unmet need for delivery platforms that realize the full potential of next-generation nucleic acid therapeutics. The in vivo usefulness of current delivery systems is limited by numerous weaknesses, including poor targeting specificity, inefficient access to target cell cytoplasm, immune activation, off-target effects, small therapeutic windows, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we characterize the safety and efficacy of a delivery platform comprising engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli SVC1) for intracellular cargo delivery. SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to allow escape of their cargo from the phagosome, and to have minimal immunogenicity. We describe SVC1's ability to deliver short hairpin RNA (shRNA), localized SVC1 administration to various tissues, and its minimal immunogenicity. To validate the therapeutic potential of SVC1, we used it to deliver influenza-targeting antiviral shRNAs to respiratory tissues in vivo. These data are the first to establish the safety and efficacy of this bacteria-based delivery platform for use in multiple tissue types and as an antiviral in the mammalian respiratory tract. We expect that this optimized delivery platform will enable a variety of advanced therapeutic approaches.

Antimicrobial resistance and genetic diversity of Staphylococcus aureus collected from livestock, poultry and humans.

Staphylococcus aureus is one of the most prominent nosocomial, community and farm acquired bacterial infections among animals and human populations. The main purpose of our study was to identify and characterize antimicrobial resistance (AMR) among Staphylococcus aureus isolated from livestock, poultry and humans and to further identify the associated genes. Staphylococcus aureus isolates from human, bovine, swine and poultry were collected from different laboratories across the United States collected between 2003 and 2016. Antimicrobial susceptibility testing for 13 antimicrobials was performed and conventional PCR was used to detect the presence of the nuc gene, mec gene, and to detect int1 gene. Associations between the presence of mec and intl and specific AMR profiles were determined. Antimicrobial resistance was detected in all four host categories, with the highest overall rates found in swine, 100% resistant to tetracycline, 88% to penicillin and 64% clindamycin. The next highest was found among humans with 81.6% of isolates resistant to penicillin followed by 44% to clindamycin and 43% to erythromycin. Among beef cattle isolates, 63.2% were resistant to penicillin, 15.8% resistant to clindamycin and 15.8% to erythromycin. No isolates from any of the hosts were resistant to linezolid. Among poultry isolates, the highest AMR was found to clindamycin, followed by erythromycin and penicillin. Among dairy cattle, highest resistance was found to penicillin, followed by chloramphenicol and gentamicin. Dairy cattle were the only host category with isolates that are resistant to trimethoprim-sulfamethoxazole. Of the 220 isolates detected by latex agglutination, 217 were confirmed to be S. aureus via PCR of the nuc gene, 21.4% were positive for the mecA gene. Swine had the highest prevalence of the mecA gene, followed by humans, poultry and beef cattle. This study has demonstrated a high occurrence of penicillin resistance among all S. aureus isolates. There were differences observed between host species with tetracycline resistance being the highest among swine isolates and clindamycin being highest in poultry isolates. No detection of oxacillin resistance was found in isolates from dairy cattle but was found in isolates from all of the other host species, 94% of which contained the mecA gene.

Metabolomic Profiles of Multidrug-Resistant Salmonella Typhimurium from Humans, Bovine, and Porcine Hosts.

Antimicrobial resistance (AMR) is a global public health threat, yet tools for detecting resistance patterns are limited and require advanced molecular methods. Metabolomic approaches produce metabolite profiles and help provide scientific evidence of differences in metabolite expressions between Salmonella Typhimurium from various hosts. This research aimed to evaluate the metabolomic profiles of S. Typhimurium associated with AMR and it compares profiles across various hosts. Three samples, each from bovine, porcine, and humans (total n = 9), were selectively chosen from an existing library to compare these nine isolates cultured under no drug exposure to the same isolates cultured in the presence of the antimicrobial drug panel ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline). This was followed by metabolomic profiling using UPLC and GC-mass spectrometry. The results indicated that the metabolite regulation was affected by antibiotic exposure, irrespective of the host species. When exposed to antibiotics, 59.69% and 40.31% of metabolites had increased and decreased expressions, respectively. The most significantly regulated metabolic pathway was aminoacyl-tRNA biosynthesis, which demonstrated increased expressions of serine, aspartate, alanine, and citric acid. Metabolites that showed decreased expressions included glutamate and pyruvate. This pathway and associated metabolites have known AMR associations and could be targeted for new drug discoveries and diagnostic methods.

Socio-ecological Factors of Zoonotic Diseases Exposure in Colorado Dairy Workers.

Objectives: Zoonotic pathogens on dairy farms are a known risk for people who work and live there. Exposure and/or transmission of Salmonella serovars, E. coli (O157; H7), Campylobacter jejuni, and Cryptosporidium parvum have been documented to occur in the dairy farm environment. Social ecological factors have been identified as determinants of preventive behaviors of people at risk of infectious diseases.Methods: This study described the effect of socio-ecological factors on selected zoonotic bacterial and protozoal diseases in 42 workers of two dairy farms.Results: Occupational exposure to Salmonella ser. Dublin, E. coli, and Campylobacter spp. was confirmed. Self-efficacy and negative workplace perceptions were risk factors for Salmonella Dublin exposure (OR = 1.43[95% CI 1.11-2.22] & 1.22 [95% CI 1.02-1.53] respectively,). Additionally, safety knowledge and risk perceptions were protective factors of exposure (OR = 0.90 [95% CI 0.79-1.00]). Positive perceptions of supervisors and coworkers was a protective factor of Campylobacter exposure (OR = 0.89 [95% CI 0.79-0.98]).Conclusion: Results indicated that the presence of a supporting organizational environment, good communication with supervisors and coworkers, and training on prevention of zoonotic diseases would potentially reduce occupational exposures to zoonotic diseases on these farms.

Genomic diversity of class I integrons from antimicrobial resistant strains of Salmonella Typhimurium isolated from livestock, poultry and humans.

INTRODUCTION: Multidrug resistance (MDR) is a serious issue prevalent in various agriculture-related foodborne pathogens including Salmonella enterica (S. enterica) Typhimurium. Class I integrons have been detected in Salmonella spp. strains isolated from food producing animals and humans and likely play a critical role in transmitting antimicrobial resistance within and between livestock and human populations. OBJECTIVE: The main objective of our study was to characterize class I integron presence to identify possible integron diversity among and between antimicrobial resistant Salmonella Typhimurium isolates from various host species, including humans, cattle, swine, and poultry. METHODS: An association between integron presence with multidrug resistance was evaluated. One hundred and eighty-three S. Typhimurium isolates were tested for antimicrobial resistance (AMR). Class I integrons were detected and sequenced. Similarity of AMR patterns between host species was also studied within each integron type. RESULTS: One hundred seventy-four (95.1%) of 183 S.Typhimurium isolates were resistant to at least one antimicrobial and 82 (44.8%) were resistant to 5 or more antimicrobials. The majority of isolates resistant to at least one antimicrobial was from humans (45.9%), followed by swine (19.1%) and then bovine (16.9%) isolates; poultry showed the lowest number (13.1%) of resistant isolates. Our study has demonstrated high occurrence of class I integrons in S. Typhimurium across different host species. Only one integron size was detected in poultry isolates. There was a significant association between integron presence of any size and specific multidrug resistance pattern among the isolates from human, bovine and swine. CONCLUSIONS: Our study has demonstrated a high occurrence of class I integrons of different sizes in Salmonella Typhimurium across various host species and their association with multidrug resistance. This demonstration indicates that multidrug resistant Salmonella Typhimurium is of significant public health occurrence and reflects on the importance of judicious use of antimicrobials among livestock and poultry.

A Cautionary Report for Pathogen Identification Using Shotgun Metagenomics; A Comparison to Aerobic Culture and Polymerase Chain Reaction for Salmonella enterica Identification.

This study was conducted to compare aerobic culture, polymerase chain reaction (PCR), lateral flow immunoassay (LFI), and shotgun metagenomics for identification of Salmonella enterica in feces collected from feedlot cattle. Samples were analyzed in parallel using all four tests. Results from aerobic culture and PCR were 100% concordant and indicated low S. enterica prevalence (3/60 samples positive). Although low S. enterica prevalence restricted formal statistical comparisons, LFI and deep metagenomic sequencing results were discordant with these results. Specifically, metagenomic analysis using k-mer-based classification against the RefSeq database indicated that 11/60 of samples contained sequence reads that matched to the S. enterica genome and uniquely identified this species of bacteria within the sample. However, further examination revealed that plasmid sequences were often included with bacterial genomic sequence data submitted to NCBI, which can lead to incorrect taxonomic classification. To circumvent this classification problem, we separated all plasmid sequences included in bacterial RefSeq genomes and reassigned them to a unique taxon so that they would not be uniquely associated with specific bacterial species such as S. enterica. Using this revised database and taxonomic structure, we found that only 6/60 samples contained sequences specific for S. enterica, suggesting increased relative specificity. Reads identified as S. enterica in these six samples were further evaluated using BLAST and NCBI's nr/nt database, which identified that only 2/60 samples contained reads exclusive to S. enterica chromosomal genomes. These two samples were culture- and PCR-negative, suggesting that even deep metagenomic sequencing suffers from lower sensitivity and specificity in comparison to more traditional pathogen detection methods. Additionally, no sample reads were taxonomically classified as S. enterica with two other metagenomic tools, Metagenomic Intra-species Diversity Analysis System (MIDAS) and Metagenomic Phylogenetic Analysis 2 (MetaPhlAn2). This study re-affirmed that the traditional techniques of aerobic culture and PCR provide similar results for S. enterica identification in cattle feces. On the other hand, metagenomic results are highly influenced by the classification method and reference database employed. These results highlight the nuances of computational detection of species-level sequences within short-read metagenomic sequence data, and emphasize the need for cautious interpretation of such results.

Investigating Effects of Tulathromycin Metaphylaxis on the Fecal Resistome and Microbiome of Commercial Feedlot Cattle Early in the Feeding Period.

The objective was to examine effects of treating commercial beef feedlot cattle with therapeutic doses of tulathromycin, a macrolide antimicrobial drug, on changes in the fecal resistome and microbiome using shotgun metagenomic sequencing. Two pens of cattle were used, with all cattle in one pen receiving metaphylaxis treatment (800 mg subcutaneous tulathromycin) at arrival to the feedlot, and all cattle in the other pen remaining unexposed to parenteral antibiotics throughout the study period. Fecal samples were collected from 15 selected cattle in each group just prior to treatment (Day 1), and again 11 days later (Day 11). Shotgun sequencing was performed on isolated metagenomic DNA, and reads were aligned to a resistance and a taxonomic database to identify alignments to antimicrobial resistance (AMR) gene accessions and microbiome content. Overall, we identified AMR genes accessions encompassing 9 classes of AMR drugs and encoding 24 unique AMR mechanisms. Statistical analysis was used to identify differences in the resistome and microbiome between the untreated and treated groups at both timepoints, as well as over time. Based on composition and ordination analyses, the resistome and microbiome were not significantly different between the two groups on Day 1 or on Day 11. However, both the resistome and microbiome changed significantly between these two sampling dates. These results indicate that the transition into the feedlot-and associated changes in diet, geography, conspecific exposure, and environment-may exert a greater influence over the fecal resistome and microbiome of feedlot cattle than common metaphylactic antimicrobial drug treatment.

Prevalence of Escherichia coli O157 Shedding in Preweaned Calves on Colorado Dairies.

To gain insight into a potential age-related predisposition for Escherichia coli pathogen shedding on dairies, this pilot study measured the prevalence of E. coli O157 (ECO157) in the feces of preweaned dairy calves. An aim of this study was to link these outcomes with the concurrent environmental presence of ECO157 and dam ECO157 shedding elucidated in a parallel study. Recto-anal mucosal swabs and a subset of fecal grab samples were collected from calves (2 to 8 weeks of age; n = 399) monthly between December 2013 and June 2014 on three dairies in northern Colorado. A subset of calf dams (n = 111) were also sampled via fecal grab. Concurrently, environmental samples were collected from locations within the vicinity of the calves: farm tractor tires, steering wheels, hutches, buckets, and gloves from the research technicians and the employees involved in calf rearing. The presence of ECO157 and virulence genes was measured in the samples and confirmed via PCR. Of the calves, only 1 (0.25%) of 399 individuals shed during the time period, and the ECO157 strain detected carried no measured virulence genes (eaeA, stx1, and stx2). No difference was seen in detection between the recto-anal mucosal swabs and the fecal grab technique. In contrast, 32% (35 of 111) of the dams shed ECO157, with 1.8% (2 of 111) of the shed isolates containing virulence genes. No ECO157 was detected in the environmental samples. These outcomes demonstrate a disparity between dam and calf ECO157 shedding and indicate that preweaned calves, managed similarly to those of this study, probably have a minor influence on dairy contamination and the transmission of ECO157.

Rapid screening for Mycobacterium tuberculosis complex in clinical elephant trunk wash samples.

Mycobacterium tuberculosis can infect and be transmitted between elephants and humans. In elephants, the 'gold standard' reference test for detection of tuberculosis is culture, which takes a minimum of eight weeks for results and has limited sensitivity. A screening test that is rapid, easily implemented, and accurate is needed to aid in diagnosis of tuberculosis in elephants. Ninety-nine clinical trunk wash samples obtained from 33 elephants were utilized to validate three molecular extraction techniques followed by a polymerase chain reaction for detection of M. tuberculosis. Diagnostic sensitivity and specificity were estimated compared to culture. Kappa coefficients were determined between molecular results and various culture categories and serological test results. An internal amplification control was developed and assessed to monitor for PCR inhibition. One molecular test (the Column method) outperformed the other two, with diagnostic sensitivity and kappa agreement estimates of 100% (CI 57-100) and 0.46 (CI 0.2-0.74), respectively, compared to culture alone. The percentage of molecular-positive/culture-negative samples was 8.4% overall. The molecular extraction technique followed by PCR provides a much-needed rapid screening tool for detection of tuberculosis in elephants. Immediate procedures can be implemented to further assess PCR-positive animals and provide personnel biosecurity. While a positive result is not a definitive test for elephant tuberculosis, the molecular test results can be used to support current diagnostic procedures applied by veterinarians for treatment decisions to prevent the spread of tuberculosis in elephants.

Characterization of the resistome in manure, soil and wastewater from dairy and beef production systems.

It has been proposed that livestock production effluents such as wastewater, airborne dust and manure increase the density of antimicrobial resistant bacteria and genes in the environment. The public health risk posed by this proposed outcome has been difficult to quantify using traditional microbiological approaches. We utilized shotgun metagenomics to provide a first description of the resistome of North American dairy and beef production effluents, and identify factors that significantly impact this resistome. We identified 34 mechanisms of antimicrobial drug resistance within 34 soil, manure and wastewater samples from feedlot, ranch and dairy operations. The majority of resistance-associated sequences found in all samples belonged to tetracycline resistance mechanisms. We found that the ranch samples contained significantly fewer resistance mechanisms than dairy and feedlot samples, and that the resistome of dairy operations differed significantly from that of feedlots. The resistome in soil, manure and wastewater differed, suggesting that management of these effluents should be tailored appropriately. By providing a baseline of the cattle production waste resistome, this study represents a solid foundation for future efforts to characterize and quantify the public health risk posed by livestock effluents.

Resistome diversity in cattle and the environment decreases during beef production.

Antimicrobial resistant determinants (ARDs) can be transmitted from livestock systems through meat products or environmental effluents. The public health risk posed by these two routes is not well understood, particularly in non-pathogenic bacteria. We collected pooled samples from 8 groups of 1741 commercial cattle as they moved through the process of beef production from feedlot entry through slaughter. We recorded antimicrobial drug exposures and interrogated the resistome at points in production when management procedures could potentially influence ARD abundance and/or transmission. Over 300 unique ARDs were identified. Resistome diversity decreased while cattle were in the feedlot, indicating selective pressure. ARDs were not identified in beef products, suggesting that slaughter interventions may reduce the risk of transmission of ARDs to beef consumers. This report highlights the utility and limitations of metagenomics for assessing public health risks regarding antimicrobial resistance, and demonstrates that environmental pathways may represent a greater risk than the food supply.

Antimicrobial Resistance Profiles in Escherichia coli O157 Isolates from Northern Colorado Dairies.

Escherichia coli O157 (EcO157) infections can lead to serious disease and death in humans. Although the ecology of EcO157 is complex, ruminant animals serve as an important reservoir for human infection. Dairy cattle are unique because they may be a source of contamination for milk, meat, and manure-fertilized crops. Foodborne dairy pathogens such as EcO157 are of primary importance to public health. Antimicrobial resistance (AMR) is a complex phenomenon that complicates the treatment of serious bacterial infections and is of increasing concern. In the face of recommended use restrictions for antimicrobial agents in livestock operations, current AMR patterns in known foodborne pathogens should be documented. The objective of this study was to document AMR patterns in EcO157 isolates from dairies in northern Colorado using antimicrobial agents commonly found on dairies and representative of medically important antimicrobial drug classes. Seventy-five EcO157 isolates were recovered from three dairies. Six isolates were resistant to at least 1 of the 10 tested antimicrobial agents: four were resistant to streptomycin, sulfisoxazole, and tetracycline; one was resistant to streptomycin and tetracycline; and one was resistant to only tetracycline. All resistant isolates were from a single dairy. Overall, a low prevalence (8%) of AMR was observed among the 75 EcO157 isolates. No significant effects on AMR profiles due to virulence genes, parity, or previous antimicrobial treatments within the current lactation period were detected. The results of this study provide background information for future comparative studies investigating AMR trends. Future studies should include more participating farms and more samples and should control for potential confounding factors of AMR that may underlie individual farm variation.

Use of Metagenomic Shotgun Sequencing Technology To Detect Foodborne Pathogens within the Microbiome of the Beef Production Chain.

Foodborne illnesses associated with pathogenic bacteria are a global public health and economic challenge. The diversity of microorganisms (pathogenic and nonpathogenic) that exists within the food and meat industries complicates efforts to understand pathogen ecology. Further, little is known about the interaction of pathogens within the microbiome throughout the meat production chain. Here, a metagenomic approach and shotgun sequencing technology were used as tools to detect pathogenic bacteria in environmental samples collected from the same groups of cattle at different longitudinal processing steps of the beef production chain: cattle entry to feedlot, exit from feedlot, cattle transport trucks, abattoir holding pens, and the end of the fabrication system. The log read counts classified as pathogens per million reads for Salmonella enterica,Listeria monocytogenes,Escherichia coli,Staphylococcus aureus, Clostridium spp. (C. botulinum and C. perfringens), and Campylobacter spp. (C. jejuni,C. coli, and C. fetus) decreased over subsequential processing steps. Furthermore, the normalized read counts for S. enterica,E. coli, and C. botulinumwere greater in the final product than at the feedlots, indicating that the proportion of these bacteria increased (the effect on absolute numbers was unknown) within the remaining microbiome. From an ecological perspective, data indicated that shotgun metagenomics can be used to evaluate not only the microbiome but also shifts in pathogen populations during beef production. Nonetheless, there were several challenges in this analysis approach, one of the main ones being the identification of the specific pathogen from which the sequence reads originated, which makes this approach impractical for use in pathogen identification for regulatory and confirmation purposes.

Inhibiting avian influenza virus shedding using a novel RNAi antiviral vector technology: proof of concept in an avian cell model.

Influenza A viruses pose significant health and economic threats to humans and animals. Outbreaks of avian influenza virus (AIV) are a liability to the poultry industry and increase the risk for transmission to humans. There are limitations to using the AIV vaccine in poultry, creating barriers to controlling outbreaks and a need for alternative effective control measures. Application of RNA interference (RNAi) techniques hold potential; however, the delivery of RNAi-mediating agents is a well-known obstacle to harnessing its clinical application. We introduce a novel antiviral approach using bacterial vectors that target avian mucosal epithelial cells and deliver (small interfering RNA) siRNAs against two AIV genes, nucleoprotein (NP) and polymerase acidic protein (PA). Using a red fluorescent reporter, we first demonstrated vector delivery and intracellular expression in avian epithelial cells. Subsequently, we demonstrated significant reductions in AIV shedding when applying these anti-AIV vectors prophylactically. These antiviral vectors provided up to a 10,000-fold reduction in viral titers shed, demonstrating in vitro proof-of-concept for using these novel anti-AIV vectors to inhibit AIV shedding. Our results indicate this siRNA vector technology could represent a scalable and clinically applicable antiviral technology for avian and human influenza and a prototype for RNAi-based vectors against other viruses.

Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples.

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.