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.

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.

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.

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.

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.

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.

Identification of a novel Neorickettsia species in a Kemp's ridley sea turtle with granulomatous nephritis and development of a quantitative PCR assay.

An adult male Kemp's ridley turtle was found dead on the coast of Kenedy County, Texas, in August 2019 with bilateral severe, diffuse granulomatous nephritis. Pan-bacterial 16S rRNA gene polymerase chain reaction (PCR) and amplicon sequencing of affected tissue indicated the presence of a Neorickettsia. Neorickettsia is a genus of obligate intracellular Alphaproteobacteria that are transmitted by digenean trematodes. For further characterization, primers were designed to amplify and sequence the groEL gene. Phylogenetic analysis found that the organism was distinct from other known species to a degree consistent with a novel species. Immunohistochemistry using an antibody directed against a Neorickettsia surface protein showed bacterial clusters within the renal granulomas. A species-specific quantitative PCR was designed and detected the organism within the liver and colon of the index case. A quantitative PCR survey of grossly normal kidneys opportunistically collected from additional stranded sea turtle kidneys detected this organism in five of 15 Kemp's ridley turtles, two of nine green turtles, and neither of two loggerhead turtles. Recognition of this novel organism in an endangered species is concerning; additional work is underway to further characterize the potential of this organism as a pathogen of sea turtles.

Transferability of ESBL-encoding IncN and IncI1 plasmids among field strains of different Salmonella serovars and Escherichia coli.

OBJECTIVES: This study aimed to sequence, assemble, and annotate three plasmids (two IncN and one IncI1) carrying the blaCTX-M-1 gene and assess their transferability rates between homologous and heterologous serovars and/or species of bacteria. METHODS: First, the plasmids were sequenced, assembled, and annotated. They were then transferred from three donor strains (Escherichia coli/IncN, S. Heidelberg/IncN, and S. Heidelberg/IncI1) into nine recipient strains (S. Enteritidis, S. Heidelberg, S. Saintpaul, S. Cero, S. Infantis, S. Braenderup, E. coli 50, and E. coli 2010). The blaCTX-M-1 gene polymerase chain reaction (PCR), plasmid isolation, and antimicrobial susceptibility testing were used on the transconjugants to confirm the successful transfer of extended-spectrum beta lactamase (EBSL) plasmids into the recipient strains. RESULTS: Both IncN plasmids were 42,407 bp in size and showed >99.4% similarity to the S. Bredeney pET1.2-IncN (GenBank accession CP043224.1), whereas the IncI1 plasmid was 107,635 bp in size and demonstrated >99.9% similarity to the E. coli pCOV33 plasmid (GenBank accession MG649046.1). Successful plasmid transfer was observed between donor ​E. coli (IncN) and all recipient strains except for E. coli 50 and between donor S. Heidelberg (IncN) and all recipient strains. Successful plasmid transfer was also observed between S. Heidelberg (IncI1) and E. coli 50. CONCLUSION: Transfer of the blaCTX-M-1 encoding IncN and IncI1 plasmids via conjugation is possible and yet occurs at different frequencies depending on the donor strain of bacteria, with S. Heidelberg (IncN) having the highest donor-dependent transfer frequency, followed by E. coli 9079 (IncN) and S. Heidelberg (IncI1).

Lactococcus lactis Delivery of Surface Layer Protein A Protects Mice from Colitis by Re-Setting Host Immune Repertoire.

Inflammatory bowel disease (IBD) is characterized by gastrointestinal inflammation comprised of Crohn's disease and ulcerative colitis. Centers for Disease Control and Prevention report that 1.3% of the population of the United States (approximately 3 million people) were affected by the disease in 2015, and the number keeps increasing over time. IBD has a multifactorial etiology, from genetic to environmental factors. Most of the IBD treatments revolve around disease management, by reducing the inflammatory signals. We previously identified the surface layer protein A (SlpA) of Lactobacillus acidophilus that possesses anti-inflammatory properties to mitigate murine colitis. Herein, we expressed SlpA in a clinically relevant, food-grade Lactococcus lactis to further investigate and characterize the protective mechanisms of the actions of SlpA. Oral administration of SlpA-expressing L. lactis (R110) mitigated the symptoms of murine colitis. Oral delivery of R110 resulted in a higher expression of IL-27 by myeloid cells, with a synchronous increase in IL-10 and cMAF in T cells. Consistent with murine studies, human dendritic cells exposed to R110 showed exquisite differential gene regulation, including IL-27 transcription, suggesting a shared mechanism between the two species, hence positioning R110 as potentially effective at treating colitis in humans.

Complete Genome Sequence of Campylobacter hepaticus Strain UF2019SK1, Isolated from a Commercial Layer Flock in the United States.

The thermophilic Campylobacter species Campylobacter hepaticus is the causative agent of spotty liver disease (SLD) in chickens. This announcement describes the complete genome sequence of C. hepaticus strain UF2019SK1, isolated from the liver of a commercial layer chicken with SLD in the United States.

Prevalence and distribution of multilocus sequence types of Staphylococcus aureus isolated from bulk tank milk and cows with mastitis in Pennsylvania.

A total of 163 S. aureus isolates; 113 from mastitic milk (MM) and 50 from bulk tank milk (BTM) (2008, 2013-2015) submitted for bacteriologic analysis at the Penn State Animal Diagnostic Laboratory were examined for their phenotypic and genotypic characteristics. Multi-locus sequence typing (MLST) analysis identified 16 unique sequence types (STs) which belonged to eight clonal complexes (CCs). Majority of the isolates were variants of CC97 (68.7%) and CC151 (25.1%). CC97 comprised of seven STs, of which two were new STs (ST3273, ST3274), while CC151 comprised of three STs of which ST3272 was identified for the first time. Several farms had more than one ST type that were either members of the same clonal complex or unrelated STs. On one farm, six different STs of both categories were seen over the years within the farm. It was observed that ST352 and ST151 were the two main clonal populations in cattle not only in Pennsylvania but also globally. Most isolates were susceptible to all the antibiotics evaluated. 6.7% of isolates showed resistance to vancomycin and penicillin. Two isolates of ST398 showed multidrug resistance (>3 antibiotics) against clindamycin, erythromycin, tetracycline, and penicillin. It was noted that 59 of 163 (36.2%) isolates encoded for enterotoxigenic genes. Enterotoxin genes seg/sei accounted for ~85% of enterotoxin positive isolates. Toxic shock syndrome gene tsst-1 alone was positive in two isolates (ST352, ST 2187). 97.5% of CC151 isolates were enterotoxin seg/sei positive. Most isolates were positive for lukED (95%) and lukAB (96.3%) leukotoxin genes. Bovine specific bi-component leucocidin lukMF' was present in 54% of isolates. A prominent observation of this study was the explicit association of lukMF' with lineages ST151 and ST352. In conclusion, the findings of the study, suggest that small number of S. aureus STs types (ST352, ST2187, ST3028, and ST151) are associated with majority of cases of bovine mastitis in Pennsylvania dairy farms. It was observed that one ST of S. aureus predominated in the herd and this ST can coexist with several other ST types of S. aureus strains. When STs were interpreted along with virulence, leucocidin genes and antimicrobial resistance, ST-variants allowed better interpretation of the S. aureus molecular epidemiologic findings specifically for tracing recurrence or persistence of infections in cow over time, among cows in the herd, and between herds in Pennsylvania.

Comparison of Antimicrobial Resistance and Pan-Genome of Clinical and Non-Clinical Enterococcus cecorum from Poultry Using Whole-Genome Sequencing.

Enterococcus cecorum is an emerging avian pathogen, particularly in chickens, but can be found in both diseased (clinical) and healthy (non-clinical) poultry. To better define differences between E. cecorum from the two groups, whole-genome sequencing (WGS) was used to identify and compare antimicrobial resistance genes as well as the pan-genome among the isolates. Eighteen strains selected from our previous study were subjected to WGS using Illumina MiSeq and comparatively analyzed. Assembled contigs were analyzed for resistance genes using ARG-ANNOT. Resistance to erythromycin was mediated by ermB, ermG, and mefA, in clinical isolates and ermB and mefA, in non-clinical isolates. Lincomycin resistance genes were identified as linB, lnuB, lnuC, and lnuD with lnuD found only in non-clinical E. cecorum; however, lnuB and linB were found in only one clinical isolate. For both groups of isolates, kanamycin resistance was mediated by aph3-III, while tetracycline resistance was conferred by tetM, tetO, and tetL. No mutations or known resistance genes were found for isolates resistant to either linezolid or chloramphenicol, suggesting possible new mechanisms of resistance to these drugs. A comparison of WGS results confirmed that non-clinical isolates contained more resistance genes than clinical isolates. The pan-genome of clinical and non-clinical isolates resulted in 3651 and 4950 gene families, respectively, whereas the core gene sets were comprised of 1559 and 1534 gene families in clinical and non-clinical isolates, respectively. Unique genes were found more frequently in non-clinical isolates than clinical. Phylogenetic analysis of the isolates and all the available complete and draft genomes showed no correlation between healthy and diseased poultry. Additional genomic comparison is required to elucidate genetic factors in E. cecorum that contribute to disease in poultry.

Detection of CTX-M-1 extended-spectrum beta-lactamase among ceftiofur-resistant Salmonella enterica clinical isolates of poultry.

Salmonella enterica resistance to extended-spectrum cephalosporins (ESC) conferred by cefotaximases (blaCTX-M) is a growing concern in the United States. Among food-producing animals, poultry are a major reservoir of ESC-resistant Salmonella. A retrospective study was carried out to further characterize 38 ceftiofur-resistant clinical Salmonella enterica isolates obtained from poultry during 2007-2018. Of the isolates tested, 31 displayed resistance to ceftriaxone and harbored blaCMY-2, whereas 7 isolates demonstrated resistance or reduced susceptibility to cefepime in addition to ceftriaxone resistance. These 7 isolates displayed extended-spectrum ß-lactamase activity, harbored blaCTX-M-1, and were recovered only from recent poultry diagnostic submissions made in 2011-2018 as opposed to the 31 isolates that were recovered in 2007-2018. Further characterization of the blaCTX-M-1 gene determined that it was located on conjugative IncN/ST1 and IncI1/ST87 plasmids in the isolates from commercial turkeys and broilers, respectively. These plasmids have been responsible for extensive spread of blaCTX-M-1 in livestock, poultry, and humans in Europe. Potential transfer of IncN and IncI1 plasmids and/or nontyphoidal Salmonella carrying these plasmids through the food chain, or by other means to humans, may result in treatment failures. Our study demonstrates the importance of further characterization of ceftiofur-resistant S. enterica isolates detected by veterinary diagnostic laboratories to identify the sources of blaCTX-M-1 and to mitigate the spread of ESC-resistant Salmonella in the poultry production pyramid.

The Escherichia coli O157:H7 carbon starvation-inducible lipoprotein Slp contributes to initial adherence in vitro via the human polymeric immunoglobulin receptor.

Escherichia coli O157:H7 is the most well-studied serotype of the enterohemorrhagic E. coli (EHEC) class of E. coli intestinal pathogens and is responsible for many outbreaks of serious food-borne illness worldwide each year. Adherence mechanisms are a critical component of its pathogenesis, persistence in natural reservoirs, and environmental contamination. E. coli O157:H7 has a highly effective virulence operon, the Locus of Enterocyte Effacement (LEE), and its encoded intimate adherence mechanism is well characterized. However, factors involved in the preceding initial attachment are not well understood. In this study, we propose a mechanism of initial adherence used by E. coli O157:H7 in vitro. We describe a bacterial protein not previously reported to be involved in adherence, Slp, and its interactions with the human host protein polymeric immunoglobulin receptor (pIgR). The human pIgR has previously been shown to act as an adherence receptor for some mucosal pathogens and is highly expressed in the intestine. Following observation of significant colocalization between E. coli O157:H7 bacteria and pIgR location on Caco-2 cells, a co-immunoprecipitation (Co-IP) assay using a human recombinant Fc-tagged pIgR protein led to the identification of this protein. Disruption of Slp expression in E. coli O157:H7, through deletion of its encoding gene slp, produced a significant adherence deficiency to Caco-2 cells at early time points associated with initial adherence. Plasmid complementation of the slp gene fully restored the wild-type phenotype. Furthermore, immunofluorescence microscopy revealed evidence that this interaction is specific to the pathogenic strains of E. coli tested and not the nonpathogenic control strain E. coli K12. Additionally, deletion of slp gene resulted in the absence of the corresponding protein band in further Co-IP assays, while the plasmid-encoded slp gene complementation of the deletion mutant strain restored the wild-type pattern. These data support the proposal that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor.

Complete Genome Sequences of 20 Nontyphoidal Salmonella Isolates from Rwanda.

Nontyphoidal Salmonella enterica strains are major foodborne pathogens with global public health importance. Foodborne salmonellosis can be life-threatening, especially in sub-Saharan Africa. We report the complete genome sequences of 20 nontyphoidal Salmonella enterica strains isolated in Rwanda. The reported 20 bacterial chromosomes and 8 plasmids each belong to 1 of 9 nontyphoidal Salmonella serotypes.

Shigatoxigenic and atypical enteropathogenic Escherichia coli in fish for human consumption

ABSTRACT Shigatoxigenic and enteropathogenic Escherichia coli with virulence and multidrug resistance profile were isolated from Nile tilapia. This study finding is of great importance to public health because they help understand this pathogen epidemiology in fish and demonstrate how these animals can transmit E. coli related diseases to humans.

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.

Prevalence of Group I Salmonella Kentucky in domestic food animals from Pennsylvania and overlap with human clinical CRISPR sequence types.

Although infrequently associated with illness in humans, Salmonella enterica, subsp. enterica serovar Kentucky is the most common non-clinical, non-human serovar reported in the United States, being largely found in poultry and poultry products, as well as being associated with cattle. This serovar is polyphyletic and can be separated into two groups, Group I and II, based on CRISPR-typing analysis. In Salmonella Kentucky isolates from human clinical samples in Pennsylvania, both lineages are equally represented. The goal of this study was to determine whether both groups were also represented in domestic food animals in Pennsylvania. We analysed the CRISPR arrays from 67 Salmonella Kentucky isolates used PCR and sequencing of CRISPR arrays or analysis of whole genome sequences to analyse the CRISPR arrays and Across a collection of 67 Salmonella Kentucky isolates that includes those collected from farms, veterinary clinical samples as well as isolates from retail meats, we show that Group I Salmonella Kentucky are the exclusive lineage present. We reveal that the specific subtype of over a quarter of these animal isolates are also found to be responsible for causing human salmonellosis in the same region over the same time period.

Shigatoxigenic and atypical enteropathogenic Escherichia coli in fish for human consumption.

Shigatoxigenic and enteropathogenic Escherichia coli with virulence and multidrug resistance profile were isolated from Nile tilapia. This study finding is of great importance to public health because they help understand this pathogen epidemiology in fish and demonstrate how these animals can transmit E. coli related diseases to humans.

The efficacy of self-disinfecting bedrail covers in an intensive care unit.

BACKGROUND: Hospital surfaces are considered important vectors in the spread of nosocomial pathogens. This study evaluated microbial counts on novel antimicrobial bedrail covers over a 2-week period in a critical care environment. METHODS: Disposable bedrail covers (Aionx Inc, Hershey, PA) made of a copper and silver polymer and capable of conducting an imperceptible surface potential, were installed in a case-control manner on a series of occupied intensive care unit beds. Seventeen bedrails were covered with the study bedrail surface, and 17 were left uncovered. Two hundred seventy-two microbial surface cultures were obtained from both study and control bedrails and analyzed for microbial growth by bacterial enumeration and speciation. RESULTS: The bedrails covered with the study surface demonstrated >80% average decrease in colony forming units across the study period of 15 days. CONCLUSIONS: These novel, detachable bedrail covers successfully demonstrated significant bacterial count reduction in an intensive care unit setting. This may have implications for acquisition of hospital-acquired infections.