INFECTION AND IMMUNITY,0019-9567/98/$04.0010

Nov. 1998, p. 5555–5560 Vol. 66, No. 11

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Diversity in Protein Synthesis and Viability of Helicobacter pyloriCoccoid Forms in Response to Various Stimuli

HIROMOTO MIZOGUCHI,1* TOSHIO FUJIOKA,1 KENJI KISHI,1 AKIRA NISHIZONO,2

REIJI KODAMA,1 AND MASARU NASU1

Second Department of Internal Medicine1 and Department of Infectious Disease Control,2

Oita Medical University, Hasama-machi, Oita 879-5593, Japan

Received 29 April 1998/Returned for modification 17 June 1998/Accepted 13 August 1998

The viability of the coccoid forms of Helicobacter pylori was evaluated by assessing protein synthesis.Metabolic labeling studies showed the synthesis of proteins and the specific protein profiles of H. pyloricoccoids produced under various conditions. Harsh conditions such as aerobiosis and starvation (lack of horseserum) in the culture did not affect the synthesis of proteins in the coccoids. Lowering of the pH to that ofgastric secretions induced expression of several proteins in the coccoids. However, the coccoids produced underprolonged microaerobic conditions exhibited a profile of acid stress-induced protein expression different fromthat induced by aerobiosis or starvation. Our data suggest that coccoid H. pylori exhibits diversity in viabilityfollowing exposure to different stresses and that the response to acid stress of coccoid H. pylori could beinvolved in infection of the host stomach.

Morphological conversion from spiral to coccoid forms hasbeen described for Helicobacter pylori cultured under severalsuboptimal conditions. These conditions include aerobiosis (4,6), alkaline pH (4, 6, 15), high temperature (22), extendedincubation (6), or treatment with a proton pump inhibitor (6)or antibiotics (3). This coccal form conversion phenomenon,which has been thought to result in a viable but nonculturableform of the bacterium (3), is not exclusive to H. pylori, as it iscommon for other enteric pathogens (16, 20, 21). Several in-vestigators have suggested that the coccoidal form of H. pylorirepresents a degenerative form with no infectious capability (7,9, 17). Others have reported that the coccoid form retains aweak metabolic activity (3, 23), important structural compo-nents (2, 13), and pathogenicity (26). Successful in vitro cultureof coccoid forms, however, has not as yet been established.Therefore, whether the coccoid form is in the process of de-generation or in the dormant stage prior to subsequent bacte-rial transmission remains an open question. Recently, success-ful infection with coccoid forms of H. pylori or Campylobacterjejuni in animal models has been reported (5, 16). These find-ings have highlighted the possible role of the coccoid forms intransmission of infection and morphological conversion of coc-coids to the spiral form. The acidity of gastric secretions isconsidered to be harmful to most organisms in the gastricenvironment. A recent study, however, suggested that acidity isessential for the establishment of colonization of the stomachby H. pylori, as brief exposure of H. pylori to a low pH increasesthe expression of heat shock proteins (hsp’s), which enhancethe attachment of the organism to the gastric epithelium(14).

The present studies were performed to investigate whethercoccoid forms of H. pylori had the capacity to synthesize pro-teins and, if so, whether coccoids produced by various proce-dures exhibited diversity in protein synthesis levels or viability.We also examined the potential morphological conversion ofthe coccoid to the spiral form following acid exposure. Our

results demonstrated protein synthesis and induction of spe-cific proteins by acid shock in the coccoid form. Under unfa-vorable conditions, this metabolism appears to be more stablein the coccoid form than in the spiral form.

Morphological change and culturability of coccoid forms. H.pylori ATCC 43504 was used in our studies. The frozen bacte-rial suspension was thawed from storage at 280°C andsmeared onto 7% horse blood–agar plates containing Mueller-Hinton agar (Becton Dickinson, Cockeysville, Md.). Cultureswere incubated under microaerobic conditions in anaerobicjars (Campypak System; BBL Microbiology Systems) with highhumidity at 37°C for 3 days. Subsequently, a portion of theculture was harvested and resuspended in 8 ml of liquid bru-cella broth (Difco, Detroit, Mich.) supplemented with 10%(vol/vol) horse serum (Gibco, Grand Island, N.Y.) and incu-bated in a microaerobic environment at 37°C for 2 days on arotary shaker.

To induce coccoid forms via various environmental condi-tions and subsequently compare their biological features, weemployed different culture conditions, such as aerobiosis, pro-longed culture, and nutrient starvation. Aerobiosis was per-formed as follows: when bacteria became subconfluent in liq-uid microaerobic culture, the culture was transferred into anaerobic atmosphere and incubated for a further 3 or 7 dayswith agitation (200 rpm). Prolonged H. pylori culture was per-formed in liquid medium for up to 20 days at 37°C, withoutsupplementation with fresh medium, under microaerobic con-ditions, and with the Campypak being changed every 3 days.For nutrient starvation conditions, bacteria in exponentialgrowth were washed three times with phosphate-buffered sa-line (PBS) (pH 7.4) and then incubated in PBS at 4°C for 3months without agitation. The culturability of coccoid popula-tions was determined by plating 100 ml of a 1- or 10-folddilution of the broth culture (adjusted to a 5 McFarland con-centration) in PBS onto blood agar plates. Plates were incu-bated at 37°C under microaerobic conditions for 3 or 7 days,respectively. Coccoid populations produced under all of theconditions assayed in this study lacked colony-forming ability.No spiral forms were detected by light microscopy in the ran-domly chosen fields of coccoid populations induced by any

* Corresponding author. Mailing address: Second Department ofInternal Medicine, Oita Medical University, Hasama-machi, Oita 879-5593, Japan. Phone: 81-97-549-4411, ext. 5804. Fax: 81-97-549-4245.

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condition used. In contrast, the 3-day microaerobic culturecontaining .95% spiral forms exhibited 108 to 109 CFU/ml.

In an attempt to clarify whether different suboptimal cultureconditions induced morphologically different coccoid forms,we compared coccoid forms induced by aerobic culture andprolonged culture with respect to ultrastructural morphology.The morphology of H. pylori incubated under various condi-

tions was determined by scanning electron microscopy. For thispurpose, samples of each culture were placed on a 0.4-mm-pore-size polycarbonate filter (Isopore Track-Etched Mem-brane Filter; Millipore, Bedford, Mass.) and fixed in 2% glu-taraldehyde in PBS for 1 h. The samples were then dehydratedfor 10 min in serial concentrations of ethyl alcohol (50, 70, 80,90, 95, and 100%) and t-butyl alcohol and subjected to critical-

FIG. 1. Morphology of coccoid forms of strain ATCC 43504 produced under various conditions, as observed by scanning electron microscopy. (A) Coexistence ofspiral (S) and coccoid forms in 5-day microaerobic culture; (B) complete spherical coccoid (C), U-shaped (U), doughnut-shaped (O), and degraded (E) forms producedby 3-day aerobiosis; (C) coccoid forms and degraded forms produced by 7-day aerobiosis; (D) coccoid forms produced by 20-day microaerobic culture.

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point drying and gold-palladium coating. The samples wereexamined by scanning electron microscopy (S 800 model elec-tron microscope; Hitachi Co., Tokyo, Japan).

Figure 1A shows coexistence of spiral and coccoid formsafter a 5-day microaerobic culture. After a 3-day aerobic cul-ture (Fig. 1B), almost all organisms were spherical and nospiral forms were observed; a few U-shaped and doughnut-shaped forms (the latter are thought to be an intermediatestate between U-shaped and completely spherical coccoid

forms) and degraded forms were also present. After a 7-dayaerobic culture (Fig. 1C), most organisms appeared degradedalthough a small subset maintained spherical forms. Prolongedculture for 20 days generated coccoid forms exclusively (Fig. 1D).Coccoids from prolonged culture exhibited a rougher surfacethan that of coccoids from a 3-day aerobiosis; the latter resembledcoccoids from a 3-day microaerobic culture. Furthermore, mostof the coccoids from prolonged culture appeared to fuse andaggregate, probably due to cell wall degeneration.

FIG. 1—Continued.

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Protein synthesis in coccoid forms. To determine whetherthe coccoid form was viable, trans-35S metabolic labeling of H.pylori with methionine and cysteine was performed, followedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Spiral or coccoid H. pylori cells (109 bacteria)were suspended in 300 ml of serum- and methionine-freeRPMI 1640 medium and then plated in 24-well plates. Thebacteria were metabolically labeled in the medium with 300mCi of trans-35S per ml at 37°C for 1 or 5 h under one of severalconditions: in a microaerobic or aerobic environment, with orwithout 2% horse serum, or in medium at pH 2.0, 3.5, 5.0, or7.4. 35S-labeled bacteria were washed three times with PBS andthen lysed in 100 ml of Laemmli sample buffer containing 5%(vol/vol) b-mercaptoethanol, 2% (wt/vol) SDS, 10% (vol/vol)glycerol, and 125 mM Tris-HCl (pH 6.8). Samples were heatedat 100°C for 5 min, and 50 ml of each sample was subjected toSDS-PAGE on a 9% acrylamide slab gel (27). The gels weredried and processed for fluorography using Kodak XAR film.

As shown in Fig. 2, H. pylori coccoids subjected to a 3-dayaerobic culture synthesized proteins. This synthetic ability wasmaintained in coccoids subjected to a 7-day aerobic culture,but the level of synthesized proteins in coccoids from the 7-dayaerobic culture was much reduced, apparently due to the de-generation of coccoid forms (Fig. 1C). Coccoid forms pro-duced ,1% of the amount of proteins synthesized by spiralforms. Furthermore, the protein profiles of coccoid and spiralforms were apparently different. Restoring the 3-day aerobi-cally cultured coccoid forms to adequate culture conditions for3 days did not lead to morphological conversion of coccoids orrestoration of the pattern of synthesized proteins.

The demonstration of DNA synthesis (by incorporation of5-bromodeoxyuridine) (3) and preservation of intact cellularstructures and certain enzymes (2, 13) in coccoid forms haveconfirmed that coccoid forms are viable but in a nonculturablestate. This viability appears to be stable under optimal condi-tions since DNA synthesis was observed in coccoids after 3months of storage in physiological saline at 4°C (3). Our datafrom the metabolic labeling study support this finding (see Fig.5B).

Stability of protein synthesis in coccoid forms. H. pylorirequires a microaerobic atmosphere and serum for in vitropropagation. We investigated the effect of these factors onmetabolism in spiral and coccoid forms of H. pylori. Figure 3demonstrates that no difference in the pattern or intensity oflabeled proteins in coccoids from microaerobic and aerobiccultures was detected by SDS-PAGE. However, the amount ofprotein produced from spiral forms markedly decreased underaerobic conditions. On the other hand, the addition of horseserum for up to 5 h during labeling did not alter the profile oramount of synthesized protein in either spiral or coccoidforms. The long-term survival of this spherical shape, evenunder harsh conditions outside the host stomach, is indicatedby the lack of inhibition of protein synthesis in coccoids underaerobic conditions (Fig. 3) and conservation of the ability tosynthesize proteins under starvation conditions for at least 3months (see Fig. 5B). However, coccoids readily lose viabilityunder high-temperature (37°C) storage conditions while at lowtemperatures (,15°C) coccoids can maintain viability for atleast 3 months (11, 22). It is thus likely that storage tempera-ture affects the viability of coccoids.FIG. 2. Protein synthesis in coccoid forms of H. pylori. Coccoid forms were

produced by 3-day (d3) and 7-day (d7) aerobic cultures. Each form was 35Slabeled for 5 h under microaerobic conditions. Samples of spiral forms werediluted to 1-, 10-, and 100-fold and subjected to SDS-PAGE. Samples of coccoidforms were undiluted.

FIG. 3. Effect of horse serum or aerobic conditions on protein synthesis in H.pylori. Each form was 35S labeled for 5 h in the presence (1) or absence (2) of2% horse serum (HS) under microaerobic conditions (lanes M) or aerobicconditions (lanes A).

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Acid shock induces the expression of proteins in coccoidforms. The acid nature of gastric secretions is a critical barrierin preventing infections in the stomach. To examine the re-sponse of H. pylori to acid stress under the condition withouturea, we exposed spiral forms and coccoids from 3-day aerobicculture to low-pH-adjusted media during metabolic labeling.The exposure of spiral forms to a pH of 5.0 enhanced theproduction of proteins with molecular masses of 68, 40, and 30kDa. In contrast, protein synthesis was diminished when pHwas lowered further to 3.5 or 2.0. In the coccoid forms, low-ering the pH to 3.5 induced expression of 90- and 68-kDaproteins and exposure to pH 2.0 induced a 35-kDa protein(Fig. 4).

In another series of experiments, we examined the stabilityof coccoids from a 3-day aerobic culture under adverse condi-tions by storing the coccoids for 20 days at 4°C in distilledwater. Exposure of this population of coccoids to pH 2.0 in-duced the same profile of proteins as that observed in coccoidsfrom a 3-day aerobic culture. However, the viability of 3-dayaerobically cultured coccoids was completely lost after incuba-tion in distilled water at room temperature (data not shown).

It is conceivable that surface urease activity enables H. pylorito survive in the gastric environment, as a urease-deficientmutant of H. pylori lacks the ability to colonize the nude mousestomach (18, 25). Since coccoid forms of H. pylori have nourease activity (13, 19), their survival in the stomach in such alow-pH environment is unlikely. However, our data show thatacid shock, even for a period of 1 h, did not inhibit the pro-duction of proteins but, conversely, induced the synthesis of

proteins (Fig. 4 and 5), which might be hsp’s (14). Members ofthe hsp family function as molecular chaperones and protectintracellular proteins from denaturation (8, 12) but are alsolikely to be relevant to cell cycle progression (24) and rear-rangement of cytoskeletal proteins (10). The preservation ofpolyphosphate granules in coccoid forms, probably as energy,supports the possibility of subsequent transformation of coc-coids into spiral forms (3). These findings suggest that gastricacidity is an essential factor for initiation of morphologicalconversion and regrowth of coccoid H. pylori. However, acidshock alone was insufficient for restoration of either morphol-ogy or growth of coccoid forms in vitro. If acid stress is aninitiator for conversion to the spiral form, additional stimuliand/or appropriate circumstances might be necessary for suchconversion.

Differential profiles and amount of proteins synthesized bycoccoid forms induced under various conditions. Many condi-tions are known to induce the coccoid form; however, little isknown about variations in biological features among coccoidsproduced by different procedures. To compare the character-istics of metabolism in coccoid forms produced by variousprocedures, we employed three different culture conditions:aerobiosis, prolonged culture, and long-term starvation at lowtemperatures. All coccoid populations produced under theseconditions exhibited protein-synthetic ability and responsive-ness to acid shock (by expression of stress proteins) despite theabsence of colony-forming ability.

In coccoids produced by long-term starvation, the pattern ofboth constitutively synthesized and acid-induced proteins (35-,68-, and 90-kDa proteins) was similar to that in coccoids fromthe 3-day aerobic culture. However, the amount of proteinssynthesized (after starvation) was much greater than in the3-day aerobic culture or prolonged culture (Fig. 4 and 5). In

FIG. 4. Induction of proteins in coccoid forms of H. pylori by acid shock.Spiral and coccoid forms produced by a 3-day aerobic culture were 35S labeledfor 1 h during exposure to acid at the indicated pH in serum- and methionine-free RPMI 1640 medium under microaerobic conditions. Proteins induced byexposure of coccoids to pH 3.5 and 2.0 are indicated by arrowheads.

FIG. 5. Comparison of profiles of proteins induced by acid exposure of coc-coid forms produced by a 20-day microaerobic culture (A) and a 3-monthincubation in PBS at 4°C (B). Coccoids were 35S labeled for 1 h in methionine-free RPMI 1640 medium under microaerobic conditions at pH 7.4 or 2.0.

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coccoids obtained from prolonged culture, only one protein of55 kDa was induced by exposure to pH 2 (Fig. 5A). Furtherincubation of coccoids from 20 to 40 days was accompanied bya loss of responsiveness to acid shock, although small amountsof proteins were still produced.

Bode et al. (3) reported that the diameter of the amoxicillin-induced coccoid is markedly smaller than that of coccoidsinduced by bismuth agents or erythromycin. Sequential alter-ations of the activity of several enzymes in the coccoid formduring extended culture have been described recently by Huaand Ho (13). These investigators showed that the activity ofsome enzymes in coccoids is fully conserved for up to 35 daysin culture, with levels as high as those observed in the spiralform, while others gradually decrease after 21 days in culture,without alteration at the DNA level. Our results showed dif-ferences in the profiles of acid-induced proteins between coc-coid forms produced by aerobiosis and prolonged culture (Fig.4 and 5). This difference in biological response may be partlyrelated to slight differences in shape between coccoids fromprolonged culture with rough surfaces and those from aerobicculture with smooth surfaces (Fig. 1). This minimal change inmorphology in coccoids in prolonged culture may mark theonset of degeneration. Thus, it is likely that coccoids exhibitvariation in viability. Recently, successful colonization by anonculturable population of mostly coccoid H. pylori cells in amouse model was reported (1, 5); however, Eaton and cowork-ers (9) could not achieve this in a gnotobiotic piglet model.These contradictory results may reflect the differential viabilityof coccoids, although strain diversity, contamination with spiralforms, and differential host species specificities should be con-sidered.

Based on the present results, we speculate that the proteinsynthesis response of coccoid H. pylori to acid stress plays animportant role in triggering conversion from the coccoid to thespiral form and also that diversity in the viability of coccoidscorrelates with their ability to cause infection. Together withfuture in vivo studies using animal models, our in vitro ap-proach evaluating the viability of coccoids may help distinguish“reversible” coccoids, capable of transition to the spiral form,from “irreversible” coccoids, which progress to cell death.

We thank K. Arita and M. Kimoto for technical assistance. We alsothank F. G. Issa, Department of Medicine, University of Sydney, Syd-ney, Australia, for careful reading and editing of the manuscript.

REFERENCES

1. Aleljung, P., H. O. Nilsson, X. Wang, P. Nyberg, T. Morner, I. Warsame, andT. Wadstrom. 1996. Gastrointestinal colonisation of BALB/cA mice by Hel-icobacter pylori monitored by heparin magnetic separation. FEMS Immunol.Med. Microbiol. 13:303–309.

2. Benaissa, M., P. Babin, N. Quellard, L. Pezennec, Y. Cenatiempo, and J. L.Fauchere. 1996. Changes in Helicobacter pylori ultrastructure and antigensduring conversion from the bacillary to the coccoid form. Infect. Immun.64:2331–2335.

3. Bode, G., F. Mauch, and P. Malfertheiner. 1993. The coccoid forms ofHelicobacter pylori. Criteria for their viability. Epidemiol. Infect. 111:483–490.

4. Catrenich, C. E., and K. M. Makin. 1991. Characterization of the morpho-logic conversion of Helicobacter pylori from bacillary to coccoid forms. Scand.

J. Gastroenterol. 26(Suppl. 181):58–64.5. Cellini, L., N. Allocati, D. Angelucci, T. Iezzi, E. Di Campli, L. Marzio, and

B. Dainelli. 1994. Coccoid Helicobacter pylori not culturable in vitro reverts inmice. Microbiol. Immunol. 38:843–850.

6. Cellini, L., N. Allocati, E. Di Campli, and B. Dainelli. 1994. Helicobacterpylori: a fickle germ. Microbiol. Immunol. 38:25–30.

7. Cole, S. P., D. Cirillo, M. F. Kagnoff, D. G. Guiney, and L. Eckmann. 1997.Coccoid and spiral Helicobacter pylori differ in their abilities to adhere togastric epithelial cells and induce interleukin-8 secretion. Infect. Immun.65:843–846.

8. Deshaies, R. B., B. Koch, M. Werner-Wasburne, E. Craig, and R. Shekman.1988. A subfamily of stress proteins facilitates translocation of secretory andmitochondrial precursor polypeptides. Nature (London) 332:800–805.

9. Eaton, K. A., C. E. Catrenich, K. M. Makin, and S. Krakowka. 1995. Viru-lence of coccoid and bacillary forms of Helicobacter pylori in gnotobioticpiglets. J. Infect. Dis. 171:459–462.

10. Gao, Y., I. E. Vainberg, R. L. Chow, and N. J. Cowan. 1993. Two cofactorsand cytoplasmic chaperonin are required for the folding of a- and b-tubulin.Mol. Cell. Biol. 13:2478–2485.

11. Gribbon, L. T., and M. R. Barer. 1995. Oxidative metabolism in non-cultur-able Helicobacter pylori and Vibrio vulnificus cells studied by substrate en-hanced tetrazolium reduction and digital image processing. Appl. Environ.Microbiol. 61:3379–3384.

12. Hendrick, J. P., and F. U. Hartl. 1993. Program of cellular biochemistry andbiophysics. Annu. Rev. Biochem. 62:349–384.

13. Hua, J., and B. Ho. 1996. Is the coccoid form of Helicobacter pylori viable?Microbios 87:103–112.

14. Huesca, M., S. Borgia, P. Hoffman, and C. A. Lingwood. 1996. Acid pHchanges receptor binding specificity of Helicobacter pylori: a binary adhesionmodel in which surface heat shock (stress) proteins mediate sulfatide rec-ognition in gastric colonization. Infect. Immun. 64:2643–2648.

15. Jones, D. M., and A. Curry. 1991. The genesis of coccal forms of Helicobac-ter, p. 29–37. In P. Malfertheiner and H. Ditschuneit (ed.), Gastritis andpeptic ulcer. Springer, Berlin, Germany.

16. Jones, D. M., E. M. Sutcliffe, and A. Curry. 1991. Recovery of viable butnon-culturable Campylobacter jejuni. J. Gen. Microbiol. 137:2477–2482.

17. Kusters, J. G., M. M. Gerrits, J. A. G. Van Strijp, and C. M. J. E. Vanden-broucke-Grauls. 1997. Coccoid forms of Helicobacter pylori are the morpho-logic manifestation of cell death. Infect. Immun. 65:3672–3679.

18. Marshall, B. J., L. J. Barret, C. Prakash, R. W. McCallum, and R. L.Guerrant. 1990. Urea protects Helicobacter (Campylobacter) pylori from thebacterial effect of acid. Gastroenterology 99:697–702.

19. Nilius, M., A. Strohle, G. Bode, and P. Malfertheiner. 1993. Coccoid-likeforms of Helicobacter pylori. Enzyme activity and antigenicity. Zentbl. Bak-teriol. 280:259–272.

20. Perez-Rosas, N., and T. C. Hazen. 1989. In situ survival of Vibrio cholerae andEscherichia coli in a tropical rain forest watershed. Appl. Environ. Microbiol.55:495–499.

21. Roszak, D. B., D. J. Grimes, and R. R. Colwell. 1984. Viable but non-recoverable stage of Salmonella enteritidis in aquatic system. Can. J. Micro-biol. 30:334–338.

22. Shahamat, M., U. Mai, C. Paszuko-Kolva, M. Kessel, and R. R. Colwell.1993. Use of radioautography to assess viability of Helicobacter pylori inwater. Appl. Environ. Microbiol. 59:1231–1235.

23. Sorberg, M., M. Nilsson, H. Hanberger, and L. E. Nilsson. 1996. Morpho-logic conversion of Helicobacter pylori from bacillary to coccoid form. Eur.J. Clin. Microbiol. Infect. Dis. 15:216–219.

24. Taira, T., T. Narita, S. M. Iguchi-Ariga, and H. Ariga. 1997. A novel G1specific enhancer identified in the human heat shock protein 70 gene. Nu-cleic Acids Res. 25:1975–1983.

25. Tsuda, M., M. Karita, M. G. Morshed, K. Okita, and T. Nakazawa. 1994. Aurease-negative mutant of Helicobacter pylori constructed by allelic exchangemutagenesis lacks the ability to colonize the nude mouse stomach. Infect.Immun. 62:3586–3589.

26. Vijayakumari, S., M. M. Khin, B. Jiang, and B. Ho. 1995. The pathogenicrole of the coccoid form of Helicobacter pylori. Cytobios 82:251–260.

27. Yokota, K., Y. Hirai, M. Haqui, S. Hayashi, H. Isogai, T. Sugiyama, E.Nagamachi, Y. Tsukada, N. Fujii, and K. Oguma. 1994. Heat shock proteinproduced by Helicobacter pylori. Microbiol. Immunol. 38:403–405.

Editor: E. I. Tuomanen

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