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1.  Escherichia coli from Crohn’s disease patient displays virulence features of enteroinvasive (EIEC), enterohemorragic (EHEC), and enteroaggregative (EAEC) pathotypes 
Gut Pathogens  2015;7(1):2.
Escherichia coli is a normal inhabitant of the gut which upon acquiring virulence factors becomes potentially able to cause diseases. Although E. coli population augments in Crohn’s disease (CD), the reason of this proliferation is not yet clear. CD associated E. coli shows features of extraintestinal pathogenic categories (ExPEC), and eventually the ability to invade cultured epithelial cells, a property observed among diarrheagenic E. coli (DEC). In this work, data on the characterization of an E. coli isolate from a CD patient reveal that, besides invasiveness, CD associated E. coli may harbor other typical DEC markers, namely those defining enterohemorragic (EHEC) and enteroaggregative (EAEC) pathotypes.
The studied strain, detected both in an ileum biopsy and stools, belonged to the B2 E. coli reference collection (EcoR) phylogroup and harbored the intimin, Shiga cytotoxin 1, and AggR transcriptional activator encoding genes (eae, stx1, aggR, respectively); displayed aggregative adherence to Hep-2 cells and an ability to enter Caco-2 cells four times as high as that of EIEC reference strain and half of invasiveness of AIEC LF82. It was able to enter and replicate in J774 macrophages with invasiveness 85 times as high as that of LF82, but with only one sixth of the intracellular proliferation ability of the later. Extracellular products with cytotoxic activity on Vero cells were detected in strain’s cultures. Preliminary analysis indicated similarity of this strain’s genome with that of O104:H4/2011C-3493.
Following its isolation from a resected CD patient, the strain was characterized by in vitro adhesion and invasion assays to Hep-2, invasion to Caco-2 cells and to J774 macrophages and tested for the ability to form biofilm and to produce Shiga cytotoxins. PCRs were carried out to identify virulence genetic markers and for EcoR phylogrouping. The strain’s genome was sequenced by means of Ion torrent PGM platform.
The detection, in a CD patient, of an E. coli combining virulence features of multiple DEC pathotypes seems not only to stress the relevance of E. coli to CD etiopathogenesis but also to indicate the existence of new and potentially more virulent strains putatively associated with this disease.
PMCID: PMC4316758  PMID: 25653719
Escherichia coli; Crohn’s disease; Invasive; MLST; Serotype; Adherence; Virulence
2.  The vesicle-associated function of NOD2 as a link between Crohn’s disease and mycobacterial infection 
Gut Pathogens  2015;7(1):1.
Although Crohn’s disease (CD) etiology remains unclear, a growing body of evidence suggests that CD may include an infectious component, with Mycobacterium avium subsp. paratuberculosis (MAP) being the most likely candidate for this role. However, the molecular mechanism of the MAP involvement in CD pathogenesis remains unclear. The polymorphism of the NOD2 gene, coding for an intracellular pattern recognition receptor, is a factor of predisposition to mycobacterial infections and CD. Recent findings on NOD2 interactions and functions provide the missing pieces in the puzzle of a NOD2-mediated mechanism common for mycobacterial infections and CD. Implications of these new findings for the development of a better understanding and treatments of CD and mycobacterial infections are discussed.
PMCID: PMC4316803  PMID: 25653718
Vesicle acidity; Crohn’s disease; Mycobacteria; Sulfonated glycolipids; Autophagy; Pattern recognition receptors
3.  Whole genome sequence and comparative genomic analysis of multidrug-resistant Staphylococcus capitis subsp. urealyticus strain LNZR-1 
Gut Pathogens  2014;6(1):45.
Staphylococcus capitis is an emerging opportunistic pathogen of humans, and found as a colonizer of the human gut. Here, we report a case of S. capitis subsp. urealyticus infection. The strain LNZR-1 was isolated from the blood culture of a patient with sigmoid colon cancer. It was found to be resistant to some important antibiotics, such as linezolid, a highly effective antimicrobial against clinically important Staphylococci pathogens. However, data on the genetic resistance mechanisms in S. capitis subsp. urealyticus are only sparsely available.
The draft genome of S. capitis subsp. urealyticus strain LNZR-1 was sequenced by using next-generation sequencing technologies. Sequence data assembly revealed a genome size of 2,595,865 bp with a G + C content of 32.67%. Genome annotation revealed the presence of antibiotic resistance genes conferring resistance against some of the tested antibiotics as well as non-tested antibiotics. The genome also possesses a lot of genes that may be related to multidrug resistance. Whole genome comparison of the LNZR-1 with five other S. capitis strains showed that some functional regions are highly homologous between the six assemblies made herein. The LNZR-1 genome has high similarity with the genomes of the strains VCU116 and CR01, although some short stretches present in the genomes of strains VCU116 and CR01 were absent in the strain LNZR-1.
The presence of a plethora of genes responsible for antibiotic resistance suggests that strain LNZR-1 could present a potential threat to human health. The comparative genomic analysis of S. capitis strains presented in this study is important for better understanding of multidrug resistance in S. capitis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13099-014-0045-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4310196  PMID: 25649186
Staphylococcus capitis subsp. urealyticus; Multidrug-resistant; Genome sequencing; Comparative genomic analysis
4.  The role of the Cronobacter sakazakii ProP C-terminal coiled coil domain in osmotolerance 
Gut Pathogens  2014;6(1):46.
We investigate the role of the C-terminal coiled coil of the secondary proline porter ProP in contributing to Cronobacter sakazakii osmotolerance.
The extended C-terminal domain of ProP1 (encoded by ESA_02131) was spliced onto the truncated C-terminal end of ProP2 (encoded by ESA_01706); creating a chimeric protein (ProPc) which exhibits increased osmotolerance relative to the wild type.
It appears that the C-terminal coiled coil domain tunes ProP at low osmolality, whereas ProP transporters lacking the coiled coil domain are more active at a higher osmolality range.
PMCID: PMC4272814  PMID: 25530808
5.  Synergistic effects of Bifidobacterium thermophilum RBL67 and selected prebiotics on inhibition of Salmonella colonization in the swine proximal colon PolyFermS model 
Gut Pathogens  2014;6(1):44.
Probiotics and prebiotics are promising strategies to counteract Salmonella prevalence in swine. In the present study, we investigated the effects of prebiotics (fructo- (FOS), galacto- (GOS) and mannan- (MOS) oligosaccharides) and the bacteriocinogenic Bifidobacterium thermophilum RBL67 (RBL67) on Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15) colonization using the PolyFermS in vitro continuous fermentation model simulating the swine proximal colon.
Material and methods
The PolyFermS model was designed with a first-stage reactor containing immobilized fecal pig microbiota. This reactor continuously inoculated five parallel second-stage reactors, a control and four treatment reactors, all operated with proximal colon conditions. FOS and GOS (5.2 g/day), and MOS (half dosage) and RBL67 (108 copy numbers/mL applied daily) were tested on the ability of N-15 to colonize reactors, inoculated with the same microbiota. Reactor effluents were collected daily and analyzed for microbial composition (quantitative PCR and 454 pyrosequencing of 16S rRNA gene pool) and main metabolites (HPLC).
RBL67 and N-15 were shown to stably colonize the system. Colonization of N-15 was strongly inhibited by FOS and GOS, whereas addition of RBL67 alone or combined with MOS showed intermediate results. However, the effect of FOS and GOS was enhanced when prebiotics were combined with a daily addition of RBL67. FOS and GOS increased the total short chain fatty acid production, especially acetate and propionate. RBL67 combined with FOS additionally stimulated butyrate production.
Our study demonstrates the suitability of the porcine PolyFermS in vitro model to study nutritional effects of pro- and prebiotics on gut microbiota composition and activity. It can further be used to monitor Salmonella colonization. The inhibition effects of FOS and GOS on N-15 colonization are partly due to an increased acetate production, while further antimicrobial mechanisms may contribute to an enhanced inhibition with prebiotic-RBL67 combinations. A future direction of this work could be to understand the anti-Salmonella effects of Bifidobacterium thermophilum RBL67 in the presence of prebiotics to unravel the mechanism of this probiotic:pathogen interaction.
Electronic supplementary material
The online version of this article (doi:10.1186/s13099-014-0044-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4215022  PMID: 25364390
Bifidobacterium thermophilum RBL67; Swine; Intestinal fermentation model; Prebiotics; Probiotics; Salmonella enterica subsp. enterica serovar Typhimurium N-15
6.  Quantitative evaluation of fungi of the genus Candida in the feces of adult patients with type 1 and 2 diabetes - a pilot study 
Gut Pathogens  2014;6(1):43.
Gastrointestinal tract microbiota, particularly bacterial microflora, seem to have a different qualitative and quantitative composition in both type 1 (T1DM) and type 2 diabetes (T2DM) mellitus cases as compared to non-diabetic individuals. So far, there are no data from diabetes research concerning the prevalence of fungi, particularly the most common genus, i.e. Candida, which are important components of human colon microflora.
We aimed to examine whether there are quantitative changes of Candida fungi in the feces of patients with T1DM and T2DM as compared to healthy controls.
Overall, we included 44 diabetic patients (27 patients with T1DM and 17 with T2DM) as well as 17 healthy, non-diabetic controls. Feces and blood samples were collected from all study individuals. DNA was isolated from fecal samples and quantitative real time PCR (qPCR) was applied in order to determine the number of fungal cells. Statistical association with selected clinical and biochemical features was examined.
There was a difference in the amount of Candida in the feces among the three examined groups (p = 0.007). Candida spp. populations in T1DM and T2DM subjects were larger as compared to controls (p = 0.017 and p = 0.037, respectively). However, no difference was found between T1DM and T2DM. No association was identified between the quantity of fungi and examined patients’ characteristics, except for negative correlation with blood lipid parameters in T2DM group.
Candida fungi appear to be more prevalent in the feces of patients with T1DM and T2DM. Their amount seems to be associated with serum lipids in T2DM patients. This initial finding requires further confirmation.
PMCID: PMC4201707  PMID: 25328543
Candida; Feces; Serum lipids; T1DM; T2DM
7.  Extremely low Helicobacter pylori prevalence in North Sulawesi, Indonesia and identification of a Maori-tribe type strain: a cross sectional study 
Gut Pathogens  2014;6(1):42.
Sulawesi in Indonesia has a unique geographical profile with assumed separation from Sundaland. Studies of Helicobacter pylori in this region are rare due to the region’s rural location and lack of endoscopy equipment. Indirect methods are, therefore, the most appropriate for measuring H. pylori infection in these areas; with the disposable gastric brush test, we can obtain gastric juice as well as small gastric tissue samples for H. pylori culture. We investigated the prevalence of H. pylori infection and evaluated human migration patterns in the remote areas of North Sulawesi.
We recruited a total of 251 consecutive adult volunteers and 131 elementary school children. H. pylori infection was determined by urine antibody test. A gastric brush test was used to culture H. pylori. We used next-generation and polymerase chain reaction based sequencing to determine virulence factors and multi-locus sequence typing (MLST).
The overall H. pylori prevalence was only 14.3% for adults and 3.8% for children, and 13.6% and 16.7% in Minahasanese and Mongondownese participants, respectively. We isolated a single H. pylori strain, termed -Manado-1. Manado-1 was East Asian type cagA (ABD type), vacA s1c-m1b, iceA1 positive/iceA2 negative, jhp0562-positive/β-(1,3) galT-negative, oipA “on”, and dupA-negative. Phylogenetic analyses showed the strain to be hspMaori type, a major type observed in native Taiwanese and Maori tribes.
Our data support that very low H. pylori infection prevalence in Indonesia. Identification of hspMaori type H. pylori in North Sulawesi may support the hypothesis that North Sulawesi people migrated from north.
PMCID: PMC4189669  PMID: 25299127
Helicobacter pylori; Maori; Indonesia
8.  Genome sequencing and genomic characterization of a tigecycline-resistant Klebsiella pneumoniae strain isolated from the bile samples of a cholangiocarcinoma patient 
Gut Pathogens  2014;6(1):40.
The relationship between Klebsiella pneumoniae and nosocomial and community-acquired infections is well known, and K. pneumoniae resistance to most antibiotics is increasing worldwide. In contrast, tigecycline remains active against many bacterial strains, and serves as a last resort for treating multi-drug resistant bacterial infections. That tigecycline nonsusceptibility among K. pneumoniae isolates has been reported worldwide is worrying. However, the mechanisms of tigecycline resistance in K. pneumoniae are less well known. We report the genome sequence and genomic characterization of tigecycline-resistant K. pneumoniae strain 5422 isolated from the bile samples of a patient with cholangiocarcinoma.
We sequenced the K. pneumoniae strain 5422 genome using next-generation sequencing technologies. Sequence data assembly revealed a 5,432,440-bp draft genome and 57.1% G + C content, which contained 5397 coding sequences. The genome has extensive similarity to other sequenced K. pneumoniae genomes, but also has several resistance-nodulation-cell division (RND) efflux pump genes that may be related to tigecycline resistance.
K. pneumoniae strain 5422 is resistant to multiple antibiotics. The genome sequence of the isolate and comparative analysis with other K. pneumoniae strains presented in this paper are important for better understanding of K. pneumoniae multi-drug resistance. The RND efflux pump genes identified in the genome indicate the presence of an antibiotic resistance mechanism prior to antibiotics overuse. The availability of the genome sequence forms the basis for further comparative analyses and studies addressing the evolution of the K. pneumoniae drug resistance mechanism and the K. pneumoniae transcriptome.
PMCID: PMC4182774  PMID: 25279004
Tigecycline-resistant; Klebsiella pneumoniae; Cholangiocarcinoma; Next-generation sequencing; Comparative genomics
9.  Association between Helicobacter pylori genotypes and severity of chronic gastritis, peptic ulcer disease and gastric mucosal interleukin-8 levels: Evidence from a study in the Middle East 
Gut Pathogens  2014;6(1):41.
The varied clinical presentations of Helicobacter pylori (H. pylori) infection are most likely due to differences in the virulence of individual strains, which determines its ability to induce production of interleukin-8 (IL-8) in the gastric mucosa. The aim of this study was to examine association between cagA, vacA-s1 and vacA-s2 genotypes of H. pylori and severity of chronic gastritis and presence of peptic ulcer disease (PUD), and to correlate these with IL-8 levels in the gastric mucosa.
Gastric mucosal biopsies were obtained from patients during esophagogastroduodenoscopy. The severity of chronic gastritis was documented using the updated Sydney system. H. pylori cagA and vacA genotypes were detected by PCR. The IL-8 levels in the gastric mucosa were measured by ELISA.
H. pylori cagA and/or vacA genotypes were detected in 99 patients (mean age 38.4±12.9; 72 males), of whom 52.5% were positive for cagA, 44.4% for vacA-s1 and 39.4% for vacA-s2; and 70.7% patients had PUD. The severity of inflammation in gastric mucosa was increased with vacA-s1 (p=0.017) and decreased with vacA-s2 (p=0.025), while cagA had no association. The degree of neutrophil activity was not associated with either cagA or vacA-s1, while vacA-s2 was significantly associated with decreased neutrophil activity (p=0.027). PUD was significantly increased in patients with cagA (p=0.002) and vacA-s1 (p=0.031), and decreased in those with vacA-s2 (p=0.011). The level of IL-8 was significantly increased in patients with cagA (p=0.011) and vacA-s1 (p=0.024), and lower with vacA-s2 (p=0.004). Higher levels of IL-8 were also found in patients with a more severe chronic inflammation (p=0.001), neutrophil activity (p=0.007) and those with PUD (p=0.001).
Presence of vacA-s1 genotype of H. pylori is associated with more severe chronic inflammation and higher levels of IL-8 in the gastric mucosa, as well as higher frequency of PUD. Patients with vacA-s2 have less severe gastritis, lower levels of IL-8, and lower rates of PUD. The presence of cagA genotype is not associated with the severity of gastritis or IL-8 induction in the gastric mucosa. The association of cagA with PUD may be a reflection of its presence with vacA-s1 genotype.
PMCID: PMC4181383  PMID: 25279005
Helicobacter pylori; cagA; vacA-s1; vacA-s2; Gastritis; Interleukin-8; Peptic ulcer disease
10.  Correlation between the genomic o454-nlpD region polymorphisms, virulence gene equipment and phylogenetic group of extraintestinal Escherichia coli (ExPEC) enables pathotyping irrespective of host, disease and source of isolation 
Gut Pathogens  2014;6:37.
The mutS-rpoS intergenic region in E. coli displays a mosaic structure which revealed pathotype specific patterns. To assess the importance of this region as a surrogate marker for the identification of highly virulent extraintestinal pathogenic E. coli (ExPEC) strains we aimed to: (i) characterize the genetic diversity of the mutS gene and the o454-nlpD genomic region among 510 E. coli strains from animals and humans; (ii) delineate associations between the polymorphism of this region and features such as phylogenetic background of E. coli, pathotype, host species, clinical condition, serogroup and virulence associated genes (VAG)s; and (iii) identify the most important VAGs for classification of the o454-nlpD region.
Size variation in the o454-nlpD region was investigated by PCR amplification and sequencing. Phylogenetic relationships were assessed by Ecor- and Multilocus sequence- typing (MLST), and a comparative analysis between mutS gene phylogenetic tree obtained with RAxML and the MLST grouping method was performed. Correlation between o454-nlpD patterns and the features described above were analysed. In addition, the importance of 47 PCR-amplified ExPEC-related VAGs for classification of o454-nlpD patterns was investigated by means of Random Forest algorithm.
Four main structures (patterns I-IV) of the o454-nlpD region among ExPEC and commensal E. coli strains were identified. Statistical analysis showed a positive and exclusive association between pattern III and the ExPEC strains. A strong association between pattern III and either the Ecor group B2 or the sequence type complexes known to represent the phylogenetic background of highly virulent ExPEC strains (such as STC95, STC73 and STC131) was found as well. RF analyses determined five genes (csgA, malX, chuA, sit, and vat) to be suitable to predict pattern III strains.
The significant association between pattern III and group B2 strains suggested the o454-nlpD region to be of great value in identifying highly virulent strains among the mixed population of E. coli promising to be the basis of a future typing tool for ExPEC and their gut reservoir. Furthermore, top-ranked VAGs for classification and prediction of pattern III were identified. These data are most valuable for defining ExPEC pathotype in future in vivo assays.
PMCID: PMC4209514  PMID: 25349632
Escherichia coli; MLST; Ecor; mutS-rpoS and o454-nlpD genomic regions
11.  Fine tuning of virulence regulatory pathways in enteric bacteria in response to varying bile and oxygen concentrations in the gastrointestinal tract 
Gut Pathogens  2014;6:38.
After entering the gastrointestinal (GI) tract on the way to their physiological site of infection, enteric bacteria encounter a remarkable diversity in environmental conditions. There are gross differences in the physico-chemical parameters in different sections of the GI tract e.g. between the stomach, small intestine and large intestine. Furthermore, even within a certain anatomical site, there are subtle differences in the microenvironment e.g. between the lumen, mucous layer and epithelial surface. Enteric pathogens must not only survive passage through the rapidly changing environments encountered at different niches of the GI tract but must also appropriately coordinate expression of virulence determinants in response to environmental cues at different stages of infection. There are some common themes in the responses of enteric pathogens to environmental cues, there are also distinct differences that may reflect differences in basic pathogenesis mechanisms. The role of bile and oxygen concentration in spatiotemporal regulation of virulence genes in selected enteric pathogens has been reviewed.
PMCID: PMC4209513  PMID: 25349633
V. cholerae; Shigella; Salmonella; Virulence; Bile; Oxygen concentration
12.  Probiotics and virulent human rotavirus modulate the transplanted human gut microbiota in gnotobiotic pigs 
Gut Pathogens  2014;6:39.
We generated a neonatal pig model with human infant gut microbiota (HGM) to study the effect of a probiotic on the composition of the transplanted microbiota following rotavirus vaccination and challenge. All the HGM-transplanted pigs received two doses of an oral attenuated rotavirus vaccine. The gut microbiota of vaccinated pigs were investigated for effects of Lactobacillus rhamnosus GG (LGG) supplement and homotypic virulent human rotavirus (HRV) challenge. High-throughput sequencing of V4 region of 16S rRNA genes demonstrated that HGM-transplanted pigs carried microbiota similar to that of the C-section delivered baby. Firmicutes and Proteobacteria represented over 98% of total bacteria in the human donor and the recipient pigs. HRV challenge caused a phylum-level shift from Firmicutes to Proteobacteria. LGG supplement prevented the changes in microbial communities caused by HRV challenge. In particular, members of Enterococcus in LGG-supplemented pigs were kept at the baseline level, while they were enriched in HRV challenged pigs. Taken together, our results suggested that HGM pigs are valuable for testing the microbiota’s response to probiotic interventions for treating infantile HRV infection.
PMCID: PMC4209515  PMID: 25349634
Microbiota; Gnotobiotic pigs; Rotavirus; Vaccine; Lactobacillus; Probiotics
13.  Phase-variable restriction/modification systems are required for Helicobacter pylori colonization 
Gut Pathogens  2014;6:35.
One mechanism utilized by bacterial pathogens for host adaptation and immune evasion is the generation of phenotypic diversity by the phasevarion that results from the differential expression of a suite of genes regulated by the activity of a phase-variable methyltransferase within a restriction modification (RM) system. Phasevarions are active in Helicobacter pylori, however there have been no studies investigating the significance of phase-variable RM systems on host colonization.
Two mutant types incapable of phase variation were constructed; a clean deletion mutant (‘DEL’) and a mutant (‘ON’) where the homopolymeric repeat was replaced with a non-repeat synonymous sequence, resulting in expression of the full-length protein. The resulting mutants were assessed for their colonisation ability in the mouse model.
Five phase-variable genes encoding either methyltransferases or members of RM systems were found in H. pylori OND79. Our mutants fell into three categories; 1, those with little effect on colonization, 2, those where expression of the full-length protein was detrimental, 3, those where both mutations were detrimental.
Our results demonstrated that phase-variable methyltransferases are critical to H. pylori colonization, suggesting that genome methylation and generation of epigenetic diversity is important for colonization and pathogenesis. The third category of mutants suggests that differential genome methylation status of H. pylori cell populations, achieved by the phasevarion, is essential for host adaptation. Studies of phase-variable RM mutants falling in the two other categories, not strictly required for colonization, represent a future perspective to investigate the role of phasevarion in persistence of H. pylori.
PMCID: PMC4209511  PMID: 25349630
Phase variation; Restriction; Modification; Phasevarion; Helicobacter pylori
14.  A draft genome of Escherichia coli sequence type 127 strain 2009-46 
Gut Pathogens  2014;6:32.
Escherichia coli are a frequent cause of urinary tract infections (UTI) and are thought to have a foodborne origin. E. coli with sequence type 127 (ST127) are emerging pathogens increasingly implicated as a cause of urinary tract infections (UTI) globally. A ST127 isolate (2009-46) resistant to ampicillin and trimethoprim was recovered from the urine of a 56 year old patient with a UTI from a hospital in Sydney, Australia and was characterised here.
We sequenced the genome of Escherichia coli 2009-46 using the Illumina Nextera XT and MiSeq technologies. Assembly of the sequence data reconstructed a 5.14 Mbp genome in 89 scaffolds with an N50 of 161 kbp. The genome has extensive similarity to other sequenced uropathogenic E. coli genomes, but also has several genes that are potentially related to virulence and pathogenicity that are not present in the reference E. coli strain.
E. coli 2009-46 is a multiple antibiotic resistant, phylogroup B2 isolate recovered from a patient with a UTI. This is the first description of a drug resistant E. coli ST127 in Australia.
PMCID: PMC4155142  PMID: 25197321
Escherichia coli 2009-46; Genome; Sequencing
15.  Whole-genome sequence assembly of Pediococcus pentosaceus LI05 (CGMCC 7049) from the human gastrointestinal tract and comparative analysis with representative sequences from three food-borne strains 
Gut Pathogens  2014;6:36.
Strains of Pediococcus pentosaceus from food and the human gastrointestinal tract have been widely identified, and some have been reported to reduce inflammation, encephalopathy, obesity and fatty liver in animals. In this study, we sequenced the whole genome of P. pentosaceus LI05 (CGMCC 7049), which was isolated from the fecal samples of healthy volunteers, and determined its ability to reduce acute liver injury. No other genomic information for gut-borne P. pentosaceus is currently available in the public domain.
We obtained the draft genome of P. pentosaceus LI05, which was 1,751,578 bp in size and possessed a mean G + C content of 37.3%. This genome encoded an abundance of proteins that were protective against acids, bile salts, heat, oxidative stresses, enterocin A, arsenate and universal stresses. Important adhesion proteins were also encoded by the genome. Additionally, P. pentosaceus LI05 genes encoded proteins associated with the biosynthesis of not only three antimicrobials, including prebacteriocin, lysin and colicin V, but also vitamins and functional amino acids, such as riboflavin, folate, biotin, thiamine and gamma-aminobutyrate. A comparison of P. pentosaceus LI05 with all known genomes of food-borne P. pentosaceus strains (ATCC 25745, SL4 and IE-3) revealed that it possessed four novel exopolysaccharide biosynthesis proteins, additional putative environmental stress tolerance proteins and phage-related proteins.
This work demonstrated the probiotic properties of P. pentosaceus LI05 from the gut and the three other food-borne P. pentosaceus strains through genomic analyses. We have revealed the major genomic differences between these strains, providing a framework for understanding the probiotic effects of strain LI05, which exhibits unique physiological and metabolic properties.
PMCID: PMC4209512  PMID: 25349631
16.  Clinical pilot study: efficacy of triple antibiotic therapy in Blastocystis positive irritable bowel syndrome patients 
Gut Pathogens  2014;6:34.
Blastocystis species are common human enteric parasites. Carriage has been linked to Irritable Bowel Syndrome (IBS). Treatment of Blastocystis spp. with antimicrobials is problematic and insensitive diagnostic methods and re-infection complicate assessment of eradication. We investigated whether triple antibiotic therapy comprising diloxanide furoate, trimethoprim/sulfamethoxazole and secnidazole (TAB) given to diarrhoea-predominant IBS (D-IBS) patients positive for Blastocystis would achieve eradication.
In a longitudinal, prospective case study 10 D-IBS Blastocystis-positive patients took 14 days of diloxanide furoate 500 mg thrice daily, trimethoprim/sulfamethoxazole 160/80 mg twice daily and secnidazole 400 mg thrice daily. Faecal specimens were collected at baseline, day 15 and 4 weeks after completion of TAB. Specimens were analysed using faecal smear, culture and polymerase chain reaction (PCR) of the 16 SSU rRNA. Patients kept a concurrent clinical diary.
Six (60%) patients cleared Blastocystis spp. after TAB, including three who had failed previous therapy. Subtypes detected were ST3 (60%), ST4 (40%), ST1 (20%) and ST7, 8 (10%); four patients had mixed ST infections. Serum immunoglobulin A (IgA) levels were low in 40% of patients. Higher rates of Blastocystis clearance were observed in patients symptomatic for less than a year (Mann–Whitney, p = 0.032, 95% confidence) with no associations found with age, previous antibiotic therapy, faecal parasite load, ST, IgA level or clinical improvement.
Clearance of Blastocystis spp. was achieved with TAB in 60% of D-IBS patients, an improvement over conventional monotherapy. Higher clearance rates are needed to facilitate investigation of the relevance of this parasite in clinically heterogenous IBS.
PMCID: PMC4209510  PMID: 25349629
Blastocystis; Therapy; Irritable bowel syndrome; PCR
17.  Anti-inflammatory effects of Lactococcus lactis NCDO 2118 during the remission period of chemically induced colitis 
Gut Pathogens  2014;6:33.
Many probiotic bacteria have been described as promising tools for the treatment and prevention of inflammatory bowel diseases (IBDs). Most of these bacteria are lactic acid bacteria, which are part of the healthy human microbiota. However, little is known about the effects of transient bacteria present in normal diets, including Lactococcus lactis.
In the present study, we analysed the immunomodulatory effects of three L. lactis strains in vitro using intestinal epithelial cells. L. lactis NCDO 2118 was administered for 4 days to C57BL/6 mice during the remission period of colitis induced by dextran sodium sulphate (DSS).
Only one strain, L. lactis NCDO 2118, was able to reduce IL-1β-induced IL-8 secretion in Caco-2 cells, suggesting a potential anti-inflammatory effect. Oral treatment using L. lactis NCDO 2118 resulted in a milder form of recurrent colitis than that observed in control diseased mice. This protective effect was not attributable to changes in secretory IgA (sIgA); however, NCDO 2118 administration was associated with an early increase in IL-6 production and sustained IL-10 production in colonic tissue. Mice fed L. lactis NCDO 2118 had an increased number of regulatory CD4+ T cells (Tregs) bearing surface TGF-β in its latent form (Latency-associated peptide-LAP) in the mesenteric lymph nodes and spleen.
Here, we identified a new probiotic strain with a potential role in the treatment of IBD, and we elucidated some of the mechanisms underlying its anti-inflammatory effect.
PMCID: PMC4126083  PMID: 25110521
Lactococcus lactis; Colitis; Cytokines; Regulatory T cells; Probiotics
18.  The genome of Bifidobacterium pseudocatenulatum IPLA 36007, a human intestinal strain with isoflavone-activation activity 
Gut Pathogens  2014;6:31.
Bifidobacterium species, including Bifidobacterium pseudocatenulatum, are among the dominant microbial populations of the human gastrointestinal tract. They are also major components of many commercial probiotic products. Resident and transient bifidobacteria are thought to have several beneficial health effects. However, our knowledge of how these bacteria interact and communicate with host cells remains poor. This knowledge is essential for scientific support of their purported health benefits and their rational inclusion in functional foods.
This work describes the draft genome sequence of Bifidobacterium pseudocatenulatum IPLA 36007, a strain isolated as dominant from the feces of a healthy human. Besides several properties of probiosis, IPLA 36007 exhibited the capability of releasing aglycones from soy isoflavone glycosides. The genome contains 1,851 predicted genes, including 54 genes for tRNAs and fie copies of unique 16S, 23S and 5S rRNA genes. As key attributes of the IPLA 36007 genome we can mention the presence of a lysogenic phage, a cluster encoding type IV fimbriae, and a locus encoding a clustered, regularly interspaced, short, palindromic repeat (CRISPR)-Cas system. Four open reading frames (orfs) encoding β-glucosidases belonging to the glycosyl hydrolase family 3, which may act on isoflavone glycosides, were encountered. Additionally, one gene was found to code for a glycosyl hydrolase of family 1 that might also have β-glucosidase activity.
The availability of the B. pseudocatenulatum IPLA 36007 genome should allow the enzyme system involved in the release of soy isoflavone aglycones from isoflavone glycosides, and the molecular mechanisms underlying the strain’s probiotic properties, to be more easily understood.
PMCID: PMC4121622  PMID: 25097668
19.  Genome sequencing of multidrug resistant novel Clostridium sp. BL8 reveals its potential for pathogenicity 
Gut Pathogens  2014;6:30.
The human gut microbiome is important for maintaining the health status of the host. Clostridia are key members of the human gut microbiome, carrying out several important functions in the gut environment. Hence understanding the role of different Clostridium species isolated from human gut is essential. The present study was aimed at investigating the role of novel Clostridium sp. isolate BL8 in human gut using genome sequencing as a tool.
The genome analysis of Clostridium sp. BL8 showed the presence of several adaptive features like bile resistance, presence of sensory and regulatory systems, presence of oxidative stress managing systems and presence of membrane transport systems. The genome of Clostridium sp. BL8 consists of a wide variety of virulence factors like phospholipase C (alpha toxin), hemolysin, aureolysin and exfoliative toxin A, as well as adhesion factors, proteases, Type IV secretion system and antibiotic resistance genes. In vitro antibiotic sensitivity testing showed that Clostridium sp. BL8 was resistant to 11 different tested antibiotics belonging to 6 different classes. The cell cytotoxicity assay confirmed the cytotoxic effect of Clostridium sp. BL8 cells, which killed 40% of the Vero cells after 4 hrs of incubation.
Clostridium sp. BL8 has adapted for survival in human gut environment, with presence of different adaptive features. The presence of several virulence factors and cell cytotoxic activity indicate that Clostridium sp. BL8 has a potential to cause infections in humans, however further in vivo studies are necessary to ascertain this fact.
PMCID: PMC4115488  PMID: 25076986
Clostridium; Virulence; Antibiotic resistance; Ion Torrent PGM™
20.  Gene clusters of Hafnia alvei strain FB1 important in survival and pathogenesis: a draft genome perspective 
Gut Pathogens  2014;6:29.
Hafnia alvei is an opportunistic pathogen involved in various types of nosocomical infections. The species has been found to inhabit food and mammalian guts. However, its status as an enteropathogen, and whether the food-inhabiting strains could be a source of gastrointestinal infection remains obscure. In this report we present a draft genome of H. alvei strain FB1 isolated from fish paste meatball, a food popular among Malaysian and Chinese populations. The data was generated on the Illumina MiSeq platform.
A comparative study was carried out on FB1 against two other previously sequenced H. alvei genomes. Several gene clusters putatively involved in survival and pathogenesis of H. alvei FB1 in food and gut environment were characterised in this study. These include the widespread colonisation island (WCI), the tad locus that is known to play an essential role in biofilm formation, a eut operon that might contribute to advantage in nutrient acquisition in gut environment, and genes responsible for siderophore production This features enable the bacteria to successful colonise in the host gut environment.
With the whole genome data of H. alvei FB1 presented in this study, we hope to provide an insight into future studies on this candidate of enteropathogen by looking into the possible mechanisms employed to survive stresses and gain advantage in competitions, which eventually leads to successful colonisation and pathogenesis. This is to serve as the basis for more effective clinical diagnosis and treatment.
PMCID: PMC4114107  PMID: 25075225
Hafnia alvei; Gut pathogen; Widespread Colonisation Island; tad; Ethanolamine utilisation; eut; Siderophore; Next generation sequencing
21.  Genome sequencing, annotation and comparative genomic analysis of Shigella dysenteriae strain SD1D 
Gut Pathogens  2014;6:28.
Shigellosis is an acute form of gastroenteritis caused by the bacteria belonging to the genus Shigella. It is the most common cause of morbidity and mortality in children. Shigella belongs to the family Enterobactericeae, which is a Gram-negative and rod shaped bacterium. In the present study, we report the draft genome of Shigella dysenteriae strain SD1D, which was isolated from the stool sample of a healthy individual.
Based on 16S rRNA gene sequence and phylogenetic analysis, the strain SD1D was identified as Shigella dysenteriae. The draft genome of SD1D consisted of 45, 93, 159 bp with a G + C content of 50.7%, 4, 960 predicted CDSs, 75 tRNAs and 2 rRNAs. The final assembly contained 146 contigs of total length 45, 93, 159 bp with N50 contig length of 77, 053 bp; the largest contig assembled measured 3, 85, 550 bp.
We have for the first time performed the whole genome sequencing of Shigella dysenteriae strain SD1D. The comparative genomic analysis revealed several genes responsible for the pathogenesis, virulence, defense, resistance to antibiotics and toxic compounds, multidrug resistance efflux pumps and other genomic features of the bacterium.
PMCID: PMC4099087  PMID: 25028600
Shigella dysenteriae; Shigellosis; G + C content; EzTaxon; RAST
22.  Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes 
Gut Pathogens  2014;6:27.
Clarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. Using the MiSeq platform, whole-genome sequencing of the 19 clinical strains and the reference strain ATCC26695 was performed to identify single nucleotide variants (SNVs) of multi-drug resistant efflux pump genes in the CLR-resistant phenotype.
Based on sequencing data of ATCC26695, over one million sequencing reads with over 50-fold coverage were sufficient to detect SNVs, but not indels in the bacterial genome. Sequencing reads of the clinical isolates ranged from 1.82 to 10.8 million, and average coverage ranged from 90.9- to 686.3-fold, which were acceptable criteria for detecting SNVs. Utilizing the conventional approach of allele-specific PCR, point mutations in the 23S rRNA gene were detected in 12 clinical resistant isolates, but not in 7 clinical susceptible isolates. All sequencing reads of CLR-resistant strains had a G mutation in an identical position of the 23S rRNA gene. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of TolC homologues, which have been implicated in multi-drug resistance, were examined. Specific SNVs were dominantly found in resistant strains.
Gene clusters of TolC homologues are involved in CLR susceptibility profiles in individual H. pylori strains. Whole-genome sequencing has yielded novel understanding of genotype-phenotype relationships.
PMCID: PMC4079918  PMID: 24995043
Whole-genome sequencing; Helicobacter pylori; Clarithromycin; Multidrug efflux; TolC homolog
23.  Co-occurrence of driver and passenger bacteria in human colorectal cancer 
Gut Pathogens  2014;6:26.
Both genetic and epigenetic alterations have been reported to act as driving forces of tumorigenesis in colorectal cancer (CRC), but a growing body of evidence suggests that intestinal microbiota may be an aetiological factor in the initiation and progression of CRC. Recently, the “driver-passenger” model for CRC has connected these different factors, but little has been done to characterize the CRC gut microbiome.
Building on the driver-passenger model, we used 454 pyrosequencing of bacterial 16S rRNA genes associated with 10 normal, 10 adenoma, and 8 tumor biopsy samples, and found 7 potential driver bacterial genera and 12 potential passenger bacterial genera (7 being pro-inflammatory and 5 anti-inflammatory). Further analysis also showed certain co-expression patterns among different clusters of bacteria that may potentially be related to the promotion or progression of gut cancers.
The present findings provide preliminary experimental evidence supporting the proposition of bacterial “driver-passenger model” for CRC, and identified potentially novel microbial agents that may be connected to risk of CRC in a Han Chinese population.
PMCID: PMC4080773  PMID: 24995042
CRC; Driver bacteria; Passenger bacteria; Co-occurrence
24.  The murine appendiceal microbiome is altered in spontaneous colitis and its pathological progression 
Gut Pathogens  2014;6:25.
Inflammatory bowel disease (comprising ulcerative colitis and Crohn’s disease) is a multifactorial disease that is extensively associated with stool microbiome changes (dysbiosis). Appendicitis and appendectomy limits subsequent colitis, clinically, and in animal models. We wanted to examine how the appendiceal and stool microbiome fared in our spontaneous colitic Winnie (Muc2−/−) mice model.
Two C57BL/6 and 10 Winnie mice at ages 12 and 15 weeks were euthanized for stool and caecal patch samples. DNA was extracted using the QIAamp DNA Stool Mini Kit then the V1-V3 hypervariable region of the 16S rRNA gene was sequenced using the Roche/454 GS FLX + pyrosequencing instrument. A Galaxy metagenomic pipeline was used to define phyla and families at sequence similarity threshold of ≥ 80%.
Bacteriodetes was decreased in 15-week Winnie mice appendices compared to corresponding stool samples (P < 0.01). Proteobacteria was increased in appendices of Winnie mice compared to corresponding stool samples (P < 0.05). The Bacteroidetes family Rikenellaceae could be identified only in 15-week-old Winnie mice appendices. A higher quantity of Acetobacteraceae (Proteobacteria phylum) was present in 15-week Winnie mice when compared to 12-week Winnie mice (P < 0.01). Helicobacteraceae (Proteobacteria phylum), which is prominent in all Winnie mice, is absent in control mice.
The appendiceal dysbiosis observed in our Winnie mice is commensurate with, and adds to extant literature data. The presence of Helicobacteraceae (Proteobacteria) only in colitic Winnie mice (but not control mice) is consistent with reports of increased Helicobacter in IBD patients. Bacteroides (Bacteroidetes) decreases may be a reflection of reduced anti-inflammatory commensal species such as B. fragilis. Further research is warranted to expand and delineate the relationship between IBD and the appendix microbiome.
PMCID: PMC4085080  PMID: 25002910
Microbiome; Appendix; Stool; Winnie; Inflammatory bowel disease; Colitis; Proteobacteria; Bacteroidetes
25.  Human enteroendocrine cell responses to infection with Chlamydia trachomatis: a microarray study 
Gut Pathogens  2014;6:24.
Enteroendocrine cells (EEC) are highly specialized cells producing signalling molecules vital to the normal functions of the gut. Recently, we showed altered protein distribution in Chlamydia infected EEC in vitro. The aim of this study was to perform a microarray analysis of the response pattern of EEC from both large and small bowel to infection in vitro, using Chlamydia trachomatis infection as a model.
Two human EEC lines: LCC-18, derived from a neuroendocrine colonic tumour, and CNDT-2, derived from a small intestinal carcinoid, were infected using cultured C. trachomatis serovar LGV II strain 434 (ATCC VR-902B). Penicillin G was used to induce persistent infection. We used microarray analysis (Affymetrix GeneChip®) for studying changes in gene expression at different stages of infection.
Twenty-four hours after active and persistent infection, 66 and 411 genes in LCC-18 and 68 and 170 genes in CNDT-2 cells, respectively showed mean expression ratios >2-fold compared to non-infected cells. These genes encoded factors regulating apoptosis, cell differentiation, transcription regulation, cytokine activity, amine biosynthesis and vesicular transport. We found significant differences in gene transcription levels between persistently infected and non-infected cells in 10 genes coding for different solute carrier transporters (SLC) and in 5 genes related to endocrine function (GABARAPL1, GRIP1, DRD2, SYT5 and SYT7).
Infected EEC cells exhibit cell-type specific patterns related to vesicular transport, secretion and neurotransmitters. EEC play a pivotal role in regulation of gut motility and an impairment of enteroendocrine function can contribute to motility disorders.
PMCID: PMC4067063  PMID: 24959205
Enteroendocrine cells; Chlamydia trachomatis; Infection; Microarray

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