Research Article: Evaluation of potassium clavulanate supplementation of Bolton broth for enrichment and detection of Campylobacter from chicken

Date Published: October 24, 2018

Publisher: Public Library of Science

Author(s): Bai Wei, Min Kang, Hyung-Kwan Jang, Anderson de Souza Sant’Ana.

http://doi.org/10.1371/journal.pone.0205324

Abstract

Culture-based detection of Campylobacter can be affected by competing flora, temperature, incubation time, and presence of blood. The presence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in poultry has become one of the most common factors interfering with the detection of Campylobacter. In the present study, we evaluated potassium clavulanate (ESBL inhibitor) as a supplement in Bolton broth (C-Bolton broth) for enrichment and detection of Campylobacter. First, we determined growth kinetics of Campylobacter in the presence of different concentrations of ESBL E. coli in C-Bolton broth during enrichment. The effects of other factors such as incubation time, incubation temperature, and presence of blood on Campylobacter detection in C-Bolton broth were also investigated. The growth of Campylobacter co-cultured at a low concentration (2 and 4 log10 CFU/mL) of ESBL E. coli was similar to that of Campylobacter alone in C-Bolton broth, and Campylobacter co-cultured at a high concentration (6 and 8 log10 CFU/mL) of ESBL E. coli showed slower growth than the pure Campylobacter culture. The Campylobacter detection limit was 1 log10 CFU/mL when mixed with 2, 4, or 6 log10 CFU/mL of E. coli and 3 log10 CFU/mL when mixed with 8 log10 CFU/mL of E. coli after 48 h enrichment in the broth. Campylobacter detection from chicken feces and litter samples was not affected by incubation time, or presence of blood in the broth. A modified procedure of enrichment in C-Bolton broth at 37°C for 24 h without blood showed a significantly (P ≤ 0.05) higher detection rate and a lower false-negative rate than the ISO 10272–1:2006 method for Campylobacter detection from chicken feces and litter samples. In summary, the present study demonstrates the efficacy of Bolton broth supplemented with potassium clavulanate in the detection of Campylobacter mixed with ESBL E. coli, and an improved procedure to detect Campylobacter from chicken feces and litter samples.

Partial Text

Campylobacteriosis is a significant and increasing problem to human health both in developed and developing countries. Campylobacter is the most common bacterial cause of gastroenteritis with 246,307 confirmed cases of campylobacteriosis in 2016 in Europe [1]. Campylobacter jejuni and C. coli account for over 99% of campylobacteriosis cases [1]. The most common clinical symptoms of Campylobacter infections include diarrhea, abdominal pain, fever, headache, nausea, and vomiting. The majority of human campylobacteriosis cases is sporadic, with poultry being the natural reservoir and thought to be the most important vehicle of infection [2].

Although various selective agar media and enrichment broths have been developed and introduced for the isolation of Campylobacter, the Bolton broth is the most common medium used to detect Campylobacter spp. [14]. However, the increasing prevalence of ESBL E. coli has become the primary factor hindering the detection of Campylobacter in Bolton broth [4]. Therefore, inhibiting ESBL E. coli using an ESBL inhibitor has attracted attention and has shown significant benefits in detecting Campylobacter in chicken products [5]. A previous study showed that Campylobacter growth was not competitively inhibited due to a low level of ESBL E. coli in Bolton broth supplemented with potassium clavulanate in artificial and natural samples [6]. However, the efficiency of this modified medium in detecting Campylobacter from environmental samples with high levels of competitive bacteria including ESBL E. coli has not been determined. Therefore, we conducted the present study to investigate the growth kinetics of Campylobacter in C-Bolton broth with competitive ESBL E. coli. Campylobacter growth has been shown to be inhibited in Bolton broth at concentrations < 3 log10 CFU/mL in the presence of competitive ESBL E. coli, with no Campylobacter recovery from the second agar plate [4]. In the present study, we used 1, 2, or 3 log10 CFU/mL of Campylobacter mixed with different concentrations of competitive ESBL E. coli.   Source: http://doi.org/10.1371/journal.pone.0205324

 

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