# Optimizing ISO standard microbiological techniques for isolating Campylobacter from poultry samples amidst challenges from extended spectrum beta lactamase producing Escherichia coli

Keya Ghosh, Tahia Ahmed Logno, Tridip Das, Pangkaj Kumar Dhar, Damer Blake, Guillaume Fournie, Fiona Tomley, Richard A Stabler, Burhan Lehri, Paritosh Kumar Biswas

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## To cite this version:

Keya Ghosh, Tahia Ahmed Logno, Tridip Das, Pangkaj Kumar Dhar, Damer Blake, et al.. Optimizing ISO standard microbiological techniques for isolating Campylobacter from poultry samples amidst challenges from extended spectrum beta lactamase producing Escherichia coli. PLoS ONE, 2025, 20 (7), pp.e0327963. 10.1371/journal.pone.0327963. hal-05213649

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RESEARCH ARTICLE

# Optimizing ISO standard microbiological techniques for isolating *Campylobacter* from poultry samples amidst challenges from extended spectrum beta lactamase producing *Escherichia coli*

Keya Ghosh<sup>1</sup>, Tahia Ahmed Logno<sup>1</sup>, Tridip Das<sup>1,2,3</sup>, Pangkaj Kumar Dhar<sup>1,2,3</sup>, Damer Blake<sup>4*</sup>, Guillaume Fournie<sup>4,5,6</sup> Fiona Tomley<sup>4</sup>, Richard A. Stabler<sup>1,7</sup>, Burhan Lehri<sup>7</sup>, Paritosh Kumar Biswas<sup>1</sup>

1 Department of Microbiology and Veterinary Public Health, Chattogram Veterinary and Animal Sciences University, Chittagong, Bangladesh, 2 School of Agricultural, Environmental and Veterinary Sciences, Faculty of Sciences and Health, Charles Sturt University, Wagga Wagga, New South Wales, Australia, 3 UKRI GCRF One Health Poultry Hub, Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh, 4 Department of Pathobiology and Population Sciences, Royal Veterinary College, London, United Kingdom, 5 INRAE, VetAgro Sup, UMR EPIA, Université de Lyon, Marcy l'Etoile, France, 6 INRAE, VetAgro Sup, UMR EPIA, Université Clermont Auvergne, Saint Genes Champanelle, France, 7 London School of Hygiene and Tropical Medicine, London, United Kingdom

* dblake@rvc.ac.uk

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OPEN ACCESS

**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](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data availability statement:** 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

## 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 *bla*<sub>TEM</sub>, *bla*<sub>CTX</sub> or *bla*<sub>SHV</sub>. 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
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.

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.

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 its 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

<table><caption>Table 1. Primers used in PCR to detect *hipO*, *glyA* and 23S rRNA gene sequences as markers for confirmation of *C. jejuni*, *C. coli* and *Campylobacter* spp. Respectively, and for the presence of *adk*, *blaTEM*, *blaSHV*, *blaCTX* genes in *E. coli*.</caption><thead><tr><th>Gene</th><th>Primer name</th><th>Primer sequence (5'-------- 3')</th><th>Amplicon size(bp)</th><th>Annealing temp</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">adk</td><td>AdkF</td><td>ATTCTGCTTGGCGCTCCGGG</td><td rowspan="2">536</td><td rowspan="2">54</td><td rowspan="2">[14]</td></tr><tr><td>AdkR</td><td>CCGTCAACTTTCGCGTATTT</td></tr><tr><td rowspan="2">blaTEM</td><td>TEM-F</td><td>GCGGAACCCCTATTTG</td><td rowspan="2">964</td><td rowspan="2">50</td><td rowspan="2">[15]</td></tr><tr><td>TEM-R</td><td>TCTAAAGTATATATGAGTAAACTTGGTCTGAC</td></tr><tr><td rowspan="2">blaSHV</td><td>SHV-F</td><td>TTCGCCTGTGTATTATCTCCCTG</td><td rowspan="2">854</td><td rowspan="2">50</td><td rowspan="2">[16]</td></tr><tr><td>SHV-R</td><td>TTAGCGTTGCCAGTGYTCG</td></tr><tr><td rowspan="2">blaCTX</td><td>CTX-F</td><td>ATGTGCAGYACCAGTAARGTKATGGC</td><td rowspan="2">593</td><td rowspan="2">60</td><td rowspan="2">[17]</td></tr><tr><td>CTX-R</td><td>TGGGTRAARTARGTSACCAGAAYCAGCGG</td></tr><tr><td rowspan="2">hipO</td><td>CJF</td><td>ACTTCTTTATTGCTTGCTGC</td><td rowspan="2">323</td><td rowspan="2">59</td><td rowspan="2">[18]</td></tr><tr><td>CJR</td><td>GCCACAACAAGTAAAGAAGC</td></tr><tr><td rowspan="2">glyA</td><td>CCF</td><td>GTAAAACCAAAGCTTATCGTG</td><td rowspan="2">126</td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td>CCR</td><td>TCCAGCAATGTGTGCAATG</td></tr><tr><td rowspan="2">23S rRNA</td><td>23S rRNA F</td><td>TATACCGGTAAGGAGTGCTGGAG</td><td rowspan="2">650</td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td>23S rRNA R</td><td>ATCAATTAACCTTCGAGCACCG</td></tr></tbody></table>

https://doi.org/10.1371/journal.pone.0327963.t001
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 37°C 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 42°C 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.

<table><caption>Table 2. Inoculum groups in the simulation study with different organisms in sterile chicken cecal matrix and their growth on mCCDA after pre-enrichment in Preston broth.</caption><thead><tr><th>Inoculum group</th><th>Contents in the inoculum matrix</th><th>C. jejuni</th><th>C. coli</th><th>E. coli</th><th>E. faecalis</th></tr></thead><tbody><tr><td>A</td><td>CCM + C. jejuni</td><td>+++</td><td></td><td>-</td><td>-</td></tr><tr><td>B</td><td>CCM + C. coli</td><td></td><td>+++</td><td>-</td><td>-</td></tr><tr><td>C</td><td>CCM + C. jejuni + C. coli</td><td>+</td><td>+++</td><td>-</td><td>-</td></tr><tr><td>D</td><td>CCM + C. coli + C. jejuni + ESBL E. coli</td><td>-</td><td>-</td><td>+++</td><td>-</td></tr><tr><td>E</td><td>CCM + C. coli + C. jejuni + E. coli ATCC25922</td><td>+</td><td>+++</td><td>-</td><td>-</td></tr><tr><td>F</td><td>CCM + E. faecalis</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

[CCM= chicken cecal matrix, +++ = highest isolation efficiency, ++ = moderate isolation efficiency, + = low isolation efficiency, - = nil].

https://doi.org/10.1371/journal.pone.0327963.t002
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**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 Mac-Conkey 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 *bla*<sub>CTX</sub> gene (n=20, 80%) followed by 18 (72%) and 2 (8%) for *bla*<sub>TEM</sub> and *bla*<sub>SHV</sub> 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).

<figure><img src="image_6.png" /><figcaption>Fig 1. Distribution of ESBL genes in 25 *E. coli* strains isolated from growth on mCCDA during selective culture for *Campylobacter* from cecal contents of chickens.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0327963.g001
<figure><img src="image_7.png" /><figcaption>Fig 2. Depicting the growth of *C. jejuni* (A) and *C. coli* (B) on mCCDA.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0327963.g002

<figure><img src="image_8.png" /><figcaption>Fig 3. 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).</figcaption></figure>

https://doi.org/10.1371/journal.pone.0327963.g003

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.

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.

<table><caption>Table 3. Tazobactam susceptibility of ESBL E. coli strains isolated from cecal swabs from live chickens with some identified ESBL genes.</caption><thead><tr><th>E. coli Isolate no</th><th>ESBL gene</th><th>MIC (mg/L)*</th><th>Bird type</th><th>Sample source</th></tr></thead><tbody><tr><td>1</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub></td><td>>128</td><td>Deshi</td><td>Live bird market</td></tr><tr><td>2</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub></td><td>>128</td><td>Sonali</td><td>Farm</td></tr><tr><td>3</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub></td><td>128</td><td>Broiler</td><td>Live bird market</td></tr><tr><td>4</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub>, bla<sub>SHV</sub></td><td>128</td><td>Deshi</td><td>Live bird market</td></tr><tr><td>5</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub>, bla<sub>SHV</sub></td><td>128</td><td>Deshi</td><td>Live bird market</td></tr><tr><td>6</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub></td><td>128</td><td>Broiler</td><td>Live bird market</td></tr><tr><td>7</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub></td><td>128</td><td>Sonali</td><td>Farm</td></tr><tr><td>8</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub></td><td>128</td><td>Broiler</td><td>Farm</td></tr><tr><td>9</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub>, bla<sub>SHV</sub></td><td>>128</td><td>Sonali</td><td>Live bird market</td></tr><tr><td>10</td><td>bla<sub>CTX</sub>, bla<sub>TEM</sub></td><td>128</td><td>Sonali</td><td>Live bird market</td></tr></tbody></table>

https://doi.org/10.1371/journal.pone.0327963.t003

<table><caption>Table 4. Determination of the level of Tazobactam that could inhibit the growth of ESBL E. coli in Preston broth and recovering C. coli, and C. jejuni on mCCDA inoculated with inoculum from Preston broth pre-enriched with the three organisms.</caption><thead><tr><th rowspan="2">Tazobactam concentration in Preston broth</th><th colspan="2">Recovery from mCCDA without Tazobactam</th><th rowspan="2">ESBL E. coli</th></tr><tr><th>C. coli</th><th>C. jejuni</th></tr></thead><tbody><tr><td>0mg/L</td><td>+</td><td>+</td><td>+++</td></tr><tr><td>1mg/L</td><td>+</td><td>+</td><td>+++</td></tr><tr><td>4mg/L</td><td>+</td><td>+</td><td>+++</td></tr><tr><td>16mg/L</td><td>+</td><td>+</td><td>+++</td></tr><tr><td>64mg/L</td><td>++</td><td>++</td><td>+++</td></tr><tr><td>128mg/L</td><td>+++</td><td>+++</td><td>+</td></tr></tbody></table>

https://doi.org/10.1371/journal.pone.0327963.t004

<footnote>* minimum inhibitory concentration of tazobactam sodium.</footnote>

<footnote>[+++ = highest isolation efficiency, ++ = Moderate, + = Low, - = Nil].</footnote>
<table><caption>Table 5. Determination of the tazobactam concentration added to mCCDA plates for direct recovery of C. coli, and C. jejuni without any pre-enrichment in Preston broth.</caption><thead><tr><th rowspan="2">Tazobactam concentration in mCCDA</th><th colspan="3">Recovery from mCCDA with Tazobactam</th></tr><tr><th>C. coli</th><th>C. jejuni</th><th>ESBL E. coli</th></tr></thead><tbody><tr><td>0mg/L</td><td>+</td><td>+</td><td>+++</td></tr><tr><td>1mg/L</td><td>++</td><td>++</td><td>+++</td></tr><tr><td>4mg/L</td><td>+++</td><td>+++</td><td>–</td></tr><tr><td>16mg/L</td><td>+++</td><td>+++</td><td>–</td></tr><tr><td>64mg/L</td><td>+++</td><td>+++</td><td>–</td></tr><tr><td>128mg/L</td><td>+++</td><td>+++</td><td>–</td></tr></tbody></table>

[+++ = highest isolation efficiency, ++ = Moderate, + = Low, – = Nil].

https://doi.org/10.1371/journal.pone.0327963.t005

<figure><img src="image_9.png" /><figcaption>Fig 4. Growth of *Campylobacter* on mCCDA plate (A) *Campylobacter* growth following inoculation from Tazobactam supplemented-Preston broth at the level of 128 mg/L, inoculated with known C. jejuni, C. coli and an ESBL E. coli strain. (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.</figcaption></figure>

https://doi.org/10.1371/journal.pone.0327963.g004

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 *bla*<sub>CTX</sub> was more frequently found than *bla*<sub>TEM</sub> and *bla*<sub>SHV</sub>, 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.
(DOCX)

**S1 Fig.** Phenotypic observation of *E.coli* on mCCDA agar.
(TIF)

**S2 Fig.** Growth of *E.coli* observed on Mac conkey agar.
(TIF)
**S3 Fig.** Growth of *E.coli* observed on EMB agar.
(TIF)

**S4 Fig.** Growth of *E.coli* observed on Blood agar.
(TIF)

**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).
(TIF)

## Author contributions

**Conceptualization:** Keya Ghosh, Paritosh Kumar Biswas.

**Data curation:** Keya Ghosh, Paritosh Kumar Biswas.

**Investigation:** Keya Ghosh, Paritosh Kumar Biswas.

**Methodology:** Keya Ghosh, Tahia Ahmed Logno, Tridip Das, Pangkaj Kumar Dhar, Damer Blake, Richard A. Stabler, Burhan Lehri, Paritosh Kumar Biswas.

**Project administration:** Keya Ghosh.

**Supervision:** Paritosh Kumar Biswas.

**Validation:** Keya Ghosh, Paritosh Kumar Biswas.

**Visualization:** Paritosh Kumar Biswas.

**Writing – original draft:** Keya Ghosh.

**Writing – review & editing:** Keya Ghosh, Tridip Das, Damer Blake, Guillaume Fournie, Fiona Tomley, Richard A. Stabler, Burhan Lehri, Paritosh Kumar Biswas.

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