Sequencing and characterization of Helcococcus ovis: a comprehensive comparative genomic analysis of virulence.

BACKGROUND: Helcococcus ovis (H. ovis) is an emerging bacterial pathogen that commonly causes opportunistic respiratory, mammary, and uterine infections across mammalian hosts. This study applied long- and short-read whole genome sequencing technologies to identify virulence factors in five H. ovis isolates with low, medium, and high virulence phenotypes. RESULTS: The resulting assemblies contained one circular chromosome ranging from 1,744,566 to 1,850,083 bp in length and had a mean GC content of 27.6%. Phylogenetic and nucleotide identity analyses found low virulence strain KG38 to be part of a clade that forms an outgroup apart from the rest of the H. ovis taxon. Assembling the first complete genomes of the species revealed major genomic rearrangements in KG38. One to six prophage regions were identified in each genome. A novel pathogenicity island was found exclusively in the two high virulence strains (KG37 and KG104), along with two hypothetical transmembrane proteins designated as putative VFs. Finally, three zinc ABC transporters and three Type-II/IV secretion systems were identified as possible virulence determinants in this species. The low virulence strain KG38 has fewer intact paralogs of these operons in its genome compared to the higher virulence isolates, which strongly suggests a role in virulence. This strain is also missing four putative virulence factors (VFs) found in other isolates associated with adherence (collagen adhesin precursor), immune evasion (choline-binding protein A and a PspA-like hypothetical protein) and cell wall synthesis (glycerol-3-phosphate cytidylyltransferase). CONCLUSIONS: In this study, we assembled reference-quality complete genomes for five H. ovis strains to identify putative virulence factors. Phylogenetic analyses of H. ovis isolates revealed the presence of a clade representing a potentially novel species within the genus Helcococcus. A novel pathogenicity island and two hypothetical transmembrane proteins were found exclusively in high-virulence strains. The identification of Zinc ABC transporters and Type-II/IV secretion systems as possible virulence determinants, along with the differences in operon content between the low and high virulence isolates, strongly suggests they also play a role in the bacterium's pathogenicity. Taken together, these findings are a valuable first step toward deciphering the pathogenesis of H. ovis infections.

High-Resolution Identification of Multiple Salmonella Serovars in a Single Sample by Using CRISPR-SeroSeq.

Salmonella enterica is represented by >2,600 serovars that can differ in routes of transmission, host colonization, and in resistance to antimicrobials. S. enterica is the leading bacterial cause of foodborne illness in the United States, with well-established detection methodology. Current surveillance protocols rely on the characterization of a few colonies to represent an entire sample; thus, minority serovars remain undetected. Salmonella contains two CRISPR loci, CRISPR1 and CRISPR2, and the spacer contents of these can be considered serovar specific. We exploited this property to develop an amplicon-based and multiplexed sequencing approach, CRISPR-SeroSeq (serotyping by sequencing of the CRISPR loci), to identify multiple serovars present in a single sample. Using mixed genomic DNA from two Salmonella serovars, we were able to confidently detect a serovar that constituted 0.01% of the sample. Poultry is a major reservoir of Salmonella spp., including serovars that are frequently associated with human illness, as well as those that are not. Numerous studies have examined the prevalence and diversity of Salmonella spp. in poultry, though these studies were limited to culture-based approaches and therefore only identified abundant serovars. CRISPR-SeroSeq was used to investigate samples from broiler houses and a processing facility. Ninety-one percent of samples harbored multiple serovars, and there was one sample in which four different serovars were detected. In another sample, reads for the minority serovar comprised 0.003% of the total number of Salmonella spacer reads. The most abundant serovars identified were Salmonella enterica serovars Montevideo, Kentucky, Enteritidis, and Typhimurium. CRISPR-SeroSeq also differentiated between multiple strains of some serovars. This high resolution of serovar populations has the potential to be utilized as a powerful tool in the surveillance of Salmonella species.IMPORTANCESalmonella enterica is the leading bacterial cause of foodborne illness in the United States and is represented by over 2,600 distinct serovars. Some of these serovars are pathogenic in humans, while others are not. Current surveillance for this pathogen is limited by the detection of only the most abundant serovars, due to the culture-based approaches that are used. Thus, pathogenic serovars that are present in a minority remain undetected. By exploiting serovar-specific differences in the CRISPR arrays of Salmonella spp., we have developed a high-throughput sequencing tool to be able to identify multiple serovars in a single sample and tested this in multiple poultry samples. This novel approach allows differences in the dynamics of individual Salmonella serovars to be measured and can have a significant impact on understanding the ecology of this pathogen with respect to zoonotic risk and public health.

Evaluation of Three Bacterial Identification Systems for Species Identification of Bacteria Isolated from Bovine Mastitis and Bulk Tank Milk Samples.

A study was conducted to evaluate Sensititre® Automated Reading and Incubation System 2x System (ARIS), API® (API), and Bruker MALDI-TOF MS (MALDI) bacterial species identification systems using 132 diverse bacterial isolates from bovine milk samples and bulk tank milk received at the Penn State Animal Diagnostic Laboratory. The results were compared with 16S rRNA gene sequence analysis, which served as the reference method for species identification. The ARIS, API, and MALDI identified 0%, 40%, and 33.4% of species classified as Gram-positive rod isolates belonging to genera Arthrobacter, Bacillus, Brachybacterium, Brevibacterium, and Corynebacterium, respectively. It was observed that 76.5%, 93.9%, and 96.9% of catalase-negative, Gram-positive cocci (n = 33; Aerococcus, Enterococcus, Lactococcus, Streptococcus) were correctly identified to the species level by ARIS, API, and MALDI, respectively, while 33.4%, 84.5%, and 97.7% of catalase-positive, Gram-positive cocci (n = 45; Kocuria, Staphylococcus) were correctly identified to their species by ARIS, API, and MALDI, respectively. A total of 48 isolates (Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Pantoea, Pasteurella, Providencia, Pseduomonas, Serratia) of Gram-negative bacteria were examined, of which 85.4%, 93.7%, and 95.8% of the isolates were correctly identified to the species level by ARIS, API, and MALDI, respectively. In our laboratory, the MALDI had the least costs associated with consumables and reagents compared to ARIS, API, and 16S rRNA identification methods. Identification of bacterial species was accomplished in <2 h using MALDI and 24 h for ARIS, API, and 16S rRNA identification systems.

Whole genome sequencing of diverse Shiga toxin-producing and non-producing Escherichia coli strains reveals a variety of virulence and novel antibiotic resistance plasmids.

The genomes of a diverse set of Escherichia coli, including many Shiga toxin-producing strains of various serotypes were determined. A total of 39 plasmids were identified among these strains, and many carried virulence or putative virulence genes of Shiga toxin-producing E. coli strains, virulence genes for other pathogenic E. coli groups, and some had combinations of these genes. Among the novel plasmids identified were eight that carried resistance genes to aminoglycosides, carbapenems, penicillins, cephalosporins, chloramphenicol, dihydrofolate reductase inhibitors, sulfonamides, tetracyclines and resistance to heavy metals. Two of the plasmids carried six of these resistance genes and two novel IncHI2 plasmids were also identified. The results of this study showed that plasmids carrying diverse resistance and virulence genes of various pathogenic E. coli groups can be found in E. coli strains and serotypes regardless of the isolate's source and therefore, is consistent with the premise that these mobile elements carrying these traits may be broadly disseminated among E. coli.

Longitudinal Monitoring of Successive Commercial Layer Flocks for Salmonella enterica Serovar Enteritidis.

The Pennsylvania Egg Quality Assurance Program (EQAP) provided the framework for Salmonella Enteritidis (SE) control programs, including the Food and Drug Administration (FDA) mandated Final Egg Rule, for commercial layer facilities throughout the United States. Although flocks with ≥3000 birds must comply with the FDA Final Egg Rule, smaller flocks are exempted from the rule. As a result, eggs produced by small layer flocks may pose a greater public health risk than those from larger flocks. It is also unknown if the EQAPs developed with large flocks in mind are suitable for small- and medium-sized flocks. Therefore, a study was performed to evaluate the effectiveness of best management practices included in EQAPs in reducing SE contamination of small- and medium-sized flocks by longitudinal monitoring of their environment and eggs. A total of 59 medium-sized (3000 to 50,000 birds) and small-sized (<3000 birds) flocks from two major layer production states of the United States were enrolled and monitored for SE by culturing different types of environmental samples and shell eggs for two consecutive flock cycles. Isolated SE was characterized by phage typing, pulsed-field gel electrophoresis (PFGE), and clustered regularly interspaced short palindromic repeats-multi-virulence-locus sequence typing (CRISPR-MVLST). Fifty-four Salmonella isolates belonging to 17 serovars, 22 of which were SE, were isolated from multiple sample types. Typing revealed that SE isolates belonged to three phage types (PTs), three PFGE fingerprint patterns, and three CRISPR-MVLST SE Sequence Types (ESTs). The PT8 and JEGX01.0004 PFGE pattern, the most predominant SE types associated with foodborne illness in the United States, were represented by a majority (91%) of SE. Of the three ESTs observed, 85% SE were typed as EST4. The proportion of SE-positive hen house environment during flock cycle 2 was significantly less than the flock cycle 1, demonstrating that current EQAP practices were effective in reducing SE contamination of medium and small layer flocks.

A novel two-component signaling system facilitates uropathogenic Escherichia coli's ability to exploit abundant host metabolites.

Two-component signaling systems (TCSs) are major mechanisms by which bacteria adapt to environmental conditions. It follows then that TCSs would play important roles in the adaptation of pathogenic bacteria to host environments. However, no pathogen-associated TCS has been identified in uropathogenic Escherichia coli (UPEC). Here, we identified a novel TCS, which we termed KguS/KguR (KguS: α-ketoglutarate utilization sensor; KguR: α-ketoglutarate utilization regulator) in UPEC CFT073, a strain isolated from human pyelonephritis. kguS/kguR was strongly associated with UPEC but was found only rarely among other E. coli including commensal and intestinal pathogenic strains. An in vivo competition assay in a mouse UTI model showed that deletion of kguS/kguR in UPEC CFT073 resulted in a significant reduction in its colonization of the bladders and kidneys of mice, suggesting that KguS/KguR contributed to UPEC fitness in vivo. Comparative proteomics identified the target gene products of KguS/KguR, and sequence analysis showed that TCS KguS/KguR and its targeted-genes, c5032 to c5039, are encoded on a genomic island, which is not present in intestinal pathogenic E. coli. Expression of the target genes was induced by α-ketoglutarate (α-KG). These genes were further shown to be involved in utilization of α-KG as a sole carbon source under anaerobic conditions. KguS/KguR contributed to the regulation of the target genes with the direct regulation by KguR verified using an electrophoretic mobility shift assay. In addition, oxygen deficiency positively modulated expression of kguS/kguR and its target genes. Taken altogether, this study describes the first UPEC-associated TCS that functions in controlling the utilization of α-ketoglutarate in vivo thereby facilitating UPEC adaptation to life inside the urinary tract.

Risk Factors Associated With Salmonella in Laying Hen Farms: Systematic Review of Observational Studies.

Salmonella contamination of laying hen flocks and shell eggs is associated with various management and environmental factors. Foodborne outbreaks of human salmonellosis have been traced back to consumption of Salmonella-contaminated shell eggs. In the present study, a systematic literature review was conducted to identify and provide an evidence-based overview of potential risk factors of Salmonella contamination of laying hens, layer premises, and shell eggs. This systematic literature search was conducted using AGRICOLA, CAB Abstracts, and PubMed databases. Observational studies that identified risk factors for Salmonella contamination of layer flocks and shell eggs were selected, and best evidence was synthesized to summarize the results. Altogether, 13 cross-sectional studies and four longitudinal studies published in English were included in the review. Evidence scores were assigned based on the study design and quality of the study to grade the evidence level. The strength of association of a risk factor was determined according to the odds ratios. In this systematic review, the presence of previous Salmonella infection, absence of cleaning and disinfection, presence of rodents, induced molting, larger flock size (>30,000 hens), multiage management, cage housing systems, in-line egg processing, rearing pullets on the floor, pests with access to feed prior to movement to the feed trough, visitors allowed in the layer houses, and trucks near farms and air inlets were identified as the risk factors associated with Salmonella contamination of laying hen premises, whereas high level of manure contamination, middle and late phase of production, high degree of egg-handling equipment contamination, flock size of >30,000, and egg production rate of >96% were identified as the risk factors associated with Salmonella contamination of shell eggs. These risk factors demonstrated strong to moderate evidence of association with Salmonella contamination of laying hens and shell eggs. Eggshells testing positive for Salmonella were 59 times higher when fecal samples were positive and nine times higher when floor dust samples were positive. Risk factors associated with Salmonella Enteritidis infection in laying hens were flock size, housing system, and farms with hens of different ages. As a summary, this systematic review demonstrated that Salmonella contamination of laying hen flocks and shell eggs in layer production systems is multifactorial. This study provides a knowledge base for the implementation of targeted intervention strategies to control Salmonella contamination of laying hen flocks and shell eggs.

Complete nucleotide sequence of pRS218, a large virulence plasmid, that augments pathogenic potential of meningitis-associated Escherichia coli strain RS218.

BACKGROUND: Escherichia coli is the most predominant Gram-negative bacterial pathogen associated with neonatal meningitis. Previous studies indicated that the prototypic neonatal meningitis E. coli (NMEC) strain RS218 (O18:K1:H7) harbors one large plasmid. Objectives of the present study were to analyze the complete nucleotide sequence of this large plasmid (pRS218) and its contribution to NMEC pathogenesis using in vitro and in vivo models of neonatal meningitis. RESULTS: The plasmid is 114,231 bp in size, belongs to the incompatibility group FIB/IIA (IncFIB/IIA), and contains a genetic load region that encodes several virulence and fitness traits such as enterotoxicity, iron acquisition and copper tolerance. The nucleotide sequence of pRS218 showed a 41- 46% similarity to other neonatal meningitis-causing E. coli (NMEC) plasmids and remarkable nucleotide sequence similarity (up to 100%) to large virulence plasmids of E. coli associated with acute cystitis. Some genes located on pRS218 were overly represented by NMEC strains compared to fecal E. coli isolated from healthy individuals. The plasmid-cured strain was significantly attenuated relative to the RS218 wild-type strain as determined in vitro by invasion potential to human cerebral microvascular endothelial cells and in vivo by mortalities, histopathological lesions in the brain tissue, and bacterial recovery from the cerebrospinal fluid of infected rat pups. CONCLUSIONS: The pRS218 is an IncFIB/IIA plasmid which shares a remarkable nucleotide sequence similarity to large plasmids of E. coli associated with cystitis. Both in vitro and in vivo experiments indicated that pRS218 plays an important role in NMEC pathogenesis.

Interdigitated silver-polymer-based antibacterial surface system activated by oligodynamic iontophoresis - an empirical characterization study.

There is a pressing need to control the occurrences of nosocomial infections due to their detrimental effects on patient well-being and the rising treatment costs. To prevent the contact transmission of such infections via health-critical surfaces, a prophylactic surface system that consists of an interdigitated array of oppositely charged silver electrodes with polymer separations and utilizes oligodynamic iontophoresis has been recently developed. This paper presents a systematic study that empirically characterizes the effects of the surface system parameters on its antibacterial efficacy, and validates the system's effectiveness. In the first part of the study, a fractional factorial design of experiments (DOE) was conducted to identify the statistically significant system parameters. The data were used to develop a first-order response surface model to predict the system's antibacterial efficacy based on the input parameters. In the second part of the study, the effectiveness of the surface system was validated by evaluating it against four bacterial species responsible for several nosocomial infections - Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis - alongside non-antibacterial polymer (acrylic) control surfaces. The system demonstrated statistically significant efficacy against all four bacteria. The results indicate that given a constant total effective surface area, the system designed with micro-scale features (minimum feature width: 20 µm) and activated by 15 µA direct current will provide the most effective antibacterial prophylaxis.

An experimental infection model for Escherichia coli egg peritonitis in layer chickens.

The present study describes an experimental infection model for avian pathogenic Escherichia coli (APEC)-induced egg peritonitis in layer chickens. First, a pilot study which consisted of two separate experiments was carried out to compare two routes of inoculations of APEC to induce peritonitis and to examine if the presence of egg yolk in the peritoneum would facilitate APEC-induced peritonitis. This study showed that the presence of egg yolk in the peritoneum facilitated the development of egg peritonitis when the APEC was inoculated via the intra-uterine (IU) route. Based on the results of the pilot study, 56-wk-old white leghorn hens were divided into two groups of five chickens, Group G (inoculated with E. coli APECO78 strain) and Group H (control). Both groups were inoculated with 2-3 ml of egg yolk via the intraperitoneal route (IP). Subsequently, hens in Group H were inoculated with only egg yolk whereas the hens in Group G were inoculated with 1 x 10(9) colony-forming units of APECO78 bacteria via the IU route. Parameters such as mortality, clinical signs (anorexia, depression, and egg production efficiency), gross lesion scores, bacterial loads in internal organs, and histopathology of ovary and oviduct were assessed to evaluate the success of the infection model. Group G showed 40% acute mortality, severe depression, and anorexia with markedly reduced egg production and developed peritonitis-associated lesions such as accumulation of yellowish caseous fluid in the peritoneum, salpingitis, and oophoritis. Histopathologically, ovarian and oviduct tissues from group G exhibited severe inflammatory changes such as infiltration of mononuclear cells and edema. Group G also showed significant bacterial loads in the peritoneum, ovary, and oviduct. Interestingly, deceased birds from group G had also developed mild perihepatitis and pericarditis with heavy bacterial loads in the internal organs. On the other hand, group H birds did not exhibit any of the clinical signs and remained healthy until the end of the experiment. To summarize, our results demonstrate that IP administration of egg yolk followed by IU inoculation of APECO78 induced peritonitis in laying hens. Experimental infection models are often required to understand the mechanisms of disease pathogenesis. Therefore, the present infection model will aid in the studies of pathogenesis of layer peritonitis caused by APEC and in evaluating vaccine candidates to control the disease.

Acute septicemia caused by Streptococcus gallolyticus subsp. pasteurianus in turkey poults.

Streptococcus gallolyticus, previously known as Streptococcus bovis biotypes I and II/2, is a well-known cause of sepsis and meningitis in humans and birds. The present case report describes an outbreak of fatal septicemia associated with S. gallolyticus subsp. pasteurianus (S. bovis biotype II/2) in 11 turkey flocks in Pennsylvania between 2010 and 2013. Affected poults were 2-3 wk of age. Major clinical observation was sudden increase in mortality among turkey poults without any premonitory clinical signs. Postmortem examination findings revealed acute septicemia with lesions such as fibrinous pericarditis, meningitis, splenic multifocal fibrinoid necrosis, hepatitis, osteochondritis, myositis, and airsacculitis. Gram-positive cocci were isolated from several organs by routine bacterial culture. Biotyping identified bacteria as streptococci, whereas 16S ribosomal RNA gene sequencing identified them as S. gallolyticus subsp. pasteurianus. Antibiotic susceptibility profiles revealed that all the strains isolated were sensitive to penicillin and erythromycin with different sensitivity profiles for other antibacterial agents tested. The present study reports the first confirmed case of acute septicemia in turkey poults caused by S. gallolyticus subsp. pasteurianus.

Host-Pathogen Interactions during Shiga Toxin-Producing Escherichia coli Adherence and Colonization in the Bovine Gut: A Comprehensive Review.

Shiga toxin-producing Escherichia coli (STEC) is a significant public health threat due to its ability to cause severe gastrointestinal diseases in humans, ranging from diarrhea to life-threatening conditions such as hemorrhagic colitis and hemolytic uremic syndrome (HUS). As the primary reservoir of STEC, cattle play a crucial role in its transmission through contaminated food and water, posing a considerable risk to human health. This comprehensive review explores host-pathogen interactions during STEC colonization of the bovine gut, focusing on the role of gut microbiota in modulating these interactions and influencing disease outcomes. We integrated findings from published transcriptomics, proteomics, and genomics studies to provide a thorough understanding of how STEC adheres to and colonizes the bovine gastrointestinal tract. The insights from this review offer potential avenues for the development of novel preventative and therapeutic strategies aimed at controlling STEC colonization in cattle, thereby reducing the risk of zoonotic transmission.

Interferon-γ/IL-2 ELISpot and mRNA Responses to the SARS-CoV2, Feline Coronavirus Serotypes 1 (FCoV1), and FCoV2 Receptor Binding Domains by the T Cells from COVID-19-Vaccinated Humans and FCoV1-Infected Cats.

The receptor binding domain (RBD) of SARS-CoV-2 (SCoV2) has been used recently to identify the RBD sequences of feline coronavirus serotypes 1 (FCoV1) and 2 (FCoV2). Cats naturally infected with FCoV1 have been shown to possess serum reactivities with FCoV1 and SCoV2 RBDs but not with FCoV2 RBD. In the current study, COVID-19-vaccinated humans and FCoV1-infected laboratory cats were evaluated for interferon-gamma (IFNγ) and interleukin-2 (IL-2 ELISpot responses by their peripheral blood mononuclear cells (PBMC) to SCoV2, FCoV1, and FCoV2 RBDs. Remarkably, the PBMC from COVID-19-vaccinated subjects developed IFNγ responses to SCoV2, FCoV1, and FCoV2 RBDs. The most vaccinated subject (five vaccinations over 2 years) appeared to produce hyperreactive IFNγ responses to all three RBDs, including the PBS media control. This subject lost IFNγ responses to all RBDs at 9 months (9 mo) post-last vaccination. However, her IL-2 responses to FCoV1 and FCoV2 RBDs were low but detectable at 10 mo post-last vaccination. This observation suggests that initially robust IFNγ responses to SCoV2 RBD may be an outcome of robust inflammatory IFNγ responses to SCoV2 RBD. Hence, the T-cell responses of vaccine immunity should be monitored by vaccine immunogen-specific IL-2 production. The PBMC from chronically FCoV1-infected cats developed robust IFNγ responses to SCoV2 and FCoV2 RBDs but had the lowest IFNγ responses to FCoV1 RBD. The constant exposure to FCoV1 reinfection may cause the IFNγ responses to be downregulated to the infecting virus FCoV1 but not to the cross-reacting epitopes on the SCoV2 and FCoV2 RBDs.

Pangenomic and biochemical analyses of Helcococcus ovis reveal widespread tetracycline resistance and a novel bacterial species, Helcococcus bovis.

Helcococcus ovis (H. ovis) is an opportunistic bacterial pathogen of a wide range of animal hosts including domestic ruminants, swine, avians, and humans. In this study, we sequenced the genomes of 35 Helcococcus sp. clinical isolates from the uterus of dairy cows and explored their antimicrobial resistance and biochemical phenotypes in vitro. Phylogenetic and average nucleotide identity analyses classified four Helcococcus isolates within a cryptic clade representing an undescribed species, for which we propose the name Helcococcus bovis sp. nov. By establishing this new species clade, we also resolve the longstanding question of the classification of the Tongji strain responsible for a confirmed human conjunctival infection. This strain did not neatly fit into H. ovis and is instead a member of H. bovis. We applied whole genome comparative analyses to explore the pangenome, resistome, virulome, and taxonomic diversity of the remaining 31 H. ovis isolates. An overwhelming 97% of H. ovis strains (30 out of 31) harbor mobile tetracycline resistance genes and displayed significantly increased minimum inhibitory concentrations of tetracyclines in vitro. The high prevalence of mobile tetracycline resistance genes makes H. ovis a significant antimicrobial resistance gene reservoir in our food chain. Finally, the phylogenetic distribution of co-occurring high-virulence determinant genes of H. ovis across unlinked and distant loci highlights an instance of convergent gene loss in the species. In summary, this study showed that mobile genetic element-mediated tetracycline resistance is widespread in H. ovis, and that there is evidence of co-occurring virulence factors across clades suggesting convergent gene loss in the species. Finally, we introduced a novel Helcococcus species closely related to H. ovis, called H. bovis sp. nov., which has been reported to cause infection in humans.

Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Associates with CRISPR Sequence Type.

Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of food-borne salmonellosis in the United States. The number of antibiotic-resistant isolates identified in humans is steadily increasing, suggesting that the spread of antibiotic-resistant strains is a major threat to public health. S Typhimurium is commonly identified in a wide range of animal hosts, food sources, and environments, but little is known about the factors mediating the spread of antibiotic resistance in this ecologically complex serovar. Previously, we developed a subtyping method, CRISPR-multi-virulence-locus sequence typing (MVLST), which discriminates among strains of several common S. enterica serovars. Here, CRISPR-MVLST identified 22 sequence types within a collection of 76 S Typhimurium isolates from a variety of animal sources throughout central Pennsylvania. Six of the sequence types were identified in more than one isolate, and we observed statistically significant differences in resistance among these sequence types to 7 antibiotics commonly used in veterinary and human medicine, such as ceftiofur and ampicillin (P < 0.05). Importantly, five of these sequence types were subsequently identified in human clinical isolates, and a subset of these isolates had identical antibiotic resistance patterns, suggesting that these subpopulations are being transmitted through the food system. Therefore, CRISPR-MVLST is a promising subtyping method for monitoring the farm-to-fork spread of antibiotic resistance in S Typhimurium.

Comparative Transcriptome Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 on Bovine Rectoanal Junction Cells and Human Colonic Epithelial Cells during Initial Adherence.

Shiga toxin-producing Escherichia coli (STEC) are notorious foodborne pathogens, capable of causing severe diarrhea and life-threatening complications in humans. Cattle, acting as both primary reservoirs and asymptomatic carriers of STEC, predominantly harbor the pathogen in their rectoanal junction (RAJ), facilitating its transmission to humans through contaminated food sources. Despite the central role of cattle in STEC transmission, the molecular mechanisms governing STEC's adaptation in the RAJ of the asymptomatic reservoir host and its subsequent infection of human colonic epithelial cells, resulting in diarrhea, remain largely unexplored. This study aims to uncover these complicated dynamics by focusing on the STEC O157:H7 serotype within two distinct host environments, bovine RAJ cells and human colonic epithelial cells, during initial colonization. We employed comparative transcriptomics analysis to investigate differential gene expression profiles of STEC O157:H7 during interactions with these cell types. STEC O157:H7 was cultured either with bovine RAJ cells or the human colonic epithelial cell line CCD CoN 841 to simulate STEC-epithelial cell interactions within these two host species. High-throughput RNA sequencing revealed 829 and 1939 bacterial genes expressed in RAJ and CCD CoN 841, respectively. After gene filtering, 221 E. coli O157:H7 genes were upregulated during initial adherence to CCD CoN cells and 436 with RAJ cells. Furthermore, 22 genes were uniquely expressed with human cells and 155 genes with bovine cells. Our findings revealed distinct expression patterns of STEC O157:H7 genes involved in virulence, including adherence, metal iron homeostasis, and stress response during its initial adherence (i.e., six hours post-infection) to bovine RAJ cells, as opposed to human colonic epithelial cells. Additionally, the comparative analysis highlighted the potential role of some genes in host adaptation and tissue-specific pathogenicity. These findings shed new light on the potential mechanisms of STEC O157:H7 contributing to colonize the intestinal epithelium during the first six hours of infection, leading to survival and persistence in the bovine reservoir and causing disease in humans.

Identification, 16S rRNA-based characterization, and antimicrobial profile of Gallibacterium isolates from broiler and layer chickens.

Gallibacterium spp., particularly G. anatis, have received much attention as poultry pathogens in recent years. We report here the presence and antimicrobial resistance profile of 69 Gallibacterium isolates obtained from 2,204 diagnostic submissions of broiler and layer chickens in 2019-2021. Gallibacterium-positive chickens had lesions primarily in the respiratory tract, reproductive tract, and related serosal surfaces. Gallibacterium spp. were initially identified based on their typical cultural characteristics on blood agar. The isolates were confirmed by a genus-specific PCR spanning 16S-23S rRNA and MALDI-TOF mass spectrometry. Phylogenetic analysis based on 16S rRNA gene sequence revealed distinct clades. Of the 69 isolates, 68 clustered with the reference strains of G. anatis and 1 with Gallibacterium genomospecies 1 and 2. Antimicrobial susceptibility testing of 58 of the 69 isolates by a MIC method showed variable responses to antimicrobials. The isolates were all susceptible to enrofloxacin, ceftiofur, florfenicol, and gentamicin. There was a high level of susceptibility to trimethoprim-sulfamethoxazole (98.0%), streptomycin (98.0%), amoxicillin (84.0%), sulfadimethoxine (71.0%), and neomycin (71.0%). All of the isolates were resistant to tylosin. There was resistance to penicillin (98.0%), erythromycin (95.0%), clindamycin (94.0%), novobiocin (90.0%), tetracycline (88.0%), oxytetracycline (76.0%), and sulfathiazole (53.0%). A high rate of intermediate susceptibility was observed for spectinomycin (67.0%) and sulfathiazole (40.0%). Our findings indicate a potential role of G. anatis as an important poultry pathogen and cause of subsequent disease, alone or in combination with other pathogens. Continuous monitoring and an antimicrobial susceptibility assay are recommended for effective treatment and disease control.

The infected and the affected: A longitudinal study of the impact of the COVID-19 pandemic on schoolchildren in Florida.

Objectives: To identify risk factors associated with symptoms of anxiety, depression, and obsessive-compulsive disorder (OCD) among children during the 1st year of the COVID-19 pandemic. Methods: A longitudinal study with three cross-sectional timepoints [April 2020 (n = 273), October 2020 (n = 180), and April 2021 (n = 116)] was conducted at a K-12 public school in Florida. Infection and sero-positivity for SARS-CoV-2 was determined by molecular and serologic approaches. Adjusted odds ratios using mixed effect logistic regression models for symptom-derived indicators of anxiety, depression, and OCD in children in April 2021 are presented; past infection and seropositivity were included in the models. Results: The prevalence of anxiety, depression, or OCD moved from 47.1, to 57.2, to 42.2% across the three timepoints during the study. By endline of the study, in April 2021, non-white children were at higher risk for depression and OCD. Risk for anxiety, depression, and OCD was associated with students who lost a family member due to COVID-19 and who were identified as at-risk in previous timepoints. Rates of SARS-CoV-2 infection and seropositivity were low and not statistically associated with assessed outcomes. Conclusions: In situations like the COVID-19 pandemic, targeted mental health interventions and screenings are needed in children and adolescents, especially among minority children.

Both Feline Coronavirus Serotypes 1 and 2 Infected Domestic Cats Develop Cross-Reactive Antibodies to SARS-CoV-2 Receptor Binding Domain: Its Implication to Pan-CoV Vaccine Development.

The current study was initiated when our specific-pathogen-free laboratory toms developed unexpectedly high levels of cross-reactive antibodies to human SARS-CoV-2 (SCoV2) receptor binding domain (RBD) upon mating with feline coronavirus (FCoV)-positive queens. Multi-sequence alignment analyses of SCoV2 Wuhan RBD and four strains each from FCoV serotypes 1 and 2 (FCoV1 and FCoV2) demonstrated an amino acid sequence identity of 11.5% and a similarity of 31.8% with FCoV1 RBD (12.2% identity and 36.5% similarity for FCoV2 RBD). The sera from toms and queens cross-reacted with SCoV2 RBD and reacted with FCoV1 RBD and FCoV2 spike-2, nucleocapsid, and membrane proteins, but not with FCoV2 RBD. Thus, the queens and toms were infected with FCoV1. Additionally, the plasma from six FCoV2-inoculated cats reacted with FCoV2 and SCoV2 RBDs, but not with FCoV1 RBD. Hence, the sera from both FCoV1-infected cats and FCoV2-infected cats developed cross-reactive antibodies to SCoV2 RBD. Furthermore, eight group-housed laboratory cats had a range of serum cross-reactivity to SCoV2 RBD even 15 months later. Such cross-reactivity was also observed in FCoV1-positive group-housed pet cats. The SCoV2 RBD at a high non-toxic dose and FCoV2 RBD at a 60-400-fold lower dose blocked the in vitro FCoV2 infection, demonstrating their close structural conformations essential as vaccine immunogens. Remarkably, such cross-reactivity was also detected by the peripheral blood mononuclear cells of FCoV1-infected cats. The broad cross-reactivity between human and feline RBDs provides essential insights into developing a pan-CoV vaccine.

Prevalence of avian-pathogenic Escherichia coli strain O1 genomic islands among extraintestinal and commensal E. coli isolates.

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include pathogens of humans and animals. Previously, the genome of avian-pathogenic E. coli (APEC) O1:K1:H7 strain O1, from ST95, was sequenced and compared to those of several other E. coli strains, identifying 43 genomic islands. Here, the genomic islands of APEC O1 were compared to those of other sequenced E. coli strains, and the distribution of 81 genes belonging to 12 APEC O1 genomic islands among 828 human and avian ExPEC and commensal E. coli isolates was determined. Multiple islands were highly prevalent among isolates belonging to the O1 and O18 serogroups within phylogenetic group B2, which are implicated in human neonatal meningitis. Because of the extensive genomic similarities between APEC O1 and other human ExPEC strains belonging to the ST95 phylogenetic lineage, its ability to cause disease in a rat model of sepsis and meningitis was assessed. Unlike other ST95 lineage strains, APEC O1 was unable to cause bacteremia or meningitis in the neonatal rat model and was significantly less virulent than uropathogenic E. coli (UPEC) CFT073 in a mouse sepsis model, despite carrying multiple neonatal meningitis E. coli (NMEC) virulence factors and belonging to the ST95 phylogenetic lineage. These results suggest that host adaptation or genome modifications have occurred either in APEC O1 or in highly virulent ExPEC isolates, resulting in differences in pathogenicity. Overall, the genomic islands examined provide targets for further discrimination of the different ExPEC subpathotypes, serogroups, phylogenetic types, and sequence types.