MATERIALS AND METHODS

Studies on the antibiotic resistance of aerobic mesophilic bacteria isolated from poultry faeces obtained from Obafemi Awolowo University Teaching and Research farms, lle-Ife, Nigeria was carried out in Microbiology Laboratory, Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria between 1st February and 27th August, 2010.

Samples: Fifteen samples each of fresh poultry faecal droppings from cockerels and layers were obtained between the hours of 7 and 8 a.m. over a period of five weeks (1st February to 8th March, 2010) from the poultry unit of the Obafemi Awolowo University Teaching and Research Farms, Ile-Ife, Nigeria. The samples were collected aseptically in sterile McCartney bottles and transported to the laboratory within 30 min for analysis.

Isolation and Identification of bacteria strains: Ten grams of faeces sample were homogenized with 90 mL of maximum recovery diluent (MRD, Oxoid) to obtain a 1:10 dilution. Successive decimal dilutions were carried out with sterile MRD. Aliquots (1000 μL) of appropriately diluted sample homogenates were pour-plated in duplicate using nutrient agar. The agar plates were allowed to set and incubated at 37°C for up to 48 h. The colony forming units of the bacteria on the plates were enumerated and representatives of the different colonies were selected according to their morphological characteristics and purified by successive sub culturing on nutrient agar and identified phenotipically based on standard methods (Harrigan and McCance, 1976; Buchannan and Gibbons, 1985).

Testing for resistance to antibiotics: The bacterial isolates were tested for resistance to 14 antibiotics produced by FONDISC (Fondoz Laboratories Ltd., Nigeria). These were: augmentin (30 μg), ceftriazone (30 μg), nitrofurantoin (200 μg), gentamycin (10 μg), cotrimoxazole (25 μg), ofloxacin (5 μg), amoxicillin (25 μg), ciprofloxacin (10 μg), tetracycline (30 μg), pefloxacin (5 μg), ofloxacin (5 μg), streptomycin (10 μg), chloramphenicol (30 μg) and erythromycin (5 μg). This testing was performed using the standard disc diffusion method (Clinical Laboratory and Standards Institute, 2006). The antibiotics susceptibility pattern of the isolates was interpreted using Progressive Diagnostics Manufacturers (PDM) Interpretative Chart.

Multiple antibiotics resistance indexing of isolates: The Multiple Antibiotic Resistance (MAR) index is defined as a/b where ‘a’ represents the number of antibiotics to which the particular isolate is resistant and ‘b’ the number of antibiotics to which the isolate is exposed (Krumperman, 1983). MAR index values higher than 0.2 are considered to have originated from high-risk sources where antibiotics are often used. MAR index values of less than or equal to 0.2 indicates a strain originated from sources where antibiotics are seldom or never used.

Statistical analysis: A two-way clustered analysis of multi-variance was used to estimate overall similarities of the bacterial resistance using their zones of inhibition. Correlation method of the similarity measure was used on the Paleontological statistics software package for education and data analysis (Hammer et al., 2001).

RESULTS

The total aerobic mesophilic counts ranged from 7.16±0.10 to 7.67±0.05 log cfu g^-1 and 6.14±0.09 to 8.64±0.04 cfu g^-1 in layers and cockerel faeces, respectively (Table 1). A total of 15 strains of bacterial belonging to the genera Alcaligenes, Corynebacterium, Staphylococcus, Bacillus, Proteus, Pseudomonas, Escherichia, Acinetobacter and Aeromonas were isolated and characterized phenotipically. The occurrence pattern of the isolates in the faeces samples is shown in Table 1.

The antibiotic susceptibility pattern and Multiple Antibiotics Resistance (MAR) index of both Gram-positive and Gram-negative bacteria isolates from poultry feces are shown in Table 2 and 3, respectively. The MAR index ranged from 0.6 to 1.0 and 0.1 to 1.0 for gram positive and gram negative bacteria isolates respectively. All the bacterial isolates showed high level MAR index (>0.2) except a strain of Aeromonas sp. which had a value of 0.1 (Table 3). The percentage antibiotic susceptibility pattern of the Gram positive bacteria isolates showed 100% resistance to streptomycin, chloramphenicol, ceftriazone, gentamycin and erythromycin; 80% resistance to amoxicillin and cotrimoxazole and 40% resistance to ofloxacin, pefloxacin and ciprofloxacin (Table 4).

Table 1:	Total aerobic mesophilic bacteria count and their occurrence in poultry faeces
Values represent the mean of three determinations±standard deviation, Isolate codes are in parenthesis

Table 2:	Antibiotic susceptibility pattern of gram positive bacteria isolated from poultry faeces
R = Resistant, S = Susceptible, I = Intermediate, MAR INDEX = Multiple Antibiotic Resistance Index; *Isolate code in parenthesis

Table 3:	Antibiotic susceptibility pattern of gram negative bacteria isolated from poultry faeces
R = Resistant; S = Susceptible; I = Intermediate; *Isolate code in parenthesis

Table 4:	Percentage antibiotics susceptibility pattern of gram positive bacteria isolated from poultry faeces
AMX = AMOXYCILLIN (25 μg), OFL = OFLOXACIN (5 μg), STR = STREPTOMYCIN (10 μg) CHL = CHLORAMPHENICOL (30 μg), CEF = CEFTRIAZONE (30 μg), GEN = GENTAMYCIN (10 μg) PEF = PEFLOXACIN (5 μg), COT = COTRIMOXAZOLE (25 μg), CPX = CIPROFLOXACIN (10 μg), ERY = ERYTHROMYCIN (5 μg)

Table 5:	Percentage antibiotics susceptibility pattern of gram negative bacteria isolated from poultry faeces
AUG = AUGMENTIN (30 μg), CRO = CEFTRIAZONE (30 μg), NIT = NITROFURANTOIN (200 μg) GEN = GENTAMYCIN (10 μg), COT = COTRIMOXAZOLE (25 μg), OFL = OFLOXACIN (5 μg) AMX = AMOXICILLIN (25 μg), CPX = CIPROFLOXACIN (10 μg), TET = TETRACYCLINE (30 μg), PFX = PEFLOXACIN (5 μg)

While the Gram negative bacterial isolates showed above 70% resistance to augmentin, nitrofurantoin, ceftriazone, gentamycin cotrimoxazole and amoxicillin; above 60% resistance to tetracycline; below 30% resistance to ciprofloxacin and below 20% resistance to ofloxacin and pefloxacin (Table 5). The two-way clustered analysis of the Gram positive bacteria isolates showed that the antibiotics susceptibility pattern of Staphylococcus sp. (4) and Bacillus alvei (6) were the most related followed by Corynebacterium kutsceri (2) and Corynebacterium ulcerans (5) (Fig. 1). The two-way clustered analysis of the Gram negative bacteria isolates deduced that the antibiotic sensitivity pattern of Acinetobacter mallei (6) and Pseudomonas fluorescens (11) were the most related (Fig. 2).

Fig. 1:	Two-way clustered analysis of the gram positive bacteria isolated from poultry faeces. B = Amoxicillin; E = Chloramphenicol; K = Erythromycin; D = Streptomycin; G = Gentamycin; I = Cotrimoxazole; H = Pefloxacin; J = Ciprofloxacin; C = Ofloxacin; F = Ceftriazone; 2 = Corynebacterium kutsceri ; 3 = Staphylococcus aureus; 4 = Staphylococcus sp.; 5 = Corynebacterium ulcerans; 6= Bacillus alvei

Fig. 2:

Two-way clustered analysis of the gram negative bacteria isolated from poultry faeces. B = Augmentin; C = Ceftriazone; K = Pefloxacin; D = Nitrofurantoin; G= Ofloxacin; I = Ciprofloxacin; H = Amoxycillin; J = Tetracycline; E = Gentamycin; F = Cotrimoxazole 2 = Escherichia coli; 3 = Acaligenes faecalis; 4 = Proteus morganii; 5 = Salmonella arizonae; 6 = Acinetobacter mallei; 7 = Acinetobacter mallei; 8 = Acaligenes faecalis; 9 = Acaligenes faecalis; 10 = Aeromonas sp.; 11 = Pseudomonas fluorescens; 12 = Aeromonass sp.

DISCUSSION

The cockerel faeces in this study contained slightly higher bacterial population than those of layer faeces, This seems to correspond with the reports of Adegunloye (2006). The average bacterial load (8.15 log cfu g^-1) of cockerel faeces in this study is slightly higher than values previously reported for poultry faeces in Nigeria (Adegunloye, 2006). This might be attributed to constant contact between feed, poultry fowl and faecal droppings (Vellinga and Van Loock, 2002). The bacteria isolated from the faecal samples were identified as Alcaligenes faecalis, Corynebacterium kutsceri, Staphylococcus aureus, Bacillus alvei, Proteus morganii, Corynebacterium ulcerans, Salmonella arizonae, Acinetobacter mallei, Staphylococcus sp. Escherichia coli, Aeromonas sp. and Pseudomonas fluorescens. Presant finding on the bacterial composition of poultry faeces is different from those of Adegunloye (2006) who only reported Staphylococcus aureus, Staph. epidermidis, Bacillus cereus and E. coli. Our identification was however based on phenotypic characters, authentic identity of species, should be confirmed by molecular identification. Many of the isolated bacteria are normal flora of intestinal tract of poultry (Esposito and Leone, 2007) while a few have been implicated in poultry diseases (Islam et al., 2003; Simon, 2005). The distribution of bacterial isolates between the layers and cockerel faecal samples was similar in this study. The observation of more Gram negative bacteria (11 strains) than Gram-positive bacteria (5 strains) in this study is in agreement with the findings of Chopra and Roberts (2001).

In this study, all the bacteria isolates showed high level of antibiotics resistance except a strain of Aeromonas sp. This result is in agreement with Cloud et al. (1985) and Muhammad et al. (2010) who reported that the abuse and misuse of antimicrobial agents for growth promotion and prevention of diseases has impressed a selective pressure that causes discovery of more resistance bacteria. This is true with the bacteria associated with poultry faeces in this study. Hence, the antibiotic selection pressure for resistance by bacteria in poultry is high and as a result their fecal flora contains high proportion of resistant bacteria. Salehi and Bonab (2006) reported that the resistance of bacteria to existing antimicrobial agents is widespread and of great concern to poultry veterinarians. The use of antimicrobial in animal feed can also lead to selection of antimicrobial resistant zoonotic enteric pathogens, which can be transferred to human through the consumption of contaminated food, or by direct animal contact.

According to Nandi et al. (2004) Gram-positive bacteria especially Corynebacterium sp. that has been found to be associated with poultry litter serves as a major reservoir of class 1 integrons (in-1). Corynebacterium kutsceri and Corynebacterium ulcerans showed 100% resistance to all the antibiotics used in this study. C. ulcerans is a veterinary pathogen, which has been implicated in pharyngeal infection mimicking classical diphtheria in humans (Gubler et al., 1990). Alcaligenes are apparently saprophytic inhabitants of the intestinal tract of vertebrates, which are involved in decomposition and mineralization processes of poultry products (Holt, 1981). Three strains of Alcaligenes faecalis (C9, C2, 1D4) showing resistance of 100, 70 and 40%, respectively to the antibiotics used were encountered in this study. The percentage of antibiotics susceptibility in Salmonella sp. isolated in this study corresponds with reports on Salmonella strains isolated from broiler flocks in Canada and India (Suresh et al., 2000).

All the isolates except Aeromonas sp. (2D2) showed MAR index > 0.2, inferring that they have arisen from high-risk sources of contamination where antibiotics are often used. This is an indication of a high presence of antibiotics selective pressure, which agrees with the report of Suresh et al. (2000). The MAR pattern of E. coli and P. fluorescens showed the same multiple antibiotic resistance pattern hence they have the identical MAR index (0.7), this is suggestive that both strains have a common origin (Kasper et al., 1990). Similar pattern of MAR was also observed for the Corynebacterium species. The development of multiple antibiotics resistance may be as a result of transfer of R factor (borne on plasmids) and E. coli are noted to carry multiple plasmids which can carry any number of multiple resistant genes (Sumathi et al., 2008).

The two-way clustered analysis of the Gram positive bacterial isolates showed that the antibiotics susceptibility pattern of Staphylococcus sp. and Bacillus alvei are the most related followed by Corynebacterium kutsceri and Corynebacterium ulcerans. This establishes the observed MAR index (1.0) for Corynebacterium sp. in this study and it is in agreement with previous report by Kasper et al. (1990). The relatedness of Proteus morganii and Acinetobacter mallei to Salmonella arizonae and Acaligenes faecalis is close but distantly followed by their relatedness to that of Staphylococcus aureus. Similar relatedness is observed in the resistance pattern of Erythromycin to Streptomycin and Ciprofloxacin to Ofloxacin. This is also used to show the relatedness in Gram negative bacterial isolates. From the analysis it can be deduced that two-way clustered analysis is a method that can be used as a taxonomic tool.

CONCLUSION

The results obtained in this study revealed that poultry (cockerel and layer) faeces contain bacteria with multiple antibiotic resistance patterns suggestive of possible horizontal gene transfer among non related bacterial isolates. The multiple antibiotic resistance index of the bacteria isolates suggest that they have arisen from sources of high level of antibiotics selective pressure resulting from non-specific, misuse or abuse of antibiotics. The cross infection of these bacteria in humans or as secondary pathogen might be of serious health concern. Therefore, there is need for a national policy which will take into cognizance the rational use of antibiotics and standard antibiotics test before veterinary antibiotics therapy is administered in poultry industry.