Bile-tolerant Helicobacter spp. are emerging human and animal pathogens. However, due to their fastidious nature, which requires nutrient-rich complex media to grow, infection with these bacteria may be underestimated. The accumulation of toxic metabolites in cultures may be one of the main obstacles for successful culture of these organisms. The present study examined various potential growth-enhancing substances for Helicobacter spp. and, furthermore, how they may affect spiral to coccoid conversion. Five Helicobacter spp. were cultured on agar and in broth media supplemented with activated charcoal, β-cyclodextrin, or porcine gastric mucin. Growth was determined by estimating the numbers of colony-forming units and colony diameter, as well as bacterial cell mass. Coccoid transformation was estimated every 24 h by both Gram and acridine-orange staining. Activated charcoal was superior in supporting growth and increased cell mass on agar and in broth media. β-Cyclodextrin delayed spiral to coccoid conversion by Helicobacter pylori and Helicobacter canis, whereas activated charcoal delayed the conversion to coccoid forms of Helicobacter hepaticus and Helicobacter bilis. The progression to coccoid forms by Helicobacter pullorum on agar media was not influenced by any growth supplement. The spiral to coccoid conversion was more rapid in broth media than on agar media. The growth enhancement observed is probably related to the capacity of activated charcoal to remove toxic compounds in culture media.
BarerM. R., GribbonL. T., HarwoodC. R.,
NwoguhC. E.1993; The viable but non-culturable hypothesis and medical bacteriology. Rev Med Microbiol 4:183–191[CrossRef]
BenaissaM., BabinP., QuellardN., PezennecL., CenatiempoY.,
FaucherJ. L.1996; Changes in Helicobacter pylori ultrastructure and antigens during conversion from the bacillary to coccoid form. Infect Immun 64:2331–2335
BradshawD. J., HomerK. A., MarshP. D.,
BeightonD.1994; Metabolic cooperation in oral microbial communities growth on mucin. Microbiology 140:3407–3412[CrossRef]
CostaK., BacherG., AllmaierG., Dominguez-BelloM. G., EngstrandL., FalkP., Garcia-del PortilloF., de PedroM. A.1999; The morphological transition of Helicobacter pylori cells from spiral to coccoid is preceded by a substantial modification of the cell wall. J Bacteriol 181:3710–3715
DonelliG., MatarreseP., FiorentiniC., DainelliB., TaraborelliT., Di CampliE., Di BartolomeoS.,
CelliniL.1998; The effect of oxygen on the growth and cell morphology of Helicobacter pylori . FEMS Microbiol Lett 168:9–15[CrossRef]
EdelsteinP. H.,
EdelsteinM. A. C.1993; Comparison of three buffers used in the formation of buffer charcoal yeast extract medium. J Clin Microbiol 31:3329–3330
ErmolaevaS., BelyiY.,
TartakovskiiI.1999; Characteristics of induction of virulence factor expression by activated charcoal in Listeria monocytogenes . FEMS Microbiol Lett 174:137–141[CrossRef]
FoxJ. G., DewhirstF. E., ShenZ.8 other authors1998; Hepatic Helicobacter species identified in bile and gallbladder tissues in Chileans with chronic cholecystitis. Gastroenterology 114:755–767[CrossRef]
FranklinC. L., BeckwithC. S., LivingstonR. S., RileyL. K., GibsonS. V., Besch-WillifordC. V.,
HookR. R.1996; Isolation of a novel Helicobacter cholecystus sp. from the gallbladder of Syrian hamsters with cholangiofibrosis and centrilobular pancreatitis. J Clin Microbiol 4:2952–2958
HazellS. L., MarkesichD. C., EvansD. J.,
GrahamD. Y.1989; Influence of media supplements on growth and survival of Campylobacter pylori . Eur J Microbiol 8:597–602[CrossRef]
KustersJ. G., GerritsM. M., Van StrijpJ. A. G.,
Vandenbroucke-GraulsC. M. J. E.1997; Coccoid forms of Helicobacter pylori are the morphologic manifestation of cell death. Infect Immun 65:3672–3679
LeeA., PhillipsM. W., O’RourkeJ. L.7 other authors1992; Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from intestinal mucosa of rodents. Int J Syst Bacteriol 42:27–36[CrossRef]
MarchiniA., d’ApolitoM., MassariP., AtzeniM., CopassM.,
OlivieriR.1995; Cyclodextrins for growth of Helicobacter pylori and production of vacuolating cytotoxin. Arch Microbiol 164:290–293[CrossRef]
MoranA. P.,
UptonM. E.1987a; Effect of medium supplements, illumination and superoxide dismutase on the production of coccoid forms of Campylobacter jejuni ATCC 29428. J Appl Bacteriol 62:43–51[CrossRef]
MoranA. P.,
UptonM. E.1987b; Factors affecting production of coccoid forms by Campylobacter jejuni on solid media during incubation. J Appl Bacteriol 62:527–537[CrossRef]
NarikawaS., KawaiS., AoshimaH., KawamataO., KawaguchiR., HikijiK., KatoM., IinoS.,
MizushimaY.1997; Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori . Clin Diagn Lab Immunol 4:285–290
NilssonH.-O., TaneeraJ., CastedalM., GlatzE., OlssonR., WadströmT.2000a; Identification of Helicobacter pylori and other Helicobacter species by PCR, hybridization and partial DNA sequencing in human liver samples from patients with primary sclerosing cholangitis or primer biliary cirrhosis. J Clin Microbiol 38:1072–1076
NilssonI., LindgrenS., ErikssonS., WadströmT.2000b; Serum antibodies to Helicobacter hepaticus and Helicobacter pylori in patients with liver disease. Gut 46:410–414[CrossRef]
NorrisC. R., MarksS. L., EatonK. A., TorabianS. Z., MunnR. J.,
SolnickJ. V.1999; Healthy cats are commonly colonized with Helicobacter heilmannii that is associated with minimal gastritis. J Clin Microbiol 37:189–194
ShahamatM., MaiH. E. U., Paszko-KolvaC., YamamotoH.,
ColwellR. R.1991; Evaluation of liquid media for growth of Helicobacter pylori. J Clin Microbiol 29:2835–2837
SignorettoC., TafiM. C.,
CanepariP., del Mar LleoM.2000; Cell wall chemical composition of Enterococcus faecalis in the viable but non-culturable state. Appl Environ Microbiol 66:1953–1959[CrossRef]
StanleyJ., LintonD., BurnensA. P., DewhirstF. E., OnS. L., PorterA., OwenR. J.,
CostasM.1994; Helicobacter pullorum sp. nov., genotype and phenotype of a new species isolated from poultry and from humans with gastroenteritis. Microbiology 140:3441–3449[CrossRef]
WalshE. J.,
MoranA. P.1997; Influence of medium composition on the growth and antigen expression of Helicobacter pylori . J Appl Microbiol 83:67–75[CrossRef]
WardJ. M., FoxJ. G., AnverM. B., HainesD. C., GeorgeC. V.,
CollinsM. L.1994; Chronic active hepatitis and associated liver tumours in mice caused by persistent bacterial infection with a novel Helicobacter species. J Natl Cancer Inst 86:1222–1227[CrossRef]
WorkuM. L., SidebothamR. L., WalkerM. M., KeshavarzT.,
KarimQ. N.1999; The relationship between Helicobacter pylori motility, morphology and phase of growth: implications for gastric colonization and pathology. Microbiology 145:2803–2811