Prevalence and Antibiotic Susceptibility of Thermophilic Campylobacter jejuni from Commercial Broiler Farms in and Around Lahore

Faiza Ghazanfar1, Masood Rabbani1*, Aamir Ghafoor2 and

Muhammad Hassan Mushtaq3

1Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore.

2University Diagnostic Laboratory, University of Veterinary and Animal Sciences, Lahore.

3Department of Epidemiology, University of Veterinary and Animal Sciences, Lahore

ABSTRACT

Campylobacter jejuni is one of the most important poultry commensal causing food-borne zoonosis in human. The present study was conducted to determine the prevalence and the antibiotic resistance profile of multiple drugs resistant (MDR) strains of thermophilic C. jejuni from broiler chickens. A total of 375 cloacal swab samples were collected by systematic sampling method of commercial broiler farms in and around Lahore and examined for the presence of thermophilic C. jejuni. Their prevalence was found to be 53% (200/375). Confirmation was done by biochemical and molecular (Polymerase chain reaction) testing methods. Phylogenetic tree analysis was also performed for genetic identification of the species. in vitro antibiotic disc diffusion method of those selected isolates was performed against 20 most common human therapeutic antibiotics. The isolates were found to be highly resistant against most of the available antibiotics leaving behind very less choice of selection for treatment. The results are as follows: ciprofloxacin (91%), clindamycin (87%), moxifloxacin (84%), telithromycin (82%), erythromycin (82%), clarithromycin (76%), nalidixic acid (76%), azithromycin (69%), ampicillin (62%), co-amoxiclav (56%), chloramphenicol (51%), tetracycline (50%), tygecycline (45%), norfloxacin (27%), gentamicin (25%), levofloxacin (24%), ofloxacin (20%), imipenum (4%), meropenum (2%) and ceftazidime (1%). Carbapenums (imipenum and meropenum) and cephalosporin (ceftazidime) was found to be highly effective against these MDR thermophilic C. jejuni isolates. Its efficacy for the treatment of campylobacteriosis should be further evaluated in clinical trials.

Article Information

Received 17 October 2022

Revised 28 October 2022

Accepted 14 November 2022

Available online 12 May 2023

(early access)

Published 24 September 2024

Authors’ Contribution

FG, MR, AG and HM conceived and designed study. FG executed the experiments. FG and MR analyzed the data. FG and MR prepared the manuscript. The manuscript is critically revised by all authors and approved this final version of manuscript.

Key words

Campylobacter jejuni, Food safety, Antibiotics, Broiler chicken, Feed water, Bacteria, Poultry

DOI: https://dx.doi.org/10.17582/journal.pjz/20221017111032

* Corresponding author: [email protected]

0030-9923/2024/0006-2511 $ 9.00/0

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Campylobacter jejuni is a most important food-borne bacteria causing enteric disease in human. It is principally considered a pathogen with raw or under cooked chicken meat being a primary source of infection in humans (Gutiérrez-Martín et al., 2011). It causes a zoonotic illness with symptoms involving watery diarrhea, discontent, abdominal distress and fever (Cantero et al., 2018). Young adults are mostly affected in developed countries while the disease is predominantly existing in children of developing countries (Omara et al., 2015). Although most cases of campylobacteriosis are self-limiting, a small proportion needs medical involvement and antibiotics are suggested in severe and prolonged cases of enteritis, sepsis or other severe and persistent aggressive infections particularly in immuno-compromised patients as well as in infants and pregnant women (Weerasooriya et al., 2022).

During the last decade, concern about this food-borne pathogen has increased mostly because of consistent isolation of antimicrobial resistant strains of thermophilic Campylobacter spp. in human and animals worldwide (Elhadidy et al., 2018; Oh et al., 2015). For decades, macrolides had been the first while fluoroquinolones had been the second choice of replacement to the remedial therapeutics of C. jejuni infections (Lehtopolku et al., 2010; Kayman et al., 2019). Resistance of C. jejuni to both of these antibiotic classes is increasing in numerous countries (Dias et al., 2022). The rise in macrolide resistance is of grave concern as macrolide-resistant strains are usually resistant to fluoroquinolones and other antibiotic groups (Sierra-Arguello et al., 2016; Aksomaitiene et al., 2019). Rapid emergence of multiple drug resistant (MDR) thermophilic Campylobacter strains is getting problematic because of the extremely limited range of treatment options in that situation (Venardou et al., 2021). It has further complicated the cure of this disease (Melero et al., 2012). It had been reported earlier that more than 94 percent of Campylobacter isolated from tourists had resistance to two or more antibiotics that are regarded as MDR (Abay et al., 2014). MDR has become a global challenge and use of antibiotics has become a threat to human health. Even in the absence of antibiotic exposure, resistant strains from other farm animals and the farm environment may enter a contamination cycle infecting animal hosts (Maćkiw et al., 2012).

The aim of the present study was to examine the prevalence of thermophilic C. jejuni and to identify antibiotics that could be useful against those strains.

Materials and Methods

Collection of samples

A total of 375 samples were taken from 25 systematically selected commercial broiler farms in and around the Lahore area. For this purpose, cloacal samples of chicken (n= 15 per broiler farm) were obtained from every 10th farm selected from the list of broiler farms, in and around Lahore that were registered in Pakistan Poultry Association (PPA). The samples were collected from 3 to 3.5 weeks old chicken from different pens in a farm by using sterile cultural swab pre-moistened in sterile water. Each individual swab was kept in the pre-sterile and pre-labeled tube containing Carry-Blair transport medium, and transported on ice-packs, followed by refrigeration at appropriate temperature. Enrichment of each individual sample tube was done by placing it on vortex for 5 min then adding 1ml of that vortex sample into 9 ml of Bolton broth. Incubation was done at 42oC in 2.5L anaerobic jar where Campy-Gen gas generating sachet (CampyGen, Oxoid) was introduced to provide microaerophillic conditions (5% O₂, 10% CO₂ and 85% N₂) for 24 h.

Isolation and identification of Campylobacter jejuni

After 24 h, enriched broth (100µl) was streaked onto separate, modified Charcoal-Cefoperazone-Deoxycholate-Agar (mCCDA) petri plates, which were incubated at 42˚C for 48 h under microaerophillic conditions. Microscopy of the suspected colonies were examined and thermophilic C. jejuni was identified by the biochemical tests of hippurate hydrolysis, indoxyl acetate hydrolysis, oxidase and catalase tests (Holt et al., 2013). Confirmation of the positive samples was done by molecular tests.

Molecular confirmation of the isolates

DNA extraction was done by commercial nucleic acid extraction kit (Vivantis, USA) as per their instructions. The quantification of DNA (ng/µl) was done using Nanodrop 2000 (Thermofisher Scientific). The purity of the extracted DNA was checked at 260/280 ratio. Conventional polymerase chain reaction was done by using organism specific PCR kit (Genetic PCR solutions™ -CamJej epPCR Test - Code1117045) according to manufacturer’s instructions. It was resolved on 1.2% TAE agarose gel to visualize the PCR amplified products. Band size of specie specific primer according to PCR kit was 242 bp. The positive samples were preserved at -70˚C in 80% nutrient broth with added 20% (v/v) glycerol for future research.

Phylogenetic studies

The PCR products were sent for sequencing to a commercial supplier (Advance Bioscience International, Pakistan). The reported sequences were downloaded from GenBank® and compared with the isolated sequence. The phylogenetic maximum likelihood tree for the C. jejuni genome was generated by using the Molecular Evolutionary Genetic Analysis (MEGA) software version 10 (X) software.

Antibiotic susceptibility testing

The C. jejuni isolates were tested by Kirby- Bauer disc diffusion method. Since, there are no antimicrobial resistance breakpoints specific of Campylobacter currently available (Lehtopolku et al., 2010). The resistance breakpoints of enteric bacteria in the family non-Enterobacteriaceae were used to determine antimicrobial resistance of Campylobacter spp. according to the guidelines of Clinical Laboratory Standards Institute (Patel et al., 2017). C. jejuni was grown on nutrient agar and then in nutrient broth, under microaerophillic conditions at 42oC for 24 h, to prepare 0.5 McFarland standard of cell density, which was obtained through spectrophotometer. 100 µl of that bacterial solution was swabbed onto the Mueller-Hinton agar plate. Known antibiotic discs were placed on that agar plate at defined distances and incubated overnight at 42˚C maintaining microaerophillic conditions. Results were interpreted by measuring the zones of inhibited growth for antibiotics corresponding to CLSI breakpoints criteria. All disc diffusion results were determined in triplicate using C. jejuni ATCC 33291 as control during this trial.

The results were interpreted as resistant or sensitive. The inhibition zone readings defined as intermediate were also classified as resistant. The strain that showed resistance to 3 or more antimicrobial agents was considered as multidrug resistant (MDR).

The concentrations of antimicrobial agents tested along with the CLSI zone diameter breakpoints interpretation used in this project are elaborated in Supplementary Table I.

Results

Prevalence of C. jejuni

Out of 375 samples, 200 were found positive for thermophilic C. jejuni. A 242 bp amplicon was indicative for the presence of C. jejuni according to the instructions provided by organism specific PCR kit (Genetic PCR solutions™ - CamJej epPCR Test-Code 1117045). Additional genomic confirmation was done by sequencing the gene commercially (Advance Bioscience International, Pakistan). After sequencing of the gene, it was blasted on (BLAST) NCBI for confirmation that resulted as (ompA) gene, which produces the outer- membrane porin protein.

Phylogenetic analysis

The results of phylogenetic analysis showed that the local isolate of C. jejuni have more similarity with reported sequences of USA and UK (Fig. 1). Our isolated sequence has a little likelihood with three sequences (FDAARGOS422, NCTC13265 and FDAARGOS 263) but maintained its uniqueness at root 99.

Antibiotic resistance in C. jejuni

The isolates showed resistance against most of the antibiotics except few. Of all 200 C. jejuni isolates, 182 (90.90%) were highly resistant against ciprofloxacin, while 164 (81.81%) were resistant against erythromycin and also resistant against azithromycin 138 (69.09%), clarithromycin 153 (76.36%), telithromycin 165 (82.35%), clindamycin 174 (87.03%), nalidixic acid 153 (76.36%), moxifloxacin 167 (83.63%), ampicillin 124 (61.81%), co-amoxiclav 111 (55.55%), chloramphenicol 102 (50.90%), tetracycline 100 (50%) and tygecycline 91 (45.45%). Resistance against norfloxacin 55 (27.27%), ofloxacin 40 (20%), levofloxacin 47 (23.63%) and gentamicin 51 (25.45%) was less that 30%. Three antibiotics which are imipenum 7 (3.63%), meropenum 4 (1.81%) and ceftazidime 2 (1.06%) showed less than 5% resistance. The details of antibiotic resistance is shown (Fig. 2).

Discussion

Food borne infections that are caused by C. jejuni, is one of the most frequently reported infections, that occurs all over the world (Kim et al., 2018). The colonization of chicken gut is usually undetectable before the age of 7 days. It occurs between the second and fourth week of rearing. It can reach the highest level at slaughter age (Rahimi et al., 2013). The epidemiology of Campylobacter in broiler production is still unclear and the linear relationship occurs between the broiler flock prevalence and probability of human infection. Thus, reducing the Campylobacter at farm can contribute in reduction of human infection.

 

 

Multiple drug resistant strains of microbes are a major problem of the era (Elhadidy et al., 2018) and in the present study, the isolates of thermophilic C. jejuni were found to be uniformly resistant against multiple groups of antibiotics. There is an increase in the percentage of Campylobacter strains that are resistant to antibiotics and it is getting more difficult to control the management of infections caused by those antibiotic resistant strains in clinics (Maćkiw et al., 2012; Hameed et al., 2020). This study also showed antibiotic resistant C. jejuni are common among broilers that can easily find their way into our food chain. We have found MDR thermophilic C. jejuni to be highly prevalent during our study which is 53% (n=200) out of the total samples (375).

The percentage of thermophilic C. jejuni isolated in this study was found to be 53% (n=200) out of the total samples (375). Although the Minimum Inhibitory Concentration (MIC) method is preferred, World Health Organization (WHO) has suggested use of disc diffusion in limited resource conditions. Numerous authors had used disc diffusion method to study antibiotic resistance among Campylobacter isolated from poultry (Maćkiw et al., 2012; Ma et al., 2014; Nobile et al., 2013).

Both macrolides and floroquinones are drugs of choice for human in systemic infections or in severe and long lasting cases of enteritis (Rahimi et al., 2013). We found the resistance to macrolide to be highly frequent as 81.81 percent of 200 C. jejuni samples was resistant against erythromycin, also the resistance was found to be 69.09 percent against azithromycin, 76.36 percent against clarithromycin and 82.35 percent against telithromycin. This frequent resistance was also reported by (Pergola et al., 2017; Elhadidy et al., 2018). Among the fluororquinolone compounds, norfloxacin was 27.27% resistant; ofloxacin was 20% and levofloxacin was 23.63%. Ciprofloxacin was found to be 90.90% resistant, moxifloxacin was 83.63% and nalidixic acid was found to be 76.36% resistant. Frequent resistance to fluoroquinolones may be most likely due to its wide use in poultry industry particularly in feed and as biological fitter in chicken (Abay et al., 2014).

Moreover, the samples were also showing resistance against lincosamides (clindamycin) to be 87.03 percent. The level of resistance we found to penicillin (ampicillin 61.81%) and penicillin combination (co-amoxiclav/ augmentin 55.55%) for C. jejuni was more than 50 percent, while in chloramphenicol (50.90%), tetracycline (50%) and tygecycline (45.45%) were found to be around 50 percent. Mostly the strains were sensitive to carbapenems (imipenem 3.63% and meropenum 1.8%) and cephalosporin (cetazidime 1.06%). It may be because of their less use in the poultry industry. Successful control with carbapenems have been reported earlier (Kim et al., 2017; Geng et al., 2019; Roberts et al., 2020; Morita et al., 2022). It is of great note that cephalosporin (ceftazidime) was highly active against all isolates.

Among the older antibiotics, gentamicin (25.45%) was effective, but its usage is restricted in case of pregnancy. Still, gentamicin may be effective against septicemia and other general infections in combination with carbapenums or cephalosporin. The in vitro antibiotic resistance activity of tetracycline was found to be 50% while tigecycline resulted as 45.45%. Tetracycline can survive longer in an environment than do other antimicrobials consequently could cause bacterial resistant against it. Tigecycline is known for its best activity particularly for the treatment of problematic skin and soft tissue infections (Lawton et al., 2018), which is well known to be one of the manifestation of extra intestinal campylobacteriosis (Kim et al., 2017; Roberts et al., 2020). Tigecycline flows largely as unchanged drug and its major course of elimination is through the feces, likely via biliary defecation route (Lehtopolku et al., 2010; Quinteros et al., 2021). Based on this, it sounds sensible to assume that tigecycline might be effective even for patients with gastroenteritis if given in combination with carbapenums or cephalosporin.

With frequent occurrence of resistant Campylobacter against different antibiotics will ultimately lead to limited choice of drugs for treatment. Thus, antibiotic susceptibility test is required before any treatment that is to be initiated in Campylobacter-infected patients. The efficacy of carbapenums and ceftazidime for the treatment of human campylobacteriosis should also be evaluated in clinical trials.

Conclusion

In conclusion, we had found high contamination in commercial broiler farms by thermophilic C. jejuni and high resistance to antimicrobial groups. The study shows the need to establish an efficient system for cautious use of antibiotics in poultry production as resistance to most of the antibiotics is increasing. We have found the antibiotic disc diffusion method to be trustworthy and prompt, to check the resistance of any antibiotic that has been selected for the patient suffering from Campylobacter infection, before any treatment. The results also show the need to develop alternate antimicrobial methods to reduce the growth and spread of MDR thermophilic C. jejuni under careful guidance.

Acknowledgement

The authors would like to pay their gratitude to the laboratory and support staff at the Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore (Pakistan) for their help.

Funding

The authors acknowledge Higher Education Commission (HEC) funded project# 7058/NRPU/R&D/HEC/16/246 entitled “Evaluation of anti- Campylobacter activity of indigenous probiotic Lactobacilli alone and in combination with different organic acids in poultry” for supporting this study.

IRB approval

The study was approved by Advanced Studies and Research Board in its meeting held on 30-09-2016 (Letter number DAS/9023).

Ethical statement

All the proceedures undertaken were in compliance with Institutional Guidelines for the Care and Use ofvEexperimental Animals of University of Veterinary and Animal Sciences, Lahore (DR/246, dated: 3-5-2016).

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20221017111032

Statement of conflict of interest

The authors have declared no conflict of interest.

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