Title: Aerobic growth of campylobacter in media supplemented with C3-monocarboxylates and C4-dicarboxylates Author
Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 2, 2012
Publication Date: June 1, 2013
Citation: Hinton Jr, A. 2013. Aerobic growth of campylobacter in media supplemented with C3-monocarboxylates and C4-dicarboxylates. Journal of Food Protection. 76:685-690. Interpretive Summary: Campylobacter are one of the major causes of human foodborne illnesses. However, growing these bacteria in the laboratory can be difficult and expensive because cultures of the bacteria must be grown in special atmospheres containing low oxygen concentrations and high carbon dioxide concentrations (microaerobic). Therefore, a new bacterial medium was formulated, and four Campylobacter isolates were tested for their ability to grow in the medium when incubated in normal breathing air (aerobic). The basal test medium was composed of tryptose, yeast extract, and a mineral-vitamin solution which was then supplemented with 4-carbon organic acids (fumarate, succinate, or malate) and various concentrations of 3-carbon organic acids (pyruvate or lactate). Media was inoculated with the Campylobacter isolates then incubated aerobically for 72 h, and growth of the bacteria was determined by measuring increases in the optical density of the cultures. Results indicated that all Campylobacter isolates could grow in the supplemented media, and growth was generally improved by increasing the concentration of 3-carbon organic acids added to the media. Results also indicated that aerobic growth of Campylobacter in the media was improved when small concentrations of agar and sodium bicarbonate were added to media. Finally, the number of Campylobacter that grew in media supplemented with fumarate and pyruvate was determined by counting the number of bacteria recovered from the media after 72 h of aerobic or microaerophilic incubation. Results indicated that the numbers of Campylobacter recovered from the medium increased by over 1 million fold during incubation and there was no difference in the number of bacteria recovered from media incubated aerobically or microaerophilically. Findings from these experiments indicate that the new medium can be used to aerobically culture Campylobacter. Therefore, the medium might provide an alternative to the current procedures that require incubating Campylobacter under microaerophilic conditions; thereby, eliminating the additional expense and training required for the use of specialized atmospheres in culturing these pathogenic bacteria.
Technical Abstract: Experiments were conducted to examine aerobic growth of Campylobacter spp. in media supplemented with C4-dicarboxylates (fumarate, succinate, or malate) and C3-monocarboxylates (pyruvate or lactate). Basal broth media composed of tryptose, yeast extract, and a mineral-vitamin solution was supplemented with 30 mM fumarate, succinate, or malate and 0 to 100 mM of lactate or pyruvate. Media was inoculated with approximately 106 colony-forming-units/ml of Campylobacter coli 33559, Campylobacter fetus 27349, or Campylobacter jejuni 33560, and Campylobacter jejuni 49349 then incubated aerobically at 37C for 72 h in a Bioscreen C Microbiology Reader. The optical density (OD) of the cultures was measured at 600 nm during incubation. The effect of adding 0 to 0.20% agar and 0 to 0.10 % sodium bicarbonate (NaHCO3) to media supplemented with 30 mM fumarate and 100 mM pyruvate on the ability of the Campylobacter spp. to grow aerobically was also determined. Finally, the number of colony-forming-units (cfu) of Campylobacter/ml recovered from media containing 30 mM fumarate, 100 mM pyruvate, 0.15% agar, and 0.05 % NaHCO3 was determined after the media was inoculated with 103 cfu/ml of Campylobacter then incubated aerobically or microaerophilically for 72 h at 37C. Results indicated that the OD of Campylobacter cultures incubated aerobically in media supplemented with mixtures of a C4-dicarboxylate and a C3-monocarboxylate was generally significantly greater than the growth of cultures incubated in media supplemented with a C4-dicarboxylate, only. The OD of cultures of all Campylobacter isolates grown in media supplemented with fumarate and pyruvate was significantly higher when agar was added to the media, and the addition of NaHCO3 to the medium produced an additional significant increase in the OD of most of the isolates during early periods of growth. There was also a 5 to 6 log increase in the number of Campylobacter recovered from media supplemented with 30 mM fumarate, 100 mM pyruvate, 0.15% agar, and 0.05% NaHCO3 that was inoculated with Campylobacter spp. and incubated aerobically or microaerophilically at 37C for 72 h. Findings indicate that media supplemented with a C4-dicarboxylate, a C3-monocarboxylate, agar, and NaHCO3 can be used to aerobically culture Campylobacter. The medium might provide an alternative to current procedures of incubating Campylobacter under microaerophilic conditions; thereby, eliminating the additional expense and training required for the use of specialized atmospheres in culturing Campylobacter.