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1.  Draft genome sequences of the type strains of Shigella flexneri held at Public Health England: comparison of classical phenotypic and novel molecular assays with whole genome sequence 
Gut Pathogens  2014;6:7.
Public Health England (PHE) holds a collection of Shigella flexneri Type strains isolated between 1949 and 1972 representing 15 established serotypes and one provisional type, E1037. In this study, the genomes of all 16 PHE Type strains were sequenced using the Illumina HiSeq platform. The relationship between core genome phylogeny and serotype was examined.
The most common target gene for the detection of Shigella species in clinical PCR assays, ipaH, was detected in all genomes. The type-specific target genes were correctly identified in each genome sequence. In contrast to the S. flexneri in serotype 5 strain described by Sun et al. (2012), the two PHE serotype 5 Type strains possessed an additional oac gene and were differentiated by the presence (serotype 5b) or absence (serotype 5a) of gtrX. The somatic antigen structure and phylogenetic relationship were broadly congruent for strains expressing serotype specific antigens III, IV and V, but not for those expressing I and II. The whole genome phylogenies of the 15 isolates sequenced showed that the serotype 6 Type Strain was phylogenetically distinct from the other S. flexneri serotypes sequenced. The provisional serotype E1037 fell within the serotype 4 clade, being most closely related to the Serotype 4a Type Strain.
The S. flexneri genome sequences were used to evaluate phylogenetic relationships between Type strains and validate genotypic and phenotypic assays. The analysis confirmed that the PHE S. flexneri Type strains are phenotypically and genotypically distinct. Novel variants will continue to be added to this archive.
PMCID: PMC3972513  PMID: 24684748
Shigella flexneri type strains; Next generation sequencing technology; Molecular serotyping
2.  Genomic characterization of a Helicobacter pylori isolate from a patient with gastric cancer in China 
Gut Pathogens  2014;6:5.
Helicobacter pylori is well known for its relationship with the occurrence of several severe gastric diseases. The mechanisms of pathogenesis triggered by H. pylori are less well known. In this study, we report the genome sequence and genomic characterizations of H. pylori strain HLJ039 that was isolated from a patient with gastric cancer in the Chinese province of Heilongjiang, where there is a high incidence of gastric cancer. To investigate potential genomic features that may be involved in pathogenesis of carcinoma, the genome was compared to three previously sequenced genomes in this area.
We obtained 42 contigs with a total length of 1,611,192 bp and predicted 1,687 coding sequences. Compared to strains isolated from gastritis and ulcers in this area, 10 different regions were identified as being unique for HLJ039; they mainly encoded type II restriction-modification enzyme, type II m6A methylase, DNA-cytosine methyltransferase, DNA methylase, and hypothetical proteins. A unique 547-bp fragment sharing 93% identity with a hypothetical protein of Helicobacter cinaedi ATCC BAA-847 was not present in any other previous H. pylori strains. Phylogenetic analysis based on core genome single nucleotide polymorphisms shows that HLJ039 is defined as hspEAsia subgroup, which belongs to the hpEastAsia group.
DNA methylations, variations of the genomic regions involved in restriction and modification systems, are the “hot” regions that may be related to the mechanism of H. pylori-induced gastric cancer. The genome sequence will provide useful information for the deep mining of potential mechanisms related to East Asian gastric cancer.
PMCID: PMC3938082  PMID: 24565107
Helicobacter pylori; Gastric cancer; Next generation sequencing; Genomic features
3.  Genome sequence and comparative analysis of a Vibrio cholerae O139 strain E306 isolated from a cholera case in China 
Gut Pathogens  2014;6:3.
Vibrio cholerae is a human intestinal pathogen and V. cholerae of the O139 serogroups are responsible for the current epidemic cholera in China. In this work, we reported the whole genome sequencing of a V. cholerae O139 strain E306 isolated from a cholera patient in the 306th Hospital of PLA, Beijing, China.
We obtained the draft genome of V. cholerae O139 strain E306 with a length of 4,161,908 bps and mean G + C content of 47.7%. Phylogenetic analysis indicated that strain E306 was very close to another O139 strain, V. cholerae MO10, which was isolated during the cholera outbreak in India and Bangladesh. However, unlike MO10, strain E306 harbors the El Tor-specific RS1 element with no pre-CTX prophage (VSK), very similar to those found in some V. cholerae O1 strains. In addition, strain E306 contains a SXT/R391 family integrative conjugative element (ICE) similar to ICEVchInd4 and SXT MO10, and it carries more antibiotic resistance genes than other closest neighbors.
The genome sequence of the V. cholerae O139 strain E306 and its comparative analysis with other V. cholerae strains we present here will provide important information for a better understanding of the pathogenicity of V. cholerae and their molecular mechanisms to adapt different environments.
PMCID: PMC3923101  PMID: 24517211
Cholera toxin prophage; Integrative conjugative elements; Antibiotic resistance genes
4.  Variations in motility and biofilm formation of Salmonella enterica serovar Typhi 
Gut Pathogens  2014;6:2.
Salmonella enterica serovar Typhi (S. Typhi) exhibits unique characteristics as an intracellular human pathogen. It causes both acute and chronic infection with various disease manifestations in the human host only. The principal factors underlying the unique lifestyle of motility and biofilm forming ability of S. Typhi remain largely unknown. The main objective of this study was to explore and investigate the motility and biofilm forming behaviour among S. Typhi strains of diverse background.
Swim and swarm motility tests were performed with 0.25% and 0.5% agar concentration, respectively; while biofilm formation was determined by growing the bacterial cultures for 48 hrs in 96-well microtitre plate. While all S. Typhi strains demonstrated swarming motility with smooth featureless morphology, 58 out of 60 strains demonstrated swimming motility with featureless or bull’s eye morphology. Interestingly, S. Typhi strains of blood-borne origin exhibited significantly higher swimming motility (P < 0.05) than stool-borne strains suggesting that swimming motility may play a role in the systemic invasion of S. Typhi in the human host. Also, stool-borne S. Typhi displayed a negative relationship between motility and biofilm forming behaviour, which was not observed in the blood-borne strains.
In summary, both swimming and swarming motility are conserved among S. Typhi strains but there was variation for biofilm forming ability. There was no difference observed in this phenotype for S. Typhi strains from diverse background. These findings serve as caveats for future studies to understand the lifestyle and transmission of this pathogen.
PMCID: PMC3922113  PMID: 24499680
Salmonella Typhi; Biofilm; Motility swarming; Swimming
5.  Prevalence of antibiotic resistance in lactic acid bacteria isolated from the faeces of broiler chicken in Malaysia 
Gut Pathogens  2014;6:1.
Probiotics are commonly used as feed additive to substitute antibiotic as growth promoter in animal farming. Probiotic consists of lactic acid bacteria (LAB), which enhance the growth and health of the animal. Probiotic also have higher possibility to become pathogenic bacteria that may carry antibiotic resistant gene that can be transmitted to other LAB species. The aim of this study was to identify the LAB species in the faeces of broiler chicken and to determine the prevalence of antibiotic resistant in LAB of broiler chicken.
Sixty faeces samples were collected from wet markets located in Klang Valley of Malaysia for the isolation of LAB using de-Mann Rogosa Sharpe medium. Thirteen species of LAB were obtained in this study and the identification of LAB was performed by using API test kit on the basis of carbohydrate fermentation profile. Antibiotic susceptibility assay was then carried out to determine the prevalence of LAB antibiotic resistance.
Lactococcus lactis subsp lactis was found in nine out of sixty faecal samples. Lactobacillus paracasei was the second common LAB species isolated from chicken faecal. No significant difference (P > 0.05) was found between the occurrence of Lactobacillus brevis, Lactobacillus curvatus, Lactobacillus plantarum, Leuconostoc lactis mesenteroides subsp mesenteroides/dectranium and Pediococcus pentosaceus isolated from 5 different locations. Most of the isolated LAB was resistant to antibiotic and high variability of the antibiotic resistance was observed among the LAB against 15 types of antibiotics. Penicillin, amoxicillin, chloramphenicol, and ampicillin had significant higher (P< 0.05) inhibitory zone than nalidixic acid, gentamycin, sulphamethoxazole, kanamycin, and streptomycin.
Many species of LAB were isolated from the faecal samples of broiler chicken that resistance to the common antibiotics used in the farm. The development of resistant against antibiotics in LAB can be attributed to the long term exposure of antibiotic as growth promoter and therapeutic agents. Thus, it is essential to advise farmer the safety measure of antibiotic application in animal farming. Additionally, the supplementation of probiotic in animal feeding also needs more attention and close monitoring.
PMCID: PMC3902413  PMID: 24447766
Lactic acid bacteria; Antibiotic resistance; Broiler chicken
6.  Role of StdA in adhesion of Salmonella enterica serovar Enteritidis phage type 8 to host intestinal epithelial cells 
Gut Pathogens  2013;5:43.
Salmonella is often implicated in foodborne outbreaks, and is a major public health concern in the United States and throughout the world. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Adhesion to epithelial cells in the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified stdA as a potential adhesion mutant of SE. Therefore, we hypothesize StdA plays a significant role in adhesion of SE to the intestinal mucosa of poultry.
Methods and results
To test our hypothesis, we created a mutant of SE in which stdA was deleted. Growth and motility were assayed along with the in vitro and in vivo adhesion ability of the ∆stdA when compared to the wild-type SE strain. Our data showed a significant decrease in motility in ∆stdA when compared to the wild-type and complemented strain. A decrease in adhesion to intestinal epithelial cells as well as in the small intestine and cecum of poultry was observed in ∆stdA. Furthermore, the lack of adhesion correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens.
These studies suggest StdA is a major contributor to the adhesion of Salmonella to the intestinal mucosa of poultry.
PMCID: PMC3877977  PMID: 24367906
Salmonella; Adhesion; StdA; Poultry
7.  Draft genome sequences of two Bifidobacterium sp. from the honey bee (Apis mellifera) 
Gut Pathogens  2013;5:42.
Widely considered probiotic organisms, Bifidobacteria are common inhabitants of the alimentary tract of animals including insects. Bifidobacteria identified from the honey bee are found in larval guts and throughout the alimentary tract, but attain their greatest abundance in the adult hind gut. To further understand the role of Bifidobacteria in honey bees, we sequenced two strains of Bifidobacterium cultured from different alimentary tract environments and life stages.
Reflecting an oxygen-rich niche, both strains possessed catalase, peroxidase, superoxide-dismutase and respiratory chain enzymes indicative of oxidative metabolism. The strains show markedly different carbohydrate processing capabilities, with one possessing auxiliary and key enzymes of the Entner-Doudoroff pathway.
As a result of long term co-evolution, honey bee associated Bifidobacterium may harbor considerable strain diversity reflecting adaptation to a variety of different honey bee microenvironments and hive-mediated vertical transmission between generations.
PMCID: PMC3878406  PMID: 24350840
Bifidobacterium; Probioiotic; Apis mellifera; Honey bee; Crop; Respiratory metabolic pathway; ROS tolerance
8.  Virulence characteristics of five new Campylobacter jejuni chicken isolates 
Gut Pathogens  2013;5:41.
Campylobacter enteritis has emerged as one of the most common forms of human diarrheal illness. In this study we have investigated the virulence potential of five new C. jejuni chicken isolates (RO14, RO19, RO24, RO29 and RO37) originated from private households in the rural regions of Banat and Transylvania in Romania. Following isolation and in vitro virulence assay, on HCT-8 cells, our results show that all the C. jejuni chicken isolates overcome the virulence abilities of the highly virulent strain C. jejuni 81-176. Motility, an important virulence factor was significantly improved in all the new chicken isolates. The ability to survive to the antimicrobial activity of the human serum, to resist to the violent attack of bile acids and to survive in the presence of synthetic antibiotics was increased in all the chicken isolates. However, these were statistically significant only for isolates RO29 and RO37. In conclusion our study shows, based on invasiveness and motility, and also on the data provided by the serum and bile resistance experiments that all the new chicken isolates are able to infect human cells, in vitro, and could potentially represent a health hazard for humans.
PMCID: PMC3866932  PMID: 24330718
9.  Serogroups, virulence genes and antibiotic resistance in Shiga toxin-producing Escherichia coli isolated from diarrheic and non-diarrheic pediatric patients in Iran 
Gut Pathogens  2013;5:39.
From a clinical perspective, it is important to know which serogroups, virulence genes and antibiotic resistance patterns are present in Shiga toxin-producing Escherichia coli strains in pediatric patients suffering from diarrheic and non-diarrheic infections. This is the first study in Iran that has comprehensively investigated the Shiga toxin-producing Escherichia coli -related infection characteristics in diarrheic and non-diarrheic pediatric patients of 0–60 months of age.
Two-hundred and twenty four diarrheic and 84 non-diarrheic stool specimens were collected from the Baqiyatallah hospital of Tehran, Iran. The stool samples were cultured immediately and those that were E. coli-positive were analyzed for the presence of antibiotic resistance genes and bacterial virulence factors using PCR. Antimicrobial susceptibility testing was performed using disk diffusion method.
One-hundred and fifty four out of 224 (68.75%) diarrheic stools and 31 out of 84 (36.90%) non-diarrheic stools harbored E. coli. In addition, children in 13–24 month-old age group had the highest incidence of infection with this bacterium (77.63%). A significant difference was found between the frequency of Attaching and Effacing Escherichia coli and Enterohaemorrhagic Escherichia coli (P =0.045). The genes encoding Shiga toxins and intimin were the most commonly detected virulence factors. Among all serogroups studied, O26 (27.04%) and O111 (18.85%) had the highest incidences in the diarrheic and non-diarrheic patients. The incidence of genes encoding resistance against sulfonamide (sul1), gentamicin (aac(3)-IV), trimethoprim (aadA1), cephalothin (blaSHV) and tetracycline (tetA) were 82.78%, 68.03%, 60.65%, 56.55% and 51.63%, respectively. High resistance levels against penicillin (100%), tetracycline (86.88%), gentamicin (62.29%) and streptomycin (54.91%) were observed. Marked seasonality in the serogroup distributions was evident, while STEC infections were more common in summer (P =0.041).
Our findings should raise awareness about antibiotic resistance in diarrheic pediatric patients in Iran. Clinicians should exercise caution when prescribing antibiotics, especially during the warmer months of the year.
PMCID: PMC3866933  PMID: 24330673
Shiga toxin-producing Escherichia coli; Diarrhea; Pediatric patients; Iran
10.  Phylogenetic analysis of Helicobacter pylori cagA gene of Turkish isolates and the association with gastric pathology 
Gut Pathogens  2013;5:33.
The cagA gene is one of the important virulence factors of Helicobacter pylori. The diversity of cagA 5′ conserved region is thought to reflect the phylogenetic relationships between different H. pylori isolates and their association with peptic ulceration. Significant geographical differences among isolates have been reported. The aim of this study is to compare Turkish H. pylori isolates with isolates from different geographical locations and to correlate the association with peptic ulceration.
Total of 52 isolates of which 19 were Turkish and 33 from other geographic locations were studied. Gastric antral biopsies collected from 19 Turkish patients (Gastritis = 12, ulcer = 7) were used to amplify the cagA 5′ region by PCR then followed by DNA sequencing.
The phylogenetic tree displayed 3 groups: A) a mix of 2 sub-groups “Asian” and “African/Anatolian/Asian/European”, B) “Anatolian/European” and C) “American-Indian”. Turkish H. pylori isolates clustered in the mixed sub-group A were mostly from gastritis patients while those clustered in group B were from peptic ulcer patients. A phylogenetic tree constructed for our Turkish isolates detected distinctive features among those from gastritis and ulcer patients. We have found that 2/3 of the gastritis isolates were clustered alone while 1/3 was clustered together with the ulcer isolates. Several amino acids were found to be shared between the later groups but not with the first group of gastritis.
This study provided an additional insight into the profile of our cagA gene which implies a relationship in geographic locations of the isolates.
PMCID: PMC3843586  PMID: 24245965
Helicobacter pylori; Phylogenetic analysis; cagA
11.  Possible ameliorative effects of antioxidants on propionic acid / clindamycin - induced neurotoxicity in Syrian hamsters 
Gut Pathogens  2013;5:32.
Propionic acid (PA) found in some foods and formed as a metabolic product of gut bacteria has been reported to mimic/mediate the effects of autism. The present study was undertaken to compare the effect of orally administered PA with that of clindamycin-induced PA-microbial producers in inducing persistent biochemical autistic features in hamsters. The neuroprotective potency of carnosine and carnitine supplements against PA toxicity was also investigated.
The following groups were studied. 1. Control group, which received phosphate buffered saline orally, 2. Propionic acid treated group which were given PA at a dose of 250 mg/kg body weight/day for 3 days orally, 3. Clindamycin treated group which received a single dose of the antibiotic orogastrically at a dose of 30 mg/kg on the day of the experiment, 4. Carnosine-treated group which were given carnosine at a dose of 10 mg/kg body weight/day orally for one week, 5. Carnitine treated group given 50 mg/kg body weight/day carnitine orally daily for one week. Group 6. Carnosine followed by PA, Group 7. Carnitine followed by PA. Dopamine, adrenaline and noradrenaline, serotonin and Gamma amino-butyric acid (GABA) were measured in the cortex and medulla of the nine studied groups.
PA administration caused significant decrease in the neurotransmitters in the brains of treated hamsters while clindamycin caused a significant decrease only in dopamine in hamster brains (cortex and medulla) and GABA in the cerebral cortex of the treated hamsters. Administration of carnosine and carnitine which are known antioxidants caused no significant changes in the levels of neurotransmitters when administered alone to hamsters. However when administered with PA both carnosine and carnitine restored the altered neurotransmitters to near normal levels.
Carnosine and carnitine may be used as supplements to protect against PA neurotoxicity.
PMCID: PMC3828401  PMID: 24188374
Autism; Clindamycin; Propionic acid; Carnosine; Carnitine; Cortex; Medulla
12.  Insights of biosurfactant producing Serratia marcescens strain W2.3 isolated from diseased tilapia fish: a draft genome analysis 
Gut Pathogens  2013;5:29.
Serratia marcescens is an opportunistic bacterial pathogen with broad range of host ranging from vertebrates, invertebrates and plants. S. marcescens strain W2.3 was isolated from a diseased tilapia fish and it was suspected to be the causal agent for the fish disease as virulence genes were found within its genome. In this study, for the first time, the genome sequences of S. marcescens strain W2.3 were sequenced using the Illumina MiSeq platform.
Several virulent factors of S. marcescens such as serrawettin, a biosurfactant, has been reported to be regulated by N-acyl homoserine lactone (AHL)-based quorum sensing (QS). In our previous studies, an unusual AHL with long acyl side chain was detected from this isolate suggesting the possibility of novel virulence factors regulation. This evokes our interest in the genome of this bacterial strain and hereby we present the draft genome of S. marcescens W2.3, which carries the serrawettin production gene, swrA and the AHL-based QS transcriptional regulator gene, luxR which is an orphan luxR.
With the availability of the whole genome sequences of S. marcescens W2.3, this will pave the way for the study of the QS-mediated genes expression in this bacterium.
PMCID: PMC3816309  PMID: 24148830
Serratia marcescens; Biosurfactant; Serrawettin; Quorum sensing; N-acyl homoserine lactone; swrA; Next generation sequencing technology
13.  Gleaning evolutionary insights from the genome sequence of a probiotic yeast Saccharomyces boulardii 
Gut Pathogens  2013;5:30.
The yeast Saccharomyces boulardii is used worldwide as a probiotic to alleviate the effects of several gastrointestinal diseases and control antibiotics-associated diarrhea. While many studies report the probiotic effects of S. boulardii, no genome information for this yeast is currently available in the public domain.
We report the 11.4 Mbp draft genome of this probiotic yeast. The draft genome was obtained by assembling Roche 454 FLX + shotgun data into 194 contigs with an N50 of 251 Kbp. We compare our draft genome with all other Saccharomyces cerevisiae genomes.
Our analysis confirms the close similarity of S. boulardii to S. cerevisiae strains and provides a framework to understand the probiotic effects of this yeast, which exhibits unique physiological and metabolic properties.
PMCID: PMC3843575  PMID: 24148866
Diarrhea; Human gut; Clostridium difficile; Enterohemorrhagic Escherichia coli
14.  Understanding the sequential activation of Type III and Type VI Secretion Systems in Salmonella typhimurium using Boolean modeling 
Gut Pathogens  2013;5:28.
Three pathogenicity islands, viz. SPI-1 (Salmonella pathogenicity island 1), SPI-2 (Salmonella pathogenicity island 2) and T6SS (Type VI Secretion System), present in the genome of Salmonella typhimurium have been implicated in the virulence of the pathogen. While the regulation of SPI-1 and SPI-2 (both encoding components of the Type III Secretion System - T3SS) are well understood, T6SS regulation is comparatively less studied. Interestingly, inter-connections among the regulatory elements of these three virulence determinants have also been suggested to be essential for successful infection. However, till date, an integrated view of gene regulation involving the regulators of these three secretion systems and their cross-talk is not available.
In the current study, relevant regulatory information available from literature have been integrated into a single Boolean network, which portrays the dynamics of T3SS (SPI-1 and SPI-2) and T6SS mediated virulence. Some additional regulatory interactions involving a two-component system response regulator YfhA have also been predicted and included in the Boolean network. These predictions are aimed at deciphering the effects of osmolarity on T6SS regulation, an aspect that has been suggested in earlier studies, but the mechanism of which was hitherto unknown. Simulation of the regulatory network was able to recreate in silico the experimentally observed sequential activation of SPI-1, SPI-2 and T6SS.
The present study integrates relevant gene regulatory data (from literature and our prediction) into a single network, representing the cross-communication between T3SS (SPI-1 and SPI-2) and T6SS. This holistic view of regulatory interactions is expected to improve the current understanding of pathogenesis of S. typhimurium.
PMCID: PMC3849742  PMID: 24079299
Salmonella typhimurium; Salmonella pathogenicity island 1 (SPI-1); SPI-2; Type VI Secretion System (T6SS); Boolean modeling; Cross-talk network
15.  Timely approaches to identify probiotic species of the genus Lactobacillus 
Gut Pathogens  2013;5:27.
Over the past decades the use of probiotics in food has increased largely due to the manufacturer’s interest in placing “healthy” food on the market based on the consumer’s ambitions to live healthy. Due to this trend, health benefits of products containing probiotic strains such as lactobacilli are promoted and probiotic strains have been established in many different products with their numbers increasing steadily. Probiotics are used as starter cultures in dairy products such as cheese or yoghurts and in addition they are also utilized in non-dairy products such as fermented vegetables, fermented meat and pharmaceuticals, thereby, covering a large variety of products.
To assure quality management, several pheno-, physico- and genotyping methods have been established to unambiguously identify probiotic lactobacilli. These methods are often specific enough to identify the probiotic strains at genus and species levels. However, the probiotic ability is often strain dependent and it is impossible to distinguish strains by basic microbiological methods.
Therefore, this review aims to critically summarize and evaluate conventional identification methods for the genus Lactobacillus, complemented by techniques that are currently being developed.
PMCID: PMC3848994  PMID: 24063519
16.  Persistent gut barrier damage and commensal bacterial influx following eradication of Giardia infection in mice 
Gut Pathogens  2013;5:26.
Recent studies of Giardia lamblia outbreaks have indicated that 40–80% of infected patients experience long-lasting functional gastrointestinal disorders after parasitic clearance. Our aim was to assess changes in the intestinal barrier and spatial distribution of commensal bacteria in the post-clearance phase of Giardia infection.
Mice were orogastrically inoculated with G. lamblia trophozoites (strain GS/M) or pair-fed with saline and were sacrificed on post-infective (PI) days 7 (colonization phase) and 35 (post-clearance phase). Gut epithelial barrier function was assessed by Western blotting for occludin cleavage and luminal-to-serosal macromolecular permeability. Gut-associated, superficial adherent, and mucosal endocytosed bacteria were measured by agar culturing and were examined by fluorescence in situ hybridization. Intracellular bacteria cultured from isolated mucosal cells were characterized by 16S rDNA sequencing. Neutrophil-specific esterase staining, a myeloperoxidase activity assay, and enzyme-linked immunosorbent assays for cytokine concentrations were used to verify intestinal tissue inflammation.
Tight junctional damage was detected in the intestinal mucosa of Giardia-infected mice on PI days 7 and 35. Although intestinal bacterial overgrowth was evident only during parasite colonization (PI day 7), enhanced mucosal adherence and endocytosis of bacteria were observed on PI days 7 and 35. Multiple bacterial strains, including Bacillus, Lactobacillus, Staphylococcus, and Phenylobacterium, penetrated the gut mucosa in the post-infective phase. The mucosal influx of bacteria coincided with increases in neutrophil infiltration and myeloperoxidase activity on PI days 7 and 35. Elevated intestinal IFNγ, TNFα, and IL-1β levels also were detected on PI day 35.
Giardia-infected mice showed persistent tight junctional damage and bacterial penetration, accompanied by mucosal inflammation, after parasite clearance. These novel findings suggest that the host’s unresolved immune reactions toward its own microbiota, due to an impaired epithelial barrier, may partly contribute to the development of post-infective gut disorders.
PMCID: PMC3765889  PMID: 23991642
Giardiasis; Post-infective intestinal dysfunction; Epithelial barrier; Tight junction; Bacterial endocytosis
17.  Comparing the genomes of Helicobacter pylori clinical strain UM032 and Mice-adapted derivatives 
Gut Pathogens  2013;5:25.
Helicobacter pylori is a Gram-negative bacterium that persistently infects the human stomach inducing chronic inflammation. The exact mechanisms of pathogenesis are still not completely understood. Although not a natural host for H. pylori, mouse infection models play an important role in establishing the immunology and pathogenicity of H. pylori. In this study, for the first time, the genome sequences of clinical H. pylori strain UM032 and mice-adapted derivatives, 298 and 299, were sequenced using the PacBio Single Molecule, Real-Time (SMRT) technology.
Here, we described the single contig which was achieved for UM032 (1,599,441 bp), 298 (1,604,216 bp) and 299 (1,601,149 bp). Preliminary analysis suggested that methylation of H. pylori genome through its restriction modification system may be determinative of its host specificity and adaptation.
Availability of these genomic sequences will aid in enhancing our current level of understanding the host specificity of H. pylori.
PMCID: PMC3751790  PMID: 23957912
Helicobacter pylori; PacBio Single Molecule; Real-Time (SMRT) technology; Clinical H. pylori; Mice-adapted
18.  The pathogenic intestinal spirochaete Brachyspira pilosicoli forms a diverse recombinant species demonstrating some local clustering of related strains and potential for zoonotic spread 
Gut Pathogens  2013;5:24.
Brachyspira pilosicoli is an anaerobic spirochaete that can colonizes the large intestine of many host species. Infection is particularly problematic in pigs and adult poultry, causing colitis and diarrhea, but it is also known to result in clinical problems in human beings. Despite the economic importance of the spirochaete as an animal pathogen, and its potential as a zoonotic agent, it has not received extensive study.
A multilocus sequence typing (MLST) method based on the scheme used for other Brachyspira species was applied to 131 B. pilosicoli isolates originating from different host species and geographical areas. A variety of phylogenetic trees were constructed and analyzed to help understand the data.
The isolates were highly diverse, with 127 sequence types and 123 amino acid types being identified. Large numbers (50-112) of alleles were present at each locus, with all loci being highly polymorphic. The results of Shimodaira-Hasegawa tests identified extensive genetic recombination, although the calculated standardized index of association value (0.1568; P <0.0005) suggested the existence of some clonality. Strains from different host species and geographical origins generally were widely distributed throughout the population, although in nine of the ten cases where small clusters of related isolates occurred these were from the same geographical areas or farms/communities, and from the same species of origin. An exception to the latter was a cluster of Australian isolates originating from pigs, chickens and a human being, suggesting the likelihood of relatively recent transmission of members of this clonal group between species.
The strongly recombinant population structure of B. pilosicoli contrasts to the more highly clonal population structures of the related species Brachyspira hyodysenteriae and Brachyspira intermedia, both of which are specialized enteric pathogens of pigs and poultry. The genomic plasticity of B. pilosicoli may help to explain why it has been able to adapt to colonize the large intestines of a wider range of hosts compared to other Brachyspira species. The identification of a clonal group of isolates that had been recovered from different host species, including a human being, suggests that zoonotic transmission by B. pilosicoli may occur in nature. Evidence for local transmission between the same host species also was obtained.
PMCID: PMC3751851  PMID: 23957888
Brachyspira pilosicoli; Spirochaete; Recombination; MLST; Zoonosis
19.  Commensal Clostridia: leading players in the maintenance of gut homeostasis 
Gut Pathogens  2013;5:23.
The gastrointestinal tract is a complex and dynamic network where an intricate and mutualistic symbiosis modulates the relationship between the host and the microbiota in order to establish and ensure gut homeostasis. Commensal Clostridia consist of gram-positive, rod-shaped bacteria in the phylum Firmicutes and make up a substantial part of the total bacteria in the gut microbiota. They start to colonize the intestine of breastfed infants during the first month of life and populate a specific region in the intestinal mucosa in close relationship with intestinal cells. This position allows them to participate as crucial factors in modulating physiologic, metabolic and immune processes in the gut during the entire lifespan, by interacting with the other resident microbe populations, but also by providing specific and essential functions. This review focus on what is currently known regarding the role of commensal Clostridia in the maintenance of overall gut function, as well as touch on their potential contribution in the unfavorable alteration of microbiota composition (dysbiosis) that has been implicated in several gastrointestinal disorders. Commensal Clostridia are strongly involved in the maintenance of overall gut function. This leads to important translational implications in regard to the prevention and treatment of dysbiosis, to drug efficacy and toxicity, and to the development of therapies that may modulate the composition of the microflora, capitalizing on the key role of commensal Clostridia, with the end goal of promoting gut health.
PMCID: PMC3751348  PMID: 23941657
Gut microbiota; Clostridia spp; Dysbiosis; Gut homeostasis
20.  Probiotic Lactobacillus rhamnosus GG mono-association suppresses human rotavirus-induced autophagy in the gnotobiotic piglet intestine 
Gut Pathogens  2013;5:22.
Human rotavirus (HRV) is the most important cause of severe diarrhea in infants and young children. Probiotic Lactobacillus rhamnosus GG (LGG) reduces rotavirus infection and diarrhea. However, the molecular mechanisms of LGG-mediated protection from rotavirus infection are poorly understood. Autophagy plays an essential role in responses to microbial pathogens. However, the role of autophagy in HRV infection and LGG treatment is unknown. We hypothesize that rotavirus gastroenteritis activates autophagy and that LGG suppresses virus-induced autophagy and prevents intestinal damage in infected piglets.
We used LGG feeding to combat viral gastroenteritis in the gnotobiotic pig model of virulent HRV infection.
We found that LGG feeding did not increase autophagy, whereas virus infection induced autophagy in the piglet intestine. Virus infection increased the protein levels of the autophagy markers ATG16L1 and Beclin-1 and the autophagy regulator mTOR. LGG treatment during viral gastroenteritis reduced autophagy marker expression to normal levels, induced apoptosis and partially prevented virus-induced tissue damage.
Our study provides new insights into virus-induced autophagy and LGG suppression of uncontrolled autophagy and intestinal injury. A better understanding of the antiviral activity of LGG will lead to novel therapeutic strategies for infant infectious diseases.
PMCID: PMC3750464  PMID: 23924832
Autophagy; Apoptosis; Diarrhea; Gnotobiotic pig; Lactobacillus rhamnosus GG; Infectious disease; Intestinal inflammation; Intestinal injury; Probiotics; Rotavirus
21.  Changing trends and serotype distribution of Shigella species in Beijing from 1994 to 2010 
Gut Pathogens  2013;5:21.
Shigella species are a common cause of acute diarrheal disease in China. In this study, we characterized the changing trends and serotype distribution of Shigella species in Beijing from 1994 to 2010. A total of 5999 Shigella strains were isolated and serotyped from the 302nd Hospital in Beijing. The annual number of Shigella isolates reached a peak (n = 1192; 19.84%) in 1996 and then decreased annually, reaching the lowest point (n = 24; 0.41%) in 2010. S. flexneri 2a and S. sonnei were the most frequently isolated Shigella, with their respective isolates making up 53.3% and 27.6% of the total. Isolates of S. flexneri 4c, 4a, and x made up 3% respectively of the total isolates. Significant decreases in percentage of S. flexneri over time were observed. S. sonnei surpassed S. flexneri 2a as the predominant serotype in 2000. Most isolates were recovered from July to September; 13.6% of the isolates were recovered from children aged 0 to 5 years, and 16% were recovered from those aged 21 to 25 years. S. flexneri 2a and 5 were recovered mostly from males (33.41%, p < 0.001; and 0.46%, p < 0.001%; respectively), whereas S. flexneri 2b and 6, and S. sonnei were most often isolated from females. Continuous monitoring of Shigella showed that all 4 species and 27 serotypes were present in Beijing, China, during the study period. The emergence of S. sonnei and the overall decreasing isolation rate of Shigella in Beijing can potentially aid in the development of vaccine and control strategies for shigellosis in the city.
PMCID: PMC3750644  PMID: 23919811
Shigellosis; Shigella; Serotype; Beijing hospital
22.  Evidence of a new metabolic capacity in an emerging diarrheal pathogen: lessons from the draft genomes of Vibrio fluvialis strains PG41 and I21563 
Gut Pathogens  2013;5:20.
Vibrio fluvialis is an emerging diarrheal pathogen for which no genome is currently available. In this work, draft genomes of two closely related clinical strains PG41 and I21563 have been explored.
V. fluvialis strains PG41 and I21563 were sequenced on the Illumina HiSeq 1000 platform to obtain draft genomes of 5.3 Mbp and 4.4 Mbp respectively. Our genome data reveal the presence of genes involved in ethanolamine utilization, which is further experimentally confirmed by growth analysis.
Combined in silico and growth analysis establish a new metabolic capacity of V. fluvialis to harvest energy from ethanolamine.
PMCID: PMC3729366  PMID: 23895343
Metabolic fitness; Eut-operon; Vibrionaceae
23.  The bioluminescent Listeria monocytogenes strain Xen32 is defective in flagella expression and highly attenuated in orally infected BALB/cJ mice 
Gut Pathogens  2013;5:19.
In vivo bioluminescence imaging (BLI) is a powerful method for the analysis of host-pathogen interactions in small animal models. The commercially available bioluminescent Listeria monocytogenes strain Xen32 is commonly used to analyse immune functions in knockout mice and pathomechanisms of listeriosis.
To analyse and image listerial dissemination after oral infection we have generated a murinised Xen32 strain (Xen32-mur) which expresses a previously described mouse-adapted internalin A. This strain was used alongside the Xen32 wild type strain and the bioluminescent L. monocytogenes strains EGDe-lux and murinised EGDe-mur-lux to characterise bacterial dissemination in orally inoculated BALB/cJ mice. After four days of infection, Xen32 and Xen32-mur infected mice displayed consistently higher rates of bioluminescence compared to EGDe-lux and EGDe-mur-lux infected animals. However, surprisingly both Xen32 strains showed attenuated virulence in orally infected BALB/c mice that correlated with lower bacterial burden in internal organs at day 5 post infection, smaller losses in body weights and increased survival compared to EGDe-lux or EGDe-mur-lux inoculated animals. The Xen32 strain was made bioluminescent by integration of a lux-kan transposon cassette into the listerial flaA locus. We show here that this integration results in Xen32 in a flaA frameshift mutation which makes this strain flagella deficient.
The bioluminescent L. monocytogenes strain Xen32 is deficient in flagella expression and highly attenuated in orally infected BALB/c mice. As this listerial strain has been used in many BLI studies of murine listeriosis, it is important that the scientific community is aware of its reduced virulence in vivo.
PMCID: PMC3720536  PMID: 23856386
Listeriosis; Flagella; Mouse infection model; Bioluminescent imaging
24.  Molecular identification of Mycobacterium avium subspecies paratuberculosis in oral biopsies of Crohn’s disease patients 
Gut Pathogens  2013;5:18.
Oral lesions may be found in patients with Crohn’s disease (CD), in a percentage up to 20%. The aim of this study was to investigate a possible relationship between Mycobacterium avium subsp. paratuberculosis (MAP) and oral lesions in CD patients. 23 oral biopsies were examined performing IS900 Nested PCR; 9 of them were positive: 8 from CD patients and 1 from a control. Our purpose is to go on with this study, amplifying the number of subjects examined and testing subjects with oral lesions related to diseases other than CD to verify the specific association between MAP and oral lesions in CD patients.
PMCID: PMC3711722  PMID: 23842143
MAP; Oral lesion Crohn; Oral granulomatous lesions; PCR IS900
25.  Neutrophil activation by Campylobacter concisus 
Gut Pathogens  2013;5:17.
Campylobacter concisus is an emerging enteric pathogen associated with prolonged diarrhoea and possibly inflammatory bowel disease in children as well as adults, but the interaction with cells of the innate immune system is unclear. The magnitude of systemic immunoglobulin response in acute infection is unknown.
Neutrophils from healthy volunteers were activated with five faecal isolates of C. concisus from patients with gastroenteritis as well as the oral reference strain C. concisus ATCC33237. Neutrophils were tested for the expression of adherence molecule CD11b by immunoflourescence and for oxidative burst response by chemiluminescence. The opsonic activity in a chemiluminescence assay was assessed with heat treated serum from patients with C. concisus infection.
A strong and dose-dependent activation of neutrophil adherence molecule CD11b and oxidative burst response was demonstrated with all six C. concisus isolates. Bacteria opsonised in heat treated serum induced an increased chemiluminescence response. Heat treated serum from patients with C. concisus infection did not have a higher opsonic activity than heat treated serum from healthy volunteers.
C. concisus has the capability to activate the innate immune system by stimulating neutrophil cells to increased adherence molecule expression and oxidative burst response, both crucial for acute inflammation. In a chemiluminescence assay the opsonic activity of heat treated serum from patients was not increased compared to heat treated control serum suggesting a weak systemic IgG response to infection.
PMCID: PMC3702423  PMID: 23819746
Campylobacter concisus; Neutrophil; Oxidative Burst; CD11b; Inflammation

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