Inactivation of Escherichia coli, Salmonella enterica, and Listeria monocytogenes using the Contamination Sanitization Inspection and Disinfection (CSI-D) device.

The Contamination Sanitization Inspection and Disinfection (CSI-D) device is a handheld fluorescence-based imaging system designed to disinfect food contact surfaces using ultraviolet-C (UVC) illumination. This study aimed to determine the optimal CSI-D parameters (i.e., UVC exposure time and intensity) for the inactivation of the following foodborne bacteria plated on non-selective media: generic Escherichia coli (indicator organism) and the pathogens enterohemorrhagic E. coli, enterotoxigenic E. coli, Salmonella enterica, and Listeria monocytogenes. Each bacterial strain was spread-plated on non-selective agar and exposed to high-intensity (10 mW/cm2) or low-intensity (5 mW/cm2) UVC for 1-5 s. Control plates were not exposed to UVC. The plates were incubated overnight at 37 °C and then enumerated. Three trials for each bacterial strain were conducted. Statistical analysis was carried out to determine if there were significant differences in bacterial growth between UVC intensities and exposure times. Overall, exposure to low or high intensity for 3-5 s resulted in consistent inhibition of bacterial growth, with reductions of 99.9-100 % for E. coli, 96.8-100 % for S. enterica, and 99.2-100 % for L. monocytogenes. The 1 s exposure time showed inconsistent results, with a 66.0-100 % reduction in growth depending on the intensity and bacterial strain. When the results for all strains within each species were combined, the 3-5 s exposure times showed significantly greater (p < 0.05) growth inhibition than the 1 s exposure time. However, there were no significant differences (p > 0.05) in growth inhibition between the high and low UVC intensities. The results of this study show that, in pure culture conditions, exposure to UVC with the CSI-D device for ≥3 s is required to achieve consistent reduction of E. coli, S. enterica, and L. monocytogenes.

Escherichia coli Isolated from Bovine Sources Encoding the Fosfomycin Resistance Gene fosA7.5.

Dairy animals are reservoirs of antimicrobial-resistant Escherichia coli that are frequently resistant to tetracycline, aminoglycoside, ß-lactam, sulfonamide, and macrolide-lincosamide-streptogramin B antibiotics. However, resistance to other classes of antimicrobials is less frequently observed, and resistance to fosfomycin is rarely observed in E. coli. In this study, we describe the genomic characteristics of E. coli encoding fosA7.5 that have been recovered from bovine sources in the United States. Most isolates only encoded the fosA7.5 gene, whereas 37% encoded at least one other resistance gene, and 25% were genotypically multidrug-resistant. Most (112 isolates, 93%) belonged to phylogenetic group B1 and were assigned to 19 sequence types (STs), the most frequently identified being ST1727, ST2307, and ST3234. Results of this study indicate that fosA-encoding E. coli from bovine sources is very rare in the United States with isolates demonstrating a high level of similarity across a broad geographic region.

Virulome analysis of Escherichia coli ST117 from bovine sources identifies similarities and differences with strains isolated from other food animals.

Escherichia coli ST117 is a pandemic extraintestinal pathogenic E. coli (ExPEC) causing significant morbidity globally. Poultry are a known reservoir of this pathogen, but the characteristics of ST117 strains from other animal sources have not been adequately investigated. Here we characterize the genomes of 36 ST117 strains recovered primarily from preweaned dairy calves, but also from older postweaned calves and lactating cows, in the context of other bovine-associated strains and strains from poultry, swine, and humans. Results of this study demonstrate that bovine-associated ST117 genomes encode virulence factors (VFs) known to be involved in extraintestinal infections, but also occasionally encode the Shiga toxin, a virulence factor (VF) involved in severe gastrointestinal infections and more frequently identified in E. coli from ruminants than other animals. Bovine-associated ST117 genomes were also more likely to encode afa-VIII (adhesins), pap (P-fimbriae), cdt (cytolethal distending toxin), and stx (Shiga toxins) than were poultry and swine-associated genomes. All of the ST117 genomes were grouped into seven virulence clusters, with bovine-associated genomes grouping into Clusters 1, 2, 4, 5, but not 3, 6, or 7. Major differences in the presence of virulence factors between clusters were observed as well. Antimicrobial resistance genes were detected in 112 of 122 (91%) bovine-associated genomes, with 103 of these being multidrug-resistant (MDR). Inclusion of genomes that differed from ST117 by one multi-locus sequence type (MLST) allele identified 31 STs, four of these among the bovine-associated genomes. These non-ST117 genomes clustered with the ST117 genomes suggesting that they may cause similar disease as ST117. Results of this study identify cattle as a reservoir of ST117 strains, some of which are highly similar to those isolated from other food animals and some of which have unique bovine-specific characteristics.

Microdiversity of Salmonella Kentucky During Long-Term Colonization of a Dairy Herd.

Salmonella enterica subsp. enterica serovar Kentucky was repeatedly isolated from a commercial dairy herd that was enrolled in a longitudinal study where feces of asymptomatic dairy cattle were sampled intensively over an 8-year period. The genomes of 5 Salmonella Kentucky isolates recovered from the farm 2 years before the onset of the long-term colonization event and 13 isolates collected during the period of endemicity were sequenced. A phylogenetic analysis inferred that the Salmonella Kentucky strains from the farm were distinct from poultry strains collected from the same region, and three subclades (K, A1, and A2) were identified among the farm isolates, each appearing at different times during the study. Based on the phylogenetic analysis, three separate lineages of highly similar Salmonella Kentucky were present in succession on the farm. Genomic heterogeneity between the clades helped identify regions, most notably transcriptional regulators, of the Salmonella Kentucky genome that may be involved in competition among highly similar strains. Notably, a region annotated as a hemolysin expression modulating protein (Hha) was identified in a putative plasmid region of strains that colonized a large portion of cows in the herd, suggesting that it may play a role in asymptomatic persistence within the bovine intestine. A cell culture assay of isolates from the three clades with bovine epithelial cells demonstrated a trend of decreased invasiveness of Salmonella Kentucky isolates over time, suggesting that clade-specific interactions with the animals on the farm may have played a role in the dynamics of strain succession. Results of this analysis further demonstrate an underappreciated level of genomic diversity within strains of the same Salmonella serovar, particularly those isolated during a long-term period of asymptomatic colonization within a single dairy herd.

Genome-Wide Analysis of Escherichia coli Isolated from Dairy Animals Identifies Virulence Factors and Genes Enriched in Multidrug-Resistant Strains.

The gastrointestinal tracts of dairy calves and cows are reservoirs of antimicrobial-resistant bacteria (ARB), which are present regardless of previous antimicrobial therapy. Young calves harbor a greater abundance of resistant bacteria than older cows, but the factors driving this high abundance are unknown. Here, we aimed to fully characterize the genomes of multidrug-resistant (MDR) and antimicrobial-susceptible Escherichia coli strains isolated from pre-weaned calves, post-weaned calves, dry cows, and lactating cows and to identify the accessory genes that are associated with the MDR genotype to discover genetic targets that can be exploited to mitigate antimicrobial resistance in dairy farms. Results indicated that both susceptible and resistant E. coli isolates recovered from animals on commercial dairy operations were highly diverse and encoded a large pool of virulence factors. In total, 838 transferrable antimicrobial resistance genes (ARGs) were detected, with genes conferring resistance to aminoglycosides being the most common. Multiple sequence types (STs) associated with mild to severe human gastrointestinal and extraintestinal infections were identified. A Fisher's Exact Test identified 619 genes (ARGs and non-ARGs) that were significantly enriched in MDR isolates and 147 genes that were significantly enriched in susceptible isolates. Significantly enriched genes in MDR isolates included the iron scavenging aerobactin synthesis and receptor genes (iucABCD-iutA) and the sitABCD system, as well as the P fimbriae pap genes, myo-inositol catabolism (iolABCDEG-iatA), and ascorbate transport genes (ulaABC). The results of this study demonstrate a highly diverse population of E. coli in commercial dairy operations, some of which encode virulence genes responsible for severe human infections and resistance to antibiotics of human health significance. Further, the enriched accessory genes in MDR isolates (aerobactin, sit, P fimbriae, and myo-inositol catabolism and ascorbate transport genes) represent potential targets for reducing colonization of antimicrobial-resistant bacteria in the calf gut.

Characterization of Antimicrobial Resistance Genes and Virulence Factors in the Genomes of Escherichia coli ST69 Isolates from Preweaned Dairy Calves and Their Phylogenetic Relationship with Poultry and Human Clinical Strains.

Escherichia coli sequence type 69 (ST69) are common causative agents of extraintestinal infections occurring in the bloodstream, cerebrospinal fluid, surgical sites, and, most frequently, the urinary tract. The objective of this study was to analyze the genomic characteristics of 45 antimicrobial-resistant Escherichia coli ST69 strains that were isolated from 28 calves on eight dairy farms in Pennsylvania, USA. The genomes were sequenced and the antimicrobial resistance genes (ARGs), virulence factors (VFs), and plasmid replicons were identified in silico. A phylogenetic analysis was conducted to compare these calf isolate genomes with poultry and human clinical E. coli ST69 genomes. In total, 23 ARGs, 45 VFs, and 15 plasmid replicons were identified. The majority of genomes (n = 36, 80%) had a multidrug-resistant (MDR) genotype and carried genes conferring resistance to antibiotics of human health significance. Phylogenetic analysis based on the core genomes revealed that calf isolates were nested within clades that included human and poultry isolates, indicating that they are not phylogenetically distinct. Results suggest that dairy calves are a reservoir of MDR E. coli ST69 strains with diverse ARG and VF profiles. This information will be helpful in assessing public health risks associated with E. coli ST69 in commercial dairy production systems.

Genomic diversity of antimicrobial-resistant and Shiga toxin gene-harboring non-O157 Escherichia coli from dairy calves.

OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) are globally significant foodborne pathogens. Dairy calves are a known reservoir of both O157 and non-O157 STEC. The objective of this study was to comprehensively evaluate the genomic attributes, diversity, virulence factors, and antimicrobial resistance gene (ARG) profiles of the STEC from preweaned and postweaned dairy calves in commercial dairy herds. METHODS: In total, 31 non-O157 STEC were identified as part of a larger study focused on the pangenome of >1000 E. coli isolates from the faeces of preweaned and postweaned dairy calves on commercial dairy farms. These 31 genomes were sequenced on an Illumina NextSeq500 platform. RESULTS: Based on the phylogenetic analyses, the STEC isolates were determined to be polyphyletic, with at least three phylogroups: A (32%), B1 (58%), and G (3%). These phylogroups represented at least 16 sequence types and 11 serogroups, including two of the 'big six' serogroups, O103 and O111. Several Shiga toxin gene subtypes were identified in the genomes, including stx1a, stx2a, stx2c, stx2d, and stx2g. Using the ResFinder database, the majority of the isolates (>50%) were determined to be multidrug-resistant strains because they harboured genes conferring resistance to three or more classes of antimicrobials, including some of human health significance (e.g., ß-lactams, macrolides, and fosfomycin). Additionally, non-O157 STEC strain persistence and transmission within a farm was observed. CONCLUSION: Dairy calves are a reservoir of phylogenomically diverse multidrug-resistant non-O157 STEC. Information from this study may inform assessments of public health risk and guide preharvest prevention strategies focusing on STEC reservoirs.

Differences between the global transcriptomes of Salmonella enterica serovars Dublin and Cerro infecting bovine epithelial cells.

BACKGROUND: The impact of S. enterica colonization in cattle is highly variable and often serovar-dependent. The aim of this study was to compare the global transcriptomes of highly pathogenic bovine-adapted S. enterica serovar Dublin and the less pathogenic, bovine-adapted, serovar Cerro during interactions with bovine epithelial cells, to identify genes that impact serovar-related outcomes of S. enterica infections in dairy animals. RESULT: Bovine epithelial cells were infected with S. enterica strains from serovars Dublin and Cerro, and the bacterial RNA was extracted and sequenced. The total number of paired-end reads uniquely mapped to non-rRNA and non-tRNA genes in the reference genomes ranged between 12.1 M (Million) and 23.4 M (median: 15.7 M). In total, 360 differentially expressed genes (DEGs) were identified with at least two-fold differences in the transcript abundances between S. Dublin and S. Cerro (false discovery rate ≤ 5%). The highest number of DEGs (17.5%, 63 of 360 genes) between the two serovars were located on the genomic regions potentially associated with Salmonella Pathogenicity Islands (SPIs). DEGs potentially located in the SPI-regions that were upregulated (≥ 2-fold) in the S. Dublin compared with S. Cerro included: 37 SPI-1 genes encoding mostly Type 3 Secretion System (T3SS) apparatus and effectors; all of the six SPI-4 genes encoding type I secretion apparatus (siiABCDEF); T3SS effectors and chaperone (sopB, pipB, and sigE) located in SPI-5; type VI secretion system associated protein coding genes (sciJKNOR) located in SPI-6; and T3SS effector sopF in SPI-11. Additional major functional categories of DEGs included transcription regulators (n = 25), amino acid transport and metabolism (n = 20), carbohydrate transport and metabolism (n = 20), energy production and metabolism (n = 19), cell membrane biogenesis (n = 18), and coenzyme transport and metabolism (n = 15). DEGs were further mapped to the metabolic pathways listed in the KEGG database; most genes of the fatty acid ß-oxidation pathway were upregulated/uniquely present in the S. Dublin strains compared with the S. Cerro strains. CONCLUSIONS: This study identified S. enterica genes that may be responsible for symptomatic or asymptomatic infection and colonization of two bovine-adapted serovars in cattle.

Genome and virulome characterization of Escherichia coli encoding multiple antimicrobial resistance genes isolated from commercial dairy operations.

OBJECTIVES: The aim of this study was to describe the genome sequences of 12 Escherichia coli isolates that encode antimicrobial resistance genes (ARGs) and were isolated from composite dairy animal faecal samples. METHODS: The isolates were recovered between 2013 and 2014 from a larger study evaluating the presence of resistance on dairy operations in Pennsylvania, USA. The draft genomes were sequenced on an Illumina NextSeq 500 platform and were assembled using SPAdes. RESULTS: In total, 69 ARGs were identified, and these were categorized into 20 unique genes conferring resistance to eight classes of antimicrobials. In order of decreasing frequency of detection, these classes were aminoglycosides, tetracyclines, sulphonamides, ß-lactams, phenicols, trimethoprim, fosfomycin, and macrolide-lincosamide-streptogramin B (MLS). Among the resistant isolates, 11 sequence types (ST) were identified, with ST86 detected twice. Although pathogenicity was not evaluated in vivo, multiple genomes encoded virulence factors involved in severe gastrointestinal and extraintestinal infections, including a single Shiga-toxigenic isolate. CONCLUSION: The results of this study demonstrate the presence of antimicrobial-resistant E. coli in dairy animal faeces encoding virulence factors involved in severe human infections.

Virulome and genome analyses identify associations between antimicrobial resistance genes and virulence factors in highly drug-resistant Escherichia coli isolated from veal calves.

Food animals are known reservoirs of multidrug-resistant (MDR) Escherichia coli, but information regarding the factors influencing colonization by these organisms is lacking. Here we report the genomic analysis of 66 MDR E. coli isolates from non-redundant veal calf fecal samples. Genes conferring resistance to aminoglycosides, ß-lactams, sulfonamides, and tetracyclines were the most frequent antimicrobial resistance genes (ARGs) detected and included those that confer resistance to clinically significant antibiotics (blaCMY-2, blaCTX-M, mph(A), erm(B), aac(6')Ib-cr, and qnrS1). Co-occurrence analyses indicated that multiple ARGs significantly co-occurred with each other, and with metal and biocide resistance genes (MRGs and BRGs). Genomic analysis also indicated that the MDR E. coli isolated from veal calves were highly diverse. The most frequently detected genotype was phylogroup A-ST Cplx 10. A high percentage of isolates (50%) were identified as sequence types that are the causative agents of extra-intestinal infections (ExPECs), such as ST69, ST410, ST117, ST88, ST617, ST648, ST10, ST58, and ST167, and an appreciable number of these isolates encoded virulence factors involved in the colonization and infection of the human urinary tract. There was a significant difference in the presence of multiple accessory virulence factors (VFs) between MDR and susceptible strains. VFs associated with enterohemorrhagic infections, such as stx, tir, and eae, were more likely to be harbored by antimicrobial-susceptible strains, while factors associated with extraintestinal infections such as the sit system, aerobactin, and pap fimbriae genes were more likely to be encoded in resistant strains. A comparative analysis of SNPs between strains indicated that several closely related strains were recovered from animals on different farms indicating the potential for resistant strains to circulate among farms. These results indicate that veal calves are a reservoir for a diverse group of MDR E. coli that harbor various resistance genes and virulence factors associated with human infections. Evidence of co-occurrence of ARGs with MRGs, BRGs, and iron-scavenging genes (sit and aerobactin) may lead to management strategies for reducing colonization of resistant bacteria in the calf gut.

Draft Genome Sequence of Escherichia coli ARS-CC7049, a Sequence Type 38 Strain Isolated from Dry Cow Feces on a Commercial Dairy Operation.

Escherichia coli is a diverse species of commensal and pathogenic strains, of which some can cause extraintestinal infections, such as sequence type 38 (ST38) strains. Here, we report the genome sequence of E. coli ARS-CC7049, an ST38 strain that was isolated from a composite fecal sample on a dairy farm.

Genomic diversity and resistome profiles of Salmonella enterica subsp. enterica serovar Kentucky isolated from food and animal sources in Ireland.

Salmonella enterica subsp. enterica serovar Kentucky is frequently isolated from poultry, dairy and beef cattle, the environment and people with clinical salmonellosis globally. However, the sources of this serovar and its diversity and antimicrobial resistance capacities remain poorly described in many regions. To further understand the genetic diversity and antimicrobial sensitivity patterns among S. Kentucky strains isolated from non-human sources in Ireland, we sequenced and analysed the genomes of 61 isolates collected from avian, bovine, canine, ovine, piscine, porcine, environmental and vegetation sources between 2000 and 2016. The majority of isolates (n = 57, 93%) were sequence type (ST) 314, while only three isolates were ST198 and one was ST152. Several isolates were multidrug-resistant (MDR) and 14 carried at least one acquired antimicrobial resistance gene. When compared to a database of publicly available ST314, four distinct clades were identified (clades I-IV), with the majority of isolates from Ireland clustering together in Clade I. Two of the three ST198 isolates were characteristic of those originating outside of the Americas (Clade ST198.2), while one was distantly clustered with isolates from South and North America (Clade ST198.1). The genomes of the two clade ST198.2 isolates encoded Salmonella Genomic Island 1 (SGI1), were multidrug-resistant and encoded polymorphisms in the DNA gyrase (gyrA) and DNA topoisomerase (parC) known to confer resistance to fluoroquinolones. The single ST152 isolate was from raw beef, clustered with isolates from food and bovine sources in North America and was pan-susceptible. Results of this study indicate that most S. Kentucky isolates from non-human sources in Ireland are closely related ST314 and only a few isolates are antimicrobial-resistant. This study also demonstrates the presence of multidrug-resistant ST198 in food sources in Ireland.

FI: The Fecobiome Initiative.

Animal husbandry has been key to the sustainability of human societies for millennia. Livestock animals, such as cattle, convert plants to protein biomass due to a compartmentalized gastrointestinal tract (GIT) and the complementary contributions of a diverse GIT microbiota, thereby providing humans with meat and dairy products. Research on cattle gut microbial symbionts has mainly focused on the rumen (which is the primary fermentation compartment) and there is a paucity of functional insight on the intestinal (distal end) microbiota, where most foodborne zoonotic bacteria reside. Here, we present the Fecobiome Initiative (or FI), an international effort that aims at facilitating collaboration on research projects related to the intestinal microbiota, disseminating research results, and increasing public availability of resources. By doing so, the FI can help mitigate foodborne and animal pathogens that threaten livestock and human health, reduce the emergence and spread of antimicrobial resistance in cattle and their proximate environment, and potentially improve the welfare and nutrition of animals. We invite all researchers interested in this type of research to join the FI through our website: www.fecobiome.com.

Genome sequences of antibiotic-resistant Escherichia coli isolated from veal calves in the USA.

OBJECTIVES: The aim of this study was to describe the genome sequences of 38 antibiotic-resistant Escherichia coli isolated from veal calves. METHODS: The isolates were recovered in 2015 from nine veal farms in the eastern USA and were screened for antibiotic susceptibility using an automated microdilution procedure. The draft genomes were sequenced on an Illumina NextSeq 500 platform and were assembled using SPAdes. RESULTS: In total, 294 resistance genes, categorised into 42 unique genes, conferring resistance to seven different antibiotic classes were detected. Extended-spectrum ß-lactamase (ESBL) genes (blaCTX-M and blaCMY) and the azithromycin resistance gene mph(A) were detected in multiple genomes. Furthermore, mutations in gyrA, parC and parE conferring resistance to fluoroquinolones were detected, as were mutations in the ampC promoter responsible for hyperproduction of ß-lactamases. We identified 25 unique sequence types (STs), including STs that are associated with extraintestinal infections. CONCLUSION: The results of this study indicate a high level of diversity among multidrug-resistant E. coli isolates from veal operations. The identification of multiple isolates encoding resistance to ß-lactams, macrolides and fluoroquinolones as well as virulence factors responsible for human infections warrants more study on the ecology of antibiotic resistance in veal operations.

Genomic Analysis of Antibiotic-Resistant and -Susceptible Escherichia coli Isolated from Bovine Sources in Maputo, Mozambique.

This study reports a genomic analysis of Escherichia coli isolates recovered from 25 bovine fecal composite samples collected from four different production units in Maputo city and around Maputo Province, Mozambique. The genomes were analyzed to determine the presence of antibiotic resistance genes (ARGs), genetic relatedness, and virulence factors known to cause diseases in humans. Whole-genome sequencing was conducted on 28 isolates using an Illumina NextSeq 500 sequencing platform. The genomes were analyzed using BLASTN for the presence of resistance genes and virulence factors, as well as to determine their phylogenetic groups, sequence types (ST), and ST complexes (ST Cplxs). The majority of the isolates (85%) were identified as members of phylogenetic groups B1, with fewer isolates identified as members of group A, and a single isolate identified as group "E/Clade I." The ST analysis demonstrated a higher level of diversity than the phylogenetic group analysis. Sixteen different STs, five ST Cplxs, and seven singleton complexes were identified. A strain identified as a novel ST (ST9215) showed a high level of similarity with an isolate recovered from a wild animal in the Gambia. Seven different ARGs were identified, with tet(B) being the most frequently detected, followed by aph(3″)-Ib, aph(6)-Id, sul2, blaTEM-1B, and dfrA1. Three isolates encoded ß-lactam-conferring point mutations in the ampC promoter (-42C>T). In total, 51 different virulence factors were identified among the genomes. This study demonstrates that E. coli from bovine sources in Mozambique encoded multiple antibiotic resistance elements, plasmids, and virulence factors. To the best of our knowledge, this is the first genomic description of antibiotic-resistant E. coli isolated from bovine sources in Mozambique.

Differences in the Microbial Community and Resistome Structures of Feces from Preweaned Calves and Lactating Dairy Cows in Commercial Dairy Herds.

Preweaned dairy calves and lactating dairy cows are known reservoirs of antibiotic-resistant bacteria. To further understand the differences in the resistomes and microbial communities between the two, we sequenced the metagenomes of fecal composite samples from preweaned dairy calves and lactating dairy cows on 17 commercial dairy farms (n = 34 samples). Results indicated significant differences in the structures of the microbial communities (analysis of similarities [ANOSIM] R = 0.81, p = 0.001) and resistomes (ANOSIM R = 0.93 to 0.96, p = 0.001) between the two age groups. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the predominant members of the communities, but when the groups were compared, Bacteroidetes and Verrumicrobia were significantly more abundant in calf fecal composite samples, whereas Firmicutes, Spirochaetes, Deinococcus-Thermus, Lentisphaerae, Planctomycetes, Chlorofexi, and Saccharibacteria-(TM7) were more abundant in lactating cow samples. Diverse suites of antibiotic resistance genes (ARGs) were identified in all samples, with the most frequently detected being assigned to tetracycline and aminoglycoside resistance. When the two groups were compared, ARGs were significantly more abundant in composite fecal samples from calves than those from lactating cows (calf median ARG abundance = 1.8 × 100 ARG/16S ribosomal RNA [rRNA], cow median ARG abundance = 1.7 × 10-1 ARG/16S rRNA) and at the antibiotic resistance class level, the relative abundance of tetracycline, trimethoprim, aminoglycoside, macrolide-lincosamide-streptogramin B, ß-lactam, and phenicol resistance genes was significantly higher in calf samples than in cow samples. Results of this study indicate that composite feces from preweaned calves harbor different bacterial communities and resistomes than composite feces from lactating cows, with a greater abundance of resistance genes detected in preweaned calf feces.

Interaction of Salmonella enterica with Bovine Epithelial Cells Demonstrates Serovar-Specific Association and Invasion Patterns.

Dairy cows are known reservoirs of Salmonella enterica and human salmonellosis has been attributed to the consumption of contaminated dairy and beef products as well as poultry meat and eggs. Although many S. enterica serovars are known to colonize the gastrointestinal tract of cattle, the interactions between dairy commensal (or persistent) and transient Salmonella serovars with bovine epithelial cells are not well understood. Association-invasion assays were used to characterize the interactions of 26 S. enterica strains from bovine origins, comprising serovars Anatum, Cerro, Dublin, Give, Kentucky, Mbandaka, Meleagridis, Montevideo, Muenster, Newport, Oranienburg, Senftenberg, and Typhimurium, with cultured bovine epithelial cells. There were significant differences in the association with and invasion of bovine epithelial cells within and across Salmonella serovars (Tukey's Honestly Significant Difference test, p < 0.05). Salmonella enterica serovar Dublin strains were the most invasive, whereas Kentucky, Mbandaka, Cerro, and Give strains were the least invasive (p < 0.05). Significant differences in motility on semisolid medium were also observed between strains from different serovars. Findings from this study demonstrate an underappreciated level of phenotypic diversity among Salmonella strains within and across serovars and serve as a baseline for future studies that may identify the molecular mechanisms of asymptomatic Salmonella carriage and bovine salmonellosis.

Metagenomic Analysis of the Microbial Communities and Resistomes of Veal Calf Feces.

Antimicrobial resistance (AMR) is a major public health concern, and dairy calves, including veal calves, are known reservoirs of resistant bacteria. To investigate AMR in the fecal microbial communities of veal calves, we conducted metagenomic sequencing of feces collected from individual animals on four commercial veal operations in Pennsylvania. Fecal samples from three randomly selected calves on each farm were collected soon after the calves were brought onto the farms (n = 12), and again, just before the calves from the same cohorts were ready for slaughter (n = 12). Results indicated that the most frequently identified phyla were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Fecal microbial communities in samples collected from the calves at the early and late stages of production were significantly different at the genus level (analysis of similarities [ANOSIM] on Bray-Curtis distances, R = 0.37, p < 0.05), but not at the phylum level. Variances among microbial communities in the feces of the younger calves were significantly higher than those from the feces of calves at the late stage of production (betadisper F = 8.25, p < 0.05). Additionally, our analyses identified a diverse set of mobile antimicrobial resistance genes (ARGs) in the veal calf feces. The fecal resistomes mostly consisted of ARGs that confer resistance to aminoglycosides, tetracyclines, and macrolide-lincosamide-streptogramin B (MLS), and these ARGs represented more than 70% of the fecal resistomes. Factors that are responsible for selection and persistence of resistant bacteria in the veal calf gut need to be identified to implement novel control points and interrupt detrimental AMR occurrence and shedding.

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.