An Improved Culture Method for Selective Isolation of Campylobacter jejuni from Wastewater

Abstract

Campylobacter jejuni is one of the leading foodborne pathogens worldwide. C. jejuni is isolated from a wide range of foods, domestic animals, wildlife, and environmental sources. The currently available culture-based isolation methods are not highly effective for wastewater samples due to the low number of C. jejuni in the midst of competing bacteria. To detect and isolate C. jejuni from wastewater samples, in this study, we evaluated a few different enrichment conditions using five different antibiotics (i.e., cefoperazone, vancomycin, trimethoprim, polymyxin B, and rifampicin), to which C. jejuni is intrinsically resistant. The selectivity of each enrichment condition was measured with Ct value using quantitative real-time PCR, and multiplex PCR to determine Campylobacter species. In addition, the efficacy of Campylobacter isolation on different culture media after selective enrichment was examined by growing on Bolton and Preston agar plates. The addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of C. jejuni. The results of 16S rDNA sequencing also revealed that Enterococcus spp. and Pseudomonas aeruginosa are major competing bacteria in the enrichment conditions. Although it is known to be difficult to isolate Campylobacter from samples with heavy contamination, this study well exhibited that the manipulation of antibiotic selective pressure improves the isolation efficiency of fastidious Campylobacter from wastewater.

Detection of Campylobacter sp. from Poultry Feces in Ouagadougou, Burkina Faso

“…Previous studies have reported that increased selective pressure in selective agar and/or enrichment broth enhances Campylobacter isolation from various samples. Kim et al, [11] reported that the addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of Campylobacter isolation from wastewater. Yoo et al [12] 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.…”

Campylobacter hepaticus, the Cause of Spotty Liver Disease in Chickens: Transmission and Routes of Infection

“…There have been no published studies regarding the epidemiology of the bacterium and how it is transmitted between and within chicken flocks. Studies on closely related species commonly isolated from poultry, Campylobacter jejuni and Campylobacter coli, have shown the presence of these bacteria in multiple sources besides poultry (15)(16)(17), including, wild birds (18,19), cattle (20), pigs (21), dogs (22), flies (23,24), darkling beetles (25), water (26), and soils (27).…”

Addition of Rifampicin to Bolton Broth to Inhibit Extended‐Spectrum β‐Lactamase‐Producing Escherichia coli for the Detection of Campylobacter

“…Yoo and others () used 10 μg/mL of rifampicin in their modified agar to suppress Pseudomonas and Ochrobactrum . Kim and others () used the same concentration of rifampicin for the inhibition of competing microbiota in wastewater. However, 10 μg/mL of rifampicin was insufficient to inhibit all the ESBL‐producing E. coli strains in Bolton broth.…”

“…Yoo and others () formulated BRS agar (Bolton agar supplemented with rifampicin and sulfamethoxazole) containing 10 μg/mL of rifampicin and 50 μg/mL of sulfamethoxazole in Bolton agar, to inhibit the growth of indigenous bacteria, including Pseudomonas and Ochrobactrum in fresh produce. Kim and others () also evaluated rifampicin as an additional supplement in Bolton broth along with polymyxin B for the selective isolation of Campylobacter from wastewater. However, they did not evaluate the effect of rifampicin on the inhibition of ESBL‐producing E. coli from chicken products which have different indigenous microbiota than other food or environmental samples.…”