Antibiotic susceptibility of attached and free-floating Helicobacter pylori

Joanne L. Simala-Granta, David Zopfb and Diane E. Taylora,*

a 141 Medical Sciences Building, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7; b Neose Technologies, Inc., Horsham, PA 19044, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori are found attached to mucous cells of the human stomach or under the mucous layer. Models mimicking the in vivo situation may be more suitable for H. pylori MIC determinations than traditional agar dilution methods. Megraud et al. (Antimicrobial Agents and Chemotherapy 1991, 35, 869–72) developed a model for measuring the susceptibility of attached and free-floating H. pylori. We have modified this model so that free-floating and attached H. pylori are treated in a more similar manner, before and after incubation with antibiotic, and performed additional controls to ensure H. pylori and tissue culture cells are not detrimentally affected and maintain their viability during the course of the experiment. We found only 10% of plate-grown H. pylori were competent for attachment to HEp-2 cells; however, all progeny of attached bacteria remained adherent. Killing curves were performed using 0, 0.001, 0.01, 0.1 and 1 mg/L amoxycillin, and 0, 0.0025, 0.0075 and 0.01 mg/L clarithromycin. H. pylori divided at concentrations <= 0.01 mg/L amoxycillin and <= 0.0025 mg/L clarithromycin. Contrary to the previous study, using our modified method we found that HEp-2 adherent and freefloating H. pylori are equally susceptible to amoxycillin (strains 26695, CCUG18943, CCUG19104 and CCUG19110) and clarithromycin (strain 26695). Therefore, we find no evidence that attachment of H. pylori to eukaryotic cells increases their resistance to antibiotics compared with non-attached bacteria. Nonetheless, these results confirm confidence in traditional MIC studies when a comparison is made between susceptible and resistant strains.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori infects approximately half of the human population, causing gastritis.1,2 In some individuals infection leads to the development of gastric or duodenal ulcers, gastric mucosal-associated lymphoid tissue lymphoma or gastric adenocarcinoma.3

H. pylori are found attached to mucous cells of the human stomach, or in the mucous layer.4,5 Although H. pylori is highly susceptible to antimicrobial agents in vitro,610 they are not easily eradicated by mono-antibiotic therapy in vivo.11 Consequently, eradication of H. pylori requires the use of combination antibiotic therapies for eradication. The Canadian Consensus suggests ‘triple’ eradication regimes be employed using a proton-pump inhibitor, amoxycillin and clarithromycin, the latter being preferable to metronidazole since in some areas of Canada up to 40% of H. pylori are metronidazole resistant.12

There are a number of potential reasons why antibiotic susceptibility may be greater in vitro than in vivo. It is possible that some antibiotics do not achieve an appropriate MIC in the mucus, fundus or corpus, or that concentrations reached do not remain high enough for a sufficient amount of time. The antibiotic may be inactivated by the acidic environment of the stomach or may be removed too soon due to stomach emptying.11,13,14 Traditional MIC studies result in the bacteria being incubated with antibiotic until the colonies grow up, which for H. pylori is several days. This is very different from the in vivo situation. In addition, the attached and free-floating counterparts of a variety of bacteria, including H. pylori, have been shown to possess morphological and biochemical differences,15,16 and it is possible that this diversity could influence antibiotic susceptibility in vivo and in vitro. For example, adherence of H. pylori to gastric epithelial cells in vitro stimulates the transcription of ice(A1) (induced by contact with epithelium).17

Since routine MIC studies have employed conditions not equivalent to the in vivo situation, it is possible that these MIC studies are not physiologically relevant. Therefore, models that mimic the in vivo situation may be better suited for MIC determinations of H. pylori, particularly since bacteria grown in vivo have shown altered antibiotic susceptibility.18,19

Megraud and coworkers20 developed a model for measuring the antibiotic susceptibility of HEp-2-attached and free-floating bacteria. Their data indicated increased amoxycillin susceptibility for free-floating H. pylori compared with attached counterparts. The goal of this study was to duplicate the results of Megraud et al. with amoxycillin and extend them to clarithromycin, after modifying their model so that free-floating and attached H. pylori were treated in a more similar manner, before and after incubation with antibiotic. A large number of controls were also performed to ensure that H. pylori and tissue culture cells were not detrimentally affected and maintained their viability during the course of the experiment. Contrary to the previous study, using this modified method, HEp-2-adherent and planktonic H. pylori were found to possess equal resistance to amoxycillin, and this observation extended to the antibiotic clarithromycin.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

The amoxycillin- and clarithromycin-susceptible H. pylori strains 26695,21 UA1182 (University of Alberta clinical isolate), 1832, CP22 (Neose clinical isolates),22 CCUG18943, CCUG19104 and CCUG19110 (Culture Collection, University of Goteborg) were used (amoxycillin was from Sigma, St Louis, MO, USA; clarithromycin was from Abbott, Mississauga, Ontario, Canada). The identity of the cultures was verified by testing for urease,23 oxidase and catalase activities,24 and by phase-contrast light microscopy for unique morphological characteristics. Bacteria were stored at –80°C in 3.7% brain–heart infusion media (BHI; Difco, Detroit, MI, USA), 0.5% yeast extract (YE; Difco), and 15% glycerol until use. H. pylori were grown on BHI/YE agar plates with 5% horse serum (Hyclone Laboratories, Logan, UT, USA) and 15 mg/L vancomycin (Eli Lilly, Indianapolis, IN, USA) and 15 mg/L amphotericin B (Sigma). Plate-grown cultures were incubated in jars in the presence of balanced nitrogen with 5% CO2 and 5% H2, or with a Campylobacter gas generating kit with platinum catalyst (Oxoid, Basingstoke, UK), resulting in a residual O2 concentration of 6% with 10% CO2. Bacteria were passaged only twice before their use for antibiotic susceptibility testing.

Tissue culture

Human gastric adenocarcinoma [AGS; American Type Culture Collection (ATCC) CRL-1739], human epidermoid larynx carcinoma (HEp-2; ATCC CCL-23) and human duodenal adenocarcinoma (Hutu-80; ATCC HTB-40) tissue cell lines were used. Tissue culture cells were thawed from frozen stock at room temperature (90% culture medium and 10% dimethylsulphoxide; Sigma) every 20 passages to decrease the potential for age-related changes during the course of our experiments.25 This was done by diluting thawed cells 1:10 in tissue culture media, pelleting cells at 300g for 5 min, and resuspending the pellet in tissue culture media. The growth medium was Ham's F12 (Gibco-BRL, Burlington, Ontario, Canada) for AGS, and minimum essential medium with Earle's salts (MEM; GibcoBRL) for HEp-2 and Hutu-80. Ten per cent fetal bovine serum (FBS; Hyclone Laboratories) was added before use. Tissue culture cells were incubated in a tissue culture incubator with 5% CO2 at 100% humidity. Cells were detached from plates by incubating with 0.25% trypsin for 3–5 min. As necessary, cells were counted using a haemocytometer after addition of trypan blue to a final concentration of 0.04%.

Antibiotic susceptibility testing

HEp-2 cells were used for studies with adherent H. pylori and split at 2.0 x 105 cells/well and incubated overnight to allow for adherence. The number of cells doubled during this period and there were 4 x 105 HEp-2 cells present in each well at time 0. The medium was removed and replaced with 1.8 mL equilibrated tissue culture medium the day of the experiment. H. pylori were routinely cultured from frozen on BHI/YE agar plates, as previous studies have demonstrated that growth in liquid culture reduces the frequency of the epithelial cell-adherent phenotype by 50%.22 The bacteria were grown for 2–3 days, subcultured to new plates, and grown for an additional day before use. Various epithelial cell lines have been shown to support binding of up to 250 H. pylori organisms per individual epithelial cell.26,27 However, we selected a multiplicity of infection of 10 to keep H. pylori numbers low enough that by the end of the experiment very little cytotoxicity had occurred, yet high enough that it would be possible to measure the killing effects of a range of antibiotic concentrations on H. pylori. To do this, the bacteria were resuspended in BHI/YE and the optical density at 600 nm (OD600) was measured after dilution. Subsequently, the bacteria were diluted in BHI/YE to OD600s of 4 and 0.4, and 200 µL was added to wells with or without HEp-2 for attached and free-floating experiments, respectively. Attachment of H. pylori to HEp-2 was allowed to proceed for 1 h as numerous studies have demonstrated that attachment of H. pylori to tissue culture cells occurs within this time frame (J. L. Simala-Grant, R. Sherburne & D. E. Taylor, unpublished data).2729 Plates with free-floating H. pylori were also stored in the tissue culture incubator in 5% CO2, 100% humidity for 1 h to ensure that the conditions for the two sets of experiments were as similar as possible. In the attached experiment, after the 1 h allotted for adherence, any free-floating H. pylori were removed by washing the tissue culture monolayer three times with MEM/10% FBS/10% BHI/YE. Antibiotic was added to wells from 1000-fold concentrated stocks in BHI (0.001, 0.01, 0.1 and 1 g/L amoxycillin, and 0.0025, 0.0075 and 0.01 g/L clarithromycin). At time 0, 2, 6 and 24 h, the wells were scraped (with a slice of rubber glued to a needle) to remove attached HEp-2 and H. pylori, and the medium was removed. The antibiotic must be removed from both free-floating and adherent bacterial samples before plating. To maintain consistency between the free-floating and attached experiments, antibiotic was removed from bacterial samples by replacement of the medium with BHI/YE twice after centrifugation at 8160 relative centrifugal force in a microcentrifuge for 10 min, a step found to have no detrimental effect on bacterial viability. The samples were resuspended, diluted in duplicate and plated in triplicate. The plates were incubated for up to 10 days, and plates with >10 and <350 colonies were counted and used for subsequent analysis. The minimum number of viable H. pylori that could be measured in a sample accurately was 200 cfu, since 0.1 mL of each 2 mL sample was plated.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We demonstrated that none of the steps employed in the model system used to measure the antibiotic susceptibility of attached and free-floating H. pylori was detrimental to the viability of the bacteria or tissue culture cells (see below and Materials and methods).

Choice of H. pylori for use in susceptibility experiments

Four strains of H. pylori (26695, 1182, 1832 and CP22) were examined for their cfu/OD600, growth characteristics, propensity to clump, binding to tissue culture wells and cytotoxicity to tissue culture cells to determine their suitability for use in antibiotic susceptibility experiments.

Since it was important to be able to add a precise and similar number of H. pylori cells for each experiment, the number of H. pylori cfu/OD600 was determined. Numerous trials revealed plate-grown H. pylori strains 26695, 1182, 1832 and CP22 to possess an average of 2 x 108 cfu/OD600. Visualization of bacteria under the light microscope revealed that essentially all bacteria were spiral shaped and motile.

Epithelial cell lines and H. pylori each grow optimally under different culture conditions. Cell culture medium is formulated to maintain pH 7.2 in a tissue culture incubator (5% CO2, 100% humidity), but we found that it becomes too alkaline (pH 7.8) for use with mammalian cells or H. pylori when equilibrated in a microaerobic environment (balanced N2, 5% CO2, 5% H2) optimized for growth of H. pylori. Fortunately, we found that H. pylori survive and grow for at least 24 h (the time required to complete an antibiotic susceptibility testing experiment) in tissue culture medium (MEM with 10% FBS) supplemented with 10% BHI/YE maintained in the tissue culture incubator. The ‘no antibiotic’ curves in Figures 1–3GoGoGo demonstrate that H. pylori incubated under these conditions divide rapidly after a short lag phase.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 1. Per cent recovery after incubation with amoxycillin of free-floating (top) and HEp-2-attached (bottom) strain 26695. H. pylori were incubated with 0 ({square}), 0.001 ({diamond}), 0.01 ({circ}), 0.1 ({triangleup}) or 1 ({blacksquare}) mg/L amoxycillin. See Materials and methods for details.

 


View larger version (17K):
[in this window]
[in a new window]
 
Figure 2. Per cent recovery after incubation with clarithromycin of free-floating (top) and HEp-2 attached (bottom) strain 26695. H. pylori were incubated with 0 ({square}), 0.0025 ({diamond}), 0.0075 ({circ}) or 0.01 ({triangleup}) mg/L clarithromycin. See Materials and methods for details.

 


View larger version (9K):
[in this window]
[in a new window]
 
Figure 3. Per cent recovery of free-floating and attached CCUG19104 with or without incubation with amoxycillin. Bacteria were incubated with (attached) or without (free-floating) HEp-2, in the presence or absence of amoxycillin. {square}, free-floating CCUG19104 with no amoxycillin; {diamond}, free-floating CCUG19104 with 0.001 mg/L amoxycillin; {circ}, HEp-2-attached CCUG19104 with no amoxycillin; {triangleup}, HEp-2-attached CCUG19104 with 0.001 mg/L amoxycillin. See Materials and methods for details.

 
To examine the propensity of bacteria to aggregate, each H. pylori strain was diluted to 106, 107 or 108 cfu/mL, incubated in cell culture medium in the tissue culture incubator for 24 h, and then examined by phase-contrast microscopy. All strains of H. pylori showed some propensity to aggregate (clumps of from two up to hundreds of bacteria) at 108 cfu/mL. Clumping was substantially diminished, with essentially all H. pylori found as single cells, at 106 and 107 cfu/mL for 1182, 1832 and 26695. However, the propensity for CP22 to form clumps was so great that this strain was eliminated from use in antibiotic susceptibility testing experiments. These results indicate that whichever strain is selected, the cfu/mL should be kept below 107.

We also examined the tendency of the four H. pylori strains to adhere non-specifically to tissue culture wells, using phase-contrast microscopy. Only H. pylori 1182 showed a high propensity for binding to the tissue culture wells, and thus this strain was eliminated for use in the antibiotic susceptibility experiments.

We investigated the effect of various densities of each strain of H. pylori on each of the tissue culture cell lines of interest. H. pylori 26695, 1182, 1832 and CP22 were diluted to 106, 107 or 108 cfu/mL and incubated with AGS, HEp-2 or Hutu-80 grown on coverslips for 24 h. The coverslips were then viewed by phase-contrast microscopy. At 108 cfu/mL 26695 caused vacuolization in all cell lines, and 1832 caused vacuolization in Hutu-80. However, cytotoxicity decreased as the cfu/mL were decreased. At 106 cfu/mL, 26695 caused vacuolization in AGS and Hutu-80, while 1832 caused vacuolization in Hutu-80. These observations suggested that strain 26695 should not be used with AGS or Hutu-80, or strain 1832 with Hutu-80 in the antibiotic susceptibility experiments for attached H. pylori, but that other pairings would be acceptable.

As a result of all the observations listed above, H. pylori strain 26695 was selected for use in antibiotic susceptibility experiments.

Removal of non-adherent H. pylori

We determined the number of washes necessary to remove non-adherent H. pylori from tissue culture wells containing confluent HEp-2 cells. Eighty-three and 5% of the cfu were found in washes one and two, respectively, while <0.5% of added bacteria were found in the third wash. These observations indicate that three washes are sufficient to remove non-adherent H. pylori. This confirmed previously published results.30

Percentage of H. pylori competent for attachment to Hep-2

The same number of attached and free-floating bacteria should be present in the attached and free-floating antibiotic susceptibility experiments when antibiotic is added. We found c. 10% of plate-grown H. pylori to be competent for attachment to HEp-2, a percentage not substantially affected by the multiplicity of infection in the range of 0.1–20. This result indicated that 10 times more H. pylori needed to be added in the attached experiment, so that at time 0, after washing to remove non-adherent H. pylori, the same number of bacteria would be present as in the experiment examining antibiotic susceptibility of free-floating H. pylori.

Since H. pylori divide during the 24 h incubation with tissue culture cells (see Figures 1–3GoGoGo), it was necessary to determine whether the progeny formed during this time were competent for attachment. If, for example, only 10% of progeny in cell culture medium were competent for attachment, the experiment would not provide an accurate measurement of antibiotic susceptibility of attached bacteria at the 24 h time point, as free-floating bacteria would outnumber the attached bacteria. To address this concern, H. pylori 26695 were incubated with HEp-2, and the non-adherent bacteria were removed by washing. After 24 h, the samples in the wells were either plated directly to determine the total number of bacteria present (6.52 ± 0.709 x 106 cfu), or washed three times to remove non-adherent bacteria and then plated (5.97 ± 1.52 x 106 cfu). Surprisingly, all bacteria present at 24 h were attached to HEp-2. This indicated that the experiment measuring antibiotic susceptibility of attached H. pylori would not be confounded by free-floating H. pylori at any point up to 24 h.

Effect of HEp-2 disruption technique on H. pylori cfu

Since the antibiotic susceptibility experiment requires the binding of multiple H. pylori per tissue culture cell, the tissue culture cells must be disrupted to ensure each H. pylori results in one colony. Tissue culture cells have been demonstrated to be lysed with 0.1–1% Triton X-100,28,31 distilled water25,32 and homogenization.20 We examined the effect of 0.1% Triton X-100, distilled water for 10 min or Ultraturax homogenization for up to 6 s on H. pylori cfu recovery. No cfu were recovered after exposure to Triton X-100. In contrast, no cfu were lost after homogenization or exposure to distilled water. Nonetheless, we found that with multiplicities of infection >1 (i.e. 3.2), samples with homogenization (1.47 ± 0.27 x 106 cfu) and no homogenization (2.58 ± 0.69 x 106 cfu) resulted in approximately the same number of cfu. This indicates that the tissue culture cells were disrupted during the dilution and spreading steps making a specific disruption technique (i.e. homogenization) unnecessary.

Effect of exposure of H. pylori to ambient concentrations of oxygen

Since it takes a finite amount of time to dilute and spread the samples from each time point when exposing the H. pylori to oxygen in ambient air, H. pylori were incubated in tissue culture media in the CO2 incubator at 37°C for 24 h, and then diluted and spread immediately, or exposed to air for 2 h before dilution and spreading. For the sample diluted and spread immediately, 1.93 ± 0.54 x 108 cfu/mL were recovered, and 1.83 ± 0.25 x 108 cfu/mL after the sample had been exposed to air for 2 h. This result indicated that if the samples are plated within 2 h after recovery at an experimental time point, there would be no loss in H. pylori cfu. As antibiotic is removed at an earlier step, any lag in dilution and spreading does not result in increased exposure to antibiotic.

Antibiotic susceptibility testing of attached versus free-floating H. pylori 26695

The results of one representative experiment of the susceptibility of free-floating and attached H. pylori strain 26695 to amoxycillin (Figure 1Go) and clarithromycin (Figure 2Go) are shown. Both adherent and free-floating H. pylori remained viable and, in fact, divided at concentrations of <=0.01 mg/L amoxycillin and <=0.0025 mg/L clarithromycin, and were killed to the same extent at higher antibiotic concentrations. These results demonstrate that H. pylori adherent to epithelial cells do not differ from H. pylori suspended in cell culture medium with respect to susceptibility to amoxycillin and clarithromycin. The small differences in the slopes in Figures 1 and 2GoGo are not significant.

Comparison of antibiotic susceptibility using CCUG H. pylori strains

To confirm that the identical susceptibilities to amoxycillin and clarithromycin for free-floating and attached H. pylori strain 26695 was not strain specific, we determined amoxycillin susceptibility of free-floating and HEp-2-attached CCUG18943, CCUG19104 and CCUG19110. The results from one representative experiment are shown. We observed no significant difference in amoxycillin susceptibility for free-floating or attached CCUG19104 (Figure 3Go) CCUG18943, CCUG19110 and 26695 (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We elected to examine amoxycillin susceptibility since the effect of this antibiotic on attached and free-floating H. pylori was examined previously by Megraud et al.20 We then proceeded to examine clarithromycin susceptibility as this is the second antibiotic recommended in ‘triple therapy’ eradication regimes.12 The results reported here show that the amoxycillin and clarithromycin susceptibilities of attached and free-floating H. pylori are virtually identical. Thus, adherence of H. pylori to mammalian tissue culture cells does not appear to confer increased antibiotic resistance on the bacteria. These results indicate that use of anti-adhesin compounds in treatment regimes along with antibiotics, to detach H. pylori from the stomach mucous cells, and thus increase antimicrobial susceptibility, is not feasible, although anti-adhesin compounds may still be useful in the eradication of H. pylori. Accordingly, an alternative explanation must be found to rationalize the wellknown resistance of H. pylori to single antibiotic treatment regimes in the face of in vitro MIC results for clinical isolates showing antibiotic susceptibility. One such explanation has been proposed by Cooreman et al.,14 who showed that amoxycillin was delivered at MBC in the stomach mucus layer in less than half of the patients examined, and when present at the MBC, remained at the MBC for <60 min. Moreover, the antibiotic did not even reach MBC in the fundus and corpus regions of the stomach. These results emphasize that the agar dilution method, although allowing a relative comparison between resistant and susceptible strains, does not equate with in vivo eradication by current drug regimes. More studies are needed to determine whether antibiotics are reaching the MBC in vivo, at which time the concentration of antibiotic found in the stomach after a typical dose can be compared with the clarithromycin susceptibility data reported here and future susceptibility results generated using this model, to indicate whether antibiotic is reaching the MBC in vivo for long enough.

Whether or not decreased growth rate of H. pylori in a specialized niche in vivo may explain unexpected difficulty in clinical eradication of the organism has not been examined. Decreased growth rate of other bacteria in biofilms has been shown to play a role in increased antibiotic resistance.18,19 If decreased growth rate of H. pylori was demonstrated in vivo, the effect of decreased growth rate on H. pylori antibiotic susceptibility could be examined in vitro using a chemostat to regulate growth rate.33

The antibiotic susceptibility results reported here are different from those reported by Megraud et al.20 Their study showed decreased amoxycillin susceptibility for attached H. pylori. This was most apparent at the 6 and 24 h time points of the killing curves. We studied strain 26695, and compared it with strains CCUG18943, CCUG19104 and CCUG19110, the latter three employed by Megraud et al.,20 in order to eliminate the possibility that differences in experimental outcomes could be due to strain-specific effects. Megraud et al.20 reported 1000-fold fewer cfu for free-floating bacteria under their experimental conditions at 24 h after exposure to 0.001 mg/L amoxycillin for CCUG18943, CCUG19104 and CCUG 19110, and this was the data point at which the greatest difference was observed between attached and free-floating organisms. As a result we elected to examine the same data point, as any difference between attached and free-floating bacteria would be most obvious under these conditions. There are, however, a number of possible explanations as to why the results reported here differ from those of Megraud et al.20 Virulence factors of H. pylori, like those of other bacteria, are tightly regulated and their expression often depends on temperature, growth conditions, partial O2 pressure and host characteristics.34 Adherence of H. pylori to sulphatide,35,36 heparin37 and tissue culture cells has been observed to increase with a decreased pH.32 In addition, the lipopolysaccaride structure of H. pylori has been shown to be dependent on the pH of the medium.38 Adherence of H. pylori to Lewis B39 and extracellular matrix proteins40 has been shown to be greater when the bacteria are in stationary phase, and plate-grown bacteria have been shown to bind better to sialic acid41,42 and heparin43 than liquid-grown H. pylori. The results reported here may differ from those of Megraud et al.20 because of differences in H. pylori growth phase, growth conditions, pH of growth medium, phase variation of bacterial strains or partial O2 pressure used.

Each step in our experimental protocols was carefully studied, and appropriate controls were performed before selecting the procedure to be followed. For example, our control experiments demonstrated that tissue culture medium equilibrates at a rather high pH (7.8) in microaerobic jars. It appears that Megraud et al.20 performed their incubations in a microaerobic jar where the cell culture medium may have become alkaline.20 Stressed cells have been observed to demonstrate aberrant MICs,6 thus if non-adherent H. pylori are more susceptible to the increased pH of the medium in the microaerobic jar, this effect may explain the differences in observed results. Examination of the no antibiotic curves in Figures 1 and 2GoGo reveals that H. pylori incubated in the absence of antibiotic are clearly dividing exponentially in our experiments. This observation provides further evidence that our culture conditions differ from those of Megraud et al.,20 where they observed only a doubling in H. pylori cfu in 24 h. Thus the H. pylori in Megraud et al.'s experiments may have been in a stressed condition.

Our procedures differed from those of Megraud et al. in other respects as well, in that we adjusted our protocols to minimize differences in handling of attached and free-floating bacteria. For example, a 1 h incubation was included for the free-floating bacteria to mimic the time required for binding of adherent bacteria to cell monolayers. Also, we removed antibiotic from attached and free-floating bacteria by the same centrifugation method.

A number of factors were considered when selecting which H. pylori strain to use. The H. pylori strain selected did not show a propensity to clump, as it was important to ensure that experiments examining attached bacteria were in fact employing single attached bacteria rather than a clump of bacteria attached by one or several H. pylori. In addition, since the readout to determine viable bacteria is by colony count in these experiments, it was necessary to ensure that the results were not confounded by a tendency for bacteria to clump (i.e. 1 cfu must equal one H. pylori, not one clump of bacteria). Furthermore, the strain of bacteria selected did not adhere to the plastic culture wells, ensuring that bacteria not removed by washing in experiments examining the antibiotic susceptibility of attached H. pylori had, in fact, adhered to tissue culture cells. Moreover, since some strains of H. pylori have been observed to cause vacuolization in tissue culture cell lines,44,45 we selected a strain that did not cause vacuolization of the tissue culture cell line chosen.

There are a number of gastric carcinoma cell lines, including Kato-III46 and AGS; however, the larynx carcinoma cell line HEp-2 was selected for use for a number of reasons. HEp-2 is a hardy adherent cell line whose growth is confluent. Unlike AGS, HEp-2 remain attached for >48 h, as well as during the washing steps necessary to remove non-adherent H. pylori. If HEp-2 are split at 2.0 x 105 cells/well (6-well), they are almost confluent at 24 h (time 0), and do not detach to any great extent by 48 h (time 24 h). Most importantly, HEp-2 was the cell line used by Megraud et al.,20 and use of the same cell line allows for comparison with their published results. Use of a different cell line would make comparison of these two sets of results difficult if not impossible.

Pilot experiments revealed that only 10% of plate-grown H. pylori were competent for attachment. These results are similar to those of Fauchere & Blaser,47 who found that the mean percentage of the original innoculum bound to HeLa cells did not vary markedly (3–14%) when the bacteria:cell ratio varied between 102 and 105. Nonetheless, we found that all progeny of adherent H. pylori generated during the course of the experiment were competent for attachment. One possible explanation for this observation is that H. pylori may be competent for adherence only before stationary phase, and a large portion of plate-grown bacteria may have already passsed the exponential growth phase. Alternatively, the population of cells may be heterogeneous genetically or epigenetically, such that selection for adherent bacteria leads to subsequent growth of a subset of the original bacterial population.

The model system reported here is an improvement over traditional MIC studies since it allows measurement of the antibiotic susceptibility of both attached and free-floating H. pylori, and the time of incubation with antibiotic can be varied. The controls performed here demonstrate that the steps involved are not detrimental to the viability of H. pylori or tissue culture cells. Using this model it is possible to incubate H. pylori with antibiotic over a wide range of time periods, thus, after determination of the length of time a drug is found at the MBC in the mucous layer of the stomach it would be possible to incubate the bacteria with antibiotic for this length of time. This is not possible with the traditional agar dilution method, where the bacteria are exposed to antibiotic until colonies grow up. Nonetheless, traditional MIC studies are preferable, and confidence in results obtained can be maintained when a comparison is to be made between susceptible and resistant strains, as traditional MIC studies are much more easily performed, and our results indicate that attachment of H. pylori does not increase the resistance to antibiotics as reported previously.20

This model, although it differs from the in vivo situation as it does not incorporate peristalsis, provides additional useful features compared with traditional MIC testing. It could be modified further to incorporate a mucous layer to determine how this affects the susceptibility of H. pylori to antibiotics. A mucous layer has been mimicked previously, employing methylcellulose when examining Campylobacter jejuni.48


    Acknowledgments
 
We would like to thank Susan Amundsen for technical assistance. This work was funded by a grant from Neose Technologies, Inc. and by the Canadian Bacterial Diseases Network (Center of Excellence Program). D.E.T. is a Medical Scientist with the Alberta Heritage Foundation for Medical Research.


    Notes
 
* Corresponding author. Tel: +1-780-492-4777; Fax: +1-780-492-7521; E-mail: diane.taylor{at}ualberta.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Ge, Z. & Taylor, D. E. (1999). Contributions of genome sequencing to understanding the biology of Helicobacter pylori. Annual Review of Microbiology 53, 353–87.[ISI][Medline]

2 . Cave, D. R. (1996). Transmission and epidemiology of Helicobacter pylori. American Journal of Medicine 100, Suppl. 5A, 12S–17S.[Medline]

3 . Montalban, C., Manzanal, A., Boixeda, D., Redondo, C., Alvarez, I., Calleja, J. L. et al. (1997). Helicobacter pylori eradication for the treatment of low-grade gastric MALT lymphoma: follow-up together with sequential molecular studies. Annals of Oncology 8, Suppl. 2, 37–9.[Abstract]

4 . Hessey, S. J., Spencer, J., Wyatt, J. I., Sobala, G., Rathbone, B. J., Axon, A. T. et al. (1990). Bacterial adhesion and disease activity in Helicobacter associated chronic gastritis. Gut 31, 134–8.[Abstract]

5 . Caselli, M., Figura, N., Trevisani, L., Pazzi, P., Guglielmetti, P., Bovolenta, M. R. et al. (1989). Patterns of physical modes of contact between Campylobacter pylori and gastric epithelium: implications about the bacterial pathogenicity. American Journal of Gastroenterology 84, 511–3.[ISI][Medline]

6 . Morgan, D. R., Fitzpatrick, P. M., David, K. L. & Kraft, W. G. (1987). Susceptibility patterns of Campylobacter pyloridis. FEMS Microbiology Letters 42, 245–8.[ISI]

7 . McNulty, C. A., Dent, J. & Wise, R. (1985). Susceptibility of clinical isolates of Campylobacter pyloridis to 11 antimicrobial agents. Antimicrobial Agents and Chemotherapy 28, 837–8.[ISI][Medline]

8 . McNulty, C. A. & Dent, J. C. (1988). Susceptibility of clinical isolates of Campylobacter pylori to twenty-one antimicrobial agents. European Journal of Clinical Microbiology and Infectious Diseases 7, 566–9.[ISI][Medline]

9 . Lambert, T., Megraud, F., Gerbaud, G. & Courvalin, P. (1986). Susceptibility of Campylobacter pyloridis to 20 antimicrobial agents. Antimicrobial Agents and Chemotherapy 30, 510–1.[ISI][Medline]

10 . Goodwin, C. S., Blake, P. & Blincow, E. (1986). The minimum inhibitory and bactericidal concentrations of antibiotics and anti-ulcer agents against Campylobacter pyloridis. Journal of Antimicrobial Chemotherapy 17, 309–14.[Abstract]

11 . Katayama, H., Nishimura, T., Ochi, S., Tsuruta, Y., Yamazaki, Y., Shibata, K. et al. (1999). Sustained release liquid preparation using sodium aliginate for eradication of Helicobacter pylori. Biological and Pharmacological Bulletin 22, 55–60.

12 . Hunt, R. & Thomson, A. B. (1998). Canadian Helicobacter pylori consensus conference. Canadian Association of Gastroenterology. Canadian Journal of Gastroenterology 12, 31–41.[ISI][Medline]

13 . Lozniewski, A., deKorwin, J. D., Muhale, F. & Jehl, F. (1997). Gastric diffusion of antibiotics used against Helicobacter pylori. International Journal of Antimicrobial Agents 9, 181–93.[ISI][Medline]

14 . Cooreman, M. P., Krausgrill, P. & Hengels, K. J. (1993). Local gastric and serum amoxicillin concentrations after different oral application forms. Antimicrobial Agents and Chemotherapy 37, 1506–9.[Abstract]

15 . Goodwin, C. S. & Armstrong, J. A. (1990). Microbiological aspects of Helicobacter pylori (Campylobacter pylori). European Journal of Clinical Microbiology and Infectious Diseases 9, 1–13.[ISI][Medline]

16 . Krogfelt, K. A. (1995). Adhesin-dependent isolation and characterization of bacteria from their natural environment. Methods in Enzymology 253, 50–3.[ISI][Medline]

17 . Peek, R. M. Jr, Thompson, S. A., Donahue, J. P., Tham, K. T., Atherton, J. C., Blaser, M. J. et al. (1998). Adherence to gastric epithelial cells induces expression of a Helicobacter pylori gene, iceA, that is associated with clinical outcome. Proceedings of the Association of American Physicians 110, 531–44.[ISI][Medline]

18 . Gilbert, P. & Brown, M. R. (1998). Biofilms and ß-lactam activity. Journal of Antimicrobial Chemotherapy 41, 571–2.[Free Full Text]

19 . Ashby, M. J., Neale, J. E., Knott, S. J. & Crichley, I. A. (1994). Effect of antibiotics on non-growing planktonic cells and biofilms of Escherichia coli. Journal of Antimicrobial Chemotherapy 33, 443–52.[Abstract]

20 . Megraud, F., Trimoulet, P., Lamouliatte, H. & Boyanova, L. (1991). Bactericidal effect of amoxicillin on Helicobacter pylori in an in vitro model using epithelial cells. Antimicrobial Agents and Chemotherapy 35, 869–72.[ISI][Medline]

21 . Akopyants, N. S., Eaton, K. A. & Berg, D. E. (1995). Adaptive mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets. Infection and Immunity 63, 116–21.[Abstract]

22 . Simon, P. M., Goode, P. L., Mobasseri, A. & Zopf, D. (1997). Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides. Infection and Immunity 65, 750–7.[Abstract]

23 . Atlas, R. M. (1993). Handbook of Microbiological Media, (Parks, L. C., Ed). CRC Press, Boca Raton, FL.

24 . Koneman, E. W., Allen, S. D., Janda, W. M., Schreckenberger, P. C. & Winn, W. C. (1997). Color Atlas and Textbook of Diagnostic Microbiology. Lipincott-Raven Publishers, Philadelphia, PA.

25 . Gaillard, J. L. & Finlay, B. B. (1996). Effect of cell polarization and differentiation on entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line. Infection and Immunity 64, 1299–308.[Abstract]

26 . Clyne, M. & Drumm, B. (1993). Adherence of Helicobacter pylori to primary human gastrointestinal cells. Infection and Immunity 61, 4051–7.[Abstract]

27 . Kamisago, S., Iwamori, M., Tai, T., Mitamura, K., Yazaki, Y. & Sugano, K. (1996). Role of sulfatides in adhesion of Helicobacter pylori to gastric cancer cells. Infection and Immunity 64, 624–8.[Abstract]

28 . Evans, D. G., Evans, D. J. Jr & Graham, D. Y. (1989). Receptor-mediated adherence of Campylobacter pylori to mouse Y-1 adrenal cell monolayers. Infection and Immunity 57, 2272–8.[ISI][Medline]

29 . Evans, D. G., Evans, D. J. Jr & Graham, D. Y. (1992). Adherence and internalization of Helicobacter pylori by HEp-2 cells. Gastroenterology 102, 1557–67.[ISI][Medline]

30 . Graham, L. L. & MacDonald, K. L. (1998). The Campylobacter fetus S layer is not essential for initial interaction with HEp-2 cells. Canadian Journal of Microbiology 44, 244–50.[ISI][Medline]

31 . Kenny, B., Lai, L. C., Finlay, B. B. & Donnenberg, M. S. (1996). EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells. Molecular Microbiology 20, 313–23.[ISI][Medline]

32 . Bukholm, G., Tannaes, T., Nedenskov, P., Esbensen, Y., Grav, H. J., Hovig, T. et al. (1997). Colony variation of Helicobacter pylori: pathogenic potential is correlated to cell wall lipid composition. Scandinavian Journal of Gastroenterology 32, 445–54.[ISI][Medline]

33 . Hassan, I. J., Stark, R. M., Greenman, J. & Millar, M. R. (1999). Activities of ß-lactams and macrolides against Helicobacter pylori. Antimicrobial Agents and Chemotherapy 43, 1387–92.[Abstract/Free Full Text]

34 . Rosenshine, I., Ruschkowski, S. & Finlay, B. B. (1996). Expression of attaching/effacing activity of enteropathogenic Escherichia coli depends on growth phase, temperature, and protein synthesis upon contact with epithelial cells. Infection and Immunity 64, 966–73.[Abstract]

35 . Huesca, M., Goodwin, A., Bhagwansingh, A., Hoffman, P. & Lingwood, C. A. (1998). Characterization of an acidic-pH-inducible stress protein (hsp70), a putative sulfatide binding adhesin, from Helicobacter pylori. Infection and Immunity 66, 4061–7.[Abstract/Free Full Text]

36 . Huesca, M., Borgia, S., Hoffman, P. & Lingwood, C. A. (1996). Acidic pH changes receptor binding specificity of Helicobacter pylori: a binary adhesion model in which surface heat shock (stress) proteins mediate sulfatide recognition in gastric colonization. Infection and Immunity 64, 2643–8.[Abstract]

37 . Chmiela, M., Ljungh, A., Rudnicka, W. & Wadstrom, T. (1996). Phagocytosis of Helicobacter pylori bacteria differing in the heparan sulfate binding by human polymorphonuclear leukocytes. Zentralblatt für Bakteriologie 283, 346–50.[ISI][Medline]

38 . McGowan, C. C., Necheva, A., Thompson, S. A., Cover, T. L. & Blaser, M. J. (1998). Acid-induced expression of an LPS-associated gene in Helicobacter pylori. Molecular Microbiology 30, 19–31.[ISI][Medline]

39 . Falk, P. G., Bry, L., Holgersson, J. & Gordon, J. I. (1995). Expression of a human {alpha}-1,3/4-fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production of LEBcontaining glycoconjugates: a potential transgenic mouse model for studying Helicobacter pylori infection. Proceedings of the National Academy of Sciences, USA 92, 1515–9.[Abstract]

40 . Ljungh, A., Moran, A. P. & Wadstrom, T. (1996). Interactions of bacterial adhesins with extracellular matrix and plasma proteins: pathogenic implications and therapeutic possibilities. FEMS Immunology and Medical Microbiology 16, 117–26.[ISI][Medline]

41 . Lelwala-Guruge, J., Ascencio, F., Kreger, A. S., Ljungh, A. & Wadstrom, T. (1993). Isolation of a sialic acid-specific surface haemagglutinin of Helicobacter pylori strain NCTC 11637. Zentralblatt für Bakteriologie 280, 93–106.[ISI][Medline]

42 . Miller-Podraza, H., Milh, M. A., Bergstrom, J. & Karlsson, K. A. (1996). Recognition of glycoconjugates by Helicobacter pylori: an apparently high-affinity binding of human polyglycosylceramides, a second sialic acid-based specificity. Glycoconjugate Journal 13, 453–60.[ISI][Medline]

43 . Wadstrom, T., Hirmo, S. & Boren, T. (1996). Biochemical aspects of Helicobacter pylori colonization of the human gastric mucosa. Alimentary Pharmacology and Therapeutics 10, Suppl. 1, 17–27.[ISI][Medline]

44 . Wilkinson, S. M., Uhl, J. R., Kline, B. C. & Cockerill, F. R. (1998). Assessment of invasion frequencies of cultured HEp-2 cells by clinical isolates of Helicobacter pylori using an acridine orange assay. Journal of Clinical Pathology 51, 127–33.[Abstract]

45 . deBernard, M., Moschioni, M., Papini, E., Telford, J. L., Rappuoli, R. & Montecucco, C. (1998). Cell vacuolization induced by Helicobacter pylori VacA toxin: cell line sensitivity and quantitative estimation. Toxicology Letters 99, 109–15.[ISI][Medline]

46 . Hemalatha, S. G., Drumm, B. & Sherman, P. (1991). Adherence of Helicobacter pylori to human gastric epithelial cells in vitro. Journal of Medical Microbiology 35, 197–202.[Abstract]

47 . Fauchere, J. L. & Blaser, M. J. (1990). Adherence of Helicobacter pylori cells and their surface components to HeLa cell membranes. Microbial Pathogenesis 9, 427–39.[ISI][Medline]

48 . Szmanski, C. M., King, M., Haardt, M. & Armstrong, G. D. (1995). Campylobacter jejuni motility and invasion of Caco-2 cells. Infection and Immunity 63, 4295–300.[Abstract]

Received 1 September 2000; returned 9 October 2000; revised 22 November 2000; accepted 19 January 2001