Analyses of energy metabolism and stress defence provide insights into Campylobacter concisus growth and pathogenicity.

Campylobacter concisus is an emerging enteric pathogen that is associated with inflammatory bowel disease. Previous studies demonstrated that C. concisus is non-saccharolytic and hydrogen gas (H2) is a critical factor for C. concisus growth. In order to understand the molecular basis of the non-saccharolytic and H2-dependent nature of C. concisus growth, in this study we examined the pathways involving energy metabolism and oxidative stress defence in C. concisus. Bioinformatic analysis of C. concisus genomes in comparison with the well-studied enteric pathogen Campylobacter jejuni was performed. This study found that C. concisus lacks a number of key enzymes in glycolysis, including glucokinase and phosphofructokinase, and the oxidative pentose phosphate pathway. C. concisus has an incomplete tricarboxylic acid cycle, with no identifiable succinyl-CoA synthase or fumarate hydratase. C. concisus was inferred to use fewer amino acids and have fewer candidate substrates as electron donors and acceptors compared to C. jejuni. The addition of DMSO or fumarate to media resulted in significantly increased growth of C. concisus in the presence of H2 as an electron donor, demonstrating that both can be used as electron acceptors. Catalase, an essential enzyme for oxidative stress defence in C. jejuni, and various nitrosative stress enzymes, were not found in the C. concisus genome. Overall, C. concisus is inferred to have a non-saccharolytic metabolism in which H2 is central to energy conservation, and a narrow selection of carboxylic acids and amino acids can be utilised as organic substrates. In conclusion, this study provides a molecular basis for the non-saccharolytic and hydrogen-dependent nature of C. concisus energy metabolism pathways, which provides insights into the growth requirements and pathogenicity of this species.

The Clinical Importance of Campylobacter concisus and Other Human Hosted Campylobacter Species.

Historically, Campylobacteriosis has been considered to be zoonotic; the Campylobacter species that cause human acute intestinal disease such as Campylobacter jejuni and Campylobacter coli originate from animals. Over the past decade, studies on human hosted Campylobacter species strongly suggest that Campylobacter concisus plays a role in the development of inflammatory bowel disease (IBD). C. concisus primarily colonizes the human oral cavity and some strains can be translocated to the intestinal tract. Genome analysis of C. concisus strains isolated from saliva samples has identified a bacterial marker that is associated with active Crohn's disease (one major form of IBD). In addition to C. concisus, humans are also colonized by a number of other Campylobacter species, most of which are in the oral cavity. Here we review the most recent advancements on C. concisus and other human hosted Campylobacter species including their clinical relevance, transmission, virulence factors, disease associated genes, interactions with the human immune system and pathogenic mechanisms.

The Growth and Protein Expression of Inflammatory Bowel Disease-Associated Campylobacter concisus Is Affected by the Derivatives of the Food Additive Fumaric Acid.

Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract with multifactorial etiology. Both dietary factors and the microbe Campylobacter concisus have been found to be associated with the condition. The current study examined the effects of sodium fumarate, a neutralized product of the food additives fumaric acid and monosodium fumarate when in the intestinal environment, on the growth of C. concisus to determine the effects of these food additives on IBD-associated bacterial species. Through culture methods and quantification, it was found that neutralized fumaric acid, neutralized monosodium fumarate, and sodium fumarate increased the growth of C. concisus, with the greatest increase in growth at a concentration of 0.4%. Further examination of 50 C. concisus strains on media with added sodium fumarate showed that greatest growth was also achieved at a concentration of 0.4%. At a concentration of 2% sodium fumarate, all strains examined displayed less growth in comparison with those cultured on media without sodium fumarate. Using mass spectrometry, multiple C. concisus proteins showed significant differential expression when cultured on media with and without 0.4% sodium fumarate. The findings presented suggest that patients with IBD should consider avoiding excessive consumption of foods with fumaric acid or its sodium salts, and that the addition of 0.4% sodium fumarate alone to media may assist in the isolation of C. concisus from clinical samples.

Genomic analysis of oral Campylobacter concisus strains identified a potential bacterial molecular marker associated with active Crohn's disease.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC). C. concisus consists of two genomospecies (GS) and diverse strains. This study aimed to identify molecular markers to differentiate commensal and IBD-associated C. concisus strains. The genomes of 63 oral C. concisus strains isolated from patients with IBD and healthy controls were examined, of which 38 genomes were sequenced in this study. We identified a novel secreted enterotoxin B homologue, Csep1. The csep1 gene was found in 56% of GS2 C. concisus strains, presented in the plasmid pICON or the chromosome. A six-nucleotide insertion at the position 654-659 bp in csep1 (csep1-6bpi) was found. The presence of csep1-6bpi in oral C. concisus strains isolated from patients with active CD (47%, 7/15) was significantly higher than that in strains from healthy controls (0/29, P = 0.0002), and the prevalence of csep1-6bpi positive C. concisus strains was significantly higher in patients with active CD (67%, 4/6) as compared to healthy controls (0/23, P = 0.0006). Proteomics analysis detected the Csep1 protein. A csep1 gene hot spot in the chromosome of different C. concisus strains was found. The pICON plasmid was only found in GS2 strains isolated from the two relapsed CD patients with small bowel complications. This study reports a C. concisus molecular marker (csep1-6bpi) that is associated with active CD.

Modulation of Gut Microbiota: A Novel Paradigm of Enhancing the Efficacy of Programmed Death-1 and Programmed Death Ligand-1 Blockade Therapy.

Blockade of programmed death 1 (PD-1) protein and its ligand programmed death ligand 1 (PD-L1) has been used as cancer immunotherapy in recent years, with the blockade of PD-1 being more widely used than blockade of PD-L1. PD-1 and PD-L1 blockade therapy showed benefits in patients with various types of cancer; however, such beneficial effects were seen only in a subgroup of patients. Improving the efficacy of PD-1 and PD-L1 blockade therapy is clearly needed. In this review, we summarize the recent studies on the effects of gut microbiota on PD-1 and PD-L1 blockade and discuss the new perspectives on improving efficacy of PD-1 and PD-L1 blockade therapy in cancer treatment through modulating gut microbiota. We also discuss the possibility that chronic infections or inflammation may impact on PD-1 and PD-L1 blockade therapy.

Campylobacter concisus Genomospecies 2 Is Better Adapted to the Human Gastrointestinal Tract as Compared with Campylobacter concisus Genomospecies 1.

Campylobacter concisus was previously shown to be associated with inflammatory bowel disease including Crohn's disease (CD) and ulcerative colitis (UC). C. concisus has two genomospecies (GS). This study systematically examined the colonization of GS1 and GS2 C. concisus in the human gastrointestinal tract. GS1 and GS2 specific polymorphisms in 23S rRNA gene were identified by comparison of the 23S rRNA genes of 49 C. concisus strains. Two newly designed PCR methods, based on the polymorphisms of 23S rRNA gene, were developed and validated. These PCR methods were used to detect and quantify GS1 and GS2 C. concisus in 56 oral and enteric samples collected from the gastrointestinal tract of patients with IBD and healthy controls. Meta-analysis of the composition of the isolated GS1 and GS2 C. concisus strains in previous studies was also conducted. The quantitative PCR methods revealed that there was more GS2 than GS1 C. concisus in samples collected from the upper and lower gastrointestinal tract of both patients with IBD and healthy controls, showing that GS2 C. concisus is better adapted to the human gastrointestinal tract. Analysis of GS1 and GS2 composition of isolated C. concisus strains in previous studies showed similar findings except that in healthy individuals a significantly lower GS2 than GS1 C. concisus strains were isolated from fecal samples, suggesting a potential difference in the C. concisus strains or the enteric environment between patients with gastrointestinal diseases and healthy controls. This study provides novel information regarding the adaptation of different genomospecies of C. concisus in the human gastrointestinal tract.

Azathioprine, Mercaptopurine, and 5-Aminosalicylic Acid Affect the Growth of IBD-Associated Campylobacter Species and Other Enteric Microbes.

Campylobacter concisus is a bacterium that is associated with inflammatory bowel disease (IBD). Immunosuppressive drugs including azathioprine (AZA) and mercaptopurine (MP), and anti-inflammatory drug such as 5-aminosalicylic acid (5-ASA) are commonly used to treat patients with IBD. This study aimed to examine the effects of AZA, MP, and 5-ASA on the growth of IBD-associated bacterial species and to identify bacterial enzymes involved in immunosuppressive drug metabolism. A total of 15 bacterial strains of five species including 11 C. concisus strains, Bacteroides fragilis, Bacteroides vulgatus, Enterococcus faecalis, and Escherichia coli were examined. The impact of AZA, MP, and 5-ASA on the growth of these bacterial species was examined quantitatively using a plate counting method. The presence of enzymes involved in AZA and MP metabolism in these bacterial species was identified using bioinformatics tools. AZA and MP significantly inhibited the growth of all 11 C. concisus strains. C. concisus strains were more sensitive to AZA than MP. 5-ASA showed inhibitory effects to some C. concisus strains, while it promoted the growth of other C. concisus strains. AZA and MP also significantly inhibited the growth of B. fragilis and B. vulgatus. The growth of E. coli was significantly inhibited by 200 µg/ml of AZA as well as 100 and 200 µg/ml of 5-ASA. Bacterial enzymes related to AZA and MP metabolism were found, which varied in different bacterial species. In conclusion, AZA and MP have inhibitory effects to IBD-associated C. concisus and other enteric microbes, suggesting an additional therapeutic mechanism of these drugs in the treatment of IBD. The strain dependent differential impact of 5-ASA on the growth of C. concisus may also have clinical implication given that in some cases 5-ASA medications were found to cause exacerbations of colitis.

Genome analysis of Campylobacter concisus strains from patients with inflammatory bowel disease and gastroenteritis provides new insights into pathogenicity.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease. C. concisus has two major genomospecies, which appear to have different enteric pathogenic potential. Currently, no studies have compared the genomes of C. concisus strains from different genomospecies. In this study, a comparative genome analysis of 36 C. concisus strains was conducted including 27 C. concisus strains sequenced in this study and nine publically available C. concisus genomes. The C. concisus core-genome was defined and genomospecies-specific genes were identified. The C. concisus core-genome, housekeeping genes and 23S rRNA gene consistently divided the 36 strains into two genomospecies. Two novel genomic islands, CON_PiiA and CON_PiiB, were identified. CON_PiiA and CON_PiiB islands contained proteins homologous to the type IV secretion system, LepB-like and CagA-like effector proteins. CON_PiiA islands were found in 37.5% of enteric C. concisus strains (3/8) isolated from patients with enteric diseases and none of the oral strains (0/27), which was statistically significant. This study reports the findings of C. concisus genomospecies-specific genes, novel genomic islands that contain type IV secretion system and putative effector proteins, and other new genomic features. These data provide novel insights into understanding of the pathogenicity of this emerging opportunistic pathogen.

Delineation of genetic relatedness and population structure of oral and enteric Campylobacter concisus strains by analysis of housekeeping genes.

Campylobacter concisus is an oral bacterium that has been shown to be associated with inflammatory bowel disease (IBD). In this study we examined clusters of oral C. concisus strains isolated from patients with IBD and healthy controls by analysing six housekeeping genes. In addition, we investigated the population structure of C. concisus strains. Whether oral and enteric strains form distinct clusters based on the sequences of these housekeeping genes was also investigated. The oral C. concisus strains were found to contain two genomospecies, which belong to the two genomospecies previously found in enteric C. concisus strains. C. concisus clusters formed based on the sequences of a single aspA gene were the same as that formed by using previously reported MLST schemes. The analysis of combined oral and enteric C. concisus strains found that enteric C. concisus strains did not form distinct clusters. Genetic structure analysis identified five subpopulations of C. concisus and showed that genetic recombination between C. concisus strains was common. However, genetic recombination was significantly less in oral strains isolated from patients with IBD than from healthy individuals. Previously reported oral and enteric intestinal epithelial invasive C. concisus strains were in cluster II and subpopulation III. Furthermore, this study shows that there are no distinct enteric C. concisus strain clusters or subpopulations.

Investigation of the effects of pH and bile on the growth of oral Campylobacter concisus strains isolated from patients with inflammatory bowel disease and controls.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD). This study examined the impact of pH and bile on the growth of oral C. concisus strains isolated from patients with IBD and controls. The growth of 58 C. concisus strains on horse blood agar (HBA) plates following exposure to media with various pH values for different time points was examined. Furthermore, the growth of C. concisus strains on HBA plates containing different concentrations of ox bile was investigated. Following exposure to pH 2 for 30 min, none of the 58 oral C. concisus strains grew on HBA plates. Following exposure to pH 3.5 for 30 min, only four of 20 oral strains examined grew on HBA plates, with a log10 c.f.u. reduction of 0.7-2.5 compared to the same strains without low pH exposure. Exposure to pH 5 for 120 min had minimal effects on C. concisus growth. Approximately half of the oral strains (55.2%, 32/58) grew on HBA containing 2% bile. Bile inhibited the growth of C. concisus in a dose- and strain-dependent manner. These data suggest that both bacterial and intestinal environmental factors may play a role in the determination of C. concisus colonization in the different parts of the gastrointestinal tract and that increased gastric pH and reduced intestinal bile may be risk factors for increased gastric and intestinal C. concisus colonization.

Examination of the Anaerobic Growth of Campylobacter concisus Strains.

Campylobacter concisus is an oral bacterium that is associated with intestinal diseases. C. concisus was previously described as a bacterium that requires H2-enriched microaerobic conditions for growth. The level of H2 in the oral cavity is extremely low, suggesting that C. concisus is unlikely to have a microaerobic growth there. In this study, the anaerobic growth of C. concisus was investigated. The growth of fifty-seven oral C. concisus strains and six enteric C. concisus strains under various atmospheric conditions including anaerobic conditions with and without H2 was examined. The atmospheric conditions were generated using commercially available gas-generation systems. C. concisus putative virulence proteins were identified using mass spectrometry analysis. Under anaerobic conditions, 92% of the oral C. concisus strains (52/57) and all six enteric strains grew without the presence of H2 and the presence of H2 greatly increased C. concisus growth. An oral C. concisus strain was found to express a number of putative virulence proteins and the expression levels of these proteins were not affected by H2. The levels of H2 appeared to affect the optimal growth of C. concisus. This study provides useful information in understanding the natural colonization site and pathogenicity of C. concisus.