Dissipation of antimicrobial resistance genes in compost originating from cattle manure after direct oral administration or post-excretion fortification of antimicrobials.

Dissipation of antimicrobial resistance genes (ARG) during composting of cattle manure generated through fortification versus administration of antimicrobials in feed was compared. Manure was collected from cattle fed diets containing (kg-1) dry matter (DM): (1) 44 mg chlortetracycline (CTC), (2) a mixture of 44 mg each of chlortetracycline and sulfamethazine (CTCSMZ), (3) 11 mg tylosin (TYL) or (4) Control, no antimicrobials. Manures were composted for 30 d with a single mixing after 16 d to generate the second heating cycle. Quantitative PCR (qPCR) was used to measure 16S rDNA and tetracycline (tet), erythromycin (erm) and sulfamethazine (sul) genes. Temperature peaks ranged from 48 to 68°C across treatments in the first composting cycle, but except for the control, did not exceed 55°C in the second cycle. Copy numbers of 16S rDNA decreased (P < 0.05) during composting, but were not altered by antimcrobials. Except tet(L), all ARG decreased by 0.1-1.6 log10 g DM-1 in the first cycle, but some genes (tet[B], tet[L], erm[F], erm[X]) increased (P < 0.05) by 1.0-3.1 log10 g DM-1 in the second. During composting, levels of tet(M) and tet(W) in CTC, erm(A), erm(B) and erm(X) in TYL, and sul(1) in CTCSMZ remained higher (P < 0.05) in fed than fortified treatments. The dissipation of ARG during composting of manure fortified with antimicrobials differs from manure generated by cattle that are administered antimicrobials in feed, and does not always align with the dissipation of antimicrobial residues.

Subtyping Escherichia coli Virulence Genes Isolated from Feces of Beef Cattle and Clinical Cases in Alberta.

Clinical outcomes of Shiga toxin (stx)-producing Escherichia coli infection are largely determined by virulence gene subtypes. This study used a polymerase chain reaction (PCR)-pyrosequencing assay to analyze single-nucleotide polymorphisms for subtyping three major virulence genes (stx1, stx2, eae) of pathogenic E. coli (O157, O26, O111, and O103) isolated from cattle over a 2-year interval (n = 465) and human clinical cases (n = 42) in western Canada. Most bovine isolates were PCR positive for at least one target virulence gene (367/465), whereas 100% of human isolates harbored eae in combination with at least one stx gene. Four Shiga toxin (1a, 2a, 2c, and 2e) and four eae (λ/γ1-eae, ɛ-eae, θ/γ2-eae, and ß-eae) subtypes were identified in over 25 distinct virulence genotypes. Among cattle isolates, every serogroup, but O103, presented a dominant genotype (O157: stx1a+stx2a+λ/γ1-eae, O26: ß-eae alone, and O111: stx1a+θ/γ2-eae). Similar patterns were found in human isolates, although it was not possible to establish a clear genotypic association between the two sources. Many O157 and non-O157 cattle isolates lacked stx genes; the absence was greater in non-O157 (75/258) and O157:non-H7 (19/40) than in O157:H7 strains (1/164). In addition, there was a greater diversity of virulence genotypes of E. coli isolated from cattle than those of human diseases, which could be due to sample characteristics (e.g., source and clinical condition). However, the majority of cattle strains had virulence profiles identical to those of clinical cases. Consequently, determining the presence of certain stx (stx1a and stx2a) and eae (λ/γ1-eae) subtypes known to cause human disease would be a valuable tool for risk assessment and prediction of disease outcome along the farm-to-fork continuum.

Composting for Biocontained Cattle Mortality Disposal and Associated Greenhouse Gas and Leachate Emissions.

Composting can be an effective means of biodegrading livestock mortalities in emergency disposal situations, such as disease outbreaks. Within the past decade, our knowledge detailing composting has increased substantially. However, research data linking the environmental impact of composting to atmospheric and terrestrial systems are limited. We investigated composting efficacy, greenhouse gas emissions, and leachate properties from two static compost piles, each containing 16 cattle mortalities, built with either beef manure (BM) or wood shavings (WS) as envelope material. Wood shavings achieved a greater maximum temperature than BM (60 vs. 50°C) and maintained higher temperatures over 200 d ( < 0.001). Greenhouse gas emissions were evaluated using a static chamber and gas chromatography. Emissions of NO ( < 0.001), CH ( < 0.01), and CO ( < 0.05) were lower from WS than BM, resulting in 3-fold lower total CO equivalent emissions. After 250 d of composting, piles were relocated, and soil cores were taken (i) from beneath the piles, (ii) adjacent to the piles where leachate had accumulated, and (iii) in a control zone without compost exposure. Elevated concentrations of ammonium ( < 0.05) and chloride ( < 0.05) were found in soil beneath both BM and WS. Microbial DNA profiles suggested that leachate from BM compost increased bacterial diversity in soil, maintaining a biological soil impact after pile removal. Degradation of bovine mitochondrial DNA fragments was monitored by polymerase chain reaction. Limited migration of genetic bovine material from compost into soil was observed. Based on the mortalities decomposition and leachate contents, both BM and WS are suitable envelope materials for composting.

Monitoring Seven Potentially Pathogenic Escherichia coli Serogroups in a Closed Herd of Beef Cattle from Weaning to Finishing Phases.

The goal of this study was to monitor Shiga toxin-producing Escherichia coli (STEC) serogroups and virulence genes in cattle (n = 30) originating from a closed herd. Fecal samples were collected (1) at weaning, (2) upon arrival to a feedlot, (3) after 30 days on feed (DOF), and (4) after 135 DOF. DNA was extracted from feces for detection of virulence and serogroup genes by polymerase chain reaction (PCR) and immunomagnetic separation and pulsed-field gel electrophoresis (PFGE) were performed to collect and subtype STEC isolates. The prevalence of each serogroup measured by PCR from weaning to 135 DOF was 23.3-80.0% for O26, 33.3-46.7% for O45, 70.0-73.3% for O103, 36.7-86.7% for O111, 56.7-6.7% for O121, 26.7-66.7% for O145, and 66.7-90.0% for O157. Total fecal samples positive for virulence genes were 87.5% for ehxA, 85.8% for stx1, 60.0% for stx2, 52.5% for eae, and 44.2% for the autoagglutinating adhesion gene, saa. The prevalence of each serogroup and virulence gene tended to increase by 135 DOF, with the exception of O121, stx2, and saa. The frequency of detection of some virulence genes was largely affected over time, most notably with saa and stx2 decreasing, and eae increasing when cattle were transitioned to concentrate-based diets. PFGE analysis of O157 and O103 fecal isolates revealed dominant pulsotypes, but the presence of identical O103 isolates, which differed in virulence profiles. Overall, this study showed that fecal shedding of E. coli serogroups and virulence-associated genes are highly variable over time as cattle move from ranch to feedlot. To mitigate STEC, it is important to understand the factors affecting both prevalence of individual serogroups and the presence of virulence factors.

Biodegradation of prions in compost.

Composting may serve as a practical and economical means of disposing of specified risk materials (SRM) or animal mortalities potentially infected with prion diseases (transmissible spongiform encephalopathies, TSE). Our study investigated the degradation of prions associated with scrapie (PrP(263K)), chronic waste disease (PrP(CWD)), and bovine spongiform encephalopathy (PrP(BSE)) in lab-scale composters and PrP(263K) in field-scale compost piles. Western blotting (WB) indicated that PrP(263K), PrP(CWD), and PrP(BSE) were reduced by at least 2 log10, 1-2 log10, and 1 log10 after 28 days of lab-scale composting, respectively. Further analysis using protein misfolding cyclic amplification (PMCA) confirmed a reduction of 2 log10 in PrP(263K) and 3 log10 in PrP(CWD). Enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhanced degradation of PrP(263K) and PrP(CWD). For field-scale composting, stainless steel beads coated with PrP(263K) were exposed to compost conditions and removed periodically for bioassays in Syrian hamsters. After 230 days of composting, only one in five hamsters succumbed to TSE disease, suggesting at least a 4.8 log10 reduction in PrP(263K) infectivity. Our findings show that composting reduces PrP(TSE), resulting in one 50% infectious dose (ID50) remaining in every 5600 kg of final compost for land application. With these considerations, composting may be a viable method for SRM disposal.

Assessing the inactivation of Mycobacterium avium subsp. paratuberculosis during composting of livestock carcasses.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities.

Biodegradation of specified risk material and fate of scrapie prions in compost.

Composting may be a viable alternative to rendering and land filling for the disposal of specified risk material (SRM) provided that infectious prion proteins (PrP(TSE)) are inactivated. This study investigated the degradation of SRM and the fate of scrapie prions (PrP(Sc)) over 28 days in laboratory-scale composters, with and without feathers in the compost matrices. Compost was mixed at day 14 to generate a second heating cycle, with temperatures exceeding 65°C in the first cycle and 50°C in the second cycle. Approximately 63% and 77% of SRM was degraded after the first and second cycles, respectively. Inclusion of feathers in the compost matrices did not alter compost properties during composting other than increasing (P < 0.05) total nitrogen and reducing (P < 0.05) the C/N ratio. However, addition of feathers enhanced (P < 0.05) SRM degradation by 10% upon completion of experiment. Scrapie brain homogenates were spiked into manure at the start of composting and extracted using sodium dodecyl sulphate followed by detection using Western blotting (WB). Prior to composting, PrP(Sc) was detectable in manure with 1-2 log(10) sensitivity, but was not observable after 14 or 28 days of composting. This may have been due to either biological degradation of PrP(Sc) or the formation of complexes with compost components that precluded its detection.

High-pressure synthesis of acentric sodium pyrocarbonate, Na2[C2O5].

The inorganic pyrocarbonate salt Na2[C2O5] crystallizes in the acentric, monoclinic space group P21 with Z = 2. It contains monovalent alkali metal cations together with isolated pyrocarbonate anions. The [C2O5]2--groups consist of two planar [CO3]2--groups which are slightly tilted with respect to each other around the bridging oxygen atom. Na2[C2O5] was synthesized in a laser-heated diamond anvil cell at 20(2) GPa by heating a mixture of Na2[CO3] + CO2 to ≈800(200) K. Its crystal structure was obtained by single crystal synchrotron X-ray diffraction and confirmed by density functional theory-based calculations in combination with Raman spectroscopy. Second harmonic generation measurements verified the acentric space group symmetry. The crystal structure is characterized by alternating layers of Na+-cations and [C2O5]2--complex anions.

Viability of Bacillus licheniformis and Bacillus thuringiensis spores as a model for predicting the fate of bacillus anthracis spores during composting of dead livestock.

Safe disposal of dead livestock and contaminated manure is essential for the effective control of infectious disease outbreaks. Composting has been shown to be an effective method of disposal, but no information exists on its ability to contain diseases caused by spore-forming bacteria, such as Bacillus anthracis. Duplicate composters (east and west), each containing 16 dead cattle, were constructed (final capacity, 85,000 kg). Spores (10(7) CFU/g manure) of Bacillus licheniformis and Bacillus thuringiensis were mixed with autoclaved feedlot manure and placed in either sterile vials or porous nylon bags. Compost temperatures in the west composter were slightly higher than in the east composter. Viable B. thuringiensis spores were reduced to ≤10(2) CFU in all samples after 112 days but were isolated from bags (west composter) at ≤10(2) and at 10(5) CFU (east composter) after 230 days. In contrast, B. licheniformis was at ≤10(2) CFU in vials (west composter) after 112 days but remained at 10(6) CFU after 230 days (east composter). Similarly, B. licheniformis in bags was not detected after 230 days in the west composter but remained at 10(7) CFU in the east composter. Our study suggests that spore viability was reduced in the west composter by exposure to compost and elevated temperatures over time. Different temperature profiles may explain why spores remained viable in the east structure but were largely rendered nonviable in the west structure. Under practical conditions, variation in composting microclimates may preclude the complete inactivation of Bacillus spores, including those of B. anthracis, during composting. However, composting may still have merit as a method of biocontainment, reducing and diluting the transfer of infectious spores into the environment.

Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics.

BACKGROUND: Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA. RESULTS: The concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments. CONCLUSION: The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.

Biodegradation of specified risk material and characterization of actinobacterial communities in laboratory-scale composters.

As a result of bovine spongiform encephalopathy in Canada, specific tissues at risk of harbouring prions are not allowed to enter the food chain. Composting may be a viable alternative to rendering and land filling for the disposal of specified risk material (SRM). Two types of laboratory-scale composters, actively-heated and ambient systems were constructed to assess the biodegradation of SRM over 30 days. A second heating cycle was generated by mixing the compost after 15 days. Compared to ambient composters, temperature profiles in actively-heated composters were above 50°C for 5 and 4 days longer in the first and second composting cycles, respectively. Degradation of SRM was similar between two composter types during two composting cycles, averaging 52.2% in the first cycle and 43.9% in second cycle. Denaturing gradient gel electrophoresis (DGGE) revealed that changes in the actinobacteria populations in the first composting cycle were of a temporal nature, whereas alterations in populations in the second composting cycle were more related to active heating of compost. Sequencing of the dominant DGGE bands showed the predominance of Corynebacterium, Promicromonospora, Pseudonocardia, and Thermobifida in the first composting cycle and Corynebacterium, Mycobacterium, Nocardia, Saccharomonospora, and Streptomyces in the second composting cycle. Active heating can alter the nature of actinobacteria populations in compost, but does not appear to have a major impact on the extent of degradation of SRM.

Genome Sequences of 104 Escherichia coli O157:H7 Isolates from Pigs, Cattle, and Pork Production Environments in Alberta, Canada.

Genome sequences of Escherichia coli O157:H7 originating from pigs are limited in the public databases. We sequenced 104 E. coli O157:H7 isolates from pig and cattle feces and pork production environments in Alberta, Canada. The information will aid studies investigating sources of E. coli O157:H7 contaminating pork and the associated environments.

Genomic analysis of Shiga toxin-producing Escherichia coli O157:H7 from cattle and pork-production related environments.

Three E. coli O157:H7 outbreaks have been attributed to contaminated pork in Alberta, Canada, recently. This study investigates the phylogenetic relatedness of E. coli O157:H7 from pigs, cattle, and pork-production environments for source attribution. Limited strain diversity was observed using five conventional subtyping methods, with most or all strains being in one subgroup. Whole-genome single nucleotide polymorphism analysis confirmed the recent ancestry of the isolates from all three sources. Most environmental isolates clustered closer with pig isolates than cattle isolates. Also, a direct link was observed between 2018-outbreak environmental isolates and isolates collected from a pig farm in 2018. The majority of pig isolates harbor only one Shiga toxin gene, stx2a, while 70% (35/50) of the cattle isolates have both stx1a and stx2a. The results show some E. coli O157:H7 strains could establish persistence on pig farms and as such, pigs can be a significant source of the organism.

Biodegradation of genetically modified seeds and plant tissues during composting.

BACKGROUND: The increasing global market of genetically modified (GM) crops amplifies the potential for unintentional contamination of food and feed with GM plants. Methods proposed for disposal of crop residues should be assessed to prevent unintended distribution of GM materials. Composting of organic material is inexpensive and location-independent. The objective of this study was to determine the effectiveness of composting for disposal of GM plants in terms of reducing seed viability and promoting the degradation of endogenous as well as transgenic DNA. RESULTS: Duplicate samples of corn kernels, alfalfa leaves, and GM canola seeds, meal and pellets were sealed in porous nylon bags and implanted in duplicate 85,000 kg (initial weight) feedlot manure compost piles. Samples were collected at intervals over 230 days of composing. Canola seeds and corn kernels were not viable after 14 days of composting with temperatures in the piles exceeding 50 degrees C. In all samples, PCR analyses revealed that plant endogenous and transgenic fragments were substantially degraded after 230 days of composting. Southern blotting of genomic DNA isolated from canola seeds identified differences in the persistence of endogenous, transgenic, and bacterial DNA. CONCLUSION: Composting GM and non-GM plant materials with manure rendered seeds non-viable, and resulted in substantial, although not complete, degradation of endogenous and transgenic plant DNA. This study demonstrates that composting could be effective for disposing of GM crops in the event of their inadvertent entry into the food or feed chain.

Escherichia coli: Physiological Clues Which Turn On the Synthesis of Antimicrobial Molecules.

Zoonotic pathogens, like Shiga toxin-producing Escherichia coli (STEC) are a food safety and health risk. To battle the increasing emergence of virulent microbes, novel mitigation strategies are needed. One strategy being considered to combat pathogens is antimicrobial compounds produced by microbes, coined microcins. However, effectors for microcin production are poorly understood, particularly in the context of complex physiological responses along the gastro-intestinal tract (GIT). Previously, we identified an E. coli competitor capable of producing a strong diffusible antimicrobial with microcin-associated characteristics. Our objective was to examine how molecule production of this competitor is affected by physiological properties associated with the GIT, namely the effects of carbon source, bile salt concentration and growth phase. Using previously described liquid- and agar-based assays determined that carbon sources do not affect antimicrobial production of E. coli O103F. However, bile salt concentrations affected production significantly, suggesting that E. coli O103F uses cues along the GIT to modulate the expression of antimicrobial production. Furthermore, E. coli O103F produces the molecule during the exponential phase, contrary to most microcins identified to date. The results underscored the importance of experimental design to identify producers of antimicrobials. To detect antimicrobials, conventional microbiological methods can be a starting point, but not the gold standard.

Whole-Genome Draft Assemblies of Difficult-to-Classify Escherichia coli O157 and Non-O157 Isolates from Feces of Canadian Feedlot Cattle.

Forty-eight Escherichia coli strains were chosen due to variable detection of stx or serogroup by PCR. Although all strains were initially determined to be Shiga toxin-producing Escherichia coli (STEC), their genomes revealed 11 isolates carrying stx 1a, stx 1b, stx 2a, and/or stx 2b Assembled genome sizes varied between 4,667,418 and 5,556,121 bp, with N 50 values between 79,648 and 294,166 bp and G+C contents between 50.3% and 51.4%.

A biosecure composting system for disposal of cattle carcasses and manure following infectious disease outbreak.

During outbreaks of infectious animal diseases, composting may be an effective method of disposing of mortalities and potentially contaminated manure. Duplicate biosecure structures containing 16 cattle (Bos taurus) mortalities (343 kg average weight) were constructed with carcasses placed on a 40-cm straw layer and overlaid with 160 cm of feedlot manure. At a depth of 80 cm (P80), compost heated rapidly, exceeding 55 degrees C after 8 d and maintained temperatures of 55 to 65 degrees C for > 35 d. Temperatures at 160 cm (P160) failed to exceed 55 degrees C, but remained above 40 degrees C for >4 mo. To investigate rates of microbial inactivation, Escherichia coli O157:H7, Campylobacter jejuni, and Newcastle disease virus (NDV) were inoculated in manure (E. coli O157:H7 and C. jejuni approximately 10(8) CFU g(-1); NDV, approximately 10(6) EID(50) g(-1)), embedded at P80 and P160 and retrieved at intervals during composting. Escherichia coli O157:H7 and NDV were undetectable after 7 d at both depths. The C. jejuni DNA was detected up to 84 d at P80 and >147 d at P160. To estimate degradation of recalcitrant substrates, bovine brain, hoof, and rib bones were also embedded at P80 and P160 and retrieved at intervals. Residues of soft tissues remained in carcasses after opening at 147 d and bovine tissue decomposition ranked as brain > hoof > bone. More than 90% dry matter (DM) of brain disappeared after 7 d and 80% DM of hoof decomposed after 56 d. High degradation of cattle carcasses, rapid suppression of E. coli O157:H7 and NDV and reduction in viable cell densities of >6 logs for C. jejuni demonstrates that the biosecure composting system can dispose of cattle carcasses and manure in an infectious disease outbreak.

Properties of an Antimicrobial Molecule Produced by an Escherichia coli Champion.

Over recent decades, the number and frequency of severe pathogen infections have been increasing. Pathogen mitigation strategies in human medicine or in livestock operations are vital to combat emerging arsenals of bacterial virulence and defense mechanisms. Since the emergence of antimicrobial resistance, the competitive nature of bacteria has been considered for the potential treatment or mitigation of pathogens. Previously, we identified a strong E. coli competitor with probiotic properties producing a diffusible antimicrobial molecule(s) that inhibited the growth of Shiga toxin-producing E. coli (STEC). Our current objective was to isolate and examine the properties of this antimicrobial molecule(s). Molecules were isolated by filter sterilization after 12 h incubation, and bacterial inhibition was compared to relevant controls. Isolated antimicrobial molecule(s) and controls were subjected to temperature, pH, or protease digestion treatments. Changes in inhibition properties were evaluated by comparing the incremental cell growth in the presence of treated and untreated antimicrobial molecule(s). No treatment affected the antimicrobial molecule(s) properties of STEC inhibition, suggesting that at least one molecule produced is an efficacious microcin. The molecule persistence to physiochemical and enzymatic treatments could open a wide window to technical industry-scale applications.

Bacteriocin Occurrence and Activity in Escherichia coli Isolated from Bovines and Wastewater.

The increasing prevalence of antimicrobial resistant (AMR) E. coli and related Enterobacteriaceae is a serious problem necessitating new mitigation strategies and antimicrobial agents. Bacteriocins, functionally diverse toxins produced by most microbes, have long been studied for their antimicrobial potential. Bacteriocins have once again received attention for their role as probiotic traits that could mitigate pathogen burden and AMR bacteria in livestock. Here, bacteriocins were identified by activity screening and whole-genome sequencing of bacteriocin-producers capable of inhibiting bovine and wastewater E. coli isolates enriched for resistance to cephalosporins. Producers were tested for activity against shiga toxin-producing E. coli (STEC), AMR E. coli, and related enteric pathogens. Multiple bacteriocins were found in 14 out of 90 E. coli isolates tested. Based on alignment within BACTIBASE, colicins M, B, R, Ia, Ib, S4, E1, E2, and microcins V, J25, and H47, encoded by identical, variant, or truncated genes were identified. Although some bacteriocin-producers exhibited activity against AMR and STEC E. coli in agar-based assays, most did not. Despite this idiosyncrasy, liquid co-cultures of all bacteriocinogenic isolates with luciferase-expressing generic (K12) or STEC E. coli (EDL933) resulted in inhibited growth or reduced viability. These abundant toxins may have real potential as next-generation control strategies in livestock production systems but separating the bacteriocin from its immunity gene may be necessary for such a strategy to be effective.

Comparison of heating block and water bath methods to determine heat resistance in Shiga-toxin producing Escherichia coli with and without the locus of heat resistance.

This study found variability in the time required for tubes of media in heating block wells to reach 60 °C, resulting in significant effects on heat resistance measurements. To determine the extent that methodology changed heat resistance measurements, we compared the heat resistance of Shiga-toxin producing Escherichia coli (STEC) strains with and without the locus of heat resistance (LHR) using both heating block and water bath methods. A total of 34 strains of STEC were used along with a generic E. coli which has been identified as heat-resistant and used as a positive control. The E. coli strains were incubated in a water bath and a heating block set at 60 °C to determine come up time to 60 °C (T0) and for 6 additional minutes (T6) to calculate the D60 value. After incubation, the colony forming units (CFU) were enumerated and mean log CFU/mL from biological replicates was calculated. To compare reductions from T0 to T6, standard deviations among replicates within heating method and correlation of the D60 values generated across methods were determined using Mixed model and Correlation analyses. Our findings indicate that the method chosen to evaluate heat resistance of E. coli can dramatically influence results as there was not a significant correlation between D60 values for the same isolate determined by water bath and heating block methods. The water bath method generates more reliable and consistent heat resistance data and should be used in future evaluations of heat resistance in E. coli. Moreover, PCR screening for the LHR would only be moderately useful for predicting phenotypic heat-resistance of E. coli. Considering water bath data only, LHR-positive STEC isolates were either moderately heat-resistant (1 to 5 log reduction) or heat-sensitive (> 5 log reduction). As LHR-negative STEC were also moderately heat-resistant, prediction of phenotypic heat resistance from genotype requires further refinement.