Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer.

IMPORTANCE: H. pylori infects half of the world population and is the leading cause of gastric cancer. We previously demonstrated that gastric cancer risk is associated with gastric microbiota. Specifically, gastric urease-positive Staphylococcus epidermidis and Streptococcus salivarius had contrasting effects on H. pylori-associated gastric pathology and immune responses in germ-free INS-GAS mice. As gastritis progresses to gastric cancer, the oncogenic transcription factor Foxm1 becomes increasingly expressed. In this study, we evaluated the gastric commensal C. acnes, certain strains of which produce thiopeptides that directly inhibit FOXM1. Thiopeptide-positive C. acnes was isolated from Nicaraguan patient gastric biopsies and inoculated into germ-free INS-GAS mice with H. pylori. We, therefore, asked whether coinfection with C. acnes expressing thiopeptide and H. pylori would decrease gastric Foxm1 expression and pro-inflammatory cytokine mRNA and protein levels. Our study supports the growing literature that specific non-H. pylori gastric bacteria affect inflammatory and cancer biomarkers in H. pylori pathogenesis.

Evaluation and comparison of pharmacokinetic profiles and safety of two extended-release buprenorphine formulations in common marmosets (Callithrix jacchus).

While sustained-release buprenorphine (BSR) is used as a long-lasting opioid analgesic in common marmosets (Callithrix jacchus), there are no published studies on pharmaceutical-grade extended-release buprenorphine options such as Ethiqa XR (EXR) for this species. However, BSR is a compounded product and has been reported to cause injection site reactions in multiple species, including marmosets. Additionally, now with the availability of EXR, a pharmaceutical-grade veterinary product, the use of BSR in laboratory animals is not compliant with the Guide for the Care and Use of Laboratory Animals (Guide) unless scientifically justified and approved by the IACUC. We compared pharmacokinetic and safety profiles of BSR (0.15 mg/kg) and EXR (0.1-0.2 mg/kg) administered subcutaneously to adult marmosets. Blood was collected by venipuncture of the saphenous vein at multiple time points (0.25-72 h) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). EXR between 0.1 and 0.2 mg/kg resulted in a dose-dependent increase in Cmax (1.43-2.51 ng/mL) and were not statistically different from BSR (1.82 ng/mL). Tmax, lambdaz, and t1/2 were not statistically different between formulations. Mean plasma buprenorphine concentrations for BSR and EXR exceeded the therapeutic threshold (0.1 ng/mL) within 0.25 h and lasted for > 72 h. Mild sedation, but neither respiratory depression nor ataxia, was observed for both formulations. BSR injection sites had significantly higher histopathological scores compared to EXR. Video recordings for monitoring drug-induced behavioral changes showed increased animal activity levels after BSR and EXR versus saline controls. Norbuprenorphine, a buprenorphine metabolite associated with respiratory depression, was detected in the plasma after BSR and EXR administration as well as by in vitro liver microsome assays. In conclusion, we recommend using EXR over BSR as a long-lasting buprenorphine analgesic in marmosets because EXR is a pharmaceutical-grade formulation that is compliant with FDA guidelines and the Guide as well as exhibits comparable PK and safety profiles as BSR.

Shotgun Metagenomics of Gastric Biopsies Reveals Compositional and Functional Microbiome Shifts in High- and Low-Gastric-Cancer-Risk Populations from Colombia, South America.

Along with Helicobacter pylori infection, the gastric microbiota is hypothesized to modulate stomach cancer risk in susceptible individuals. Whole metagenomic shotgun sequencing (WMS) is a sequencing approach to characterize the microbiome with advantages over traditional culture and 16S rRNA sequencing including identification of bacterial and non-bacterial taxa, species/strain resolution, and functional characterization of the microbiota. In this study, we used WMS to survey the microbiome in extracted DNA from antral gastric biopsy samples from Colombian patients residing in the high-risk gastric cancer town Túquerres (n = 10, H. pylori-positive = 7) and low-risk town of Tumaco (n = 10, H. pylori-positive = 6). Kraken2/Bracken was used for taxonomic classification and abundance. Functional gene profiles were inferred by InterProScan and KEGG analysis of assembled contigs and gene annotation. The most abundant taxa represented bacteria, non-human eukaryota, and viral genera found in skin, oral, food, and plant/soil environments including Staphylococus, Streptococcus, Bacillus, Aspergillus, and Siphoviridae. H. pylori was the predominant taxa present in H. pylori-positive samples. Beta diversity was significantly different based on H. pylori-status, risk group, and sex. WMS detected more bacterial taxa than 16S rRNA sequencing and aerobic, anaerobic, and microaerobic culture performed on the same gastric biopsy samples. WMS identified significant differences in functional profiles found between H. pylori-status, but not risk or sex groups. H. pylori-positive samples were significantly enriched for H. pylori-specific genes including virulence factors such as vacA, cagA, and urease, while carbohydrate and amino acid metabolism genes were enriched in H. pylori-negative samples. This study shows WMS has the potential to characterize the taxonomy and function of the gastric microbiome as risk factors for H. pylori-associated gastric disease. Future studies will be needed to compare and validate WMS versus traditional culture and 16S rRNA sequencing approaches for characterization of the gastric microbiome.

The Epidemiology of Invasive, Multipleantibiotic-resistant Klebsiella pneumoniae Infection in a Breeding Colony of Immunocompromised NSG Mice.

Klebsiella pneumoniae (Kp) is a gram-negative opportunistic pathogen that causes severe pneumonia, pyelonephritis, and sepsis in immunocompromised hosts. During a 4-mo interval, several NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) breeders and pups in our facilities were diagnosed with Kp infections. An initial 6 adult and 1 juvenile NSG mice were submitted for necropsy and histologic examination because of acute onset of diarrhea and death. The evaluation revealed typhlocolitis in 2 of the mice and tritrichomoniasis in all 7. Escherichia coli positive for polyketide synthase (pks+) and Kp were isolated from the intestines. Given a history of sepsis due to pks+ E. coli in NSG mice in our facilities and determination of its antimicrobial susceptibility, trimethoprim-sulfamethoxazole (TMP-SMX) was administered to the colony in the drinking water for 4 wk. After this intervention, an additional 21 mice became ill or died; 11 of these mice had suppurative pneumonia, meningoencephalitis, hepatitis, metritis, pyelonephritis, or sepsis. Kp was cultured from pulmonary abscesses or blood of 10 of the mice. Whole-genome sequencing (WGS) indicated that the Kp isolates contained genes associated with phenotypes found in pore-forming Kp isolates cultured from humans with ulcerative colitis and primary sclerosing cholangitis. None of the Kp isolates exhibited a hyperviscous phenotype, but 13 of 14 were resistant to TMP-SMX. Antimicrobial susceptibility testing indicated sensitivity of the Kp to enrofloxacin, which was administered in the drinking water. Antibiotic sensitivity profiles were confirmed by WGS of the Kp strains; key virulence and resistance genes to quaternary ammonia compounds were also identified. Enrofloxacin treatment resulted in a marked reduction in mortality, and the study using the NSG mice was completed successfully. Our findings implicate intestinal translocation of Kp as the cause of pneumonia and systemic infections in NSG mice and highlight the importance of identification of enteric microbial pathogens and targeted antibiotic selection when treating bacterial infections in immunocompromised mice.

Gastric Non-Helicobacter pylori Urease-Positive Staphylococcus epidermidis and Streptococcus salivarius Isolated from Humans Have Contrasting Effects on H. pylori-Associated Gastric Pathology and Host Immune Responses in a Murine Model of Gastric Cancer.

In populations with similar prevalence of Helicobacter pylori infection, cancer risk can vary dramatically. Changes in composition or structure of bacterial communities in the stomach, either at the time of exposure or over the course of H. pylori infection, may contribute to gastric pathology. In this study, a population of 37 patients from the low-gastric-cancer-risk (LGCR) region of Tumaco, Colombia, and the high-gastric-cancer-risk (HGCR) region of Túquerres, Colombia, were recruited for gastric endoscopy. Antral biopsy specimens were processed for histology and bacterial isolation. Fifty-nine distinct species among 26 genera were isolated by aerobic, anaerobic, and microaerobic culture and confirmed by 16S rRNA analysis. Urease-positive Staphylococcus epidermidis and Streptococcus salivarius were frequently isolated from gastric biopsy specimens. We asked whether coinfection of H. pylori with urease-positive S. salivarius and/or S. epidermidis had a demonstrable effect on H. pylori-induced gastritis in the germfree (GF) INS-GAS mouse model. Coinfections with S. salivarius and/or S. epidermidis did not affect gastric H. pylori colonization. At 5 months postinfection, GF INS-GAS mice coinfected with H. pylori and S. salivarius had statistically higher pathological scores in the stomachs than mice infected with H. pylori only or H. pylori with S. epidermidis (P < 0.05). S. epidermidis coinfection with H. pylori did not significantly change stomach pathology, but levels of the proinflammatory cytokine genes Il-1ß, Il-17A , and Il-22 were significantly lower than in H. pylori-monoinfected mice. This study demonstrates that non-H. pylori urease-positive bacteria may play a role in the severity of H. pylori-induced gastric cancer in humans. IMPORTANCE Chronic infection with H. pylori is the main cause of gastric cancer, which is a global health problem. In two Colombian populations with high levels of H. pylori prevalence, the regional gastric cancer rates are considerably different. Host genetic background, H. pylori biotype, environmental toxins, and dietary choices are among the known risk factors for stomach cancer. The potential role of non-H. pylori gastric microbiota in gastric carcinogenesis is being increasingly recognized. In this study, we isolated 59 bacterial species from 37 stomach biopsy samples of Colombian patients from both low-gastric-cancer-risk and high-gastric-cancer-risk regions. Urease-positive S. epidermidis and S. salivarius commonly cultured from the stomachs, along with H. pylori, were inoculated into germfree INS-GAS mice. S. salivarius coinfection with H. pylori induced significantly higher gastric pathology than in H. pylori-monoinfected mice, whereas S. epidermidis coinfection caused significantly lower H. pylori-induced proinflammatory cytokine responses than in H. pylori-monoinfected mice. This study reinforces the argument that the non-H. pylori stomach microflora play a role in the severity of H. pylori-induced gastric cancer.

Characterization of genotoxin-encoding Escherichia coli isolated from specific-pathogen free cats with impaired fertility.

Escherichia coli strains encoding colibactin (pks), hemolysin-associated cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt) are associated with intestinal inflammation and cancer, urinary tract infection, and septicemia in susceptible hosts. Over a 2-year period, an inbred laboratory colony of specific-pathogen free (SPF) cats (∼25) presented with resorptions, stillbirths, and pyometras in >50 % of pregnancies. Hemolytic E. coli were cultured from vaginal and preputial swabs of clinically normal, intact males, healthy kittens, and placenta and fetal tissues of a dam with reproductive disorders. We hypothesized cats from this colony were colonized with cytotoxin-encoding E. coli. 27 E. coli isolates were cultured from 20 fresh feces representing the majority of cats with and without fertility failures. Two E. coli isolates were also cultured from vaginal swabs from the same cat. 22 isolates (75.9 %) demonstrated hemolysis on blood agar. Twelve isolates (41.4 %) were pks+, 14 (48.3 %) were cnf+, and 10 (34.5 %) were cdt+ by PCR. Serotypes and virulence factor profiles were consistent with the extraintestinal E. coli (ExPEC) pathotype. Antibiotic resistance to cephalothin was exhibited in 13/14 representative isolates. Whole genome sequence analysis of 3 representative isolates confirmed the hemolysin-associated cnf, cdt, and the pks gene island. Representative isolates were cytotoxic to cervical epithelial cells in vitro. This study indicated ExPEC were present in SPF cats with a history of reproductive failure. While causality cannot be established, it is probable ExPEC was associated with impaired reproductive health and breeding success. Since treatment of the colony with cefovecin, reproductive performance has appreciably improved.

Helicobacter pylori Antimicrobial Resistance and Gene Variants in High- and Low-Gastric-Cancer-Risk Populations.

Colombia, South America has one of the world's highest burdens of Helicobacter pylori infection and gastric cancer. While multidrug antibiotic regimens can effectively eradicate H. pylori, treatment efficacy is being jeopardized by the emergence of antibiotic-resistant H. pylori strains. Moreover, the spectrum of and genetic mechanisms for antibiotic resistance in Colombia is underreported. In this study, 28 H. pylori strains isolated from gastric biopsy specimens from a high-gastric-cancer-risk (HGCR) population living in the Andes Mountains in Túquerres, Colombia and 31 strains from a low-gastric-cancer-risk (LGCR) population residing on the Pacific coast in Tumaco, Colombia were subjected to antibiotic susceptibility testing for amoxicillin, clarithromycin, levofloxacin, metronidazole, rifampin, and tetracycline. Resistance-associated genes were amplified by PCR for all isolates, and 29 isolates were whole-genome sequenced (WGS). No strains were resistant to amoxicillin, clarithromycin, or rifampin. One strain was resistant to tetracycline and had an A926G mutation in its 16S rRNA gene. Levofloxacin resistance was observed in 12/59 isolates and was significantly associated with N87I/K and/or D91G/Y mutations in gyrA Most isolates were resistant to metronidazole; this resistance was significantly higher in the LGCR (31/31) group compared to the HGCR (24/28) group. Truncations in rdxA and frxA were present in nearly all metronidazole-resistant strains. There was no association between phylogenetic relationship and resistance profiles based on WGS analysis. Our results indicate H. pylori isolates from Colombians exhibit multidrug antibiotic resistance. Continued surveillance of H. pylori antibiotic resistance in Colombia is warranted in order to establish appropriate eradication treatment regimens for this population.

Cytotoxic Escherichia coli strains encoding colibactin, cytotoxic necrotizing factor, and cytolethal distending toxin colonize laboratory common marmosets (Callithrix jacchus).

Cyclomodulins are virulence factors that modulate cellular differentiation, apoptosis, and proliferation. These include colibactin (pks), cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt). Pathogenic pks+, cnf+, and cdt+ E. coli strains are associated with inflammatory bowel disease (IBD) and colorectal cancer in humans and animals. Captive marmosets are frequently afflicted with IBD-like disease, and its association with cyclomodulins is unknown. Cyclomodulin-encoding E. coli rectal isolates were characterized using PCR-based assays in healthy and clinically affected marmosets originating from three different captive sources. 139 E. coli isolates were cultured from 122 of 143 marmosets. The pks gene was detected in 56 isolates (40%), cnf in 47 isolates (34%), and cdt in 1 isolate (0.7%). The prevalences of pks+ and cnf+ E. coli isolates were significantly different between the three marmoset colonies. 98% of cyclomodulin-positive E. coli belonged to phylogenetic group B2. Representative isolates demonstrated cyclomodulin cytotoxicity, and serotyping and whole genome sequencing were consistent with pathogenic E. coli strains. However, the presence of pks+, cnf+, or cdt+ E. coli did not correlate with clinical gastrointestinal disease in marmosets. Cyclomodulin-encoding E. coli colonize laboratory common marmosets in a manner dependent on the source, potentially impacting reproducibility in marmoset models.

Campylobacter taeniopygiae sp. nov., Campylobacter aviculae sp. nov., and Campylobacter estrildidarum sp. nov., Novel Species Isolated from Laboratory-Maintained Zebra Finches.

Zebra finches (Taeniopygia guttata) are laboratory animal species commonly used for modeling neurobiology and learning. Historically, using bacterial culture, biochemical analysis, and 16S ribosomal RNA gene sequencing, bacterial isolates from feces of finches housed at Massachusetts Institute of Technology had been presumptively diagnosed as Campylobacter jejuni, which is commonly isolated from both domestic and wild birds. Although the zebra finches were not clinically affected, C. jejuni is a known zoonotic pathogen that causes gastroenteritis in humans worldwide. Human transmission is predominantly foodborne and associated with the consumption of contaminated poultry; however, humans can also become infected from contact with C. jejuni-infected reservoir hosts. Because C. jejuni-infected finches pose a risk to research personnel, a study was undertaken to investigate the prevalence and taxonomic identification of Campylobacter spp. present in the finch colony. Campylobacter spp. were isolated from a total of 26 finch fecal samples collected in 2003, 2010, and 2017. 16S ribosomal RNA sequencing of all isolates determined that they shared 99% identity with either C. jejuni or Campylobacter lari. Sixteen of the isolates were subjected to further biochemical characterization and atpA and rpoB gene sequence analysis. Based on these analyses, three clusters of Campylobacter species were identified. The draft whole-genome sequences were determined for one representative isolate from each cluster. A pan-genomic phylogenetic tree, average nucleotide identity, digital DNA-DNA hybridization, and orthologous gene analyses indicated that each isolate was its own novel species, distinct from C. jejuni and other avian Campylobacter species. We have named these novel species Campylobacter taeniopygiae, Campylobacter aviculae, and Campylobacter estrildidarum, and in each novel species, we identified virulence genes suggesting their pathogenic and zoonotic potential.

Helicobacter monodelphidis sp. nov. and Helicobacter didelphidarum sp. nov., isolated from grey short-tailed opossums (Monodelphis domestica) with endemic cloacal prolapses.

In a search for potential causes of increased prolapse incidence in grey short-tailed opossum colonies, samples from the gastrointestinal tracts of 94 clinically normal opossums with rectal prolapses were screened for Helicobacter species by culture and PCR. Forty strains of two novel Helicobacter species which differed from the established Helicobacter taxa were isolated from opossums with and without prolapses. One of the Helicobacter species was spiral-shaped and urease-negative whereas the other Helicobacter strain had fusiform morphology with periplasmic fibres and was urease-positive. 16S rRNA gene sequence analysis revealed that all the isolates had over 99 % sequence identity with each other, and were most closely related to Helicobacter canadensis. Strains from the two novel Helicobacter species were subjected to gyrB and hsp60 gene and whole genome sequence analyses. These two novel Helicobacter species formed separate phylogenetic clades, divergent from other known Helicobacter species. The bacteria were confirmed as novel Helicobacter species based on digital DNA-DNA hybridization and average nucleotide identity analysis of their genomes, for which we propose the names Helicobacter monodelphidis sp. nov. with the type strain MIT 15-1451T (=LMG 29780T=NCTC 14189T) and Helicobacter didelphidarum sp. nov with type strain MIT 17-337T (=LMG 31024T=NCTC 14188T).

Draft Genome Sequences of Novel Campylobacter Species Isolated from Nonhuman Primates.

Campylobacter species are being increasingly isolated and associated with disease in humans and animals. Here, we describe four draft genome sequences of Campylobacter species from nonhuman primates. These include Campylobacter troglodytis, isolated from wild chimpanzees, and two likely new Campylobacter species isolated from a lemur, common marmoset, and cotton-top tamarin.

Natural Transmission of Helicobacter saguini Causes Multigenerational Inflammatory Bowel Disease in C57/129 IL-10-/- Mice.

Cotton-top tamarins (CTTs) are an ideal model of human inflammatory bowel disease (IBD) because these animals develop multigenerational, lower bowel cancer. We previously isolated and characterized a novel enterohepatic Helicobacter species, Helicobacter saguini, from CTTs with IBD and documented that H. saguini infection in germfree C57BL IL-10-/- mice recapitulates IBD, suggesting that H. saguini influences IBD etiopathogenesis. In this study, we utilized a germfree IL-10-/- model to illustrate that H. saguini infection can naturally transmit and infect four generations and cause significant intestinal inflammatory pathology. Additionally, whole-genome sequencing of representative H. saguini isolates from each generation of IL-10-/- mice revealed gene mutations suggestive of multigenerational evolution. Overall, these results support that specific bacterial species with pathogenic potential, like H. saguini, are transmissible microorganisms in the etiopathogenesis of IBD in CTTs and reinforces the importance of specific microbiota in the pathogenesis of IBD in humans.IMPORTANCE While family history is a significant risk factor for developing inflammatory bowel disease (IBD), it is unclear whether the microbiome from parents is a transmissible influence on disease in their offspring. Furthermore, it is unknown whether IBD-associated microbes undergo genomic adaptations during multigenerational transmission and chronic colonization in their hosts. Herein, we show that a single bacterial species, Helicobacter saguini, isolated from a nonhuman primate species with familial IBD, is transmissible from parent to offspring in germfree IL-10-/- mice and causes multigenerational IBD. Additionally, whole-genome sequence analysis of H. saguini isolated from different mouse generations identified microevolutions in environmental interaction, nutrient metabolism, and virulence factor genes that suggest that multigenerational transmission may promote adaptations related to colonization and survival in new hosts and chronic inflammatory environments. The findings from our study highlight the importance of specific bacterial species with pathogenic potential, like H. saguini, as transmissible microorganisms in the etiopathogenesis of IBD.

Draft Genome Sequence of a Mycobacterium porcinum Strain Isolated from a Pet Cat with Atypical Mycobacterial Panniculitis.

A fast-growing Mycobacterium species was cultured from draining, purulent lesions on the caudal abdomen of a 12-year-old male domestic long-haired cat. Whole-genome sequencing identified the organism as Mycobacterium porcinum.

Intestinal colonization of genotoxic Escherichia coli strains encoding colibactin and cytotoxic necrotizing factor in small mammal pets.

Escherichia coli encoding colibactin (clb), cytolethal distending toxin (cdt), and hemolysin-associated cytotoxic necrotizing factor (cnf) are associated with various intestinal and extra-intestinal diseases in humans and animals. Small mammal pets are not evaluated for genotoxin-encoding E. coli. Thus, the prevalence of such strains is unknown. The objective of this study was to isolate and characterize genotoxin-encoding E. coli from healthy and ill small mammal pets examined at a veterinary clinic and at two animal adoption centers. E. coli isolates were cultured from fecal samples and biochemically characterized. A total of 65 animals, including mice, rats, rabbits, guinea pigs, and hedgehogs, were screened. Twenty-six E. coli isolates were obtained from 24 animals. Twelve of the 26 isolates (46.2 %) were PCR-positive for the pks genes clbA and clbQ. Two isolates (7.7 %) were PCR-positive for cnf. All isolates were PCR-negative for cdt. All genotoxin-encoding isolates belonged to the pathogen-associated phylogenetic group B2. Representative genotoxin-encoding isolates had serotypes previously associated with clinical disease in humans and animals. Isolates encoding pks or cnf induced megalocytosis and cytotoxicity to HeLa cells in vitro. Although most isolates were obtained from healthy pets, two guinea pigs with diarrhea had pks-positive isolates cultured from their feces. Whole genome sequencing on four representative isolates confirmed the presence of pks and cnf genes and identified other virulence factors associated with pathogenicity in animals and humans. Our results suggest that small mammalian pets may serve as a reservoir for potentially pathogenic E. coli and implicate a zoonotic risk.

Draft Genome Sequences of Klebsiella pneumoniae Strains Isolated from Immunocompromised NOD-scid Gamma Research Mice.

Thirteen Klebsiella pneumoniae isolates cultured from feces, intestines, liver, lungs, and blood from immunocompromised NOD-scid gamma (NSG) mice with clinical illness, housed at a biomedical research institute, were sequenced using Illumina MiSeq technology for elucidation of pathogenic potential and genes encoding antibiotic resistance.

Genotoxic Escherichia coli Strains Encoding Colibactin, Cytolethal Distending Toxin, and Cytotoxic Necrotizing Factor in Laboratory Rats.

Although many Escherichia coli strains are considered commensals in mammals, strains encoding the cyclomodulin genotoxins are associated with clinical and subclinical disease in the urogenital and gastrointestinal tracts, meningitis, and inflammatory disorders. These genotoxins include the polyketide synthase (pks) pathogenicity island, cytolethal distending toxin (cdt), and hemolysin-associated cytotoxic necrotizing factor (cnf). E. coli strains are not excluded from rodents housed under SPF conditions in academic or vendor facilities. This study isolated and characterized genotoxin-encoding E. coli from laboratory rats obtained from 4 academic institutions and 3 vendors. A total of 69 distinct E. coli isolates were cultured from feces, rectal swab, nares, or vaginal swab of 52 rats and characterized biochemically. PCR analysis for cyclomodulin genes and phylogroup was performed on all 69 isolates. Of the 69 isolates, 45 (65%) were positive for pks, 20/69 (29%) were positive for cdt, and 4 (6%) were positive for cnf. Colibactin was the sole genotoxin identified in 21 of 45 pks+ isolates (47%), whereas cdt or cnf was also present in the remaining 24 isolates (53%); cdt and cnf were never present together or without pks. All genotoxin-associated strains were members of pathogen-associated phylogroup B2. Fisher exact and χ² tests demonstrated significant differences in genotoxin prevalence and API code distribution with regard to vendor. Select E. coli isolates were characterized by HeLa cell in vitro cytotoxicity assays, serotyped, and whole-genome sequenced. All isolates encoding cyclomodulins induced megalocytosis. Serotypes corresponded with vendor origin and cyclomodulin composition, with the cnf+ serotype representing a known human uropathogen. Whole-genome sequencing confirmed the presence of complete pks, cdt, and hemolysin-cnf pathogenicity islands. These findings indicate that genotoxin-encoding E. coli colonize laboratory rats from multiple commercial vendors and academic institutions and suggest the potential to contribute to clinical disease and introduce confounding variables into experimental rat models.

Gamma-glutamyltranspeptidase expression by Helicobacter saguini, an enterohepatic Helicobacter species isolated from cotton top tamarins with chronic colitis.

BACKGROUND: Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H. saguini infection in gnotobiotic IL-10-/- mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H. saguini expresses an enzymatically active GGT homologue with virulence properties. EXPERIMENTAL PROCEDURES: Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H. saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments. RESULTS: Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H. saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H. saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H. saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells. CONCLUSIONS: This study shows that H. saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.

Characterization of Campylobacter jejuni, Campylobacter upsaliensis, and a novel Campylobacter sp. in a captive non-human primate zoological collection.

BACKGROUND: The aim of this study was to longitudinally investigate the prevalence and characterization of Campylobacter spp. from non-human primates primate (NHP) with a history of endemic diarrhea housed at Como Park Zoo. METHODS: Fecal samples from 33 symptom-free NHP belonging to eight different species were collected weekly for 9 weeks. Species-level characterization and phylogenetic analysis of isolates included biochemical testing and 16S rRNA sequencing. RESULTS: Campylobacter spp. were isolated from the feces of 42% (14/33) of the primates. Three Campylobacter spp. (C upsaliensis, C jejuni, and novel Campylobacter sp.) were identified from three NHP species. A possible positive host Campylobacter species-specificity was observed. However, no statistical association was observed between the isolation of Campylobacter spp. and age and sex of the animal. CONCLUSIONS: The study revealed the value of conducting repeated fecal sampling to establish the overall prevalence of Campylobacter in zoo-maintained NHP; it also importantly identifies a novel Campylobacter sp. isolated from white-faced saki monkeys.

Comparative genomics analysis to differentiate metabolic and virulence gene potential in gastric versus enterohepatic Helicobacter species.

BACKGROUND: The genus Helicobacter are gram-negative, microaerobic, flagellated, mucus-inhabiting bacteria associated with gastrointestinal inflammation and classified as gastric or enterohepatic Helicobacter species (EHS) according to host species and colonization niche. While there are over 30 official species, little is known about the physiology and pathogenic mechanisms of EHS, which account for most in the genus, as well as what genetic factors differentiate gastric versus EHS, given they inhabit different hosts and colonization niches. The objective of this study was to perform a whole-genus comparative analysis of over 100 gastric versus EHS genomes in order to identify genetic determinants that distinguish these Helicobacter species and provide insights about their evolution/adaptation to different hosts, colonization niches, and mechanisms of virulence. RESULTS: Whole-genome phylogeny organized Helicobacter species according to their presumed gastric or EHS classification. Analysis of orthologs revealed substantial heterogeneity in physiological and virulence-related genes between gastric and EHS genomes. Metabolic reconstruction predicted that unlike gastric species, EHS appear asaccharolytic and dependent on amino/organic acids to fuel metabolism. Additionally, gastric species lack de novo biosynthetic pathways for several amino acids and purines found in EHS and instead rely on environmental uptake/salvage pathways. Comparison of virulence factor genes between gastric and EHS genomes identified overlapping yet distinct profiles and included canonical cytotoxins, outer membrane proteins, secretion systems, and survival factors. CONCLUSIONS: The major differences in predicted metabolic function suggest gastric species and EHS may have evolved for survival in the nutrient-rich stomach versus the nutrient-devoid environments, respectively. Contrasting virulence factor gene profiles indicate gastric species and EHS may utilize different pathogenic mechanisms to chronically infect hosts and cause inflammation and tissue damage. The findings from this study provide new insights into the genetic differences underlying gastric versus EHS and support the need for future experimental studies to characterize these pathogens.

Helicobacter pylori-infected C57BL/6 mice with different gastrointestinal microbiota have contrasting gastric pathology, microbial and host immune responses.

C57BL/6 (B6) mice from Taconic Sciences (Tac) and the Jackson Laboratory (Jax) were infected with H. pylori PMSS1 (Hp) for 16 week; there was no significant difference in the gastric histologic activity index between Hp infected Tac and Jax B6. However, the degree of gastric mucous metaplasia and Th1-associated IgG2c levels in response to Hp infection were increased in Tac mice over Jax mice, whereas the colonization levels of gastric Hp were higher by 8-fold in Jax B6 compared with Tac B6. Additionally, mRNA expression of gastric Il-1ß, Il-17A and RegIIIγ were significantly lower in the infected Tac compared to the infected Jax mice. There were significant differences in the microbial community structures in stomach, colon, and feces between Jax and Tac B6 females. Differences in gastric microbial communities between Jax and Tac B6 females are predicted to affect the metagenome. Moreover, Hp infection perturbed the microbial community structures in the stomach, colon and feces of Jax mice, but only altered the colonic microbial composition of Tac mice. Our data indicate that the GI microbiome of Tac B6 mice is compositionally distinct from Jax B6 mice, which likely resulted in different pathological, immunological, and microbial responses to Hp infection.