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 0.45 μm pore-size membrane, which was then incubated in Preston enrichment broth. Four variables were compared: water sample volume (2000 mL vs. 500 mL), enrichment broth volume (25 mL vs. 100 mL), enrichment incubation duration (24 h vs. 48 h), 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 2000 mL sample volume (68.5% vs. 43.0%, p < 0.0001) and the 25 mL 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 48 h 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 2000 mL sample volume and a single enrichment broth of 25 mL PB incubated for 48 h.

Introduction

Campylobacter jejuni is the leading reported cause of bacterial enteritis in developed countries (Altekruse et al., 1999). In 2004 in Canada, campylobacter enteritis was the leading notifiable enteric food- and waterborne disease, with 9345 reported cases (http://dsol-smed.phac-aspc.gc.ca). In Quebec province alone, nearly 3000 cases of diarrheal illness are attributed annually to Campylobacter enteritis, more than the combined total caused by Salmonella and Shigella species, Escherichia coli O157:H7 and Yersinia enterocolitica (Comité provincial sur les entérites à Campylobacter au Québec, 1999). Thomas et al. (2006) recently concluded that even these numbers appear to represent a substantial underestimate of the public health burden of this enteric pathogen and that for every case of campylobacter infection reported in Canada each year, there are an additional unreported 23 to 49 cases.

Raw milk, untreated surface water, and poultry have all been well-documented as sources of campylobacter outbreaks (Allerberger et al., 2003, Blaser et al., 1983, Hutchinson et al., 1985, Jones and Roworth, 1996, Miller and Mandrell, 2005, Olson et al., 2008, Pebody et al., 1997, Said et al., 2003, Skirrow et al., 1981, Stehr-Green et al., 1991, Vogt et al., 1982). Nevertheless, most clinical cases appear as isolated, sporadic infections for which the source is rarely identified (Blaser, 1997). Identifying the sources and routes of transmission of campylobacteriosis is essential for developing effective, targeted preventive measures.

There is ample opportunity for Campylobacter spp. to contaminate environmental water, including streams, rivers, and lakes. The members of the genus colonize a wide variety of hosts, from domestic animals to wild birds, and thus an extensive burden of organisms is excreted in animal feces (Altekruse et al., 1999, Blaser et al., 1983). Other potential sources include discharges from wastewater treatment plants.

Testing for indicator organisms (typically thermotolerant coliforms or E. coli) has generally been considered to reflect adequately the presence of enteric pathogens; consequently, campylobacters have not been explicitly monitored in water. However, multiple studies, albeit often limited in scope, have reported conflicting results regarding the correlation between the presence of E. coli and Campylobacter spp. in environmental water (Arvanitidou et al., 1995, Bolton et al., 1987, Brennhovd et al., 1992, Carter et al., 1987, Dorner et al., 2007, Eyles et al., 2003, Horman et al., 2004, Martikainen et al., 1990, Moore et al., 2001, Obiri-Danso and Jones, 1999, Savill et al., 2001, Skjerve and Brennhovd, 1992, Stelzer et al., 1989, Till et al., 2008).

The primary challenges in detecting Campylobacter spp. in water are (a) the small numbers of organisms present; (b) their intrinsic fastidious requirements and slow growth rate; and (c) the presence of a significant proportion of organisms that may be injured or have difficulty adapting to in vitro conditions (Jones et al., 1991, Rollins and Colwell, 1986). This study evaluated alternative protocols for the detection of Campylobacter spp. in environmental water, examining four key variables: water sample volume (2000 mL vs. 500 mL), enrichment broth volume (25 mL vs. 100 mL), enrichment incubation duration (24 h vs. 48 h), and number of enrichment passages (one vs. two). Culture results were also compared to three PCR methods for detecting Campylobacter in environmental water.