Showing papers in "Gut Pathogens in 2016"

Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples

Abstract: The advent and use of highly sensitive molecular biology techniques to explore the microbiota and microbiome in environmental and tissue samples have detected the presence of contaminating microbial DNA within reagents. These microbial DNA contaminants may distort taxonomic distributions and relative frequencies in microbial datasets, as well as contribute to erroneous interpretations and identifications. We herein report on the occurrence of bacterial DNA contamination within commonly used DNA extraction kits and PCR reagents and the effect of these contaminates on data interpretation. When compared to previous reports, we identified an additional 88 bacterial genera as potential contaminants of molecular biology grade reagents, bringing the total number of known contaminating microbes to 181 genera. Many of the contaminants detected are considered normal inhabitants of the human gastrointestinal tract and the environment and are often indistinguishable from those genuinely present in the sample. Laboratories working on bacterial populations need to define contaminants present in all extraction kits and reagents used in the processing of DNA. Any unusual and/or unexpected findings need to be viewed as possible contamination as opposed to unique findings.

Alterations of the gut microbiome in Chinese patients with systemic lupus erythematosus

Abstract: Systemic lupus erythematosus (SLE) in patients from Spain is associated with intestinal dysbiosis. This study explores whether the alteration of the gut microbiome in SLE patients from China is consistent with the intestinal dysbiosis of SLE patients from Spain. The depletion of Firmicutes and the enrichment of Bacteroidetes in SLE patients from China were consistent with the SLE patients from Spain. Furthermore, we found that nine genera of gut microbiota were SLE-related microorganisms in Chinese subjects. Genera Rhodococcus, Eggerthella, Klebsiella, Prevotella, Eubacterium, Flavonifractor and Incertae sedis were significantly enriched, while genera Dialister and Pseudobutyrivibrio were significantly depleted in SLE patients. Receiver operating characteristic analysis indicated that the nine genera have the potential to distinguish SLE patients from healthy controls. Comparing the dysbiosis of the gut microbiome among SLE patients from China or Spain, may indicate that the gut microbiome profiles of SLE patients are more influenced by disease than ethnicity.

Systematic review of time trends in the prevalence of Helicobacter pylori infection in China and the USA.

Abstract: It has been suggested that the prevalence of Helicobacter pylori infection has stabilized in the USA and is decreasing in China. We conducted a systematic literature analysis to test this hypothesis. PubMed and Embase searches were conducted up to 19 January 2015. Trends in the prevalence of H. pylori infection over time were assessed by regression analysis using Microsoft Excel. Overall, 25 Chinese studies (contributing 28 datasets) and 11 US studies (contributing 11 datasets) were included. There was a significant decrease over time in the H. pylori infection prevalence for the Chinese studies overall (p = 0.00018) and when studies were limited to those that used serum immunoglobulin G (IgG) assays to detect H. pylori infection (p = 0.014; 20 datasets). The weighted mean prevalence of H. pylori infection was 66 % for rural Chinese populations and 47 % for urban Chinese populations. There was a significant trend towards a decreasing prevalence of H. pylori infection for studies that included only urban populations (p = 0.04; 9 datasets). This trend was no longer statistically significant when these studies were further restricted to those that used serum IgG assays to detect H. pylori infection, although this may have been because of low statistical power due to the small number of datasets available for this analysis (p = 0.28; 6 datasets). There were no significant trends in terms of changes in the prevalence of H. pylori infection over time for studies conducted in the USA. In conclusion, the prevalence of H. pylori infection is most likely decreasing in China, due to a combination of increasing urbanization, which we found to be associated with lower H. pylori infection rates, and possibly also decreasing rates of H. pylori infection within urban populations. This will probably result in a gradual decrease in peptic ulcer and gastric cancer rates in China over time.

Gut microbiome alterations in patients with stage 4 hepatitis C

Abstract: Hepatitis C virus (HCV) causes debilitating liver diseases, which may progress to cirrhosis and cancer, and claims 500,000 annual lives worldwide. While HCV epidemiology, pathophysiology, and therapy are being deeply studied, rare attention is given to reciprocal interactions between HCV infection , HCV-induced chronic liver diseases, and the human gut microbiome. As Egypt has the world’s highest prevalence of HCV infections, we launched this study to monitor differences in the gut microbial community composition of Egyptian HCV patients that may affect, or result from, the patients’ liver state. To this end, we analyzed stool samples from six stage 4-HCV patients and eight healthy individuals by high-throughput 16S rRNA gene sequencing using Illumina MiSeq. Overall, the alpha-diversity of the healthy persons’ gut microbiomes was higher than those of the HCV patients. Whereas members of phylum Bacteroidetes were more abundant in HCV patients, healthy individuals had higher abundance of Firmicutes, Proteobacteria, and Actinobacteria. Genus-level analysis showed differential abundance of Prevotella and Faecalibacterium (higher in HCV patients) vs. Ruminococcus and Clostridium (healthy group), indicating that the higher abundance of Bacteroidetes in HCV patients is most likely due to Prevotella overabundance. The probiotic genus, Bifidobacterium, was only observed in the microbiotas of healthy individuals. To the best of our knowledge, this study provides a first overview of major phyla and genera differentiating stage 4-HCV patients from healthy individuals and suggests possible microbiome remodeling in chronic hepatitis C, possibly shaped by bacterial translocation as well as the liver’s impaired role in digestion and protein synthesis. Future studies will investigate the microbiome composition and functional capabilities in more patients while tracing some potential biomarker taxa (e.g., Prevotella, Faecalibacterium vs. Bifidobacterium).

Bacteriophage infections of microbiota can lead to leaky gut in an experimental rodent model

Abstract: Increased intestinal permeability and translocation of gut microbiota from the intestinal lumen to the systemic circulation predispose patients to various diseases and may be one of the main triggers thereof. The role of microbiota in increased intestinal permeability is under intensive investigation. Here, we studied alterations in the host and increased intestinal permeability as a direct effect of treatment with a bacteriophage cocktail. After 10 days of challenge, the rats showed weight loss, messy hair, and decreased activity. Additionally, they displayed a significantly elevated lactulose:mannitol ratio and the level of circulating immune complexes. To our knowledge, this study demonstrates for the first time that increased intestinal permeability may be induced by bacteriophages that affect the microbiota.

Helicobacter pylori strains harboring babA2 from Indian sub population are associated with increased virulence in ex vivo study.

Abstract: The babA2 gene along with the cagA and vacA of Helicobacter pylori has been considered as a risk factor for the disease outcome in certain populations. This study was aimed to understand the role of babA2 of H. pylori with the background of cagA and vacA in disease manifestations in Indian sub population. A total of 114 H. pylori strains isolated from duodenal ulcer (DU) (n = 53) and non-ulcer dyspepsia (NUD) patients (n = 61) were screened for the prevalence of these virulence markers by PCR. The comparative study of IL-8 production and apoptosis were done by co-culturing the AGS cell line with H. pylori strains with different genotypes. Adherence assay was performed with babA2 positive and negative strains. Two isogenic mutants of babA2 were constructed and the aforesaid comparative studies were carried out. PCR results indicated that 90.6 % (48/53), 82 % (50/61) and 73.6 % (39/53) strains from DU patients were positive for cagA, vacA, and babA2, respectively. Whereas the prevalence of these genes in NUD subjects were 70.5 % (43/61); 69.8 % (37/53), and 65.6 % (39/61), respectively. Although adherence to AGS cells was comparable among strains with babA2 positive and negative genotypes, but the triple positive strains could induce highest degree of IL-8 production and apoptosis, followed by the cagA −/vacA −/babA2 + strains and triple negative strains, respectively. The wild type strains showed significantly higher IL-8 induction as well as apoptosis in ex vivo than its isogenic mutant of babA2. PCR study demonstrated that there was no significant association between the distribution of babA2 genotype or of triple positive strains and disease outcome in this sub population. The adherence assay showed that there was no significant difference in the extent of adherence to AGS cells among babA2 positive and negative strains. But the ex vivo study indicated that the triple positive or even the babA2 only positive strains are involved in increased virulence. The wild type strains also exhibited increased virulence compared to the babA2 mutant strains. This inconsistency demonstrated that bacterial genotype along with host genetic polymorphisms or other factors play important role in determining the clinical manifestation of H. pylori infections.

Antimicrobial resistance of Campylobacter isolates from small scale and backyard chicken in Kenya

Abstract: Thermophilic Campylobacter species are a major cause of bacterial foodborne diarrhoea in humans worldwide. Poultry and their products are the predominant source for human campylobacteriosis. Resistance of Campylobacter to antibiotics is increasing worldwide, but little is known about the antibiotic resistance in Campylobacter isolated from chicken in Kenya. In this study, 35 suspected Campylobacter strains isolated from faeces and cloacal swabs of chicken were tested for their susceptibility to seven antibiotics using a broth microdilution assay and molecular biological investigations. Overall, DNA of thermophilic Campylobacter was identified in 53 samples by PCR (34 C. jejuni, 18 C. coli and one mix of both species) but only 35 Campylobacter isolates (31 C. jejuni and 4 C. coli) could be re-cultivated after transportation to Germany. Isolates were tested for their susceptibility to antibiotics using a broth microdilution assay. Additionally, molecular biological detection of antibiotic resistance genes was carried out. C. jejuni isolates showed a high rate of resistance to nalidixic acid, tetracycline and ciprofloxacin of 77.4, 71.0 and 71.0 %, respectively. Low resistance (25.8 %) was detected for gentamicin and chloramphenicol. Multidrug resistance in C. jejuni could be detected in 19 (61.3 %) isolates. Resistance pattern of C. coli isolates was comparable. Resistance to ciprofloxacin was confirmed by MAMA–PCR and PCR–RFLP in all phenotypically resistant isolates. The tet(O) gene was detected only in 54.5 % of tetracycline resistant C. jejuni isolates. The tet(A) gene, which is also responsible for tetracycline resistance, was found in 90.3 % of C. jejuni and in all C. coli isolates. Thirteen phenotypically erythromycin-resistant isolates could not be characterised by using PCR–RFLP and MAMA–PCR. To the best of our knowledge, this study is the first report about resistance to antibiotics in thermophilic Campylobacter originating from chicken in Kenya. Campylobacter spp. show a high level of resistance to ciprofloxacin, nalidixic acid and tetracycline but also a remarkable one to chloramphenicol and gentamicin and they are multidrug resistant. Resistance to antibiotics is a global public health concern. In Kenya, resistance surveillance needs further attention in the future. Efforts to establish at least a National Laboratory with facilities for performing phenotypic and genotypic characterization of thermophilic Campylobacter is highly recommended.

Modeling human enteric dysbiosis and rotavirus immunity in gnotobiotic pigs

Abstract: Rotavirus vaccines have poor efficacy in infants from low- and middle-income countries. Gut microbiota is thought to influence the immune response to oral vaccines. Thus, we developed a gnotobiotic (Gn) pig model of enteric dysbiosis to study the effects of human gut microbiota (HGM) on immune responses to rotavirus vaccination, and the effects of rotavirus challenge on the HGM by colonizing Gn pigs with healthy HGM (HHGM) or unhealthy HGM (UHGM). The UHGM was from a Nicaraguan infant with a high enteropathy score (ES) and no seroconversion following administration of oral rotavirus vaccine, while the converse was characteristic of the HHGM. Pigs were vaccinated, a subset was challenged, and immune responses and gut microbiota were evaluated. Significantly more rotavirus-specific IFN-γ producing T cells were in the ileum, spleen, and blood of HHGM than those in UHGM pigs after three vaccine doses, suggesting HHGM induces stronger cell-mediated immunity than UHGM. There were significant correlations between multiple Operational Taxonomic Units (OTUs) and frequencies of IFN-γ producing T cells at the time of challenge. There were significant positive correlations between Collinsella and CD8+ T cells in blood and ileum, as well as CD4+ T cells in blood, whereas significant negative correlations between Clostridium and Anaerococcus, and ileal CD8+ and CD4+ T cells. Differences in alpha diversity and relative abundances of OTUs were detected between the groups both before and after rotavirus challenge. Alterations in microbiome diversity and composition along with correlations between certain microbial taxa and T cell responses warrant further investigation into the role of the gut microbiota and certain microbial species on enteric immunity. Our results support the use of HGM transplanted Gn pigs as a model of human dysbiosis during enteric infection, and oral vaccine responses.

Molecular epidemiology, virulence determinants and antimicrobial resistance of Campylobacter spreading in retail chicken meat in Central China.

Abstract: Campylobacter species are the major food-borne pathogens which could cause bacterial gastroenteritis in humans. Contaminated chicken products have been recognized as the primary vehicles of Campylobacter transmission to human beings. In this study, the prevalence of Campylobacter in retail chicken meat in Central China was investigated, and the isolates were further characterized using molecular approaches and tested for antibiotic resistance. A total of 302 chicken samples purchased from April 2014 to April 2015 were tested. The level of Campylobacter contamination was enumerated by most probable number-PCR (MPN-PCR). The Campylobacter positive rate was 17.2% (52/302), with bacterial count varying from 3.6 to 360 MPN/g in positive samples. A total of 52 Campylobacter strains, including 40 Campylobacter jejuni and 12 Campylobacter coli, were isolated from the positive samples. To examine the genetic diversity of the isolates, multilocus sequence typing (MLST) technology was applied, which identified 23 sequence types (STs) belonging to seven clonal complexes (CCs) and unassigned. Among them, the dominant CCs of C. jejuni included CC-353 and CC-464, and the dominant CCs of C. coli were CC-828 and CC-1150. Antibiotic resistance analysis showed that all of the isolates were resistant to norfloxacin and ciprofloxacin. 23 virulence-associated genes were tested in the isolates, which showed that the number of virulence-associated genes detected in the C. jejuni isolates ranged from 16 to 21, while in most of the C. coli isolates ranged from 12 to 16. Virulence-associated genes, flaA, flgB, flgE2, fliM, fliY and cadF were detected in all isolates. VirB11, however, was not detected in any of the isolates. To the best of our knowledge, this is the first report on the contamination level and molecular biological features of Campylobacter strains in retail chicken meat in Central China, which showed high genetic diversity and remarkable antibiotic resistance. This study provided scientific data for the risk assessment and evaluation of Campylobacter contamination in retail chicken products.

Impacts of resistant starch and wheat bran consumption on enteric inflammation in relation to colonic bacterial community structures and short-chain fatty acid concentrations in mice

Abstract: Identifying the connection among diet, the intestinal microbiome, and host health is currently an area of intensive research, but the potential of dietary fiber (DF) consumption to ameliorate intestinal inflammation has not been extensively studied. We examined the impacts of the DFs, wheat bran (WB) and resistant starch (RS) on host enteric health. A murine model of acute Th1/Th17 colitis (i.e. incited by Citrobacter rodentium) was used. Diets enriched with RS increased weight gain in mice inoculated with C. rodentium compared to mice consuming a conventional control (CN) diet. Short-chain fatty acid (SCFA) quantities in the cecum and distal colon were higher in mice consuming DFs, and these mice exhibited higher butyrate concentrations in the distal colon during inflammation. Histopathologic scores of inflammation in the proximal colon on day 14 post-inoculation (p.i.) (peak infection) and 21 p.i. (late infection) were lower in mice consuming DF-enriched diets compared to the CN diet. Consumption of WB reduced the expression of Th1/Th17 cytokines. As well, the expression of bacterial recognition and response genes such as Relmβ, RegIIIγ, and Tlr4 increased in mice consuming the RS-enriched diets. Furthermore, each diet generated a region-specific bacterial community, suggesting a link between selection for specific bacterial communities, SCFA concentrations, and inflammation in the murine colon. Collectively, data indicated that the consumption of DF-rich diets ameliorates the effects of C. rodentium-induced enteritis by modifying the host microbiota to increase SCFA production, and bacterial recognition and response mechanisms to promote host health.

Antimicrobial resistance and integron gene cassette arrays in commensal Escherichia coli from human and animal sources in IRI

Abstract: Background The human and animal intestinal tract harbors a complex community of microbes which enables bacteria to inherit antibiotic resistance genes. The aims of this study were to investigate clonality, antimicrobial resistance, prevalence and gene cassette arrays of class I and II integrons among commensal Escherichia coli from human and animals.

Differences in host breed and diet influence colonization by Campylobacter jejuni and induction of local immune responses in chicken

Abstract: Chickens are regarded as the main reservoir for human campylobacteriosis. Little is known about the interaction between Campylobacter jejuni (C. jejuni) and chickens. This interaction may be influenced by the stage of maturation of the immune system, developing gut microbiota composition and other factors including breed and diet. Our aim was to investigate the impact of breed, and diet on C. jejuni colonization and host immune responses in chickens. Birds were inoculated with 104 colony forming units (CFU) of C. jejuni or diluent at one (Exp. 1) or 22 (Exp. 2) days post hatch. We compared local immune cell subpopulations, cytokine expression levels, and gut microbiota composition between broiler-type (BT) and layer-type (LT) birds fed with either commercial broiler feed (bf) or layer feed (lf). Lower colonization rates were observed in the older age group independent of breed and diet. Independent of breed, birds fed with bf showed higher CFU of C. jejuni compared to lf-fed groups. Campylobacter jejuni-inoculation had a significant effect on lymphocyte numbers and cytokine expression levels in BT birds independent of feeding strategy (p < 0.05). These effects were not detected in LT birds, only LT birds fed with bf showed a significant increase in IL-8-expression at 7 days post C. jejuni inoculation compared to LT-control birds (p < 0.05). Diet influenced gut microbiota composition in a comparable manner between BT and LT birds, but changes in microbiota composition associated with C. jejuni inoculation varied between breeds. Diet and breed influenced C. jejuni colonization, immune responses and microbiota composition to a different extent comparing between LT and BT birds. The mechanisms behind these differences have to be elucidated further. Our results suggest that selection for more resistant breeds in combination with adapted feeding strategies may help to reduce Campylobacter colonization levels in commercial poultry in the future.

Optimized cultivation of Campylobacter concisus from gut mucosal biopsies in inflammatory bowel disease

Abstract: Background Campylobacter concisus is a commensal of the human oral flora that has been linked to prolonged diarrhea and inflammatory bowel disease (IBD). It has been detected more often from intestinal biopsies in patients with IBD compared to healthy controls using PCR-based techniques, whereas the number of C. concisus culture-positive biopsies in previous studies has been very limited. Determining the rate of viable isolates present in the gut mucosa is of great importance when evaluating the role in different disease presentations. We therefore investigated a novel two-step cultivation procedure combining anaerobic and microaerobic incubation from several gut mucosal sites to improve isolate yield, and compared this to PCR results, from IBD patients and healthy controls.

Prevalence and antibiotic resistance of bacterial pathogens isolated from childhood diarrhea in Beijing, China (2010–2014)

Abstract: Diarrhea is one of the main causes of morbidity and mortality among children less than 5 years of age worldwide, and its causes vary by region. This study aimed to determine the etiologic spectrum, prevalent characteristics and antimicrobial resistance patterns of common enteropathogenic bacteria from diarrheagenic children in Beijing, the capital of China. Stool samples were collected from 2524 outpatients who were aged 0–5 years in Beijing, China during 2010–2014. Microbiological methods, real-time PCR and antimicrobial susceptibility test were used to identify the bacterial causes and antimicrobial resistance patterns in the isolates. Of the 2524 patients screened, we identified the causes of 269 cases (10.7 %) as follows: diarrheagenic Escherichia coli (4.6 %), Salmonella (4.3 %), Shigella (1.4 %) and Vibrio parahaemolyticus (0.4 %). Atypical EPEC, Salmonella enteritidis, Shigella sonnei and serotype O3:K6 were the most common serogroups or serotypes of the four etiological bacteria. The prevalence of pathogens was correlated with age, season and clinical symptoms. The highest proportion of all causative bacteria was found in children aged 3–5 years and in summer. The clinical symptoms associated with specific bacterial infection, such as fever; abdominal pain; vomiting; and watery, mucus, and bloody stool, were observed frequently in diarrheal patients. Salmonella showed moderate rates of resistance (40–60 %) to ampicillin, nalidixic acid, streptomycin and sulfisoxazole. Resistance to at least three antimicrobials was found in 50 % of isolates. Of the top three serotypes in Salmonella, high-level antimicrobial resistance to single and multiple antibiotics was more common among Salmonella typhimurium and Salmonella 1, 4, [5], 12:i:- than among S. enteritidis. More than 90 % of Shigella isolates showed more alarming resistance to most antibiotics, with a widened spectrum compared to Salmonella. Constant antibiotic surveillance is warranted because the bacteria were highly resistant to various antimicrobials. Our study contributes to the strengthening of the existing surveillance system and provides aid for effective prevention and control strategies for childhood diarrhea.

Examination of the effects of Campylobacter concisus zonula occludens toxin on intestinal epithelial cells and macrophages

Abstract: Campylobacter concisus is a Gram-negative bacterium that is associated with inflammatory bowel disease (IBD). Some C. concisus strains carry zonula occludens toxin (zot) gene which has polymorphisms. This study investigated the effects of C. concisus Zot on intestinal epithelial cells and macrophages using cell line models. Campylobacter concisus zot 808T gene, a polymorphism that is associated with active IBD, was cloned and expressed in Escherichia coli. The effects of C. concisus Zot on intestinal epithelial barrier were examined using Caco-2 cell model. Apoptosis induced by C. concisus Zot in Caco-2 cells was assessed by measuring the levels of caspase 3/7. The production of pro-inflammatory cytokines induced by C. concisus Zot in HT-29 cells and in THP-1 macrophage-like cells was measured using ELISA kits. Whether exposure to C. concisus Zot can affect the responses of macrophages to E. coli K12 was also investigated. Campylobacter concisus Zot caused prolonged intestinal epithelial barrier damage, induced intestinal epithelial cell apoptosis, induced epithelial production of TNF-α and IL-8 and upregulated TNF-α in THP-1 macrophage-like cells. Pre-exposure to C. concisus Zot significantly enhanced the production of TNF-α and IL-8 as well as phagocytosis by THP-1 macrophage-like cells in response to E. coli K12. This study suggests that C. concisus Zot may have enteric pathogenic potential by damaging intestinal epithelial barrier, inducing intestinal epithelial and macrophage production of proinflammatory cytokines in particular TNF-α and enhancing the responses of macrophages to other enteric bacterial species.

Genotyping and antibiotic resistance of thermophilic Campylobacter isolated from chicken and pig meat in Vietnam

Abstract: Campylobacter species are recognized as the most common cause of foodborne bacterial gastroenteritis in humans. In this study nine Campylobacter strains isolated from chicken meat and pork in Hanoi, Vietnam, were characterized using molecular methods and tested for antibiotic resistance. The nine isolates (eight C. jejuni and one C. coli) were identified by multiplex PCR, and tested for the presence or absence of 29 gene loci associated with virulence, lipooligosaccharide (LOS) biosynthesis and further functions. flaA typing, multilocus sequence typing and microarray assay investigation showed a high degree of genetic diversity among these isolates. In all isolates motility genes (flaA, flaB, flhA, fliM), colonization associated genes (cadF, docB), toxin production genes (cdtA, cdtB, secD, secF), and the LOS biosynthesis gene pglB were detected. Eight gene loci (fliY, virB11, Cje1278, Cj1434c, Cj1138, Cj1438c, Cj1440c, Cj1136) could not be detected by PCR. A differing presence of the gene loci ciaB (22.2 %), Cje1280 (77.8 %), docC (66.7 %), and cgtB (55.6 %) was found. iamA, cdtC, and the type 6 secretion system were present in all C. jejuni isolates but not in C. coli. flaA typing resulted in five different genotypes within C. jejuni, MLST classified the isolates into seven sequence types (ST-5155, ST-6736, ST-2837, ST-4395, ST-5799, ST-4099 and ST-860). The microarray assay analysis showed a high genetic diversity within Vietnamese Campylobacter isolates which resulted in eight different types for C. jejuni. Antibiotic susceptibility profiles showed that all isolates were sensitive to gentamicin and most isolates (88.8 %) were sensitive to chloramphenicol, erythromycin and streptomycin. Resistance rates to nalidixic acid, tetracycline and ciprofloxacin were 88.9, 77.8 and 66.7 %, respectively. To the best of our knowledge, this study is the first report that shows high genetic diversity and remarkable antibiotic resistance of Campylobacter strains isolated from meat in Vietnam which can be considered of high public health significance. These preliminary data show that large scale screenings are justified to assess the relevance of Campylobacter infections on human health in Vietnam.

Whole genome sequencing of “Faecalibaculum rodentium” ALO17, isolated from C57BL/6J laboratory mouse feces

Abstract: Intestinal microorganisms affect host physiology, including ageing. Given the difficulty in controlling for human studies of the gut microbiome, mouse models provide an alternative avenue to study such relationships. In this study, we report on the complete genome of “Faecalibaculum rodentium” ALO17, a bacterium that was isolated from the faeces of a 9-month-old female C57BL/6J mouse. This strain will be utilized in future in vivo studies detailing the relationships between the gut microbiome and ageing. The whole genome sequence of “F. rodentium” ALO17 was obtained using single-molecule, real-time (SMRT) technique on a PacBio instrument. The assembled genome consisted of 2,542,486 base pairs of double-stranded DNA with a GC content of 54.0 % and no plasmids. The genome was predicted to contain 2794 open reading frames, 55 tRNA genes, and 38 rRNA genes. The 16S rRNA gene of ALO17 was 86.9 % similar to that of Allobaculum stercoricanis DSM 13633T, and the average overall nucleotide identity between strains ALO17 and DSM 13633T was 66.8 %. After confirming the phylogenetic relationship between “F. rodentium” ALO17 and A. stercoricanis DSM 13633T, their whole genome sequences were compared, revealing that “F. rodentium” ALO17 contains more fermentation-related genes than A. stercoricanis DSM 13633T. Furthermore, “F. rodentium” ALO17 produces higher levels of lactic acid than A. stercoricanis DSM 13633T as determined by high-performance liquid chromatography. The availability of the “F. rodentium” ALO17 whole genome sequence will enhance studies concerning the gut microbiota and host physiology, especially when investigating the molecular relationships between gut microbiota and ageing.

Dysfunctional gut microbiota and relative co-abundance network in infantile eczema.

Abstract: Infantile eczema is an immunological disease that is characterized by itchy and dry skin. Recent studies have suggested that gut microbiota (GM) plays a role in the development and progression of eczema. To further evaluate this potential link, we collected feces from 19 infants with eczema and 14 infants without eczema and analyzed the molecular discrepancies between the two groups using 16S rDNA analysis. Bacteroidaceae and Deinococcaceae were significantly enriched in eczema infants, and Bacteroidaceae was potentially involved in autoimmune diseases by promoting the Th17 (T helper cell 17) secretion of IL-17 (interleukin-17). In the infants without eczema, the co-abundance network featured three core nodes: Clostridiaceae, Veillonellaceae, and Lactobacillaceae, all of which were lacking in the infants with eczema. Furthermore, our data suggested that Enterobacteriaceae was the core of the co-abundance network for the diseased subjects. GM is closely connected to the human immune system, and the dysbiotic GM network plays a role in eczema. This study furthered our understanding of the dynamic GM network and its correlation to the occurrence of eczema.

Global investigation of composition and interaction networks in gut microbiomes of individuals belonging to diverse geographies and age-groups.

Abstract: Factors like ethnicity, diet and age of an individual have been hypothesized to play a role in determining the makeup of gut microbiome. In order to investigate the gut microbiome structure as well as the inter-microbial associations present therein, we have performed a comprehensive global comparative profiling of the structure (composition, relative heterogeneity and diversity) and the inter-microbial networks in the gut microbiomes of 399 individuals of eight different nationalities. The study identified certain geography-specific trends with respect to composition, intra-group heterogeneity and diversity of the gut microbiomes. Interestingly, the gut microbial association/mutual-exlusion networks were observed to exhibit several cross-geography trends. It was seen that though the composition of gut microbiomes of the American and European individuals were similar, there were distinct patterns in their microbial interaction networks. Amongst European gut-microbiomes, the co-occurrence network obtained for the Danish population was observed to be most dense. Distinct patterns were also observed within Chinese, Japanese and Indian datasets. While performing an age-wise comparison, it was observed that the microbial interactions increased with the age of individuals. Furthermore, certain bacterial groups were identified to be present only in the older age groups. The trends observed in gut microbial networks could be due to the inherent differences in the diet of individuals belonging to different nationalities. For example, the higher number of microbial associations in the Danish population as compared to the Spanish population, may be attributed to the evenly distributed diet of the later. This is in line with previously reported findings which indicate an increase in functional interdependency of microbes in individuals with higher nutritional status. To summarise, the present study identifies geography and age specific patterns in the composition as well as microbial interactions in gut microbiomes.

Duck gut viral metagenome analysis captures snapshot of viral diversity

Abstract: Ducks (Anas platyrhynchos) an economically important waterfowl for meat, eggs and feathers; is also a natural reservoir for influenza A viruses. The emergence of novel viruses is attributed to the status of co-existence of multiple types and subtypes of viruses in the reservoir hosts. For effective prediction of future viral epidemic or pandemic an in-depth understanding of the virome status in the key reservoir species is highly essential. To obtain an unbiased measure of viral diversity in the enteric tract of ducks by viral metagenomic approach, we deep sequenced the viral nucleic acid extracted from cloacal swabs collected from the flock of 23 ducks which shared the water bodies with wild migratory birds. In total 7,455,180 reads with average length of 146 bases were generated of which 7,354,300 reads were de novo assembled into 24,945 contigs with an average length of 220 bases and the remaining 100,880 reads were singletons. The duck virome were identified by sequence similarity comparisons of contigs and singletons (BLASTx E score, <10−3) against viral reference database. Numerous duck virome sequences were homologous to the animal virus of the Papillomaviridae family; and phages of the Caudovirales, Inoviridae, Tectiviridae, Microviridae families and unclassified phages. Further, several duck virome sequences had homologous with the insect viruses of the Poxviridae, Alphatetraviridae, Baculoviridae, Densovirinae, Iflaviridae and Dicistroviridae families; and plant viruses of the Secoviridae, Virgaviridae, Tombusviridae and Partitiviridae families, which reflects the diet and habitation of ducks. This study increases our understanding of the viral diversity and expands the knowledge about the spectrum of viruses harboured in the enteric tract of ducks.

The Salmonella pathogenicity island 13 contributes to pathogenesis in streptomycin pre-treated mice but not in day-old chickens

Abstract: Salmonella enterica serovar Enteritidis (S. Enteritidis) is a human and animal pathogen that causes gastroenteritis characterized by inflammatory diarrhea and occasionally an invasive systemic infection. Salmonella pathogenicity islands (SPIs) are horizontally acquired genomic segments known to contribute to Salmonella pathogenesis. The objective of the current study was to determine the contribution of SPI-13 to S. Enteritidis pathogenesis. We deleted the entire SPI-13 (∆SPI-13) from the genome of S. Enteritidis CDC_2010K_0968 strain isolated from a human patient during the 2010 egg-associated outbreak in the US. The kinetics of infection of the wild-type parent and the ∆SPI-13 were compared in orally challenged day-old chickens and streptomycin pre-treated mice. The degree of intestinal inflammation and the survival of mutant strain within the avian (HD11) and murine (RAW264.7) macrophages were also determined. The deletion of the SPI-13 resulted in impaired infection kinetics of S. Enteritidis in streptomycin pre-treated mice which was characterized by significantly lower (P < 0.05) viable counts in the ceca, liver and spleen, impaired ability to induce intestinal inflammation and reduced survival within murine macrophages. Conversely, there were no significant differences in the infection kinetics of ∆SPI-13 in day-old chickens in any of the organs tested and the survival of ∆SPI-13 within chicken macrophages remained unaltered. The results of this study show that SPI-13 contributes to the pathogenesis of S. Enteritidis in streptomycin pre-treated mice but not in day-old chickens and raises the possibility that SPI-13 may play a role in pathogenesis and the host adaptation/restriction of Salmonella serovars.

The efficacy of thuricin CD, tigecycline, vancomycin, teicoplanin, rifampicin and nitazoxanide, independently and in paired combinations against Clostridium difficile biofilms and planktonic cells

Abstract: Thuricin CD is a two-component antimicrobial, belonging to the recently designated sactibiotic subclass of bacteriocins. The aim of this study was to investigate the effects of thuricin CD, as well as the antibiotics, tigecycline, vancomycin, teicoplanin, rifampicin and nitazoxanide when used independently and when combined at low concentrations on the viability of Clostridium difficile 20291 R027, TL178 R002, Liv022 R106, DPC6350 and VPI10463 biofilms and planktonic cells. On the basis of XTT (2,3-bis[2-methyloxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide)-menadione biofilm viability assays, we found that thuricin CD was effective against biofilms of R027, Liv022 R106 and DPC6350 when used independently while nitazoxanide and rifampicin were also potent against biofilms of R027 and DPC6350, when applied on their own. Tigecycline was found to be effective against R027 and DPC6350 biofilms, whereas teicoplanin and vancomycin when used independently were only effective against DPC6350 biofilms. The efficacies of the antibiotics rifampicin, tigecycline, vancomycin and teicoplanin against C. difficile 20291 R027 biofilms were significantly potentiated when combined with thuricin CD, indicating effective antimicrobial combinations with this sactibiotic against R027 biofilms. However, the potency of nitazoxanide against R027 biofilms was significantly diminished when combined with thuricin CD, indicating an ineffective combination with this sactibiotic against R027 biofilms. Paired combinations of thuricin CD along with these five antibiotics were effective at diminishing the viability of DPC6350 biofilms. However, such combinations were largely ineffective against biofilms of TL178 R002, Liv022 R106 and VPI10463. To the best of our knowledge, this is the first study to highlight the activity of a sactibiotic bacteriocin against biofilms and the first to reveal the potency of the antibiotics tigecycline, teicoplanin and nitazoxanide against C. difficile biofilms. On the basis of this study, it is apparent that different strains of C. difficile possess varying abilities to form biofilms and that the sensitivities of these biofilms to different antimicrobials and antimicrobial combinations are strain-dependent. Since the formation of relatively strong biofilms by certain C. difficile strains may contribute to increased cases of antibiotic resistance and recurrence and relapse of C. difficile infection, the findings presented in this study could provide alternative strategies to target this pathogen.

Case–control study of diarrheal disease etiology in individuals over 5 years in southwest China

Abstract: Acute diarrhea is one of the major public health problems worldwide. Most of studies on acute diarrhea have been made on infants aged below 5 years and few efforts have been made to identify the etiological agents of acute diarrhea in people over five, especially in China. 271 diarrhea cases and 149 healthy controls over 5 years were recruited from four participating hospitals between June 2014 and July 2015. Each stool specimen was collected to detect a series of enteric pathogens, involving five viruses (Rotavirus group A, RVA; Norovirus, NoV; Sapovirus, SaV; Astrovirus, As; and Adenovirus, Ad), seven bacteria (diarrheagenic Escherichia coli, DEC; non-typhoidal Salmonella, NTS; Shigella spp.; Vibrio cholera; Vibrio parahaemolyticus; Aeromonas spp.; and Plesiomonas spp.) and three protozoa (Cryptosporidium spp., Giardia lamblia, G. lamblia, and Blastocystis hominis, B. hominis). Standard microbiological and molecular methods were applied to detect these pathogens. Data was analyzed using Chi square, Fisher-exact tests and logistic regressions. The prevalence of at least one enteric pathogen was detected in 29.2% (79/271) acute diarrhea cases and in 12.1% (18/149) in healthy controls (p < 0.0001). Enteric viral infections (14.4%) were the most common in patients suffering from acute diarrhea, followed by bacteria (13.7%) and intestinal protozoa (4.8%). DEC (12.5%) was the most common causative agent in diarrhea cases, followed by NoV GII (10.0%), RVA (7.4%) and B. hominis (4.8%). The prevalence of co-infection was statistically higher (p = 0.0059) in the case group (7.7%) than in the healthy control (1.3%). RVA–NoV GII (3.0%) was the most common co-infection in symptomatic cases. DEC was the most predominant pathogen in diarrhea cases, but it was largely overlooked because the lack of laboratory capacities. Because of the high prevalence of co-infections, it is recommended the urgent development of alternative laboratory methods to assess polymicrobial infections. Such methodological improvements will result in a better prevention and treatment strategies to control diarrhea illness in China.

Oxidative stress due to Mycobacterium avium subspecies paratuberculosis (MAP) infection upregulates selenium-dependent GPx activity

Abstract: This study was designed to determine the relationship between Mycobacterium avium subspecies paratuberculosis (MAP) infection and selenium-dependent glutathione peroxidase (GPx) activity, in the blood of humans and cattle infected with MAP. MAP infection status and GPx activity were determined in sera from 42 cattle, a group of 27 patients with Crohn’s disease and 27 of their healthy biological relatives, and a group of 66 subjects with various diseases other than Crohn’s disease and 34 non-related healthy subjects. GPx activity was significantly higher overall in the case of MAP infection in both humans and cattle. The mean value for GPx activity was 1.59 ± 0.65 units/ml in MAP positive cattle compared to 0.46907 ± 0.28 units/ml in healthy cattle sera, where a unit was defined as one mmol/minute (P < 0.01). The mean value of the GPx activity in MAP negative humans clinical sera was 0.42367 ± 0.229 units/ml compared to 0.80941 ± 0.521 in MAP positive sera in a study comparing Crohn’s disease patients to their healthy relatives. The mean activity in MAP negative humans was 0.4702 ± 0.1299 compared to 0.6510 ± 00.1665 units/ml in positive samples in a randomized field study of 100 subjects. This study demonstrated a strong correlation between MAP and the elevation of GPx activity. This was especially evident in Crohn’s patients, which further supports the association of MAP and Crohn’s disease. GPx activity may also be used to predict MAP infection status and to show that Crohn’s disease patients who are infected with MAP have higher tendency to develop oxidative stress than Crohn’s disease patients who are negative for the bacteria.

Comparative genomic analysis of Klebsiella pneumoniae subsp. pneumoniae KP617 and PittNDM01, NUHL24835, and ATCC BAA-2146 reveals unique evolutionary history of this strain

Abstract: Klebsiella pneumoniae subsp. pneumoniae KP617 is a pathogenic strain that coproduces OXA-232 and NDM-1 carbapenemases. We sequenced the genome of KP617, which was isolated from the wound of a Korean burn patient, and performed a comparative genomic analysis with three additional strains: PittNDM01, NUHL24835 and ATCC BAA-2146. The complete genome of KP617 was obtained via multi-platform whole-genome sequencing. Phylogenetic analysis along with whole genome and multi-locus sequence typing of genes of the Klebsiella pneumoniae species showed that KP617 belongs to the WGLW2 group, which includes PittNDM01 and NUHL24835. Comparison of annotated genes showed that KP617 shares 98.3 % of its genes with PittNDM01. Nineteen antibiotic resistance genes were identified in the KP617 genome: bla OXA-1 and bla SHV-28 in the chromosome, bla NDM-1 in plasmid 1, and bla OXA-232 in plasmid 2 conferred resistance to beta-lactams; however, colistin- and tetracycline-resistance genes were not found. We identified 117 virulence factors in the KP617 genome, and discovered that the genes encoding these factors were also harbored by the reference strains; eight genes were lipopolysaccharide-related and four were capsular polysaccharide-related. A comparative analysis of phage-associated regions indicated that two phage regions are specific to the KP617 genome and that prophages did not act as a vehicle for transfer of antimicrobial resistance genes in this strain. Whole-genome sequencing and bioinformatics analysis revealed similarity in the genome sequences and content, and differences in phage-related genes, plasmids and antimicrobial resistance genes between KP617 and the references. In order to elucidate the precise role of these factors in the pathogenicity of KP617, further studies are required.

Zonula occludens toxins and their prophages in Campylobacter species

Abstract: We previously showed that zonula occludens toxin (Zot) encoded by Campylobacter concisus zot 808T gene has the potential to initiate inflammatory bowel disease. This Zot protein caused prolonged intestinal epithelial barrier damage, induced intestinal epithelial and macrophage production of tumor necrosis factor-α and enhanced the responses of macrophages to other microbes. In order to understand the potential virulence of Zot proteins in other Campylobacter species, in this study we examined their presence, similarities, motifs and prophages. The presence of Zot proteins in Campylobacter species was examined by searching for the Zot family domain in multiple protein databases. Walker A and Walker B motifs in Zot proteins were identified using protein sequence alignment. A phylogenetic tree based on Campylobacter zot genes was constructed using maximum-likelihood method. Campylobacter Zot proteins were compared using protein sequence alignment. The zot-containing prophages in Campylobacter species were identified and compared with known prophage proteins and other viral proteins using protein sequence alignment and protein BLAST. Twelve Zot proteins were found in nine Campylobacter species/subspecies. Among these Campylobacter species, three species had two Zot proteins and the remaining six species/subspecies had one Zot protein. Walker A and Walker B motifs and a transmembrane domain were found in all identified Campylobacter Zot proteins. The twelve Campylobacter zot genes from the nine Campylobacter species/subspecies formed two clusters. The ZotCampyType_1 proteins encoded by Cluster 1 Campylobacter zot genes showed high similarities to each other. However, ZotCampyType_2 proteins encoded by Cluster 2 Campylobacter zot genes were more diverse. Furthermore, the zot-containing Campylobacter prophages were identified. This study reports the identification of two types of Campylobacter Zot proteins. The high similarities of ZotCampyType_1 proteins suggest that they are likely to have similar virulence. ZotCampyType_2 proteins are less similar to each other and their virulent properties, if any, remain to be examined individually.

Host, pathogen and the environment: the case of Macrobrachium rosenbergii, Vibrio parahaemolyticus and magnesium

Abstract: Macrobrachium rosenbergii is well-known as the giant freshwater prawn, and is a commercially significant source of seafood. Its production can be affected by various bacterial contaminations. Among which, the genus Vibrio shows a higher prevalence in aquatic organisms, especially M. rosenbergii, causing food-borne illnesses. Vibrio parahaemolyticus, a species of Vibrio is reported as the main causative of the early mortality syndrome. Vibrio parahaemolyticus infection in M. rosenbergii was studied previously in relation to the prawn’s differentially expressed immune genes. In the current review, we will discuss the growth conditions for both V. parahaemolyticus and M. rosenbergii and highlight the role of magnesium in common, which need to be fully understood. Till date, there has not been much research on this aspect of magnesium. We postulate a model that screens a magnesium-dependent pathway which probably might take effect in connection with N-acetylglucosamine binding protein and chitin from V. parahaemolyticus and M. rosenbergii, respectively. Further studies on magnesium as an environment for V. parahaemolyticus and M. rosenbergii interaction studies will provide seafood industry with completely new strategies to employ and to avoid seafood related contaminations.

Calcium binding protects E-cadherin from cleavage by Helicobacter pylori HtrA

Abstract: The cell adhesion and tumor suppressor protein E-cadherin is an important factor in the establishment and maintenance of epithelial integrity. E-cadherin is a single transmembrane protein, which consists of an intracellular domain (IC), a transmembrane domain (TD), and five extracellular domains (EC). EC domains form homophilic interactions in cis and trans that require calcium binding to the linker region between the EC domains. In our previous studies, we identified the serine protease high temperature requirement A (HtrA) from the human pathogen and class-I carcinogen Helicobacter pylori (H. pylori) as a bacterial E-cadherin-cleaving protease that targets the linker region of the EC domains, thereby disrupting gastric epithelial integrity. However, it remains unclear how calcium binding to the E-cadherin linker regions affects HtrA-mediated cleavage. Investigating the influence of calcium on the HtrA-mediated cleavage of recombinant E-cadherin (rCdh1) in vitro, we tested different concentrations of calcium ions and the calcium chelator ethylenediaminetetraacetic acid (EDTA). Calcium efficiently reduced HtrA-mediated E-cadherin fragmentation. Conversely, the addition of EDTA strongly increased cleavage, resulting in a ladder of defined E-cadherin fragments. However, calcium ions did not affect HtrA oligomerization and protease activity as monitored by degradation of the universal protease substrate casein. Finally, addition of ethyleneglycol-bis-tetraacetic acid (EGTA) slightly enhanced E-cadherin cleavage during H. pylori infection of gastric epithelial cells. Our results suggest that calcium blocks HtrA-mediated cleavage by interfering with the accessibility of calcium-binding regions between the individual EC domains, which have been identified as cleavage sites of HtrA.

Serotype epidemiology and multidrug resistance patterns of Salmonella enterica infecting humans in Italy.

Abstract: Background Salmonella enterica is the zoonotic agent most frequently responsible for foodborne infections in humans worldwide. In this work the presence of S. enterica was investigated in 734 unique enteropathogenic isolates collected from human patients between 2011 and 2012.

Genome sequence of OXA-23 producing Acinetobacter baumannii IHIT7853, a carbapenem-resistant strain from a cat belonging to international clone IC1

Abstract: Multidrug resistance in Acinetobacter baumannii has dramatically increased in recent years worldwide. Thus, last-line antibiotics like carbapenems are increasingly being used which in turn further augments selection pressure for resistant strains. Resistance to carbapenems in A. baumannii is frequently mediated by carbapenemases, particularly OXA-23 and OXA-58. Carbapenemase-producing bacteria are mainly described in human patients and the intestinal tract represents a common source for such pathogens. In this study, we sequenced and analyzed the genome of A. baumannii IHIT7853, a carbapenem-resistant, OXA-23 producing strain isolated from cystitis in a cat in 2000 in Germany. Phylogenetic analysis revealed that IHIT7853 belonged to the globally distributed international clone IC1 and MLST type ST1/ST231 (Pasteur/Oxford MLST scheme). A phylogenetic tree based on the maximum common genome of 18 A. baumannii isolates placed IHIT7853 close to human clinical isolates, such as the multidrug-resistant (MDR) outbreak strain AYE that was isolated from a patient with pneumonia and cystitis in 2001 in France. The OXA-23 plasmid sequence could be determined as 53,995 bp in size, possessing resistance genes strA and strB in addition to bla OXA-23. The analysis of the genome of IHIT7853 reveals that companion animals carry MDR A. baumannii that resemble relevant clonal lineages involved in severe infections in humans. As urinary tract infections are often caused by bacteria that reside in the intestinal tract, future studies should unveil, if the animal gut serves as a source for MDR A. baumannii.