Subtractive sequence-mediated therapeutic targets from the conserved gene clusters of Campylobacter hyointestinalis and computational inhibition assessment.

Campylobacter hyointestinalis is a causative agent of enteritis, proctitis, human gastroenteritis, and diarrhea. Reported transmission is from pigs to humans. Link with gastrointestinal carcinoma has also been established in non-Helicobacter pylori patients carrying this strain. The genome size of the strain LMG9260 is 1.8 MB with 1785 chromosomal and seven plasmid proteins. No therapeutic targets have been identified and reported in this bacterium. Therefore, subtractive computational screening of its genome was carried out for the purpose. In total, 31 such targets were mined and riboflavin synthase was utilized for screening natural product inhibitors against it. Among more than 30,000 screened natural compounds from the NPASS library, three (NPC472060, NPC33653, and NPC313886) were prioritized to have the potential to be developed into new antimicrobial drugs. Dynamics simulation assay along with other relevant parameters like absorption, toxicity, and distribution of the inhibiting compounds were also predicted and NPC33653 was identified as having the best drug-like properties among the prioritized compounds. Thus, it has potential to be pursued further for the inhibition of riboflavin synthesis in C. hyointestinalis for subsequent obstruction of its growth and survival.Communicated by Ramaswamy H. Sarma.

Pangenome analysis reveals genetic isolation in Campylobacter hyointestinalis subspecies adapted to different mammalian hosts.

Campylobacter hyointestinalis is an emerging pathogen currently divided in two subspecies: C. hyointestinalis subsp. lawsonii which is predominantly recovered from pigs, and C. hyointestinalis subsp. hyointestinalis which can be found in a much wider range of mammalian hosts. Despite C. hyointestinalis being reported as an emerging pathogen, its evolutionary and host-associated diversification patterns are still vastly unexplored. For this reason, we generated whole-genome sequences of 13 C. hyointestinalis subsp. hyointestinalis strains and performed a comprehensive comparative analysis including publicly available C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii genomes, to gain insight into the genomic variation of these differentially-adapted subspecies. Both subspecies are distinct phylogenetic lineages which present an apparent barrier to homologous recombination, suggesting genetic isolation. This is further supported by accessory gene patterns that recapitulate the core genome phylogeny. Additionally, C. hyointestinalis subsp. hyointestinalis presents a bigger and more diverse accessory genome, which probably reflects its capacity to colonize different mammalian hosts unlike C. hyointestinalis subsp. lawsonii that is presumably host-restricted. This greater plasticity in the accessory genome of C. hyointestinalis subsp. hyointestinalis correlates to a higher incidence of genome-wide recombination events, that may be the underlying mechanism driving its diversification. Concordantly, both subspecies present distinct patterns of gene families involved in genome plasticity and DNA repair like CRISPR-associated proteins and restriction-modification systems. Together, our results provide an overview of the genetic mechanisms shaping the genomes of C. hyointestinalis subspecies, contributing to understand the biology of Campylobacter species that are increasingly recognized as emerging pathogens.

Updating the genomic taxonomy and epidemiology of Campylobacter hyointestinalis.

Campylobacter hyointestinalis is a member of an emerging group of zoonotic Campylobacter spp. that are increasingly identified in both gastric and non-gastric disease in humans. Here, we discovered C. hyointestinalis in three separate classes of New Zealand ruminant livestock; cattle, sheep and deer. To investigate the relevance of these findings we performed a systematic literature review on global C. hyointestinalis epidemiology and used comparative genomics to better understand and classify members of the species. We found that C. hyointestinalis subspecies hyointestinalis has an open pangenome, with accessory gene contents involved in many essential processes such as metabolism, virulence and defence. We observed that horizontal gene transfer is likely to have played an overwhelming role in species diversification, favouring a public-goods-like mechanism of gene 'acquisition and resampling' over a tree-of-life-like vertical inheritance model of evolution. As a result, simplistic gene-based inferences of taxonomy by similarity are likely to be misleading. Such genomic plasticity will also mean that local evolutionary histories likely influence key species characteristics, such as host-association and virulence. This may help explain geographical differences in reported C. hyointestinalis epidemiology and limits what characteristics may be generalised, requiring further genomic studies of C. hyointestinalis in areas where it causes disease.

A PCR-RFLP assay to detect and type cytolethal distending toxin (cdt) genes in Campylobacter hyointestinalis.

Campylobacter hyointestinalis is considered as an emerging zoonotic pathogen. We have recently identified two types of cytolethal distending toxin (cdt) gene in C. hyointestinalis and designated them as Chcdt-I and Chcdt-II. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay that can differentiate Chcdt-I from Chcdt-II. When the PCR-RFLP assay was applied to 17 other Campylobacter strains and 25 non-Campylobacter strains, PCR products were not obtained irrespective of their cdt gene-possession, indicating that the specificity of the PCR-RFLP assay was 100%. In contrast, when the PCR-RFLP assay was applied to 35 C. hyointestinalis strains including 23 analyzed in the previous study and 12 newly isolated from pigs and bovines, all of them showed the presence of cdt genes. Furthermore, a restriction digest by EcoT14-I revealed that 29 strains contained both Chcdt-I and Chcdt-II and 6 strains contained only Chcdt-II, showing 100% sensitivity. Unexpectedly, however, PCR products obtained from 7 C. hyointestinalis strains were not completely digested by EcoT14-I. Nucleotide sequence analysis revealed that the undigested PCR product was homologous to cdtB but not to Chcdt-IB or Chcdt-IIB, indicating the presence of another cdt gene-variant. Then, we further digested the PCR products with DdeI in addition to EcoT14-I, showing that all three cdt genes, including a possible new Chcdt variant, could be clearly differentiated. Thus, the PCR-RFLP assay developed in this study is a valuable tool for evaluating the Chcdt gene-profile of bacteria.

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari.

In this study, we devised a multiplex PCR assay based on the gene of cytolethal distending toxin (cdt) B subunit to simultaneously detect and discriminate Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari. Species-specific PCR products were successfully obtained from all 38 C. jejuni, 12 C. fetus, 39 C. coli, 22 C. upsaliensis, 24 C. hyointestinalis, and 7 C. lari strains tested. On the other hand, no specific PCR products were obtained from other campylobacters and bacterial species tested (41 strains in total). The proposed multiplex PCR assay is a valuable tool for detection and descrimination of 6 major Campylobacter species, that are associated with gastrointestinal diseases in humans.

Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin.

Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals.

A new variant of cytolethal distending toxin in a clinical isolate of Campylobacter hyointestinalis.

Increasing numbers of Campylobacter hyointestinalis have been isolated from humans and animals with gastroenteritis, although the virulence mechanism of this species remains largely unknown. Here, we show that C. hyointestinalis isolated from a patient with diarrhoea in Thailand produced a novel variant of cytolethal distending toxin (CDT). Sequencing of a 13 965 bp genomic region of C. hyointestinalis carrying the genes coding for Ch-CDT revealed three ORFs of 798, 804 and 537 bp, which code for the Ch-CdtA, Ch-CdtB and Ch-CdtC subunits, respectively. The deduced amino acid sequence of Ch-CdtA showed ∼38.9 % homology with the CdtA of Campylobacter coli, but sequences of Ch-CdtB and Ch-CdtC were homologous to CdtB (65.7 %) and CdtC (33.1 %) of Campylobacter upsaliensis, respectively. Filter-sterilized sonic lysate of C. hyointestinalis demonstrated distension and death of HeLa cells by arresting the cell cycle at the G(2)/M phase and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. Rabbit antiserum raised against recombinant Ch-CdtB was not reactive against the recombinant CdtB protein of Campylobacter jejuni. A reconstituted Ch-CDT holotoxin prepared using each of the recombinant subunit proteins demonstrated distension and death of HeLa cells, suggesting that the C. hyointestinalis isolate indeed produced functionally active Ch-CDT. Furthermore, the immunological distinctiveness of the Ch-CDT produced by C. hyointestinalis and the increasing prevalence of the species in patients and animals with gastroenteritis suggest that this species may be an important emerging zoonotic pathogen.

The origin of non-H. pylori-related positive Giemsa staining in human gastric biopsy specimens: A prospective study.

BACKGROUND: Upper gastrointestinal endoscopically biopsied specimens are usually sent for the diagnosis of Helicobacter pylori infection. The study aimed to determine the relationship between the origin of positive Giemsa staining and the grade of gastritis based on the updated Sydney system. METHODS: Gastric biopsy specimens taken at the lesser curvature and greater curvature sides of the corpus and greater curvature side of the antrum were stained with H&E, Giemsa, anti-East Asian-specific antibody and anti-H. pylori antibody stains. Pyrosequencing analysis was performed in cases that showed discrepancy between the Giemsa and anti-H. pylori antibody staining. RESULTS: Seventy-two out of 150 cases (48%) stained positive for anti-H. pylori antibody, of which 68 (94.4%) stained positive for anti-East Asian-specific antibody stain. Twelve of the 20 cases with discrepant results for Giemsa and anti-H. pylori antibody stains exhibited Campylobacter hyointestinalis infection. The grades of neutrophil activity (p<0.001) and chronic inflammation (p<0.001) were lower for Campylobacter infection than for East Asian CagA H. pylori-related infection. CONCLUSION: C. hyointestinalis is the most common cause of non-H. pylori-related Giemsa positive infection, and is associated with lower grades of neutrophil activity and chronic inflammation than East Asian CagA H. pylori-related infection.

Cryptic plasmids isolated from Campylobacter strains represent multiple, novel incompatibility groups.

Three small, cryptic plasmids from the multi-drug-resistant (MDR) Campylobacter coli strain RM2228 and one small, cryptic plasmid from the MDR Campylobacter jejuni strain RM1170 were sequenced and characterized. pCC2228-1 has some similarity to Firmicutes RepL family plasmids that replicate via a rolling-circle mechanism. pCC2228-2 is a theta-replicating, iteron-containing plasmid (ICP) that is a member of the same incompatibility (Inc) group as previously described Campylobacter shuttle vectors. The other two ICPs, pCC2228-3 and pCJ1170, represent a second novel Inc group. Comparison of the four plasmids described in this study with other characterized plasmids from C. jejuni, C. coli, C. lari, and C. hyointestinalis suggests that cryptic plasmids in Campylobacter may be classified into as many as nine Inc groups. The plasmids characterized in this study have several unique features suitable for the construction of novel Campylobacter shuttle vectors, e.g., small size, absence of many common multiple-cloning site restriction sites, and Inc groups not represented by current Campylobacter shuttle plasmids. Thus, these plasmids may be used to construct a new generation of Campylobacter shuttle vectors that would permit transformation of environmental Campylobacter isolates with an existing repertoire of native plasmids.

Effects of subtherapeutic administration of antimicrobial agents to beef cattle on the prevalence of antimicrobial resistance in Campylobacter jejuni and Campylobacter hyointestinalis.

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (> or =94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.