Campylobacter is the major bacterial cause of foodborne infection, annually accounting for approximately 166 million foodborne illnesses around the world (Kirk et al., 2015). In addition to the clinical symptoms of gastroenteritis, Campylobacter is the major risk factor of Guillain–Barré syndrome (GBS), a neurological disorder causing muscular paralysis, as a post-infection complication (Hughes and Cornblath, 2005). Among pathogenic Campylobacter species, C. jejuni and C. coli are most frequently associated with human infection (Kaakoush et al., 2015). Thus far, the consumption of contaminated poultry is the primary cause of developing human campylobacteriosis (Whiley et al., 2013).

Despite the well-known fastidious nature of Campylobacter (Silva et al., 2011), Campylobacter is isolated from environmental sources, such as lake, river, sea, and sewage, suggesting that environmental water is a possible vehicle that transmits Campylobacter to humans (Jones, 2001). C. jejuni is the pathogenic species that is mainly related to water-borne campylobacteriosis worldwide (Pitkanen, 2013). In Canada, Campylobacter outbreaks caused by cross contamination related with meltwater and heavy rainfall are problematic to public health (Millson et al., 1991; Clark et al., 2003). However, the isolation of Campylobacter implicated in water-borne outbreak appear to be challenging, not only due to rapid loss in culturability of isolates from the environment (Wingender and Flemming, 2011) and from stool samples (Bullman et al., 2012), but also due to the time gap between the initial infection and outbreak investigation (Hanninen et al., 2003; Jakopanec et al., 2008). Therefore, regular monitoring system of water resources by using culture-based methods is likely to underestimate the prevalence of Campylobacter spp. in the environment. This might mislead our understanding of the role played by the environmental sources in human infection and possibly the contamination of food chain by Campylobacter, even though Campylobacter is most frequently detected in animal fecal samples (29.7%), untreated human sewage (25.6%), and surface water (26.6%), according to a study in Alberta, Canada, among the three major foodborne pathogens, including Campylobacter, Salmonella, and Escherichia coli O157:H7 (Jokinen et al., 2011).

Various culture supplements have been examined to improve selective isolation of Campylobacter spp. (Corry et al., 1995). For example, ISO method 2005 has been applied for the detection of thermo-tolerant campylobacters from water, and alternative culture-based methods in combination with molecular end-point confirmation (Hokajarvi et al., 2013; Pitkanen, 2013). Sample volume, incubation time, enrichment volume, passage of enrichment, and PCR-primer specificity all play an important role (Levesque et al., 2011) and enrichment procedures as well (Rosef et al., 2001). Khan et al. (2009, 2013) compared two methods (i.e., centrifugation vs. membrane filtration) for the isolation and detection of Campylobacter from agriculture watersheds, and reported the effect of incubation temperature on the detection rates and the type of dominant Campylobacter species detected from water samples. However, wastewater samples are even more challenging than samples from agricultural watersheds due to the relatively low number of Campylobacter in comparison with the high levels of microbial competitors and PCR inhibitors in wastewater (Koenraad et al., 1997; Abulreesh et al., 2005; Schrader et al., 2012).

To overcome the limitations in traditional culture-based methods for the detection of foodborne pathogens, molecular methods, such as PCR, have become practical and widely used due to the speed and reproducibility (Law et al., 2015). Recently, direct quantitative PCR was applied to the detection of Campylobacter in river water and showed the possibility of an alternative method for Campylobacter detection (Van Dyke et al., 2010). Nevertheless, the detection validation of Campylobacter with culture-based methods would still be necessary to reveal the direct correlation between a clinical illness and its etiological agents.

In this study, we present amendments to existing culture methods to improve the enrichment and isolation of Campylobacter spp. from wastewater. We used raw sewage influent samples since they are contaminated more heavily than effluent samples. By adopting different incubation temperatures and several antibiotics, to which C. jejuni is intrinsically resistant, we developed an improved enrichment method to recover culturable C. jejuni from wastewater samples. The efficiency of Campylobacter isolation was evaluated using quantitative real-time PCR (qRT-PCR) targeting genus-specific 16S rDNA primers, and a second end-point multiplexed PCR with species-specific primers. By using 16S rDNA amplicon sequencing, in addition, we identified the major bacteria in wastewater that compete with Campylobacter under the selective enrichment conditions.

In this study, we improved the efficacy of C. jejuni isolation from wastewater by increasing antibiotic selective pressure in the enrichment step. The addition of rifampicin, polymyxin B, or both to the enrichment media affected the C_t values of qRT-PCR results (Figure 2). According to the distribution of C_t values, the addition of the antibiotic(s) decreased C_t values, meaning that antibiotic supplements improved the growth of Campylobacter. In particular, rifampicin significantly reduced C_t values (Figure 2). A few studies have thus far reported that increased selective pressure enhances Campylobacter isolation from food. Yoo et al. (2014) reported that the addition of rifampicin (10 μg/ml) or polymyxin B (5 IU/ml) to Bolton agar (Bolton agar with Bolton supplement) restrained the growth of non-Campylobacter without any inhibition of C. jejuni and C. coli in fresh produce foods. Chon et al. (2013) demonstrated that the addition of high concentrations of polymyxin B to the mBolton supplement in enrichment procedure improved the efficiency of C. jejuni and C. coli recovery and suppressed background competing bacteria. Consistently, our results showed that the supplementation with additional antibiotics improved the efficacy of C. jejuni isolation even from heavily contaminated wastewater samples. In addition, we also identified bacterial populations that compete with Campylobacter under the four different selective enrichment conditions. The inputs of Campylobacter entering the influent of wastewater treatment facilities in this study would be primarily from sewage effluent in Calgary and also possibly from wildlife, such as migrating birds (Cody et al., 2015). Depending on the treatment procedure, the incidence rate of Campylobacter in sewage effluent can be altered, and cross contamination between water resources and sewage is associated with water-borne Campylobacter outbreaks (Jones, 2001; Pitkanen, 2013).

The 16S rDNA amplicon sequencing analysis of individual colonies from the enrichment plates revealed that Escherichia, Pseudomonas, Klebsiella, and Enterococcus were the major competing bacteria in C. jejuni isolation from wastewater (Table 3). Baylis et al. (2000) identified competitor organisms in foods by using Preston and Bolton selective supplement media, showing that Yersinia, Enterobacter, Escherichia, Enterococcus, Pseudomonas, and Klebsiella are representative competitors. This is quite similar to our results from the BN enrichment conditions. Escherichia were frequently isolated in BN (Table 3), presumably because extended-spectrum beta-lactamase (ESBL)-producing E. coli may reduce the selectivity of Bolton supplement and consequently E. coli growth would suppress Campylobacter (Moran et al., 2011). Although the supplementation of additional antibiotic(s) suppressed the overgrowth of competing bacteria and enriched Campylobacter, Enterococcus survived well in the presence of five different antibiotics as it was frequently isolated with Campylobacter (Table 3). The survival of Enterococcus in the presence of vancomycin (20 μg/mL) in Bolton supplement indicated that Enterococcus isolated from the enrichment broth is vancomycin-resistant enterococci (VRE), a drug-resistant strain of serious public health concern (Cetinkaya et al., 2000). This study aimed at developing an improved culture method to isolate Campylobacter from wastewater, and we used the influent samples, not the effluent, since the influent is more contaminated than the effluent. Therefore, the results do not provide the information about the level of Campylobacter contamination in the effluent that may have a direct impact on public health compared to the influent data.

In this study, we demonstrated that antibiotic selective pressure and culture temperature are the critical factors for C. jejuni isolation from raw sewage. The BN, BNR, BNP, and BNRP conditions showed similar MPN values at 42°C; however, only BNRP showed reasonable MPN values and BNR and BNP showed relatively lower MPN numbers at 37°C compared to those at 42°C (data not shown). The results exhibited that culture temperature also plays an important role in the selective enrichment of C. jejuni. Humphrey et al. showed the effect of antibiotics and temperature on the recovery rate in cold-damaged C. jejuni. The sub-lethally injured cells are more sensitive to antibiotics in 43°C than 37°C, affecting the restoration of C. jejuni (Humphrey, 1986). In previous studies, Humphrey et al. also suggested that pre-incubation at 37°C for 4–18 h followed 42 or 37°C incubation for 48 h would be beneficial to the recovery of Campylobacter in comparison with 42°C (Humphrey, 1989; Humphrey and Muscat, 1989). Whereas Khan et al. (2013) demonstrated that the detection frequency of Campylobacter spp. was higher at 37°C in BN than 42°C, C. jejuni was detected more frequently at 42°C than at 37°C. Consistently, our results suggested that 42°C seems to enhance C. jejuni growth in raw sewage samples.

The additional antibiotic(s) plus an increased incubation temperature (i.e., 42°C) improved the isolation rates of C. jejuni and C. coli from heavily contaminated raw sewage samples. The addition of rifampicin and polymyxin B specifies the selective enrichment of thermo-tolerant Campylobacter spp., such as C. jejuni and C. coli, the major human pathogenic species (Kaakoush et al., 2015). Based on our findings, increased antibiotic selective pressure and culture temperature are the key parameters impacting the success in C. jejuni isolation from heavily contaminated wastewater samples. Additionally, rifampicin appears to be effective in improving the selectivity of Campylobacter enrichment for PCR-based quantitative methods, whereas both rifampicin and polymyxin B are required to suppress competing bacterial growth and improve the selectivity of C. jejuni isolation with culture-based methods.

Design of the project: JK, NA, NN, and BJ; Performance of the experiments: JK, EO, GB, and SB; Data analysis: JK, EO, GB, SB, LC, NA, NN, and BJ; Writing of the manuscript: JK, NA, and BJ.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.