research article concomitant colonization of helicobacter...

Research ArticleConcomitant Colonization of Helicobacter pylori inDental Plaque and Gastric Biopsy

Amin Talebi Bezmin Abadi1 Ashraf Mohabati Mobarez1

Omid Teymournejad2 and Mona Karbalaei1

1 Department of Bacteriology Faculty of Medical Sciences Tarbiat Modares University PO Box 14115-111 Tehran Iran2Department of Microbiology School of Medicine University of Medical Science Babol Iran

Correspondence should be addressed to Ashraf Mohabati Mobarez mmmobarezmodaresacir

Received 11 May 2014 Revised 26 June 2014 Accepted 1 July 2014 Published 9 July 2014

Academic Editor Nongnuch Vanittanakom

Copyright copy 2014 Amin Talebi Bezmin Abadi et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Frequently reportedH pylori antimicrobial therapy failures suggest that there might be a different niche where the bacteria can staysafe Current study aims to examine potential role of oral colonization ofH pylori to feed reinfection after primary therapyHoweverpatients who were admitted to the gastroscopy section were chosen and gastric biopsy and dental plaque specimens were collectedMolecular and biochemical tests were applied to confirm H pylori identity in different colonization niches Results showed that888 of dyspeptic patients had epigastric pains with nocturnal awakening when they were hungry (119875 = 0023) All patients whoreceived therapy already were again H pylori positive while they are still carryingH pylori in dental plaque (119875 = 0001) MoreoverH pylori infection was sought in 100 of gastric biopsyrsquos dyspeptic patients who had ulcerated esophagitis and erosive duodenitisand who were H pylori positive and 75 of dyspeptic patients with duodenum deformity had this bacterium in gastric biopsies(119875 = 0004) Present study showed that only successful eradication of gastricH pylori cannot guarantee prevention of reinfectionConclusively a new strategy which indicates concomitant eradication in oral and gastric colonization can result in clearance of Hpylori infection

1 Introduction

Helicobacter pylori (H pylori) is a Gram-negative spiral andmotile bacterium that is present in the human stomach ofapproximately half of the worldrsquos population [1] H pyloriis an important gastrointestinal pathogen that is stronglyassociated with gastritis as well as peptic ulcer disease Thereis strong evidence that H pylori has an undeniable role inoccurrence of gastric abnormality atrophic inflammationand gastric cancer [2] Colonization begins in childhoodhowever little is known about its timing and actual route ofbacterial transmission [3 4] Recent findings are indicatinga narrow link between oral and gastric colonization of Hpylori [5] Frequently failed antibiotic therapy to cure Hpylori infection suggests that there might be certain differentsites where the organism can survive [1 6] To date theexact mechanism of transmission of H pylori is not fullyunderstood a crucial fact which implies on unknown routes

and reservoir locations that are still undiscovered Indeeddefeated therapeutic approaches to cure gastric H pyloriinfection triggered a thought that different locality mightbe involved in reinfection of this persistent bacterium [6]Nonetheless alarm symptoms and endoscopic finding aresuch approach to detect causes of dyspepsia [1 7] Dyspepsiais a kind of discomfort in center upper abdomen and can beaffected by many factors such as peptic ulcer and gastroe-sophageal reflux [1] Alarm symptoms have poor diagnosisfor etiology in dyspeptic patients [8] and endoscopy is aninvasive technique for diagnosis of gastritis but that will notbe enough for detecting the H pylori [9] As such thereare noninvasive tests such as urea breast test (UBT) andstool and blood tests which can be done in alignment withendoscopic method [10] With regard to the diagnosis thenoninvasive tests are often used [10] The aim of our studywas to investigate the relationship between alarm symptomsand endoscopic findings withH pylori colonization in dental

Hindawi Publishing CorporationJournal of PathogensVolume 2014 Article ID 871601 4 pageshttpdxdoiorg1011552014871601

2 Journal of Pathogens

Table 1 Relationship of clinical symptoms and frequency of H pylori detected in dental plaque and gastric biopsyrsquos dyspeptic patients

Clinical symptoms Dental plaque PCR ureC Gastric biopsyculture culture

Epigastric pain without nocturnal awakening while patient is hungry (119899 = 82) 34 (414) 62 (726) 30 (366)Epigastric pain with nocturnal awakening while patient is hungry (119899 = 18) 15 (833) 15 (833) 16 (888)Bloating after meal without nocturnal awakening (119899 = 61) 39 (639) 55 (901) 34 (557)Bloating after meal with nocturnal awakening (119899 = 9) 5 (555) 6 (666) 6 (666)Reflux without nocturnal awakening (119899 = 31) 15 (484) 24 (774) 15 (484)Reflux with nocturnal awakening (119899 = 8) 6 (750) 7 (875) 6 (750)Previously infected with H pylori and cured (119899 = 24) 18 (750) 24 (100) 13 (542)

plaque and gastric biopsy isolated from Iranian dyspepticpatients

2 Materials and Methods

21 Questionnaire In this survey patients who admittedto the gastroscopy section at Baghiatallah hospital TehranIran were chosen for our examination Pain with or withoutnocturnal awakening while patient is hungry bloating aftermeal with or without nocturnal awakening reflux with orwithout nocturnal awakening and endoscopic finding suchas ulcerated or nonulcerated esophagitis antral gastritiserosive gastritis gastric ulcer duodenitis duodenum ulcererosive duodenitis and duodenum deformity were applied asincluding criteria

22 Sampling Gastric biopsy and dental plaque specimenswere collected from each dyspeptic subject Dental plaqueand biopsyrsquos sample were carried in thioglycolate broth(Merck Germany) and then sent to the laboratory in less thanfour hours after gastric endoscopy Two samples from antrumwere shipped immediately after endoscopic procedures tomicrobiology and pathology labs for detecting H pyloriidentity

23 Bacterial Isolation Dental plaques and gastric biopsieswere cultured for one week in anaerobic jar with gas pack C(Merck Germany) and presence of H pylori was confirmedby biochemical tests catalase oxidase urease and gramstaining In brief all samples were cultured in Brucellaagar (Merck Germany) supplemented with 7 fetal calfserum (Gipso USA) 10 sheep blood (Jahad DaneshgahiTehran University Tehran Iran) polymyxin B trimetoprimamphotericin B and vancomycin after homogenization

24 Polymerase Chain Reaction (PCR) 22DNA extractionwas performed by genomic DNA extraction kit (BioneerSouth Korea) followed by polymerase chain reaction (PCR)test with specific forward and reverse primers of ureCgene Forward 51015840-CCCTCACGCCATCAGTCCCAAAAA-31015840 and reverse 51015840-AAGAAGTCAAAAACGCCCCAAAAC-31015840 Total volume of reactions was 25 120583L and solution included25 120583L of 10x buffer (PH 84) containing 100mM trisHCl500mM KCl 2mM MgCl

2 02mM dNTP 15 U Taq DNA

polymerase 25 120583L bacterial DNA and 02mM primer Sub-sequently 30 cycles performed at 94∘C for 5minutes (primarydenaturation) 94∘C for 1 minute 55∘C for 1 minute and 72∘Cfor 1 minute and final extension cycle performed at 72∘C for10 minutes

25 Statistical Analysis We used SPSS version 180 softwarefor statistical analysis 119875 values less than 005 were regardedas statistically significant

3 Results

Correlation between alarm symptoms such as epigastricpain when people are hungry with and without nocturnalawakening bloating after meal with and without nocturnalawakening and reflux with and without awakening with Hpylori detection from dental plaque and gastric biopsy hasbeen shown in Table 1 Additionally we observed recurrenceof H pylori infection among different samples which arelisted in Table 1 All patients who received therapy alreadywere again H pylori positive while they were still carryingH pylori in their dental plaque (119875 = 0001) Correla-tion between endoscopic findings such as ulcerated andnonulcerated esophagitis antral and erosive gastritis gastriculcer duodenum ulcer duodenitis erosive duodenitis andduodenum deformity with H pylori isolation from dentalplaques and gastric biopsies was shown in Table 2

4 Discussion

H pylori colonization affects more than half of the worldrsquospopulation and thus it is the most persistent bacterial infec-tion worldwide Various clinical manifestations are reportingabout this persistence colonization [1] Alarm symptoms ofdyspepsia were found in 20ndash40 of community thus thisdisorder causes the annualmillions of admission to the clinics[2] Unfortunately alarm symptoms have no good prognosisfor H pylori infection but clinicians can apply this items todecide on who should receive early endoscopic service [11]In current investigation we compared the predictive value ofalarm symptoms with detection of H pylori in dental plaqueand gastric biopsy Actually our research was the first whichrevealed that there is relation between epigastric pains andnocturnal awakening while patient is hungry Interestingly

Journal of Pathogens 3

Table 2 Relationship of endoscopic findings and H pylori preva-lence in dental plaque and gastric biopsyrsquos dyspeptic patients

Endoscopic findings Dental plaque PCR ureC Gastric biopsyculture culture

Ulcerated esophagitis(119899 = 3) 1 (333) 2 (666) 3 (1000)

Nonulceratedesophagitis (119899 = 6) 6 (1000) 6 (1000) 0 (00)

Antral gastritis(119899 = 21) 12 (571) 17 (810) 12 (571)

Erosive gastritis(119899 = 25) 25 (1000) 25 (1000) 25 (1000)

Gastric ulcer (119899 = 8) 8 (1000) 8 (1000) 8 (1000)Duodenitis (119899 = 24) 14 (583) 20 (833) 17 (708)Duodenum ulcer(119899 = 19) 15 (789) 16 (842) 12 (631)

Erosive duodenitis(119899 = 12) 12 (1000) 12 (1000) 12 (1000)

Duodenum deformity(119899 = 16) 16 (1000) 16 (1000) 12 (750)

in 1989 for the first time Krajden et alisolated cultured Hpylori from the dental plaques a finding which disclosedlikely crucial role of oral cavity to feed the infectious loadof stomach [12] Undoubtedly oral H pylori is more difficultto be eradicated than gastric H pylori so a new adoptedstrategy to eradicate gastric H pylori must be consideredto target gastric H pylori as well Taken together oral Hpylori seems to be the main source of gastric H pylori forinfection and reinfection models Remarkably all patientswho received antibiotics were alreadyH pylori positive whilethey were carryingH pylori in their dental plaque (119875 lt 005)(Table 1) To date many studies had showed that H pylori indental plaque is a possible reservoir of recurrence of gastricinfection and route of entrance of these bacteria to stomachfrom mouth [13] Furthermore some endoscopic findingssuch as ulcerated esophagitis erosive gastritis gastric ulcererosive duodenitis and duodenum deformity had strongcorrelation withH pylori isolation from gastric biopsies (119875 lt005) (Table 1) All of gastric biopsyrsquos dyspeptic patients whohad ulcerated esophagitis erosive gastritis gastric ulcer anderosive duodenitis were H pylori positive with cultivationand 75 of dyspeptic patients with duodenum deformity hadthis bacterium in gastric biopsies (Table 2) There are someinvestigations that compared between endoscopic findingsand detection rate of H pylori In a study from the Republicof Georgia it has been indicated that 78 and 58 of patientswho had gastritis and peptic ulcer were H pylori positive(119875 lt 005) [14] In an investigation in southern Iran70 and 86 of dyspeptic patients who had gastritis andduodenal ulcer were reported as H pylori positive in theirstomach subsequently (119875 gt 005) The exact mechanism ofH pylori reinfection in stomach is under debate but currentnovel findings showed that a prospective study which cancheck genomic content of both oral and gastric H pylori candisclose more about unclear transmission route Currently

there are various diagnostic tests to detect the infection withthis mysterious microorganism but there is no commonlyacknowledged ldquogold standardrdquo yet [1] Similarly our resultsshowed that we might need to consider the current strategiesto detect H pylori in blood or biopsy samples In otherwords significant presence ofH pylori in oral cavity calls forurgent new assays to be tracked and likely possible eradicativeintervention However we are still away from actual factsabout this transmission Thus more research is needed toestablish the exact transmission model of the infection ofhuman population Conclusively our study showed that onlysuccessful eradication of gastric H pylori cannot guaranteeprevention of reinfection Indeed a new strategy which indi-cates concomitant eradication in oral and gastric colonizationcan cause prevention of H pylori infection

Disclosure

The content of this paper is sole responsibility of the authorand necessarily represents personal prospective Moreoverthe funding agencies had no role in the decision to publishor the preparation of the paper

Conflict of Interests

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

References

[1] J G Kusters AHM vanVliet and E J Kuipers ldquoPathogenesisof Helicobacter pylori infectionrdquo Clinical Microbiology Reviewsvol 19 no 3 pp 449ndash490 2006

[2] Y Yamaoka ldquoMechanisms of disease helicobacter pylori viru-lence factorsrdquoNature Reviews Gastroenterology and Hepatologyvol 7 no 11 pp 629ndash641 2010

[3] X Wan D Tang X Zhang et al ldquoExploratory study oforal mucosal colonization of human gastric Helicobacter pyloriin micerdquo International Journal of Clinical and ExperimentalMedicine vol 7 no 3 pp 523ndash529 2014

[4] M E Martin and V Jay Solnick ldquoThe gastric microbialcommunity Helicobacter pylori colonization and diseaserdquo GutMicrobes vol 5 no 3 2014

[5] X Didelot S Nell I Yang S Woltemate S van der Merweand S Suerbaum ldquoGenomic evolution and transmission ofHelicobacter pylori in two South African familiesrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 110 no 34 pp 13880ndash13885 2013

[6] A Talebi BezminAbadi ldquoTherapy ofHelicobacter pylori presentmedley and future prospectiverdquo BioMed Research Internationalvol 2014 Article ID 124607 7 pages 2014

[7] L Checchi P Felice C Acciardi et al ldquoAbsence ofHelicobacterpylori in dental plaque assessed by stool testrdquo The AmericanJournal of Gastroenterology vol 95 no 10 pp 3005ndash3006 2000

[8] Q Song B Haller D Ulrich A Wichelhaus G Adler andG Bode ldquoQuantitation of Helicobacter pylori in dental plaquesamples by competitive polymerase chain reactionrdquo Journal ofClinical Pathology vol 53 no 3 pp 218ndash222 2000

[9] M Voutilainen T Mantynen K Mauranen I Kunnamo andM Juhola ldquoIs it possible to reduce endoscopy workload using


age alarm symptoms andH pylori as predictors of peptic ulcerand oesophagogastric cancersrdquoDigestive and Liver Disease vol37 no 7 pp 526ndash532 2005

[10] E Garza-Gonzalez G I Perez-Perez H J Maldonado-Garzaand F J Bosques-Padilla ldquoA review of Helicobacter pyloridiagnosis treatment andmethods to detect eradicationrdquoWorldJournal of Gastroenterology vol 20 no 6 pp 1438ndash1449 2014

[11] S J Czinn ldquoHelicobacter pylori infection detection investiga-tion and managementrdquo Journal of Pediatrics vol 146 no 3 ppS21ndashS26 2005

[12] S KrajdenM Fuksa J Anderson et al ldquoExamination of humanstomach biopsies saliva and dental plaque for Campylobacterpylorirdquo Journal of Clinical Microbiology vol 27 no 6 pp 1397ndash1398 1989

[13] N Tarkhashvili R Beriashvili N Chakvetadze et al ldquoHelico-bacter pylori infection in patients undergoing upper endoscopyRepublic of Georgiardquo Emerging Infectious Diseases vol 15 no3 pp 504ndash505 2009

[14] H Cai W Li X Shu K Peng Y Zhang andM Jiang ldquoGeneticvariation of Helicobacter pylori in the oral cavity and stomachdetected using TA-cloning in children with chronic gastritisrdquoPediatric Infectious Disease Journal vol 33 no 1 pp e1ndashe6 2014

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Table 1 Relationship of clinical symptoms and frequency of H pylori detected in dental plaque and gastric biopsyrsquos dyspeptic patients

Clinical symptoms Dental plaque PCR ureC Gastric biopsyculture culture

Epigastric pain without nocturnal awakening while patient is hungry (119899 = 82) 34 (414) 62 (726) 30 (366)Epigastric pain with nocturnal awakening while patient is hungry (119899 = 18) 15 (833) 15 (833) 16 (888)Bloating after meal without nocturnal awakening (119899 = 61) 39 (639) 55 (901) 34 (557)Bloating after meal with nocturnal awakening (119899 = 9) 5 (555) 6 (666) 6 (666)Reflux without nocturnal awakening (119899 = 31) 15 (484) 24 (774) 15 (484)Reflux with nocturnal awakening (119899 = 8) 6 (750) 7 (875) 6 (750)Previously infected with H pylori and cured (119899 = 24) 18 (750) 24 (100) 13 (542)

plaque and gastric biopsy isolated from Iranian dyspepticpatients

2 Materials and Methods

21 Questionnaire In this survey patients who admittedto the gastroscopy section at Baghiatallah hospital TehranIran were chosen for our examination Pain with or withoutnocturnal awakening while patient is hungry bloating aftermeal with or without nocturnal awakening reflux with orwithout nocturnal awakening and endoscopic finding suchas ulcerated or nonulcerated esophagitis antral gastritiserosive gastritis gastric ulcer duodenitis duodenum ulcererosive duodenitis and duodenum deformity were applied asincluding criteria

22 Sampling Gastric biopsy and dental plaque specimenswere collected from each dyspeptic subject Dental plaqueand biopsyrsquos sample were carried in thioglycolate broth(Merck Germany) and then sent to the laboratory in less thanfour hours after gastric endoscopy Two samples from antrumwere shipped immediately after endoscopic procedures tomicrobiology and pathology labs for detecting H pyloriidentity

23 Bacterial Isolation Dental plaques and gastric biopsieswere cultured for one week in anaerobic jar with gas pack C(Merck Germany) and presence of H pylori was confirmedby biochemical tests catalase oxidase urease and gramstaining In brief all samples were cultured in Brucellaagar (Merck Germany) supplemented with 7 fetal calfserum (Gipso USA) 10 sheep blood (Jahad DaneshgahiTehran University Tehran Iran) polymyxin B trimetoprimamphotericin B and vancomycin after homogenization

24 Polymerase Chain Reaction (PCR) 22DNA extractionwas performed by genomic DNA extraction kit (BioneerSouth Korea) followed by polymerase chain reaction (PCR)test with specific forward and reverse primers of ureCgene Forward 51015840-CCCTCACGCCATCAGTCCCAAAAA-31015840 and reverse 51015840-AAGAAGTCAAAAACGCCCCAAAAC-31015840 Total volume of reactions was 25 120583L and solution included25 120583L of 10x buffer (PH 84) containing 100mM trisHCl500mM KCl 2mM MgCl

2 02mM dNTP 15 U Taq DNA

polymerase 25 120583L bacterial DNA and 02mM primer Sub-sequently 30 cycles performed at 94∘C for 5minutes (primarydenaturation) 94∘C for 1 minute 55∘C for 1 minute and 72∘Cfor 1 minute and final extension cycle performed at 72∘C for10 minutes

25 Statistical Analysis We used SPSS version 180 softwarefor statistical analysis 119875 values less than 005 were regardedas statistically significant

3 Results

Correlation between alarm symptoms such as epigastricpain when people are hungry with and without nocturnalawakening bloating after meal with and without nocturnalawakening and reflux with and without awakening with Hpylori detection from dental plaque and gastric biopsy hasbeen shown in Table 1 Additionally we observed recurrenceof H pylori infection among different samples which arelisted in Table 1 All patients who received therapy alreadywere again H pylori positive while they were still carryingH pylori in their dental plaque (119875 = 0001) Correla-tion between endoscopic findings such as ulcerated andnonulcerated esophagitis antral and erosive gastritis gastriculcer duodenum ulcer duodenitis erosive duodenitis andduodenum deformity with H pylori isolation from dentalplaques and gastric biopsies was shown in Table 2

4 Discussion

H pylori colonization affects more than half of the worldrsquospopulation and thus it is the most persistent bacterial infec-tion worldwide Various clinical manifestations are reportingabout this persistence colonization [1] Alarm symptoms ofdyspepsia were found in 20ndash40 of community thus thisdisorder causes the annualmillions of admission to the clinics[2] Unfortunately alarm symptoms have no good prognosisfor H pylori infection but clinicians can apply this items todecide on who should receive early endoscopic service [11]In current investigation we compared the predictive value ofalarm symptoms with detection of H pylori in dental plaqueand gastric biopsy Actually our research was the first whichrevealed that there is relation between epigastric pains andnocturnal awakening while patient is hungry Interestingly













Disclosure




References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























Disclosure




References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article concomitant colonization of helicobacter...

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