Prevalence of Fluoroquinolone-Resistant Campylobacter Species in Iran: A Systematic Review and Meta-Analysis

Background Campylobacter species are one of the main causes of bacterial food poisoning worldwide. Recently, WHO reported that the emergence of fluoroquinolone-resistant Campylobacter species is becoming a public health issue around the world. The aim of the present systematic review and meta-analysis was to evaluate the prevalence of the antimicrobial susceptibility patterns of Campylobacter species, especially fluoroquinolone-resistant strains isolated from human and animal origins in Iran. Methods Using related keywords and without date and language limitations, a comprehensive literature search was conducted in PubMed, Scopus, ISI Web of Knowledge, Google Scholar, and SID to identify relevant studies on the prevalence of the antimicrobial susceptibility patterns of Campylobacter species in Iran. Results A total of 34 reports (9 in Persian and 25 in English) were selected based on inclusion and exclusion criteria. Disk diffusion, E-test, and agar dilution were common methods used for antimicrobial susceptibility testing. The antibiotic resistance profiles of Campylobacter species against fluoroquinolones were as follows: 53.6%, 41.8%, and 0% to ciprofloxacin for C. jejuni, C. coli, and C. lari, respectively, 24.3% and 25.1% to enrofloxacin for C. jejuni and C. coli, respectively, 59.6% and 49.2% to nalidixic acid for C. jejuni and C. coli, respectively, and 87.3% and 64.7% to ofloxacin for C. jejuni and C. coli, respectively. Conclusion Our findings revealed a high prevalence of fluoroquinolone-resistant Campylobacter species in Iran. This calls for the use of more effective antibiotics with low resistance rates including aminoglycosides, chloramphenicol, and imipenem.


Introduction
e genus Campylobacter includes small, motile, and curved Gram-negative bacteria [1][2][3]. ese rod-shaped bacteria are thermophilic, 30°C to 46°C, and microaerophilic, 5% O 2 , and belong to the family Campylobacteraceae [4]. Campylobacter is a zoonotic pathogen that is colonized in the intestinal tract of domestic and wild animals and birds and can infect human through consumption of contaminated water, different foods such as raw or uncooked meat, unpasteurized milk, and contact with infected animals or (rarely) human [4,5]. Poultry, cattle, sheep, pigs, birds, dogs, and cats are common reservoir hosts for human infections [2]. is foodborne bacterial pathogen is the major cause of bacterial gastroenteritis and septicemia in humans in both developing and developed countries [1,2]. In developed countries, Campylobacter bacteria are the most important causative agents for gastrointestinal infection [6]. It is estimated that between 400 and 500 million individuals become infected with Campylobacter species in the world annually [4,5]. e most common species associated with bacterial gastroenteritis in human are Campylobacter jejuni and Campylobacter coli as well as Campylobacter fetus associated with systemic infections [2,4]. Additionally, in some cases, these enteric pathogens are associated with two immune-related late complications, i.e., Guillain-Barré syndrome and reactive arthritis [1,2]. e severity of Campylobacter infections varies from a mild and self-limiting illness to severe infections [5]. For the treatment of self-limiting intestinal infections, the replacement of fluids and electrolytes is enough, while severe extraintestinal Campylobacter infections such as septicemia, endocarditis, and septic thrombophlebitis should be treated with appropriate antibiotics [2,7]. Macrolides, including erythromycin and azithromycin, and fluoroquinolones are considered as the drugs of choice for infected patients [2,4,5]. Alternative treatments are tetracyclines and gentamicin [2,4,5]. Additionally, Campylobacter strains are susceptible to amoxicillin/clavulanic acid, imipenem, aminoglycosides, chloramphenicol, and clindamycin [2]. However, in recent years, antimicrobial resistance of Campylobacter species has been increased in both developing and developed countries and is becoming a global problem [8]. It is noteworthy that resistance to penicillins, cephalosporins, and sulfonamides has emerged [2]. On the other hand, in February 2017, the World Health Organization (WHO) announced that fluoroquinolone-resistant Campylobacter species are growing globally, calling for a priority to find effective antibiotics [9]. erefore, the aim of the present systematic review and meta-analysis was to evaluate the antimicrobial susceptibility patterns of Campylobacter species, especially fluoroquinolone-resistant strains, isolated from human and animal origins in Iran. ere was no date and language limitation for searching, and related keywords used were antibiotic resistance, Campylobacter species (C. jejuni, C. coli, and C. lari), and Iran. A number of missed studies were obtained by reviewing the list of references and searching for journals. e meta-analysis was performed step by step based on the PRISMA recommendations [10].

Literature Search and Selection
Inclusion or exclusion criteria for assessing eligibility in the study were all types of Persian-and English-language articles which had enough data on the prevalence of the resistance patterns of campylobacters, in species level, to different antibiotics in Iran. Review articles, case reports, abstracts of articles, and duplicates were excluded. Articles evaluating the resistance patterns of campylobacters only at the genus level or the resistance genes and those studies with unclear results were excluded.

Data Extraction of Articles.
After completely reviewing all included studies by two authors, needed information was extracted and placed in Tables 1-3 based on organism   species type. e quality of data was evaluated based on the Newcastle-Ottawa scale adapted for cross-sectional studies (data has not been shown). e checklist of items was based on three criteria including selection (representativeness of the sample, sample size, nonrespondents, and ascertainment of the exposure) (maximum 5 stars), comparability (comparability of outcome groups) (maximum 2 stars), and outcome (assessment of the outcome and statistical test) (maximum 3 stars).
Data obtained from eligible studies include publishing year, location of the study, number of strains, origin of samples, methods used for antimicrobial susceptibility testing, and antibiotic resistance profiles of C. jejuni, C. coli, and C. lari.

Meta-Analysis.
e data for the quantitative data synthesis were transferred to the Comprehensive Meta-Analysis (CMA) software (Biostat, Englewood, NJ). Resistance rates of C. jejuni, C. coli, and C. lari were calculated for each antibiotic as a percentage and expressed as 95% confidence intervals (95% CIs). I 2 statistic was used to evaluate the existed heterogeneity, and considering the percent of inconsistency among studies, pooling of data was performed using fixed-or random-effects models. e assessment of publication bias was done using funnel plots. Figure 1, a total of 1299 articles were obtained from five databases (PubMed, Scopus, ISI Web of Knowledge, Google Scholar, and SID). According to the presented inclusion or exclusion criteria in Figure 1, 1249 articles were removed and the eligibility of 50 remained articles was evaluated. Among them, 16 studies did not meet inclusion criteria because of reporting the resistance patterns of campylobacters only in the genus level or had inadequate information, while 34 articles (9 in Persian and 25 in English) had complete data and were included in our meta-analysis. e characteristics of the 34 included studies are summarized in Tables 1-3. e main data was extracted from 3 studies from Ahvaz [11][12][13], 2 studies from Hamadan [14,15], 4 studies from Isfahan [16][17][18][19], 1 study from Kerman [20], 1 study from Kurdistan [21], 2 studies from Mashhad [22,23], 1 study from Mazandaran and Golestan [24], 1 study from Rafsanjan [25], 5 studies from Shahrekord [13,18,[26][27][28], 2 studies from Semnan [29,30], 4 studies from Shiraz [31][32][33][34], 8 studies from Tehran [35][36][37][38][39][40][41][42], 2 studies from Tonekabon [34,43], 1 study from Yazd [19], and 1 study from Zahedan [44]. Disk diffusion, E-test, and agar dilution were the most common methods used to evaluate antibiotic-resistant Campylobacter species in Iran (Tables 1-3). Additionally, the most common Campylobacter species for which their antibiotic resistance has been evaluated were C. jejuni, C. coli, and C. lari. e origins of Campylobacter species were human and animal fecal samples as well as food samples with animal origin including milk, dairy products, and animal meats like poultry, cattle,        International Journal of Microbiology sheep, camels, beef, water buffalo, ducks, and geese. A random-effects model was used for pooling data on the prevalence of antibiotic resistance of Campylobacter species due to the presence of high heterogeneity (I 2 > 25%). ere was some evidence of publication bias (Figures 2 and 3).

Characteristics of C. jejuni Antibiotic Resistance.
In our presented meta-analysis, a total of 34 studies determined the prevalence of C. jejuni antibiotic resistance (Table 1).

Characteristics of C. lari Antibiotic Resistance.
A total of 4 studies investigating the prevalence of C. lari antibiotic resistance were included in the meta-analysis (

Discussion
Food-borne illnesses caused by Campylobacter species as well as other bacteria related to food poisoning can be    prevented by avoiding food contamination and growth of bacteria through proper food preparation and proper cooking as well as avoidance of contamination of water sources and consuming pasteurized dairy products [2,45]. However, the main problem is food contamination with drug-resistant pathogens, which is a major threat to public health [46]. Antibiotic resistance genes can be transferred among food-borne pathogens, and this makes the treatment of severe infections difficult [46]. Today, fluoroquinoloneresistant Campylobacter species have turned into a global concern [9]. Fluoroquinolones are selective drugs in the treatment of campylobacteriosis; however, an increasing trend of resistance in Campylobacter species isolated from human and animal origins has been reported in the USA and Canada (19-47%), European countries (17-99%), and Africa and Asia (>80%) [5]. According to the present study, the resistance of Campylobacter species isolated from human and animal origins to quinolones and fluoroquinolones including ciprofloxacin, nalidixic acid, enrofloxacin, and ofloxacin was also prevalent in Iran and varied from 0% to 87.3% (Tables 1-3). Efflux pumps, CmeABC, and single point mutations in DNA gyrase A (GyrA) such as C257 T mutation, the most frequent mutation, are involved in chromosomally mediated quinolone resistance in Campylobacter species [5].
Macrolides are also recommended as another selective antibiotic class for the treatment of campylobacteriosis [2,5]. e resistance rate to erythromycin in Campylobacter species isolated from human and animal samples in Iran was low (6.4%, 13%, and 7.4% for C. jejuni, C. coli, and C. lari, respectively). e frequency of erythromycin resistance in Iran was higher than that in Turkey [47], Ethiopia [48], Canada [49], Australia [50], and the Czech Republic [51] and lower than that of South Africa [52], Malaysia [53], Italy [54], and China [55]. e target modifications through point mutations in the 23S rRNA gene such as A2074C, A2074G, and A2075G mutations, modifying L4 and L22 ribosomal proteins along with CmeABC efflux pump, are three main mechanisms complicated in macrolide resistance in Campylobacter species [5].
Tetracyclines and gentamicin have also importance in Campylobacter infection therapy as second-line antibiotics [2,4,5]. e tet(O) gene, which encodes a ribosomal protection protein, and CmeABC multidrug efflux pump are associated with tetracycline resistance in Campylobacter species [5]. Additionally, aminoglycoside-modifying enzymes play an important role in aminoglycoside resistance in Campylobacter species [5]. Based on the results of this study, C. jejuni and C. coli antibiotic resistance rates to tetracycline were much higher than gentamicin. Similar results were observed in the studies reported from Turkey [47], Italy [54], South Korea [56], and Poland [57]. Moreover, similar to our results, high rates of tetracycline resistance were reported in the studies of Turkey [47], Canada [49], South Africa [52], Malaysia [53], Italy [54], China [55], South Korea [56], and Poland [57]. Noteworthy, the determination of Campylobacter species susceptibility patterns against other antibiotics has received less attention. One reason could be attributed to the high sensitivity of bacteria to these antibiotics. For example, in the current study, antibiotic resistance pattern to protein synthesis inhibitors was low. On the other hand, resistance to protein synthesis inhibitors was lower than cell growth inhibitors and folic acid metabolism inhibitors.
As shown in other studies, the efficacy of cell growth inhibitor antibiotics against Campylobacter species is limited [5]. Our study also showed that among the three evaluated Campylobacter species, antibiotic resistance to β-lactam antibiotics, especially penicillins and cephalosporins, was high. On the other hand, the resistance rate to imipenem was lower than meropenem. e intrinsic resistance and β-lactamase enzymes are two main mechanisms of resistance to β-lactam antibiotics in Campylobacter species [5]. e intrinsic resistance is also the main resistance mechanism of Campylobacter species against vancomycin and folic acid metabolism inhibitors [5]. Our results confirmed high resistance rates to these antibiotics probably due to intrinsic resistance.

Conclusion
In accordance with the WHO report on fluoroquinoloneresistant Campylobacter species in the world and the urgent need to develop new antibiotics, our meta-analysis showed a high prevalence of resistance of Campylobacter species isolated from human and animal origins to quinolones and fluoroquinolones in Iran. On the other hand, compared to penicillins, cephalosporins, and sulfonamides,

International Journal of Microbiology
Campylobacter species were susceptible or showed low resistance rates to aminoglycosides, chloramphenicol, and imipenem. erefore, these antibiotics could be recommended for the treatment of campylobacteriosis in Iran. We recommend monitoring antibiotic-resistant Campylobacter species through continuous drug sensitivity monitoring and investigating resistance mechanisms, especially against fluoroquinolones, to prevent further expansion of resistant species in Iran.

Data Availability
ere are no original data associated with this review.

Conflicts of Interest
e authors declare that there are no conflicts of interest.