Antimicrobial Usage, Susceptibility Profiles, and Resistance Genes in Campylobacter Isolated from Cattle, Chicken, and Water Samples in Kajiado County, Kenya

Campylobacter organisms are the major cause of bacterial gastroenteritis and diarrhoeal illness in man and livestock. Campylobacter is growingly becoming resistant to critically crucial antibiotics; thereby presenting public health challenge. This study aimed at establishing antimicrobial use, susceptibility profiles, and resistance genes in Campylobacter isolates recovered from chicken, cattle, and cattle-trough water samples. The study was conducted between October 2020 and May 2022 and involved the revival of cryopreserved Campylobacter isolates confirmed by PCR from a previous prevalence study in Kajiado County, Kenya. Data on antimicrobial use and animal health-seeking behaviour among livestock owners (from the same farms where sampling was done for the prevalence study) were collected through interview using a pretested semistructured questionnaire. One hundred and three isolates (29 C. coli (16 cattle isolates, 9 chicken isolates, and 4 water isolates) and 74 C. jejuni (38 cattle isolates, 30 chicken isolates, and 6 water isolates)) were assayed for phenotypic antibiotic susceptibility profile using the Kirby–Bauer disk diffusion method for ampicillin (AX), tetracycline (TE), gentamicin (GEN), erythromycin (E), ciprofloxacin (CIP), and nalidixic acid (NA). Furthermore, detection of genes conferring resistance to tetracyclines (tet (O), β-lactams (blaOXA-61), aminoglycosides (aph-3-1), (fluoro)quinolones (gyrA), and multidrug efflux pump (cmeB) encoding resistance to multiple antibiotics was detected by mPCR and confirmed by DNA sequencing. The correlation between antibiotic use and resistance phenotypes was determined using the Pearson's correlation coefficient (r) method. Tetracyclines, aminoglycosides, and β-lactam-based antibiotics were the most commonly used antimicrobials; with most farms generally reported using antimicrobials in chicken production systems than in cattle. The highest resistance amongst isolates was recorded in ampicillin (100%), followed by tetracycline (97.1%), erythromycin (75.7%), and ciprofloxacin (63.1%). Multidrug resistance (MDR) profile was observed in 99 of 103 (96.1%) isolates; with all the Campylobacter coli isolates displaying MDR. All chicken isolates (39/39, 100%) exhibited multidrug resistance. The AX-TE-E-CIP was the most common MDR pattern at 29.1%. The antibiotic resistance genes were detected as follows: tet (O), gyrA, cmeB, blaOXA-61, and aph-3-1 genes were detected at 93.2%, 61.2%, 54.4%, 36.9%, and 22.3% of all Campylobacter isolates, respectively. The highest correlations were found between tet (O) and tetracycline-resistant phenotypes for C. coli (96.4%) and C. jejuni (95.8%). A moderate level of concordance was observed between the Kirby–Bauer disk diffusion method (phenotypic assay) and PCR (genotypic assay) for tetracycline in both C. coli (kappa coefficient = 0.65) and C. jejuni (kappa coefficient = 0.55). The study discloses relatively high resistance profiles and multidrug resistance to antibiotics of critical importance in humans. The evolution of the multidrug-resistantCampylobacter isolates has been linked to the use and misuse of antimicrobials. This poses a potential hazard to public and animal health, necessitating need to reduce the use of antibiotics in livestock husbandry practice coupled with stringent biosecurity measures to mitigate antimicrobial resistance.


Introduction
Campylobacters are widely distributed as a normal fora in the gut of both domestic and wild animals, and are also found in environmental samples, including surface water, soil, and feeds [1]. Te incidence of campylobacters in environmental sources is mainly related to fecal contamination. Poultry are the main reservoirs though bovine, swine, shoats, dog, and cat have also been recognized as other probable reservoirs for human disease. Cattle-derived isolates can infect poultry painting a picture that cattle could be a source of infections to chicken [2], and vice versa.
Of the more than 25 species in the genus Campylobacter, C. jejuni and C. coli have been reported to cause major public health burden globally. Campylobacter jejuni and C. coli accounted for 98% of cases reported in humans in 2015-2017 in the USA [3], with the WHO projecting that Campylobacter causes 37,600 fatalities/year globally [4]. Tis burden is even higher than the burden caused by salmonellosis [5]. Campylobacter is the major cause of food-borne infections in man through ingestion of raw and/or poorly cooked contaminated food of animal origin (be it beef, pork, or chicken meat), and consumption of contaminated water and raw milk. In addition, cross-contamination of fast foods during preparation, besides coming into contact with faeces from sick humans and companion animals has also been reported as risk factors [6,7]. Campylobacter illness in humans manifests itself as episodes of gastroenteritis accompanied by abdominal pain, biliousness, unsettled stomach, pyrexia, and watery diarrhoea and/or dysentery [8]. However, in infants and in patients with lowered immunity, Campylobacter jejuni is associated with postinfection sequelae including Guillain-Barre syndrome and/or Miller Fischer Syndrome (demyelinating neuropathies afecting peripheral nerves), reactive arthritis, meningitis, myocarditis [9], and fatal septicaemic infection.
Although Campylobacter infections in humans are sporadic and often self-limiting, antimicrobial therapy is indicated in severe and prolonged cases of enteritis, immunosuppressed individuals, and/or in young children. Macrolides (erythromycin) and fuoroquinolones (FQs) (ciprofoxacin) are considered the last resort drugs in clinical cases requiring therapy. However, other classes of antibiotics including aminoglycosides (gentamicin), tetracyclines, lincosamide (clindamycin), and penicillin (ampicillin) can be prescribed as substitute medication for the management of septicaemic campylobacteriosis. However, over the decades, several studies in Kenya and beyond, have reported an increase in infections caused by multidrug-resistant (MDR) Campylobacter [10][11][12].
Tere is little information on antibiotic susceptibility profles of Campylobacter strains emanating from food animals in Kenya; and even then, the few available studies are in humans. In addition, no previous studies have been conducted in Kajiado County on antibiograms of thermotolerant Campylobacter species from food animals, despite the high dependency and/or consumption of animal protein in this county. Te few animal-based studies conducted in other regions in Kenya have only focused on resistance profles displayed by chicken Campylobacter isolates [12][13][14], without investigating the resistance situation in cattle and their respective environment. It is worth noting that, phenotypic antibiotic resistance may be caused by many diferent genetic determinants which may present particular epidemiological characteristics [15]. Of particular concern are the genetic determinants encoding MDR [16], especially when disseminated with AMR phenotypes. Furthermore, evaluation of genetic determinants of resistance is vital for elucidating and controlling antimicrobial resistance, i.e., it can be used to reliably predict resistant phenotypes. Terefore, it is of paramount importance to delve into the genetic mechanisms linked to antibiotic resistance in Campylobacter species. Te genetic determinants of antimicrobial resistance in Campylobacter have been characterized exquisitely in studies conducted in other countries. Tey showed that resistance genetic determinants in Campylobacter are mediated by the following: (1) existence of tet (O), tet (M), and/or tet (A) genes which are responsible for resistance against tetracyclines [17]; (2) point mutations in the gyrA and 23S rRNA genes which contributes to FQs and macrolide resistance, respectively; (3) an efux pump (cmeABC) which reduces the intracellular concentration of antimicrobials; works synergistically with other resistance mechanisms and contributes to the resistance to multiple antibiotics; and (4) presence of "naturally" occurring resistance genes against β-lactams (e.g., ampicillin), mainly due to the ubiquitousness of the bla OXA-61 gene [18,19]. In addition, alleles of other genes associated with resistance to aminoglycoside (e.g., aminoglycoside 3′-phosphotransferase gene (aph-3-1)) have also been reported [20].
Te emergence and spread of AMR among Campylobacter spp. in the livestock sector and human health contexts have been linked to the overuse or inappropriate usage of antimicrobial drugs. Antimicrobials are used to treat sick animals (therapeutic purposes), prevent livestock diseases (both prophylactic and metaphylactic purposes), and enhance growth. However, any application of antimicrobials, whether considered curative or not, deliberate or otherwise, exposes both pathogenic bacteria and gut commensals to varying concentrations for varying times [21]. Tis creates 2 International Journal of Microbiology a selective pressure that can result in evolution and spread of resistance or an increase in the abundance of resistant bacteria, especially where a resistant subpopulation exists [21]. As such, there is an urgency to control antimicrobial resistance (AMR) amid the rampant failure in veterinary and/or human medicines. Te scourge of antimicrobial resistance in Kajiado and Kenya at large is further compounded by the collapse of public services in the 1980s, including veterinary services. With privatization of veterinary services, delivery of animal health services, more so in arid and semiarid countries, has become a nightmare. Alternatives to this new reality include engaging "communitybased animal health workers" (CAHWs) in treating animals [22]. CAHWs lack continuous training on/or up-to-date know-how on antimicrobial use (AMU) and treatment guidelines, and may end up prescribing inappropriate antimicrobial therapy, including the controlled antimicrobials for humans and animals. While in some developed countries including Australia and Korea; use of fuoroquinolones (FQs) and gentamicin in livestock including poultry was banned over a decade ago [20,23]; the same antibiotics continue to be used in livestock in Kenya. Furthermore, Kajiado County is dominated by the Maasai, one of Kenya's major pastoralists, who are known to self-treat and/or engage unskilled people to treat their sick animals with antibiotics. Here, the resistance begins. As such, there is a need to monitor antimicrobial use (AMU) practice, so as to minimize the development of AMR. However, signifcant knowledge gaps exist on the exact quantities, frequency, and types of antimicrobials being used in cattle and chicken production systems at farm level in Kajiado and Kenya at large. As a result of the widespread resistance to multiple antibiotic classes, it is no surprise that the World Health Organization has listed fuoroquinolone-resistant Campylobacter as a high priority pathogen; with the objective of more research and development of new antibiotics [24]. In the wake of these glaring realities and scarce published data on AMU and AMR in Kenya, this study aimed to investigate antimicrobial use, susceptibility profles, and resistance genes in Campylobacter isolates from chicken, cattle, and water in Kajiado County, Kenya.

Study Area, Design, and Selection of Production Systems.
A feld and laboratory-based cross-sectional study design was conducted between October 2020 and May 2022 in Kajiado County, located south of Nairobi, Kenya ( Figure 1). Te county has well-established smallholder mixed-livestock (cattle and poultry) production systems. Tese production systems were chosen based on the fact that (1) poultry production is the highest consumer of antimicrobials; (2) there is sketchy information on antimicrobial use in cattle production systems and environmental samples (water).

Origin of Campylobacter
Isolates. Campylobacter isolates used in this study were obtained from a previous study on seasonal prevalence of thermophilic Campylobacter from chicken cloacal swabs, cattle rectal swabs, and water samples from cattle-troughs in Kajiado County, Kenya [25]. Tese isolates were cryopreserved in pure colonies in tryptone soya broth (Hi-media) with 30% glycerol and in the respective genomic DNA in a deep freezer at −20°C. In this study, 119 Campylobacter species (29 C. coli (16 cattle isolates, 9 chicken isolates, and 4 isolates from water samples isolates) and 90 C. jejuni (42 isolates from bovine, 42 isolates from chicken, and 6 water isolates)) from the prevalence study were used.

Survey on Antibiotic Use (AMU) and AMR Awareness.
Data on antimicrobial use were collected through administration of semistructured questionnaire in the same farms where sampling was done for the prevalence study. Farm owners/respondents were requested to avail any drugs or used drug containers/sachets kept at the house/farm; these were then recorded accordingly. In farms that indicated to have used antibiotics but had disposed of the container/ sachet, the respondents were asked if they could recall the drugs used by their trade name. Te survey also concentrated on local disease histories, animal health-seeking behaviours, and AMR awareness.

Phenotypic Antibiotic Susceptibility Profle Using Kirby-Bauer Difusion Method.
Te antimicrobial susceptibility of C. jejuni and C. coli isolates was established using the Kirby-Bauer disc difusion technique on plates containing Mueller-Hinton agar augmented with 10% defbrinated ovine blood (MHBA): strictly in accordance with the procedures of the Clinical and Laboratory Standards Institute (CLSI) [26]. Standard antimicrobial impregnated disks (HiMedia Mumbai, India) containing 6 diferent antibiotics at the given concentration were used as follows: (1)  PCR-confrmed C. jejuni and C. coli isolates from cryopreserved stocks in tryptone soya broth (HiMedia) with 30% glycerol were defrosted and then revived by direct plating on blood agar plates augmented with selective supplement (SR0167 E, Oxoid ® ) and 10% lysed ovine blood.
Ten, the inoculated plates were incubated for 36 hours at 42°C under microaerobic conditions. Of 119 Campylobacter isolates, 103 (29 C. coli (16 isolates from cattle, 9 isolates from chicken, and 4 isolates from water samples) and 74 C. jejuni (38 isolates from bovine, 30 isolates from chicken, and 6 isolates from water)) were recovered. However, 16 C. jejuni isolates (4 from bovine and 12 from International Journal of Microbiology chicken) could not be recovered from TSB-glycerol stocks. Colonies of previously revived Campylobacter isolates were emulsifed in physiological saline and then diluted to a turbidity equivalent to that of the 0.5 McFarland standards. Fresh uninoculated MHBA plates were initially dried in an incubator at 35°C with the lid removed for 15 minutes prior to inoculation. Sterile swabs were then used to seed the suspension onto MHBA plates, to produce confuent growth. Te inoculum was allowed to dry for 5 minutes, then, antibiotic discs were placed on the plate. Te seeded plates were microaerobically incubated overnight at 42°C. C. coli (ATCC 33559) and C. jejuni (NCTC 11168) were used as positive controls.
Te inhibition zone diameters around antibiotic (ciprofoxacin, erythromycin, and tetracycline) discs were measured, recorded, and then construed as sensitive and/ or resistant, following [26] breakpoints guidelines for infrequently isolated or fastidious organisms (M45) including C. jejuni and C. coli. Since CLSI's M45 (third edition) have no interpretive criterion for inhibition diameters for ampicillin, nalidixic acid, and gentamicin for C. jejuni and C. coli, the breakpoints provided by CLSI [27], (M100S) for the Enterobacteriaceae family was used instead.
Cryopreserved DNA was defrosted and then amplifed in a fnal reaction volume of 25 μL in a BIO-RAD, T100 ™ Termal Cycler (Singapore). Te reaction mixture contained   [11]. Te amplifcation conditions for the gyrA gene (a 235-bp product) were as follows: an initial primary denaturation at 95°C for 5 minutes, 30 cycles at 95°C for 50 seconds, annealing at 53°C for 30 seconds, and 72°C for 1 min, followed by a fnal extension at 72°C for 7 minutes [30]. DNAse/RNAse free water (BioConcept) was used as a negative control. Te amplicons were resolved by electrophoresis on a 1.5% agarose gel stained with ethidium bromide in Tris-Borate-EDTA (TBE) bufer; run at 60 V for 60 minutes, and then, visualised under ultraviolet light using the GelMax ® 125 imager (UVP, Cambridge UK).

DNA Sequencing.
A representative of positive amplicons (two C. jejuni and one C. coli for each antimicrobial resistance gene) generated with each primer was purifed using QIAquick PCR Purifcation Kit (Qiagen) and commercially Sanger-sequenced in both directions at Inqaba Biotechnologies, Pretoria, South Africa. Te forward and reverse sequences were edited, aligned, and assembled in consensus sequences using BioEdit software. Nucleotide sequences were subjected to BLASTn search tool (https:// www.ncbi.nlm.nih.gov/BLAST), for confrmation of genes detected.

Data
Handling and Analysis. Data were analyzed with statistical software R version 3.6.1. Te diference was signifcant when p < 0.05. Cohen's kappa coefcient was used to assess the concordance between phenotypic antibiotic susceptibility and genotypic expression of resistance genes. According to McHugh [31], a kappa value of 0-0.2 indicates nonagreement, 0.21-0.39 (minimal level of agreement), 0.4-0.59 (weak level of agreement), 0.60-0.79 (moderate level of agreement), 0.80-0.90 (strong level of agreement), and above 0.90 (almost perfect level of agreement). A kappa value of 1 (100%) indicates total concordance between the two antibiotic susceptibility tests. Te correlation between AMU and the occurrence of resistance was determined by Pearson's correlation coefcient (r) method. Furthermore, a 95% confdence interval was also determined for antibiotic resistance rates. All analyses were considered statistically signifcant at P < 0.05.
Based on recall of antibiotic use in the last 6 months, 76.4% (42/55) of the farmers reported that they had used antibiotics mainly for treatment and prevention. Tetracyclines, aminoglycosides (streptomycin and gentamicin), and β-lactams-based antibiotics were the most commonly used antimicrobials to treat sick cattle and/or chicken (Table 1; Supplementary Figure S1). Antimicrobial use was generally higher in chicken production systems than in cattle for most of antibiotics apart from aminoglycosides and β-lactams (penicillins).
Some farmers (10/55, 18.2%) indicated using nonconventional medications such as herbs like Aloe vera, leaves of Tithonia diversifolia (Supplementary Figure S2), and chilli pepper among other "mitishamba" and/or "dawa za kienyeji" to relieve respiratory distress, diarrhoea, and other related sick-bird syndrome cases in chicken.
As for C. coli, all the isolates were resistant to ampicillin (100%), followed by resistance to tetracycline (96.6%), erythromycin (93.1%), and ciprofoxacin (69%); few strains were resistant to nalidixic acid and gentamicin (each at 10.3%). Tetracycline resistance in C. coli was seen more frequently in isolates from chicken and water samples (each at 100%). Similarly, C. coli resistance ciprofoxacin was prevalent in isolates from water samples and chicken at 100% and 77.8%, respectively. C. coli isolates from chicken International Journal of Microbiology  6 International Journal of Microbiology and cattle swabs showed the highest resistance to erythromycin at 100% and 93.8%, respectively. Although no resistance to gentamicin was observed in any of the Campylobacter isolates from water samples; C. coli isolates from chicken recorded a relatively high resistance to gentamicin at 22.2%. Likewise, ampicillin resistance was the most prevalent in C. jejuni, with levels of 100%, followed by resistance to tetracycline (97.3%), erythromycin (68.9%), ciprofoxacin (60.8%), and nalidixic acid (45.9%); few strains were resistant to gentamicin (1.3%). C. jejuni isolates from chicken showed a high rate of resistance to tetracycline and ciprofoxacin with 100% and 83.3%, respectively. Conversely, C. jejuni from cattle were highly resistant to erythromycin (76.3%) and gentamicin (15.8%), whereas those from water samples were 100% resistant to tetracycline, 66.7% resistance to ciprofoxacin and 50% resistance to nalidixic acid.

Multiple Drug Resistance and
Resistance Patterns of C. coli and C. jejuni. Campylobacter isolates that were resistant to three or more classes of antibacterial agents were designated multidrug resistant (MDR). Ninety-nine of 103 (96.1%) isolates (29 (100%) C. coli and 70 (94.6%) C. jejuni) displayed MDR. In addition, the highest MDR was found among chicken isolates, with 100% (n = 39) MDR, regardless of the drug tested and/or Campylobacter species. Overall, a total of 14 diferent multiple drug resistance profles were exhibited by Campylobacter species from cattle, chicken, and water samples are shown in Table 3. Te most frequent MDR profles of the isolates from diferent sources were resistant to AX-TE-E-CIP (29.1%), AX-TE-NA-CIP (18.4%), and AX-TE-E (16.5%).

Correlation between the Use of Various Antimicrobials and the Phenotypic Resistance among Campylobacter Isolates.
Pearson correlation demonstrated highly signifcant (p < 0.01) positive correlations between antimicrobial use at the farm level and the phenotypic antibiotic resistance profles for various drugs investigated in this study (Table 4). Te highest positive correlations exist between the usage of tetracycline and its resistance at 31.4%. Beta-lactams and macrolide use showed positive correlation with resistance to erythromycin at 29.6% and 25.6%, respectively.

Detection of Genes Conferring Resistance, and Concordance between Resistance Phenotypes and Genotypes.
Te occurrence of assayed genes conferring resistance to tetracyclines (tet (O)), β-lactams/ampicillin (bla OXA-61 ),     Figure 4 illustrates the fndings which indicate that C. coli isolates, as well as C. jejuni isolates, demonstrated more or less similar occurrence of antimicrobial resistance genes.

GenBank Accession Numbers.
Te partial sequences for some of the isolates from this study have been deposited in the GenBank database and assigned accession numbers: OQ389471, OQ389472, and OQ389473 for the gyrA gene; OQ390085 and OQ390086 for tet (O) gene; OQ421183 and OQ421184 for bla OXA-61 gene. Consensus sequences obtained from cmeB and aph-3-1 genes were too short with many gaps and as such were rejected on submission to GenBank.

Discussion
Te world is at the verge of tipping over due to the adverse efects of AMR; with the latter emerging and spreading at a rate that by far surpasses development of newer drugs. It is notable that macrolide-fuoroquinolone-resistant bacterial pathogens particularly Campylobacter spp., have increased dramatically [32]. Fluoroquinolones and macrolides are prescribed as the frst priority drugs for the treatment of human campylobacteriosis, and as such, increasing resistance trends pose a public health hazard.
Campylobacter species are naturally resistant to β-lactam antibiotics, including ampicillin [11]. None of the Campylobacter isolates in this study were susceptible to ampicillin, translating into 100% "acquired" resistance. Previous studies in other African countries including Tanzania and Morocco have reported resistance rate to this antibiotic at 63% and 95.2%, respectively [11,33]. Te high ampicillinresistant phenotypes in this study might be due to the reported usage of β-lactams (including amoxicillin or a combination of procaine penicillin and dihydrostreptomycin sulphate or cloxacillin and ampicillin) among farmers in the treatment of bacterial infections such as mastitis in cattle.
Tetracycline is relatively inexpensive and highly efective against a wide range of microorganism; thus, it has been frequently used in livestock husbandry practices [34]. Terefore, it is not surprising that more than 97% of the isolates (96.6% for C. coli and 97.3% for C. jejuni) in this study were resistant to tetracycline. Te results found in this study are comparable to a study conducted recently in various Kenyan counties, including Kajiado County [14]. Beyond Kenya, similar fndings were reported in studies carried out in Spain [35], Tunisia [36], South Korea [37], and China [38].
Furthermore, the results demonstrated that the resistance rate among Campylobacter isolates recovered from livestock and water samples to erythromycin was 75.7%, including 93.1% for C. coli and 68.9% for C. jejuni. Tis resistance rate is somewhat worrying in contrast to previous fndings from the outskirts of Tika, a city in Central Kenya International Journal of Microbiology [12]. Te fnding is consistent with the study by Asmai et al. [33] who also reported a high phenotypic Campylobacter resistance rate of 92.8% to erythromycin. Going by the fndings of this study, macrolide (erythromycin) would no longer be considered as an alternative therapy in systemic campylobacter infections in man. Ciprofoxacin, a fuoroquinolone, is one of the frst line antibiotics in the treatment of clinical campylobacteriosis in man. Notably, signifcant 63.1% ciprofoxacin-resistant isolates (69% C. coli and 60.8% C. jejuni) compared to strains resistant to nalidixic acid at 35.9% (10.3% C. coli and 45.9% C. jejuni) were reported in this study. Te observed resistance to ciprofoxacin is comparable to other studies in Kenya [12], Ethiopia [39], and Poland [40]. Te relatively low resistance to nalidixic acid observed in this study is in contrast with those on Campylobacter isolates from backyard chicken in Central Kenya, where resistance to nalidixic acid was observed at 77.4% [12]. Te level of resistance to   nalidixic acid observed in this study is however concordant with fndings found in studies from other regions: Poland [41], Tanzania [7], South Africa [42], and the USA [43]. Te low resistance to nalidixic acid may be as a result of a decrease in the use of quinolones including nalidixic acid, over most sought-after fuoroquinolones (such as ciprofoxacin) for curative or prophylactic purposes. Te overall resistance for gentamicin was low (11.7%) with C. jejuni isolates portraying slightly higher (12.2%) resistance than C. coli (10.3%). Te fndings concord with reports from other African and European states. For instance, in Tanzania, 11.8% of the Campylobacter isolates from dressed beef carcasses and raw milk in Tanzania were resistant to gentamicin [11]. In North African countries such as Morocco, 7.1% of the isolates from poultry were gentamycin-resistant [33]. Low resistance to gentamicin was also been observed in Spain, where 12.1% and 14.7% of C. coli strains from cattle and broilers were resistant [35]. Te relatively low resistance could possibly be due to restricted use for systemic infections [44], and also due to the fact that there are no oral formulations to be administered in drinking water or feeds for use in livestock production.
However, the results of phenotypic and genotypic assays of resistance to various antibiotics were partially concordant; moderate level of agreement being observed only in tetracycline. A similar observation was also reported by Kashoma et al. [11]. Tis deduces that other factors beyond this study, including the occurrence of other molecular determinants that encode resistance could be involved.
Te tet (O) gene is the most common ribosomal protection mechanism mediating Campylobacter resistance to tetracycline. However, other genes such as tet (A), tet (K), tet (B), and multidrug efux, have also been reported. Almost all the tetracycline-resistant phenotypes were shown to harbour the tet (O) gene at 93.1%. Tis is higher in this study than the percentage of the same gene in chicken samples in a report by Nguyen et al. [12]. However, similar results to this study have been reported in China [38].
Te gyrA gene was confrmed in 61.2% of the isolates, including 62.1% C. coli and 60.8% C. jejuni in this study. Te substitution of threonine to isoleucine (Tr86Ile region) in the gyrA genome confers cross-resistance to both quinolones (nalidixic acid) and fuoroquinolones (ciprofoxacin). However, Ge et al. [45] reported upper-level resistance to ciprofoxacin linked to a mutation in the Tr86Ile region of the gyrA genome. Te results of this study further revealed that low nalidixic acid-resistant phenotypes possessing gyrA genome compared to the ciprofoxacin-resistant phenotypes possessing gyrA genome. Te discrepancies in the gyrA gene detection rate for ciprofoxacin and nalidixic acid resistance could further be explained by the fact that occurrence of point mutation in the Tr86Ala region of the gyrase subunit A gene (by substitution of threonine to alanine) has been linked with high nalidixic acid-resistant and low ciprofoxacinresistant C. jejuni [45]. Indeed, more molecular studies are needed to explore gyrA gene sequences and other antibiotic resistance genes incriminated in Campylobacter spp. resistance to nalidixic acid and ciprofoxacin.
Despite the high resistance to ampicillin reported in this study, β-lactam conferring gene (bla OXA-61 ) was detected in only 36.9% of all Campylobacter isolates (44.8% in C. coli and 33.8% in C. jejuni), suggesting that other means of acquired ampicillin resistance could be involved. Comparable fndings were reported by Kashoma et al. [11], where 52.6% and 28.1% of C. coli and C. jejuni strains, respectively, were found to harbour the bla OXA-61 gene. Undeniably, other genetic determinants including modifcations in outer membrane porins and/or decreased afnity of penicillin-binding protein (PBP) and efux pump are most likely involved [11,46].
More than 22% of the strains were found to possess the aph-3-1 gene. Obviously, gentamicin-resistant phenotypes cannot be elucidated by aph-3-1 gene. However, our fndings were much higher than previous reports in Africa [11]. Yet Hailu et al. [43] reported 100% detection rate amongst Campylobacter isolates from dairy cattle and chicken manure in the USA.
Multidrug resistance (MDR) presents a public health threat by limiting antibacterial agents to choose from for curative therapy. Almost all the Campylobacter isolates (>96%) in this study were resistant to three or more of the six tested antibacterial agents; with C. coli and C. jejuni reported 100% and 94.6% MDR, respectively. Ampicillin-tetracycline-erythromycin-ciprofoxacin (AX-TE-E-CIP) and AX-TE-NA-CIP were the most common MDR patterns in both C. coli and C. jejuni. Te MDR rate reported in this study is much higher than what has been reported in some European nations; for instance, in Poland, where MDR for Campylobacter isolates from raw chicken meat was 7% [40]. However, the fndings of this study are concordant with some studies in other African countries: 95% of the Campylobacter isolates from broiler in Morocco displayed drug resistance to ≥3 drugs [33]; 95.5% of isolates from livestock (cattle and shoat), poultry, human, and water in Ethiopia [39]; 94.7% of the strains from poultry in Ghana [47], and 32.5% of in Campylobacter isolates from beef cattle in South Africa [42]. Te observed discrepancies in MDR in Campylobacter may possibly be explained by the following: (1) level of intensifcation and type of production system; (2) the introduction and implementation of legislation to minimize antimicrobial use in livestock in European countries. In underdeveloped nations including Kenya, there are laws and rules on antimicrobials use in food animals; however, enforcement is done to a limited extent or practically nonexistent. Consequently, higher resistances to most antimicrobial agents tested may be due to the relatively unrestricted use of antimicrobial agents in animal treatment that is practiced in most of the developing countries [48]. In this study, extensive use of antimicrobial drugs was observed in this study, with tetracyclines, aminoglycosides, and β-lactams being commonly used. Excessive use of these antibiotics in livestock has also been reported in other studies [49,50]. Moreover, antibiotic usage was positively correlated with the high level of resistance to tetracyclines and erythromycin amongst Campylobacter isolates in this study.
In this study, extensive misuse of antimicrobials was observed in this study, where 56.4% of farmers treated their animals themselves without the prescription or advice from a qualifed veterinarian. Tis fnding agrees with Chepkwony [13], who reported that 67.5% of livestock owners admitted injecting drugs into their animals themselves without professional consultation. Although the self-reported use of antibiotics among farmers in this study precluded establishment of diagnosis and dosage regime; there is a possibility that antibiotics are often administered in absence of a confrmatory diagnosis, or antibiotic susceptibility testing in response to various clinical syndromes or illnesses, some of which are caused by nonbacterial pathogens such as foot and mouth disease, lumpy skin disease, or tick-borne diseases. Terefore, inadequate veterinary skills and accessibility is of great concern and could accelerate antibiotic overuse in livestock; thus, they may be linked with the evolution of MDR Campylobacter isolates in the county.
Finally, where the use of fuoroquinolones among other antibiotics in food production is banned, the frequency of Campylobacter-resistant isolates is relatively low. For instance, Australia, where administration of fuoroquinolones in food animals is prohibited, recorded Campylobacter strains susceptible to ciprofoxacin recovered from pigs in 2004 [51]. However, years later, fuoroquinolone-resistant Campylobacter isolates emerged and were detected among Australian chickens, even in the absence of fuoroquinolone application [52]. Tese fuoroquinolone-resistant Campylobacter isolates might have emerged from outside and brought into Australian chicken by people, vectors, or wild birds [52]. Tese fndings dramatically underline the critical role of biosecurity in the overall fght against antimicrobial resistance. Consequently, even as nations call for a policy on minimizing application of antimicrobials in livestock; stringent farm biosecurity measures come handy in the overall fght against antimicrobial resistance.

Conclusions
In this study, extensive use of antimicrobial drugs was observed in this study, with tetracyclines, aminoglycosides, and β-lactams being commonly used. Application of antibiotics in cattle and poultry production systems was positively correlated with the high level of resistance to tetracyclines and erythromycin. Tis highlights the signifcance of the warranted application of antibacterial agents in the said production systems in the county. Regarding antimicrobial resistance, almost all isolates (96.1%) displayed MDR, with C. coli expressed greater resistance to three or more of the assayed antimicrobials. Tis might further limit treatment options for Campylobacter infections. A high level of resistance to ampicillin, tetracycline, erythromycin, and ciprofoxacin was found among the Campylobacter strains. As such, none of the priority drugs in Campylobacter infections therapy can be prescribed in the county. Chicken-derived Campylobacter strains showed greater resistance; this could be due to the widespread use of antibiotics in the poultry production system compared to the cattle production system. Te tet (O), gyrA, and cmeB were the most frequently detected genes, while the occurrence of bla OXA-61 and aph-3-1 was signifcantly lower (p < 0.05).

Recommendations
Furthermore, molecular studies should include all the cryptic antibiotic resistance genes and plasmids in C. jejuni and C. coli strains to give insights on their transmission and possible transfer to other Campylobacter strains. Te existing national action plan on AMR spearheaded by the ministries of health and agriculture, livestock, and fsheries in Kenya must strengthen the surveillance programs and policies advocating for a reduction in unwarranted use of antibiotics. Moreover, the veterinary directorate at the county and national governments ought to be on the fore-front in managing and implementing appropriate biosecurity measures aimed at fghting antimicrobial resistance. Screening of alternative treatment, e.g., use of medicinal plant extracts (Aloe vera, Tithonia diversifolia, and chilli pepper) needs to be encouraged, in efort to reduce usage of antibiotics.

Data Availability
All the data relating to this study are available on mail request to the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.