Determination of the optimal culture conditions for detecting thermophilic campylobacters in environmental water.

ABSTRACT

This study evaluated alternative protocols for culturing thermophilic campylobacters in environmental water. All samples were filtered through a sterile 0.45µm pore-size membrane, which was then incubated in Preston enrichment broth. Four variables were compared water sample volume (2000mL vs. 500mL), enrichment broth volume (25mL vs. 100mL), enrichment incubation duration (24h vs. 48h), and number of enrichment passages (one vs. two). In addition, DNA extracts were prepared from all final broths and analyzed using three rRNA PCR assays. River water was collected at 3 sampling sites weekly for 9 weeks. Among these 27 collections, 25 (93%) yielded Campylobacter spp. under at least one of the 16 culture conditions. By univariate analysis, yields were significantly better for the 2000mL sample volume (68.5% vs. 43.0%, p<0.0001) and the 25mL enrichment broth volume (64.5% vs. 47.0%, p<0.0004). Neither of the enrichment period had a significant effect, although there was a trend in favor of 48h incubation (59.5% vs. 52.0%, p=0.13). The three PCR methods gave concordant results for 66 (33%) of the culture-negative samples and 103 (50%) of the culture-positive samples. Compared with culture results, Lubeck's 16S PCR assay had the best performance characteristics, with a sensitivity of 82% and a specificity of 94%. Of the 12 culture-negative samples positive by Lubeck's PCR assay, 11 (92%) samples were also positive by Denis' 16S PCR assay, suggesting that in these cases the culture might have been falsely negative. Based on our results, we conclude that the optimal conditions for detecting Campylobacter spp. in natural waters include 2000mL sample volume and a single enrichment broth of 25mL PB incubated for 48h.