Identification of equine cecal bacteria producing amines in an in vitro model of carbohydrate overload

Abstract

Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO(4); 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2- to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.

FIG. 1.

Effects of carbohydrate overload on numbers of streptococci (a), lactobacilli (b), and gram-negative anaerobes (c) in equine cecal contents incubated anaerobically in vitro. Cecal contents were divided into aliquots and incubated for 24 h with the inclusion of either inulin (1 g/100 ml; •) or corn starch (1 g/100 ml; ▴) or without added carbohydrate (control; ▪). Bacterial numbers in each aliquot at 6-h intervals were determined by serial dilution of cecal contents in sterile reduced physiological saline plated onto selective growth medium. Each value represents the geometric mean ± SEM (error bars) of estimates taken from 10 separate experiments. *, significant difference compared with control values (two-way repeated measures analysis of variance with Bonferroni's post hoc test).

FIG. 2.

Effects of virginiamycin and calcium hydrogen phosphate on numbers of Streptococcus (a) and Lactobacillus (b) organisms in equine cecal contents induced by incubation with starch or inulin. Aliquots of cecal contents were incubated with carbohydrate in the presence or absence of virginiamycin (VM) (1 mg/100 ml) or calcium hydrogen phosphate (CaHPO₄) (30 mg/100 ml). Bacterial numbers were measured at 0 and 24 h and expressed as CFU/milliliter, and the magnitude of the change over this period was calculated by dividing the number of bacteria present at 24 h by the initial figure (geometric mean + SEM [error bars] of estimates taken from four separate experiments). Abbreviations atop error bars: a, significant differences compared with control; b, significant difference compared to starch or inulin alone (one-way analysis of variance with Fisher's multiple comparison.)