American bison (Bison bison) were nearly hunted to extinction by the 20th century. This rapid decline and the fragmented nature of remnant populations pose challenges to their recovery and resilience, as does human-facilitated admixture between bison subspecies and cattle (Bos taurus). To contextualize current diversity, we sequenced 115 ancient and 45 modern bison genomes from the past ~20,000 years. Our results show that past bison populations were connected, in contrast to structured modern herds. Modern wood bison (B. b. athabascae), a northerly distributed subspecies, carry plains bison (B. b. bison) ancestry from 1920s translocations, and many sampled bison lack cattle ancestry. Our findings reveal the legacy of human impacts on bison and highlight the applicability of ancient DNA for guiding wildlife restoration.

The occurrence, taxonomic identity, drug resistance pattern and virulence traits of cultivable environmental Leptospira inhabiting the Indian subcontinent remain largely unexplored. Here, we describe the isolation and characterization of six novel strains of Leptospira from environmental samples and decode their drug resistance and virulence traits using in silico and in vitro approaches. Among six new isolates of Leptospira, two strains were identified to represent L. interrogans (SA-L3 and SA-L4), originating from sewage that flows proximal to a wildlife enclosure in Mangalore. We also found a strain each of L. levettii (SA-E5), L. kmetyi (SA-E7), L. koniambonensis (SA-E9) and Leptospira sp. (SA-E8, a putative L. andrefontaineae-like new species of Leptospira) within a ~ 5000 sq. ft. farmland area at Halageri, Uttara Kannada District, India. All isolates exhibited hemolysis and harbored genes encoding diverse sphingomyelinases. All strains were resistant to vancomycin, rifamycin, aztreonam, lincomycin, nalidixic acid, minocycline and troleandomycin and carried genes for a multitude of proteins conferring drug-resistant mechanisms. Leptospira sp. SA-E8 exhibited superior virulence traits and significant biofilm formation in vitro. Comparative genomics revealed remarkable interspecies variations in genome size (4.0‒4.8 Mbp), GC content (35.1‒44.8), coding sequences (3823‒5296) and RNA (39‒43). All strains exhibited the oxidation of diverse organic acids and were equipped with genetic machinery for ketone body oxidation. Our study demonstrated the occurrence of genetically distant Leptospira in environmental samples collected from India and provided evidence for conserved multidrug resistance and virulence, offering new dimensions to the theranostics of Leptospirosis. We suggest the inclusion of L. levettii SA-E5, L. kmetyi SA-E7, L. koniambonensis SA-E9 and Leptospira sp. SA-E8 in the array of leptospiral antigens being used in sero-epidemiological studies to establish their seroprevalence in humans, rodents and livestock.

American plains bison (Bison bison bison, bison hereafter) experienced an extreme demographic bottleneck in the late 1800s. The species has since rebounded but is primarily managed as small and isolated herds due to habitat and sociopolitical limitations. Thus, reintroducing bison and allowing herds to achieve as much of their natural dynamics as possible is a major conservation goal. Concerns about genetic diversity loss in small, isolated herds and the persistence of cattle-origin variants from historical crossbreeding efforts have made genetic analysis an important part of bison conservation. The limitations of the current conservation genetic tools which are based on traditional markers such as microsatellites and mitochondrial DNA sequences, may be overcome with genome-wide genotyping panels commonly developed for agricultural species. Bison reintroduction in the grasslands at American Prairie began in 2005. Genetic analysis on these herds has yet to be conducted. We used the Illumina 777K Bovine genotyping panel to obtain data from 197 bison and 179 domestic cows to understand the current population genetic state of bison at American Prairie and gain insight on cattle (Bos taurus) introgression. Overall, bison at American Prairie currently have relatively high genetic diversity, low inbreeding, and no obvious signs of cattle introgression. A more comprehensive evaluation of introgression, likely including whole-genome sequence data, would clarify this finding. These results can serve as a baseline for future comparison as part of a genetic monitoring framework.

Introduction: Detection of pathogens associated with bovine respiratory disease (BRD) typically involves several laboratory tools, with results limited to a defined list of targets. This study adapted a previously reported method for metagenomic sequencing of nasal swabs to describe sequencing data from BRD associated viruses. Changes in virus composition were identified between arrival to a feedlot and 14 days on feed (DOF). These data were also assessed for the simultaneous characterization of bacteria and antimicrobial resistance genes (ARGs). Methods: Nasal swabs were obtained from fall-placed calves (FPC) and yearlings (YRL) from western Canadian commercial feedlots. Evidence of respiratory viruses were identified by sampling 380 animals during processing on arrival to the feedlot and again after 14 DOF using Nanopore metagenomic sequencing. Results: Twenty-one distinct viruses from 12 viral families were identified, with multiple viruses detected in most samples. In FPC arrival samples, the most common BRD associated viruses were bovine rhinitis B virus (BRBV; 46%), bovine coronavirus (BCoV; 32%), influenza D virus (IDV; 17%), bovine respiratory syncytial virus (BRSV; 8.5%), and bovine parainfluenza virus 3 (BPIV-3; 4.2%). The prevalences of bovine herpesvirus type 1 (BoHV-1; 2.7%), BPIV-3 (12%), BRSV (26%), and IDV (51%) were higher in 14 DOF samples compared to arrival samples (p < 0.05). Bovine viral diarrhea virus 1 (BVDV-1) and 2 (BVDV-2) were rarely detected at either time. The most prevalent viruses detected in YRL arrival samples were BRBV (42%), BRSV (39%), BPIV-3 (20%), IDV (16%), BCoV (12%), and BVDV-2 (7.5%). The prevalences of BRSV (60%), BPIV-3 (39%), and BVDV-2 (17%) were higher in 14 DOF samples than arrival samples (p < 0.05). BRSV (OR 7.0, 1.7-29) and BPIV-3 (OR 5.7, 1.5-21) were more likely to be detected in arrival samples from YRL than FPC (p = 0.01). In 14 DOF samples, BPIV-3 (OR 4.9, 1.3-19, p = 0.02) and BVDV-2 (OR 13, 2.0-83, p = 0.01) were identified more frequently in YRL than FPC. These data allowed the identification of respiratory bacteria and 33 ARGs in parallel with assessment of the viral components. The most prevalent bacteria detected in FPC at arrival were Mannheimia haemolytica (35%), Histophilus somni (35%) and Pasteurella multocida (23%). Detection of M. haemolytica increased at 14 DOF (p = 0.02), while P. multocida detection decreased (p = 0.03). At both arrival and 14 DOF in YRL, M. haemolytica was the most prevalent bacterium, followed by P. multocida and H. somni with no significant differences between arrival and 14 DOF samples. ARGs were detected more frequently in the 14 DOF samples than at arrival for both FPC (p = 0.03) and YRL (p = 0.01). The most commonly detected ARGs were associated with resistance to lincosamides and aminoglycosides; however, ARGs associated with other antimicrobials used in cattle including tetracyclines were also identified. Discussion: Changes in the prevalence of BRD associated viruses early in the feeding period reflect transmission and the potential risk of developing the disease. Frequent detection of BCoV, BRSV, and BPIV-3 in newly arrived feedlot cattle suggests the need for improved vaccination before shipping or limitations in existing commercial vaccine preparations.

Previous studies have described the potential emergence of treatment-resistant pathogenic E. coli in municipal sewage. This study sought to characterize a group of chlorine-tolerant Klebsiella pneumoniae species complex (KpSC) isolates based on antimicrobial resistance genes (ARGs), phylogenetic relationships to clinical strains, and other genomic traits associated with pathogenicity. Sewage samples collected from various wastewater treatment plants in Alberta, Canada, were treated with chlorine bleach at doses sufficient to reduce coliform bacteria by 4 log10 (i.e., 99.99% reduction), yielding 21 chlorine-tolerant KpSC isolates. Comparative genomics was used to characterize these chlorine-tolerant KpSC isolates against publicly available genomes from clinical and non-clinical KpSC strains. Chlorine-tolerant KpSC isolates belonged to 15 different sequence types and included Klebsiella pneumoniae, Klebsiella quasipneumoniae, and Klebsiella variicola, which are among the most common and clinically relevant KpSC phylogroups. Phylogenetically, chlorine-tolerant isolates from sewage were genetically diverse, clustering with clinical genomes according to species and sequence type, and reflecting significant strain diversity. Of the 21 chlorine-tolerant sewage isolates, eight resolved into a clade with one or more exclusively clinical genomes. Six isolates differed by < 80 single nucleotide polymorphisms from at least one clinical isolate, implying high genetic similarity to clinical strains. Virulence gene profiles were also remarkably similar, albeit the resistomes of the chlorine-tolerant sewage isolates lacked many relevant ARGs frequently detected in related clinical genomes. Collectively, these data suggest that chlorine-tolerant KpSC sewage isolates may be clinically important.

IntroductionThe risk to humans and animals from antimicrobial resistance (AMR) has increased the emphasis on antimicrobial stewardship in food animal agriculture. Current stewardship recommendations include increasing diagnostic laboratory testing to inform antimicrobial use for bovine respiratory disease (BRD) management in beef feedlot production, yet the performance of newer molecular and sequencing-based diagnostic tests in commercial settings remains poorly characterized.MethodsUsing nasopharyngeal swabs collected from commercial feedlot calves as part of Canadian surveillance, this study evaluated diagnostic laboratory testing approaches for detecting key bacterial BRD pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasmopsis bovis) and associated AMR genes. Bayesian latent class models (BLCMs) were applied to compare traditional culture and antimicrobial susceptibility testing (AST) or qPCR with long-read metagenomic sequencing and recombinase polymerase amplification (RPA). Differences in detection of target bacteria and phenotypic or genotypic AMR were assessed across the early feeding period and between age cohorts.ResultsThis represents the first large-scale field evaluation of a recently developed, long-read metagenomic sequencing protocol implemented by a commercial laboratory for detecting BRD bacteria and AMR in respiratory samples (n = 760) collected by private veterinarians from western Canadian beef feedlots. Detection patterns for BRD bacteria and AMR using culture/AST and metagenomics were often similar between fall-placed calves and yearlings, but with differences from RPA. Detection of BRD bacteria had low sensitivity (< 65% for most organisms/tests), but higher specificity (>90% for all organisms/tests). Detection of macrolide and tetracycline resistance had low but variable sensitivity, with higher estimates for AST compared to metagenomics and RPA, and higher but variable specificity (>90% for most resistance outcomes/tests). Despite not using any targeted enrichment, metagenomic sequencing detected M. bovis although with a sensitivity lower than qPCR or RPA. Estimates of predictive value were most informative across the largest range of prevalence for AST, followed by metagenomics and then RPA.DiscussionThis work demonstrates the potential for large scale implementation of long-read metagenomic sequencing to support antimicrobial stewardship and AMR surveillance for feedlot cattle. The estimates of clinical diagnostic performance and predictive values provide evidence-based guidance for three different laboratory tests for BRD management.

Bacteriophages (phages) are being investigated as potential biocontrol agents for the suppression of bacterial diseases in cultivated crops. Jumbo bacteriophages, which possess genomic DNA larger than 200 kbp, generally have a broader host range than other phages and therefore would be useful as biocontrol agents against a wide range of bacterial strains. Thus, the characterization of novel jumbo phages specific for agricultural pathogens would be of importance for the development of phage biocontrol strategies. Herein, we demonstrate that phage S13 requires Burkholderia glumae flagella for its attachment and infection and that loss of B. glumae flagella prevents S13 cellular lysis. As flagella is a known virulence factor, loss of flagella results in a surviving population of B. glumae with reduced virulence. Further experimentation demonstrates that phage S13 can protect rice plants from B. glumae-sponsored destruction in a rice seedling model of infection.IMPORTANCEBacterial plant pathogens threaten many major food crops and inflict large agricultural losses worldwide. B. glumae is a bacterial plant pathogen that causes diseases such as rot, wilt, and blight in several food major crops including rice, tomato, hot pepper, and eggplant. B. glumae infects rice during all developmental stages, causing diseases such as rice seedling rot and bacterial panicle blight (BPB). The B. glumae incidence of rice plant infection is predicted to increase with warming global temperatures, and several different control strategies targeting B. glumae are being explored. These include chemical and antibiotic soil amendment, microbiome manipulation, and the use of partially resistant rice cultivars. However, despite rice growth amelioration, the treatment options for B. glumae plant infections remain limited to cultural practices. Alternatively, phage biocontrol represents a promising new method for eliminating B. glumae from crop soils and improving rice yields.

American bison were pushed to the brink of extinction by the 20th century. This bottleneck and the fragmented nature of remnant populations pose challenges to their resilience, as does human-facilitated admixture between bison subspecies and with cattle. To contextualize current diversity, we sequenced 115 ancient and 45 modern bison genomes from across North America dating back within the last ~20,000 years. Past bison populations were highly connected, in contrast to structured modern herds. Modern wood bison carry plains bison ancestry from 1920s translocations, while many bison lack cattle ancestry that has previously been believed to be ubiquitous. Our findings reveal the legacy of human impacts on bison in the context of modern conservation and highlight the applicability of ancient DNA for guiding wildlife restoration. ### Competing Interest Statement The authors have declared no competing interest. Genome Prairie, https://ror.org/013s3gf12, Genome Prairie-GAPP Round 21–6338 U.S. National Science Foundation, https://ror.org/021nxhr62, DEB 1754451 Howard Hughes Medical Institute, https://ror.org/006w34k90

The involvement of Cronobacter, which is frequently associated with meningitis and necrotizing enterocolitis, in human colorectal cancer remains unexplored. In this study, we isolate and characterize a novel strain of C. malonaticus designated PO3 from a fecal sample of a colon cancer patient and demonstrate its proliferative effects on colorectal cancer both in vitro and in vivo. The secretome of PO3 significantly promoted cell proliferation, as evidenced by increased cell viability, fluorescence intensity, and Ki-67 expression, without inducing cell death. Furthermore, using high-resolution mass spectrometry (HRMS), we identified a novel tryptic oncopeptide designated P506, in the PO3 secretome that promotes colorectal cancer. Synthetic P506 further stimulated human colorectal adenocarcinoma cell line HT-29 cell proliferation in a dose-dependent manner. Experiments with the BALB/c mouse model in vivo revealed that both the PO3 secretome and P506 contributed to the development of colorectal polyps and associated histological changes, including dysplasia and altered colonic architecture. These findings suggest that P506, a potent peptide from the PO3 secretome, may have oncogenic potential, promoting colorectal cancer progression.

This study explored the potential of circulatory serum metabolite profiles to increase understanding of the physiology of feed efficiency and identify biomarkers to predict residual feed intake (RFI) in lactating Holsteins. Serum metabolite profiles were compared in high (n = 20) and low RFI (n = 20) cows at early, mid, and late lactation stages. The low RFI cows had decreased (P < 0.05) concentrations of dodecanoylcarnitine, dodecenoylcarnitine, dodecanedioylcarnitine, tetradecanoylcarnitine, succinic acid, trimethylamine N-oxide, betaine, and increased concentrations of p-Hydroxyhippuric acid, hydroxysphingomyeline C16:1, phosphatidylcholine diacyl C40:6, and glutarylcarnitine at early lactation. A similar comparison at mid lactation stage showed altered serum concentrations of 26 metabolites that fall into the categories of acyl carnitines, glycerophospholipids, biogenic amines, amino acids, and organic acids. At late lactation, fewer sets of metabolites were significantly affected by RFI grouping. Receiver operator curve analyses identified p-Hydroxyhippuric acid as the top biomarker at early lactation and acetylornithine at mid and late lactation. Models based on sets of serum metabolites in early, mid, and late lactation stages predicted RFI with a validation coefficient of determination of 0.54, 0.68, and 0.64, respectively. This study demonstrated the potential of circulatory serum metabolites as biomarkers and predictors of RFI in lactating dairy cows.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) infection is one of the global healthcare concerns. Here, we report the phenotypic and genotypic characterization of a novel multi-host Staphylococcus phage RuSa1, isolated from wastewater samples derived from a spotted sambar deer (Rusa unicolor) enclosure located at Mangalore, India. Clinical MRSA strains (n = 18) susceptible to RuSa1 were genetically and phenotypically diverse as determined by DNA fingerprinting and in vitro culture assays. RuSa1 displayed a latent period and burst size of 10 min and 50 PFU, respectively, and exhibited efficient biofilm removal activities against S. aureus ATCC BAA-44. The phage exhibited moderate UV stability (3 min) and high titre at 4–37 °C and pH 5‒9. RuSa1 possessed a linear double-stranded genomic DNA with a length of 140 kb. The genome contained 30.18% GC composition and shared 82.0‒94.9% sequence similarity with eleven authentic species of Kayvirus recognized by the International Committee on Taxonomy of Viruses based on VIRIDIC analysis. RuSa1 established distinct phyletic lineage in the maximum likelihood phylogenetic analysis of DNA encoding structural proteins and lacked genes that confer lysogeny. Based on the genotypic, phylogenetic and phenotypic data, RuSa1 is proposed to be a lytic phage and a new species of Kayvirus with a potential therapeutic ability against staphylococcal infections.

Background/Objectives: Long-read metagenomic sequencing can assign antimicrobial resistance genes (ARGs) to speciated bacterial reads. This study evaluated whether metagenomic data from respiratory bacteria derived from feedlot calves sampled in the early feeding period were associated with subsequent bovine respiratory disease (BRD) treatment and phenotypic antimicrobial resistance (AMR) at treatment. Methods: Deep nasopharyngeal swabs (DNPSs) obtained at arrival processing (1 day on feed; DOF), 13 DOF, and the time of BRD treatment were cultured and subjected to antimicrobial susceptibility testing (AST) and long-read metagenomic sequencing. Analyses focused on macrolide (mphE-msrE, EstT) and tetracycline (tet(H)) ARGs within reads assigned to Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, or Bibersteinia trehalosi. Generalized estimating equations assessed associations between metagenomic results from 1 and 13 DOF and subsequent BRD treatment risk and AST outcomes at treatment, at both the individual animal (calf) and pen levels. Results: Calf-level detection of H. somni at 13 DOF was associated with a greater BRD treatment risk between 14 and 45 DOF. An increased pen prevalence of either M. haemolytica or P. multocida at 13 DOF was associated with a greater BRD treatment risk from 14 to 45 DOF. At 13 DOF, detections of mphE-msrE, EstT, or tet(H) in target bacteria were associated with corresponding phenotypic AMR at BRD treatment. Similarly, a higher pen-level prevalence of mphE-msrE or EstT at 13 DOF was also associated with increased macrolide resistance at BRD treatment. Conclusions: The results from long-read metagenomic sequencing of DNPSs collected at 13 DOF were associated with both BRD risk and AMR at treatment. These findings align with prior culture-based results and support the potential utility of pen-level metagenomic testing for AMR surveillance and informing antimicrobial selection in feedlots.

Porcine reproductive and respiratory syndrome (PRRS) severely impacts global swine production. While studies have revealed host genetic factors and molecular mechanisms of fetal response to PRRSV infection, the relationship between fetal genotype, thymic transcription and PRRS susceptibility remains unclear. This study integrates genomic and thymic transcriptomic analyses of fetal pigs from a maternal PRRSV-2 challenge model. Fetuses were categorized by susceptibility to PRRS, following maternal inoculation at gestation day 84–86 into four groups: complete resistance (CR), partial resistance (PR), viable susceptible (VS), and meconium-stained susceptible (MS). CR and PR fetuses exhibited 0 or less than 4 viral loads in serum and thymus, respectively, despite maternal exposure, while VS and MS fetuses had high viral loads with MS showing signs of compromise. Thymic transcriptome analyses revealed a strong interferon response and downregulation of cell cycle and DNA repair pathways in VS and MS fetuses compared to CR and PR fetuses. Severe infection (MS) was further associated with downregulation of genes involved in early thymocyte development, suggesting disrupted T cell development in the thymus. Interaction expression quantitative trait loci (ieQTLs) were identified, where the association between genetic variants and expression levels of differentially expressed genes (DEGs) varied by fetal PRRS susceptibility. Notably, ieQTLs were identified for genes critical for T cell development, including TMEM98 (involved in Th1 cell differentiation) and PTCRA (expressed in early stages of thymocyte development). These findings highlight the complex interplay between fetal genotype, thymic gene expression, and PRRS susceptibility, offering potential genetic markers for breeding programs for replacement gilts.

Early lactation in dairy cows is characterized by negative energy balance and compromised immune function that could lead to metabolic or inflammatory diseases. Circulating biochemical blood variables are increasingly used as indicators of metabolic and inflammatory diseases in many species. This study aimed to estimate genetic parameters and identify candidate genes and quantitative trait loci (QTL) associated with serum proteins (total protein, albumin, globulin, and albumin-to-globulin ratio), liver enzymes (gamma-glutamyltransferase [GGT], aspartate-amino-transferase [AST], glutamate dehydrogenase [GLDH]) and other serum variables (glucose, urea, nonesterified fatty acids [NEFA], β-hydroxybutyric acid [BHBA], and cholesterol). The study population consisted of genotyped lactating Holsteins (938 cows with 80,709 single nucleotide polymorphisms [SNPs]) and serum concentrations of biochemical variables sampled at 2-14 days in milk (DIM) from 11 commercial farms in Alberta. The heritability of the serum variables ranged from 0.04 to 0.35, with cholesterol the most heritable (0.35 ± 0.07), while both glucose and urea were the least heritable (0.04 ± 0.05). Strong genetic correlations were observed between NEFA and GGT (0.78 ± 0.34), AST (0.74 ± 0.29), and BHBA (0.70 ± 0.26). Genome-wide association studies (GWAS) identified 45 and 7 SNPs associated with GGT and cholesterol concentrations, respectively. Candidate genes and QTLs within 100 kb up- and downstream of significant SNPs were detected for GGT and cholesterol. Multiple candidate genes and QTLs in the identified regions are implicated in pathways influencing metabolic disorders, production, and fertility in lactating dairy cows. Overall, the results showed low-to-moderate heritability and identified candidate genes and QTL regions associated with serum GGT and cholesterol. These results have potential utility in efforts to enhance the resilience of dairy cows through genetic selection.

Background/Objectives: Long-read metagenomic sequencing can detect bacteria and antimicrobial resistance genes (ARGs) from bovine respiratory samples, providing an alternative to culture and antimicrobial susceptibility testing (C/S). This study applied Bayesian latent class models (BLCMs) to estimate the sensitivity (Se) and specificity (Sp) of long-read metagenomic sequencing compared to C/S for detecting Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, as well as associated macrolide and tetracycline resistance potential. Methods: Deep nasopharyngeal swabs were collected from fall-placed feedlot calves at arrival, 13, and 36 days on feed across two years and two metaphylaxis protocols. Samples underwent C/S and long-read metagenomic sequencing. BLCMs were used to estimate Se and Sp for the detection of bacteria and potential for antimicrobial resistance (AMR). Results: Se and Sp for detecting respiratory bacteria by metagenomics were not significantly different than culture, with four exceptions. For the 2020 samples, Se for M. haemolytica was lower than culture, and Sp for H. somni was lower, while in both 2020 and 2021 samples, Se for P. multocida was higher for metagenomics than culture. The estimated Se and Sp of metagenomics for the detection of msrE-mphE, EstT, and tet(H) within bacterial reads were either not significantly different or were lower than AST, with Sp > 95% with one exception. Conclusions: This study provided BLCM-based estimates of clinical Se and Sp of metagenomics and C/S without assuming a gold standard in a large pen research setting. These findings demonstrate the potential of long-read metagenomics to support bovine respiratory disease diagnostics, AMR surveillance, and antimicrobial stewardship in feedlot cattle.

We report the draft genome sequence of Leptospira sp. SA-E8, isolated from a farmland soil in India. SA-E8 shared 88.8% OrthoANI and 36.2% dDDH scores with L. andrefontaineae PZF11-2T, indicating distinct taxonomic placement of the isolate. In silico analysis predicted potential drug resistance and hemolytic traits in SA-E8.

Extraintestinal pathogenic Escherichia coli (ExPEC) are responsible for a variety of human infections (urinary tract infections, septicemia, meningitis) and can be routinely isolated from treated sewage. However, the phenotypic properties mediating treatment resistance in ExPEC remain elusive. Herein, we examined heat-resistance in presumptive wastewater ExPEC (W-ExPEC) surviving sewage chlorination or full-scale wastewater treatment. Interestingly, heat-resistance in W-ExPEC was triggered by exposure to hypo-osmotic conditions (i.e., sterile distilled water), resulting in a 10- to 1,000-fold increase in heat-resistance compared to cells exposed to iso-osmotic conditions (i.e., phosphate buffered saline). Remarkably, hypo-osmotic induction of heat resistance occurred extremely fast, in as little as 30 s, and was reversible, demonstrating the phenotypic plasticity of this stress response. Hypo-osmotic stressed W-ExPEC strains survived 58 °C temperatures for up to 20 min – this compared to the clinical reference ExPEC strain, CTF073, which became non-culturable after only 5 min of exposure at this temperature (>8 log10 decline in culturability). The upper thermotolerance level of W-ExPEC (defined as the temperature where culturability was lost after 5 min of exposure) was 62 °C, compared to 58 °C for CFT073. The finding that osmotic stress acts as an anticipatory inducer of heat resistance in W-ExPEC is novel, providing some insights into the possible mechanisms triggering a treatment resistant phenotype in W-ExPEC. The evolution of treatment resistance is worrying prospect for public health, given that waste treatment is a hallmark of infectious disease control in modern society.

Although it is well understood that mitochondrial DNA (mtDNA) deletion mutations cause incurable diseases and contribute to aging, little is known about the transcriptional products that arise from these DNA structural variants. We hypothesized that mitochondrial genomes containing deletion mutations express chimeric mitochondrial RNAs. To test this, we analyzed human and rat RNA sequencing data to identify, quantitate, and characterize chimeric mitochondrial RNAs. We observe increased chimeric mitochondrial RNA frequency in samples from patients with mitochondrial genetic diseases and in samples from aged humans. The spectrum of chimeric mitochondrial transcripts reflects the known pattern of mtDNA deletion mutations. To test the hypothesis that mtDNA deletions induce chimeric RNA transcripts, we treated 18 month old and 34 month old rats with guanidinopropionic acid to induce high levels of skeletal muscle mtDNA deletion mutations. With mtDNA deletion induction, we demonstrate that the chimeric mitochondrial transcript frequency also increases and correlates strongly with an orthogonal DNA-based mutation assay performed on identical samples. Further, we show that the frequency of chimeric mitochondrial transcripts predicts expression of both nuclear and mitochondrial genes central to mitochondrial function, demonstrating the utility of these events as metrics of age-induced metabolic change. Mapping and quantitation of chimeric mitochondrial RNAs provide an accessible, orthogonal approach to DNA-based mutation assays, offer a potential method for identifying mitochondrial pathology in widely accessible data sets, and open a new area of study in mitochondrial genetics and transcriptomics.

Abstract A common dietary strategy to prevent diarrhea in weaned pigs is feeding low protein diets in the period immediately following weaning. The goal of this intervention is to decrease dietary protein sufficiently to reduce pathogen proliferation and protein fermentation without negatively impacting growth performance. One potential alternative to low protein diets is provision of dietary fiber. However, many studies examining the role of protein and fiber on gastrointestinal microbiota and post-weaning diarrhea are complicated by the presence of other substrates, including polyphenols and antinutritional factors in complex ingredients. To assess the role of increased protein and fiber on gut microbiota and host metabolism, semi-purified diets differing in crude protein (CP) and crude fiber (CF) were fed to weaned pigs (n = 40; 28 d of age). Diets with high protein (HP; CP 24.3%, CF 6.4%), high fiber (HF; CP 18.9%, CF 9.4%), or both (HFHP; CP 24.9%, CF 9.5%) were compared with a control (CON; CP 18.1%, CF 6.3%) diet with industry standard crude protein and fiber levels (n = 10/group). Growth performance and diarrhea prevalence were measured alongside gut microbiota composition and metabolites. Pigs fed the HP and HFHP diets had significantly improved feed efficiency compared with both CON and HF diets. Increased protein fermentation, measured through production of biogenic amines, was observed in HP fed animals (P < 0.001). These effects were mitigated by addition of fiber in the HFHP fed pigs. Despite increased markers of protein fermentation, post-weaning diarrhea incidence was not greater in HP fed animals and no increases in gastrointestinal pathogen abundance were detected. Metabolomic and transcriptomic analyses identified critical alterations in host metabolism and cecal transcriptome in the CON compared with the HP fed animals. While all diets were formulated to meet the dietary requirements of weaned pigs, diets with lower protein levels (CON and HF) induced alteration in transcripts from the serine synthesis pathways (P < 0.001) and integrated stress response (P < 0.001) in cecal tissue alongside increases in metabolic pathways related to lysine degradation (P = 0.005). These results challenge the current practice of low protein feeding following weaning by demonstrating a detrimental effect of low protein diets on intestinal cell function and muscle accretion. This suggests that with careful ingredient selection, increased dietary protein post-weaning could better support pig health and performance compared with current industry standard diets.