Development of a Quantitative PCR Assay for <i>Arcobacter</i> spp. and its Application to Environmental Water Samples

Abstract

Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction−1 and sometimes detected as few as 3 copies reaction−1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3–9.1 log copies 100 mL−1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.

Prevalence of Arcobacter and Other Pathogenic Bacteria in River Water in Nepal

“…Diverse groups of potential pathogenic bacteria, such as Arcobacter, Acinetobacter, and Prevotella, were detected by NGS in all three regions of the Bagmati River during a one-year sampling period. In this study, the abundance ratio of the class Epsilonproteobacteria was higher than that of class Betaproteobacteria, whereas Gammaproteobacteria had the highest abundance ratio in previous studies [4,14]. This may be attributed to differences in the time and place of sample collection.…”

“…Arcobacter spp. were quantified by SYBR Green-based qPCR using a reaction volume of 25 µL containing 2.0 µL of template DNA, 12.5 µL of a MightyAmp for Real Time (SYBR Plus) (Takara Bio), 0.1 µL of 50 pmol/µL each of Arco-F and Arco-R-rev primers [14]. The thermal conditions were as follows: at 98 • C for 2 min, followed by 35 cycles at 98…”

“…In Nepal, only a few studies using next-generation sequencing (NGS) and quantitative PCR (qPCR) have detected Arcobacter in the river and groundwater samples [4,14]. The abundance ratios and concentrations of Arcobacter were highest in river water samples in these studies [4,14]. NGS has been a useful tool in obtaining large numbers of sequences to determine microbial diversity and community structure, as explained in a previous study that was conducted by Caporaso et al (2012) [17].…”

“…Arcobacter in river water samples has been linked to fecal contamination from animal or human sources [11,13]. In Nepal, only a few studies using next-generation sequencing (NGS) and quantitative PCR (qPCR) have detected Arcobacter in the river and groundwater samples [4,14]. The abundance ratios and concentrations of Arcobacter were highest in river water samples in these studies [4,14].…”

“…Arcobacter, which is widely recognized as an emerging waterborne and foodborne pathogen [10][11][12], has been detected in river water samples in Japan, Nepal, and Spain [4,[13][14][15][16]. Arcobacter in river water samples has been linked to fecal contamination from animal or human sources [11,13].…”