Original Articles Open Access
Copyright ©The Author(s) 2001. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 15, 2001; 7(2): 254-258
Published online Apr 15, 2001. doi: 10.3748/wjg.v7.i2.254
Virulence and potential pathogenicity of coccoid Helicobacter pylori induced by antibiotics
Fei-Fei She, Dong-Hui Su, Jian-Yin Lin, Lin-Ying Zhou
Fei-Fei She, Dong-Hui Su, Department of Microbiology, Fujian Medical University, Fuzhou 350004, Fujian Province, China
Jian-Yin Lin, Department of Molecular Medicine, Fujian Medical University, Fuzhou 350004, Fujian Province, China
Lin-Ying Zhou, Laboratory of Electron Microscope, Fujian Medical University, Fuzhou 350004, Fujian Province, China
Fei-Fei She, graduated from Fujian Medical University as a postgraduate in 1991, now associate professor of microbiology and immunology, specialized in molecular biology of pathogen, having 15 papers published.
Author contributions: All authors contributed equally to the work.
Supported by the Natural Science Foundation of Fujian Province, China, No. 95A003
Correspondence to: Fei-Fei She, Department of Microbiology, Fujian Medical University, Fuzhou 350004, Fujian Province, China
Telephone: +86-591-3569309
Received: November 15, 2000
Revised: February 22, 2000
Accepted: December 30, 2000
Published online: April 15, 2001


AIM: To explore the virulence and the potential pathogenicity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to antibiotic.

METHODS: Three strains of H. pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid from by exposure to metronidazole. Both spiral and coccoid form of H. pylori were tested for the urease activity, the adherence to Hep 2 cells and the vacuolating cytotoxicity to Hela cells, and the differences of the protein were analysed by SDS-PAGE and Western blot. The mutation of the genes including ureA, ureB, hpaA, vacA and cagA, related with virulence, was detected by means of PCR and PCR-SSCP.

RESULTS: In the coccoid H. pylori, the urease activity, the adherence to Hep 2 cells and the vacuolating cytotoxicity to Hela cells alldecreased. In strain F44, the rate and index of adherence reduced from 70.0% ± 5.3% to 33% ± 5.1% and from 2.6 ± 0.4 to 0.96 ± 0.3 (P < 0. 01), respectively. The invasion of coccoid H. pylori into Hep 2 cell could be seen under electronmicroscope. SDS-PAGE showed that the content of the protein with the molecular weight over Mr 74000 decreased, and the hybriditional signal in band Mr 125000 weakened, while the band Mr 110000 and Mr 63000 strengthened in coccoid H.pylori as shown in Western blot. The results of PCR were all positive, and PCR-SSCP indicated that there may exist the point mutation in gene hpaA or vacA.

CONCLUSION: The virulence and the proteins with molecular weight over Mr 74000 in coccoid H.pylori decrease, but no deletion exists in amplification fragments from ureA, ureB, hpaA, vacA and cagA genes, suggesting that coccoid H.pylori may have potential pathogenicity.

Key Words: Helicobacter pylori/drug, Helicobacter pylori/pathogenicity, antibiotics/pharmacology, polymerase chain reaction


Helicobacter pylori is an important pathogen that causes chronic gastritis and peptic ulcer and may be a risk factor for gastric carcinoma[1-9]. The organism exists in two forms, a spiral form and a coccoid form. The coccoid H.pylori, which can be induced by increased oxygen tension, extended incubation and exposured to antibiotics[10-14], is nonculturable but alive [12,15-17], and could be induced to revert to a virulent spiral form in vivo[11]. Therefore, the coccoid H. pylori has been suspected to play a role in the transmission of the bacteria and to be partly responsible for recrudescence of infection after antimicrobiol treatment. However, the pathogenesis of coccoid H.pylori remains unclear. In this study, three strains of coccoid H.pylori transformed from spiral form by exposure to metronidazole, were tested for the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxin activities to Hela cells, the differences of the protein were analysed by SDS-PAGE and Western blot, and mutation of the genes involving ureA, ureB, hpaA, vacA and cagA was detected by means of PCR and PCR-SSCP, in order to explore the virulence and the potential pathogenicity of coccoid H.pylori.

Bacterial strains

Three strains (F44, F45 and F49 ) of H.pylori were isolated from gastric biopsy specimens of confirmed peptic ulcer patients. The isolates were spiral shape, positive for catalase, oxidase, urease, and cagA and vacA gene. Stock cultures were maintained in defatted milk at -80 °C.


The Hep-2 cells and Hela cells were maintained in 1640 medium supplemented with 10% fetal calf serum, 200 IU/mL penicillin and 50 μg/mL streptomycin at 37 °C in 5% CO2-95% air, and recultivated once or twice a week.

Cultivation of H.pylori and induction of coccoid forms

The stored strains of H.pylori were cultivated on Brucella agar with 5% sheep blood at 37 °C for 2-3 days under microaerophilic conditions (5% O2; 10% CO2; 85% N2). After subculturing, the bacteria were harvested and suspended in Brucella broth with 10% fetal calf serum, and the suspension was divided into two parts, one as spiral H.pylori, the other added with metronidazole at a concentration of 1/2 of MIC values (MIC = 32 mg/L-64 mg/L) at 37 °C under the microaerophilic conditions for a few days until coccoid forms reaching 100%(about 2-3 days).

Examination of urease activity

According to the manufacturer’s instructions of the kit detecting fastly urease activity (Sanqiang Company), the suspension of H.pylori (1010 cfu/mL) was added into the testing well in volume of 5 uL, and on the basis of reacting colors, the urease activity was divided into five grades (“+++ +”“+++”, ”++”,”+”and “-”).

Assay for adherence to Hep-2 cells

To assay bacterial adherence, Hep-2 cells were grown to confluence on coverslips in culture flask, and the suspension of H.pylori (108 cfu/mL) were added in a total volume of 0.5mL for Hep-2 cultures and allowed to adhere for 3.5 h at 37 °C in 5% CO2 95% air. Cultures were washed and stained with Wright-Giemsa stain, and both the amount of cells adhereed by bacteria and bacteria adhereing to cells were counted among one hundred cells under the light microscope. In addition, the culture with strain F44 was scraped using a glass club, centrifuged, and the pellet was embedded in Epoxy 618, then the ultrathin sections were cut and examined under a Hu-12A transmission electron microscope.

Assay for cytotoxicity to Hela cells

To assay vacuolating cytotoxicity of H.pylori, Hela cells were grown to confluence in 96-well plates (2 × 104 cells/well at the time of infection), the suspension of bacteria were swung, centrifuged, and the cell-free supernatants were concentrated 20-fold using polyethylene glycol Mr 20000, then sterilized by passage through a 0.25 μm pore-size filter. The concentrated supernatants at the consistency shown in Table 1 were added in a total volume of 0.1 mL to Hela cells, and Brucella broth served as a negative control. After incubation for 48 h, cells were stained and observed under phase microscope. Wells, in which 50% or more cells were vacuolated, were defined as showing a cytotoxic effect[18].

Table 1 Vacuolating activity of H.pylori to Hela cells.
Dilution of concentrated supernatantsSpiral form
Coccoid form
No dilution+++---
SDS-PAGE of whole cell proteins

The suspension of H.pylori (6 × 1010 cfu/mL) were centrifuged, washed once with 0.5 mol/L Tris (pH 7.4), and the pellets were suspended in extraction buffer consisting of 0.75% Tris, 2% sodium dodecyl sulfate, 5% dithiothreitol, 10% glycerol and 0.1% bromophenol blue. The homogenate was heated for 5 min in a boiling water bath and frozen at -20 °C until use. According to Sambrook J et al[19], the protein solutions (10 μL) were electrophoresed on SDS-polyacrylamide gels with the stacking and the separating gels containing 5% and 10% acrylamide, respectively, and the gels were stained with Coomassie blue.

Western blot

According to Sambrook J et al[19], after SDS-PAGE, the proteins were blotted onto a nitrocellulose membrane under a constant current of 100 mA for 7 h, and the antigenic profiles were studied by Western blotting using serum from the patients infected with strain F44 (diluted at 1/50).

PCR and PCR-single-strand conformational polymorphism (PCR-SSCP)

PCR primers and the size of the corresponding PCR products are shown in Table 2[20-22]. The suspension of bacteria were centrifuged, and the pellets were resuspended in distilled water(105 cfu/mL), heated in a boiling water bath for 1min to obtain the DNA template, which was added into PCR reaction system in volume of 1/5. PCR was performed as follows: denaturation at 95 for 5 min, followed by 30 cycles of denaturation (94 °C for 0.5 min);annealing (52 °C for 1 min); and extension (72 °C for 1 min), and final extension at 72 °C for 7 min. The PCR products were electrophoretically separated on 0.2% agarose gel, and stained with ethidium bromide.

Table 2 Oligonucleotide primers used for PCR reactions.
Gene amplifiedPrimer sequenceSize of PCR product

SSCP was performed as follows: the mixture consisting of 5 μL of PCR product and 3 μL of loading buffer (95% formamide, 200 mmol/L EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol) was heated at 100 °C for 5 min to denature double-stranded DNA and then plunged into ice for 5 min, and electrophoresed on 50% polyacrylamide gel under a constant voltage of 300v for 7 h, the gel was stained with 0.2% silve nitrate.

Statistical analysis

Analysis of data was performed using the Student t test. A value of P < 0.05 was regarded as statistically significant.

Urease activity

As shown in Table 3, the urease activity of coccoid H. pylori, which was transformed from spiral form by exposure to antibiotic in subinhibitory concentrations, decreased.

Table 3 Urease activity of H.pylori.
StrainUrease activity
Spiral formCoccoid form
Adherence to Hep-2 cells

According to the following formula, the rate and the index of adherence were calculated:

The rate of adherence = the amount of cell adherenced by bacteria/100 × 100%

The index of adherence = the amount of bactria adhereing to cells/100

For each coverslip five-fields (one field containing of one hundred cells) were counted, and the mean of all fields is shown in Table 4. As compared with the spiral forms (P < 0.01), the adherence of coccoid forms to Hep-2 cells decreased. In addition, invasion of coccoid H. pylori into cell could be seen under electron microscope (Figure 1).

Figure 1
Figure 1 Invasion of coccoid H. pylori into Hep-2 cell. Transmission electron microscope, × 15 000
Table 4 Adherence test of H.pylori to Hep-2 cells.
Rate of adherence
Index of adherence
Spiral form70.0 ± 5.373.0 ± 5.172.6 ± 4.52.60 ± 0.43.1 ± 0.52.90 ± 0.4
Coccoid form33.0 ± 4.340.1 ± 3.735.5 ± 4.10.96 ± 0.31.0 ± 0.30.98 ± 0.4
P< 0.01< 0.01< 0.01< 0.01< 0.01< 0.01
Vacuolating activity to Hela cells

As shown in Table 1, vacuolating activity of coccoid H. pylori to Hela cell impaired after Hela cells were incubated with different concentrations of supernatants of coccoid H.pylori for 48 h.

SDS-PAGE and Western blot

SDS-PAGE patterns are illustrated in Figure 2A. The content of the proteins with molecular weight over Mr 74000 decreased, especially the band at Mr 125000 was presented with deletion in coccoid H. pylori. The protein patterns of the three strains of coccoid H. pylori were similar. Western blot patterns showed that the hybriditional signal in band Mr 125000 weakens, meanwhile, strengthens in band Mr 110000 and Mr 63000 in all coccoid H. pylori as illustrated in Figure 2B.

Figure 2
Figure 2 A. SDS-PAGE pattern of whole cell proteins of H. pylori, B. Western blot pattern of the proteins of H. pylori. a. marker; b.d.f. spiral forms of H.pyloriF44, F45 and F49, respectively; c.e.g. coccoid forms of H.pylori F44, F45 and F49, respectively.

The result of PCR for strain F44 is illustrated in Figure 3. The genes detected by PCR, which included ureA, ureB, hpaA, vacA and cagA, were all positive in both spiral and coccoid H.pylori. The patterns of SSCP showed that there could exist point mutation in vacA gene of strain F44 and F45, as well as in hpaA gene of strain F49 in coccoid forms. The pattern of SSCP for strain F44 is illustrated in Figure 4.

Figure 3
Figure 3 The results of PCR of H. pylori F44. a. PCR marker 100bp lader; b.c. ureA gene; d.e. ureB gene; f.g. hpaA gene; h.i. vacA gene; j.k. cagA gene; b.d.f.h.j. spiral forms of H.pylori; c.e.g.i.k. coccoid forms of H.pylori.
Figure 4
Figure 4 The pattern of PCR-SSCP of H. pylori F44. a.b. vacA gene; c.d. ureA gene; e.f. cagA gene; g.h. hpaA gene; a.c.e.g. spiral forms of H. pylori; b.d.f.h. coccoid forms of H.pylori.

H.pylori can convert into coccoid forms after induced by antibiotics in vitro or in viro[12,23,24]. It has been reported that coccoid H.pylori is able to colonize and to produce gastric alterations in the suitable animal model[11]. Costas M et al[25]compared the pre-with post-treatment isolates of H.pylori from the same patients by using 1-D SDS PAGE of proteins and considered that recurrent patients were not reinfected with a different strain but that there was recrudescence of the pre-treatment strain. Thus, it is reasonable to suppose that the viability of the coccoid form may account for the wide number of relapses in patients. However, as yet, the pathogenesis of the coccoid form remains unclear.

The putative pathogenic determinants of H.pylori can be divided into two major groups[26]: maintenance factors, which allow the bacterium to colonize and remain within the host, and virulence factors, which contribute to the pathogenetic effects of the bacterium. Both urease activity and adherence to epithelia cells of H.pylori are important maintenance factors[27-30]. In this study, It is shown that both urease activity and adherence to Hep-2 cell of coccoid H.pylori decreased, suggesting that virulence related to colonization in coccoid H.pylori reduced. Vacuolating cytotoxin produced by about 50%-60% of H.pylori strains[18,31-33] is one of the important virulence factors[34]. Infection with cytotoxin-producing H.pylori strains was more prevalent among patients with peptic ulcer disease[35-37]and gastric carcinoma[18,38-42] than among patients with gastritis alone. In our study, the decrease of vacuolating cytotoxicity in coccoid H. pylori was found, which may be related with the reduction of the volume of Mr 87000 VacA and Mr 125000 CagA proteins determining vacuolating cytotoxicity [34,43] by SDS-PAGE. These findings indicate that the coccoid H.pylori is less efficient in the colonizational virulence and vacuolating cytotoxicity,which may make it unlikely to induce an inflammatory response. Thus the alleviation of clinical symptom of the patients after antimicrobiol treatment does not necessarily mean eradication of H.pylori, it may also result from the convertion to coccoid form. As shown in the assay for adherence, a few coccoid H.pylori still adhered to Hep-2 cells, even invated into them, which indicates that coccoid H.pylori is likely to sustain in the host, thus making the recrudescence of infection possible.

In order to display the hereditary background of coccoid H.pylori, the genes related with virulence mentioned above (involving ureA, ureB, hpaA, vacA and cagA) were detected by PCR and PCR-SSCP. No deletion was in these genes, but there only existed the point mutation in genes hpaA or vacA. These data demonstrate that the coccoid H.pylori may revert into an infectious spiral form under the appropriate conditions and result in recudescence of infection, suggesting that coccoid H.pylori may have potential pathogenicity.

According to the features of coccoid H.pylori, it may escape the techniques usually applied for their detection such as cultivation and assay for urease activity. Because of no deletion in genes mentioned above in coccoid H.pylori, we can use PCR to detect these genes instead of conventional methods to determine whether the bacteria have been completely eliminated after treatment.

Some studies showed that the synthesis of some proteins of coccoid H.pylori increased such as 62KDa and > 94 KDa proteins[16,44,45]. In this study, the results of Western blot showed that the antigenic fraction with molecular weight in Mr 110000 and Mr 63000 was detected more intensively in all three strains of coccoid H.pylori, as compared with spiral H.pylori. The fractions may be the one degraded from a high-molecular-mass antigen. Further studies are required to determine whether these antigenic proteins have special functions.


Edited by Ma JY

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