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.

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.

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.

Growth Inhibition and Alternation of Virulence Genes of Salmonella on Produce Products Treated with Polyphenolic Extracts from Berry Pomace.

ABSTRACT: Organic farming, including integrated crop-livestock farms and backyard farming, is gaining popularity in the United States, and products from these farms are commonly sold at farmers' markets, local stores, and roadside stalls. Because organic farms avoid using antibiotics and chemicals and because they use composted animal waste and nonprofessional harvesting and packaging methods, their products have an increased risk of cross-contamination with zoonotic pathogens. This study sets out to evaluate the efficiency of new postharvest disinfection processes using natural berry pomace extracts (BPEs) as a means to reduce the bacterial load found in two common leafy greens, spinach and celery. Spinach and celery were inoculated with a fixed bacterial load of Salmonella Typhimurium and later were soaked in BPE-supplemented water (wBPE) for increasing periods of time, at two different temperatures (24 and 4°C). The remaining live bacteria were quantified (log CFU per leaf), and numbers were compared with those on vegetables soaked in water alone. The relative expression of virulence genes (hilA1/C1/D1, invA1/C1/E1/F1) of wBPE-treated Salmonella Typhimurium was determined. For spinach, there was a significant (P < 0.05) reduction of Salmonella Typhimurium: 0.2 to 1.2 log CFU/mL and 0.5 to 5 log CFU/mL at 24 and 4°C, respectively. For celery, there was also a significant (P < 0.05) reduction of Salmonella Typhimurium at either 24 or 4°C. The changes in relative expression of virulence genes of Salmonella Typhimurium isolated from spinach and celery varied depending on the treatment conditions but showed a significant down-regulation of inv genes when treated at 24°C for 1,440 min (P < 0.05). After seven uses, the total polyphenolic compounds in wBPE remained at an effective concentration. This research suggests that soaking these vegetables with BPE-containing water at lower temperatures can still reduce the Salmonella Typhimurium load enough to minimize the risk of infection and alter virulence properties.

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.

Physicochemical, mechanical, and molecular properties of nonlysogenic and p22-lysogenic Salmonella typhimurium treated with citrus oil.

The aim of this study was to evaluate the phenotypic and genotypic properties of nonlysogenic Salmonella Typhimurium (ST(P22-)) and lysogenic Salmonella Typhimurium (ST(P22+)) in the presence of sublethal concentrations (SLC2D) of citrus essential oils (CEOs), which were used to evaluate antimicrobial susceptibility, cell surface hydrophobicity, autoaggregation ability, bacterial motility, lysogenic conversion, gene expression patterns, and antibiofilm formation. The SLC2D values of non-heat-treated (N-CEO) and heat-treated (H-CEO) CEO in an autoclave at 121°C for 20 min were 2.0 to 2.1 mg/ml against ST(P22-) and 1.7 to 1.9 mg/ml against STP(22+). The rates of injured ST(P22-) and ST(P22+) cells treated with SLC2D of N-CEO and H-CEO ranged from 67 to 83%. The hydrophobicity and autoaggregation were decreased to 2.5 and 19.5% for ST(P22-) and 4.7 and 21.7% for ST(P22+), respectively, in the presence of N-CEO. A noticeable reduction in the swarming motility was observed in ST(P22-) with N-CEO (14.5%) and H-CEO (13.3%). The numbers of CEO-induced P22 were 5.40 log PFU/ml for N-CEO and 5.65 log PFU/ml for H-CEO. The relative expression of hilA, hilC, hilD, invA, invC, invE, invF, sirA, and sirB was down-regulated in ST(P22-) and ST(P22+) with N-CEO and H-CEO. The numbers of adherent ST(P22-) and ST(P22+) were effectively reduced by more than 1 log in the presence of CEO. These results suggest that CEO has potential to be used to control bacterial attachment, colonization, and invasion.