Antibiograms and plasmid profiles of Pasteurella multocida isolates from cattle in North Central Nigeria

The Pasteurellaceae family comprises the genera Actinobacillus, Mannhemia, Bibersteinia, Histophilus and Pasteurella. It is a group of Gram negative, non-motile organisms that includes many pathogenic species for birds, mammals and humans (10). Pasteurella multocida is an economically important bacterial pathogen of domestic animals. Hemorrhagic septicemia is an acute septicemic pasteurellosis, caused by Pasteurella multocida serotypes B and E, which mainly affects cattle and water buffaloes. The disease occurs in many parts of the world, predominantly in the tropics (4). Plasmid profile analysis is a useful tool in epidemiological studies (12); it has been used in several studies to learn more about the pathogenicity and virulence mechanisms of P. multocida (11). Sequence comparisons show that the antibiotic resistance genes found in plasmids exhibit a high degree of sequence homology to the corresponding genes found in a great variety of gram-negative bacteria (9). Mechanisms of antibiotic resistance in bacteria are varied and include target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic accumulation. Acquisition of genes needed to expand the various mechanisms is greatly aided by a variety of promiscuous gene transfer systems, such as bacterial conjugative plasmids, transposable elements and integron systems that facilitate genes from one deoxyribonucleic acid (DNA) system to another, and from one bacterial cell to another, not necessarily one related to the gene donor (3). Over a period of time P. multocida changes its antibiogram and develops resistance to chemotherapeutic agents in use. Therefore, for chemotherapy to be effective, drugs to which the organism has been found sensitive need to be administered during the early phase of the disease (8). This study was conducted to examine the antibiograms and plasmid profiles of P. multocida isolated from cattle in North Central Nigeria.


■ INTRODUCTION
The Pasteurellaceae family comprises the genera Actinobacillus, Mannhemia, Bibersteinia, Histophilus and Pasteurella.It is a group of Gram negative, non-motile organisms that includes many pathogenic species for birds, mammals and humans (10).Pasteurella multocida is an economically important bacterial pathogen of domestic animals.Hemorrhagic septicemia is an acute septicemic pasteurellosis, caused by Pasteurella multocida serotypes B and E, which mainly affects cattle and water buffaloes.The disease occurs in many parts of the world, predominantly in the tropics (4).Plasmid profile analysis is a useful tool in epidemiological studies (12); it has been used in several studies to learn more about the pathogenicity and virulence mechanisms of P. multocida (11).Sequence comparisons show that the antibiotic resistance genes found in plasmids exhibit a high degree of sequence homology to the corresponding genes found in a great variety of gram-negative bacteria (9).Mechanisms of antibiotic resistance in bacteria are varied and include target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic accumulation.Acquisition of genes needed to expand the various mechanisms is greatly aided by a variety of promiscuous gene transfer systems, such as bacterial conjugative plasmids, transposable elements and integron systems that facilitate genes from one deoxyribonucleic acid (DNA) system to another, and from one bacterial cell to another, not necessarily one related to the gene donor (3).Over a period of time P. multocida changes its antibiogram and develops resistance to chemotherapeutic agents in use.Therefore, for chemotherapy to be effective, drugs to which the organism has been found sensitive need to be administered during the early phase of the disease (8).This study was conducted to examine the antibiograms and plasmid profiles of P. multocida isolated from cattle in North Central Nigeria.

■ MATERIALS AND METHODS
Pasteurella multocida isolates were obtained from the bank of bacteriology division, National Veterinary Research Institute, Vom, Nigeria and had been isolated from the lungs, liver and spleen of zebu cattle that were asymptomatic carriers after necropsy in North Central Nigeria in 2012.

Summary
Eighteen strains of Pasteurella multocida isolated from zebu cattle at the Veterinary Research Laboratory, National Veterinary Research Institute, Vom, in 2012, were screened for antimicrobial susceptibility and presence of plasmids.Among the 18 isolates studied, 13 (72%) were susceptible to sulfamethoxazole/trimethoprim, 8 (44%) to gentamicin, 8 to amoxicillin / clavulanic acid, 7 (39%) to ciprofloxacin, 7 to chloramphenicol, 5 (28%) to ampicillin, 1 (5.6%) to oxacillin, and 1 to vancomycin.All the isolates were resistant to tetracycline and erythromycin.They all harbored a 5 kb plasmid.Three isolates of serotype E had an additional 3 kb plasmid and one had an additional 6 kb plasmid, but none carried all three plasmids.

Plasmid DNA detection
Isolates were grown overnight at 37°C in 3 ml BHI broth for plasmid isolation.Plasmid DNA was extracted using Plasmid DNA extraction Kit (Thermo Fishers, USA) according to the manufacturer's instructions.The presence of plasmid DNA was detected by agarose gel electrophoresis.

Agarose gel electrophoresis
Plasmid DNA was resolved by electrophoresis in submerged horizontal agarose slab gel (0.9%) in trisborate buffer.The required amount of agarose (Sigma, Aldrich, UK) was dissolved and the DNA ethidium bromide complex gel was visualized on a 320 nm UV transilluminator.

■ RESULTS
The results on the antimicrobial susceptibility of the eighteen isolates of P. multocida presented in Table I show a high level of resistance and multiresistance.In particular all isolates were resistant to tetracycline and erythromycin.However, the lowest resistance was observed to sulfamethoxazole/trimethoprim (5/18) and streptomycin (7/18).
The eighteen isolates of P. multocida were also examined for the presence of plasmid DNA.All isolates indicated the presence of one or more plasmids.They all harbored a plasmid of about 5 kb molecular weight (Table II).Three isolates had an additional 3 kb plasmid and one had an additional 6 kb plasmid (Figure 1).

■ DISCUSSION
In clinical management of the disease, the antibiotic sensitivity assay serves as a guide to choose the correct antibiotic to be used in the field (5).Bacterial organisms over a period of time change their antibiogram patterns and develop resistance against commonly used chemotherapeutic agents.Amoxicillin is an aminopenicillin-type broad-spectrum antibiotic that acts against many Gram-positive and Gram-negative aerobic and anaerobic bacteria.Amoxicillin is a bactericidal, cell-wall active agent that inhibits bacterial cell-wall synthesis by binding to penicillin-binding proteins and inhibits cross-linking of bacterial peptidoglycan.Clavulanic acid is a beta-lactam antibiotic, structurally related to penicillin.It possesses the ability to inactivate a wide range of beta-lactamase enzymes commonly found in microorganisms resistant to penicillin and cephalosporin.In this study, the number of strains resistant to amoxicillin / clavulanic acid (10, i.e. 55.5%) was lower than that resistant to penicillin (17, i.e. 94.4%).

■ CONCLUSION
All isolates harbored a similar plasmid of 5 kb.Three isolates from serotype E had an additional 3 kb plasmid and one isolate had an additional 6 kb plasmid, but none of the isolates carried all three plasmids.All the isolates were resistant to tetracycline and erythromycin.However, the lower resistance of the isolates to sulfamethoxazole/trimethoprim and streptomycin may suggest that they are more effective and may be used for the treatment of P. multocida, the agent involved in hemorrhagic septicemia.In Nigeria the use of antimicrobial drugs in animals is not regulated.Therefore farmers can purchase them without prescriptions.The indiscriminate use of antimicrobials by farmers may be responsible for the high levels of multiple resistances.There is a need to educate cattle owners on the dangers of indiscriminate use of drugs.