Occurrence of Campylobacter species in beef cattle and local chickens and their antibiotic profiling in Ibadan , Oyo State

Food animals like cattle and poultry are often regarded as reservoirs for Campylobacter infections in human. This study investigated the occurrence of Campylobacter coli in cattle and local chickens and their antibiotic susceptibility to commonly used antibiotics in Ibadan, Oyo State, Nigeria. A total of 250 samples comprising 100 rectal swabs, 100 gall bladder contents from cattle and 50 cloacal swabs from local chickens that were apparently healthy, were subjected to standard microbiological identification and antibiotic susceptibility tests. Overall, 51 (20.4%) C. coli were isolated including 34/100 (34%) from rectal swabs, 12/100 (12%) from gall bladders and 5/50 (10%) from the cloaca. All the isolated C. coli displayed multiple antibiotic resistances to between 4 and 10 of the antibiotics tested showing up to 40 different resistance patterns. The cattle C. coli displayed a high frequency of resistance to erythromycin and ciprofloxacin, while all the chicken isolates were resistant to erythromycin, the drug of choice for the treatment of the Campylobacter infections in Nigeria. This investigation carried out in apparently healthy animals identified cattle and local chickens as potential reservoir hosts for C. coli infection in the study area.


INTRODUCTION
Campylobacter is a Gram-negative, spiral shaped, obligate microaerophilic, motile bacterium, having up to 23 species documented in the NCBI taxonomy division (Moolhueijzen et al., 2009).Morphologically, they are helical or curved shaped with long spiral forms which resemble spirochaetes superficially.Campylobacter species are motile by means of flagella which are usually single at one or both poles (Barrow and Feltham, 1993;Moolhueijzen et al., 2009).Campylobacteriosis, an important bacteria zoonoses is caused by species from the Genus Campylobacter (Tambur et al., 2013).The Thermophilic species such as Campylobacter jejuni, C. coli, C laris, and C. upsaliensi are the most common causative agents of human diseases (Tambur et al., 2013).
Campylobacter species, particularly C. jejuni and C. coli are commonly traced to foodborne illnesses in the United States and worldwide (CDC, 2013;Scallan et al., *Corresponding author.E mail: peculiarj@yahoo.com. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0International License 2011).For instance, they accounted for approximately 35% of laboratory confirmed foodborne illnesses within the FoodNet surveillance areas in the United States in 2012 (CDC, 2013).C. jejuni and C. coli were mostly reported during the period with C. jejuni responsible for 80-90% of human infections (CDC, 2013;Nachamkin and Blaser, 2000).Based also on European Food safety Authority report for 2010, there were 212064 confirmed cases of campylobacteriosis, making it to be the most reported zoonosis in European Union during the period (Anonymous, 2010).Campylobacter was reported to be mainly distributed in poultry; however cattle, pigs, sheep and pet animals were also acknowledged as the possible sources of Campylobacter infection (Anonymous, 2010;2012a).The prevalence of the bacteria in retail fresh broilers meat in EU region varied between 3.1 to 58.8% depending on the member of State as from 2006 (Anonymous, 2010;2012).Most Campylobacteriosis in New-Zealand around 2005 were attributable to C. jejuni and only around 10% were associated with C. coli (Moore et al., 2005).
These organisms are known to colonize different hosts including human and other animals with varying degrees of virulence (Fouts et al., 2005).Although chickens have been its most frequently identified reservoir for human infection, Campylobacter species have been isolated from other sources such as the faeces of healthy cattle (Humphrey et al., 2007;Baserisehalehi et al., 2007;Mohammed et al., 2009;Salihu et al., 2009).Cattle strains can infect poultry suggesting cattle as possible reservoir for poultry infections (Ziprin et al., 2003).The organism may also be carried asymptomatically by a wide range of animals and excreted into the environment in faeces (EPIDAT, 2005;Moore et al., 2005).Humans can thus be infected by several non-human hosts through consumption of contaminated water, or from food animals and their products (Rodrigues et al., 2001;Kapperud et al., 2003;Stanley and Jones, 2003;Teunis et al., 2005).However, contamination during food processing has been identified as the most important means of Campylobacter infections and the characteristics of the organism such as motility, ability to adhere to intestinal mucosa, capability to invade enterocytes as well as toxin production have been associated with its pathogenicity (Datta et al., 2003;Dasti et al., 2010).
Campylobacteriosis is usually a self-limiting disease and thus do not usually require antimicrobial treatment (Wieczorek et al., 2012).In some cases however such as septiceamic form of the disease characterized by severe and prolonged enteritis, in immune-compromised or young patients, antimicrobial therapy may be required; and in such cases, macrolides (erythromycin) and quinolones/ fluoroquinolones (ciprofloxacin, nalidixic acids) are usually the drugs of choice (Skirrow and Blaser, 2000;Engberg et al., 2001;Wieczorek et al., 2012).
According to Lehtopolku (2011), multidrug resistance in Campylobacter is associated with resistance to the drug of choice like the macrolides and fluoroquinolones for the treatment of the life threatening infections, whereas those resistant to three or more group of antimicrobial agents apart from the macrolides could be referred to as multiple drug resistant organisms (Lehtopolku, 2011).The multidrug resistant Campylobacter is often associated with the presence of the CmeABC multidrug efflux pump (Lehtopolku, 2011).There have been various reports of multidrug resistance Campylobacter species in different parts of the world.For instance, 2.2% incidence of multidrug resistance Campylobacter species was reported between 1989 and 1993 in North India (Prasad et al., 1994).From the same region there was an increase to 30.6% among C. jejuni and C. coli in 2002jejuni and C. coli in and 90% for 2008jejuni and C. coli in (Jain et al., 2005;;Chen et al., 2010).In China, 76.8% incidence of multidrug resistant C. coli was reported, and the strains showed 19 different multiple antimicrobial patterns (Qin et al., 2011).
In the Northern Nigeria, Salihu et al. (2009) documented the prevalence of 65.1% for C. jejuni, 23.0% for C. coli, 7.9% for C. laris, 3.2% for C. hyointestinalis and 0.8% for C. fetus.This paper reports the occurrence of Campylobacter species in beef cattle and local chicken and their antibiotic sensitivity in Ibadan, Oyo State, Southwestern Nigeria.

Sample collection/location
A total of 250 samples comprising of 100 rectal swabs and 100 swab samples of gall bladder contents from slaughtered cattle in Municipal abattoir Bodija, Ibadan Oyo State and 50 cloacal swabs from local chickens at Abadina Community, University of Ibadan and from Igbo oloyin area of Ibadan were collected.Ibadan, the biggest city in the South Western Nigeria, hosts the biggest cattle market and abattoir in the region.Cattle and local chickens were sampled by insertion of a sterile swab (Global swab ® ) into the rectums and cloaca, respectively.Each swab was placed in Amies charcoal transport medium (Oxoid CM 0425 ® ) and transported to laboratory within 5 hours in ice packs.The laboratory analysis of the sample was carried out at the Nigerian Institute of Science Laboratory Technology (NISLT), Ibadan.

Bacteriological processing
The samples were analysed for the thermotolerant Campylobacter species as earlier described (Skirrow and Benjamin, 1980;Georges-Courbot et al., 1986;Karmali et al., 1986;Barrow and Feltham, 1993).The cattle rectal swabs, gall bladder contents and chicken cloacal swabs were inoculated in duplicates onto modified charcoal cefoperazone deoxycholate agar (MCCDA Oxoid CM0739 ® , and incubated microaerobically at 25C (to allow for the growth of Campylobacter fetus) and 42C respectively, for 48 h.The microaerophilic environment of 5% O2, 10% CO2, and 85% N2 was produced using Campygen sachet (Oxoid CN0025A ® ) inside an anaerobic jar.The suspected Campylobacter colonies were Gram -stained and subjected to further biochemical tests: catalase and oxidase tests, urease production, H2S production, nalidixic acid and cephalothin sensitivity tests, growth at 42C and hippurate hydrolysis (Gerhardt et al., 1984).Each isolate was stored at -80C in a peptone broth with 15% glycerol for further analysis.

Hippurate hydrolysis
The test was carried out to differentiate between C. coli and C. jejuni.A large loopful of suspected Campylobacter colonies were scraped from the MCCDA plates and mixed with hippurate solution to form a very cloudy suspension, the tube was incubated in water bath at 37 o C for 2 h.Subsequently, 0.2 mL of ninhydrin reagent was added without shaking the tubes and incubated at 37C for 10 min.Formation of a deep purple colour due to glycine formation from hippurate hydrolysis indicated presence of C. jejuni, while absence of colour formation indicated presence of C. coli (Gerhardt et al., 1984).

Bacterial processing
All the plates incubated at 25C for possible isolation of Campylobacter fetus showed no growth.The positive plates of local chicken cloacal swabs (1 from Abadina and 4 from Igbo oloyin) and cattle rectal swabs/ gall bladders incubated at 42C showed the characteristic small, grey, butyrous, moist, flat and spreading colonies.The isolates were Gram-negative and curved rods.
Biochemically, isolates were oxidase-and catalasepositive.Isolates were motile and H 2 S-negative.All the isolates produced negative reactions for hippurate hydrolysis and suggestive of C. coli.All the isolates were susceptible to 30 µg cephalothin (Figure 1) and resistant to 30 µg nalidixic acid (Figure 2).
A total of 63% of C. coli from cattle were susceptible to ofloxacin followed by ceftriazone (36%).However, there were high resistance of 84.8 and 82.6% for ciprofloxacin and erythromycin, respectively (Table 1).The organisms that produced 17 to 27 mm clearing zones for 10 µg of ciprofloxacin and 18 to 22 mm for 5 µg of erythromycin were adjudged susceptible, whereas all the isolates considered resistant did not produce any clearing zones.
Likewise, from the local chickens there was a 100% susceptibility to ofloxacin followed by 60% susceptibility to ciprofloxacin, but the 5 isolates from the local chicken cloacal were 100% resistant to amoxicillin, streptomycin, chloramphenicol, ceftriazone, gentamycin and erythromycin (Table 2).
The 40 different multiple antibiotics resistance patterns exhibited by the isolates from cattle and chickens are shown in Table 3.In cattle, there were five different resistance patterns for 10 antimicrobial agents, 3 patterns for 9, 5 patterns for 8, 17 patterns for 7, 10 patterns for 6, 2 patterns for 5 and 1 pattern for 4 antimicrobial agents.
For the local chickens; there was 1 pattern for resistance to 9 antimicrobial agents, 3 patterns for 7, and 1 pattern for 6.   (2005) who earlier documented the organism to be a commensal in the various organs of healthy cattle.This study shows that gall bladders of cattle harbor Campylobacter and may result in contamination of carcass during unhygienic slaughtering and subsequent transmission to human beings.Wild birds, domestic and companion animals are known as reservoirs for Campylobacter species, and they shed the organisms in faeces contaminating the environment (Akitoye et al., 2002).Occurrence of 10% C. coli from apparently healthy local chickens is noteworthy.In Nigeria, local chickens are found within households, hence, they are important economically and constitute a source of transmission of Campylobacter organisms to human.One report showed that strains isolated from human and chickens were phenotypically and genotypically correlated, confirming that chickens are an important source of human campylobacteriosis in developing countries including Nigeria (Adegbola et al., 1990).
The antibiotic sensitivity test revealed low susceptibility by these C. coli to most of the 10 antibiotics studied.The cattle C. coli isolates exhibited low susceptibility to ciprofloxacin and erythromycin, while all the chicken C. coli were resistant to amoxicillin, streptomycin, chloramphenicol, ceftriazone, gentamycin and erythromycin; those resistant Campylobacter species to erythromycin and ciprofloxacin conform to the definition of multidrug resistance (Lehtopolku, 2011) because they are resistant to the drug of choice for treating Campylobacter infections when need be.The observed 18 to 22 mm clearing zone for the erythromycin susceptible C. coli in this study is comparable to those of Gaudreau et al. (2007) where susceptible C. coli had a clearing zones of ≥ 15 mm at erythromycin MIC ≤ 4 mg/L.The ciprofloxacin susceptibility in this study was based on clearing zones of 17 to 27 mm which is slightly different from ≥ 25 mm zone of clearing around 5 µg ciprofloxacin as reported by the same author (Gaudreau et al., 2007).
A better susceptibility was however observed for ofloxacin both in cattle and chicken isolates.The antibiotics resistance in this study is similar to that of Sammarco et al. (2010) who found Campylobacter coli isolated from chicken and beef meat to be resistant to most antibiotics tested in Italy.Chatre et al. (2010) in France also documented an upward trend in resistance of Campylobacter species isolated from cattle to commonly used antibiotics notably quinolones, aminoglycosides and penicillins.The antibiotics resistance exhibited by C. coli observed in this investigation also agrees with observations from other parts of the world, as observed from food and water sources as well as from clinical samples reported in Europe (Moore et al., 2001;San'enz et al., 2000); Canada (Gaudreau and Gilbert, 1998), and the United States (CDC, 2000).
Fluoroquinolone, like ciprofloxacin and erythromycin are often regarded as the drugs of choice for treatment of patient with severe campylobacteriosis, while tetracycline, doxycycline, and chloramphenicol are sometimes listed  (Luangtongkum et al., 2009;Jong et al., 2009).The low susceptibility of the C. coli to ciprofloxacin calls for concern.However, such a phenol-menon suggests the misuse/abuse of the drug by most livestock farmers and dealers without proper prescription by professionals in Nigeria (Unpublished data).Prudent use of the commonly used antibiotic tested in this study, particularly those drugs of choice for treatment of Campylobacter infection is recommended.

Table 2 .
Antimicrobial Susceptibilities of local chicken isolates.
Muz et al. (1992)attle gall bladders in this study was lower than 47% reported in a previous study byMuz et al. (1992)and 35.6% Acik and Cetinkaya (2005) outside, Nigeria.The C. coli recovered from gall bladders and faecal samples agreed with those Acik and Cetinkaya

Table 3 .
Antibiotic resistance patterns of Campylobacter coli isolated from Cattle and local chickens.