First report of phenotypic and genotypic (blaOXA-61) beta-lactam resistance in Campylobacter jejuni from broilers in Indonesia

Background and Aim: Campylobacter is a zoonotic bacterium that is a major source of foodborne diseases. In humans, most cases of campylobacteriosis are caused by Campylobacter jejuni. Poultry is the main reservoir of Campylobacter for humans, because Campylobacter is part of the normal flora of the digestive tract of poultry. Antimicrobial resistance to several antibiotics in Campylobacter isolated from humans and food animals has increased rapidly. Beta-lactam is an antibiotic with a high prevalence of resistance in Campylobacter. This study aimed to investigate phenotypic and genotypic (blaOXA-61) beta-lactam resistance in C. jejuni from broilers in Indonesia. Materials and Methods: A total of 100 samples of broiler intestinal contents were obtained from 10 broiler farms in Pasuruan Regency, Indonesia. Campylobacter jejuni was identified using conventional and polymerase chain reaction (PCR)-based methods. Phenotypic detection of beta-lactam resistance was performed using an antimicrobial susceptibility test with antibiotic disks of aztreonam, ampicillin, and amoxicillin-clavulanic acid. Genotypic detection by PCR was performed using the blaOXA-61 gene, which encodes beta-lactamase. Results: Campylobacter jejuni was identified in 23% of the samples. Phenotypically, 100% (23/23) and 73.9% (17/23) C. jejuni isolates had high resistance to aztreonam and ampicillin, respectively, but all isolates were susceptible to amoxicillin–clavulanic acid. Genotypically, all isolates carried blaOXA-61, indicated by the presence of a 372-bp PCR product. Conclusion: Campylobacter jejuni is highly resistant to beta-lactams and is a serious threat to human health. Resistance to beta-lactams should be monitored because beta-lactamase genes can be transferred between bacteria. Public awareness must also be increased on the importance of using antibiotics rationally in humans and animals.


Introduction
Campylobacter is a zoonotic pathogen that is a major source of foodborne bacterial illness worldwide [1].In humans, campylobacteriosis is caused by Campylobacter jejuni, whereas the remainder is mostly caused by Campylobacter coli [2].Recently, the incidence and prevalence of campylobacteriosis have been increasing in both developing and developed countries [1].According to the Centers for Disease Control and Prevention, campylobacteriosis affects >1.5 million people (excluding undiagnosed and unreported cases) in the United States every year [3].Campylobacteriosis is generally self-limiting with fever, stomach cramps, and bloody diarrhea.However, campylobacteriosis can become chronic and cause reactive arthritis, Guillain-Barré syndrome, Miller-Fisher syndrome, urinary tract infection, irritable bowel, sepsis, and certain neuropathies.[4].
Poultry is the main source of Campylobacter transmission to humans, because it is a part of the normal flora in the digestive tract of poultry [5].Campylobacter spp., especially C. jejuni, can be found in high numbers in ceca broilers at the farmer level and on carcasses in poultry slaughterhouses and meat markets [6].Campylobacter can be transmitted from poultry through activities that may expose humans to poultry excrement, or by handling and consuming contaminated meat [7].In Indonesia, 61.9% of chicken meat was reported to contain Campylobacter and most isolates (41.07%) were C. jejuni [8].In South Asia, the incidence of antimicrobial Available at www.veterinaryworld.org/Vol.16/November-2023/5.pdf resistance in Campylobacter isolated from humans and food animals has increased during the past decade, especially in countries with widespread use of antibiotics in livestock [9][10][11].Campylobacter spp.are highly resistant to beta-lactam antibiotics [11].Campylobacter strains that produce beta-lactamase were more resistant to ticarcillin, amoxicillin, and ampicillin than beta-lactamase-negative strains.This is attributed to the bla OXA-61 gene, which encodes Cj0299-a putative periplasmic amino acid class D beta-lactamase [12].The overuse of antibiotics in human and animal populations has increased the number of antimicrobial-resistant bacteria [13].Antimicrobial resistance poses an additional risk because infection with antibiotic-resistant Campylobacter leads to longer hospitalizations, higher rates of treatment failure, and increased morbidity and mortality [14].
In Indonesia, information regarding antimicrobial resistance in C. jejuni is limited; therefore, this study aimed to investigate phenotypic and genotypic (bla OXA-61 ) beta-lactam resistance in C. jejuni from broilers.An antimicrobial susceptibility test was used for phenotype detection, and polymerase chain reaction (PCR) for bla OXA-61 was used for genotype detection.

Ethical approval
Animal ethical approval was obtained from the Ethical Clearance Committee of the Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Indonesia (ethics no.: 86-KKE/2022).

Study period and location
The study was conducted from October 2022 to December 2022 at the Balai Besar Veteriner Wates Yogyakarta and the Institute of Tropical Disease Airlangga University.

Sample collection
A total of 100 samples of broiler intestinal contents were obtained from ten broiler farms in Pasuruan Regency, Indonesia.Sampling was performed by slaughtering broilers and dissecting the abdomen to obtain the contents of the small intestine.Samples were placed in sterile plastic and stored in a cool box on the way to the laboratory.

Isolation and identification
Campylobacter isolation was performed using a modified BAM 2001 method [15].One loop pellet was streaked on modified charcoal cefoperazone deoxycholate selective medium containing charcoal cefoperazone deoxycholate selective supplement (Oxoid SR 155E, England) and incubated at 42°C for 48 h under microaerophilic conditions.Isolates were confirmed as Campylobacter by microscopic examination, Gram staining, and biochemical tests for catalase and oxidase.

DNA extraction
QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and QIAprep Spin Miniprep Kit (Qiagen) were used to extract genomic and plasmid DNA, respectively, according to the manufacturer's recommendations.

Polymerase chain reaction assay
The PCR master mix contained 5 μL DNA template, 1 μL each primer, 0.5 μL nuclease-free water, and 12.5 μL PCR master mix (Promega, USA), containing Taq DNA polymerase, dNTPs, MgCl 2 , and reaction buffer.The final volume of the reaction mixture was 20 μL.
The PCR products were separated by electrophoresis in 1.5% agarose gel (Invitrogen, USA), supplemented with RedSafe Nucleic Acid Staining Solution gel dye (Intron, South Korea).The size of the PCR products was evaluated using a 100-bp DNA marker.The gel was electrophoresed for 30 min at a constant voltage of 100 V and visualized under ultraviolet light.

Phenotypic detection of beta-lactam resistance using an antibiotic sensitivity test
All C. jejuni isolates were analyzed for antimicrobial resistance on Mueller-Hinton agar plates (Oxoid CM 0337b, England), supplemented with 5% defibrinated sheep blood, containing ampicillin (10 μg), aztreonam (30 μg), and amoxicillin-clavulanic acid (30 μg) disks.The medium was incubated at 42°C for 24 h under microaerophilic conditions [18].The bacterial susceptibility to antibiotics was interpreted by measuring the inhibition zone and using the Clinical Laboratory Standard Institute table as a standard [19].

Results and Discussion
In this study, samples were collected from the small intestine, especially from parts of the jejunum, because the results for C. jejuni isolation were good.Campylobacter jejuni mainly colonizes the ceca and small intestine [20].Campylobacter jejuni can colonize the intestinal tract, especially at the junction between the ceca, small intestine (jejunum), and large intestine, where their number is high (≥10 8 Colonyforming unit/g) [21].
In Indonesia, the prevalence of C. jejuni in poultry is 23%.The prevalence rates were similar to our study in Jordan 17% [22], Ecuador 18.9% [23], Benin (West Africa) 23.4% [24], and India 24% [25].Polymerase chain reaction after selective enrichment is recommended for identifying Campylobacter in drinking water, dairy products, aquatic habitats, and poultry products.In other countries, prevalence rates vary, which is probably influenced by variations in sampling design and testing technique [25].
In this study, all C. jejuni isolates were resistant to aztreonam (100%), which is consistent with results from Lebanon [12] and Jordan [26].Most C. jejuni are resistant to ampicillin (74%), which is the most commonly used antibiotic in poultry [27].This result is similar to studies in South China 70.7% [28], but lower than the incidence in several countries with a range of 81.8%-100% [26,29,30].The combination of amoxicillin-clavulanic acid is the most efficient against C. jejuni based on the results of this study, because all C. jejuni isolates were susceptible to amoxicillin-clavulanic acid.These results are similar to studies in the Lebanon [12] and United Kingdom [31].In contrast, a study in Tunisia showed that C. jejuni was 22.7% resistant to amoxicillin-clavulanic acid [29].
This study focuses on discovering the gene (bla OXA-61 ) in C. jejuni, a new gene discovered in Indonesia.Polymerase chain reaction detected the bla OXA-61 gene (372-bp PCR product) in all 23 C. jejuni isolates (Figure -3).Six C. jejuni isolates that were susceptible to ampicillin also carried the bla OXA-61 gene.Most C. jejuni strains can produce beta-lactamase, which inactivates beta-lactam by hydrolyzing the lactam ring [32].Beta-lactamase is divided into four classes according to Ambler's classification: class A (KPC and most extended-spectrum β-lactamase), class B (MBL), class C (AmpC beta-lactamase), and class D (OXA).The bla OXA-61 gene belongs to class D and is widely encoded in integrons [33].Class D beta-lactamase OXA-61 is the most frequently identified in Campylobacter [34].
In this study, all C. jejuni isolates were phenotypically resistant to aztreonam and genotypically possessed the bla OXA-61 gene, indicating a strong correlation between the two.Consistently,  class D beta-lactamase, which consists only of OXA, can hydrolyze cephalosporins and aztreonam and has carbapenemase activity [35].The ability to hydrolyze monobactam (aztreonam) is shared by class A and class C, but not class B, beta-lactamases [33,36].Molecular investigation of antimicrobial resistance in the tested isolates showed a strong correlation between the antibiotic resistance phenotype and the genotypes and mutations coding for antibiotic resistance [18].In this study, all isolates that were phenotypically resistant to ampicillin were detected by the bla OXA-61 gene, which encodes antimicrobial resistance to beta-lactams.Most ampicillin-resistant Campylobacter isolates from poultry carried the bla OXA-61 gene, according to other studies conducted in the UK [31] and Brazil [37].
However, despite the strong correlation between antimicrobial resistance to beta-lactams and bla OXA-61 , 6 ampicillin-susceptible C. jejuni isolates possessed this gene.Our results are supported by a report in which 59% of ampicillin-susceptible isolates carried blaOXA-61 [38], indicating that bla OXA-61 is poorly expressed in ampicillin-susceptible isolates, and therefore, less beta-lactamase is produced compared with resistant isolates [39].This could be because there is a relationship between ampicillin susceptibility and the presence of G-T transversion in the bla OXA-61 promoter, only isolates that are resistant to ampicillin have G-T transversion [38].Alfredson and Korolik [34] (GenBank accession number AY587956) identified bla OXA-61 in ampicillin-resistant C. jejuni isolates with G-T transversion.A large-scale study is warranted to evaluate the association between G-T transversion and high-level blaOXA-61-mediated beta-lactam resistance in C. jejuni isolates [38].
CmeABC efflux pumps contribute to beta-lactamase resistance and could explain the ampicillin susceptibility of C. jejuni strains carrying bla OXA-61 [12].In a study, ampicillin susceptibility increased 32-fold after CmeB insertion mutagenesis in C. jejuni strain 81-176136 and other strains [40].In another study utilizing the NCTC 11168 strain, the CmeB mutant was four times more susceptible to ampicillin than parental strain, and cmeB overexpression increased ampicillin resistance four folds [41].
In this study, all C. jejuni isolates susceptible to amoxicillin-clavulanic acid carried bla OXA-61 , indicating that this combination strongly inhibits beta-lactamase and can be used as an alternative to   fluoroquinolone, tetracycline, and erythromycin, which C. jejuni is resistant to [31,39].In C. jejuni, the expression of beta-lactamase, which causes resistance to amoxicillin, ampicillin, and ticarcillin, can be inhibited with tazobactam, clavulanic acid, and sulbactam [12].Antimicrobial resistance in pathogenic microorganisms, especially C. jejuni, is a global health challenge.According to the Centers for Disease Control and Prevention, drug-resistant strains of Campylobacter infect >300,000 individuals each year, making treatment a major health challenge and economic burden.Antimicrobial resistance in bacteria can be generated by antibiotic overuse by humans, antibiotic use in animal feed or veterinary treatment, and increased industrial waste in the environment [26,32,42].Bacterial resistance to antimicrobials has evolved in a variety of ways; in most situations, bacteria exposed to antibiotics discover ways to avoid or resist antimicrobial agents [31,40,43,44].

Conclusion
Our results show that C. jejuni has high resistance to beta-lactams and is a serious threat to human health.Beta-lactam resistance should be monitored because beta-lactamase genes can be transferred between bacteria.Public awareness of the importance of using antibiotics rationally in humans and animals must be increased.More studies are needed to understand antibiotic resistance to and develop diagnostic media for Campylobacter spp.To the best of our knowledge, this is the first study to genotypically identify beta-lactam resistance in C. jejuni from Indonesian poultry.Our findings highlight the importance of a surveillance and monitoring system and risk analysis for C. jejuni prevalence and resistance in poultry and other animal feeds.

Figure- 2 :
Figure-2: Results of the antimicrobial susceptibility test of Campylobacter jejuni isolate.
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Table - 2
: Results of isolation, identification, and PCR test to detect Campylobacter jejuni.