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Journal of Clinical Microbiology, May 2006, p. 1650-1658, Vol. 44, No. 5
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.5.1650-1658.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Nutritional Requirements and Antibiotic Resistance Patterns of Helicobacter Species in Chemically Defined Media

Traci L. Testerman,1* P. Brian Conn,2 Harry L. T. Mobley,3 and David J. McGee1

Louisiana State University Health Sciences Center, Department of Microbiology and Immunology, Shreveport, Louisiana 71130-3932,1 University of South Alabama, Biology Department, Mobile, Alabama,2 University of Michigan, Department of Microbiology and Immunology, Ann Arbor, Michigan3

Received 24 June 2005/ Returned for modification 16 September 2005/ Accepted 10 March 2006


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ABSTRACT
 
The growth of the gastric pathogen Helicobacter pylori in the absence of serum remains challenging, and nutritional requirements have only partially been defined, while almost nothing is known about nutritional requirements of other Helicobacter spp. Although previous data showed that H. pylori grows in the chemically defined medium F-12, but not in other tissue culture media examined, the specific components responsible for growth were not entirely understood. Here we describe the optimization of amino acids, metals, and sodium chloride for H. pylori. Iron, zinc, and magnesium were critical for growth; copper was not required. Optimization of sodium chloride was further beneficial. Nutritional requirements and antibiotic resistance patterns of several other Helicobacter spp. revealed that all except H. felis grew in serum-free, unsupplemented F-12. All Helicobacter spp. were resistant to at least six antimicrobial agents when cultured in the presence of serum. However, in the absence of serum, H. pylori, H. mustelae, and H. muridarum became sensitive to polymyxin B and/or trimethoprim. Much of the data were obtained using a convenient ATP assay to quantify growth. H. pylori has surprisingly few absolute requirements for growth: 9 amino acids, sodium and potassium chloride, thiamine, iron, zinc, magnesium, hypoxanthine, and pyruvate. These data suggest that H. pylori and other Helicobacter spp. are not as fastidious as previously thought. The data also suggest that chemically defined media described herein could yield the growth of a wide range of Helicobacter spp., allowing a more detailed characterization of Helicobacter physiology and interactions with host cells.


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INTRODUCTION
 
The expanding Helicobacter genus currently contains more than 35 species, with newly recognized species being isolated from a wide range of hosts, such as dolphins and birds (6, 8). All Helicobacter species are characterized as fastidious, and most are associated with gastric or extragastric diseases. Nutritional requirements of the human gastric pathogen, H. pylori, are not completely understood, and less is known about nutritional requirements of other Helicobacter species. One important member of the Helicobacter genus, H. heilmannii, has not yet been cultured in vitro. This organism colonizes humans and several other species and may cause diseases similar to those attributed to H. pylori (11, 16). Comparison of nutritional requirements and antibiotic resistance profiles of other Helicobacter species may produce reasonable predictions about the metabolic properties of H. heilmannii. This information could facilitate future cultivation of this important bacterium.

The existence of two complete H. pylori genome sequences is a valuable tool for predicting metabolic pathways (1, 24), but in silico studies are not yet reliable enough to provide a complete picture of nutritional requirements. An in silico model based on known and putative genes for metabolic enzymes predicted a requirement for 8 to 9 amino acids, oxygen, phosphate, and thiamine but did not make any predictions on metal requirements (21). Furthermore, the aforementioned study predicted that purines (adenine, guanine, and hypoxanthine) should not be required for the growth of H. pylori, in contrast to previously published findings (19), and did not provide insight into the relative concentrations of nutrients required for optimal growth.

Several published studies focused primarily on amino acid requirements of H. pylori (17, 19, 20, 22). Additional studies revealed that H. pylori prefers to use amino acids rather than sugars as carbon sources (17). Our discovery that Ham's F-12 (referred to hereinafter as F-12), a commonly used tissue culture medium, supports the growth of H. pylori in the absence of serum or protein, opened up many possibilities for H. pylori research but also raised new questions about the nutritional requirements of the organism (23). Although F-12 supports more-robust and consistent H. pylori growth than other chemically defined media, the addition of fetal bovine serum (FBS) dramatically improves growth, suggesting that further optimization or supplementation with defined nutrients is necessary. A better understanding of H. pylori nutritional requirements could elucidate the role of serum in H. pylori growth, and these findings may be applicable to other fastidious, serum-requiring organisms as well. We therefore embarked upon a study to determine which components of F-12 medium are essential or beneficial and to determine whether alterations in the F-12 formula might further improve growth. We further sought to compare growth requirements of other Helicobacter species with those of H. pylori.

H. pylori is resistant to some antimicrobial agents, such as trimethoprim and polymyxin B, that kill many other gram-negative organisms. When these antibiotics are combined with antifungal agents and antimicrobials specific for gram-positive organisms, it is possible to inhibit the vast majority of gastric and fecal microbial floras from animal stomachs and feces (15). Such inhibition is of tremendous value in attempts to isolate H. pylori from sources in which it may be present in small numbers.

H. pylori and most other Helicobacter species achieve growth densities too low for reliable spectrophotometric measurement in protein-free, chemically defined media, presenting a challenge for quantifying growth. Moreover, counts of CFU are time-consuming and labor intensive, and dilution counts are impossible for species, such as H. cinaedi, that do not form isolated colonies on plates. H. pylori organisms frequently aggregate when cultured in the presence of serum, potentially reducing the accuracy of viable counts. Other means of assessing the growth and viability of Helicobacter species were therefore needed.

In this study, we present an alternative method of quantifying viable Helicobacter organisms. A commercially available ATP assay kit permits rapid and quantitative measurement of growth. Our studies show that several Helicobacter species can be grown in chemically defined media. We also demonstrate that H. pylori and other Helicobacter species have similar antibiotic resistance patterns when cultured in the presence of serum but that H. pylori, H. mustelae, and H. muridarum become sensitive to trimethoprim and/or polymyxin B when cultured without serum. The data presented in this study will allow investigators to formulate custom media for their particular applications and allow a more detailed characterization of Helicobacter physiology, gene regulation, and interactions with host cells.


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MATERIALS AND METHODS
 
Bacterial strains and cultivation. Bacterial strains used in this study are listed in Table 1. H. pylori was cultured in a humidified environment at 37°C with 5% CO2. Non-H. pylori Helicobacter species were cultured in an atmosphere of 5% O2, 10% CO2 unless otherwise noted. Campylobacter agar (Becton Dickinson, Sparks, MD) containing 10% defibrinated sheep blood (Quad Five, Ryegate, MT) (CBA) was used for routine cultivation of Helicobacter species, and bacteria were passaged every 2 to 3 days. Broth cultures were grown in 25-cm2 tissue culture flasks containing 5 to 7 ml of medium or in 24-well plates containing 1 ml per well. Our original stock of H. pylori strain 26695 (from K. Eaton) is nonmotile, but a spontaneous motile variant (26695m) was isolated in our laboratory.


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  		TABLE 1. Strains used in this study

Determination of nutritional requirements. Care was taken not to increase the total volume of each culture by more than 5%. When the volume of added components exceeded 2% of the initial volume, equivalent volumes of water were added to wells as a control. Amino acid stock solutions were prepared in distilled, deionized water at concentrations 100-fold higher than those found in Ham's F-12 (catalog no. SH30026.02; HyClone, Logan, UT), with pH adjustments made as necessary to improve solubility. Sterile stock solutions of other chemicals to be tested were prepared in distilled, deionized water at concentrations 1,000 times greater than the working concentration when possible. Compounds with limited solubility were prepared at the highest feasible concentrations. Final medium formulations had pH values similar to that of F-12 based on the color of the phenol red indicator. Other tissue culture media used were RPMI 1640 with 2.05 mM L-glutamine (catalog no. SH30027.01; HyClone); Dulbecco's modified Eagle's medium (DMEM) with high glucose, L-glutamine, sodium pyruvate, and pyridoxine HCl (catalog no. 11995-065; Invitrogen, Carlsbad, CA); and McCoy's 5a with L-glutamine and 25 mM HEPES (catalog no. 12330-031; Invitrogen). Experimental media were tested in at least three independent experiments to ensure reproducibility. Trace element solutions A and B (catalog no. 99-182 and 99-175, respectively; Mediatech, Herndon, VA) were used to assess the need for additional metals. Bovine serum albumin (BSA) was from Sigma-Aldrich (catalog no. A-7906; low-fatty acid BSA, catalog no. A-7030; Sigma-Aldrich, St. Louis, MO).

Assessment of growth. H. pylori was cultured in F-12 supplemented with 1% heat-inactivated FBS. An aliquot of the culture was pelleted at 16,000 x g at room temperature for 1 min and resuspended in phosphate-buffered saline (PBS) or medium, as indicated in the text. Twenty-four-well plates containing 1-ml aliquots of F-12 were inoculated with 10 µl of the washed bacteria per well. Unmodified F-12 with and without 1% FBS were used as controls, and experimental media were tested in duplicate wells. Plates were incubated at 37°C in an atmosphere of 5% CO2 for 18 to 30 h. Helicobacter species unable to grow in an atmosphere of 5% CO2 were instead incubated in jars containing CampyPak Plus (Becton Dickinson, Sparks, MD) envelopes to generate a microaerobic atmosphere or in a humidified incubator maintained at 5% O2, 10% CO2, and 85% N2. Growth under each test condition was quantified by using a bioluminescent-ATP assay, as described below.

Numbers of CFU were assessed by performing serial 10-fold dilutions in PBS. Aliquots (50 µl) of each dilution were spotted in duplicate onto predried CBA plates and incubated at 37°C in an atmosphere of 5% CO2 for 4 to 5 days.

ATP assay. Growth under each test condition was quantified using a bioluminescent-ATP assay (CellTiter-Glo, catalog no. G7571; Promega), with sterile F-12 or a derivative thereof serving as a blank. Light production was measured on a Turner TD20e luminometer (Promega). For routine growth assessments, relative light unit values were compared only to other values within the same experiment, making determination of ATP concentrations unnecessary. Validation has been performed by numerous comparisons between colony counts and ATP values (data not shown).

Resistance to antimicrobial agents. Helicobacter species were tested for their abilities to form isolated colonies on CBA plates containing all of the following: vancomycin, 10 µg/ml; trimethoprim, 10 µg/ml; amphotericin B, 5 µg/ml; polymyxin B, 10 µg/ml; bacitracin, 30 µg/ml; and flucytosine, 5 µg/ml. Plates were incubated in an atmosphere of 5% CO2 or in a microaerobic environment generated by using a CampyPak Plus system, as noted in the tables. Susceptibility to individual antibiotics was assessed in F-12 medium with no serum or with FBS. Growth in the presence of antibiotics was assessed by microscopic examination (Olympus IMT-2 phase-contrast microscope) and ATP content.


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RESULTS
 
Validation of an ATP assay to assess Helicobacter growth. Since spectrophotometric analysis of cells cultured in chemically defined media is not sufficiently sensitive to evaluate Helicobacter species growth, a sensitive, rapid method of quantifying growth was sought. We have found the luciferase-based CellTiter-Glo ATP assay kit to be a rapid and reliable means for assessing the viability of H. pylori and for comparing levels of growth of H. pylori in experimental media. Although the assay kit was originally designed for use with eukaryotic cells, we have found that the lysis buffer successfully lyses H. pylori. Comparisons between plate counts and relative light units obtained by the ATP assay kit revealed excellent correlation between the two methods, especially when bacterial densities were above 104 CFU/ml. ATP assays performed on a single, serially diluted culture yields a straight line, with an r2 of >0.9 (data not shown). Figure 1 shows one sample experiment comparing ATP and CFU measurements obtained from 10 independent cultures. ATP concentrations as low as 10 pM can be detected (data not shown). Background luminescence (blank) values result mostly from the assay reagent unless the medium contains ATP. Various types of media or solutions, including PBS, F-12, and Mueller-Hinton broth, do not significantly influence background luminescence. Brain heart infusion broth increases background luminescence slightly, likely due to trace ATP from heart tissue. The ATP assay is extremely reliable for detecting growth differences between samples assayed on the same day and has good day-to-day reproducibility. If one wishes to make comparisons between samples assayed on different days with absolute confidence, an ATP standard can be included. This ATP assay also works with other Helicobacter species and is thus used throughout the study to assess viability.


Figure 1
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  		FIG. 1. Validation of ATP assay to measure the viability of H. pylori. H. pylori 26695m was cultured overnight in an atmosphere of 5% CO2. Parallel samples were diluted and plated on Campylobacter blood agar or assayed for ATP content. ATP results are expressed as relative light units (rlu). Each point on the graph represents an independent culture in various experimental media. One ATP measurement was performed with each culture, and colony counts from duplicate spots were averaged.

It should be noted that the data presented were obtained with an older luminometer. A recently purchased Turner Biosystems model 20/20n luminometer yields blank and experimental relative light units approximately 1,000-fold greater than those yielded by the older model. These higher numbers are intuitively more satisfying but do not seem to influence the sensitivity of the assay.

Amino acid requirements of H. pylori. In addition to confirming amino acid requirements, we hoped to identify specific amino acid combinations and/or concentrations which would lead to improved growth of H. pylori compared to that achieved in F-12. Experiments were carried out using a basal medium lacking amino acids but containing the compounds listed in Table 2, which were at the same concentrations as those in F-12. To a concentrated stock of this medium, each of the 20 amino acids was separately added to different H. pylori cultures. Water was added as necessary to bring the final concentrations of nutrients to levels found in F-12. We found that arginine, histidine, leucine, methionine, phenylalanine, valine, alanine, cysteine, and proline were absolutely required for growth in the absence of serum. A medium, TT14 (Table 3), containing the above-named amino acids was used as a basis for investigating further amino acid additions. Sulfate (as MgSO4) could not substitute for cysteine (data not shown). Our findings largely agree with those reported by other investigators (Table 4), but we found that the combination of tryptophan and isoleucine is very beneficial for H. pylori growth when added to TT14 (Fig. 2). Addition of neither tryptophan nor isoleucine alone to TT14 yielded any improvement in growth (data not shown). Glutamine, glutamate, and serine have been reported to be used as carbon sources by H. pylori (22). We found, however, that none of these three amino acids augments growth, unless tryptophan and isoleucine are also present (Fig. 2). Four additional H. pylori strains (Table 1) displayed slight, but reproducible, growth in TT14 and increased growth when tryptophan, isoleucine, and glutamic acid were added, suggesting that the reported growth requirements are not unique to 26695m. Aspartate and asparagine are also used as carbon sources by H. pylori (22), but we found no growth augmentation by either of these amino acids when added to TT14 plus tryptophan and isoleucine or by supplementation of F-12 with aspartate and asparagine (twice above the original F-12 concentration). A doubling of the concentrations of all amino acids similarly failed to boost growth. In contrast with a previous report (19), we did not find any growth inhibition by glycine or lysine. Since alterations of amino acid composition in chemically defined medium derivatives failed to achieve better H. pylori growth than that obtained in standard F-12, the amino acid composition of F-12 does not appear to limit growth.


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  		TABLE 2. Non-amino acid components of the chemically defined medium F-12a


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  		TABLE 3. Comparison of components in derivatives of synthetic chemically defined media


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  		TABLE 4. Comparison of H. pylori amino acid requirements in chemically defined medium derivatives


Figure 2
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  		FIG. 2. Effect of amino acid composition on the growth of H. pylori in chemically defined media. Medium TT14 contains the essential elements listed in Table 2, along with the amino acids listed in Table 3. Additional amino acids were added as indicated at final concentrations equal to those found in F-12. Wells were inoculated with washed 26695m and incubated overnight in an atmosphere of 5% O2, 10% CO2. Growth was assessed by assaying for ATP. Data represent ATP measurements from duplicate cultures of each medium. rlu, relative light units.

Salts and metals required by H. pylori. We formulated a medium, TT17 (Table 3), based on F-12 that lacked metals and salts other than NaCl to test the importance of each element individually. Concentrated stock solutions of each metal or salt (500x to 1,000x) were added to TT17, yielding final concentrations that were the same as that of F-12. Bacteria were resuspended in TT17 prior to inoculation, but no further precautions were taken to prevent the carryover of elements from F-12-grown cultures. We consistently achieved some growth in the absence of calcium, although ATP values tended to be about 50% of those obtained with all elements present (Fig. 3). We could not detect significant growth in the absence of zinc, iron, magnesium, or KCl (Fig. 3). The zinc requirement is consistent with our previous report, showing that decreased zinc concentrations reduce growth (23). We have not detected any difference in growth when copper is omitted, even after passaging bacteria several times in the absence of copper. We cannot rule out a requirement for trace copper to be present as a contaminant in other compounds, but copper supplementation of media is clearly not required for the growth of H. pylori. Addition of two trace element solutions did not impact growth, suggesting that other elements, such as manganese, selenium, vanadium, and molybdenum, are not required in greater than trace amounts.


Figure 3
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  		FIG. 3. Effect of metals on the growth of H. pylori in chemically defined media. Medium TT17 contains essential elements and all 20 amino acids but lacks all metals and salts except sodium chloride (Table 3). TT17+all is TT17 plus Zn, Ca, Mg, KCl, and Fe. Cultures were inoculated with washed 26695m and incubated overnight in an atmosphere of 5% CO2. Growth was assessed by assaying for ATP. Data represent ATP measurements from duplicate cultures of each medium. rlu, relative light units.

Optimal sodium chloride concentration for growth of H. pylori. Optimal sodium chloride concentrations were determined by varying the concentration of sodium chloride in our chemically defined medium derivative TT8 without altering concentrations of other salts. H. pylori grew optimally at a salt concentration equal to 80% of the normal F-12 concentration, or 104 mM (Fig. 4). Growth declined below that obtained in F-12 when NaCl concentrations were less than 60% of the salt present in F-12. Equimolar KCl concentrations cannot replace NaCl (data not shown). Similarly, dilution of standard F-12 with water to 80% strength to reduce sodium chloride concentration resulted in a slight boost in growth, in spite of concomitant reductions in other nutrients (data not shown).


Figure 4
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  		FIG. 4. Effect of NaCl on the growth of H. pylori in chemically defined media. 26695m was cultured in chemically defined medium TT8 with various NaCl concentrations. Cultures were inoculated with washed 26695m and incubated overnight in an atmosphere of 5% CO2. Growth was assessed by assaying for ATP. Data represent ATP measurements from duplicate cultures of each sodium chloride concentration. rlu, relative light units.

Optimal iron concentration. F-12 contains 3 µM ferrous sulfate (FeSO4). We initially supplemented F-12 medium with additional FeSO4, to a concentration of 75 µM. This supplementation resulted in improved H. pylori growth (data not shown). To ensure that the growth effect was due to iron and not sulfate, we performed experiments in which comparable molar concentrations of ammonium sulfate or magnesium sulfate were added. Neither of these compounds resulted in growth augmentation at any concentration tested (up to 75 µM) (data not shown). The insolubility of ferrous sulfate made further increases in iron concentration difficult. For this reason we switched to the more soluble ferric chloride and ferrous chloride. Both forms of iron stimulated growth, with maximum culture density occurring at ferrous or ferric iron concentrations between 50 and 100 µM (Fig. 5). Although 10 mM stock solutions of FeCl2 and FeCl3 can be prepared, both tended to cause precipitation when added to F-12 medium, especially at concentrations above 50 µM. Therefore, we are uncertain whether the decline in growth seen at iron concentrations above 100 µM is due to iron toxicity or to the coprecipitation of other key elements in the medium. Visible precipitates are more abundant when FeCl2 is used than when FeCl3 is used. Interestingly, the effect of additional iron disappeared when 1% serum was added to the medium (data not shown).


Figure 5
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  		FIG. 5. Effect of ferrous and ferric iron on the growth of H. pylori in chemically defined media. H. pylori was cultured in F-12 supplemented with additional iron as FeCl2 or FeCl3 in an atmosphere of 5% CO2. Growth was assessed after 24 h by measurement of ATP content. Data represent ATP measurements from duplicate cultures of each iron concentration. rlu, relative light units.

Vitamin and other requirements. The only vitamin found to be required for the growth of H. pylori was thiamine, and only trace amounts were necessary. For example, when H. pylori was passaged from thiamine-containing media to thiamine-free chemically defined media, H. pylori initially grew. However, the organism did not successfully passage unless thiamine was added. F-12 contains approximately 1 µM thiamine. Although a dose-response curve was not generated, less than 1 µM thiamine is clearly sufficient, since carryover from the previous F-12 culture is enough to permit growth for one passage. No other vitamin or combination of vitamins was found to influence growth. We found no benefit from the addition of lipoic acid, linoleic acid, or putrescine. We examined the requirement for hypoxanthine and pyruvate at oxygen concentrations ranging from 5% to 19% and found these components to be required under all growth conditions (data not shown).

Modification of other tissue culture media. H. pylori does not grow in any unsupplemented tissue culture medium tested except F-12 (23). Armed with a more complete picture of H. pylori nutritional requirements, we supplemented several other tissue culture media with the required components that were either lacking or at insufficient levels to determine if growth would occur. Supplements were added in concentrations matching those in F-12. McCoy's 5a medium (Table 5) yielded nearly as much growth as F-12 when iron, zinc, hypoxanthine, and pyruvate were added and when these four plus alanine were added to RPMI 1640 (data not shown). Some growth could be obtained in DMEM supplemented with iron, zinc, hypoxanthine, alanine, proline, and cysteine, although growth in supplemented DMEM remained lower than that obtained in F-12. Medium 199 did not yield any growth, even with addition of iron, zinc, hypoxanthine, and pyruvate. Medium 199 contains Tween 80, which is toxic to H. pylori at the concentrations found in this medium (data not shown), suggesting that Tween 80 may be the ingredient inhibiting growth in medium 199.


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  		TABLE 5. Comparison of tissue culture media

Requirements of non-H. pylori Helicobacter species. Since no chemically defined media have been described for other Helicobacter spp., it was of interest to establish whether F-12 would support the growth of some of these other species. We have successfully cultured H. cinaedi, H. mustelae, H. muridarum, and H. hepaticus in F-12 chemically defined medium, both with and without serum (Table 6). Each species was passaged at least three times without serum to eliminate effects of serum carried over from the original culture. Some species reached higher densities in F-12 than did H. pylori (Table 6). Cultures were followed for several days to rule out effects of possible growth rate differences between species, and growth was assessed by ATP assay, visible turbidity, and microscopic examination of cultures. H. cinaedi reached a slightly higher density when cultured in the presence versus the absence of serum in some experiments. H. felis is the only species tested that is unable to grow in F-12 without serum supplementation. This species grows in 5% CO2, 5% O2-10% CO2, or in a microaerobic environment generated by using a CampyPak in an anaerobic jar (Campy jar). H. cinaedi and H. hepaticus could not be reproducibly cultured in an atmosphere of 5% CO2 (Table 6). These species required a lower oxygen concentration, such as 5% O2, or a Campy jar. While H. pylori, H. felis, H. muridarum, and H. mustelae could be cultured either in 5% CO2 or at lower oxygen concentrations, growth was typically better under low oxygen tension. Although serum is required for H. felis and dramatically increases the density of H. pylori and H. mustelae cultures, the same was not true for H. muridarum and H. hepaticus, which were ambivalent to the presence of serum (Table 6).


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  		TABLE 6. Growth characteristics of Helicobacter species in F-12 chemically defined mediaa

We compared ATP contents of cultures grown in unsupplemented F-12 to that of F-12 cultures supplemented with ferrous iron at a concentration of 50 µM. Unlike H. pylori, none of the other species showed a growth benefit in the presence of additional iron. H. felis was unable to grow in the absence of serum even when F-12 was supplemented with extra iron (data not shown).

Antibiotic resistance in the presence or absence of serum. All of the Helicobacter species used in this study grew on CBA containing vancomycin, trimethoprim, bacitracin, polymyxin B, flucytosine, and amphotericin B. Similarly, all species thrived in F-12 containing all of these antimicrobials, as long as serum was also present. In the absence of serum, H. pylori surprisingly became sensitive to both polymyxin B and trimethoprim, as assessed by ATP and microscopic observation of cultures for morphology (coccoid versus bacillary) and motility. H. muridarum showed sensitivity to polymyxin B in the absence of serum, while H. mustelae showed sensitivity to trimethoprim when cultured without serum. H. cinaedi and H. hepaticus did not become sensitive to either of the above antibiotics in the absence of serum. H. felis could not be tested due to the serum requirement for growth. While antibiotic sensitivity increased when some Helicobacter species were cultured without serum, this observation did not correlate with serum growth benefit. For example, Helicobacter species that grow better in the presence of serum (H. pylori and H. mustelae) are not more sensitive to antibiotics in the absence of serum than those species (H. hepaticus and H. muriadarum) whose growth is ambivalent to the presence of serum.

Helicobacter spp. incorporate cholesterol into their membranes (7, 9). We therefore hypothesized that cholesterol scavenged from serum might influence antibiotic sensitivity, especially to polymyxin B. To test this hypothesis, we cultured H. pylori in F-12 in the absence or presence of cholesterol, BSA, or fatty acid-free BSA. Addition of 1 mg/ml BSA or low-fatty acid BSA to F-12 cultures restored the ability of H. pylori to grow in the presence of trimethoprim but not polymyxin B. Commercially available BSA contains a number of other serum proteins as contaminants (23), so we cannot say whether the albumin itself or a contaminant is responsible for the restoration of growth in the presence of trimethoprim. Cholesterol, added alone or in the presence of BSA, had no impact on the susceptibility of H. pylori to either trimethoprim or polymyxin B (data not shown).


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DISCUSSION
 
For the majority of Helicobacter researchers who are interested primarily in improving the growth of this organism, these studies provide practical applications. A chemically defined medium can easily be prepared by diluting commercially available Ham's F-12 medium to 80% strength with water and supplementing it with 50 µM FeCl3. Further growth improvement can be achieved by the addition of 200 µg/ml ß-cyclodextrin (discussed in reference 23). Growth in medium containing extra iron and ß-cyclodextrin yields growth 20 to 70% better than that in unsupplemented F-12, as assessed by the ATP assay. For those not requiring serum-free medium, the use of 80% F-12 with 1 to 5% serum yields robust and reliable growth. Growth in F-12 derivatives containing 5% serum rivals growth in brain heart infusion broth or Mueller-Hinton broth containing 5% serum after 24 h but falls behind after 48 h.

We introduced the use of an ATP assay as a convenient, reliable method of assessing the viability of Helicobacter cultures. Assessment of Helicobacter growth and viability via ATP measurements has proven both more reliable and more sensitive than spectrophotometric optical density measurements and far more rapid than plating and enumeration of CFU. Examinations of the time courses of cultures have shown that ATP levels parallel viable counts during both the growth and the death phases of broth cultures. This property is advantageous for investigators wishing to assess the viability of a culture to be used for in vivo experiments, since optical densities do not accurately reflect the number of viable organisms in the culture. The ATP assay was especially useful in studies of H. cinaedi, which grows as a thin film on blood agar and does not form isolated colonies.

Our studies indicate that iron is the growth-limiting nutrient for H. pylori in F-12-derived minimal media. We were surprised to find how dramatically growth could be improved by increasing the iron concentration above that of F-12, especially since F-12 contains more iron than most other tissue culture media. It is unclear why H. pylori appears to require high concentrations of iron in chemically defined, protein-free media. The benefit of elevated iron concentrations disappears in the presence of serum (data not shown). The lack of an iron effect in the presence of serum may be due simply to the greatly increased growth seen in the presence of serum, which could mask any effect of iron. Although H. pylori is not known to produce siderophores, the organism is reported to possess numerous other iron acquisition systems (26, 27, 28). It is possible that iron acquisition differs in chemically defined, protein-free media from serum-containing media used in previous studies of iron uptake (4, 26-28). This difference may have in vivo relevance. Tight junctions between epithelial cells in intact gastric tissue are unlikely to leak significant amounts of serum, thereby making mucus overlying healthy tissue a serum-free microenvironment. Reduction of ferric iron to the ferrous form by gastric acid may make ferrous iron originating from dietary sources more abundant in the gastric environment than elsewhere in the host, where it is sequestered in various biomolecules. Dhaenens and colleagues reported that extragastric Helicobacter species are able to use a wider variety of iron sources than are gastric Helicobacter species (4). This may help to partially explain the specific adaptation of H. pylori for the stomach, where few microbial floras reside. The requirement for ample amounts of dietary iron is supported by an in vivo study by Koga and colleagues (12). Those authors found that improved colonization of piglets by H. pylori occurred when the animals' diet was supplemented with additional iron (12). The high-iron requirement of H. pylori could also explain why this organism does not colonize the intestine, where other bacterial species would rapidly scavenge iron. Since proton pump inhibitors block acid secretion by parietal cells, this could result in decreased dietary iron being available for H. pylori and help to explain why proton pump inhibitors remain an effective part of many treatment modalities.

Detailed investigations of amino acid requirements revealed several interesting amino acid preferences for H. pylori. Amino acids essential for the growth of H. pylori in our chemically defined medium derivatives were similar to those reported previously (19, 20), but the addition of the nonessential amino acids tryptophan and isoleucine together improves growth, whereas neither of those amino acids alone has any effect. Either glutamine or glutamate boosts H. pylori growth as long as both tryptophan and isoleucine are also present in the medium (Fig. 2). In contrast to a previous report (19), no H. pylori growth inhibition was noted in the presence of glycine and lysine. Increasing the concentrations of all amino acids at once, or of some individual amino acids, such as tryptophan, isoleucine, glutamate, or glutamine, above levels found in F-12 did not further improve growth. Therefore, we conclude that amino acids are not growth limiting in F-12 or the F-12 derivatives described.

Gastric juice contains all 20 amino acids, and some are present at concentrations of over 50 µM (13); thus, amino acids can serve as dependable carbon and nitrogen sources for H. pylori in the host. Chemically defined medium derivatives of F-12 used in this study (TT8, TT14, TT17) lacked glucose, and no consistent benefit of glucose was observed (data not shown), confirming the findings of Mendz and Hazell which suggest that amino acids are preferred as carbon sources (17).

A thorough understanding of H. pylori nutritional requirements will allow physiologically relevant coculture experiments to be conducted with tissue culture cells. Currently, the field uses tissue culture media that are optimized for eukaryotic cells but poorly optimized for H. pylori, which can certainly affect experimental outcomes. F-12 and the derivatives reported here provide chemically defined medium alternatives that can be optimized for the growth of both H. pylori and tissue culture cells, more accurately reflecting the in vivo situation. Most tissue culture media lack certain amino acids, such as alanine, which H. pylori requires. Most tissue culture media also lack zinc. By adding missing amino acids and other components that are either absent or present at suboptimal concentrations, such as iron, hypoxanthine, and pyruvate, we were able to obtain at least limited growth in several media. Of these other media, supplemented RPMI 1640 and McCoy's 5a media proved most suitable for the growth of H. pylori.

Treatment of H. pylori infections has been complicated by the organism's inherent resistance to many antibiotics. This resistance pattern is useful, however, in cultivating the organism from animal stomachs, which naturally harbor small numbers of other organisms. Our experiments suggest that antibiotic resistance is an inherent trait that is widespread in the Helicobacter genus. All species tested could be cultured in the presence of trimethoprim, bacitracin, polymyxin, vancomycin, and the antifungal agents flucytosine and amphotericin B. These antimicrobial agents are sufficient to block the growth of normal microbial floras in rodent stomachs and feces (15, 23). In the course of these studies, we noticed, however, that H. pylori becomes sensitive to polymyxin B and trimethoprim when serum is absent from the culture medium. We found that in the absence of serum, H. mustelae and H. muridarum also have increased sensitivity to trimethoprim and polymyxin B, respectively. It is possible that serum alters the permeability of the outer membrane in Helicobacter species, leading to a decreased uptake of certain antibiotics. While H. pylori is known to incorporate host cholesterol into its membranes (7, 9), we have no evidence supporting a role for cholesterol in antibiotic sensitivity. Bovine serum albumin could restore the growth of H. pylori in the presence of trimethoprim, but this was true even when albumin with a low-fatty acid (low-cholesterol) content was used. Our data suggest that caution must be used when interpreting antimicrobial resistance profiles of Helicobacter species, because results will depend on the media employed.

The data in this study suggest that H. pylori has surprisingly few absolute requirements for growth: 9 amino acids, sodium and potassium chloride, thiamine, iron, zinc, magnesium, hypoxanthine, and pyruvate. Since multiple Helicobacter spp. grow in this minimal medium, the data cast considerable doubt over the widely held notion that Helicobacter spp. are nutritionally fastidious. Numerous experiments failed to reveal any benefit of individual vitamins or groups of vitamins other than thiamine. Similarly, we found no benefit of other additives, such as lipoic acid or putrescine. Indeed, a "less is more" philosophy should be applied to the growth of H. pylori in chemically defined, protein-free media.

To date, little has been published regarding the nutritional requirements of other Helicobacter species. Studies performed with five gastric and nongastric Helicobacter spp. indicate that the nutritional requirements of those species are largely similar to the requirements of H. pylori, with a few key exceptions. All Helicobacter species tested grew in F-12 without serum or other supplementation except H. felis. We have thus far been unable to culture H. felis in protein- or serum-free medium. On the other hand, some species reached much higher densities in F-12 than H. pylori, as evidenced by increased ATP content and microscopic examination of cultures. None of the other Helicobacter species displayed increased growth in response to additional iron, and some species seemed oblivious to the presence of serum. It is interesting to note that the species whose growth is improved by serum are gastric helicobacters, whereas those whose growth is not improved by serum are considered extragastric. Future work will focus on further defining subtle differences in the abilities of non-H. pylori Helicobacter spp. to grow in chemically defined medium derivatives.

Some epidemiological studies have suggested the presence of H. pylori in contaminated water supplies (14), and H. pylori DNA has been amplified from well water, saliva, and feces (2, 3, 10, 25), yet the bacterium has rarely been cultured from these sources (5). F-12 with serum is superior to Mueller-Hinton broth with serum for the recovery of H. pylori from frozen stocks (23). Therefore, chemically defined media may be superior for reinitiating the growth of Helicobacter spp. from a variety of stressful environments, such as feces or environmental sources. In summary, the results described above will be useful for research on a wide range of Helicobacter species but may also improve the chances of cultivating the human pathogen H. heilmannii and other newly discovered Helicobacter species. A thorough understanding of nutritional requirements and physiology could also shed light on the perplexing question of H. pylori transmission.


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ACKNOWLEDGMENTS
 
We especially thank J. Solnick and K. Beckwith for generously providing Helicobacter strains.

This work was supported by Public Health Service grants R01 CA101931 (to D.J.M.) and F32 DK59709-01 (to T.L.T.) from the National Institutes of Health and by a grant from the University of South Alabama Undergraduate Research Program (to P.B.C.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Louisiana State University Health Sciences Center—Shreveport, Room 2-221, Shreveport, LA 71130. Phone: (318) 675-8143. Fax: (318) 675-5764. E-mail: tteste{at}lsuhsc.edu. Back


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Journal of Clinical Microbiology, May 2006, p. 1650-1658, Vol. 44, No. 5
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.5.1650-1658.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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