In ternationa l Scholars Journa ls African Journal of Pig Farming ISSN 2375-0731 Vol. 6 (12), pp. 001-009, December, 2018. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Prevalence, haemolysis and antibiograms of Campylobacters isolated from pigs from three farm settlements in Venda region, Limpopo province, South Africa P. O. Uaboi-Egbenni1*, P. O. Bessong2, A. Samie3 and C. L. Obi4 1 Department of Microbiology, University of Venda, P.M.B. 5050, Thohoyandou, Limpopo Province, South Africa. 2 Department of Microbiology, University of Venda, P.M.B. 5050, Thohoyandou, Limpopo Province, South Africa. 3 Department of Microbiology, University of Venda, P.M.B. 5050, Thohoyandou, Limpopo province, South Africa. 4 Academic Affairs and Research, Walter Sisulu University, Mthatha, Eastern Cape, South Africa. Accepted 14 May, 2018 We investigated the prevalence, haemolytic activities and antibiotic susceptibility profiles of Campylobacter species isolated from pigs in three farms in the Venda region, Limpopo province, South Africa. During the period of investigation, which spanned over one year, 450 faeces samples from pigs comprising 150 each from the three farms were collected and appropriately transported to the base laboratory at the Department of Microbiology, University of Venda for analysis. In total, the prevalence rate of Campylobacter isolates from all three farms was 30.2%, whereas, the rates of 25 - 26% were recorded for farms X, Y and Z, respectively. Out of the isolated Campylobacter species 10 (12.5%) were identified as Campylobacter jejuni and 70(87.5%) as Campylobacter coli. Of the Campylobacter isolates from the farms, 23, 22 and 35 were β-haemolytic from farms X, Y and Z, respectively. Susceptibility of the isolates to 12 antibiotics was determined by disc diffusion technique. The overall rate of resistance determined in this study to most of the antibiotics was 84.2%. Most Campylobacter isolates from farm X (< 50%) were resistant to ciprofloxacin, cefexime, and erythromycin, while > 50% were resistant to tetracycline, (54.5%) to vancomycin, (100%) to ampicillin and methicillin. The rates of resistance to these antibiotics were not significantly different from farms X and Y (p < 0.005). However, most isolates from farm Y were more resistant to ciprofloxacin (57.1%) than isolates from farm X (36.4%) and Z (40.7%). Resistance rates to tetracycline by C. coli isolated from farm Y (48%), farm Z (45.5%) and farm X (44.4%) were noted. Similar trend was observed for C. coli from farm Y (40%), farm Z (31.8%) and farm X (22.2%) for ciprofloxacin. All C. coli from farm Z showed higher level of resistance to ampicillin (100%), Y (64%) and X (55.6%). The C. jejuni isolates though few were markedly resistant to fluoroquinolone and the macrolide as well as aminoglycoside antibiotics. Most of the isolates were resistant to at least seven (7) of the antibiotics tested. These observations indicate high levels of resistance to antibiotics commonly used in the swine industry (erythromycin and tetracycline) among the C. coli and C. jejuni isolates from pigs. Erythromycin and ciprofloxacin are drugs for treatment of human campylobacteriosis. The high prevalence rate of resistance to these drugs among C. coli and C. jejuni isolates from pig faeces is of public health significance. Key word: Campylobacter, campylobacteriosis, haemolytic, susceptibility, resistance, profiles. INTRODUCTION Campylobacter, mainly Campylobacter jejuni and *Corresponding author. E-mail: uaboiegbenni@yahoo.com. Tel: +27 15 962 8186. Campylobacter coli, are recognized as major causes of acute bacterial food-borne gastroenteritis (World Health Organization, 2002). The incidence of human campylo- bacteriosis in the European Union has increased over the past years and recently exceeded that of Salmonella in many countries. Although campylobacteriosis is usually a self-limiting diarrheal disease, severe complications such as septicemia, reactive arthritis, and Guillain-Barre syndrome sometimes occur (Humphrey et al., 2007). C. jejuni colonize the intestinal tracts of a large number of mammals and birds. Broilers are often carriers of C. jejuni. In Thailand, Campylobacter species were isolated from 12% of various food samples including pork, chicken and vegetables in Bangkok (Rasrinual et al., 1988; Young, 2005), 40% poultry ceca in India (Das et al., 1996), and 68-100% of poultry samples from retail markets in Taiwan (Shih, 2000). Reports on Campylobacter in pigs revealed higher proportions of C. coli than C. jejuni, whether they have enteritis or not (Harvey et al., 1999; Steinhauserova et al., 2001). In the U.S., Campylobacter species were isolated from 76% gilts, 100% of pregnant sows, 57.8% of newborn piglets, and 100% of weaning pigs (Young et al., 2000). In the Netherlands, fifty-percent of piglets were infected with the same serotypes at seven days of age (Weijtens et al., 1997). The average number of Campylo- bacter colonizing the gut decreased toward the end of the rearing period (Weijtens et al., 1999). A study in Belgium reported the prevalence of Campylobacter sp. on pig carcasses at slaughterhouses to be 2% (Korsak et al., 1998), and Campylobacter was found in 1.3% of samples from pork from a retail market in U.S. (Duffy et al., 2001). Although the poultry industry has often be considered primarily responsible for human campylobacteriosis, the pork industry has equally been identified as a potential source of human infection (Allos, 2001; Berndtson et al., 1996). Uaboi-Egbenni (2008) also documented the prevalence of campylobacters in dogs and guinea-fowl in Nigeria. The gastrointestinal tracts of other food animal species have also been shown to be frequently colonized with campylobacters, particularly, C. jejuni and C. coli (Minihan et al., 2004). Reported rates of Campylobacter carriage in food animals have varied widely between studies (Busato et al., 1999). The high prevalence of campylobacters in pigs has been reported previously in numerous studies and dressed pig carcasses have been shown to be more frequently contaminated than either beef or sheep (Nesbakken et al., 2003). This is most likely attributable to the fact that pig carcasses undergo a communal scalding process combined with the fact that the skin remains on the carcass following all of the dressing procedures (Moore et al., 2005). Thermotolerant campylobacters (C. jejuni/C. coli) constitute the most frequent cause of intestinal infections worldwide. The clinical spectrum of Campylobacter enteritis ranges from a watery, nonbloody, noninflammatory diarrhea to a severe inflammatory diarrhoea with abdominal pain and fever. Disease is less severe in developing countries than in developed countries. The disease is characterized by bloody stool, fever, and abdominal pain that is often more severe than that observed in Shigella and Salmonella infections. In developing countries the features reported are watery stool, fever, abdominal pain, vomiting, dehy- dration, asthenia, anorexia and presence of faecal leukocyte; patients are also often underweight and malnourished (Rao et al., 2001; Coker et al., 2002). Stools remain positive for several weeks. Treatment appears to be beneficial if it is administered early enough in the course of the disease (Salazar-Lindo et al., 1986). The recommended drugs are erythromycin, or amoxicillin or a fluoroquinolone (ciprofloxacin, nor- floxacin) or tetracycline, provided the bacterium has not acquired a resistance. However, the emergence of antibiotic resistant strains has further opened a new dimension as to how to combat the disease together with the emerging resistant strains. In 1988, the emergence of fluoroquinolone resistance in Campylobacter strains was first reported in Spain, and since then the emergence of fluoroquinolone resistance has been identified in many countries, including Finland, the Netherlands, England, and Canada (Berndston et al., 1996), but not in South Africa. In Spain, 100% of Campylobacter strains isolated from broilers and pigs were found to be resistant to fluoroquinolones (Berndtson et al., 1996). There is a dearth of information and research on the prevalence, epidemiology and antibiograms of campylo- bacters in pigs in South Africa in spite of the reports in Europe and other parts of the world on pigs as potential sources of infections for humans. This study therefore ascertained the prevalence, antibiotic susceptibility profiles and haemolytic activities of campylobacters isolated from pigs in three farms in the Venda region, Limpopo province of South Africa in order to provide updated information and data on the research subject. MATERIALS AND METHODS Collection of faeces A total of four hundred and fifty (450) freshly voided faeces comprising one hundred and fifty from each of the three farms were collected at random from pigs in sterile 50 ml plastic containers and were adequately kept in coolers packed with crushed ice, stored at 4°C and immediately transported to the laboratory for analysis. The faeces were immediately processed. About 2 gm of the samples was transferred to 6 ml of sterile phosphate-buffered saline and left to emulsify at room temperature for 10 - 20 min to release the bacteria. The suspension and bacteria were used directly for the identification of Campylobacter. The three (3) sampling sites are separated from each other by about 200 km but are located in the same province. The three farms were code named X, Y, and Z for ethical reasons. Isolation and identification of Campylobacter sp. by conventional culture methods Ten microlitres of the faecal suspension was spread on the surface of a charcoal cefoperazone deoxycholate agar plates (CM 739 [Oxoid] with cefoperazone supplement SR 155E). The plates were incubated under microaerophilic conditions employing the Cam- pygen gas generating kit (Oxoid CM025) at 42°C for 48 h. Colonies suspected to be Campylobacter were further purified on blood agar plates (Blood Agar Base No.2 (Oxoid) supplemented with 5% sterile laked horse blood). All the isolates were characterized by their catalase, oxidase reactions, hydrogen sulphide production, indoxyl acetate, hippurate and susceptibility to nalidixic acid by standard procedures (On and Holmes, 1991, 1992). The resulting isolates were subsequently stored at -80°C in brain heart infusion broth with 15% glycerol until further investigation. Confirmation of presumptive positive Campylobacter strains Presumptive identification of Campylobacter isolates was done using the Dryspot Campylobacter test kit (Oxoid Basingstoke, Hampshire England). The test is specific for pathogenic Campylo- bacter strains belonging to C. jejuni, C. coli, Campylobacter upsaliensis, and Campylobacter lari. However, the other Campylobacter species are not included in this identification scheme. The manufacturer’s instructions were strictly adhered to in this procedure. Briefly, 48 h cultures of suspected Campylobacter showing the usual cultural characteristics were selected. The reagents were removed from the refrigerator and allowed to attain room temperature according to the manufacturer’s instructions. The extraction tubes were placed in test tube rack. Thereafter, one drop of extraction reagent 1 was added into the extraction tube. Sufficient growth of the suspected Campylobacter culture was removed to fill the inner diameter of a sterile loop. These cells were thoroughly suspended in a drop of reagent 1. The loop was allowed to stay in this reagent for 3 min. Two drops of extraction reagent 2 was added to the previous mixture and mixed thoroughly using the retained loop. With the aid of a paddle pastette provided with the kit, 1 drop (50 µl) of the neutralized extract was placed onto the test circle and a drop onto the control circle. With the aid of the flat end of the paddle pastette, the extract was mixed into the dry control reagent spot until completely suspended to cover the reaction area. The same pastette was used to repeat this procedure for the test reagent. The test cards were rocked for about 3 min. This process was repeated for all suspected Campylobacter isolates. Agglutination under normal lighting condition indicated that the organism was Campylobacter. The presumptive positive campylobacters were further subjected to Mast diagnostic Campylobacter kits consisting of urease, indoxyl acetate and hippurate test solution. Briefly, 24 h cultures of the Campylobacter were inoculated into the urease and hippurate test solutions according to the manufacturer’s instructions. These were then incubated for 4 h for colour development. For urease, development of pink colour was indicative of urease enzyme production (C. lari), while development of pink colour in hippurate solution indicated production of hippuricase enzyme (C. jejuni). In the case of indoxyl acetate solution, change of colour from colourless to blue/purple was indicative of the presence of C. jejuni or C. coli. The indoxyl acetate strips were impregnated with wet cultures and allowed to stay for 3 - 5 min. Development of purple colour within this period was indicative of positive reaction (C. jejuni and C. coli) (Popovic-Uroic et al., 1990; On and Holmes, 1992; Elviss et al., 2008). Blood haemolysis test To ascertain pathogenicity of isolates, the identified Campylobacter sp. were subjected to haemolytic test according to the procedure of Samie et al. (2007). Briefly, a 24 h broth culture of Campylobacter sp. were cultured by spread plate method on Columbia agar supplemented with defibrinated sheep red blood cells. Plates were incubated at 37 ± 2°C for 24 h. Thereafter, plates were observed for complete, partial and no haemolysis. Antimicrobial agents The antibiotics tested in this study were: Trimethroprim (2.5 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), tetracycline (30 µg), ampicillin (10 µg), erythromycin (15 µg), streptomycin (10 µg), methicillin (µg), cefexime (30 µg), imipenem (µg), kanamycin (30 µg) and vancomycin (30 µg) (Oxoid, Unipath Ltd, Basingstoke, England). Antimicrobial sensitivity testing The method of Gaudreau and Gilbert (1997) was used. Briefly, the confirmed Campylobacter isolates were inoculated into plates of Mueller-Hinton agar supplemented with 5% sheep red blood cells in plates carrying a maximum of five (5) antimicrobial discs. All plates were incubated at 35°C under a microaerophilic atmosphere obtained with a gas generator envelope (Oxoid, Unipath Ltd, Basingstoke, England) for 24 h. The resulting zone diameters were measured with a graduated metre rule. Preparation of bacterial genome Genomic DNA was obtained by the whole-cell lysate method as described by Marshall et al. (1999). Briefly, cells from a 24 - 48 h culture grown on Columbia blood agar were resuspended in sterile distilled water to an optical density of 2.5 at 540 nm. The suspensions were boiled 100°C for 20 min in Eppendorf tube. The resulting templates were either used immediately for polymerase chain reaction (PCR) or were kept at 4°C for up to 1 month. PCR identification of Campylobacter strains In order to ascertain if the dryspot positive Campylobacter isolates were genuinely campylobacters, they were subjected to PCR identification using the general primers for the identification of Campylobacter. These primers are also specific for other members of the campylobacteriaceae (Helicobacter and Arcobacter). However, Arcobacter and Helicobacter sp. show negative reaction to the Campylobacter dryspot kit. Hence, any amplification of the primer sequences at the 1,004-bp fragment within the coding region of 16S rRNA confirmed such isolates as Campylobacter sp. and not Helicobacter or Arcobacter sp. The PCR-restriction fragment length polymorphism (RFLP) method used in this study was as previously described by Marshall et al. (1999). Briefly, amplification was done in 50-µl reaction volume containing 5 µl of whole-cell lysate, 1 µl each primer, 10x buffer (Roche), 1.5 mM MgCl2, 200 µM each deoxynucleotide (Roche) and 5U Taq DNA polymerase (Roche). The PCR amplification was performed with a – thermocycler (ESCO Swift Mini Thermal Cycler Version 1.0, ESCO Technologies, Philadelphia U.SA). The samples were subjected to an initial dena- turation for 2 min at 95°C, followed by 30 amplification cycles, each consisting of 94°C for 30 s, 52°C for 30 s, and 72°C for 90 s. A final primer extension at 72°C for 10 min was included. Oligonucleotides primers employed in this study were CAH16S 1a (5’ – AAT ACA TCA AAG TCG AAC GA – 3’) and CAH16S 1b (TTA ACC CAA CAT CTG ACG AC – 3’), respectively. The oligonucleotides used in this study were synthesized by Roche Applied Science (Manheim, Germany). RESULTS Cultural studies A total of four hundred and fifty (450) freshly voided c a m p y lo b a c te r is o la te s p e r fa rm N u m b e r o f 90 76.9 80 70 60 55.3 50 50 Farm X Farm Y 40 30 Farm Z 30 21 19 20 12.8 10 2 3 5 0 0 0 5.3 7.9 0 0 0 0 C. jejuni C. coli C. lari % C. jejuni % C. coli % C. lari Campylobacters isolated from pig faeces in farms X, Y and Z Fig.1 : Shows the number of C. jejuni , C. coli, C. lari and their percentages isolated from three pig farms in South Africa 100 89.7 90 80 70 60.5 60 57.9 Farm X 42.1 50 38 38 39 39 Farm Y 40 Farm Z 30 23 22 16 26.3 13.2 20 10.3 10 4 5 10 0 0 0 0 0 N o o f C am py lo ba ct er is o la te sp er fa rm N o of -β ha em ol yt ic N o of - αh ae m ol yt ic % - h a e m o ly ti c β % - h a e m o ly t ic α N o no n- ha em ol yt ic % no n- ha em ol yt ic Fig. 2: Results of haemolysis of sheep red blood cells by Campylobacter isolates faecal samples were processed for the detection of campylobacters over the course of the study. Of the 138 presumptive isolates from pigs on mCCDA from the farms, 25.6% (115 out of 450) were confirmed as campylobacters. Figures 1 and 2 show the percentage incidence of Campylobacter strains from pig faeces in the three (3) farms. From, there was an equal percentage of occurrence of campylobacters in farms X and Y with a value of 25.3%. However, there was a higher incidence of campylobacters among pigs in farm Z (26%) than in farms X and Y. It must be noted here that some strains that could not be resuscitated did not form part of the analysis and subsequent studies. With the aid of the Campylobacter dryspot and Mast diagnostic kits, 2(5.3%) of the isolates from farm X were identified as C. jejuni, while 21 (55.3%) were identified as C coli. In farm Y, 3 (7.9%) were C. jejuni, while 19 (50%) were C. coli. In farm Z, 5 (12.3%) were C. jejuni, while 30 (76.9%) were C. coli. Overall, the prevalence of C. jejuni among pigs in the three farms was 10 (12.5%), while it was 70 (87.5%) for C. coli (Figure 2). From this finding, there was a preponderance of C. coli compared with C. jejuni in all P e rc e n ta g e s u s c e p ti b il it y p ro fi le 100% 0 0 31.8 45. 5 50 50 90% 100 100 10 0 100 10 0 10 0 10 0 10 0 80% 68.2 54. 5 50 50 70% 0 4 0 0 0 0 0 0 FARM Z R FARM Z S 60% 40 48 48 64 64 52 40 60 72 FARM Y R 50% 96 10 0 10 0 40% 60 52 52 48 60 40 FARM Y S 36 36 28 FARM X R 0 0 30% 22.2 44. 4 33.3 11.1 11.1 55.6 77.8 55. 6 FARM X S 20% 88.9 10 0 10 0 88. 9 10 0 10 0 77.8 66.7 10% 55. 5 44.4 44. 4 0% 0 22.2 0 0 CIP TE CFM IPM VA AMP CN K MET E W NA Antimidrobials to which C.coli isolates were exposed Fig. 3: Percentage susceptibility profile of Campylobacter coli exposed to 12 antibiotics (Key: S = Susceptibility; R = Resistance; CIP= Ciprofloxacin; TE= Tetracycline; CFM = Cefexime; IPM = Imipenem; VA= Vancomycin; AMP = Ampicillin; CN= Gentamycin; K= Kanamycin; MET= Methicillin; E=Erythromycin; W= Trimethoprim; NA= Nalidixic acid). farms. Blood haemolysis Of the 115 Campylobacter strains from the pigs tested for sheep blood haemolysis, 23 were β-haemolytic, 10 were α-haemolytic and 5 non-haemolytic in farm X, 22 were β- haemolytic, 16 were α-haemolytic for farm Y, 35 were β- haemolytic and 4 were α-haemolytic for farm Z (Figure 3). Antimicrobial resistance Sixty-six isolates were tested for their antibiotic sus- ceptibility profiles. Most Campylobacter isolates from farm X (< 50%) were resistant to ciprofloxacin, cefexime, and erythromycin, while > 50% were resistant to tetra-cycline (54.5%), vancomycin (100%), ampicillin (54.5%), kanamycin (63.6%), and methicillin (100%). In farm Y, ≥ 50% were resistant to ciprofloxacin (57.1%), tetracycline (50%), cefexime (53.6%), vancomycin (67.9%), gentamycin (53.6%), methicillin (75%), and erythromycin (53.6%), except imipenem and kanamycin which were 10.7 and 46.4% resistant, respectively. In farm Z, > 50% but < 100% were resistant to tetracycline, gentamycin and erythromycin, while 40.7% were resistant to ciprofloxacin. Approximately 100% of Campylobacter species in this farm were resistant to cefexime, vancomycin, kanamycin, and methicillin. Resistance to most of the antibiotics tested was more pronounced among isolates from farm Z. Majority of the isolates from farm Y were more resistant to ciprofloxacin (57.1%) than isolates from farm X (36.4%) and farm Z (40.7%) (Figure 4 and 5). There were no significant differences in resistance profiles amongst Campylobacter isolates in the farms (p < 0.005). Study of individual species resistance showed that in farm X, all C. jejuni isolates were resistant to Ciprofloxacin, tetracycline, cefexime, imipenem, vancomycin, gentamycin, kanamycin and methicillin. Of the three C. jejuni isolates from farm Y, ≤ 100% were resistant to all the antimicrobials except tetracycline (33.3%) and nalidixic acid in which all were susceptible. In farm Z, ≤ 100% were resistant to 10 of the antimicrobials except ciprofloxacin to which 80% were resistant and nalidixic acid to which 100% were susceptible. For C. coli, in farm X < 50% were resistant to ciprofloxacin, tetracycline, cefexime, imipenem and erythromycin except vancomycin, ampicillin, gentamycin, kanamycin, methicillin and trimethoprim to which ≤ 100% were resistant. In farm Y, < 50% of C. coli were resistant to ciprofloxacin, tetracycline, cefexime, imipenem and kanamycin, while ≤ 100% were resistant to vancomycin, ampicillin, gentamycin, methicillin, erythromycin and trimethoprim. In farm Z, ≤ 50% were resistant to ciprofloxacin, tetracycline, gentamycin and erythromycin, while all C. coli isolates (100%) were resistant to cefexime, vancomycin, ampicillin, kanamycin, methicillin and trimethoprim. Analysis of the zone diameters was done using CLSI/NCCLS (2002) for enterobacteriaceae. The PCR micrographs of the DNA from Campylobacter strains from sheep are as indicated in. The purified DNA from the Campylobacter strains amplified at the 1004 bp, which is the specific region for the conserved 16S rRNA for members of the genus Campylobacter. The bands formed were confirmed as those for Campylobacter. Specific identification by the Mast diagnostic kits 100% 0 20 90% 80 100 100 100 100 100 100 100 100 100 100 80% 80 FARM Z R 70% 20 0 0 0 0 0 0 0 0 0 FARM Z S 60% 66.7 33.3 66.7 66.7 66.7 FARM Y R 50% 100 100 100 100 100 100 100 66.7 FARM Y S 40% 33.3 33.3 33.3 33.3 0 0 0 0 0 0 0 FARM X R 30% 50 50 20% 100 100 100 100 100 100 100 100 100 100 FARM X S 10% 50 50 0% 0 0 0 0 0 0 0 0 0 0 CIP TE CFM IPM VA AMP CN K MET E W NA Antimicrobials to which Campylobacter jejuni were exposed Fig.4: Percentage susceptibility profile of Campylobacter coli exposed to 12 antibiotics (Key: S = Susceptibility; R = Resistance; CIP= Ciprofloxacin; TE= Tetracycline; CFM = Cefexime; IPM = Imipenem; VA= Vancomycin; AMP = Ampicillin; CN= Gentamycin; K= Kanamycin; MET= Methicillin; E=Erythromycin; W= Trimethoprim; NA= Nalidixic acid). 1004bp 1004bp Fig.5: PCR products of amplified DNA from pig Campylobacter isolates aligning at the 1004bp of a 1.9kb ladder (a) pig and (b). (a) Lane 1= 1.9kb ladder; lane 2,3,4,5,6,7, amplified bands of DNA from pig Campylobacter strains; (b) lane 1= 1.9kb DNA ladder; lanes 2,3,4,5,6,7 are amplified bands of DNA from pig (farm Y) Campylobacter strains. differentiated the isolates into C. jejuni and C. coli. DISCUSSION Elucidating the shedding patterns and prevalence of Campylobacter strains in the faeces of farm animals is a prerequisite for effective healthcare strategy against zoonotic infections. In the current study, the prevalence of campylobacters from pigs and their antimicrobial susceptibility pattern was determined. Of the 450 fresh faecal samples processed for all farms, 84.6% were identified as campylobacters. There was an equal pre- valence (25.3%) among pigs’ faeces in farm X and Y, but a slightly higher predominance in faeces from farm Z. In an epidemiological study carried out on 95 pigs originating from eight farms, Weijtens et al. (1993) found 11% of the stomach samples of pigs to be positive for Campylobacter, whereas, 85% of faeces were positive. A prevalence of campylobacters in pigs ranging from 63 - 100% has been reported in other studies (Weijtens et al., 1993; Saenz et al., 2000; Young et al., 2000; Pezzotti et al., 2003; Payot et al., 2004). In our findings, none of the farms was free from Campylobacter. There were no significant differences in the prevalence of Campylo- bacter sp. in the three farms (X, Y, Z) with the average prevalence value at 25.3, 25.3 and 26% respectively (p < 0.005). Weijtens et al. (1993, 1999) reported high counts of shedding of Campylobacter genotypes in faeces of pigs. In terms of prevalence, C. coli were more predominant than C. jejuni in all farms. Farm Z had a higher incidence of C. jejuni and C. coli than the rest farms. This report is in line with previous reports which revealed that swine were predominantly contaminated by C. coli, whereas, C. jejuni were more frequently isolated from poultry (Aarestrup et al., 1997; Weijens et al., 1999; Van Looveren et al., 2001; Pezzotti et al., 2003). The reason for this dis- crepancy in the distribution pattern of C. jejuni and C. coli in swine has not been elucidated. However, it is not implausible that host adaptation or differences in rearing practices may be significant factors. Quite a large population of Campylobacter isolates from pig faeces where β-haemolytic on 5% sheep redblood cells. This observation is in line with the report of Samie et al. (2007). This is a confirmation that the Campylobacter strains could be pathogens of medical significance. The predominance of C. jejuni in human infections has led to the disregard of the impact that less prevalence species like C. coli can play in human infections. Nevertheless, a recent survey by Tam et al. (2003) showed that the morbidity resulting from C. coli has actually been underestimated. The PCR micrographs of the DNA from Campylobacter strains from all animals were as shown in Figure 1. The purified DNA from the Campylobacter strains amplified at the 1004 bp, which is the specific region for the conserved 16S rRNA for members of the genus Campylobacter. The bands formed are in line with those of Marshall et al. (1999). After exposure to twelve (12) antibiotics, several strains showed multiple resistance to most of the antibiotics used. The resistance patterns displayed by Campylobacter isolates from all farms to fluoroquinolone (ciprofloxacin) and macrolides (erythromycin) classified as second line and first line antimicrobials are of particular importance, since patients suffering from campylobacteriosis are usually treated with these antimicrobials agents. In the study, 22.2, 40 and 31.1% of C. coli were resistant to ciprofloxacin, while 11.1, 60 and 50% of C. coli were resistant to erythromycin in farm X, Y and Z, respectively. For ampicillin, 55.6, 40 and 100% of C. coli were resistant to this antimicrobial in farm X, Y and Z, respectively. Norma et al. (2007) in their study in Canada observed a high incidence of C. coli resistant to ciprofloxacin and erythromycin. The resistance of C. coli to ampicillin as exemplified in this study was higher than those reported from France and Denmark (20 and 17%), respectively but lower than that reported from Spain (65.9%) for farm X, Y but not for farm Z (100%). Resistance to β-lactam antimicrobials in pathogenic bacteria develops through bacterial conjugation. This resistance-transfer mechanism is very crucial because it permits genetic exchange of information between species of bacteria (Davis and Conner, 1994; EFSA, 2007; Norma et al., 2007). In South Africa, data on anti-microbial susceptibility of Campylobacter isolated from pigs are scanty. Due to high prevalence of resistance shown by Campylobacter sp. to β-lactam antimicrobials (ampicillin) it is no longer recommended for use in farms (Davis and Conner, 1994). In this study, we observed resistance frequently to antimicrobials most commonly used in swine industry, tetracycline and erythromycin. Different resistance patterns were observed for one aminoglycoside antibiotic for all isolates tested for C. coli. 77.8, 52 and 50% were resistant to gentamycin in farms X, Y and Z, respectively. However, the report of Norma et al. (2007) in Canada showed a low level of resistance (0.2%) of Campylo- bacter coli to gentamycin. The recommendation of gentamycin as an alternative therapeutic antimicrobial against human campylobacteriosis by Fernandez et al. (1994) in Chile should be considered with caution judging from the high prevalence of resistance to gentamycin observed in this study. Sato et al. (2004) and Inglis et al. (2005) observed high levels of resistance to tetracycline (45%), which is in line with our observation of ≥ 50%. Saenz et al. (2000) in their study reported no erythro- mycin resistance among C. jejuni isolates from broilers and high levels of resistance C. coli isolates from pigs (81.1%) compared to those from humans (34.5%). However, in our study, one C. jejuni isolate out of two was resistant to erythromycin in farm X; all three C. jejuni isolates in farm Y were resistant to erythromycin, while one of the five C. jejuni isolate in farm Z was resistant to erythromycin. In addition, more resistance was associated with C. coli than with C. jejuni. A study in Quebec found that 61% of C. coli isolated from pigs but none of C. coli isolates from chickens were resistant to erythromycin (Guevrement et al., 2006). Resistance of Campylobacter species to antibiotics may suggest the widespread use of antibiotics as growth promoters in farm animals (Piddock et al., 2003; Pezzotti et al., 2003; Moore et al., 2001). Resistance profiles to tetracycline and fluoroquinolones were very high in our study and this is consistent with other reports (Saenz et al., 2000; Norma et al., 2007). This may have stemmed from the fact that these antibiotics have been used in the swine industry for several years. All the Campylobacter isolates were susceptible to nalidixic acid in this study, so that cross-resistance between nalidixic acid and ciprofloxacin was not found in all the quinolone susceptible Campylobacter strains. This finding is however in contrast to the observation of Saenz et al. (2000) who found cross-resistance between ciprofloxacin and nalidixic acid resistant strains in Spain. Moore et al. (2005) mentioned the use of mobile genetic elements in campylobacters as mechanism for the extrusion of antibiotics out of the bacterial cell. Considering the feeding habits of pigs, it is plausible that they can ingest commensals carrying resistant genes from the feeds, facilitating transfer to pathogenic bacteria in the lumen of farm animals. Such pathogenic acquisition of resistant genes from exogenous source can result in global prevalence of resistant pathogens. Angulo et al. (2004) and Blake et al. (2003) made similar arguments regarding acquisition of exogenous genes by lumen pathogens in farm animals. Surveillance and monitoring of antimicrobial use must be done to ensure the controlled use of antibiotics. The use of antibiotics as growth promoters and prophylaxis for animals should be carefully evaluated and monitored because acquisition of antibiotic resistant strains of campylobacters by man through ingestion of animal’s food have serious health implications. . 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