INTRODUCTION

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Helicobacter pylori is a genetically diverse bacterial species that chronically infects the stomachs of more than half of all people worldwide. Its long-term carriage is a major cause of chronic gastritis and peptic ulcer disease and is an early risk factor for gastric cancer (for reviews see references 5, 8, 28, and 32). Resistance to metronidazole (MTZ) is common and is important clinically as a primary cause of failure of MTZ-based anti-Helicobacter therapies (for reviews see references 10, 15, and 24). Frequencies of clinical isolates that are MTZ resistant range from only 10% in Japan (25) to 90% or more in India (26), and up to 50% or more of strains in the United States and Western Europe also are resistant (frequency varies among countries) (8, 23). These geographic differences probably reflect frequencies of MTZ use against other, mostly parasitic and anaerobic, infections and thus inadvertent MTZ exposure of resident H. pylori strains. Recent studies have implicated mutations in the chromosomal genes rdxA (HP0954) and frxA (HP0642) in the development of resistance (7, 9, 14, 16, 30, 35). These genes encode related nitroreductases that can convert MTZ from a harmless prodrug to products such as hydroxylamine that are both bactericidal and mutagenic (9, 29).

There has been disagreement about the quantitative contributions of rdxA and frxA to MTZ susceptibility and resistance. On the one hand, Kwon and associates had concluded that inactivation of either gene by itself could make any typical H. pylori strain resistant to MTZ (Mtz^r) (21), and that following frxA inactivation, growth on MTZ-containing agar was not associated with mutation of rdxA (20). In contrast, we had concluded that rdxA inactivation is usually or always needed for a Mtz^s strain to become Mtz^r (16, 17). Two types of Mtz^s strains were distinguished, however, based on relative levels of FrxA nitroreductase activity. Most common were strains in which rdxA inactivation by itself was sufficient to cause resistance to moderate levels of MTZ (type I strains). Once rdxA had been inactivated in such type I strains, additional mutations that inactivated frxA (the second nitroreductase gene) resulted in higher levels of resistance, but frxA inactivation by itself had little, if any, effect on MTZ susceptibility if rdxA was still functional. Other strains, designated type II, required inactivation of both frxA and rdxA to become Mtz^r (16, 17). In contrast to Kwon et al. (21), we had not found any Mtz^s clinical isolate that was rendered Mtz^r by frxA inactivation alone.

Different protocols were used by Kwon et al. and ourselves to assess MTZ susceptibility or resistance. Their experiments relied on en masse growth responses after spotting aliquots of dense cultures (21), with resistance defined operationally as growth on the selective MTZ-containing medium (a traditional method for determining the minimal inhibitory concentrations [MIC] of antibacterial agents). Because up to about 0.01% of cells in fresh culture may form Mtz^r colonies on MTZ-containing medium (due to the mutagenic effects of MTZ, followed by selection for resistance [16, 29, and results of this work]), we considered a strain to be resistant only if the efficiencies of colony formation were unaffected by inclusion of MTZ in the medium. That is, cultures of interest were diluted, aliquots of each dilution were spotted, and the numbers of colonies formed at appropriate dilutions were scored on MTZ-containing and on MTZ-free media (12, 13).

The experiments presented here tested several possible explanations for the apparently different results and interpretations of Kwon et al. and ourselves, including (i) regulation of FrxA synthesis or activity by a quorum-sensing (cell density-dependent) mechanism, (ii) use of fundamentally different types of strains by our two groups, and (iii) use of dense rather than dilute cultures to score MTZ resistance, where interpretation of results with dense cultures might have been complicated by the mutagenicity of products of MTZ activation (21). Northern blot hybridization analyses were also carried out and showed that frxA mRNA and perhaps also rdxA mRNA were more abundant in type II than in type I strains.

	MATERIALS AND METHODS

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Bacterial strains and general methods. The Mtz^s H. pylori strains used here and their origins are listed in Table 1. H. pylori strains were grown on brain heart infusion agar (Difco) supplemented with 7% horse blood, 0.4% Isovitalex, and the antibiotics amphotericin B (8 µg/ml), trimethoprim (5 µg/ml), and vancomycin (6 µg/ml) (referred to here as BHI agar), essentially as described previously (16, 17). MTZ was added when needed at concentrations appropriate to each experiment, as detailed below. Rifampin-resistant mutants were selected on medium with 5 µg of rifampin per ml. H. pylori cultures were incubated at 37°C under microaerobic conditions (5% O₂, 10% CO₂, 85% N₂).

Determination of MTZ susceptibility and resistance. Frozen bacterial cultures were streaked on MTZ-free BHI agar and incubated for 3 days. Cells from one or a few colonies from these initial plates were then restreaked on fresh MTZ-free BHI agar and incubated for one more day. The resulting exponentially growing cells were suspended in phosphate-buffed saline (PBS) buffer; a series of 10-fold dilutions of these cell suspensions was prepared, and 10 µl of each dilution was spotted on freshly prepared BHI agar containing various concentrations of MTZ (0, 0.2, 0.5, 1.5, 3, 5, 8, 16, 32, or 64 µg/ml). A strain was considered to be susceptible to concentrations of MTZ that caused at least a 10-fold decrease in the efficiency of colony formation by individual cells (efficiency of plating, or EOP). In our hands this use of aliquots from dilute bacterial cultures was more sensitive and reliable for scoring MTZ susceptibility and resistance of H. pylori than the traditional agar dilution method (16), which relies on growth versus nongrowth after spotting suspensions of at least >10⁵ cells on MTZ-containing medium. The results of a large clinical trial have led others to also conclude that neither agar dilution nor the popular Etest method is reliable for determining MTZ susceptibility or resistance in H. pylori (despite prior approval of agar dilution by the National Committee for Clinical Laboratory Standards) (27).

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Viability and emergence of Mtz^r mutants in high-density cultures. Exponentially growing wild-type and frxA mutant H. pylori cells (1 × 10⁹ to 2 × 10⁹ cells) were suspended in PBS buffer and spread in 4- to 5-cm-diameter circular areas on MTZ-containing (8 µg/ml) BHI medium. Each day cells were scraped from an untouched area within (not at the edges of) these large patches of bacterial growth and were suspended in PBS buffer at an optical density at 600 nm (OD₆₀₀) of 1.0. Ten-microliter volumes of suspension were then spotted directly or after serial dilution on MTZ-free or MTZ-containing BHI plates (0 and 8 µg of MTZ/ml) to measure overall bacterial titer (survival) and titer of resistant mutants. Mtz^r bacteria used for rdxA sequencing were obtained as pools or after purification of single colonies, as appropriate, on BHI agar with 8 µg of MTZ per ml.

mRNA analyses. To prepare RNA for reverse transcriptase (RT)-PCR (Fig. 1) or Northern blots (Fig. 2 and 3), cells growing exponentially on BHI agar were subcultured and grown for another 16 h, collected, and washed three times with 10% glycerol in distilled water at 0°C. Total RNA was isolated and prepared for RT-PCR and Northern hybridization essentially as described previously (2, 17) using a Qiagen RNeasy kit and treatment of RNA preparations with RNase-free DNase I (RT-PCR) or by phenol-chloroform and isoamyl extraction and ethanol precipitation (Northern hybridization). To check RNA quality, a few micrograms from each sample was electrophoresed in agarose, and the ethidium bromide-stained gel was inspected to make sure that the ratio of intensities of 23S and 16S rRNAs was about 2:1 and that there was little, if any, accumulation of the small RNA fragments (probably degradation products) that run just ahead of the 0.2-kb RNA size marker (Perfect RNA Markers, 0.2 to 10 kb; Novagen, Madison, Wis.).

View larger version (45K): [in this window] [in a new window]   FIG. 1.   RT-PCR amplification of rdxA and frxA gene segments from total RNA of type I and type II strains. The strains tested are as follows: lane 1, HK192; lane 2, 26695; lane 3, HUP57; lane 4, R10; lane 5, X47; lane 6, SS1. The primers frxRT-F2 and frxART-R2 were used to amplify frxA gene segments (334 bp). The primers rdxRT-F and rdxRT-R2 were used to amplify rdxA gene segments (238 bp).

View larger version (62K): [in this window] [in a new window]   FIG. 2.   DNA sequence of probe affects efficiency of detection of transcripts. RNAs from seven strains were electrophoresed and blotted for Northern analysis in triplicate and probed with rdxA probe DNAs prepared from each of three different strains. The strains used for RNA (and in three cases, DNA probe) preparation were as follows: lane 1, 26695; lane 2, HK192; lane 3, HUP57; lane 4, SS1; lane 5, R10; lane 6, X47; lane 7, 2667 (strains in lanes 1 to 3 are type I and strains in lanes 4 to 7 are type II). 23S and 16S rRNA were visualized after being stained with methylene blue (23S rRNA shown in bottom panel) to ensure that the RNA was of good quality and that equal amounts were loaded in each lane.

View larger version (83K): [in this window] [in a new window]   FIG. 3.   frxA and rdxA mRNA transcript levels in type I and type II strains detected with strain-specific probes. Each of the 18 RNA transcripts (rdxA and frxA in each of nine strains) was detected with a probe generated by PCR from genomic DNA from the corresponding strain and DIG labeling. Small differences in amounts or activity of labeled probe were compensated for by using the probe DNA itself as an internal standard and varying exposure for times, ranging from 1 to 5 s, in order to allow direct comparison of amounts of frxA and of rdxA mRNA among the strains tested. The three type I strains tested in this way were as follows: lane 1, 26695; lane 2, HK192; lane 3, HUP57. The six type II stains tested were as follows: lane 4, SS1; lane 5, R10; lane 6, X47; lane 7, 2600; lane 8, 2667; lane 9, 2714.

	RESULTS

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We tested several possible explanations for the apparently different results and proposed mechanisms of MTZ resistance: frxA inactivation is a major cause of MTZ resistance (21) versus frxA inactivation by itself rarely, if ever, confers resistance (16).

Effect of frxA on mutation frequency. One possible explanation of previous results (20, 21) holds that frxA inactivation rendered H. pylori highly mutable when grown at high cell density, conceivably by perturbing metabolite balances that, in turn, decreased the fidelity of DNA replication or potency of DNA repair (see, e.g., reference 11). This was tested by measuring frequencies of rifampin-resistant mutants (which generally result from point mutations in an RNA polymerase gene [13]) in cultures of five wild-type MTZ-susceptible strains and in their isogenic frxA knockout mutant derivatives. Table 3 shows that frxA inactivation had no significant effect on the frequency of spontaneous mutants, and furthermore that it decreased the yield of MTZ-induced mutants several-fold. Thus, the postulated development of a Mtz^r phenotype by frxA inactivation (21), if correct, would not be due to H. pylori entry into a hypermutable state.

Kinetics of MTZ killing at high density. Several MTZ-susceptible wild-type strains and their frxA knockout mutant derivatives were used to test a model in which incubation at high cell density would render H. pylori tolerant or resistant to MTZ phenotypically, without additional mutation. This entailed inoculation of MTZ-susceptible bacteria at high cell density on MTZ-containing medium, scraping them from the agar surface after one or more days of incubation, and determining bacterial titers on media with and without MTZ. The results (Table 4) showed that all cells that would be considered MTZ-susceptible genetically (unable to form isolated colonies on MTZ-containing medium) were also killed eventually during incubation at high density with MTZ. The kinetics of cell killing depended on both background genotype and whether frxA was functional or not. For example, strain 26695 (type I) was killed more rapidly than HUP57 (also type I) and X47 (type II) (compare titers in Table 4 for day 1, 0 µg of MTZ per ml).

Mutation in rdxA needed for frxA mutant strains to become Mtz^r. We next tested the suggestion (20) that Mtz^r derivatives of frxA knockout mutant strains selected initially at high cell density rarely, if ever, carried new mutations in rdxA. Mtz^r mutants were selected by spreading cells from dense cultures of frxA knockout mutant derivatives of four type I lineages and one type II lineage on MTZ-containing agar, as above, and incubating them for three days. Mtz^r colonies formed by single cells were obtained later by streaking diluted aliquots of the dense suspensions of Mtz^r cells on new MTZ-containing agar plates. rdxA was PCR amplified from cell suspensions incubated on MTZ-containing agar and from single Mtz^r colonies, and the products were sequenced. Table 5 shows that each Mtz^r single colony isolate contained a sequence change in rdxA, resulting variously in frameshifts, translation stop codons, or amino acid additions or replacements that would also likely inactivate the gene. In contrast, the rdxA sequence profiles obtained after selecting confluent lawns of Mtz^r mutant bacteria matched those from the wild-type parent (data not shown). This might be due to the presence of numerous independent Mtz^r mutants in each bacterial pool. In this case, the sequence change in any single mutant allele would be obscured by the many other sequences containing unchanged nucleotides at that site (only when cultures contain just one or a few abundant mutants can each allele be detected reliably). Alternatively, it might reflect DNA extraction before new Mtz^r mutants had adequately outgrown their Mtz^s siblings (e.g., as shown in Table 4). Either interpretation might explain the seeming wild-type rdxA sequence in H. pylori collected from MTZ-containing agar that was reported previously (20).

Type II Mtz^s strains and frxA. Three of the clinical isolates whose initial studies had led Kwon and colleagues (20) to conclude that frxA inactivation by itself caused MTZ resistance, strains 2600, 2667, and 2714 (kindly provided by D. H. Kwon and D. Y. Graham) were studied and found to each be highly susceptible to MTZ. In particular, as with type II reference strains from elsewhere, Mtz^r mutant colonies were found at frequencies of 10⁸ on medium with 8 µg of Mtz per ml (see Table 6). In contrast, Mtz^r mutant colonies were found in cultures of type I strains at frequencies of about 10⁴ (16, 17). There was some variability among type II strains in survival on medium with lower levels of MTZ (e.g., with 3 µg of MTZ per ml, ~10⁶ with each of D. H. Kwon's strains and five of the seven other type II strains, but ~10⁸ with the other two; Table 6). This variability is in accord with the considerable genetic diversity among H. pylori strains, as will be detailed below.

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frxA mRNA is more abundant in type II than in type I cells. Initially we sought to use RT-PCR (as in reference 17) to test the hypothesis that frxA mRNA is more abundant in type II than in type I strains. Although results with one type II and one type I strain (SS1 and 26695) had supported this view (17 and Fig. 1), results with other strains were erratic. In particular, no frxA RT-PCR product was detected from strains R10 and X47, using rdxA mRNA as an internal standard (RT-PCR with frxA and rdxA primers together), despite mutational evidence showing that acquisition of Mtz^r in these strains depended on inactivation of frxA as well as rdxA (Table 7). Various ratios of frxA and rdxA RT-PCR products were obtained from several other type II strains, and one type II strain did not seem to yield an rdxA RT-PCR product at all (see Fig. 1). These unexpected results were obtained repeatedly in trials with RNA preparations from different exponentially growing cultures of the same strain. Accordingly, much of the variation among strains in RT-PCR results was ascribed provisionally to effects of sequence (e.g., secondary structure in single-stranded nucleic acids) on efficiency of amplification rather than differences in levels of target mRNAs.

	DISCUSSION

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The present experiments, initiated to test two alternative views of how H. pylori becomes resistant to MTZ, have given new insights into the genetic diversity of this species. Two types of MTZ-susceptible strains were identified by genetic (mutational) and molecular (Northern blot) tests. Those designated type I needed inactivation only of rdxA to become resistant, apparently because only the rdxA nitroreductase gene was well expressed. Type II strains, in contrast, needed inactivation of both rdxA and the related frxA gene to become resistant, apparently because each gene was highly active. These two Mtz^s strain types were also distinguished by frequencies of MTZ-resistant mutants: about 10⁴ and 10⁸ in cultures of type I and type II strains, respectively, reflecting the need to inactivate just one gene (rdxA) versus two genes (rdxA and frxA) to become resistant. Our tests also showed that frxA inactivation slowed the killing by MTZ of type I strains with functional rdxA genes and increased the resistance of type I derivatives that were already mutated in rdxA (although frxA inactivation by itself did not confer MTZ resistance if rdxA remained functional). These subtle effects of frxA inactivation implied that frxA can be expressed in type I strains, although too weakly for detection in our Northern blots. Given the initial aim of our study, it is noteworthy that these results were also obtained with strains used by another group (21) that had interpreted that frxA inactivation by itself could render H. pylori MTZ resistant. The present results suggest that their interpretation is incorrect.

We ascribe the differences in interpretation to technical aspects of how susceptibility versus resistance was scored. We monitored the susceptibility or resistance of individual cells to MTZ by diluting cultures sufficiently to count colonies formed on media with and without MTZ. This quantitative plating method, although common in bacterial genetics, contrasts with traditional practice in clinical microbiology of scoring (i) growth responses of spots of dense cell suspensions on medium containing the antibiotic (as in reference 21) or (ii) zones of growth inhibition near antibiotic-containing disks or Etest strips laid on dense bacterial lawns. The special value of quantitative plating tests when scoring MTZ resistance stems from the mutagenicity of MTZ and consequently the high frequency of MTZ-resistant mutants (10⁴) when mutation in only one gene is needed. This high frequency is enough to sometimes give a misleading appearance of resistance in traditional qualitative en masse growth tests (as in reference 21), a problem that may be exacerbated by the delayed killing of strains that are MTZ susceptible but contain frxA null alleles. Such traditional MTZ resistance tests were also deemed unreliable in a recent large clinical trial (27).

A second technical feature of our experiments also merits comment: the need for strain-specific hybridization probes when comparing transcript levels in different strains. Although initial RT-PCR with strains 26695 (type I) and SS1 (type II) had demonstrated frxA mRNA only in SS1 (17) as expected, RT-PCR with other type II strains often revealed little, if any, frxA product. Erratic results were also obtained when Northern hybridization with a single probe was used to monitor levels of rdxA mRNA in several strains (Fig. 2). These problems can be ascribed to sequence diversity among H. pylori strains (typically some 4 to 6% in loci such as rdxA and frxA) and consequent differences in relative efficiencies of amplification and hybridization between imperfectly matched probe and transcript in Northern blots. Accurate Northern blot results were obtained by generating strain-specific gene probes for each strain of interest, labeling each probe individually, and using each in a separate hybridization. Internal DNA calibration standards were included in each hybridization to allow valid quantitative interstrain comparisons.

Much remains to be learned about the metabolic phenomena that affect MTZ susceptibility and resistance, including the mechanisms underlying the diversity among strains in apparent levels of the related nitroreductases studied here and the actual roles of each nitroreductase during human gastric infection. In particular, although strains with highly expressed frxA genes as defined here seem to be uncommon (10% of Mtz^s isolates) in many populations (16), we have recently found that about half of Mtz^s strains from Lithuania are of this type (G. Dailide, D. Kersulyte, D. Dailidiene, J. Miciuleviciene, and D. E. Berg, unpublished data). Neither the factors underlying this geographic difference nor its generality (i.e., in other Baltic or Northern European countries) are known. That the type I versus type II difference might be adaptive and significant physiologically, however, is suggested by a recent finding that frxA mutants of the type II strain SS1 grow less well in mice than does the wild type (J. Y. Jeong, D. Dailidiene, A. K. Mukhopadhyay, and D. E. Berg, unpublished data). We are attracted to the possibility that the observed differences in gene or enzyme activity may often be significant physiologically for H. pyloriperhaps affecting bacterial nutrition and the persistence of infection in various human hosts.