Modified ammonia electrode method to investigate D-asparagine breakdown by Campylobacter strains

Abstract

An ammonia electrode method has been developed for investigating the deamination of amino acids by bacteria. It consists of incubating a standard inoculum of organisms in an amino acid solution and then measuring the amount of ammonia evolved by the electrode. Two hundred and twelve Campylobacter strains (118 C. jejuni and 94 C. coli) were tested for their ability to break down D-asparagine by this method. Organism control (bacterial suspension in buffer alone) values ranged from 0.44 to 2.0 (mean 0.93 ± 0.24) ammonia concentration (AC) units (one AC unit is equal to 10-5 mol of ammonia per liter), whereas test values ranged from 0.60 to 46.0 units. Test ACs of <2 units (97 strains) were considered negative, whereas ACs of ≥10 (77 strains) were considered positive for D-asparaginase; 38 (18%) strains with ACs between 2 and 10 units were provisionally assigned an intermediate status. The amount of ammonia produced by strains with ACs of ≥10 increased greatly when the inoculum size was increased, whereas this was not a feature of strains with ACs of <2 units. The presence or absence of an inoculum effect was instrumental in classifying strains with intermediate ACs and allowed a breakpoint to be defined. When the ammonia electrode method was repeated, 97.6% of the 212 strains gave the same positive or negative reaction that they did on the first occasion. Thus the test was highly reproducible. Five strains (all porcine C. coli from Germany) were unclassifiable because they repeatedly gave either a weak-positive or negative reaction. Overall, 12.7% of C. jejuni strains and 86.2% of C. coli strains were positive for D-asparaginase. The ammonia electrode method was found to be simple and reliable for separating strains on the basis of D-asparaginase activity.