Research Article Culturable Bacterial Microbiota of the...

Research ArticleCulturable Bacterial Microbiota of the Stomach of Helicobacterpylori Positive and Negative Gastric Disease Patients

Yalda Khosravi1 Yakhya Dieye2 Bee Hoon Poh3 Chow Goon Ng4 Mun Fai Loke1

Khean Lee Goh5 and Jamuna Vadivelu1

1 Department of Medical Microbiology Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia2 Vice-chancellorrsquos Office University of Malaya 50603 Kuala Lumpur Malaysia3 BP Diagnostics Centre Sdn Bhd 30250 Ipoh Perak Malaysia4Department of Microbiology Yong Loo Lin School of Medicine National University of Singapore Singapore 1175455 Department of Medicine Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Jamuna Vadivelu jamunaummcedumy

Received 12 May 2014 Revised 16 June 2014 Accepted 16 June 2014 Published 3 July 2014

Academic Editor Fernando Rodrigues

Copyright copy 2014 Yalda Khosravi et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Human stomach is the only known natural habitat of Helicobacter pylori (Hp) a major bacterial pathogen that causes differentgastroduodenal diseases Despite this the impact of Hp on the diversity and the composition of the gastric microbiota has beenpoorly studied In this study we have analyzed the culturable gastric microbiota of 215Malaysian patients including 131Hp positiveand 84Hp negative individuals that were affected by different gastric diseases Non-Hp bacteria isolated from biopsy samples wereidentified bymatrix assisted laser desorption ionization-time of flightmass spectrometry based biotyping and 16SrRNA sequencingThe presence ofHp did not significantly modify the diversity of the gastric microbiota However correlation was observed betweenthe isolation of Streptococci and peptic ulcer disease In addition as a first report Burkholderia pseudomalleiwas also isolated fromthe gastric samples of the local population This study suggested that there may be geographical variations in the diversity of thehuman gastric microbiome Geographically linked diversity in the gastric microbiome and possible interactions between Hp andother bacterial species from stomach microbiota in pathogenesis are proposed for further investigations

1 Introduction

A luxurious microbial flora that is important to the healthand well-being of the host inhabits human gastrointesti-nal tract Gut microbiota contributes to several functionsincluding energy harvest and storage from the diet [1]development and regulation of the gut-associated mucosalimmune system [2] regulation of the central nervous sys-tem [3] detoxification of xenobiotics and carcinogens andprotection against colonization by pathogens [4] The gutmicrobiota is acquired early after birth and is shaped byseveral factors including diet [5] genetic background andenvironment [6] Its composition and complexity can bemodified by physiological changes such as aging [5] andpregnancy [7] Fluctuations of the gut microbiota can alsoresult from antibiotic treatment metabolic immunological

or infectious diseases [6] In particular chronic infectiousand noninfectious diseases can imprint long-lasting changesof the gut microbiota that greatly impact gut homeostasisand can favor the development of other diseases [4] Theanalysis of the gut microbiota and its variation is emergingas a medical approach that will be used in the prevention ortreatment of diseases

Helicobacter pylori (Hp) is a major pathogen that hasbeen associated with humans for over 60000 years [8] Itis estimated that more than half of the world population isinfected by Hp [9] However Hp infection remains asymp-tomatic in the majority of the cases In a small proportion ofindividuals the infection leads to different diseases includingpeptic ulcers chronic atrophic gastritis gastric cancers andgut lymphoma [10 11] Interestingly Hp is also believedto confer on its host a protection against certain diseases

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 610421 10 pageshttpdxdoiorg1011552014610421

2 The Scientific World Journal

including allergies inflammatory disorders [12] and tubercu-losis [13] Hp pathogenesis and its interactions with the gut-associatedmucosal immune system have been widely studied[14] Studies of human patients and investigations in animalmodels including mouse gerbil and monkey have generateda substantial amount of knowledge on the acute phase ofHp infection establishment of colonization activation of thegut-associated mucosal immune system and immune escapestrategies that lead to chronic colonization [14] Humanstomach is the only known natural habitat for Hp and onceestablished this bacterium is usually the predominant speciesof the gastric microbiota [15] However little is known aboutthe modification of the gastric microbiota that results fromchronic Hp infection and the interactions of this bacteriumwith other members of the gastric ecosystem Study of thegastric microbiota has been delayed by the belief prior toHp discovery that human stomachwas a hostile environmentthat could not support colonization by microorganisms Fewstudies have analyzed the composition of the human gastricmicrobiota in healthy individuals and in patients sufferingfrom different diseases These studies used high-throughputmolecular approaches including metagenomics [15ndash18] ter-minal restriction fragment length polymorphism (T-RFLP)[19] and microarray [20] that are powerful techniques thatenable the capture of DNA sequences from most of thebacteria present in the stomach including both culturable andnonculturable species However one important limitationin these studies is the small number of human samples(4ndash23 individuals) making it difficult to generate statisticallysignificant conclusionsWith regard toHp even fewer studieshave analyzed the impact of this bacterium on the compo-sition of the gastric microbiota Recently Hu et al used acultivation-based approach to isolate non-Hp species fromgastric biopsies of 103 patients infectedwithHp and identifiedmost of the bacteria at the species level using MALDI-TOFmass spectrometry-based biotyping [21] This approach hasthe limitation of omitting nonculturable bacteria that arepredominant in the stomach [22] However the accuracyof mass spectrometry biotyping in bacterial identificationpermits rapid analysis of large number of samples

In this study we wanted to gain further insight intothe impact of Hp colonization on the composition and thediversity of human gastric microbiota We used the massspectrometry biotyping technology to identify bacteria thatwere cultured from stomach biopsies of 215 patients including131 Hp-positive and 84 Hp-negative subjects The patientswere affected by different gastric diseases and belongedto different ethnic groups present in Malaysia Beside theinfluence of Hp infection the approach taken in this studyhas the potential to provide an insight into the contribution ofother factors such as ethnicity and disease to the compositionand the diversity of the gastric microbiota

2 Materials and Methods

21 Study Population Gastric biopsy samples were obtainedbetween 2011 and 2013 from patients referred for endoscopyexamination at the University Malaya Medical Center

(UMMC Kuala Lumpur Malaysia) Biopsy samples weretaken from the antrum and body of the stomach for eachpatient This study was approved by the UMMC MedicalEthics Committee and written consent was obtained frompatients before being included in the study

22 Bacterial Growth and Identification Fresh gastric biopsytissues were homogenized and simultaneously inoculatedonto nonselective and selective chocolate agar plates (supple-mentedwith 5horse blood and in the case of selectivemediaantibiotics included trimethoprim (5120583gmL) vancomycin(10 120583gmL) nalidixic acid (20120583gmL) and amphotericin B(5 120583gmL)) All the antibiotics were from Sigma-Aldrich Cor-poration (St Louis MO USA)The agar plates were simulta-neously incubated at 37∘C under humidified condition with10 carbon dioxide and under ambient air condition Allwell-isolated colonies were selected and cultured for furtherstudies Hp colonies formed after at least three days ofgrowth on selective plates in our culture conditions and wereconfirmed by positivity for urease catalase and oxidase testsAdditionally Gram staining and microscopic analysis wasperformed to verify the presence of Gram-negative spiralrod bacteria Non-Hp bacterial species (hereafter termed asother bacteria) were identified using a combination of colonymorphology mass spectrometry and 16SrRNA sequencing

23 Colony Morphology Other colonies were picked andcategorized according to morphological features includingsize texture pigmentation hemolysis shape appearancemargin and elevation At least one colony from each mor-photype present on each plate was selected The colonieswere restreaked onto non-selective plates and after sufficientgrowth stored at minus80∘C in a brain heart infusion (BHI) broth(Sigma-Aldrich USA) supplemented with 04 (wtvol)yeast extract and 20 (volvol) glycerol

24 Mass Spectrometry Ethanolformic acid extraction andmass spectrometry was performed as recommended byBruker Daltonics GmbH (Breme Germany) Briefly 1 to 2well-isolated colonies (or a few colonies in the case of a smallcolony size) of similarmorphotype were suspended in 300 120583Lof ultrapure (type 1) water (EMD Millipore CorporationBillerica MA USA) to which 900 120583L of pure ethanol wereaddedThe samples were centrifuged (13000timesg 2min)Thesupernatants were decanted and the pellets were air-driedOnce dried 50 120583L of 70 formic acid (Fluka AnalyticalHPLC grade) and 50 120583L of acetonitrile (Friedemann SchmidtChemical HPLC grade) were added and the samples werecentrifuged again Subsequently 1 120583L of the supernatant wasspotted onto a MSP 96 target polished steel BC microScoutTarget (Bruker Daltonics GmbH) and allowed to completelyair-dry before overlaid with 1 120583L of fresh 120572-cyano-4-hydroxy-cinnamic acid (HCCA Bruker Daltonics GmbH) matrixsolution The samples were dried before analysis on theMicroflex LRF MALDI-TOF Mass Spectrometry Systemequipped with a 60Hz nitrogen laser and microScout ionsource (Bruker Daltonics GmbH) The parameter settingswere as follows delay 12719 pts ion source 20 kV ion

The Scientific World Journal 3

source 1834 kV lens voltage 9 kV mass range 2ndash20 kDaSpectra were obtained in the positive linear mode after 240shots RawMALDI-TOF spectra were analyzed with MALDIBiotyper 31 (Bruker Daltonics) using the default settingsThethreshold for peak acceptance was a signal-to-noise ratio of 3Peaks with amass-to-charge (119898119911) ratio differencegt250 ppmwere considered to be identical The peak lists generatedwerematched against the Biotyper reference library using theintegrated pattern matching algorithm Identification scorecriteria used based on manufacturer were as follow a scoreof ge2000 showed the identification in species-level a scoreof 1700 to 1999 showed identification to the genus leveland a score of lt1700 indicated an unreliable identificationDendrograms were created using the correlation distancemeasure and the average linkage algorithm settings of theBiotyper software to examine the diversity and relatednessof isolates within the species based on protein fingerprinting[23]

25 16SrRNA Gene Sequencing Bacterial samples unable tobe resolved by Biotyper up to the species level were analysedby 16SrRNA gene sequencing Genomic DNA was preparedusing RTP Bacteria DNA Mini Kit (STRATEC BiomedicalAG Berlin-Buch Germany) according to the manufacturerrsquosrecommendations The hyper variable regions V3 and V6of the 16SrRNA gene were PCR-amplified using universalprimers V3f (51015840-CCAGACTCCTACGGGAGGCAG-31015840)V3r(51015840-CGTATTACCGCGGCTGCTG-31015840) and V6f (51015840-TCG-ATGCAACGCGAAGAA)V6r (51015840-ACATTTCACAACAC-GAGCTGACGA) respectively [24] The respective 203 and124 bp PCR products were purified using the Wizard SV Geland PCR Clean-Up System (Promega Corporation Madi-son WI USA) and sequences were determined using theABI PRISM DNA sequencer (Perkin Elmer Inc WalthamMA USA) Nucleotide sequences were analysed using theNational Center for Biotechnology Information BLAST soft-ware (httpwwwncbinlmnihgov)

26 Statistical Analysis Statistical analyses were performedusing the IBM SPSS 220 software The diversity of the non-Hp culturable microbiota (number of species isolated) inthe different groups (Hp-positive versus Hp-negative ethnicgroups diseases etc) was analysed using independent t-test or one-way ANOVA The prevalence of Hp and non-Hpbacteria in the different groups was analysed using Fischerrsquosexact chi-squared test A 119875 value le 005 was considered asstatistically significant

3 Results

31 Patient Demographic and Clinical Data We analyzedthe gastric biopsy samples from 215 patients who presentedfor endoscopy at the University of Malaya Medical CentreThe patients consisted of 102 (474) and 113 (526) menand women respectively with a median age of 59 years(range 14 to 85 years) The ethnic distribution of the patientscorresponded to 18 Malay (84) 91 Chinese (423) and92 Indian (438) the three main ethnic groups in Malaysia

Table 1 Demographic data prevalence of Helicobacter pylori andmean number of other microbial species isolated from gastricdisease patients

Hp positive119873 ()

Hp negative119873 () Non-Hp

GenderMale 61 (598) 41 (402) 213Female 70 (619) 43 (381) 176

RaceIndian 56 (609) 36 (391) 215Chinese 55 (604) 36 (396) 16Malay 12 (667) 6 (333) 228Other 8 (571) 6 (429) 221

DiseaseNUD 112 (605) 73 (395) 19PUD 17 (773) 5 (227) 168GC 2 (25) 6 (75) 338

Hp Helicobacter pylori 119873 number of biopsies percentage in therespective group Non-Hp mean number of non-Hp species isolated in therespective group

The remaining 14 patients (65) belonged to minor ethnicgroups present in the country One hundred eighty-fivesubjects (876) were diagnosed with nonulcer dyspepsia(NUD) 22 (102) with peptic ulcer disease (PUD) and 8(37) with gastric cancer (GC) (Table 1)

32 Prevalence and Distribution of Helicobacter pylori inGastric Antrum and Body Hpwas successfully cultured fromsamples of 131 (609) individuals including 605 of theNUD (112185) 773 of the PUD (1722) and 25 (28)of the GC patients (Table 1) Biopsies from which Hp couldnot be cultured were further analyzed by microscopic exam-ination after hematoxylin-eosin staining by an independentconsultant histopathologist Samples from which Hp failedto be cultured and that did not show the bacterium inmicroscopy were considered as Hp-negative in this studyPrevalence of Hp was independent of the age gender orethnicity of the patients In contrast significant differenceswere noted between the three disease groups (Table 1) Hppresence was comparable in NUD (773 positive samples)and PUD (605 positive samples) patients while prevalencein both groups was significantly higher than in GC subjects(25 positive samples) This result is consistent with theknown progressive disappearance of Hp from gastric tissuesduring carcinogenesis [25]Hp can colonize different regionsof the stomach mainly the antrum and the corpus Wewanted to know how Hp was distributed between these twogastric areasThemajority (618) of theHp-positive patientsharbored the bacteria in both the antrum and the corpuswhile 26 and 115 were colonized only at the antrum orthe body respectively These figures make a total of 878and 733 of the positive subjects colonized in the antrumand the body respectivelyHp distribution in the antrum and


Table 2 Other microbial species identified by Biotyper

Microorganism Number of positive biopsies H pylori positive119873 ()

H pylori negative119873 ()

119875 value

Streptococcus 121 80 41 0091S119898119894119905119894119904dagger 37 23 (622) 14 (378)S parasanguinis 32 23 (719) 9 (281)S anginosus 24 14 (583) 10 (417)S salivarius 9 5 (556) 4 (444)S constellatus 4 3 (75) 1 (25)S gallinaceus 3 2 (667) 1 (333)S vestibularis 3 2 (667) 1 (333)S gallolyticus 2 1 (50) 1 (50)S gordonii 2 2 (100) 0S peroris 2 2 (100) 0S sanguinis 2 2 (100) 0S australis 1 1 (100) 0

Neisseria 44 33 33 0038N flavescens 21 16 (762) 5 (238)N perflava 11 7 (636) 4 (364)N subflava 7 7 (100) 0N macacae 4 2 (50) 2 (50)N mucosa 1 1 (100) 0

Klebsiella 42 23 19 0382K pneumoniae 41 23 (561) 18 (439)K oxytoca 1 0 1 (100)

Lactobacillus 42 27 15 0725L fermentum 32 21 (656) 11 (344)L paracasei 4 3 (75) 1 (25)L johnsonii 2 1 (50) 1 (50)L reuteri 2 1 (50) 1 (50)L salivarius 2 1 (50) 1 (50)

Rothia 22 17 5 0111R mucilaginosa 20 16 (80) 4 (20)R dentocariosa 2 1 (50) 1 (50)

Bacillus 14 12 2 0783B cereus 10 10 (100) 0B licheniformis 2 1 (50) 1 (50)B firmus 1 1 (100) 0B subtilis 1 0 1 (100)

Acinetobacter 10 6 4 0512A baumannii 9 6 (667) 3 (333)A schindleri 1 0 1 (100)

Staphylococcus 9 5 4 mdashS hominis 6 2 (333) 4 (667)S aureus 1 1 (100) 0S capitis 1 1 (100) 0S epidermidis 1 1 (100) 0


Table 2 Continued



119875 value

Candida 8 4 4 mdashC albicans 5 2 (40) 3 (60)C glabrata 2 2 (100) 0C tropicalis 1 0 1 (100)

Actinomyces 5 4 1 mdashA odontolyticus 3 2 (667) 1 (333)A graevenitzii 1 1 (100) 0A oris 1 1 (100) 0

Corynebacterium 5 5 0 mdashC simulans 4 4 (100) 0C argentoratense 1 1 (100) 0

Gemella 5 2 3 mdashG haemolysans 2 1 (50) 1 (50)G morbillorum 2 1 (50) 1 (50)G sanguinis 1 0 1 (100)

Others 91Escherichia 119888119900119897119894Dagger 24 15 (652) 8 (348) 0822Burkholderia 119901119904119890119906119889119900119898119886119897119897119890119894dagger 15 10 (667) 5 (333) 0786Morganella morganii 13 9 (643) 5 (357) 1000Lysinibacillus fusiformis 9 3 (333) 6 (667) mdashPseudomonas aeruginosa 6 2 (333) 4 (667) mdashMicrococcus luteus 6 3 (50) 3 (50) mdashPaenibacillus urinalis 3 1 (333) 2 (667) mdashKocuria palustris 3 1 (333) 2 (667) mdashEnterobacter aerogenes 3 1 (333) 2 (667) mdashMoraxella osloensis 2 1 (50) 1 (50) mdashCitrobacter koseri 2 0 2 (100) mdashArthrobacter castelli 1 0 1 (100) mdashBrevibacillus parabrevis 1 0 1 (100) mdashYersinia enterocolitica 1 0 1 (100) mdashGranulicatella adiacens 1 1 (100) 0 mdashHaemophilus parainfluenzae 1 1 (100) 0 mdash

daggerIdentified by 16SrRNA sequencing Daggerlactose-fermenting organism on MacConkey agar 119875 value le 005 is considered statistically significant 119873 number ofpositive biopsies percentage depending on Hp status

the body was independent of the age gender ethnicity anddisease of the patients

33 Species Identification of Non-Helicobacter pylori BacteriaTo analyze the influence of Hp colonization on the compo-sition and diversity of gastric microbiota we isolated 552colonies from the gastric biopsies and attempted to makespecies identification by MALDI-TOF Mass Spectrometrybiotyping Most of these colonies were successfully identifiedat the species level (Table 2) However 43 colonies (from 37patients) belonging to the genus Streptococcus could not be

discriminated between the species S pneumonia S mitisand S oralis Sequencing of the hyper variable regions V3and V6 of the 16SrRNA gene showed that the 43 Strepto-coccus colonies corresponded with S mitis Additionally weanalyzed the 16SrRNA gene sequences of 18 colonies (from15 patients) initially identified as Burkholderia fongorumby biotyping The Brucker reference library used in thisstudy to identify the colonies contained limited data fromBurkholderia bacteria that could not discriminate betweenspecies of this genus [26] The results confirmed that thecolonies actually corresponded to B pseudomallei Thirtycolonies (from 23 patients) were assigned by biotyping as


either E coli or Shigella species To unequivocally identifythese bacteria we grew the cells on McConkey agar Inthis medium lactose-fermenting bacteria (eg E coli) formred colonies while nonlactose fermenter bacteria (Shigella)form white colonies The results showed that the 30 coloniescorresponded to E coli Overall by combining biotyping16SrRNA gene sequencing and growth onto MacConkeymedium we were able to identify at the species level all theother microbial colonies isolated from the biopsies

34 Predominant Non-Helicobacter pylori Bacteria Isolatedfrom the Stomach of Gastric Diseases Patients In total 64other microbial species representing 3 bacterial phyla wereidentified (Table 2) Firmicutes (270 colonies from 208 biop-sies) and Proteobacteria (220 colonies from 160 biopsies)were predominant while Actinobacteria (53 colonies from 43biopsies) was less frequent Additionally 9 colonies from 8biopsies corresponded to Candida species (fungal phylum ofAscomycota) At the genus level Streptococci were the mostprevalent (126 positive biopsies 586) followed by Neisseria(44 positive biopsies) Klebsiella (41 positive biopsies) andLactobacilli (41 positive biopsies) thatwere each isolated fromsim20 of the samples Escherichia coli (23 positive biopsies)and Rothia mucilaginosa (20 positive biopsies) were presentin sim10 of the patients The predominant species (Table 2)comprised several human oral and upper respiratory tractcommensals (Streptococcus parasanguinis S mitis S salivar-iusNeisseria flavescensN perflava andRmucilaginosa) andmembers of the gut microflora (Lactobacillus fermentum andE coli) Interestingly a few human pathogens or opportunisticpathogens were also isolatedThese included Klebsiella pneu-monia Streptococcus anginosus Burkholderia pseudomalleiBacillus cereus and Acinetobacter baumannii

35 Helicobacter pylori Colonization Did Not SignificantlyAffect theDiversity of theGastricMicrobiota Up to 12 non-Hpbacteria could be isolated from individual biopsies (Table 3)No non-Hp cells could be isolated from 274 (59) of thepatients while 1 and 2 non-Hp bacteria were cultured from233 and 219of the samples respectivelyThese figures aresimilar to those reported from a previous study that analyzedgastric bacteria concurrent toHp infection using an approachsimilar to the one undertaken in this study [21] The meannumber of non-Hp species in single individuals was 193and did not significantly differ between Hp-positive and Hp-negative patients (Table 1)This observation is consistent withprevious findings suggesting that although Hp colonisationmight modify the gastric microflora it does not significantlyaffect its diversity [15] Similarly the number of non-Hp cellsisolated from biopsies did not significantly differ dependingon the age disease gender or race of the patients (Table 1)

36 Disease and Ethnicity Have a Greater Impact ThanHelicobacter pylori on the Composition of Gastric MicrobiotaTo analyze the influence of Hp on the composition of gastricmicrobiota in gastric disease patients we compared the pres-ence of individual non-Hp bacterial species in Hp-positiveand Hp-negative subjects Remarkably Bacillus cereus was

Table 3 Number of othermicrobial species isolated from individualbiopsies


Hp negative119873 () Total

0 30 (229) 29 (345) 59 (274)1 31 (237) 19 (226) 50 (233)2 32 (244) 15 (179) 47 (219)3 20 (153) 6 (71) 26 (121)4 3 (23) 6 (71) 9 (42)5 4 (31) 5 (60) 9 (42)6 3 (23) 2 (24) 5 (23)7 4 (31) 0 4 (19)8 2 (15) 0 2 (09)9 2 (15) 1 (12) 3 (14)12 0 1 (12) 1 (05)Total (119873) 131 84 215119873 number of gastric samples in the respective group percentage in therespective group

the only species that was significantly associated with thepresence of Hp (Table 4) with all patients in which thisbacterium was isolated (119899 = 10) being also colonized byHp B cereus forms a group of versatile bacteria that canadapt to various ecological niches [27] Human-associatedB cereus mainly causes food poisoning however we do notknow what would be if there is any the significance of itsassociation with Hp infection Analysis of the presence ofnon-Hp bacteria in the disease groups showed that 50 and25 of the GC patients contained Klebsiella pneumoniae andAcinetobacter baumannii respectively (Tables 5 and 7) Thisprevalence was significantly higher than that in the NUDand PUD groups (Tables 5 and 7) although these resultsshould be consideredwith caution because of the low numberof GC compared to NUD and PUD patients Neverthelessthey are noteworthy given the modification of the diversityand composition of the microbiota in human gastric cancerpatients [19 28] Interestingly chi-square analyses showedthatKlebsiella pneumoniae andAcinetobacter baumanniiwerealso differently present in the four ethnic groups (Tables 6 and8) Both bacteria were significantly less prevalent in Chinese(119899 = 91) than in Indian (119899 = 92) (Tables 6 and 8) These twoethnic groups represented themajority of our population andwere similar in size making statistical analyses reliable Noneof the other bacterial species isolated in this study showeda significantly different prevalence among the four ethnicgroups Overall these results suggest that even though Hpmight influence the presence or absence of bacterial speciesother factors have a greater impact on the composition ofhuman gastricmicrobiota Interestingly Streptococcus specieswere isolated significantly more frequently from PUD thanNUD patients (119875 value lt 0005) (Table 9) Dendrograms ofS mitis and S parasanguinis based on protein fingerprintingwere illustrated in Figures 1(a) and 1(b) respectively todemonstrate the diversity of these microorganisms isolatedfrom the human stomach


S mitis UM090

S mitis UM014

S mitis UM040

S mitis UM034

S mitis UM012

S mitis UM043

S mitis UM035

S mitis UM199

S mitis UM080

S mitis UM033

S mitis UM036

S mitis UM116

S mitis UM140

S mitis UM015

S mitis UM029

S mitis UM093

S mitis UM096

S mitis UM121

S mitis UM081

S mitis UM003

S mitis UM135

S mitis UM130

S mitis UM160

S mitis UM049

S mitis UM136

S mitis UM148

S mitis UM046

S mitis UM064

S mitis UM103

S mitis UM112

S mitis UM092

S mitis UM105

S mitis UM062

S mitis UM057

S mitis UM110

S mitis UM070

S mitis UM111

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level

(a)

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level

S parasanguinis UM035













S parasanguinis UM010S parasanguinis UM215
















(b)

Figure 1 Dendrograms for clinical isolates of (a) S mitis and (b) S parasanguinis generated based on protein fingerprinting using Biotyper31


Table 4 Prevalence of Bacillus cereus according to Helicobacterpylori status

B cereuspositive

B cereusnegative Total

Hp positive 10 121 131Hp negative 0 84 84Total 10 205 215

Table 5 Prevalence of Klebsiella pneumoniae according to diseasegroup

Kp pos119873 ()

Kp neg119873 () Total

NUD 35 (1890) 150 (8110) 185 (100)PUD 2 (910) 20 (9090) 22 (100)GC 4 (50) 4 (50) 8 (100)Total 41 (1910) 174 (8090) 215 (100)KpKlebsiella pneumonia pos positive neg negative119873 number of biopsy percentage in the respective group NUD nonulcer dyspepsia PUDpeptic ulcer disease GC gastric cancer

Table 6 Prevalence of Klebsiella pneumoniae according to ethnicgroup

Kp pos119873 ()


Indian 23 (25) 69 (75) 92 (100)Chinese 9 (990) 82 (9010) 91 (100)Malay 3 (1670) 15 (8330) 18 (100)Other 6 (4290) 8 (5710) 14 (100)Total 41 (1910) 174 (8090) 215 (100)KpKlebsiella pneumonia pos positive neg negative119873 number of biopsy percentage in the respective group

Table 7 Prevalence of Acinetobacter baumannii according todisease group

Ab pos119873 ()

Ab neg119873 () Total

NUD 6 (32) 179 (9680) 185 (100)PUD 1 (450) 21 (9550) 22 (100)GC 2 (25) 6 (75) 8 (100)Total 9 (420) 206 (9580) 215 (100)Ab Acinetobacter baumannii pos positive neg negative 119873 number ofbiopsy percentage in the respective group NUD nonulcer dyspepsiaPUD peptic ulcer disease GC gastric cancer

4 Discussion

In this study we have isolated and identified bacterialspecies from gastric biopsies of 131 Hp-positive and 84 Hp-negative Malaysian residents suffering from gastric diseasesand belonging to different ethnic groups We identified 552bacterial colonies that belonged to 61 species 27 generaand 3 phyla The predominant phylum was Proteobacte-ria (220 colonies) followed by Firmicutes (270 colonies)

Table 8 Prevalence ofAcinetobacter baumannii according to ethnicgroup

Ab pos119873 ()


Indian 5 (5040) 87 (9460) 92 (100)Chinese 0 (0) 91 (100) 91 (100)Malay 3 (1670) 15 (8330) 18 (100)Other 1 (710) 13 (9210) 14 (100)Total 9 (420) 206 (9580) 215 (100)Ab Acinetobacter baumannii pos positive neg negative 119873 number ofbiopsy percentage in the respective group

Table 9 Prevalence of Streptococcus sp according to disease group

Streptococcuspos119873 ()

Streptococcusneg119873 ()

Total 119875 value

NUD 98 (53) 87 (47) 185 (100) 0003PUD 19 (84) 3 (14) 22 (100)119873 number of biopsies percentage in the respective groups119875 valuele 005is considered statistically significant

while Actinobacteria were rare (53 colonies) At the specieslevel Streptococci were predominant (121 positive biopsies)followed by Neisseria (44 positive biopsies) Klebsiella (42positive biopsies) and Lactobacilli (42 positive biopsies)(Table 2) Results from this study are consistent with previousstudies that reported that Proteobacteria Firmicutes Acti-nobacteria and Bacteroidetes were the predominant phylain human gastric microbiota [15ndash17 19ndash21] We did notisolate Bacteroidetes since we did not use anaerobic growthconditions We could identify up to 12 different bacterialspecies in individual samples The mean number of non-Hpbacteria per patient was 193 a low number explained by theapproach taken in this study that aimed to gain a qualitativerather than a quantitative insight into the composition ofthe gastric microbiota Comparison of the data from Hp-positive versusHp-negative samples suggests thatHpdoes notsignificantly affect neither the diversity nor the compositionof human gastric microbiota It should be noted that incontrast to previous studies we did not use PCRamplificationof genetic materials in the detection of Hp Our attemptwas to primarily determine whether Hp directly affects thediversity or composition of the gastric microbiotaThereforewe reasoned that such an effect would require the presenceof Hp cells in a significant number Although this view isa hypothesis and not a demonstrated fact we believe it tobe very likely During acute infection Hp cells that arrivein the gastric lumen rapidly swim away from this harshenvironment toward the epithelium [29 30]The spiral shapeof the bacterium allows it to efficiently penetrate the looseexternal and the dense internal mucus layers [31 32] Ithas been shown that the majority of the Hp cells residein the internal mucus layer that is firmly attached to theepithelial cells [33 34] This site is the natural niche ofHp where the bacteria efficiently proliferate as shown by


many studies establishing that when present Hp is thepredominant species in human gastric tissues [15 16 35]Therefore we considered as negative those samples in whichthis bacterium could not be neither cultured nor detectedafter thorough microscopic examination The impact of Hpon the composition and diversity of the gastric microbiota ispoorly understood Consistent with our results Bik et al whoused a metagenomic approach found no influence of Hp inthe diversity gastric microbiota [15] Similarly Maldonado-Contreras et al did not report a variation in the numberof non-Hp present but found that Hp presence resulted indifferences in the relative abundance of several phyla [20]These observations should be considered with caution giventhat the mentioned studies included a small number ofhuman subjects

Interestingly Burkholderia (15 cases) Bacillus (14 cases)and Acinetobacter (10 cases) species were isolated from thestomach of Malaysian subjects but not in similar studieson subjects from USA China Venezuela Bangladesh andRwanda [15 20 21] Melioidosis caused by B pseudomalleiis predominately a disease of tropical climates especially inSoutheast Asia and northernAustralia where it is widespreadThe bacterium is found in contaminated water and soilThusthere may be variations in the gastric microbiome betweenpopulations from different geographical regions Goodyearet al demonstrated inmice thatB pseudomallei preferentiallypersists in the stomach following oral inoculation and servesas a reservoir for dissemination of infection to extraintestinalsites [36] To our knowledge this study is the first report ofB pseudomallei isolation from the stomach of asymptomatichuman subjects The implication of B pseudomallei presencein the human stomach deserves further investigation

Streptococci are frequently harbored in the oral cavityThe high isolation rate (average of 565) in this studysuggests that these bacteria may be able to colonize thestomach rather than be just transient bacteriaDespite relativesmall number of peptic ulcer disease patients involved inthis study significant correlation between the isolation ofstreptococci and peptic ulcer disease was demonstratedMolecular interaction between streptococci andHpmay playa role in ulcer disease and deserves further investigation

Metagenomics and microarray approaches investigateinto the diversity and relative quantitation of both cultur-able and nonculturable bacteria In contrast the approachadopted byHu et al and our study only took into account bac-teria cultured under defined laboratory conditions Howeverthis approach was adopted because it allows further in vitroinvestigation to be carried out on non-Hp isolates to examinethe role of coinfections by Hp and non-Hp

Hp infection typically becomes chronic induces a strongimmune response affects the production of important gastrichormones including ghrelin that acts in the central nervoussystem and modifies the gastric pH [25] Each of theseeffects can potentially influence the gastric microbiota It islikely that during the evolution of Hp infection to chronicityseveral recompositions of the gastric microbiota occur Forexample changes of gastric pH can lead to colonization bybacterial species from the oral mucosa the upper-respiratorytract or the intestines that cannot persist in the healthy

stomachrsquos environment Concurrently there might also be atdifferent periods species that progressively disappear becausethe changes make the gastric milieu unfavorable for theirpersistenceThis is exemplified byHp itself that progressivelydisappear in gastric tissues during carcinogenesis [25] Over-all the changes that take place in the gastric environmentduring Hp infection are complex and involve several factorswith Hp presence being one of them Each factor wouldhave a minor impact and the combination of all woulddetermine not only the composition of the gastric microbiotabut also the progress toward different diseases Consistentwith this idea Maldonado-Contreras et al reported that theHp status accounted for 28of the total variance in the gastricmicrobiota of 12 individuals they analyzed [20] Studies oflarge-scale human populations using throughput methodsare needed to better understand the influence of the differentfactors that contribute to shaping of the gastric microbiotaSuch studies can be validated by approaches like the onetaken in this study with the ultimate goal of identifyingbacterial species that could be used as microbial markers thatinform about the progress toward differentHp-related gastricdiseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors wish to thank the staff of the Endoscopy Unitat the University of Malaya Medical Centre (UMMC) for allthe assistance provided in the process of sample collection forthis studyThis study was supported by University of Malaya-Ministry of Education (UM-MoE) High Impact Research(HIR) Grant UMC6251HIRMoECHAN02 (account noH-50001-A000013)

References

[1] GMusso R Gambino andM Cassader ldquoInteractions betweengut microbiota and host metabolism predisposing to obesityand diabetesrdquo Annual Review of Medicine vol 62 pp 361ndash3802011

[2] H Chung S J Pamp J A Hill et al ldquoGut immune maturationdepends on colonization with a host-specific microbiotardquo Cellvol 149 no 7 pp 1578ndash1593 2012

[3] J F Cryan andTGDinan ldquoMind-alteringmicroorganisms theimpact of the gut microbiota on brain and behaviourrdquo NatureReviews Neuroscience vol 13 no 10 pp 701ndash712 2012

[4] I Sekirov S L Russell L C Antunes and B B Finlay ldquoGutmicrobiota in health and diseaserdquo Physiological Reviews vol 90no 3 pp 859ndash904 2010

[5] M J Claesson I B Jeffery S Conde et al ldquoGut microbiotacomposition correlates with diet and health in the elderlyrdquoNature vol 488 no 7410 pp 178ndash184 2012

[6] C A Lozupone J I Stombaugh J I Gordon J K Jansson andR Knight ldquoDiversity stability and resilience of the human gutmicrobiotardquo Nature vol 489 no 7415 pp 220ndash230 2012


[7] O Koren J K Goodrich T C Cullender et al ldquoHost remod-eling of the gut microbiome and metabolic changes duringpregnancyrdquo Cell vol 150 no 3 pp 470ndash480 2012

[8] Y Moodley B Linz R P Bond et al ldquoAge of the associationbetween Helicobacter pylori and manrdquo PLoS Pathogens vol 8no 5 Article ID e1002693 2012

[9] M M Khalifa M M Sharaf and R K Aziz ldquoHelicobacterpylori a poor manrsquos gut pathogenrdquo Gut Pathogens vol 2 no1 article 2 2010

[10] P Correa and J Houghton ldquoCarcinogenesis of Helicobacterpylorirdquo Gastroenterology vol 133 no 2 pp 659ndash672 2007

[11] A Y Wang and D A Peura ldquoThe prevalence and incidenceofHelicobacter pylori-associated peptic ulcer disease and uppergastrointestinal bleeding throughout the worldrdquo Gastrointesti-nal Endoscopy Clinics of North America vol 21 no 4 pp 613ndash635 2011

[12] I C Arnold I Hitzler andAMuller ldquoThe immunomodulatoryproperties of Helicobacter pylori confer protection againstallergic and chronic inflammatory disordersrdquo Front Cell InfectMicrobiol vol 2 no 10 2012

[13] S Perry B C de Jong J V Solnick et al ldquoInfection withHelicobacter ploryi is associated with protection against tuber-culosisrdquo PLoS ONE vol 5 no 1 Article ID e8804 2010

[14] N R Salama M L Hartung and A Muller ldquoLife in thehuman stomach persistence strategies of the bacterial pathogenHelicobacter pylorirdquo Nature Reviews Microbiology vol 11 no 6pp 385ndash399 2013

[15] E M Bik P B Eckburg S R Gill et al ldquoMolecular analysis ofthe bacterial microbiota in the human stomachrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 103 no 3 pp 732ndash737 2006

[16] A F Andersson M Lindberg H Jakobsson F Backhed PNyren and L Engstrand ldquoComparative analysis of human gutmicrobiota by barcoded pyrosequencingrdquo PLoS ONE vol 3 no7 Article ID e2836 2008

[17] X Li G L Wong K To et al ldquoBacterial microbiota profiling ingastritis without Helicobacter pylori infection or non-steroidalanti-inflammatory drug userdquo PLoS ONE vol 4 no 11 ArticleID e7985 2009

[18] J C Stearns M D J Lynch D B Senadheera et al ldquoBacterialbiogeography of the human digestive tractrdquo Scientific Reportsvol 1 article 170 2011

[19] J Dicksved M Lindberg M Rosenquist H Enroth J KJansson and L Engstrand ldquoMolecular characterization of thestomach microbiota in patients with gastric cancer and incontrolsrdquo Journal of Medical Microbiology vol 58 no 4 pp509ndash516 2009

[20] A Maldonado-Contreras K C Goldfarb F Godoy-Vitorinoet al ldquoStructure of the human gastric bacterial community inrelation to Helicobacter pylori statusrdquo ISME Journal vol 5 no4 pp 574ndash579 2011

[21] Y Hu L H He D Xiao et al ldquoBacterial flora concurrentwith Helicobacter pylori in the stomach of patients with uppergastrointestinal diseasesrdquo World Journal of Gastroenterologyvol 18 no 11 pp 1257ndash1261 2012

[22] JQin R Li J Raes et al ldquoAhumangutmicrobial gene catalogueestablished by metagenomic sequencingrdquo Nature vol 464 no7285 pp 59ndash65 2010

[23] J Goldie S J O Veldhuyzen van Zanten S Jalali et alldquoOptimization of a medium for the rapid urease test fordetection of Campylobacter pylori in gastric antral biopsiesrdquo

Journal of Clinical Microbiology vol 27 no 9 pp 2080ndash20821989

[24] S Chakravorty D Helb M Burday N Connell and D AllandldquoA detailed analysis of 16S ribosomal RNA gene segments forthe diagnosis of pathogenic bacteriardquo Journal of MicrobiologicalMethods vol 69 no 2 pp 330ndash339 2007

[25] M J Blaser and J C Atherton ldquoHelicobacter pylori persistencebiology and diseaserdquo Journal of Clinical Investigation vol 113no 3 pp 321ndash333 2004

[26] S A Cunningham and R Patela ldquoImportance of usingBrukerrsquos security-relevant library for biotyper identificationof Burkholderia pseudomallei brucella species and francisellatularensisrdquo Journal of Clinical Microbiology vol 51 no 5 pp1639ndash1640 2013

[27] E J Bottone ldquoBacillus cereus a volatile human pathogenrdquoClinical Microbiology Reviews vol 23 no 2 pp 382ndash398 2010

[28] F Aviles-Jimenez F Vazquez-Jimenez R Medrano-GuzmanA Mantilla and J Torres ldquoStomach microbiota compositionvaries between patients with non-atrophic gastritis and patientswith intestinal type of gastric cancerrdquo Scientific Reports vol 4no 4202 2014

[29] K A Eaton S Suerbaum C Josenhans and S KrakowkaldquoColonization of gnotobiotic piglets by Helicobacter pylorideficient in two flagellin genesrdquo Infection and Immunity vol 64no 7 pp 2445ndash2448 1996

[30] K M Ottemann and A C Lowenthal ldquoHelicobacter pylori usesmotility for initial colonization and to attain robust infectionrdquoInfection and Immunity vol 70 no 4 pp 1984ndash1990 2002

[31] M Bonis C Ecobichon S Guadagnini M C Prevost and IG Boneca ldquoA M23B family metallopeptidase of Helicobacterpylori required for cell shape pole formation and virulencerdquoMolecular Microbiology vol 78 no 4 pp 809ndash819 2010

[32] L K Sycuro Z Pincus K D Gutierrez et al ldquoPeptidoglycancrosslinking relaxation promotes helicobacter pylorirsquos helicalshape and stomach colonizationrdquo Cell vol 141 no 5 pp 822ndash833 2010

[33] S Schreiber M Konradt C Groll et al ldquoThe spatial orientationof Helicobacter pylori in the gastric mucusrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 14 pp 5024ndash5029 2004

[34] T Shimizu T Akamatsu A Sugiyama H Ota and T Kat-suyama ldquoHelicobacter pylori and the surface mucous gel layerof the human stomachrdquo Helicobacter vol 1 no 4 pp 207ndash2181996

[35] L Engstrand and M Lindberg ldquoHelicobacter pylori and thegastric microbiotardquo Best Practice and Research Clinical Gas-troenterology vol 27 no 1 pp 39ndash45 2013

[36] A Goodyear H Bielefeldt-Ohmann H Schweizer and S DowldquoPersistent gastric colonization with Burkholderia pseudomalleiand dissemination from the gastrointestinal tract followingmucosal inoculation of micerdquo PLoS ONE vol 7 no 5 ArticleID e37324 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


including allergies inflammatory disorders [12] and tubercu-losis [13] Hp pathogenesis and its interactions with the gut-associatedmucosal immune system have been widely studied[14] Studies of human patients and investigations in animalmodels including mouse gerbil and monkey have generateda substantial amount of knowledge on the acute phase ofHp infection establishment of colonization activation of thegut-associated mucosal immune system and immune escapestrategies that lead to chronic colonization [14] Humanstomach is the only known natural habitat for Hp and onceestablished this bacterium is usually the predominant speciesof the gastric microbiota [15] However little is known aboutthe modification of the gastric microbiota that results fromchronic Hp infection and the interactions of this bacteriumwith other members of the gastric ecosystem Study of thegastric microbiota has been delayed by the belief prior toHp discovery that human stomachwas a hostile environmentthat could not support colonization by microorganisms Fewstudies have analyzed the composition of the human gastricmicrobiota in healthy individuals and in patients sufferingfrom different diseases These studies used high-throughputmolecular approaches including metagenomics [15ndash18] ter-minal restriction fragment length polymorphism (T-RFLP)[19] and microarray [20] that are powerful techniques thatenable the capture of DNA sequences from most of thebacteria present in the stomach including both culturable andnonculturable species However one important limitationin these studies is the small number of human samples(4ndash23 individuals) making it difficult to generate statisticallysignificant conclusionsWith regard toHp even fewer studieshave analyzed the impact of this bacterium on the compo-sition of the gastric microbiota Recently Hu et al used acultivation-based approach to isolate non-Hp species fromgastric biopsies of 103 patients infectedwithHp and identifiedmost of the bacteria at the species level using MALDI-TOFmass spectrometry-based biotyping [21] This approach hasthe limitation of omitting nonculturable bacteria that arepredominant in the stomach [22] However the accuracyof mass spectrometry biotyping in bacterial identificationpermits rapid analysis of large number of samples

In this study we wanted to gain further insight intothe impact of Hp colonization on the composition and thediversity of human gastric microbiota We used the massspectrometry biotyping technology to identify bacteria thatwere cultured from stomach biopsies of 215 patients including131 Hp-positive and 84 Hp-negative subjects The patientswere affected by different gastric diseases and belongedto different ethnic groups present in Malaysia Beside theinfluence of Hp infection the approach taken in this studyhas the potential to provide an insight into the contribution ofother factors such as ethnicity and disease to the compositionand the diversity of the gastric microbiota

2 Materials and Methods

21 Study Population Gastric biopsy samples were obtainedbetween 2011 and 2013 from patients referred for endoscopyexamination at the University Malaya Medical Center

(UMMC Kuala Lumpur Malaysia) Biopsy samples weretaken from the antrum and body of the stomach for eachpatient This study was approved by the UMMC MedicalEthics Committee and written consent was obtained frompatients before being included in the study

22 Bacterial Growth and Identification Fresh gastric biopsytissues were homogenized and simultaneously inoculatedonto nonselective and selective chocolate agar plates (supple-mentedwith 5horse blood and in the case of selectivemediaantibiotics included trimethoprim (5120583gmL) vancomycin(10 120583gmL) nalidixic acid (20120583gmL) and amphotericin B(5 120583gmL)) All the antibiotics were from Sigma-Aldrich Cor-poration (St Louis MO USA)The agar plates were simulta-neously incubated at 37∘C under humidified condition with10 carbon dioxide and under ambient air condition Allwell-isolated colonies were selected and cultured for furtherstudies Hp colonies formed after at least three days ofgrowth on selective plates in our culture conditions and wereconfirmed by positivity for urease catalase and oxidase testsAdditionally Gram staining and microscopic analysis wasperformed to verify the presence of Gram-negative spiralrod bacteria Non-Hp bacterial species (hereafter termed asother bacteria) were identified using a combination of colonymorphology mass spectrometry and 16SrRNA sequencing

23 Colony Morphology Other colonies were picked andcategorized according to morphological features includingsize texture pigmentation hemolysis shape appearancemargin and elevation At least one colony from each mor-photype present on each plate was selected The colonieswere restreaked onto non-selective plates and after sufficientgrowth stored at minus80∘C in a brain heart infusion (BHI) broth(Sigma-Aldrich USA) supplemented with 04 (wtvol)yeast extract and 20 (volvol) glycerol

24 Mass Spectrometry Ethanolformic acid extraction andmass spectrometry was performed as recommended byBruker Daltonics GmbH (Breme Germany) Briefly 1 to 2well-isolated colonies (or a few colonies in the case of a smallcolony size) of similarmorphotype were suspended in 300 120583Lof ultrapure (type 1) water (EMD Millipore CorporationBillerica MA USA) to which 900 120583L of pure ethanol wereaddedThe samples were centrifuged (13000timesg 2min)Thesupernatants were decanted and the pellets were air-driedOnce dried 50 120583L of 70 formic acid (Fluka AnalyticalHPLC grade) and 50 120583L of acetonitrile (Friedemann SchmidtChemical HPLC grade) were added and the samples werecentrifuged again Subsequently 1 120583L of the supernatant wasspotted onto a MSP 96 target polished steel BC microScoutTarget (Bruker Daltonics GmbH) and allowed to completelyair-dry before overlaid with 1 120583L of fresh 120572-cyano-4-hydroxy-cinnamic acid (HCCA Bruker Daltonics GmbH) matrixsolution The samples were dried before analysis on theMicroflex LRF MALDI-TOF Mass Spectrometry Systemequipped with a 60Hz nitrogen laser and microScout ionsource (Bruker Daltonics GmbH) The parameter settingswere as follows delay 12719 pts ion source 20 kV ion





3 Results















119875 value










Table 2 Continued



119875 value





















S mitis UM090

S mitis UM014

S mitis UM040

S mitis UM034

S mitis UM012

S mitis UM043

S mitis UM035

S mitis UM199

S mitis UM080

S mitis UM033

S mitis UM036

S mitis UM116

S mitis UM140

S mitis UM015

S mitis UM029

S mitis UM093

S mitis UM096

S mitis UM121

S mitis UM081

S mitis UM003

S mitis UM135

S mitis UM130

S mitis UM160

S mitis UM049

S mitis UM136

S mitis UM148

S mitis UM046

S mitis UM064

S mitis UM103

S mitis UM112

S mitis UM092

S mitis UM105

S mitis UM062

S mitis UM057

S mitis UM110

S mitis UM070

S mitis UM111

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level

(a)

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level






























(b)




B cereuspositive




Kp pos119873 ()




Kp pos119873 ()




Ab pos119873 ()



4 Discussion



Ab pos119873 ()






Total 119875 value












Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results















119875 value










Table 2 Continued



119875 value





















S mitis UM090

S mitis UM014

S mitis UM040

S mitis UM034

S mitis UM012

S mitis UM043

S mitis UM035

S mitis UM199

S mitis UM080

S mitis UM033

S mitis UM036

S mitis UM116

S mitis UM140

S mitis UM015

S mitis UM029

S mitis UM093

S mitis UM096

S mitis UM121

S mitis UM081

S mitis UM003

S mitis UM135

S mitis UM130

S mitis UM160

S mitis UM049

S mitis UM136

S mitis UM148

S mitis UM046

S mitis UM064

S mitis UM103

S mitis UM112

S mitis UM092

S mitis UM105

S mitis UM062

S mitis UM057

S mitis UM110

S mitis UM070

S mitis UM111

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level

(a)

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level






























(b)




B cereuspositive




Kp pos119873 ()




Kp pos119873 ()




Ab pos119873 ()



4 Discussion



Ab pos119873 ()






Total 119875 value












Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





119875 value










Table 2 Continued



119875 value





















S mitis UM090

S mitis UM014

S mitis UM040

S mitis UM034

S mitis UM012

S mitis UM043

S mitis UM035

S mitis UM199

S mitis UM080

S mitis UM033

S mitis UM036

S mitis UM116

S mitis UM140

S mitis UM015

S mitis UM029

S mitis UM093

S mitis UM096

S mitis UM121

S mitis UM081

S mitis UM003

S mitis UM135

S mitis UM130

S mitis UM160

S mitis UM049

S mitis UM136

S mitis UM148

S mitis UM046

S mitis UM064

S mitis UM103

S mitis UM112

S mitis UM092

S mitis UM105

S mitis UM062

S mitis UM057

S mitis UM110

S mitis UM070

S mitis UM111

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level

(a)

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level






























(b)




B cereuspositive




Kp pos119873 ()




Kp pos119873 ()




Ab pos119873 ()



4 Discussion



Ab pos119873 ()






Total 119875 value












Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 2 Continued



119875 value





















S mitis UM090

S mitis UM014

S mitis UM040

S mitis UM034

S mitis UM012

S mitis UM043

S mitis UM035

S mitis UM199

S mitis UM080

S mitis UM033

S mitis UM036

S mitis UM116

S mitis UM140

S mitis UM015

S mitis UM029

S mitis UM093

S mitis UM096

S mitis UM121

S mitis UM081

S mitis UM003

S mitis UM135

S mitis UM130

S mitis UM160

S mitis UM049

S mitis UM136

S mitis UM148

S mitis UM046

S mitis UM064

S mitis UM103

S mitis UM112

S mitis UM092

S mitis UM105

S mitis UM062

S mitis UM057

S mitis UM110

S mitis UM070

S mitis UM111

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level

(a)

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level






























(b)




B cereuspositive




Kp pos119873 ()




Kp pos119873 ()




Ab pos119873 ()



4 Discussion



Ab pos119873 ()






Total 119875 value












Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












S mitis UM090

S mitis UM014

S mitis UM040

S mitis UM034

S mitis UM012

S mitis UM043

S mitis UM035

S mitis UM199

S mitis UM080

S mitis UM033

S mitis UM036

S mitis UM116

S mitis UM140

S mitis UM015

S mitis UM029

S mitis UM093

S mitis UM096

S mitis UM121

S mitis UM081

S mitis UM003

S mitis UM135

S mitis UM130

S mitis UM160

S mitis UM049

S mitis UM136

S mitis UM148

S mitis UM046

S mitis UM064

S mitis UM103

S mitis UM112

S mitis UM092

S mitis UM105

S mitis UM062

S mitis UM057

S mitis UM110

S mitis UM070

S mitis UM111

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level

(a)

MSP dendrogram

1000 900 800 700 600 500 400 300 200 100 0

Distance level






























(b)




B cereuspositive




Kp pos119873 ()




Kp pos119873 ()




Ab pos119873 ()



4 Discussion



Ab pos119873 ()






Total 119875 value












Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



B cereuspositive




Kp pos119873 ()




Kp pos119873 ()




Ab pos119873 ()



4 Discussion



Ab pos119873 ()






Total 119875 value












Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Acknowledgments


References














































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Culturable Bacterial Microbiota of the...

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