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Pak. J. Agri., Agril. Engg., Vet. Sci., 2010 Pak. J. Agri., Agril. Engg., Vet. Sci., 2010, 26 (2): 80-86 IN VITRO ANTIMICROBIAL SUSCEPTIBILITY OF
PASTEURELLA MUTOCIDA
D. H. Kalhoro1, A. B. Kalhoro, R. Rind1, S. Kalhoro2 and H. Baloch1 1Department of Veterinary Microbiology, Sindh Agriculture University, Tandojam, 2Department of Animal Products Technology, Sindh Agriculture University, ABSTRACT
Antimicrobial sensitivity of Pasteurella multocida was determined. Different
media, Muller Hinton agar, Nutrient agar and Brain Heart Infusion agar were
used to study the suitable medium for the antibiotic sensitivity. All media were
observed equal y good for the use with various representative antibiotics. Of
these seven from quinolones group, four from cephalosporins group, four from
pencillin group, three from aminoglycoside group, one from tetracycline group,
and eight from miscellaneous group were tested. Pasteurella multocida was
sensitive to enrofloxacin, oxytetracycline, chloramphinicol, ampicillin and their
zones of sensitivity against the Pasteurella multocida was recorded as 22, 22,
21 and 20 mm in diameter, respectively.
Keywords: Antimicrobial susceptibility, Enrofloxacin, Pasteurella multocida.
INTRODUCTION
Pasteurella multocida is a non-motile, gram-negative, coccobacillus that is found in the nasopharynx and gastrointestinal tract of many wild and domesticated animals. It is divided into various serotypes on the basis of nomenclature. The serotypes are cause of pasteurellosis in cattle, buffalo, sheep, goat, wild animals and fowl cholera in poultry. The causative agent plays a role in the respiratory disease complex of cattle (Rimler and Glisson, 1997). Pasteurella multocida toxin acts intracellularly and inhibits differentiation, particular in bone cells, where it prevents the formation of mineralized bone nodules in-vitro. The toxin is the causative agent of a porcine disease that is characterized by bone resorption. Injection of very low doses of toxin leads to proliferative effects, but at higher dose it is lethal (Lax and Grigoriadis, 2001). Haemorrhagic septicaemia is a highly contagious fatal disease of cattle and buffaloes with great economic importance in tropics and sub-tropics, including Corresponding author: drdildarkalhoro@gmail.com Pak. J. Agri., Agril. Engg., Vet. Sci., 2010 Pakistan. It is an acute inflammatory disease of cattle, but buffaloes are more susceptible. In the absence of proper treatment and preventive measures, it gives classical losses to farmers as it has high morbidity and mortality in bovines. Most cases are acute resulting in death within 8 to 24 hours after onset. Animal first evinces dullness, reluctant to move, fever, salivation, and serous nasal discharge. Oedematous swelling is frequently seen in the throat region and spreading to the parotid region, neck and brisket and animal dies within hours due to respiratory distress. Because of rapid course (8 to 24 hours) of disease it is important to use antimicrobial agents which are very effective. Various sulphonamides, tetracycline, penicillin and chloramphenicol are effective if administered early (Aiello, 1998). Berge et al. (2006) studied the antimicrobial susceptibility of isolates of Pasteurella multocida (n = 28) and Mannheimia haemolytica (39) to amoxicillin-clavulanic acid, ceftiofur, ciprofloxacin, florfenicol, and tetracycline and minimum inhibitory concentrations of florfenicol. All isolates were susceptible to amoxicillin-clavulanic acid, ceftiofur, ciprofloxacin, and florfenicol. Only 5% (4/77) of isolates were resistant to tetracycline. Stefan et al. (2004) investigated 154 Pasteurella multocida and 148 Mannheimia haemolytica strains from respiratory tract infections in cattle for their MICs of spectinomycin and other antimicrobial agents. A total of 93.5% of the Pasteurella multocida and 98.6% of the Mannheimia haemolytica strains were susceptible to spectinomycin. The proposed study was designed to determined susceptibility of Pasteurella multocida to various antibiotics.
MATERIALS AND METHODS
Pasteurella multocida type B-6, the causative agent of hemorrhagic septicemia
was obtained from Veterinary Research Institute (VRI) Lahore to investigate
antimicrobial susceptibility. The glassware like test tubes, flasks, peteri dishes
were placed in sterilizing oven at 180oC for 2 hours. Instruments such as
inoculating loops, scissors and forceps were sterilized by flaming with the help of
gas burner. Furthermore, media and reagents were also sterilized in an
autoclave at temperature of 121oC with 15lb pressure for 15 minutes. Different
media, Muller Hinton agar, Nutrient agar and Brain Heart Infusion agar were
used to study the suitable medium for the antibiotic sensitivity.
Pak. J. Agri., Agril. Engg., Vet. Sci., 2010 Drug susceptibility was adopted as proposed by Bauer et al. (1966). The antibiotics used were from five groups, Quinolones, Cephalosporins, Pencillins, Amino glycosides, Tetracycline and Miscellaneous group. Table-1. Influence of media on zone of inhibition (diameter in mm) of various antibiotics used against Pasteurella multocida. Zone of inhibition (mm)
Antibiotics used
MHA BHI NA
A. Quinolones
B. Cephalosporins
C. Pencillins
D. Aminoglycosides
E. Tetracycline
F. Miscellaneous
MHA = Muller Hinton Agar BHI = Brain Heart Infusion Agar NA = Nutrient Agar Pak. J. Agri., Agril. Engg., Vet. Sci., 2010 Table 2. The mean sensitivity of Pasteurella multocida type 1(B6) against various Antibiotics used
inhibition (mm)
Type 1(B6)
A. Quinolones
B. Cephalosporins
C. Pencillins
D. Aminoglycosides
E. Tetracycline
F. Miscellaneous
Sulphamethoxazole/trimethoprim SXT25 12 54.54% Pak. J. Agri., Agril. Engg., Vet. Sci., 2010 Drug susceptibility method

Three to five colonies of Pasteurella multocida were selected and picked-up by sterilized wire loop and inoculated into 5ml of normal saline. Sterile cotton swab was dipped into the suspension and was rotated several times with a firm pressure on the inside wall of the tube and swab was streaked-over the entire agar surface in three different directions to ensure an even distribution of the inoculum. After drying the plates for 3-5 minutes, the antibiotics disks were placed and gently pressed to ensure complete contact with the agar surface. Plates were inverted and were incubated 37 oC for 24 hours. After 24 hours of incubation, plates were examined and diameters of the zones of inhibition was measured. The disks not inhibiting growth were recorded as resistant while those inhibited growth were recorded as sensitive. (Bauer, 1966).

Plate 1: Shows antimicrobial sensitivity zones on different media
a.
RESULTS AND DISCUSSION
During present study Pasteurella multocida was observed highly sensitive to enrofloxacin, oxytetracycline, chlorampheniol, ampicillin, nalidixic acid cefuroxime sodium, ciprofloxacin and oflaxacin. Results of in-vitro susceptibility of Pak. J. Agri., Agril. Engg., Vet. Sci., 2010 Pasteurella multocida type 1(B6) against various antibiotics are summarized in
Tables 1 and 2, Plate 1. Diker et al. (1994) investigated the in-vitro susceptibility
of Pasteurella multocida and found sensitive to chloramphenicol and resistance
to lincomycin. Harmansdorfe et al. (1998) who tested antimicrobial agents
against Pasteurella multocida and found it highly sensitive to enrofloxacin,
chloramphenicol, ampicillin, penicillin G, and cephalothin, by agar diffusion and
recorded them as most effective drugs against strains of Pasteurella multocida.
Mortensen et al. (1998) performed in-vitro activity of Pasteurella multocida and
found cefuroxime, ampicillin and amoxicillin as effective drugs against
Pasteurella multocida. Hanan et al. (2000) also tested antibiotics against
Pasteurella multocida and observed ciprofloxacin to be highly effective drug.
Shivachandra et al. (2004) tested twenty antibiotics against Pasteurella multocid
and found highly susceptible to enrofloxacin, chloramphenicol, lincomycin and
doxycycline. It is very clear from the present study that the findings recorded
about the antimicrobial sensitivity of the Pasteurella multocida are in accordance
to the results demonstrated by above workers in their studies.

CONCLUSION
On the basis of the present study, the author conclude that Pasteurella multocida
was observed highly sensitive to enrofloxacin, oxytetracycline, chloramphinicol,
ampicillin and their zone of sensitivity was recorded as 22, 22, 21 and 20 mm in
diameter, respectively.

REFERENCES

Aiello, S. E. 1998. The Merck Veterinary Manual. 8th ed., Merck and Co. Inc., N.J., USA. pp 537-539 Berge, A. C., W. M. Sischo and A. L. Craiqmill. 2006. Antimicrobial susceptibility patterns of respiratory tract pathogens from sheep and goats. J. Am.Vet. Med. Assoc., 229 (8): 1279-1288. Bauer, A. W., M. M. Kirby, J. C. Sherris and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45: 493-496. Diker, K. S., M. Akan and R. Haziroglu. 1994. Antimicrobial susceptibility of Pasteurella haemolytica and Pasteurella multocida isolated from pneumonic ovine lungs. Vet Rec., 134 (23): 597-598. Hanan, M. S., E. M. Riad and N. A. El-khouly. 2000. Antibacterial efficacy and pharmacokinetic studies of ciprofloxacin on Pasteurella multocida infected rabbits. Dtsh.Tierarztl. Wochenschr, 107 (4): 151-155. Pak. J. Agri., Agril. Engg., Vet. Sci., 2010 Harmansssdorfer, S and J. Bauer. 1998. Resistance of bovine and porcine Pasteurella to florfenicol and other antibiotics. Berl Munch, Tierarztl. Wochenser, 111 (11-12): 422-426. Lax, A. J. and A. E., Grigoriadis. 2001. Pasteurella multocida toxin: the
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Mortensen, J. E., O. Giger and G. L. Rodgers. 1998. In-vitro activity of oral antimicrobial agents against clinical isolates of Pasteurella multocida. Diagn. Microbiol. Infect. Dis. 30 (2): 99-102. Rimler, R. B. and J. R. Glisson. 1997. Fowl cholera in Diseases of Poultry, 10th edn. eds Calneck, B.W., Barnes, H.J., Beard, C.W., McDougald, L.R. and Saif, Y. M. pp.143-159. Iowa: Iowa State University, Press. Shivachandra, S. B., A. A. Kumar, A. Biswas, A. Ramakrishnan, V. P. Singh and S. K. Srivastava. 2004. Antibiotic sensitivity patterns among Indian strains of avian Pasteurella multocida. Trop. Anim. Health. Prod. 36 (8): 743-50. Stefan, S., K. Corinna, A. S. Salmon and L. J. Watts. 2004. In vitro activities of spectinomycin and comparator agents against Pasteurella multocida and Mannheimia haemolytica from respiratory tract infections of cattle. J. Antimicrob. Chemother, 53: 379-382. (Received 09 December, 2010; Revised 03 August, 2011)

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