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THE 13C-UREA BREATH TEST FOR EARLY ASSESSMENT OF HEUCOBACTERPYLORI ERADICATION.FRaznlL- R.M.Zagarl. S.Fossl. P.Pozzato. P. Smonl, A.Roda. E. Roda. Cattedra dlGastroanterologla. University of Bdogna. Bologna Italy.

Introduction. The 13C_urea breath test (1304151) is an accurate method to noninvasrvely detect the actual presence of Helicobacter py4ol (H.p.) Infection. Forassessment of eradication, 13C-UBT Is currently performed, as suggested for otheraccurate methods such as histology. urease test and culteur, four weeks aftertreatment withdrawl in order to avoid fase negatve results due to partialsuppression of the Infection and subsequent recolonization. However there are atpresent no studies considering the posailty that an accuate medhod such as13C-UBT could detect true eradication at an eadlr gage. Aim. To detemilnewhether 13CUEBT is capable to assess H.p. adiation euller than theconventionally adopted four week Intenral after the end of tre ent. Methoda. 58patients (31 males, 27 femaes; range age 2549. mean age 48 yms) with non.ulcerdyspepsia and H.p. Infection, partecipating in an ongoing mndomied double blind,double dummy eradication study, underwnt upper Gl endoscopy and wereevaluated by urease test, histology and culture before treatment and four weeksafter withdrawing medicadions. 13CUBT was perfomed (Euopean standardprotocol, positive result - excess 6 13CO2 excretion>5 per ml) before tretmentand every week for four weeks after withdawig modki ons Resuts. in 23 out of58 patients H.p. eradication was established at four wees after Uteatm bynegative urease test, histoogy. cultu and 13uT (e s 613002excretdon-1.65±tO.17(mean:±SE) per ml). In l 23 pae in whom medicationwas assessed at four weeks after tremnt he 13 BT was dy negative atone week (excess 6 13C02 mmcreton- 1.720.18 (men±SE) per ml). And bt 3patients in whom successful eradicaon wee not achived the C wa POsitivesince the first week (excess 6 13C02 excrtion- 21.49*3.22 (menSE) per ml). 3patients had a negative tes at one week (aec '3C rd on- 1.5±0.7(mean±SE) per mil) which homwer tumed to be poeit at the ond weekevaluation (excess 6 13C02 eCreo 11.5±28 (en±SE) per m .Conclusions: 13C%UBT can assess Helkcobcter pylod eradlcaton as soon as oneweek after treatment withdrawl. When perormed at two weel It sh the samesentMity and specificity of the test performed at four weeks after withdrawingmedications.

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A SIMPLIFIED, RELIALEA UREA BREATH TEST.

M Buckley, H Hamilton, S Heattie, c OMorain.Dept. of Gastroenterology, heath/Adelaide Hospitals, Trinity College,Dublin, Ireland.

INlTRODCTION. Non-invasive tests to detect E.pylori are necessary forepideiological studies, invmstigation of dyspepsia In younger patientsand in primry cars and for follow-up after eradication treat_mnt. The13 Carbon urea breath test (uST) is highly sensitive and specific.However, the current recomended protocol for the UBST, including anovernight fast, the use of large reservoir bags and mltiple positionalchanges and sampl collections rdr the test unsuitable forwidespred use.ADS. The aim of this study was to assess the sensitivity andspecificity of a modified, simple OUT.PATINT8 AND METHODS. Subjects undergoing endoscopy for investigationof dyspepeia or after a course of eradication therapy were recruited.Hon-fasting patients were adeinistered a standard motility inhibitingliquid al (soels calogen +SOmls Ensure). Imsdiately after, breathaples were collected in duplicate in 20ls vacutainers via straws.Subjects then received 100 mgs of Carbon urea dissold in 100 mls ofwater. After sitting for 30 aimites, repeat breath amples werecollected in vacutainsrs. An exces of 13C02 excretion of 5 per ml wastakn as a positive result. The umT results were cqpard with antralad corpus histology (x4), antral an corpus culture (x2) nd antralCO test. The 'gold standard, was defid by the results of any two ofthe biopsy bad thods.REULTS. 169 subjects wre recruited (98 male), ma age 42 ye(ran 17-79). 89 subjects had a positive UTg 85 true positives a 4false positivs. S0 subjects ha angative OUT 76 true negative ad2 false ngative. Therefore the sestivity was 97.7% ad thespecificity was 95.1%.CXcuLUSIcH. Thiis modified OU is user-friendly, idel for us in aprimry oar setting and is as reliable as other mthods for the non-invasiv detection of E.pyloril

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ACTIVATION OF HELICOBACTER PYLORI BOUNDPLASMINOGEN BY TISSUE-TYPE PLASMINOGENACTIVATOR (tPA) - a virulence trait in peptic oker disease.Martina RINGNtR, Asa LJUNGH and Torkel WADSTROM.University of Lund, Department of Medical Microbiology, Lund,Sweden

Plasminogen is a plasma- and extracellular matrix protein, importantin haemostasis as a main mediator of fibrinolysis. Activators such astissue-type plasminogen activator (tPA), splits one peptide bond of thenative plasminogen creating the active plasmin. Receptor boundplasminogen facilitates activation to plasmin and protects themolecule from inactivation. Several bacterial pathogens bindplasminogen, followed by activation to plasmin, which is assumed tobe an important virulence mech4nism for tissue penetration.

Binding of plasminogen to Helicobacterpvlori was studied using12'I-labelled plasminogen. Activation of H. pylori cell boundplasminogen was performed by incubating plasminogen with thebacteria in presence of tPA. Conversion of plasminogen to plasminwas detected in a spectrophotometer at 405nm after adding thechromogenic substrate S-2251 (Chromogenix, Molndal, Sweden).

All of the H. pylori strains tested bound 1251-plasminpgen. H.pylori strain 17874 (NCTC 11637), was selected for furthercharacterisation of the plasminogen binding. Inhibition of theinteraction between H. pylorl and plasminogen was observed afterpreincubation of the bacterial cells with non-labelled plasminogen,plasmin, lysine and the lysine analogue e-amino caproic qcid. Ourfindings clearly showed that H. pylori binds specifically the loopstructures of the plasminogen molecule. Plasminogen fragments,containing the loop structures, did also inhibited the plasminogenbinding to H. pylori. Plasminogen bound to H. pylori was activated toplasmin in the presence of tPA. No activation was observed whenplasminogen or tPA were incubeted with bacteria alone.

Formation of Helicobacter pylori-bound plasmin may beimportant to provide a powerful proteolytic mechanism for tissuepenetration of peptic ulcers, since plasmin degrades not only fibrin butalso matrix proteins such as collagens and fibronectin.

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DNA SEQUENCE OF CAGII, A NEW MULTIGENE LOCUSIMPLICATED IN H. PYLORI VIRULENCES. Clifton,l B. Roe,l D. Kersulyte,2 N. S. Akopyants,2 E. S.Drazek3 and D. E. B 1w1Dept. Chem. and Biochem., Univ.Oklahoma, Norman, OK, 2Dept. Molec. Microbiol., WashingtonUniv. Med Sch., St. Louis, MO, and 3Dept Bact. Diseases, WalterReed Army Inst of Research, Washington, DC

The ~21 kb cagII ("second cytotoxin associated gene") locus(Akopyants et al., this meeting), like the previously described vacA(vacuolating cytotoxin) and cagA loci, seems to be characteristic ofthe most virulent H. pylori strains, i.e. those strains routinelyrecovered fromn patients with peptic ulcers or gastric cancer and lessoften from asymptomatic carriers. To help identify functionsencoded by this locus, a cosmid carrying the entire cagiI regionplus flanking H. pylori DNA (39 kb) was sequenced via a shotgun-based automated DNA sequencing strategy. To ensure highaccuracy, all regions were sequenced at least three times, and atleast once on each strand. Open reading frames and sequencemotifs of interest were identified using the standard internet-basedBLAST and BLOCKS programs. The caglI region is gene-rich, asis typical of bacterial genomes, and 64% A+T, which is quitetypical for H. pylori. Several inferred cagIl region proteins exhibithigh levels of homology to proteins found previously in otherspecies. Apparent homologs include: cell surface proteins ofBordetella, E. coli, Neisseria andV. cholera that participate in pilusassembly and/or DNA transfer; the malarial MESA protein thatbinds the cytoskeleton and is found on infected erythrocytesurfaces; a NalH antiporter from E. coli; a regulator of dipeptidepermease operon excpression in B. subtilis; a restrictionendonuclease from E. coili; and the IS200 transposable element of S.typhimurium. The present cagII DNA sequence and its openreading frames will help guide further mutational, gene excpressionand histopathologipc studies to better characterize cagll-encodedproteins, and tests of our excpectation that some of them will affectthe exctent of tissue damage, inflammatory or immune responses, orhost cell proliferation during H. pylori infection, and thereby helpdetennine the severity of disease.

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THE H. PYLORI FLAGELLAR BIOSYNTHESIS REGULATORYPROTEIN FIbA AFFECTS THE EXPRESSION OF FLAGELLARCOMPONENTS ON THE TRANSCRIPTIONAL LEVEL AND ISPROBABLY A MEMRANE PROTEIN. Andr6 Schmitz, ChristineJosenhans, Sebasdan Suerbaum. Med. Microbiol. & Immunol.,Ruhr-University, D-44780 Bochum, Germany

We have recently reported the cloning and sequencing of a H. pylongene (fibA) that encodes a homolog of the conservedLcrD/InvA/FlbF family of proteins involved in the regulation offlagellar biogenesis and of other virulence factors.Isogenic mutants of H. pylon in this gene were constructed by genedisruption with a kanamycin resistance cassette and allelicreplacement. Characterization of these mutants by motility testing,Western Blotting and electron microscopy revealed that the mutantshad lost the ability to express all flagellar structural proteins knownso far (FlaA an FlaB flagellins, hook protein), that they werecompletely aflagellate (including a loss of flagellar sheaths) andnonmotile. To study the mechanism of this effect of a singlemutation on the expression of several genes that are unlinked on thechromosome and regulated by different promotors, RNA dot blotstudies were performed. Total RNA was isolated from H. pylon N6and the isogenic fibA mutant and hybridized with PCR-generatedprobes specific for mRNAs encoding: a) FlaA flagellin, b) FlaBflagellin, c) FlgE hook protein, d) the FlbA protein itself, and e)16S rRNA. In the wild-type strain, all mRNAs could be detected inamounts consistent with the expression levels of the respectiveproteins. In thefibA mutant, no mRNA for FlaA or FlaB flagellinnor the mRNA for the hook protein could be detected, indicatingthat FlbA is involved in the coordinated transcription of these three(and probably other motility-associated) genes.In order to study the FlbA protein further, theflbA gene was PCR-cloned into the expression vector pQE30. Attempts to obtain a highlevel of expression of the recombinant protein were badly toleratedby the E. coli host cells, indicating that high concentrations of theproteins are toxic for E. coli. A possible reason for this toxicitycould be the interaction of the FIbA protein with membranes,consistent with the strong hydrophobicity of the N-terminal part ofFlbA.

H PYLORI USES UREA FOR AMINO ACID SYNTHESIS ATNEUTRAL pH.

C Williams. T. Preston, C. Slater, M Hossack, K.E.L. McColl.University Department of Medicine and ..Therapeutics, WesternInfirmary, Glasgow and IBLSURRC, East Kilbride, Scotland.

H pylori has the highest urease activity of all known bacteria. Itsenzymatic production of ammonia protects the organism from aciddamage by gastric juice. We have investigated the possibility thatthe urease activity may also allow the bacterium to utilise urea as anitrogen source for the synthesis of amino acids.

An NCTC strain of H pylori (11038) was incubated for 5 min at37°C with 50 mM urea enriched with 5 atom % N urea in thepresence of either 0.9% NaCI pH6, or 0.2 M citrate pH6. Thespensions were then centrifuged and washed twice to remove any'N absorbed to the surface of the bacteria. E coli NCTC 9001 wasused as a urease negative control. 'IN enrichment of the washedorganism was detected by isotope ratio mass spectrometry. Hpylorishowed intracellular incorporation of N in the presence of pH6citrate buffer. There was no significant incorporation of N by Hpylori in unbuffered saline or by E Coli in either pH6 citrate bufferor unbuffered saline.

Further studies were undertaken to determine the intracellular fateof the urea - nitrogen by means of gas chromatography/massspectlmetry analysis. These were performed following incubationwith N enriched SmM urea in the presence of either 0.2 M citratebuffer pH6 or 0.2 M acetate bulfeypH6. Following 5 minincubation with either buffer there was N enrichment of glutamate,glutamine, phenylalanine, aspartate and alanine.

Conclja,ion At pH and urea concentrations typical of the gastricmucosal surfaceHpylori utilises exogenous urea as a nitrogensource for amino arid synthesis. The ammonia produced by Hpylori urease activity thus facilitates the organism's nitrogenmetabolism at neutral pH as well as protecting it from acid damageat low pH.

REPORTER GENE ANALYSES SHOW THAT EXPRESSION OFBOTH H. PYLORI FLAGELLINS IS DEPENDENT ON THEGROWTH PHASE. Christine Josenhans1, Agnes Labigne2,Sebastian Suerbauml. Med. Microbiol. & Immunol., Ruhr-University, D-44780 Bochum, Germany (1) and Institut Pasteur,Unite des Enterobacteries, INSERM U389, Paris, France (2).

Motility is thought to be an important virulence factor of H. pylon.Complex flagella, consisting of two different flagellin subunits, FlaAand FlaB, are necessary for full motility in this organism.Expression of the genetically unlinked flaA and flaB genes isregulated by different promoters, sigma28 (fla) and sigma54 (aB).To address the question, whether flax and flaB expression isenvironmentally regulated in order to assure optimal motility, weconstructed transcriptional fusions of the flaA and flaB promoterswith a promotorless chloramphenicol acetyl transferase (cat) reportergene cassette. The flaA-cat fusion plasmid was obtained by directcloning of the cat cassette into the unique BamHI site of the flagene. The flaB-cat fusion plasmid was constructed by transposonmutagenesis with the Tn3-Kmcat system. The transcriptional fusionswere introduced into H. pylon by electroporation-mediated allelicexchange. Expression of CAT was determined by ELISA aftergrowth of H. pylon cat-fusion mutants under different conditions.The level offlaA gene expression was about tenfold higher than thatof flaB, consistent with previous findings about the differentamounts of the two flagellin subunits in H. pylon flagella. Weobserved a strong influence of the growth phase on the expression ofboth FlaA and FlaB. In the early logarithmic growth phase, onlysmall amounts of both flagellins were produced. At mid-log phase,we observed a 10- to 20-fold increase in expression of bothflagellins. CAT-expression from the flaA and flaB promotersremained at this high level well into the stationary phase. TheflaA/1flaB expression ratio was also subject to variation during thegrowth phase. The use of cat-fusion mutants will permit to definefurther environmental influences on motility and on the regulation ofother H. pylon virulence factor genes.

GR12231 1X CAN SIGNIFICANTLY REDUCE THEEMERGENCE OF H. PYLORP STRAINS RESISTANT TOANTIBIOTICSAMcljrc, SR McDowellGlaxo Research and Development Limited, Glaxo MedicinesResearch Centre, Stevenage, Herts SGI 2NY

Bismuth compounds have been shown clinically to lower theacquisition of nitroimidazole resiance. This study was performed tofind out if this is as a result of reducing bacterl load and hencemutation rate or if it is as a direct reslut on decreasing spontaneousmutation rate from sensitive to resistant. Three different classes ofantibiotic were investigated in the form of metronidazole,clarithromycin and streptomycin Two H. pylon strains werecontinuously sub-clured on media either containing or withoutGR122311X ranitidine bismuth citrate, (8 [±g/ml). One organismwas known to readily convert to metronidazole resistance. Afterpassages 1, 5, 8, 14, 17 and 22 reistance rates were calculated. Thiswas done by plating samples onto media conin clarithromycin(0.06 tg/ml), metronidazole (10 gLg/ml) and streptomycin (10 gg/ml)and dividing the nmber of res nt colonies by the total viablecount. Statistical analysis ofresuls was performed using a two sidedunpaired t-test except one anaWysis when mutation was iatedwith concentration which utised a two sided paired t-test. Reultsshowed GR12231 IX to signifitly reduce the rate of cquisition o'tresistance for both srains againt metronidazole, producing P-valuesof 0.007 and 0.014. GR122311X reduced the nce rate toclarithromycin in one strain P-value 0.037. Reistance tostreptomycin was unalterd.

GR12231 IX can reduce emergence of resistant rains, directlyafting mutation rate even in sains that readily convert toreistance.:

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ERADICATION OF H. PYLOPJ WITHOUT ANTIBIOTICS MONOCLONAL ANTIBODY RECOGNITION AND GENE

A AA McColm, J Bagshaw, C Donnelly, J Wallis CLONING OF A 22 KDA SURFACE-ASSOCIATED PROTEIN OF

Gaxo Reearch and Development Limited, Glaxo Medicines HELICOBACTER PYLORIReearch Centre, Stevenage, Herts SG1 2NY A Jones, TJ Hellyer, M-LA Baillon, EJ Wallington, CW Penn. Schoolof Biological Sciences, University of Birmingham, UK

The viability of H. pylon is draically affected by the balance A monoclonal antibody was derived from a mouse immunised with

between pH, H. pylon and urea. This study assessed the effects of H.pylori NCTC 11637; it reacted with a protein of 22 kDa, associated

alteing the paramers both in vtro and in vo using an with surface-derived fractions obtained by agitation of whole cells with

achlorhydric mouse model. H. pykrl was subjeced to a range of pH ballotini or by Triton-X 100 detergent solubilisation of surface materialc.~dphosphate .ffm betwem O-SOmM urea. At pH 5 from whole cells. The antibody was used to screen immunologically a

citrate/phosphate WEffrs containing between 0-50mM Urea. At pH 5 gene library prepared from genomic DNA of H. pylori strain NCTCH. pylor could not tolerate wrea; after 10 mintes inubation 2.5mM 11637, partially digested with Sau3AI, size fractionated to

caused a 90%/ drop in viability and 50mM urea produced a 99% ckill. approximately 3-8 kbp, and ligated into the BamHI site of phage lambda

HSD/ICR mice colonised with H. pylon were treaed with pH 4.5, 5 Zap Express (Stratagene). An average insert size of approximately 3

and 5.5 citrate/phosphate buffer containing 0-500mM urea bd for 4 kbp was achieved. Screening of plaque replicas from the amplified

days. Significat H. pylon reduction was observed in all pH gwp. library on nitrocellulose with either polyclonal rabbit antiserum to aThedays.ree of n.

edictionwuproportional to the ureaco grwpsclinical isolate (strain Roberts), or a pool of monoclonal antibodies to aThe degree of eradication was proportional to the urea concentration range of antigens ofNCTC 11637, yielded numerous antigen-administerd with the highest eradication (100/o) occurring in the ph expressing clones. Plasmids (pBK-CMV derivatives) were excised

5.5, 500mM urea ttment group. from antibody-reactive lambda clones, and cultures harbouring theplasmids analysed by SDS-PAGE and immunoblotting. Multiple clones

This study demonstates that altering pH may be the method by which expressing certain antigens including urease were identified. Amongdrugs that preventacidse~on are able to aid m H

these were a number of clones expressing an abundant doublet band ofdrugs that prevent acid secretion are able to aid in H. pylor antigen at 22-24 kDa, reactive with the monoclonal antibody described

eradication. The consequent pH rise places the organism in an above; the lower Mr form of the recombinant protein was identical inenvironment it is unable to tolerate, consequenly allowing H. pylon mobility on SDS-PAGE to the protein expressed by H. pylori. It was

to become more suscepble to antibiotics. This is currently being assumed that the higher Mrform of the protein represented an

explored with standard agents such as amoxycillin. unprocessed molecule not yet exported from the cell, consistent with themature form being a surface-associated, exported protein. Therecombinant protein was expressed abundantly by a number ofindependent clones (it was clearly visible by Coomassie blue staining),with or without IPTG induction of the lac promoter in the vector, andthe size of the inserted DNA fragment ranged from approximately 2 to 5kbp. The larger clones expressed an additional antigen of approximately30 kDa. Screening by PCR with primers based on sequences encodinga ferritin-like protein, a 20 kDa lipoprotein, a 20.5 kDa adhesin and anadditional neuraminic acid-binding adhesin was in all cases negative.Sequence data for the protein, and implications for its predicted natureand function, will be presented.

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DETERMINATION OF MIC AND NBC OF 14 AGENTS AGAINST85 CLINICAL ISOLATES OF HZLICOBACTER PYLORI - AN

INTERNATION&L MLTICENTRE STUDYD Tompkins, C McNulty, S Hazell, CE Nord, LeedsUK, Gloucester UK, Sydney Australia, HuddingeSweden.Introduction Most sensitivity data is fromsingle centres and does not use liquid medium ordetermine MBCs.Aim To determine the MICs and MBCs of 85clinical isolates of Helicobacter pylori (Hp) to14 drugs in a multicentre study using a singleprotocol. The reproducibility of results wasdetermined by transferring strains betweencentres.Methods 4 centres each selected 25 Hp strains.An inoculum of 5x10' cfu of each strain inisosensitest broth with 5% horse serum was addedto prepared microtitre trays containinglyophilized drugs. MBCs were determined bysubculture of loMl onto laked blood medium. 3 ofthe 25 isolates from each laboratory were sent toeach of the other 3 laboratories.Results MIC50, MIC90, MBC5O, and MBC90 valueswere calculated for each of the following agents;amoxycillin, benzylpenicillin, cefuroxime,clarithromycin, 140H- clarithromycin,clarithromycin:140H-clarithromycin, Denol,erythromycin, lansoprazole, metronidazole,minocycline, ofloxacin, omeprazole, tetracycline.Lansoprazole was more active than omeprazole.Clarithromycin was more active than its 140Hmetabolite, but the latter still had excellentactivity (MIC90 0.125mg/1). The MICs weresimilar to those previously described forantimicrobials, except for ofloxacin in which theMIC9O was higher in this study. Thereproducibility of results will be presented indetail.

GENE CLONING OF A FLAGELLAR SHEATH PROTEIN OFHEUCOBACTER PYLORIA Jopes, CJ Luke, A Cockayne, C Constantinidou, DJ Reynolds,CW Penn. School of Biological Sciences, University ofBirmingham, UK

A gene library was prepared from genomic DNA of H. pylori strainNCTC 11637, partially digested with Sau3AI, size fractionated to -

approximately 2-10 kbp, and ligated into the BamHI site of phagelambda Zap Express (Stratagene). An average insert size ofapproximately 3 kbp was achieved. Screening of plaque replicasfrom the amplified library on nitrocellulose with either polyclonalrabbit antiserum to a clinical isolate (strain Roberts), or a pool ofmonoclonal antibodies to a range of antigens of NCTC I 1637,yielded numerous antigen-expressing clones. Plasmids (pBK-CMVderivatives) were excised from antibody-reactive lambda clones, andcultures harbouring the plasmids were analysed by SDS-PAGE andimmunoblotting. Multiple clones expressing certain antigensincluding urease were identified. With difficulty, plaques wereidentified which were wealdy reactive with a monoclonal antibodywhich decorated the flagellar sheath of native H. pylori (1) andidentified a 29 kDa protein on immunoblots. The protein was alsoshown to partition into the detergent phase of a Triton X- 100 two-phase system, indicating its hydrophobic nature and possibly that itis a lipoprotein. Excised plasmids from independently derivedlambda clones expressing the 29kDa protein had inserts ofapproximately 3-6 kbp. The antigen was expressed very strongly inrecombinant Escherichia coli bearing these plasmids, both in thepresence and absence of IPTG induction of the lac promoter in thevector. The recombinant protein was prominent on coomassie blue-stained SDS-PAGE gels and strongly reactive with the anti-sheathprotein monoclonal antibody, showing an identical mobility onSDS-PAGE with the protein expressed by H. pylori. The intactprotein was blocked to N-termiinal amino acid sequencing,consistent with it being a lipoprotein. Restriction map and sequencedata for the sheath protein, and implications for its predicted natureand function, will be presented. The protein, known to be presentin all H. pylori strains examined, may have potential as a diagnosticantigen.

1. CJ Luke, CW Penn (1995) Microbiology 141, 597-604.

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DEVELOPMENT OF A QUANTITATIVE ASSAY FORADHERENCE OF H.pyloni. RPH Logan, A Cockayne, A Robins, SPBormello, CJ Hawkey. Division of Gastroenterology and Institute ofInfections & Immunity, University Hospital, Nottingham NG7 2UH.

Introduction: Adherence may be an important virulence factor forH.pylon. Current methods available for quantitation of adherence in-vitro could he improved. Viable counting and direct microscopy are

time consuming and liable to observer error. To overcome theseproblems a new direct fluorescent labelling technique and quantitativeFACS assay for assessing adherence has been validated.Methods: H.pvlon (type strains and clinical isolates) H.mustelae,H.cinnediae and H.fenneliae were grown microaerobically in brothculture for 24 h. Isolates were fluorescently labelled by incubation withcarboxyfluorescein diacetate succinimidyl ester (CFDA-SE) at 37°C.After washing in PBS to remove excess CFDA-SE, bacteria wereresuspended in PBS (Sml) and standardised numbers of bacteria co-

incubated (ratio 10: 1) with AGS or CaCo2 cells at 37°C for up to 24 h.After washing to remove non-adherent bacteria, cells were detachedfrom the tissue culture flats with EDTA (2mM), fixed withformaldehyde for flow cytometry or microscopy and quahtitated byreference to fluorescence of bacteria alone. Filamentous actin stainingwas performed on fixed cells following permeabilisation with Triton X-i100 (0.1%) and incubation with TRITC-phalloidin. All assessmentswere blinded. Results: All l.pylon strains adhered to AGS cells. Theproportion of cells with bound bacteria varied from 40-99% and numberof bacteria per cell from 1-50, and both correlated closely withmicroscopy (r=0.97, n=20 and r=0.89, n=16 respectively). ForH. mustelae H. cinnediae and H.fenneliae the proportion of cells withbound bacteria varied from 5-15% and the mean number of bacteria percell was < 1. Time course studies demonstrated saturation of bindingto AGS cells by H.pylori within 45 minutes. Specificity of binding wasdemonstrated by the pattern of actin staining.Conclusion: FACS analysis of using CFDA-SE in co-incubation withthe gastric epithelial cell line AGS provides a specific, sensitive andquantitative in-vitro method for investigating adherence of H.pylori.

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ADHESIN-RECEPTOR SPECIFICITY OF HELICOBACTER Sp.RPH Logan, A Cockayne, A Robins, CJ Hawkey, SP Borriello.Institute of Infections & Immunity and Division of Gastroenterology,University Hospital, Nottingham NG7 2UH,

Introduction: H.pyloni agglutinates red blood cells (RBC) and this canbe blocked by N-acetylneuramin-lactose (NAML). We designedexperiments to investigate whether similar binding explains H.pylori'sgastric epithelial tropism.Methods: H.pylori (type strains and clinical isolates) H.muselae,H.cinnediae and H.fenneliae were grown microaerobically in brothculture and on chocolate agar. Bacteria, fluorescently labelled withcarboxyfluorescein diacetate succinimidyl ester (CFDA-SE), were co-incubated (ratio 10:1) with AGS cells for flow cytometry. In otherexperiments washed human RBC (Group 0) were co-incubated withbacteria under saturating conditions in a 96 well microtitre plate andhaemagglutination quantitated using an automated spectrophotometricmethod. Bacteria pre-incubated with NAML or hog gastric mucin(0.5%) were used for blocking studies. To identify H.pylori adhesins,bacterial sonicates were incubated with formaldehyde fixed suspensionsof epithelial cells and RBC. Following washing, bound bacterialproteins were eluted with I.5M NaCI, dialysed and analysed by SDS-PAGE or Western blotting.Results: Haemaggluttination was greater for bacteria grown on agarcompared to broth (mean +sem absorbance = 24.3 +5.2 vs 33.6 +3.6, p=0.07). H.pylori and H.musrelae, but not other Helicobacter sp,bound to AGS cells and agglutinated RBC. However there was nocorrelation between heamaggluttination and adherence to AGS cells(r=0.08). Moreover NAML blocked haemagglutination but notadherence to AGS cells, whereas hog gastric mucin reduced adherenceto AGS cells by 44% +10% sem (p=0.033) but did not blockhaemagglutination. SDS-PAGE demonstrated a major 30 kDa adhesinto RBC but not to AGS cells, which in contrast had a variety ofdifferent adhesins. Conclusions: Adherence of H.pylori to AGS andRBC do not appear to be related. The in-vivo relevance ofhaemagglutination remains unestablished.

CHEMOTACTIC RESPONSE TO MUCUS AND ACID BYHELICOBACTER PYLORI. RPH Logan, A Cockayne, CJ Hawkey, SPBorriello. Div of Gastroenterology and Institute of Infection &Immunity, University Hospital, Nottingham NG7 2UH, UK.

Introduction: It is not known why Helicobacterpylori (H.pylori) onlyinfects gastric type epithelium. We therefore assessed the influence ofgastric mucus and acid on H.pyloni using a novel method for assessingchemotaxis.Methods: Isolates of Helicobacter spp. (H.pylori NCTC 11637, a

clinical isolate 231 and H.mustelae NCTC 12198) were grown in liquidmedium (RPMI 1640 supplemented with 4% Isosensitest broth and 5%FCS). Bacteria were pelleted, washed and resuspended and celldensities standardised by optical density measurements at 600 nm. Thetips of capillary tubes (filled with chemotaxin or control buffer) wereinserted vertically into microtitre wells containing suspensions ofHelicobacter spp. at pH 7. After microaerobic incubation at 30C for1 h bacteria in the upper 2/3rds of each tube were counted by culture ofserial dilutions.Results: All isolates tested showed chemotaxis to both hog and humangastric mucus and acid. The response to hog gastric mucin was dose(threshold 0.05%) and time (threshold 15 min with o.5%) dependent andthe taxin was resistant to pronase and trypsin and boiling at 100°C for1 h (which enhanced the response). H.pyloni was not chemotactic toIH'j alone but chemotaxis to hog gastric mucin was enhanced by acid(optimal pH 5.7-6.0). From pH 6.6 chemotaxis to 1H I alone was seen

with H.mustelae.

Conclusions: Chemotaxis towards gastric mucus under mildly acidconditions may contribute to the gastric epithelial tropism ofHelicobacter spp.

DETECTION OF Helicobacter pylori CYTOTOXIN -ASSOCIATED PROTEIN BY SMALL FORMAT POLYACRYLAMIDEGEL ELECTROPHORESIS AND APPLICATION TO IDENTIFYINGPOTENTIALLY ULCEROGENIC STRAINS. A. Hurtado,R.J Owen. National Collection of Type Cultures,Central Public Health Laboratory, London, England.

The product of the H.pylori cagA gene, the 125 kDaCagA protein, is an important antigenic marker forduodenal ulcer disease, and we have studied a fastelectrophoretic method to detect the CagA proteinin bacterial extracts to identify potentiallyulcerogenic strains.

Whole cell protein extracts were prepared from 24isolates of H.pylori with known vacuolatingcytotoxin activity and were anyalysed by one-dimensional SDS PAGE in small sized gel format.Presence of the CagA gene was tested by PCRamplification.

The CagA protein was detected electrophoreticallyin 18/24 strains. Its presence correlated closelywith the cagA gene detected by PCR but not withpublished results on cytotoxin activity. CagAproteins of the cytotoxigenic strains were all thesame size whereas the proteins of the non-cytotoxigenic strains were more heterogeneous insize.

Electrophoresis of whole cell protein extracts ofH.pylori was a simple, economic and fast methodof detecting in vitro expression of the CagAprotein and could be used as an alternative toimmunoblotting. Although the presence of CagAprotein did not consistently correlate with afunctionally active cytotoxin, it may benevertheless an easily determined marker foridentifying potentially ulcerogenic strains.

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THE HSPA ANTIGEN IS HIGHLY CONSERVED AMONGHELICOBACTER PYLORI CLINICAL ISOLATES AND ITSUSE IN SEROLOGICAL AND PROTECTION STUDIES ISWARRANTED.Imad KANSAU and Agnts LABIGNE, Institut Pasteur, Unitc INSERMU389, Paris (France)

We previously cloned the gene responsible for the expression ofthe GroES-homolog (HspA) of H. pylori (HP) strain 85P, and showedthat the HspA had two domains: a 89-aniino acid (AA) N-terminal domain(domain A), highly homologous to the bacterial GroES proteins, and a29-AA C-terminal domain (B), unique to HP, responsible for nickelbinding. We have shown that HspA was capable of eliciting a humoralimmune response in 39.5 % of HP-infected patients, and that the Adomain was responsible for this response. Finally, the MalE-HspArecombinant protein derived from the 85P isolate was shown to be aprotective antigen in mice. In this study, we investigated the degree ofconservation of the two domains of HspA among HP isolates to confirmtheir usefulness in scrological and protective studies.

The hspA genes from 25 independent clinical isolates wereamplified by PCR using primers targetting the complete gene. Theresulting 344 bp-fragment was sequenced by a direct sequencing PCRtechnique and both the nucleotide sequence and the deduced amino acidsequence were compared to those of the 85P HP isolate. For each strain,the nucleotide sequence of a 294 bp-intemal fragment of the ureC gene,known to be highly polymorphic, was also determined.

Unexpectedly, the DNA region encoding the A domain of HspAwas conserved among all the isolates over a 200 bp stretch of DNA, asituation never observed so far in HP. In contrast the DNA fragmentencoding the B domain ofHspA displayed some polymorphism; over the110 nucleotides at the 3' end of hspA, the different isolates exhibitedbetween 36 to 97 % of identity, a situation similar to that found for theureC gene. This DNA polymorphism was not correlated to a majorantigenic variation since the sequence in AA of the B domain was highlyconserved (83 to 100 % of identity with the same number of cysteine andhistidine residues). Whilst, two antigenic variants in the B domain wereobserved, no correlation with the serological response was found.

The results indicate that the bimodal serological response againstHspA observed in HP-infected patients is associated with the host-response rather than with antigenic variations in HspA. Moreover, thehigh degree of conservation of HspA supports its use as a protectiveantigen.

A RANDOM INSERTION MUTAGENESIS SYSTEM FORTHE INACTIVATION OF HELICOBACTER PYLORI GENEUSING A CONSTRUCTION DERIVED FROM THE GRAMPOSITIVE CONJUGATIVE TRANSPOSON Tnl545.AgnisABIGNE, Catherine CHEVALIER and Hilde de REUSE, InstitutPasteur, Unit6 INSERM U389, Paris (France)

Although the precise or random inactivation of genes ofH. pylorican be achieved using a shuttle mutagenesis approach, there is a need fora genetic system pemiitting the direct delivery of transposable elements inH. pylori. The aim of the study was to determine whether a recombinantgenetic system involving the well known functions of the integration-excision system* of the TnlS45 conjugative transposon could be usefulfor the random mutagenesis of H. pylori. Whilst previous attempts todeliver the native TnlS4S into H. pylori have been unsuccessful, wethought to engineer a recombinant transposable element that wouldintegrate more efficiently than the native transposon, by avoiding theexcision step of the transposition process. A minireplicon (pILL800, 2.7kb) was constructed by ligating the Campylobacter kanamycin cassette(1.4 kb) to a 1 kb fragment harboring the pBR322 replication function,and to the recombining attachment sites (att) of the Tn 1545 transposon;this site corresponds to a 341 bp fragment generated by geneamplification. The minireplicon behaves as a transposable element whenthe TnlS45-encoded integrase which recognizes the "att" site, is providedin trans by complementation*. The next step was to construct a strain ofH. pylori in which the gene encoding the integrase would beconstitutively expressed. The integrase encoding gene, deleted of its ownpromoter, was cloned in E. coli as a transcriptional fusion into the flaAgene of H. pylori. Immediately downstream of the int gene, apromoterless cassette encoding the chloramphenicol acetyl transferase,was fused to theflaA-int transcript. This construction was introduced inH. pylori by electroporation and clones in which the original flaA genewas replaced by the flaA-int-cat cassette were selected (N6-int-cat). TheseH. pylori expressing constitutively the TnlS45 integrase were thenelectroporated with the p11800 replicon and insertions of the kanamycinmarkcr were selected.Once the N6-lm-cat strain is available, the procedurefor the isolation of mutants is very fast; because of the absence of themajor restriction sites within the pILL800 replicon, the HP gene in whichinsertion took place can be easily identified by restricting the Km-HPchromosome, ligating the resulting fragments, transforming Ecoli cells,and selecting for kanamycin transformants.*P Trieu-Cuot et al., Mol. Microbiol., 1993,8:179-185

H. FELUS ISOLATES OF DIFFERING VIRULENCE: BACTERIALVIRULENCE FACTORS AND HOST RESPONSE AS MEDIATORSOF THE INFECIION PROCESSR. L. Ferrro and A. Labigne. Unitd des Ent6robactcries (INSERMU389), Institut Pasteur, 28 Rue du Dr Roux, Paris 75724, FRANCE.

Numerous pathogenic determinants of gastric Helicobacter spp havebeen identified, though the contribution of these deterninants to virulencein vivo has not, for the most part, been elucidated. We have observedthat the passage of H. felis bacteria in mice renders the bacteria highlyvirulent. The aim of the study was to compare H. felis isolates of varyingvirulence, and to analyse the immune responses of mice to these isolatesduring the early phases of gastric murine infection.

H. felis isolates of varying virulence were obtained from H. felisATCC49179 by continuous passage in vitro ("high pass" isolate) or,alternatively, by passage of the strain in mice with few subcultures invitro ("low pass" isolate). It was shown that the low pass in vivoadapted H.felis bacteria had a minimum infectious dose that was 100-fold lower than that of the isolate that had been subcultured continuouslyon artificial media. Analysis of total extracts of these isolates by SDS-PAGE and by immunoblotting against rabbit antisera raised against wholecell H.felis extracts revealed antigenic differences between the isolates.Sera fromn mice that had been inoculated with either isolate reacted weaklyto cell extracts at 2 wks post-infection. A proportion of those mice thathad been infected with the less virulent high pass H. felis isolate,however, developed strong antibody responses to a 31 kDa protein. Serafrom mice that had been orogastrically immunised with whole cell H. felissonicates and cholera toxin also reacted strongly with this protein.Extraction of cells with the nonionic detergent Tween X- 1 14 resulted inthe partitioning of the 31 kDa protein into the detergent phase. Results todate suggest that this protein is likely to be a membrane-associatedlipoprotein. Interestingly, this protein is expressed by the more virulentlow pass isolate but does not appear to be recognised during the earlyphases of the murine infection.

The results suggest that virulent H. felis (compared to bacteria thathave been adapted to artificial media) express additional factors whichpermit a more effective colonisation of mouse gastric mucosa. Moreover,the ability of virulent Helicobacters to modulate a poor or reducedimmune response in the host may be a determinant in the initial steps ofpathogenesis.

SCREENING FOR H. PYLORI GENES THAT ENCODEALTERNATE NICKEL TRANSPORT SYSTEMS NECESSARYFOR SYNTHESIS OF CATALYTICALLY ACTIVE UREASE.R. Gamner, P. Bauerfeind, K. Hendricks, C. Clayton*, and H. Mobley.University of Maryland School of Medicine, Baltimore. Maryland, USAand *Glaxo Research and Development, Greenford, Middlesex, UK

The finding that NixA (high affinity nickel transport protein)-deficient mutants of H. pylori, constructed by allelic exchange, retainedsignificant urease activity suggested that this species possesses altematenickel (Ni2,) transport systems which can supply Ni2, to the ureasemetalloenzyme. Using the phenotypic selection with which nixA wasisolated, we screened an H. pylori 7APII genomic library for clonescapable of producing active urease when cotransformed into E. coli(pHP808) which carries the entire H. pylori urease gene cluster. Among27 urease-positive cotransformants isolated on urea segregation agar,three clones were consistently urease-positive, displayed distinctrestriction endonuclease profiles, and did not hybridize with DNA probesfor urease, nixA, hspA, or a nikD-homologue. These clones, whencotransformed with pHP808, produced urease activities of 0.8, 0.2, and0.1 pmol NH.Jmin/mg protein compared to 14.0 for E. coli(pHP202/pHP808), the nixA/urease cotransformant. We conclude thatthese gene bank clones, which had lower MIC's to NiC12 than the vectorcontrol, function to increase cytosolic Ni2 concentration allowingsynthesis of catalytically active urease.

Using a different strategy to isolate urease-enhancing clones.analysis of random nucleotide sequence of H. pylori chromosomal DNArevealed an open reading frame that predicts a polypeptide with limitedamino acid sequence similarity to NikD, a component of an ATP-dependent Ni2O transport system in E. coli. Based on this sequence, a400-bp "nikD" fragment was PCR-anplified and used as a probe toidentity two EZAPII clones that carried these sequences. We concludethat we have isolated clones derived from the H. pylori genome that mayencode the altemate nickel transport system that partially masks the effectof the mutation in nixA.

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ALLELIC EXCHANGE MUTAGENESIS OF NIXA IN HEUcOBACTER PYLORIRESULTS IN REDUCED NICKEL TRANSPORT AND REDUCED UREASEACTIVITY.P. Bauerfeind, R. Garner, and H.L.T. Mobley. University ofMaryland School of Medicine, Baltimore, Maryland

NixA was shown to be necessary for full activity ofrecombinant H. pylori urease (a metalloenzyme) in E. co/i,independent of growth conditions. We hypothesized that this wasdue to increased nickel (Ni2*) transport. Ni2* transport wasmeasured in E. coli SE5000 containing either the NixA clone, thevector, or an in-frame Bal31 deletion mutant. nixA was found toencode a high affinity Ni2* transporter, with a VMAX Of 1750±220pmol/min/1 o8 cells and a KT of 11.3+2.4 nM in E.coli SE5000. Todetermine whether the NixA NF+ transport protein was essential forthe production of catalytically active urease in H. pylori, a NixA-negative mutant was constructed by insertion of a kanamycin-resistance cassette (aphA) into the nixA open reading frame. Thisconstruct was electroporated into H. pylori ATCC 43504 andUMAB41. Allelic exchange and disruption of nixA were confirmedin KanR isolates of H. pylori by PCR. Rates of Ni2* transport werereduced in the nixA mutant of H. pylori as compared to the parentstrain. Transport rates were decreased > 40% at low Ni2+concentrations (7-200 nM in the transport medium) but decreasedby only 16% at higher Ni2' concentrations. While a nixA mutationin H. pylori reduced urease activity by 30% (140±70 jmolNHJmin/mg prot in the mutant vs. 240±100 i±mol NHJmin/mg protin the parent (p=0.037)), the knockout of nixA did not completelyabolish urease activity. We conclude that the NixA tranportsystem has a high NPF affinity providing Ni2* transport at low Ni2'concentrations. Redundant systems for NiE transport, however,are present in H. pylori which may provide sufficient Ni+ forproduction of an active urease in the absence of a functional NixAtransport system. Supported by Schweizensche Stiftung fOr med.biol. Stipendien and NIH Grant A123328.

METABOLIC PATHWAYS IN METRONIDAZOLESENSITIVE AND RESISTANT STRAINS OFHELJCOBACTER PYLORI. P.. Hoffman. A. Goodwin, J.Johnsen, S. Veldhuyzen van Zanten. Depts. of Medicine &Microbiology, Dalhousie University, Halifax, Nova Scotia.B3H 4H7.

Helicobacter pylori (Hp) is a gram negative microaerophillicvibrio that exhibits a strictly respiratory form of metabolism.These organisms are also susceptible to the redox active drugmetronidazole (Met). Resistance to Met is the majordeterminative factor in the failure of triple therapy to eradicateHp infection. In the present study, we have examined themetabolic activities of Met-sensitive and -resistant strainsgrown in the presence or absence of 1/2 MIC of Met. Cell-freeextracts were prepared from bacteria harvested from abiphasic brucella-medium based system supplemented with2.5% human serum. All Hp strains were found to possess acomplete Krebs cycle and an active pyruvate:oxidoreductase(Pyr Ox/Rdase) measured with benzyl viologen as electronaccepter. Met-resistant strains grown in the presence of 18uglml Met exhibited no detectable Pyr Ox/Rdase activity.Coordinate with an absence of Pyr Ox/Rdase, was a 2-folddecrease in citrate synthase activity (529-241 nmol/min/mgprotein) and an increase in malate synthase. To establish agenetic basis for the observed repression of Pyr Ox/Rdase, anisogenic Metl-strain Hpl 107 was constructed by transformingMets strain Hp500 with DNA from MetR strain Hp439. Theenzyme profile of strain Hpl 107 was identical to the parentalHp500 strain with the exception of Pyr Ox/Rdase which waslike MetR strain Hp439. Spontaneous MetR mutants of Hp500were not isolated in this study suggesting multiple, linked locimay be associated with Met resistance.

FLNE STRUCTURE AND ANTIGENS OF COCCOID FORMS OFHELICOBACTER PYLORI.M. BENAISSA (1,3), P. BABIN (2), N. QUELLARD (2), B.FERNANDEZ (2), M.F. DRILLEAU (1), Y. CENATIEMPO (3) andJ.L. FAUCHERE* (1); Laboratoires de Microbiologie A(1) etd'Anatomo-Pathologie (2) and Institut de Biologie Moleculaire (3),University of Poitiers France.

After several days of in vitro culture, H. pylori (Hp) convert frombacillary to coccdid forms which sianification (degenerative forms ordormant staae) is controversial. By transmission electon-microscopy(TEM), we studied the coccoid conversion of the Hp strain 88Disolated from a patient suffering from a peptic ulcer. Bacteria weregrown on Colombia blood agar at 37°C under microaerobicconditions. Transmission electron microscopy carried out dailyallowed us to precise the evolution of the fine structure of Hp duringthe coccoid conversion. After 4 days, coccoid forms becamepredominant. Ingrowth of periplasmic space lead to coccoid cells inwhich the parental bacillary forms were still visible, surrounded bythe inner membrane and bent under a torsion created by the globularform of the outer membrane. At day 7, only coccoid forms were seenkeeping a double membrane system, flagella polar membrane andinvagination structures which, in other bacterial genera, are designedto oxidation of toxic components. Subculture of the coccoids provideTEM data which favour the hypothesis that the coccoid forms areable to revert to bacillary dividing forms under certain culturalconditions. A decrease of coccoid diameter and a densification of thecytoplasmic material were shown and, at day 2, dividing bacteriawere seen with an important fraction of dense periplasmic materialalso evidenced during the coccoid conversion. The evolution of theantigenic profiles during coccoid conversion of six Hp strains, wasstudied by western blot using different sera from patientsseropositive for Hp and known to be colonised for at least 6 month.Certain high molecular weight antigenic fractions non detected inbacilli are detected more and more intensely from day 2 to day 7 ofthe coccoid conversion. On the other hand, an antigenic fraction of30 kDa is only detected in the bacillary forms. These results suggestthat coccoid form of Hp may be viable. Both bacilli and coccoidsmay be present in vivo. Coccofds being more immunogenic may bein close contact with mucosal cells while bacilli may be the formsadapted to the transit in the mucus.

DIVERSITY IN METABOLIC-ENZYME PROFILES OFHFLCOBACTER PYLORI. M.F. Go, V. Kapur, D.Y. Graham, J.M.Musser. Depts. ofMedicine and Pathology, V.A. Medical Center andBaylor College ofMedicine, Houston, TX, USA.

H. pylori is strongly linked to peptic ulcer disease and gastric cancer.Research has focussed on genetic, scroepidemiologic, and hostphysiologic assays ofthis bacterium. Few studies have investigated itsmetabolism to identify physiologic factors that may be important for itssurvival in the unique ecological niche ofthe human stomach. Aim:To examine the expression of a panel of metabolic enzymes to assessthose enzymes that may be important in Hp metabolism. Methods: Hpisolates were cutured from gastric biopsies from 73 individuals withDU, GU, or histologic gastritis alone. Isolates were cultured inisosensitest broth supplemented with 10% horse serum at 370C, 12%CO2 for three days. At the time of harvest, all cultures demonstratedrod or spiral morphology on Gram stain and were posifive for urease,catalase, and oxidase. After centrifugation the cells were lysed andeach isolate was examined for the expression of 17 housekeepingenzymes utilizing horizontal starch gel electrophoresis. Reults:Although 17 metabolic wymes normally found in many microbialorganisms were assayed, only six were electrophoreticallydistinguishable. Sixty-three percent of strains had activity for all sixenzyme, but at least one metabolic ezyme activity was absent intwenty-eight (37%/) ofthe strains. Moreover, in seventeen (60%) ofthese twenty-eight strains, there was no glucose 6-phosphatedehydrogenase activity detected. Fiften strains did not have detectableacvity for glutamate dehydrogenase, isocitrate dehydrogenase,indophenol oxidase, nucleoside phosphorylase, or adenylate kinase.Condusions: Compared to most human microbial organisms that havebeen exained,Hp exhibits a large degree ofgenetic heterogeneity inaddition to extensive phenotypic diversity in the expression ofmanycommon metabolic enzymes. The apparnt-absce ofimportanthousekeeping enzymes suggests that Hp has a very unusual biology.

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DISCREPANCIES BETWEEN TEi RESULTS OF DETECTlON OF vacA BY PCR AND CORRELATION WITHANTIMICROBIAL SUSCEPTIBILITY TESTING OF cagA IN HEJICOBACTER PYLORI STRAINS ISOLATEDHEC0BACTERPYLORIUSING E-TEST COMPARED TO FROM PATIENTS WrIH DIFFERENT DISEASES. C. Birac, A.BROTH MICRODILUTION OR DISK DIFFUSION. C.Y. PM, Labigne, H. Lamouliatte, F. M6graud. Laboratoire de Bact6riologie,J.E. ClanTicge, R Reddy, R. Flammil, D .G. E3ran D.Y. G> . Hopital Pellegrin, Bordeaux; Institut Pasteur, Paris, France.VAMC and Baylor College ofMedicine, Houston, TX and Abbott Although several studies have shown that thevacA gene is present

Labs, Abbott Park, IL, USA. in all Helicobacterpylori isolates, it is not always expressed.It would be of interest to be able to determine the VacA status of

The epsilometer agar diffuiion gradient test (E-Test; AB Biodisk, strains (producing or not) using a test as simple as PCR. Recently,Solna, Sweden) was compred to broth microdilution and paper disk Cover et al., reported a correlation between the presence of adi..lidon for the antimic.ob. ... testing ofH.pylopi. A conserved central domain of the vacA gene with the VacA+difluusion for the antimicrobialsusceptiblity testingofH. pylon. A phenotype. In this study, we investigated the usefullness of

collection of 122 clMcal isolates strains ofHp was tested for seivity detemiination of the VacA phenotype using PCR with the primers

to metrodazole, ampicin and clazithromycin All strains were tested described by these authors, and the results were correlated with theon Mueler Hinton Agar supplemnated with 5% sheep blood. Plaes presence of the cagA gene.were incubated at 37 C in microaerophilic atmospher and readings Two hundred strains were tested, 100 isolated from duodenal ulcerwere done after 5 days of incubation according to _re's (DU) patients and 100 from non-ulcer dyspepsia (NUD) patients.The vacA gene was detected by PCR targeting either the 5-end (setinstruction. Strains were considered resistant if the minimum inhibitory N°1) and the mid-portion of the vacA gene (set N°2), and the cagA

concentration(MIC) was >8 lg/imL for metronidazole or ampicillin and gene was detected by using another two sets of primers. In addition,>2 pg/mL for clarithromycn. E-test MICs were easy to interpret. colony hybridization was performed on all strains using the twoMICs deteined by broth microdilution and E-te wer highly vacA PCR products as a probe.

As expected, the vacA gene was detected in all strains byreproduci'ble with replicate resuls being within one 2-fold dilton. The hybridization. Among the 146 cagA positive strains, 112 (76.7%)correlation between MICa by the broth microdilution method and the E- were positive with vacA set NO1 and 44 (30%) were positive with

test was good for clari and amp, with 96.7% and 98.2% respectively the vacA set N°2. Among the 50 cagA negative strains, 32 (64%)being within I and 2 two-fold dilution. However, for metronidazole and 5 (10%) were positive, respectively. All strains which testedonly 80.3% were withiin and 2 two-fiold dilution and 12.3% ofE-test positive for vacA with set N° 2 also tested positive for vacA with setresults did not correate to the broth mod. Discepancis occured N°1.readts did not correlate to dw broth method. Di epames occurred

If one assumes, based on several reports, that there is a highwith E-test results betwee 416 pWiL. Inall caca the orgnuim association between the CagA+ phenotype with the VacA+

tested more rest by the E-test naking it appear that metronidle phenotype, we can conclude that the results obtained with theresisance was more prvalent in a population than it might actaly be. primers proposed do not correlate with the CagA phenotype andDisk diffusion results correlated with the broth microdiludon in al therefore not with the VacA phenotype either. In any case, acases. We conclude that when E-testing for MIC ofmetronidazole of correlation of the results with the expression of the toxin stillH. pylon yields relts in the 4-16 pg/mL range, the MIC should be remains to be dctmined.reevaluated by another method.

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MUTATIONS IN 23S RIBOSOMAL RNA CONFERCLARITHROMYCIN RESISTANCE IN HELICOBACTER PYLORI.J. Versdouic, K. Kibler, S. Small, D.Y. Graham, MRF. Go.Departments ofMedicine, V.A. Medical Center and Baylor Coilege ofMedicine, Houston, TX, USA.

Claithromyc a macrolide trnation inhibitor, has been incorporatedsuccessully into anti-Helicobacterpylori (Hp) combinationantimicrobial regimens for ulcer disease and lymphoma chemothrpywith cure rates exceeding 90%. However, domycin resistanceoccurs in 4% to 5% ofpatment and >15% ofpost-tratmentfailures. Hp isolates were tested in vitro for darthromycin ssceptbilityby microbroth dilution, Kirby-Bauer disk dffision, agar dilution, andthe E-Test. Conserved regions ofthe 23S rRNA gene were PCR-amplified and subjected to cycle DNA sequencng. Purified genomicDNAs oftwenty-four Hp isolates from teny-two patients weranalyze. Two adjacent A-G mutaions c rrin clarihrmycresisance were identified. Mutations were independently confimd byrapid Bsal-mediated retriction digestion of23S rRNA amplicons. Fourclauithromycin-resiant Hp isolats from three diffent patients eachcontained an A-+G transwiton mutation wiin a conserved loop of23SrRNA. Twenty ciomycin- Hp isolates from twentydiffere patiet displayed no polymorphisms in this conserved loop of23S rRNA. One patien yielded sensitive, wild type isolates prior toantinicrobialtetmn an i munt isola afterratment.rep-PCR DNA fir rinting sconimed sig in infection ofthis patient and ion ofthe stance-conferring mutation. Thisevidence sugget that point mutations in 23S rRNA alter the nbosomaltarget ofclaithromy insuch that this antibiotic interacts ls de ielywith Hp nbosomes and allows suvival ofHp mutant dones. This is thefirst report ofa mutat cuing resstance to an ntimicrobialcommonly used ip anti-Hp eatnt and enables rapidmolecular s g ofclaritromyci suseptli.

ELUCIDATION OF HELICOBACTER PYLORI METABOLISM BYRANDOM GENOME SEQUENCING.CL Claytonl, A Tay2. C O'Donnell1 and PA Chalki. 1BiologyDivision, Glaxo Research and Development Ltd. Greenford Road.Greenford, Middlesex, UB6 OHE UK. 2Institute of Molecular andCell Biology, National Universitv of Singapore. 10 Kent RidgeCrescent, Singapore 051 1.

A knowledge of the metabolism and substrate utilisation bv H.plorimight help in the design of new drugs to treat infections. Randomlibraries of H. pylori NCTC 11637 genomic DNA were created inpBluescript vector and clones sequenced automatically. Blast Xanalysis of the sequence data has revealed H.pylori to possesssignificant homologues (p<1.0 x 10-5) of enzymes involved in energymetabolism, biosynthesis and uptake/efflux of molecules. Genesencoding enzymes of anaerobic metabolism such as fumaratereudctase and hydrogenase have been detected as well as pyruvateferredoxin/flavodoxin oxidoreductases which probably play key rolesin the metabolism of pyruvate. Hpylori contained homologues of theelectron transport associated enzymes: NADPH. NADHdehydrogenases and a microaerophilic inducable high affinitv cbterminal oxidase, confirming a respiratory type of metabolism.Catabolic glucokinase, phosphogluconate dehydratase, KDPGAldolase (Entner Doudoroffpathway enzymes) and pentose phosphatepathway/TCA cycle homologues were found. A number ofbiosynthetic gene homologues involved in purine, pyrimidine, aminoacid, porphyrin, fatty acid, lipid and peptidoglycan biosynthesis werealso detected and has enabled pathways to be constructed. Randomgenome sequencing has confirmed previous biochemical studies onH.pylori and has further elucidated the enzymes/pathways involved inthe metabolism of this important pathogen. The detection of Hpylorienzyme homologues provides new perspectives in the physiology ofthe organism and has implications for the active search to developappropriate therapies for this bacterium.

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INFECTION BY A MIXED POPULATION, INCLUDING FORCAGA, OF HELJCOBACTER PYLORI. A van der Ende,E.A.J. Rauws2, M. Fellers, C.J.J. Mulder2, G.N.J. Tytgae, andJ. Dankert'. Academic Medical Center, University ofAmsterdam, 'Dept. of Medical Microbiology, and 2he Dept. ofGastroenterology, Amsterdam, The Netherlands.

Chronic active gastritis was present in eight out of ninemembers of a duodenal ulcer disease family. The subjectshad endoscopic evidence of actual (n = 4) or previous (n = 3)duodenal ulcer. Hehcobacter pylon strains, isolated from eightout of nine members, were investigated for their similarity byPCR based RAPD fingerprinting of the bacterial chromosomalDNA using two different RAPD primers and compared withthe RAPD fingerprints of the isolates of three unrelatedsubjects. It was shown, that the strains from the familymembers are related, but display subtypic variation. Cytotoxinassociated gene A (cagA) as shown by PCR, was onlypresent in 6 of the 8 isolates. Analysis of 10 colonies from theprimary culture plates from the biopsy specimens of eachsubject by PCR fingerprinting demonstrated that all subjects,except one, harboured at least 2 and up to 4 different H.pylon subtypes. Three subjects harboured a mixture of cagApositive and cagA negative H. pylon subtypes as shown byPCR. Four other subjects harboured a mixture of only cagApositive or only cagA negative subtypes. Southem blottingand hybridization using a cagA probe confirmed these results.The proportion of cagA negative colonies on the primaryculture plates was determined by colony hybridization andvaried between patients from 10 to 100%. These resultsindicate that genotypic comparison based upon a pure cultureof one of the colonies from the primary culture plate from thebiopsy specimen is questionable. The coexistence of bothcagA positive and negative H. pylon within the sameindividual, suggests little selection pressure on cagA.

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A NEW TYPING METHOD FOR HELICOBACTER PYLORI: ENDLABELLING OF ENDONUCLEASE DIGESTED DNAFRAGMENTS. N.E.M. van Doom'. L. van Kempen'. F. Namavar',E.P. s'an Rees2. R.H.A. Plasterk3 and J. de Graaff'. Departments ofMedical Microbiology' and Celbiology & Immunology2, VrijeUniversiteit and The Netherlands Cancer Institute3. Amsterdam, TheNetherlands.

Variation amongst strains of Helicobacter pylori has been examined byseveral DNA based methods including restriction endonuclease analysisof whole chromosomal DNA (REA), Southern blot hybridization witha rihosomal DNA probe (ribotyping) and Polymerase Chain Reactionwith random primers (RAPD). However these technics have somelimitations. Although the REA method gives a large number of bands,comparison of the bandpatterns is sometimes difficult due to the lowresolution of the agarose gel. Furthermore the amount of bands that arecompared is low (ribotyping) or differ in intensity (RAPD). We presenta new typing method, wich has already been applied to ActinobacillusactinonZvcetemcomitans, that overcomes these difficulties. Totalgenomic DNA of H. pylori was isolated and digested with therestriction endonuclease BglII. DNA fragments were labelled with [a--PldATP using Klenow DNA polymerase and the labelled fragmentswere separated on a 6% polyacrylamide gel with 8M urea (sequencinggel). With this method aproximately 25 bands, 100-400 bases in lenght,were suitable for co)mparison, due to the high resolution of the gel. Allisolates tested had a few bands in common. In case of A.actinornvcetrencomlitaflS the endlabelling method proved to be morediscriminating than REA and ribotyping. A large pannel of H. pylonisolates is now under study in cluster analysis to characterizerelationships among these isolates and wether there is an correlationwith respect to clinical symptomatology.

271

HELICOBACTER PYLORI (Hp) INFECTION IN ASYMPTOMATICSARDINIAN SHEPHERDSM.P. Dore, D. Vaira, L. Cugia, A. Atzei, N. Figura, G. Pisanu, G.Massarelli, G. Realdi. Istituto di Clinica Medica and AnatomiaPatologica, University of Sassari and University of Bologna and Siena,Italy.

It has been suggested that Hp infection could be a zoonosis on the basisof its isolation in the stomach of pigs and monkeys and on the basis ofa higher prevalence of the infection, compared to control population, incategories at risk, such as veterinary surgeons, butchers andslaughtery-house workers. We undertook a study of 150 consecutivesubjects and their families living in the North of Sardinia, whose workinvolved close contact with farm animals. All subjects wereasymptomatic. A questionnaire with social and clinical data was filled infor each subjects and a blood sample was taken at place of work, afterinformed consent was obtained. Sera were tested for IgG against Hp byELISA (sonicated antigens, titres determined at optical density 470nm). Ten subjects were also investigated for Cag-A systemic antibodyresponse by immunoblotting. Upper gastrointestinal endoscopy wasperformed in 40 subjects. Reult..: Antibody to Hp was detected in 93%of the population studied, reaching 98% when shepherds alone wereconsidered (see table).Subjects positive for IgG anti-Hp and tested byimmunoblotting also showed a positive reaction for antibody to Cag-Aprotein. Acute or chronic gastritis was found on histologicalexamination in all subjects submitted to endoscopy. All these subjectsalso showed Hp on microscopy and a positive CP test, with very rapidreaction (within 3 seconds), suggesting a strong bacterial charge in thestomach. Prevalence of infection in family members was similar to thatof our control population of dyspeptic patients (73%).

NO Hp+ (%) PDyspeptic patients 710 497 70 --Shepherds and family members 150 140 93 0.03Shepherds alone 120 118 98 0.02Family members not in contactwith animals 30 22 73 n.s.Our study showed that subjects in direct contact with farm animals maybe considered a category at risk, given the significantly higherprevalence of infection, and would further suggest that Hp infection isan antropozoonosis, where the enviroment could be the carrier betweenman and animals.

272

DETERMINATION OF HELICOBACTER PYLORI EXPOSURE(HPE),SERUM GASTRIN (G) AND GASTRIN RELEASINGPEPTIDE (GRP) IN PATIENTS WITH COLORECTALCARCINOMA (CC). A. Elfant, P. Saniour, L. Mdndez,T. Spiegel, H. Fried, B. Howe, T. O'Dorisio, N.Marcon, S. Peikin. UMDNJ-Robert Wood JohnsonMedical School,Camden, New Jersey, Ohio StateUniversity, Columbus, Ohio, The WellesleyHospital-Toronto, Ontario.B -okar:n G is a tumor promoter for CC. CC andHPE are associated with elevated G in somestudies but the cause of the increase is notknown. In one study (Lambert) HPE was associatedwith colon polyps. In the present study, wereport the largest series examining HPE, serum G,GRP and CC. ethods: Academic referral centers inCanada and the USA recruited 80 patients with CCas well as matched controls. G and GRP (pg/ml)were measured by RIA. HPE was assessed byserology. Values are reported as mean ± standarderror of the mean. Reaultsi 42.5% of CC patientswere HP+ compared to 40.6% of controls (NS). 54%of Canadian subjects were HP+ compared to 31.7%of US subjects (X2=6.48, p<0.05). Histologicgrade and tumor stage were not affected by HPE. Gand GRP values in the CC (67.5 ± 11.8,229 ± 21.4)and control groups (81.5 ± 15.5,234.2 ± 18.5)were not significantly different. G and GRPvalues were also not significantly different inthe HP+ (73.6 ± 11.5,239 ± 18.8) and HP-(73.6 ±11.5,221.7 ± 24.5) subjects. Correlationcoefficients of G vs GRP in the CC group (r=0.1),the HP+ group (n=42,r=.12), the HP+ group withelevated G (G > 122 pg/ml,n=5,r=.03) and thetotal group (r=Q.02) were not significant.Cg..iilgn: No apparent association existsbetween HPE and CC. G and GRP are not elevated inpatients with CC, nor in subjects who are HP+compared to controls. GRP is not the cause of theelevated G observered in some HP+ subjects. Theprevalence of HPE is significantly greater inCanada than the USA.

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