Sample handling factors affecting the enumeration of lactobacilli and cellulolytic bacteria in equine feces

Authors: HARLOW, BRITTANY - UNIVERSITY OF KENTUCKY; LAWRENCE, LAURIE - UNIVERSITY OF KENTUCKY; Flythe, Michael

Submitted to: Journal of Equine Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/24/2015
Publication Date: 7/28/2015
Citation: Harlow, B.E., Lawrence, L.M., Flythe, M.D. 2015. Sample handling factors affecting the enumeration of lactobacilli and cellulolytic bacteria in equine feces. Journal of Equine Veterinary Science. 35(9):744-748. doi: http://dx.doi.org/10.1016/j.jevs.2015.07.011.

Interpretive Summary: Recently there has been a tremendous research effort to understand the biology and roles of beneficial bacteria. Ruminants (goats, sheep, cattle) have long been models for studying how bacteria can benefit their animal host. Horses are not ruminants, but they rely on bacteria in the hindgut to help them digest grasses and other fibrous forages. A growing number of equine researchers are interested in exploring the gastrointestinal microbiology of the horse, particularly beneficial bacteria. The methods for cultivation of disease-causing bacteria are well established, but few have worked with beneficial bacteria from horses. In addition, horse researchers prefer to study the gastrointestinal bacteria by using feces because they can be collected non-invasively over a long period of time. However, different researchers have used a variety of sample handling procedures, which could impact the number of bacteria detected in equine feces. The goal of this study was to determine the effects of storage time and temperature on how many beneficial bacteria could be counted in fecal samples. Two types of bacteria were enumerated, cellulolytic bacteria and lactobacilli. Cellulolytic bacteria enable horses to breakdown cellulose, an abundant carbohydrate in grasses. Lactobacilli are well-known beneficial microorganisms that are also found in humans and other animals. Cellulolytic bacteria were enumerated on two different media types. The performance of these two media types was comparable. The cellulolytic bacteria died rapidly outside of the horses, but survival was better when the fecal samples were held close to horse body temperature (37 ºC). When fecal samples were held at refrigerator temperature (4 ºC) all the cellulolytic bacteria were dead in 24 hours. Lactobacilli were enumerated on two commercially available media types, MRS and Rogosa Agars. MRS was not sufficiently selective in our study (it permitted the growth of mold). Rogosa Agar was selective, and revealed that lactobacilli were also impacted by fecal storage time and temperature. The number of lactobacilli also decreased when the storage temperature was refrigerator or room temperature (25 ºC). The number of lactobacilli were steady for up to 4 hours when held at body temperature, but increased from 4 to 24 hours. These results demonstrate that storage time and temperature are important to consider when enumerating lactobacilli and cellulolytics from feces. The research will benefit equine scientists who want to examine the effects of an experimental treatment (e.g. a dietary change) on gastrointestinal bacteria. The potential impacts of the study are improved sample handling that will lead to more accurate reports on lactobacilli and cellulolytic bacteria in horses. More broadly, rapid enumeration after sampling will lead to more accurate evaluation of beneficial bacteria in general. Consistent sample handling is not only important within a study, but storage time and temperature must also be reported so that results between studies can be compared.

Technical Abstract: The objectives were to compare media types and evaluate the effects of fecal storage time and temperature on the enumeration of cellulolytic bacteria and lactobacilli from horses. Fecal samples were collected from horses (n = 3) and transported to the lab (CO2, 37 ºC, 0.5 h). The samples were assigned to 1 of 4 storage temperatures: initial (no storage), 37 ºC, room temperature (RT; 22-24 ºC), or 4 ºC. The initial samples were enumerated 5 times to assess repeatability. Feces stored at each temperature were used to enumerate cellulolytics (rich or defined media) and lactobacilli (Rogosa SL or MRS agar) over a 24 h time course. All media were repeatable within media type. The cellulolytic media types produced similar results. After 2 h, the cellulolytics lost >99% viability in RT and 4 °C, and 90% viability in 37 °C (P < 0.05). By 24 h of storage, 105-106-fold fewer cellulolytics were observed in 37 ºC and RT (P < 0.05). There were no detectable cellulolytics in 4 ºC at 24 h. Viable counts on MRS were not reported due to insufficient selectivity. There were fewer lactobacilli in RT and 4 ºC after 2 h (P < 0.05). From 2-4 h, 37 ºC storage was similar to initial and after 8 h had more lactobacilli than initial that continued to increase (P < 0.05). These results demonstrate that storage time and temperature are important to consider when enumerating lactobacilli and cellulolytics from feces.