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Enterotoxigenic Escherichia coli (ETEC) are significant pathogen in both cattle and pigs, causing diarrhea in these animals and leading to economic losses in the livestock industry. Understanding the dissimilarity in genotype, antimicrobial resistance (AMR), and virulence between bovine and swine ETEC is crucial for development of targeted preventive and therapeutic approaches for livestock. However, a comprehensive study on this area remains lacking. Here, we performed whole-genome sequencingbased analyses of bovine (n = 554) and swine (n = 623) ETEC collected in the United States over a 53-year period. We identified distinct ETEC genotypes (fimH type, O antigen, H antigen, sequence type) in cattle and pigs. Furthermore, specific AMR and virulence profiles were associated with bovine and swine ETEC. Compared to swine ETEC, bovine ETEC were less diverse in genotypes and had a significantly (P < 0.001) lower number of AMR genes per isolate but higher co-occurrence of Shiga toxin and enterotoxin genes. Our results provide an overview of the key genomic differences between bovine and swine ETEC in the United States, which might be attributed to host adaptation and antibiotic usage practice. Ongoing surveillance and research are essential to monitor the genetic diversity and AMR patterns of ETEC in different host species. [ABSTRACT FROM AUTHOR]

Salmonella enterica and Escherichia coli are two pathogenic bacteria of worldwide importance that can infect the gastrointestinal tract. Contamination in the food supply chain is an area of concern. Animal feed may be supplemented with essential trace elements, which function as nutritional additives to promote growth & health and optimize production. Bacteria have acquired many metal resistance genes to adapt to the exposure of metals. In this study, our objectives were to evaluate in S. enterica and E. coli, the correlation between the resistance genotype and phenotype to certain heavy metals, and the ability of conjugative plasmids to transfer antimicrobial resistance genes (AMRGs) and heavy metal resistance genes (HMRGs). A total of 10 strains, five S. enterica and five E. coli, were used for this study. Minimal inhibitory concentrations (MICs) were determined for heavy metals: copper, silver, arsenic, and tellurite. The tested isolates showed resistance to copper (5/10; 50%), arsenic (7/10; 70%), and silver (9/10; 90%). Cohen's Kappa statistics were used to analyze genotype to phenotype agreements. Among the 10 strains sampled, the accordance between geno- and phenotypic heavy metal resistance was fair for copper (kappa = 0.4), none to slight for arsenic (kappa = 0.19) and tellurite (kappa = 0), and no agreement for silver (kappa = -0.19). The transfer of HMRGs was determined in a conjugation experiment performed for all five Salmonella strains as donors using mixed broth cultures. Transconjugants were obtained only from the genotypically tellurite-resistant strain PSU-3260, which yielded a transfer frequency of 10-³ transconjugants per donor. In such strain, the tellurite-resistant genes reside on an IncHI2-type plasmid that shares high DNA sequence identity with known HMRG-disseminating Salmonella plasmids. Our results indicated no considerable correlation between the geno- and phenotypic resistance towards heavy metals in the sampled S. enterica and E. coli. The necessity of research in this area is supported by the lack of standardized protocols and MIC clinical breakpoints for heavy metals. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium is typically considered a host generalist; however, certain isolates are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium isolates from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most isolates originating from domestic animal sources, and that genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the United States are likely host-adapted, and the representative genomic data set examined in this study can improve source prediction and facilitate outbreak investigation. [ABSTRACT FROM AUTHOR]

Non-typhoidal Salmonella is a common cause of gastroenteritis worldwide, but current non-typhoidal Salmonella surveillance is suboptimal. Here, we evaluated the utility of wastewater monitoring to enhance traditional surveillance for this foodborne pathogen. In June 2022, we tested raw sewage collected twice a week from two treatment plants in central Pennsylvania for non-typhoidal Salmonella and characterized isolates using whole-genome sequencing. We recovered 43 Salmonella isolates from wastewater samples, differentiated by genomic analysis into seven serovars: 16 Panama (37.2%), 9 Senftenberg (20.9%), 8 Baildon (18.6%), and 3 or fewer of four other serovars. We assessed genetic relatedness and epidemiologic links between these wastewater isolates with those from patients with salmonellosis. All S . Baildon isolates from wastewater were genetically similar to those associated with a known contemporaneous salmonellosis outbreak. S . Baildon from wastewater and 42 outbreak-related isolates in the national outbreak detection database had the same core genome multilocus sequence typing, and outbreak code differed by zero or one single polynucleotide polymorphism. One of the 42 outbreak-related isolates was obtained from a patient residing in the wastewater sample collection catchment area, which serves approximately 17000 people. S . Baildon is a rare serovar (reported in <1% cases nationally, over five years). Our study underscores the value of monitoring sewage from a defined population to supplement traditional surveillance methods for the evidence of Salmonella infections and to determine the extent of outbreaks.IMPORTANCEDuring the COVID-19 pandemic, monitoring for SARS-CoV-2 in wastewater was highly effective in identifying the variants of concern earlier than clinical surveillance methods. Here, we show that monitoring domestic sewage can also augment traditional reporting of foodborne illnesses to public health authorities. Our study detected multiple Salmonella enterica serovars in samples from two wastewater treatment plants in central Pennsylvania. Using whole-genome sequencing, we demonstrated that the isolates of variant S . Baildon clustered with those from a foodborne salmonellosis outbreak that occurred in a similar time frame. Cases were primarily from Pennsylvania, and one individual lived within the wastewater treatment catchment area. This study highlights the effectiveness of domestic sewage testing as a proactive public health strategy to track and respond to infectious disease outbreaks., Competing Interests: The authors declare no conflict of interest.

Shiga toxin - producing Escherichia coli (STEC) are major foodborne pathogens that result in thousands of hospitalizations each year in the United States. Cattle, the natural reservoir, harbor STEC asymptomatically at the recto-anal junction (RAJ). The molecular mechanisms that allow STEC and non-STEC E. coli to adhere to the RAJ are not fully understood, in part because most adherence studies utilize human cell culture models. To identify a set of bovine-specific E. coli adherence factors, we used the primary RAJ squamous epithelial (RSE) cell-adherence assay to coculture RSE cells from healthy Holstein cattle with diverse E. coli strains from bovine and nonbovine sources. We hypothesized that a comparative genomic analysis of the strains would reveal factors associated with RSE adherence. After performing adherence assays with historical strains from the E. coli Reference Center ( n = 62) and strains newly isolated from the RAJ ( n = 15), we used the bioinformatic tool Roary to create a pangenome of this collection. We classified strains as either low or high adherence and using the Scoary program compiled a list of accessory genes correlated with the "high adherence" strains. While none of the correlations were statistically significant, several gene clusters were associated with the high-adherence phenotype, including two that encode uncharacterized proteins. We also demonstrated that non-STEC E. coli strains from the RAJ are more adherent than other isolates and can outcompete STEC in coculture with RSEs. Further analysis of adherence-associated gene clusters may lead to an improved understanding of the molecular mechanisms of RSE adherence and may help develop probiotics targeting STEC in cattle., Importance: E. coli strains that produce Shiga toxin cause foodborne illness in humans but colonize cattle asymptomatically. The molecular mechanisms that E. coli uses to adhere to cattle cells are largely unknown. Various strategies are used to control E. coli in livestock and limit the risk of outbreaks. These include vaccinating animals against common E. coli strains and supplementing their feed with probiotics to reduce the carriage of pathogens. No strategy is completely effective, and probiotics often fail to colonize the animals. We sought to clarify the genes required for E. coli adherence in cattle by quantifying the attachment to bovine cells in a diverse set of bacteria. We also isolated nonpathogenic E. coli from healthy cows and showed that a representative isolate could outcompete pathogenic strains in cocultures. We propose that the focused study of these strains and their adherence factors will better inform the design of probiotics and vaccines for livestock.

Enterotoxigenic Escherichia coli (ETEC) are significant pathogen in both cattle and pigs, causing diarrhea in these animals and leading to economic losses in the livestock industry. Understanding the dissimilarity in genotype, antimicrobial resistance (AMR), and virulence between bovine and swine ETEC is crucial for development of targeted preventive and therapeutic approaches for livestock. However, a comprehensive study on this area remains lacking. Here, we performed whole-genome sequencing-based analyses of bovine ( n = 554) and swine ( n = 623) ETEC collected in the United States over a 53-year period. We identified distinct ETEC genotypes ( fimH type, O antigen, H antigen, sequence type) in cattle and pigs. Furthermore, specific AMR and virulence profiles were associated with bovine and swine ETEC. Compared to swine ETEC, bovine ETEC were less diverse in genotypes and had a significantly ( P < 0.001) lower number of AMR genes per isolate but higher co-occurrence of Shiga toxin and enterotoxin genes. Our results provide an overview of the key genomic differences between bovine and swine ETEC in the United States, which might be attributed to host adaptation and antibiotic usage practice. Ongoing surveillance and research are essential to monitor the genetic diversity and AMR patterns of ETEC in different host species., Importance: Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea represent one of the most economically important diseases in the livestock industry. By analyzing over a thousand livestock-derived ETEC samples in the United States, our study unveiled a clear distinction in ETEC's genetic traits (i.e., genotypes, antimicrobial resistance [AMR], and virulence profiles) that might be tied to the different use of antibiotics in cattle and pigs, and the bacteria's adaptation to their specific animal hosts. This understanding is crucial for tailoring preventive and therapeutic strategies. It also highlights the significance of ongoing surveillance and research into the evolution of bacterial pathogens like ETEC in livestock by using advanced techniques such as whole-genome sequencing., Competing Interests: The authors declare no conflict of interest.