The Role of Methanogenic Archaea in Inflammatory Bowel Disease-A Review.

Methanogenic archaea are a part of the commensal gut microbiota responsible for hydrogen sink and the efficient production of short-chain fatty acids. Dysbiosis of methanogens is suspected to play a role in pathogenesis of variety of diseases, including inflammatory bowel disease (IBD). Unlike bacteria, the diversity of archaea seems to be higher in IBD patients compared to healthy subjects, whereas the prevalence and abundance of gut methanogens declines in IBD, especially in ulcerative colitis. To date, studies focusing on methanogens in pediatric IBD are very limited; nevertheless, the preliminary results provide some evidence that methanogens may be influenced by the chronic inflammatory process in IBD. In this review, we demonstrated the development and diversity of the methanogenic community in IBD, both in adults and children.

Methanogenic Archaea in the Pediatric Inflammatory Bowel Disease in Relation to Disease Type and Activity.

The inflammatory bowel disease (IBD) is associated with gut microbiota dysbiosis; however, studies on methanogens-especially those focused on children-are extremely limited. The aim of this study was to determine the abundance of total methanogenic archaea and their three subgroups: Methanobrevibacter (Mb.) smithii, Methanosphaera (Ms.) stadtmanae, and Methanomassiliicoccales, in the feces of children with both active and inactive Crohn's disease (CD) and ulcerative colitis (UC). The results of a quantitative real-time PCR were cross-referenced with the disease type (CD vs. UC) and activity assessed with the use of Pediatric Crohn's Disease Activity Index (PCDAI) and Pediatric Ulcerative Colitis Activity Index (PUCAI) indices, and fecal calprotectin (FCP) concentration, and compared with controls. There was a significant decrease in the number of total methanogens in CD and UC compared to controls. The prevalence of total methanogens was also lower in UC compared to controls. Furthermore, patients from the inactive UC group were colonized by a lower number of Mb. smithii, and demonstrated the most pronounced positive correlation between the number of Ms. stadtmanae and the FCP concentration. Our results demonstrate that gut methanogens are related to the type and activity of pediatric IBD.

Microorganisms Involved in Hydrogen Sink in the Gastrointestinal Tract of Chickens.

Hydrogen sink is a beneficial process, which has never been properly examined in chickens. Therefore, the aim of this study was to assess the quantity and quality of microbiota involved in hydrogen uptake with the use of real-time PCR and metagenome sequencing. Analyses were carried out in 50 free-range chickens, 50 commercial broilers, and 54 experimental chickens isolated from external factors. The median values of acetogens, methanogens, sulfate-reducing bacteria (SRB), and [NiFe]-hydrogenase utilizers measured in the cecum were approx. 7.6, 0, 0, and 3.2 log10/gram of wet weight, respectively. For the excreta samples, these values were 5.9, 4.8, 4, and 3 log10/gram of wet weight, respectively. Our results showed that the acetogens were dominant over the other tested groups of hydrogen consumers. The quantities of methanogens, SRB, and the [NiFe]-hydrogenase utilizers were dependent on the overall rearing conditions, being the result of diet, environment, agrotechnical measures, and other factors combined. By sequencing of the 16S rRNA gene, archaea of the genus Methanomassiliicoccus (Candidatus Methanomassiliicoccus) were discovered in chickens for the first time. This study provides some indication that in chickens, acetogenesis may be the main metabolic pathway responsible for hydrogen sink.

Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples.

Methanogenic archaea are an important component of the human and animal intestinal microbiota, and yet their presence is rarely reported in publications describing the subject. One of the methods of quantifying the prevalence of methanogens is quantitative real-time PCR (qPCR) of the methanogen-specific mcrA gene, and one of the possible reasons for detection failure is usually a methodology bias. Here, we refined the existing protocol by changing one of the primers and improving the conditions of the qPCR reaction. As a result, at the expense of a slightly lower yet acceptable PCR efficiency, the new assay was characterized by increased specificity and sensitivity and a wider linear detection range of 7 orders of magnitude. The lowest copy number of mcrA quantified at a frequency of 100% was 21 copies per reaction. The other validation parameters tested, such as reproducibility and linearity, also gave satisfactory results. Overall, we were able to minimize the negative impacts of primer dimerization and other cross-reactions on qPCR and increase the number of not only detectable but also quantifiable stool samples-or in this case, chicken droppings.

Selection and Optimization of High-Yielding DNA Isolation Protocol for Quantitative Analyses of Methanogenic Archaea.

Methanogenic archaea are a functionally important component of the intestinal microbiota of humans and animals, participating in the utilization of detrimental hydrogen produced during gut fermentation. Despite this, archaeal DNA has rarely been found in intestinal microbiome analyses, which prompts the need to optimize detecting procedures of these microorganisms, including the DNA isolation step. Three commercially available kits for DNA isolation and one extra purification kit that removes PCR inhibitors were evaluated on chicken droppings. In addition, different variants of mechanical lysis and a double elution were tested to ensure the maximum efficiency of DNA isolation from archaea as well as bacteria. A quantitative real-time PCR was used to monitor the optimization progress. As a result, the combination of the selected Genomic Mini AX Bacteria+ kit with a 2-min-long sonication by ultrasonic probe and enzymatic pretreatment gave excellent extraction efficiency rates for DNA of methanogenic archaea (an approximate 50-fold increase compared to the standard enzymatic lysis described by the producer) and, at the same time, provided optimal protection of DNA extracted from bacteria susceptible to enzymatic lysis. The presented results indicate that the optimized protocol allows for highly efficient extraction of total DNA, which is well-suited for quantitative microbial analyses by real-time PCR.

The application of the loop-mediated isothermal amplification (LAMP) method for diagnosing Enterococcus hirae-associated endocarditis outbreaks in chickens.

BACKGROUND: Enterococcus hirae is considered a part of the normal intestinal biota of several domestic animals, including poultry. However, this species is also associated with infective endocarditis in chickens, a disease that leads to unexpected deaths and serious economical losses. Enterococcus hirae is identified predominantly with the use of conventional bacteriological methods, biochemical tests and PCR. Rapid, sensitive and specific methods for detecting E. hirae in clinical samples are required in poultry production. The aim of this study was to use the Loop-Mediated Isothermal Amplification (LAMP) for the identification and quantification of E. hirae in heart samples from broiler chickens. RESULTS: The specificity of the LAMP method was confirmed for 7 enterococcal strains and 3 non-enterococcal strains. E. hirae was detected in all of the 22 analyzed clinical bacterial isolates and in all of the 9 heart samples. Three sets of primers supported the detection of E. hirae with high sensitivity and specificity within one hour. The highest detection rate of a LAMP product was approximately 7 min for an E. hirae strain and 12 min for a positive heart sample. The detection limit for the E. hirae ATCC 10541 standard was 1.3 × 102 CFU (43.4 fg) or 13.8 copies of the E. hirae genome equivalent per reaction. The reaction was 10-fold more sensitive than conventional species-specific PCR. The LAMP assay supported the determination of the E. hirae load in chicken hearts with endocarditis in field cases. The average number of E. hirae cells in hearts was 5.19 × 107 CFU/g of tissue, and the average number of E. hirae genome equivalents in hearts was 5.51× 106 copies/g of tissue. Bacterial counts were significantly higher in the LAMP assay than in the standard plate count. CONCLUSIONS: The LAMP assay is a useful diagnostic tool and an effective alternative to conventional methods for the detection of this enterococcal species. The sodA-based LAMP assay supported direct identification of E. hirae from pure cultures and heart samples without previous bacterial cultivation. This is the first study to apply the LAMP method for the purpose of diagnosing E. hirae-associated endocarditis in poultry.

Phage Therapy in Bacterial Infections Treatment: One Hundred Years After the Discovery of Bacteriophages.

The therapeutic use of bacteriophages has seen a renewal of interest blossom in the last few years. This reversion is due to increased difficulties in the treatment of antibiotic-resistant strains of bacteria. Bacterial resistance to antibiotics, a serious problem in contemporary medicine, does not implicate resistance to phage lysis mechanisms. Lytic bacteriophages are able to kill antibiotic-resistant bacteria at the end of the phage infection cycle. Thus, the development of phage therapy is potentially a way to improve the treatment of bacterial infections. However, there are antibacterial phage therapy difficulties specified by broadening the knowledge of the phage nature and influence on the host. It has been shown during experiments that both innate and adaptive immunity are involved in the clearance of phages from the body. Immunological reactions against phages are related to the route of administration and may vary depending on the type of bacterial viruses. For that reason, it is very important to test the immunological response of every single phage, particularly if intravenous therapy is being considered. The lack of these data in previous years was one of the reasons for phage therapy abandonment despite its century-long study. Promising results of recent research led us to look forward to a phage therapy that can be applied on a larger scale and subsequently put it into practice.

Prevalence and genetic diversity of Rhodococcus equi in wild boars (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) in Poland.

BACKGROUND: Rhodococcus equi is now considered an emerging zoonotic pathogen. Sources and routes of human infection remain unclear but foodborne transmission seems to be the most probable way. Strains of pig or bovine type are most often isolated from human cases and moreover R. equi is present in submaxillary lymph nodes of apparently healthy pigs and wild boars intended for human consumption. The aim of this study was to estimate the prevalence of R. equi in submaxillary lymph nodes in wild boars, roe deer and red deer. RESULTS: Samples were collected from 936 animals and 27 R. equi strains were isolated, from 5.1 % of wild boars (23/452), 0.7 % of red deer (2/272) and 0.9 % of roe deer (2/212). Genetic diversity of all 27 isolates was studied using VspI-PFGE method, resulting in the detection of 25 PFGE patterns and four PFGE clusters. PFGE patterns of the isolates were compared with virulence plasmid types and no concordance was observed. CONCLUSIONS: R. equi was present in wild animal tissues and consumption of the game may be a potential source of R. equi infection for humans. To the authors' best knowledge, this is the first epidemiological report of R. equi prevalence in tissues of roe deer and red deer. However, risk associated with wild ruminant consumption seems marginal. Investigation of R. equi transmission between animals and humans based exclusively on types of virulence plasmids seems to be insufficient to identify sources of R. equi infection for people.