Occurrence of Florfenicol and Linezolid Resistance and Emergence of optrA Gene in Campylobacter coli Isolates from Tunisian Avian Farms

Campylobacter species, especially C. coli and C. jejuni, have been associated with a range of human gastrointestinal diseases. During the last two decades, due to the irrational use of antibiotics in poultry farms, high rates of antimicrobial resistance have been globally reported in C. coli and C. jejuni isolates. Recently, acquired linezolid-resistance mechanisms have been reported in Campylobacter spp. isolates, which is a cause of concern to human health. In this study, we performed a retrospective analysis of 139 C. coli isolates previously collected from broilers (n = 41), laying hens (n = 53), eggs (n = 4), and environment (n = 41) to detect acquired genes implicated in linezolid resistance. Isolates were tested for their susceptibility to antimicrobial agents using the Kirby–Bauer disk diffusion assay. Chloramphenicol- and linezolid-resistant isolates were subjected to PCR screening for the following genes: fexA, fexB, floR, RE-cmeABC, cfrA, and optrA. The genetic relatedness of eight multidrug-resistant isolates was determined by multilocus sequence typing (MLST). Among the 139 C. coli isolates, high rates of resistance (57.55%–100%) were detected toward nalidixic acid, ciprofloxacin, erythromycin, azithromycin, ampicillin, chloramphenicol, linezolid, and kanamycin. Among 135 chloramphenicol-resistant isolates, the optrA, cfr, fexA floR, RE-cmeABC, and fexB genes were detected in 124 (124/135, 91.85%), 108 (80%), 105 (77.7%), 64 (47.4%), 56 (41, 48%), and 27 (20%) isolates, respectively. In addition, the majority of isolates harbored more than one of these genes. The selected eight isolates belonged to the same sequence type ST13450, which is a new sequence type (ST), not belonging to ST828 and ST1150 complexes. In conclusion, the emergence of optrA gene in Campylobacter spp. isolates makes this genus an optrA reservoir and vector to other pathogens such as Staphylococcus aureus and Enterococcus spp., which is a cause of concern for human and animal health.


Introduction
Campylobacter species have been associated with a range of human gastrointestinal diseases called campylobacteriosis, which is one of the four leading causes of diarrheal diseases worldwide [1].Furthermore, reports of occasionally lethal extra-gastrointestinal infections such as reactive arthritis, irritable bowel syndrome, Guillain-Barré syndrome (GBS), and Miller Fisher syndrome, have also been recorded [2,3].Among the thirty-two species, C. jejuni and C. coli are the most common species that cause campylobacteriosis [1].Te main sources of infection for humans are meat products, particularly fresh and frozen chicken meat, and water [4].
Te main reservoirs of Campylobacter species are poultry, domestic animals, and wild animals [1,4,5].Te majority of Campylobacter infections are self-limiting and normally do not require antibiotic therapy; however, patients who are immunocompromised or have severe and prolonged symptoms beneft from proper antibiotic therapy.For diarrhea caused by Campylobacter infection, fuoroquinolones (ciprofoxacin), macrolides (azithromycin, erythromycin, and clarithromycin), and tetracyclines are the preferred treatments; however, worldwide high rates of resistance to those antibiotics have been reported in C. coli and C. jejuni from humans as well as food-producing animals, thereby threatening public health.
Concerning tetracycline resistance, the tet(O) gene, encoding a ribosomal protection protein, is often found in tetracycline-resistant Campylobacter isolates.Interestingly, tet (O) gene has been also reported in tetracyclinesusceptible C. jejuni and C. coli isolates [9].In addition, it seems that CmeABC efux pump plays an important accessory role in mediating resistance to tetracycline among Campylobacter isolates [10].
Te irrational use of antimicrobial agents in poultry production has enhanced selection of several other antimicrobial resistance mechanisms in Campylobacter.Florfenicol is a broad-spectrum antibiotic that is widely used in animals for both growth promotion and therapeutic reasons, which was the cause of increased rates of forfenicol resistance in Campylobacter spp. of animal origin in several countries like China [11].Previously, only two transferable resistance proteins were known to give low-level forfenicol resistance (MIC ≤16 mg/L) in Campylobacter: the resistanceenhancing efux pump CmeABC (RE-CmeABC) and the 23S rRNA methyltransferase Cfr(C).However, recently, the emergence of forfenicol exporter gene fexA in C. coli and C. jejuni isolates has been reported in China [12,13].Campylobacter's fexA gene was frequently linked to mobile genetic elements and multidrug resistance genomic islands (MDRGIs) containing tet(O)-catA-fexA-tet(L)-optrA genes, which led to the co-transfer of fexA and other critically relevant antibiotic resistance genes as well as an increase in the emergence of multidrug-resistant Campylobacter [13,14].Interestingly, exclusively in China, fexA-positive C. coli and C. jejuni strains have been reported in chickens, ducks, goose, pigs, pigeon meat, human, and environment, indicating high ability of fexA gene and/or fex-positive isolates to spread within and between various ecosystems [11,14,15].Te cfr gene, which encodes 23S rRNA methyltransferase, confers resistance to fve antimicrobial classes, namely, phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A, known as PhLOPS A phenotype.Among the fve known types of the cfr gene family (cfr, cfr(B), cfr(C), cfr(D), and cfr(E)) in various genera, the plasmid-mediated multidrug resistance gene cfr(C) gene has been reported in C. coli and C. jejuni isolates from porcine, chicken, and cattle origin [16][17][18].
More recently, the Gram-positive (Enterococcus spp., Staphylococcus spp., and Streptococcus spp.) oxazolidinone (linezolid and tedizolid) resistance gene optrA has also been identifed in C. jejuni and C. coli isolates [19,20].Te optrA gene codes for an ABC-F protein which confers resistance by ribosome protection to not only oxazolidinones but also to fuorinated and non-fuorinated phenicols [19].
In this study, we performed a retrospective analysis and described the emergence of the RE-CmeABC, fexA, and optrA genes in C. coli from poultry in Tunisia.[21][22][23][24][25].All farms use an intensive foor hen rearing system with bird numbers ranging from 2000 to 18,000 hens per house.

Genetic Relatedness of Isolates by Multilocus Sequence Typing (MLST).
Eight multidrug-resistant (MDR) isolates were selected to determine their clonal lineage by MLST.PCR amplicons identifying seven allele loci (aspA, glnA, gltA, glyA, pgm, tkt, and uncA) were obtained for each isolate by using the primers provided in PubMLST database (https://pubmlst.org/organisms/campylobacter-jejunicoli/primers).After sequencing of PCR products, ST profles were assigned by submitting the sequences to the PubMLST database using the submission database.

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International Journal of Microbiology International Journal of Microbiology

Results and Discussion
Globally, the rate of antibiotic resistance in Campylobacter isolates has increased over time.Of particular concern is resistance to macrolides and fuoroquinolones, which are the cornerstones of treatment for human infections.In addition, C. coli and C. jejuni isolates have showed high ability to acquire multiple resistance genes encoding resistance particularly toward tetracyclines, aminoglycosides, and fuorinated and non-fuorinated phenicols.Te continuing application of antibiotics in livestock, especially chicken, exacerbates this issue [33].
Although several molecular mechanisms of antimicrobial resistance are specifc to Campylobacter genus, several studies have showed the acquisition of antimicrobial resistance genes from Gram-positive genus, especially from Enterococcus spp., Staphylococcus spp., and Streptococcus spp.[33].
Ciprofoxacin resistance in Campylobacter arises quickly after exposure to antibiotics in the fuoroquinolone class, including enrofoxacin, which is unlawfully and excessively used in poultry production in Tunisia.Unfortunately, ofcial statistics regarding the use of antibiotics by veterinarians or the amount of antibiotics sold for use in livestock are lacking in Tunisia, which makes it more difcult to draw a comprehensive knowledge on the potential relationship between the use of antibiotics and the emergence of antibiotic resistance in livestock production.Te issue of fuoroquinolone resistance in Campylobacter poses a public health risk since fuoroquinolones, especially ciprofoxacin, are the preferred empirical treatment choices for campylobacteriosis in humans [28,37].Furthermore, a great deal of data points to a direct transfer of fuoroquinolone resistance from poultry to humans [38].As a result, the use of this antibiotic in chicken production was banned in 2005 [39].
Treatment for human campylobacteriosis primarily consists of erythromycin and azithromycin.Consequently, the observed high frequency of resistance to erythromycin (100%) and to azithromycin (96.4%) in avian C. coli isolates is also a cause of concern to human health.Indeed, evident spreading of resistant isolates to humans through the food chain or environment has been reported [33].
Te high resistance frequency toward chloramphenicol (n � 137, 98.56%) is somehow expected; in fact, despite its ofcial restriction, farmers still use it in poultry production because of its afordable cost.In the European Union (EU), the use of chloramphenicol in veterinary medicine is currently restricted to pets and non-food-producing animals.Te EU banned its use in animals raised for food in 1994.Protection of the consumer from probable negative efects resulting from chloramphenicol residues in food animal carcasses was the primary justifcation for this restriction.Chloramphenicol has been employed in human medicine over years; however, its fuorinated derivative forfenicol is one of the most frequently used antimicrobials in the treatment of animals (for respiratory and intestinal infections) and food animal production.Resistance to chloramphenicol is mediated by several mechanisms, where some of them mediate cross-resistance to forfenicol [40].
Interestingly, linezolid (belonging to oxazolidinone family) is the antibiotic of the last resort for treating clinical infections caused by MDR Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae, and vancomycinresistant Enterococcus species [41].However, linezolid has not been approved for use in the livestock or poultry industries [41,42].Terefore, the high prevalence of linezolid resistance (60.43%) detected in this study would be associated to other factors rather than linezolid use [43].
Te optrA gene codes resistance to linezolid, forfenicol, and chloramphenicol [13,19].In Tunisia, this gene has been previously reported in Enterococcus spp.isolates recovered from wastewater, chicken feces, retail chicken meat, and neutropenic patients [44][45][46][47].As mentioned above, oxazolidinones are not used in livestock or poultry industries; therefore, the acquisition of the optrA gene by the C. coli isolates might not be directly linked to selective pressure by linezolid or other oxazolidinones [13].Previous Tunisian studies have reported optrA-harboring enterococci colonizing the intestine of chickens; consequently, it is plausible that avian C. coli isolates are able to acquire the optrA gene from avian optrA-positive enterococci isolates by horizontal transfer of mobile genetic elements [45,47].Indeed, Frye et al. [48] have highlighted that C. coli and Enterococcus coisolated from swine fecal samples had tet(O) and aphA-3 genes detected in common suggesting horizontal exchange of antimicrobial resistance genes between these bacteria or there may be a common source of those genes in the swine environment.Tis is also argued by the genetic studies on 4 International Journal of Microbiology the genetic environments of optrA in C. coli and C. jejuni showing that multidrug resistance genomic islands (MDRGIs) containing optrA gene were most likely not indigenous to Campylobacter (GC content 31%) but derived from other bacteria with a higher GC content, such as E. faecalis and E. faecium (E.faecalis T5 (GenBank accession no.KB944666.1)and E. faecium ZY2 (GenBank accession no.CP039729.1)with 37.37% and 37.94%, respectively) [11,19,20].Te genetic studies showed also that the optrA regions found in the C. coli genomes closely resembled regions previously found on E. faecalis plasmids [13].In addition, as OptrA encodes also resistance to forfenicol and chloramphenicol, the emergence of this mechanism of resistance to oxazolidinones and phenicols might be a logical response to the excessive use of phenicols in poultry production.
As an intestinal commensal of chickens, Campylobacter under selective pressure by phenicols has developed other molecular mechanisms to escape the bactericidal efect of these antibiotics.One of those mechanisms is the export of chloramphenicol or forfenicol from the bacterial cell mediated by either specifc transporters and/or multidrug transporters.In Campylobacter, fexA, foR, and RE-cmeABC genes encoding those transporters have been increasingly reported [11,14,15,19,33].However, to the best of our knowledge, this is the frst report of fexB in Campylobacter [15,19].
Others drivers of multi-antimicrobial resistance in Campylobacter are cfr and RE-cmeABC genes mediating resistance to phenicols-lincosamides-oxazolidinones-pleuromutilins-streptogramin A (PhLOPS A phenotype) and phenicols-macrolides-fuoroquinolones-tetracyclines by multidrug efux pump.Among the fve types of the cfr gene family that have been reported: cfr, cfr(B), cfr(C), cfr(D), and cfr(E), only cfr(C) has been identifed in Campylobacter [49].Te CFR(C) protein showed 55.1% or 54.9% identity to the original Cfr (GenBank accession no.CAC04525.1)from Staphylococcus sciuri and Cfr(B) (GenBank accession no. AKV84429.1)from E. faecium, respectively [50].In our study, the cfr gene was found in 108 chloramphenicolresistant isolates, which is to the best knowledge its frst report in Campylobacter.Previous studies showed that RE-cmeABC was signifcantly linked with multidrug resistance among C. jejuni and C. coli [12,51].Although this resistant form of efux pump has been identifed globally in C. jejuni since at least 2014 [12], this study is one of the frst reports of the RE-cmeABC form of the pump in C. coli in Tunisia.
It is worthy to note that the distribution of the investigated resistance genes, except optrA gene, was dependent to the origin of the isolates.Te fexA and cfr genes were signifcantly more prevalent in isolates collected from layer hens, eggs, and environment than those isolates from broiler hens.However, fexB gene was more prevalent in isolates from broiler hens.Diference of gene distribution was also observed for RE-cmeABC gene which was more prevalent in isolates from broiler hens and environment.Similarly, the fexB was more prevalent in isolates from broiler hens than those from other origins.Tis fnding may be related to the dissemination of particular C. coli clones or specifc plasmids harboring those genes in each ecological niche.
In order to better understand the epidemiology of the phenicol-resistant isolates, eight isolates were selected (two from each origin) according to the occurrence of the majority of genes and their antimicrobial resistance profles and their MLST was determined (Table 3).In Table 3, genes encoding other resistance markers (quinolones/fuoroquinolones, tetracyclines, CmeABC efux system, and macrolides) and virulence factors were performed in our previous studies [21][22][23][24][25].All isolates belonged to the same sequence type ST13450, which is a new ST.Te singleton ST13450 is closely related to ST828 and ST1150 complexes, which account for the great majority of strains found in agriculture and human disease [52].Tis fnding is not surprising since it is well documented that C. coli from various origins showed a low genetic heterogeneity in contrast to C. jejuni [52,53].International Journal of Microbiology

Conclusion
Tis study highlights the high ability of C. coli to acquire several genes encoding antimicrobial resistance especially to clinically relevant antibiotics.Te distribution of those genes between isolates of various origins seems to be origin depending, which highlights possible circulation of specifc C. coli clones or plasmids.Furthermore, it seems that heavy antibiotic use in avian farms drives the selection and the spread of MDR isolates.Te emergence of optrA gene in Campylobacter spp.isolates makes this genus an optrA reservoir and vector to other pathogens such as Staphylococcus aureus and Enterococcus spp., which is a cause of concern for human and animal health.

Figure 1 :
Figure 1: Frequencies of antimicrobial resistance detected in the 139 C. coli isolates.

Table 1 :
Primers used for PCR reactions.