Department of Medical Microbiology, Dermatology and Infection, Lund University, Lund Sölvegatan 23,SE-22362, Sweden1
Department of Microbiology, National University of Ireland, Galway, Ireland2
Author for correspondence: Anthony P. Moran. Tel: +353 91 524411 ext 3163. Fax: +353 91 525700. e-mail: anthony.moran{at}nuigalway.ie
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ABSTRACT |
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Keywords: Helicobacter pylori, coccoid forms, coccoid conversion, culture media
Abbreviations: CCUG, Culture Collection of the University of Gothenburg
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INTRODUCTION |
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However, despite being isolated from an ever-increasing number of hosts, most Helicobacter spp. remain difficult to culture from clinical samples. Various solid and broth culture media have been designed to optimize growth for the production of various antigens (Shahamat et al., 1991 ; Marchini et al., 1995
; Walsh & Moran, 1997
), to facilitate metabolic and enzyme studies, and to prevent accumulation of toxic metabolites that inhibit growth in the stationary phase (Hazell et al., 1989
). However, culture media for Helicobacter spp. remain poorly developed with the exception of H. pylori.
In addition, the related phenomenon of spiral to coccoid conversion has been investigated in H. pylori (Donelli et al., 1998 ; Worku et al., 1999
) but not in other helicobacters. H. pylori cells may exist in two morphological variants (Benaissa et al., 1996
): a bacillary form with a spiral or helical shape and a coccoid form which usually appears after several days of culture. This conversion generally occurs after 610 days of culture in stationary phase (Chan et al., 1994
; Benaissa et al., 1996
). Several factors may influence the spiral to coccoid conversion by H. pylori, such as acid pH stress, oxygen, temperature, nutritional starvation (West et al., 1990
; Cellini et al., 1994
; Donelli et al., 1998
; Worku et al., 1999
) and, thus, potentially culture media composition may influence the rate of spiral to coccoid conversion by Helicobacter spp. It has been speculated that the conversion to coccoid forms may be important for maintaining viability and survival of H. pylori outside the host (West et al., 1990
), as well as in recrudescence of infection and treatment failure of patients with peptic ulcer disease (Benaissa et al., 1996
). On the other hand, other microaerophilic bacteria produce non-culturable coccoid forms resembling those of helicobacters (Moran, 1997
) and these have been shown to be a degenerate cell form which is undergoing cellular degradation (Moran & Upton, 1986
) in response to oxygen toxicity and metabolites in culture media (Moran & Upton, 1987a
, b
). Likewise, compared to spiral forms, it has been shown that coccoid forms of H. pylori have impaired genomic DNA, total amounts of DNA and RNA are reduced, there is loss of membrane potential, and levels of intracellular ATP, indicative of metabolic state, are significantly reduced, collectively indicating a process of cellular degeneration (Kusters et al., 1997
; Moran, 1997
; Narikawa et al., 1997
).
The present study was undertaken to establish optimized culture media for the growth of Helicobacter hepaticus, Helicobacter bilis, Helicobacter pullorum and Helicobacter canis through the addition of ß-cyclodextrin, porcine mucin and activated charcoal, using H. pylori for comparison. Improvements in the ability to culture these emerging pathogens would allow a more precise diagnosis of infection. Furthermore, we also examined the effect of culture conditions on the conversion of spiral to non-culturable coccoid forms.
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METHODS |
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Agar media composition and culture conditions.
Two types of agar media were used in the present study: (i) GAB-Camp agar (GCA) for culturing H. pylori (Soltesz et al., 1992 ), and (ii) Brucella agar (BA) (Becton Dickinson) to culture H. hepaticus, H. pullorum, H. canis and H. bilis. These media were modified by supplementation with 5% (v/v) sheep blood, 5% (v/v) inactivated horse serum (Gibco-BRL), 1% (v/v) IsoVitalex (Becton Dickinson) and 1% (v/v) haemin. Each strain was cultured on GCA or BA without antibiotics as indicated above at 37 °C for 24 days under microaerobic conditions (5% O2, 5% CO2, 4% H2 and 81% N2; or 10% O2, 5% CO2 and 85% N2) and subcultured under the same conditions. Colonies were harvested, suspended in normal saline and adjusted to an OD540 of 1·0, equivalent to a concentration of 5x108 c.f.u. ml-1, which was subsequently diluted, to a density of 104 c.f.u. ml-1 for each strain. An aliquot of this dilution (100 µl) was applied to agar media containing different supplements. These included various concentrations of 2,6-di-O-methylcyclodextrin (Cyclolab, Budapest, Hungary), porcine gastric mucin (type III from porcine stomach) (Sigma) and two types of activated charcoal added, with particle sizes of 1·2 mm (Sigma) and 24 mm (Rudolph Grave-Stockholm, Sweden) (Table 1
). The 2,6-di-O-methylcyclodextrin was filter-sterilized before addition to sterile media, whereas activated charcoal and porcine mucin were added prior to autoclaving at 121 °C for 20 min. The suitability of each medium for each Helicobacter spp. was evaluated by measuring both the number of c.f.u. in triplicate and the mean diameters of the colonies. All inoculations were performed in duplicate and evaluations of each medium were repeated.
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Calculation of percentage of coccoid forms.
Bacterial cells from both agar and broth media were examined by Gram and acridine-orange staining at a magnification of 1000x, at 24 h intervals. Duplicate sets of inoculated media were examined. The percentage of cells with spiral or coccoid morphology was estimated microscopically by counting 100 cells in two samples of each bacterial suspension (all data are shown as the mean percentage). Bacterial cells were stained using standard Gram-staining techniques and also acridine-orange staining. Prior to either staining, cells were suspended to a standard OD540 of 1·0. For acridine-orange staining, cells (1 µl) were mixed with 9 µl acridine orange (1·0 mg PBS ml-1, pH 7·2) on a microscope slide, and immediately examined under a fluorescent microscope (Zeiss Axioskop G 42-110e).
Cell lysate.
The protein content of cells harvested from broth media, supplemented with activated charcoal, ß-cyclodextrin, or porcine gastric mucin, was examined as an indication of growth enhancement. Cells (5 ml) grown in broth media were collected following 72 h culture, washed in PBS and lysed by sonication in an ice bath, with 50% of pulse at 30 s intervals for 4 min (Labsonic U, B. Braun Diessel Biotech Inc.). Following centrifugation (12000 g, 20 min, 4 °C), the protein content of the cell-free fractions was determined using a Coomassie brilliant blue protein assay (G250 dye reagent, Bio-Rad). Duplicate sets of broth media were inoculated and, hence, determinations of protein content were made on two independent samples.
Electron microscopy.
Cells were prefixed by application of 2·5% glutaraldehyde directly to agar plates. Subsequently, the cells on the plates were rinsed three times with Millonig phosphate buffer. A portion of growth equivalent to 1 cm2 was then removed from the agar plate and transferred to a sterile container. The colonies were dehydrated with 25%, 50%, 75% and 98% ethanol (2x20 min) respectively, followed by an overnight dehydration step with absolute ethanol, and were subsequently freeze-dried. The cells were mounted on metal stubs and sputter coated using gold/palladium. The samples were examined in a Philips 515 scanning electron microscope.
Statistical anlaysis.
For analysis of quantitative growth in various media and comparison of independent groups of data, the MannWhitney U-test was used. The TukeyKramer multiple comparisons test was applied when comparing the level of protein content in supplemented and control media. A value of P<0·05 was considered significant.
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RESULTS |
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As shown in Fig. 1(b), the conversion of H.canis started on day 4 on various agar media. On control media and BA supplemented with porcine gastric mucin, conversion to coccoid forms was complete after 6 days of culture, whereas the conversion was complete on BA supplemented with activated charcoal on day 7 and on BA supplemented with ß-cyclodextrin on day 9. On the other hand, on BA blood-free agar, coccoid forms were dominant only at day 11.
On control media, 20% coccoid forms occurred after 2 days of culture of H. pullorum and spiral to coccoid conversion was complete on day 4. On supplemented media, the transformation from spiral to coccoid forms by H. pullorum appeared on day 4 with 15% coccoid forms; on days 5 and 6 the percentages of coccoid forms were 40% and 75%, respectively. The spiral to coccoid conversion was complete on day 7 on all supplemented agar media, and on BA blood-free agar 1 day later.
The conversion to coccoid forms by H. bilis on all agar media started on day 7 with 10% coccoid forms occurring. In BA control media, BA with activated charcoal, and BA blood-free agar, the spiral to coccoid conversion occurred at a conversion rate of 5% daily and was complete after 26 days of culture. Although the conversion on BA supplemented with ß-cyclodextrin or porcine gastric mucin began similarly after 7 days of culture, the daily conversion rate was higher (7%) and was complete after 21 days of incubation. The spiral to coccoid conversion by H. hepaticus occurred 1 day after appearance of visible colonies and was complete on control media and BA supplemented with ß-cyclodextrin 2 days later. On BA supplemented with activated charcoal, complete conversion to coccoid forms occurred after one further day.
Growth and coccoid forms of bile-tolerant Helicobacter spp. in broth culture
Unlike the other helicobacters examined, H. canis was successfully subcultured from BHI. Moreover, H. canis grown on BHI supplemented with 1% (w/v) activated charcoal gave higher numbers of c.f.u. ml-1 (Fig. 2b) and the highest protein content (Table 2
) compared to the other media. This protein content was significantly greater (P<0·01) than that of cells grown on control media. In addition, compared to control media, the number of c.f.u. in charcoal-supplemented broth was significantly higher (P<0·05) at day 2 and on subsequent days of incubation, but was not significantly different (P>0·05) in the other supplemented media. Approximately 20% of the total bacterial cells examined in control media and BHI supplemented with porcine gastric mucin had converted completely to coccoid forms after 2 days of culture, and 1 day later the coccoid percentage had increased to 60% and 50%, respectively. Complete conversion occurred in both media by day 4. In BHI supplemented with activated charcoal, the spiral to coccoid conversion started after 3 days of incubation, when 20% coccoid forms occurred, and was complete on day 6. In BHI supplemented with ß-cyclodextrin, the conversion started on day 3, when 20% coccoid forms were present; 50% coccoid forms occurred on day 5, and conversion was complete after 7 days of culture.
H. pullorum and H. hepaticus began conversion with 20% coccoid forms after 2 days of culture; 60% coccoid forms were present at day 3 in all media, except that supplemented with 1% (w/v) activated charcoal, where 40% coccoid forms were present. Moreover, 1 day later, conversion to coccoid forms was complete in all media except for that supplemented with charcoal, where conversion to coccoids was complete on day 5. The spiral to coccoid conversion of H. bilis in BHI supplemented with ß-cyclodextrin or porcine mucin was slow compared to the other species; it began on day 4 and was complete on day 8 of incubation. In control media and BHI supplemented with activated charcoal, coccoid forms predominated after 12 days, whereas in BHI supplemented with porcine mucin or ß-cyclodextrin the spiral to coccoid conversion started on day 4 and finished on day 8. Moreover, compared to growth on other supplemented media, H. bilis cultured on charcoal-supplemented media had the highest protein content, and this was significantly higher (P<0·05) than that obtained when H. bilis was cultured in control media (Table 2).
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DISCUSSION |
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Both activated charcoal and ß-cyclodextrin have no nutrient value and therefore the effect could be related to their ability to remove toxic compounds present in cultures such as hydrogen peroxide and superoxide ions (Marchini et al., 1995 ; Buck & Smith, 1987
). Moreover, our results indicate that activated charcoal has a higher adsorption capacity than ß-cyclodextrin. The adsorption properties of activated charcoal depend on the number of fine pores giving a large inner surface area for adsorption. This may explain why greater growth enhancement was observed with activated charcoal having a 0·15 mm pore size than that with a 24 mm pore size.
Mucin preparations have been shown previously to enhance growth of several oral bacteria (Bradshaw et al., 1994 ) and Bacteroides fragilis (Roberton & Stanley, 1982
). Furthermore, H. pylori has surface mucin-binding proteins, in common with the bile-tolerant Helicobacter strains studied in this investigation (J. Taneera, S. Hynes & T. Wadström, unpublished data). However, in this study, in contrast to activated charcoal, porcine gastric mucin did not affect the growth kinetics of bile-tolerant Helicobacter spp. Although blood is the major iron source for some micro-organisms and enhances their growth, bile-tolerant Helicobacter spp., with the exception of H. hepaticus, were capable of growth on blood-free media. This indicates that blood is not essential for their growth, although it did enhance the growth of all these helicobacters, similar to previous observations with H. pylori (Walsh & Moran, 1997
). Our data also indicated that hydrogen is an essential growth factor for bile-tolerant helicobacters, but the mechanism by which these organisms utilize hydrogen is poorly understood.
Spiral to coccoid conversion has been observed under certain stress conditions for several Gram-negative pathogens such as Vibrio spp., Escherichia coli, Shigella spp., Salmonella enteritidis, Campylobacter spp., Aeromonas spp. and Legionella spp. (Moran & Upton, 1987a ; Barer et al., 1993
). Furthermore, culture media supplemented with activated charcoal or ß-cyclodextrin delayed the conversion to coccoid forms by all the helicobacters we examined. In general, a spiral to coccoid conversion was more rapid in broth than on solid media, most likely due to the dissolved toxic substances enhancing the coccoid transformation (Moran & Upton, 1987b
; Coudron & Statton, 1995
). The ability to delay the spiral to coccoid conversion may be beneficial in the production of cell surface antigens for immunodiagnostic tests since such conversion has been shown to induce changes in the cell wall composition of H. pylori and other bacteria (Costa et al., 1999
; Signoretto et al., 2000
).
Collectively, our data have shown that activated charcoal enhances growth of five Helicobacter spp. to a greater extent than ß-cyclodextrin or porcine gastric mucin in various culture media and, furthermore, a delay in spiral to coccoid conversion was observed in media supplemented with either activated charcoal or ß-cyclodextrin. Thus, addition of charcoal by increasing the growth yields and reducing conversion to coccoid forms could aid diagnostic laboratories in the ability to isolate and grow these helicobacters. Moreover, the ability to delay conversion to coccoid forms in helicobacters would be of benefit to various analyses carried out on these bacteria. Optimal growth conditions which would allow routine culturing of pathogenic helicobacters from liver and bile specimens would be important for a diagnosis and correlation with disease which, at present, rely on molecular biology or serological techniques for detection. In addition to improved diagnostics, optimal conditions for subculture of intestinal helicobacters would allow far greater research in this area where, to date, low numbers of isolates from each species have been available for study.
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ACKNOWLEDGEMENTS |
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Received 26 June 2001;
revised 9 November 2001;
accepted 15 November 2001.
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