https://orcid.org/0000-0002-0443-3124
Figures
Abstract
Isolation of zoonotic Campylobacter species has been standardized through the ISO 10272:2017 protocol. However, application of the protocol in a LMIC country failed to isolate Campylobacter due to extended-spectrum beta-lactamase (ESBL) producing Escherichia coli overgrowth during the Campylobacter selective enrichment phase. The aim of the study was to identify the contaminants and explore ways to mitigate them. A set of 25 non-Campylobacter contaminants isolated from chicken cecal samples grown on modified charcoal-cefoperazone-deoxycholate agar (mCCDA) during Campylobacter isolation were included. All isolates were screened for species identification and the presence of selected ESBL producing genes. Minimum inhibitory concentrations of tazobactam were measured using a microbroth dilution technique. The Campylobacter isolation protocol was then modified to inhibit the contaminants by adding the required tazobactam supplement to Preston broth or to mCCDA. All contaminants were found to be E. coli carrying at least one of the ESBL-producing genes blaTEM, blaCTX or blaSHV. The MIC of tazobactam sodium for ESBL-producing E. coli strains grown in Preston broth was at least 128 mg/L. Preston broth supplemented with tazobactam at 128 mg/L inhibited the growth of ESBL-producing E. coli but did not inhibit the growth of C. jejuni or C. coli. Interestingly, mCCDA plates supplemented with tazobactam at a much lower concentration of 4 mg/L could also prevent growth of ESBL-producing E. coli even without broth enrichment, increasing the efficiency of isolation of Campylobacter. Direct inoculation of cecal materials to mCCDA supplemented with tazobactam at 4 mg/L was recommended as the most cost-effective way to conduct Campylobacter surveillance targeting the cecal matrix instead of directly following ISO 10272:2017 protocol.
Citation: Ghosh K, Logno TA, Das T, Dhar PK, Blake D, Fournie G, et al. (2025) Optimizing ISO standard microbiological techniques for isolating Campylobacter from poultry samples amidst challenges from extended spectrum beta lactamase producing Escherichia coli. PLoS One 20(7): e0327963. https://doi.org/10.1371/journal.pone.0327963
Editor: Dwij Raj Bhatta, Tribhuvan University, NEPAL
Received: December 31, 2024; Accepted: June 24, 2025; Published: July 31, 2025
Copyright: © 2025 Ghosh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript.
Funding: No specific awards or grant numbers. Actually the authors worked under a project during her postgrad research. The project is called “One Health Poultry Hub” under United Kingdom Research Innovation (UKRI) Global Challenge Research Fund (GCRF). They were providing necessary research funds and other resources to conduct a surveillance for this research work to the principal investigator only.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Campylobacter are gram-negative, microaerophilic, non-spore-forming bacteria that resemble a spiral, curved, or rod shape [1]. Poultry, major reservoirs of Campylobacter, play a key role in the high global occurrence of human campylobacteriosis. Preliminary contamination of broiler (chickens reared for meat) flocks occurs through horizontal transmission where the organism commonly acts as a commensal, probably supported by their thermotolerant features [2]. Campylobacter colonization of poultry can be determined by isolation from feces. Detection of Campylobacter carriage before slaughter can help limit the pathogen’s spread to the human food chain. However, isolation of Campylobacter is challenging compared to many other pathogens linked to food-borne illness due to its fragile and complex nature, microaerophilic atmospheric requirements, sluggish growth rate, low bacterial numbers, and fastidious growth requirements [1,3].
The International Organization for Standardization protocol for Microbiology of the food chain horizontal method for detection and enumeration of Campylobacter spp. [4] is a standard optimized protocol used globally to isolate Campylobacter from products intended for human consumption. The protocol can also be applied to feeds intended for animals, and to environmental samples from areas of food production and food handling. The protocol uses Bolton or Preston broths to enrich Campylobacter, followed by modified charcoal cefoperazone deoxycholate agar (mCCDA) as a second selective medium [4,5]. Cefoperazone, a beta-lactam antibiotic, is used to prevent the growth of competing flora since Campylobacter is intrinsically resistant. Common selective agents also used in conventional Campylobacter agars include Cefoperazone, Cycloheximide, Trimethoprim, Rifampicin, Vancomycin and Polymyxin B [6] Possible contaminating or co-existing organisms in the sample matrix can only grow when they can hydrolase these antibiotic or resist it by any other mechanism(s). Recently, detection and selective culture of Campylobacter has become more difficult due to the high occurrence of antimicrobial resistant bacteria, specifically extended spectrum β-lactam resistant E. coli, which has been identified as a viable contaminant in studies using mCCDA and enrichment broths [7–9]. When ESBL producers are a problem, the use of suitable beta lactamase inhibitors like Tazobactum, Polymixin B, Potassium clavulanic acid and Triclosan, is recommended [7,9,10–13]. However, among all beta lactamase inhibitors, Tazobactum was found to be most effective to inhibit ESBL activity due to it’s chemical stability and cheaper cost [13].
This study aimed to characterize contaminating organisms that can grow in Preston broth or on mCCDA during surveillance for Campylobacter from chicken cecal contents following the ISO 10272:2017 protocol and to modify the protocol for efficient isolation of Campylobacter by inhibiting the growth of these challenging organisms.
Materials and methods
Samples
This study was part of the UKRI Global Challenges Research Fund (GCRF) One Heath Poultry Hub, an impact-driven development research programme working in Bangladesh, India, Sri Lanka and Vietnam (www.onehealthpoultry.org/). As part of surveillance for the zoonotic pathogen Campylobacter spp. within poultry, 25 non-Campylobacter isolates recovered from mCCDA after initial enrichment of chicken cecal contents in Preston broth were selected for study.
Confirmation of contaminants
Non-Campylobacter isolates grown on mCCDA were initially streaked onto MacConkey agar and incubated at 37°C for 24 hours aerobically. After the incubation, large pink colonies were present. Suspected E. coli isolates were inoculated onto EMB agar and incubated at 37°C for 24 hours aerobically. The presence of colonies defined by a metallic sheen supported identification as E. coli, confirmed subsequently by PCR to detect the housekeeping gene adk (adenylate kinase) [14].
Screening of ESBL producing genes
All the 25 isolates were tested for the presence of genes responsible for producing blaTEM, blaSHV, blaCTX ESBL separately with uniplex PCR, using the primers as shown in (Table 1).
Simulation study with Campylobacter strains
In house reference strains of C. jejuni and C. coli used in this study were provided by the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). According to Campylobacter methodology UNI EN ISO 10272–1:2017, a loopful of each of the strains preserved was inoculated into Preston broth (Oxoid, UK, prepared by adding Modified Preston Campylobacter Selective Supplement, Campylobacter Growth Supplement, and Lysed horse blood according to the manufacturer’s instruction) and incubated at 37°C for 5 hours followed by 42°C for 48 hours under microaerophilic condition [4]. The incubated Preston broth was then inoculated onto mCCDA agar (Oxoid, UK; prepared by adding CCDA Selective Supplement (SR0155, Oxoid, UK) to Campylobacter Blood-Free Selective Agar Base (CM0739, Oxoid, UK, according to the manufacturer’s instructions) and blood agar(Oxoid Ltd, UK) incubated under microaerophilic conditions at 42°C for 48 hours. The presumptive growth of Campylobacter on mCCDA was examined by Gram’s staining. Finally, C. jejuni and C. coli were identified by PCR to detect the hipO and glyA gene and internal control to detect Campylobacter 23S RNA gene respectively. The primer sequences used to detect them are shown in (Table 1).
For the simulation study, poultry cecal material collected from chickens was dried and then sterilized by autoclaving at 121°C for 15 minutes. The sterility of the cecal content matrix was verified by inoculating it onto blood agar and by finding no bacterial growth after 48 hours of aerobic or microaerophilic incubation. Then, the sterile matrix was divided into six inoculum groups, referred to as A, B, C, D, E and F, and inoculated by 0.5 McFarland standard of specific bacterial cultures (Table 2). Here, In house reference strain of Enterococcus fecalis and E.coli ATCC25922 were used in this study as control. One loopful of inoculum from each of group was then inoculated into Preston broth, incubated 5 hours at 37⁰C followed by 42°C for 48 hours under microaerophilic condition. A loopful of enriched Preston broth culture from each group was then inoculated onto mCCDA and incubated at 42⁰C for 48 hours. The growth yielded from each of the inoculum groups on mCCDA was recorded. Three similar replication tests with the same samples were also performed in this simulation study.
Minimum inhibitory concentration of tazobactam sodium to ESBL producing E. coli
A total of 10 randomly selected ESBL producing E. coli strains were used to assess the minimum inhibitory concentration of Tazobactam sodium using the broth micro dilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [19]. Cation adjusted Muller Hinton Broth (MHB) II (Oxoid, UK) and Tazobactam sodium (Sigma-Aldrich, Saint Louis, MO, USA) were used in broth microdilution. E. coli ATCC25922 was used as a negative control. MIC values were interpreted following CLSI guidelines [19].
Determination of Tazobactam concentration in Preston broth and mCCDA
Preston broth was prepared with Tazobactam at different concentration: 1, 4, 16, 64, 128 mg/L and inoculated with C. jejuni, C. coli, and/or an ESBL E. coli strain with inoculum turbidity of 0.5 McFarland standard (equivalent to growth of 1–2 × 108 CFU/mL). After incubation under microaerophilic condition at 42°C for 48 hours, a sub-sample from each tube was inoculated onto a mCCDA plate, incubated at 42°C for a further 48 hours under microaerophilic condition. The growth yielded after incubation were then evaluated. mCCDA was also prepared with Tazobactam at five different concentrations: 1, 4, 16, 64 and 128 mg/L, and inoculated separately with sub-samples of the same C. jejuni, C. coli and/or ESBL E. coli isolate at 0.5 McFarland standard. After incubation at 42°C for 48 hours microaerophilic, possible growth of Campylobacter was observed.
Ethics statement
This study was approved [CVASU/Dir (R&E) EC/2020/165/2/1] by the Ethics Committee of Chattogram Veterinary and Animal Sciences University (CVASU), Bangladesh. No protected species were sampled. Chickens were humanely killed at a designated establishment by cervical dislocation, under animal welfare guidelines.
Results
Confirmation of contaminants on mCCDA
Whitish finely granular colonies were observed on mCCDA and confirmed as colonies of E. coli phenotypically on MacConkey and EMB agar and by PCR.
ESBL gene screening of E. coli isolates
All contaminant E. coli isolates were PCR positive for at least one of the tested ESBL genes (Fig 1). The highest frequency was observed for blaCTX gene (n = 20, 80%) followed by 18 (72%) and 2 (8%) for blaTEM and blaSHV respectively.
Verification of C. jejuni and C. coli strains for simulation study
The C. jejuni strain collected from icddr,b produced small dry colonies on mCCDA while the C. coli strain produced comparatively larger, flat, watery and gray-colored colonies (Fig 2). PCR confirmed the presence of any Campylobacter spp., C. jejuni and C. coli as grown on different media (Fig 3).
(M = 100 bp DNA ladder, NTC = No template control; NC = Negative control; S1 = C. jejuni from blood agar; S2 = C. jejuni from mCCDA; S3 = C. jejuni from Preston broth; S4 = C. coli from Blood agar; S5 = C. coli from mCCDA).
Simulation study
Simulation of selective culture for Campylobacter from chicken cecal contents in each replicate test in the presence of ESBL E. coli confirmed that only E. coli was detected on mCCDA (Table 2). Interspersed colonies characteristic of C. jejuni or C. coli, were not observed, confirming ESBL producing E. coli could mask the growth of Campylobacter. Neither E. coli ATCC25922 strain, an E. coli strain pan-susceptible to antimicrobials, nor Enterococcus faecalis were capable of growing on mCCDA after pre-enrichment through Preston broth. We found similar result in three replicate tests in this study. Colonies characteristic of C. jejuni and/or C. coli were seen when the cecal matrix inoculated with either/both were inoculated onto mCCDA after pre-enrichment using Preston broth.
MIC of Tazobactam sodium
The MIC of Tazobactam for 10 ESBL E. coli strains found to host at least two ESBL genes was ≥ 128 mg/L for all (Table 3). Surprisingly, the MIC of Tazobactam to 7 of the 10 isolates was quite high, 128 mg/L, and for three other strains it was > 128.
Verification of different concentrations of Tazobactam in Preston broth and in mCCDA
We assessed the addition of Tazobactam sodium into Preston broth by using six different concentrations (1, 4, 16, 64 and 128 mg/L) (Table 4). Although, growth of E. coli on mCCDA was seen for all the inoculums except 128 mg/L, no inhibitory effect of Tazobactam on C. jejuni or C. coli was observed. Interestingly, direct inoculation onto mCCDA supplemented with Tazobactam without enrichment found that ESBL E. coli failed to grow at concentrations of 4 mg/L or higher (Table 5, Fig 4). No inhibition of Campylobacter growth was detected following direct inoculation of mCCDA including Tazobactam concentrations up to 128 mg/L.
(B) The growth of Campylobacter on mCCDA which was supplemented with Tazobactam at a concentration of 4 mg/L and inoculated directly with the same microorganisms without any pre-enrichment in Preston broth.
Following confirmation of inhibition of ESBL E. coli but not Campylobacter by Tazobactam in mCCDA at 4 mg/L we deployed the revised protocol in Campylobacter surveillance from cecal samples collected from chickens. Based on the modified protocol applied, a total of 85 samples were investigated where 16 (18.8%) were found to be positive for C. coli, and 15 (17.65%) for C. jejuni, with no detection of ESBL E. coli.
Discussion
The presence of Campylobacter in chicken feces poses a considerable risk of contamination to chicken meat leading to human infection. Effective isolation of Campylobacter for surveillance and diagnosis can be challenging due to overgrowth of non-Campylobacter contaminants on selective agar, e.g., ESBL producing E. coli. Supplementation of selective media using a beta lactamase inhibitor is one option to improve detection of Campylobacter from poultry cecal samples.
In this study, glossy white colonies were found to grow on mCCDA which obscured any Campylobacter present during selective culture of cecal content from chickens after pre-enrichment in Preston broth. These colonies were identified as ESBL producing E. coli, consistent with previous reports by others [8,9,11]. Cefoperazone, a selective substance in mCCDA media used to culture Campylobacter, contains a β-lactam ring that can be degraded by β-lactamase enzymes produced by beta lactamase producing E.coli, reducing the efficacy of selection [4,7,13]. ESBL producing E. coli grow faster than Campylobacter, including under microaerophilic circumstances [11], overgrowing and masking Campylobacter growth on mCCDA even after pre-enrichment culture in Preston broth. Among ESBL coding sequences blaCTX was more frequently found than blaTEM and blaSHV, reinforcing it as the most prevalent ESBL type in E. coli from poultry in all geographical areas [7,8,15,16,20–22]. The aberrant use of antibiotics in poultry, especially third-generation cephalosporins, could be linked to the acquisition and spread of ESBL genes in E. coli of poultry [23].
To test strategies to control ESBL E. coli during selective culture for Campylobacter reference C. coli and C. jejuni isolates were successfully recovered utilizing Preston broth and plating onto mCCDA in accordance with [4]. However, when mixed with an ESBL E. coli growth of Campylobacter was masked on mCCDA despite pre-enrichment in Preston broth, confirming the hypothesis that Campylobacter could not be recovered using this protocol when the original material, poultry cecal contents, is contaminated with ESBL E. coli. Contamination with E. faecalis did not prevent Campylobacter detection due to the inhibitory effects of cefoperazone in the mCCDA media [11]. Previous studies have suggested whenever there is a possibility of any ESBL producing organism(s) they need be nullified using a beta lactamase inhibitor like Tazobactum [11–13].
The breakpoint for an organism to be considered resistant to tazobactam is 4 mg/L [19]. The ESBL E. coli strains identified in this study as contaminants obscuring Campylobacter surveillance had MIC values of 128 mg/L to Tazobactam in broth culture. Such a high MIC in E. coli strains circulating in poultry could have been influenced by previous exposure to antimicrobials including ESBL inhibitors, but this was not known from the present study. Similarly high MICs have been reported for Tazobactam in E. coli from diverse sources in different geographical areas [24,25]. Culture of C. coli and C. jejuni in broth was unaffected by the presence of Tazobactam at this concentration. Culture on mCCDA revealed a lower MIC, with 4 mg/L inhibitory to ESBL E. coli but not Campylobacter, in line with previous findings that tazobactam up to 10 mg/L had no inhibitory impact on Campylobacter on mCCDA, while 1 mg/L suppressed ESBL producing E. coli [13]. In contrast, We found the concentration of Tazobactam as low as 128 mg/L that could be used in Preston broth to inhibit the growth ESBL producing E. coli strains circulating in commercial poultry in Bangladesh, enabling the isolation of Campylobacter later on mCCDA as part of sub-culturing selectively. The higher concentration of tazobactam in pre-enrichment broth was not corroborated with previous findings where 4 mg/L was enough to suppress ESBL E.coli in Bolton broth [26].
Here, the current study suggests that pre-enrichment in Preston broth can be removed from the existing ISO protocol and directly plating raw samples onto mCCDA supplemented with Tazobactam at a concentration of 4 mg/L would improve Campylobacter isolation efficiency along with the inhibition of ESBL producing E.coli. This modification offers a streamlined protocol compared to previous studies where classical pre-enrichment in Preston broth and selective enrichment on mCCDA were suggested [7,13,27–30]. When pre-enrichment is unavoidable due to very low Campylobacter occurrence, knowledge defining the Tazobactam MIC of the circulating E. coli strains can be used to refine the antibiotic concentration required to distinguish between the bacteria.
Conclusion
Carriage of ESBL producing E. coli in poultry cecal contents poses a challenge to surveillance for Campylobacter using the ISO 10272:2017 protocol. The contrast between Tazobactam concentrations required to control ESBL E. coli in Preston broth compared to on mCCDA is an important consideration when using liquid or solid media for selective culture. Because Tazobactam is relatively expensive, mCCDA supplemented with Tazobactam at a concentration of 4 mg/L can be used as a direct inoculating solid media for effective isolation of Campylobacter in surveillance targeting poultry cecal content.
Supporting information
S1 Table. Raw results of ESBL producing gene detection.
https://doi.org/10.1371/journal.pone.0327963.s001
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S1 Fig. Phenotypic observation of E.coli on mCCDA agar.
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S2 Fig. Growth of E.coli observed on Mac conkey agar.
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S3 Fig. Growth of E.coli observed on EMB agar.
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S4 Fig. Growth of E.coli observed on Blood agar.
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S5 Fig. Original image of PCR products showing the specific amplicons for 23S rRNA (650 bp), hipO (323 bp), and glyA gene (126 bp) gene fragments targeting Campylobacter spp., C. jejuni and C. coli respectively.
(M = 100 bp DNA ladder, NTC = No template control; NC = Negative control; S1 = C. jejuni from blood agar; S2 = C. jejuni from mCCDA; S3 = C. jejuni from Preston broth; S4 = C. coli from Blood agar; S5 = C. coli from mCCDA).
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