2010, Vol. 5 No. 1, Article 52

Methicilline Resistant Status of Staphylococcus Species
Isolated from Mastitic Bovines

Muhammad Imran, Sajjad Ur Rahman, Muhammad Abubakar*¹, Murtaz ul Hassan²,
Aaysha Riaz² and Manzoor Hussain²

Department of Veterinary Microbiology, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad.
¹National Veterinary Laboratory, Park Road, Islamabad.
²Faculty of Veterinary Sciences, PMAS Arid Agriculture University, Rawalpindi.

*Corresponding Author; e-mail address: [email protected]

ABSTRACT

Bacteriological examination of milk samples (n=100) from mastitic buffaloes and cows revealed presence of Staphylococcus aureus in 52 and Staphylococcus epidermidis in 16 samples. Out of these 30.76% of Staphylococcus aureus and 25% of Staphylococcus epidermidis isolates were found positive for beta-lactamase production through nitrocefin test. The isolates were subjected to antimicrobial susceptibility testing through disk diffusion using oxacillin 1µg and cefoxitin 10µg antibiotic disks. Out of total 68 staphylococcal isolates, twenty two (32.35%) were resistant to these disks hence declared as methicilline-resistant.

KEY WORDS

Staphylococcus, beta-lactamase, methicilline.

INTRODUCTION

The staphylococci are the predominant pathogens in subclinical and chronic bovine mastitis globally. Staphylococcus aureus infection results in damage to developing mammary tissue, reduction in milk yield and failure to reach maximum milk production potential. (Guidry et al., 1998).
Coagulase negative staphylococci (CNS) can cause 8.7 % loss in milk production from a 305 day milk yield (Timms and Schultz, 1987). Beta-lactam antibiotics are frequently used in intra mammary infusion (IMI) therapy. Bacterial resistance to beta-lactam mechanisms includes production of beta-lactamases and production of a low-affinity penicillin-binding protein, PBP2a (Odd and Maeland, 1997). The latter, designated as methicilline-resistance (MR), precludes therapy with any of the currently available beta-lactam antibiotics, and may predict resistance to several classes of antibiotics besides beta-lactams among all staphylococci. MR is encoded by the mecA gene (Archer and Climo, 1994). Certain CNS isolates have the ability to form biofilms that may interfere with local defenses and impair the activity of bacteriostatic agents such as macrolides when temporary or permanent synthetic devices are implanted.
The National Committee for Clinical Laboratory Standards (NCCLS), now called the Clinical and Laboratory Standards Institute (CLSI), recommends the cefoxitin or oxacillin disc screen test for the detection of methicilline-resistant staphylococcus as standard methods along with PCR for the detection of mecA gene responsible for encoding penicillin binding protein in staphylococcus spp. (CLSI, 2005).
Buffaloes and cows are the major dairy animals in Pakistan. Field surveys of major livestock diseases in Pakistan have indicated that mastitis is the major problem in the country (Cady et al., 1983; Ajmal, 1990). Indiscriminate use of antibiotics have led to the development of resistant strains and resulted in increase in the cost of mastitis therapy. The present study was aimed at finding out the prevalence of CNS in sub-clinical and clinical cases of mastitis in bovines, application of nitrocefin (chromogenic cephalosporin) test to check the MR status of CNS isolates and evaluate the susceptibility of CNS against commonly prescribed drugs of choice under field conditions.

MATERIALS AND METHODS

The milk samples were collected from the mastitic udders of bovine according to the National Mastitis Council guidance. A total of 100 milk samples were collected from various private livestock farms around Faisalabad. Blood agar was used as a general purpose medium and was prepared by using the procedure illustrated by Sullia and Shantharam, (1998). The agar was autoclaved at 121°C for 15 minutes at 15 lbs/sq. inch pressure. It was cooled to 45-50°C, then aseptically added 50 ml of 5% sterile defibrinated ovine blood. After gentle mixing, the media was dispensed in sterile Petri plates for further use. Mannitol salt agar was prepared as a selective as well as differential medium for the isolation and purification of staphylococci (Sullia and Shantharam, 1998). The medium was autoclaved at 121°C for 15 minutes at 15 lbs/sq. inch pressure.
Identification of culture isolates: Gram staining was performed for micrographic identification. Catalse and coagulase tests were also performed as described by NMC, 2004.
Detection of methicilline resistant status of isolates: Coagulase negative staphylococci were tested for B-lactamase production by the nitrocefin test. A loopful of milk sample was smeared on the surface of nitrocefin disc and the results were recorded as colour change from pale yellow to pink or red.
Oxacilline (OX-1, 1ug oxoid, UK) was included for detection of methicilline resistance because it was more stable than methicilline and provided more reliable results of CNS (NCCLS, 1999). Other discs used for the antimicrobial susceptibility of methicilline-resistant CNS were following;
1. Enrofloxacin (ENR-5, 5µg Oxoid U.K.)
2. Amoxycillin (AMOX –25, 25µg Oxoid U.K.)
3. Chloramphenicol (CHLOR-10, 10 µg Oxoid U.K.)
4. Norfloxacin (NOR-10, 10µg Oxoid U.K.).
After screening of methicilline resistant CNS, they were subjected to antimicrobial disk diffusion testing using the same technique and antibiotic discs. The zones of inhibition of each antibiotic disc were recorded and interpreted referring to Zone Diameter Interpretive Standards of CLSI.

RESULTS & DISCUSSION

Staphylococci (0.5-1.5µm) occurred singly, in pairs, tetrads, short chains and irregular grape-like clusters. produced golden yellow colony pigmentation and haemolytic zone of beta Hemolysis. This was due to the beta toxin produced by staphylococcus cell but the spp. of S. epidermidis did not show haemolysis on surface of ovine blood agar plates.
Mannitol salt agar was used as a selective as well as differential medium for isolation of staphylococci. Only staphylococci could tolerate 7.5 % NaCl concentration in the medium. The colonies of mannitol-fermenter were golden yellow and they changed the colour of the agar from red to golden yellow. Most of CNS isolates were mannitol non-fermenter and they gave white colonies on mannitol salt agar. The pattern of growth shown by the isolated staphylococci on blob agar and mannitol salt agar was similar as described by Freeman (1979) and Power (1988).
Gram’s staining revealed the morphological characteristics of isolated species of staphylococci. The cocci form that was much more uniform in size occurring in irregular often grape like clusters and was stained darkly by crystal violet was considered as S. aureus. Isolates found singly, in pairs or in bunch and stained lightly were considered to be of CNS. The results were in accordance with those described by Leslie et al. (1998). All the isolated staphylococci produced Catalase enzyme when their colonies were mixed with H₂O₂ on clean glass slides. This character of staphylococci has also been described by Bisen and Kavita, (1998).
The efficacy of results for detection of methicilline resistance obtained obtained during present study were in line with the observations of Boyce et al. (1984) and Boubaker et al. (2004) who compared two oxacillin disc methods with a cefoxitin disc diffusion test for detection of methicilline resistant staphylococci using PCR for mecA as reference method.
Methicilline/oxacillin-resistant staphylococci were heterogeneous in their expression of resistance to β-lactam agents and the test conditions had a major effect on the expression and the detection of resistance. The gold standard method for the detection of resistance mediated by mecA is PCR, which is most commonly used as a reference method at present. In the present study, enrofloxacin was found most effective against methicilline-resistant staphylococci (Table 2). The results obtained were similar to those obtained by Ganiere et al. (2001) who examined and compared the minimum inhibitory concentrations (MICs) of enrofloxacin against 393 Staphylococcus intermedius strains isolated in France from canine pyodermas.
The resistance of amoxycillin to methicilline-resistant staphylococci observed in the present study, were in agreement with results obtained by Lee, (2003). Antimicrobial susceptibility tests of mecA-positive MRSA strains were performed by the disk diffusion method. All isolates were resistant to members of the penicillin family, such as ampicillin, oxacillin, and penicillin.
Nitrocefin discs were used for the detection of beta-lactamase production by staphylococcus isolated from bovine IMI by Watts and Salmon (1997) and Gentilini et al. (2002). They used the isolated CNS for antibiotic sensitivity against various antibiotics. Similar method was adopted in this study and almost similar results were obtained. Mastitis milk sample were also directly used for detection of beta-lactamase produce by coagulase negative staphylococci.
The result of invitro sensitivity showed that enrofloxacin was most effective followed by norfloxacin and chloramphenicol. Amoxicillin was found ineffective. The aspect of methicilline resistance needs be kept in mind for successful treatment of staphylococcal mastitis .

REFERENCES

1. Ajmal, M., 1990. Livestock Wealth of Pakistan. Proc. 3rd. Int. Cong. Vet. Med. Assoc. U. G.C., Islamabad.

2. Archer, G. L. and M. W. Climo,1994. Antimicrobial susceptibility of coagulase negative staphylococci. Antimicrob. Agents chemother. 38: 2231-2237.

3. Bisen, P.S. and K. Verma, 1998. Handbook of Microbiology .CBS Publishers and Distributors, New Delhi.

4. Boubaker, B. B., I. R. Ben Abbes, H. Ben Abdallah, K. Mamlouk, F. Mahjoubi, A. Kammoun, A. Hammami and S. Ben Redjeb,2004. Evaluation of a cefoxitin disk diffusion test for the routine detection of methicilline-resistant Staphylococcus aureus. Clin. Microb. Infec. Vol. 10(8): 749-772.

5. Cady, R. A., S. K. Shah, E. C. Schermerhorn and R. E. McDowell, 1983. Factors affecting performance of NiliRavi Buffaloes in Pakistan. J. Dairy Sci. 16: 578-586.

6. CLSI, 2005. Performance standards for antimicrobial susceptibility testing. CLSI approved standard M100-S15. Clinical and Laboratory Standards, Insti. Wayne.

7. Cruickshank, R., J. P. Duguid, B. P. Marmion and R. H. A. Swain. 1975. Medical Microbiology. 12th Ed. Churchill Living Stone. Edinburgh London and Newyork.

8. Freeman, B. A., 1979. Burrow’s Textbook of Microbiology.21st Ed. W.B. Saunders Company. pp. 429-435.

9. Gentilini E., G. Denamiel, A. Betancor, M. Rebuelto, M.Rodriguez Fermepin and R. A. De Torres,2002. Antimicrobial Susceptibility of Coagulase-Negative Staphylococci Isolated from Bovine Mastitis in Argentina. J. Dairy Sci.85:1913-1917.

10. Guidry, A., A. Fattom, P. Atul, S. Shepherd and J. Lohuis. 1998. Serotyping scheme for Staphylococcus aureus isolated from cows with mastitis. Am. J. vet. Res. 59: 1537-1539.

11. Leslie, C., A. Balows and M. Sussma, 1998. Topley & Wilson’s Microbiology and Microbial Infections. 9th Ed.

12. Power, D. A. 1988. Manual of BBL® products and laboratory procedures, Sixth Edition, Becton Dickinson Microbiology Systems, Cockeysville, MD.

13. Odd, G. B. and J. A. Maeland, 1997. Mechanisms of methicilline resistance in Staphylococci. AMPIS. 105: 264-276.

14. Sullia S. B. and S. Shantharam, 1998. General Microbiology. 1st Ed. Oxford & IBH Publishing Co. New Delhi. pp.549-562.

15. Timms, L. L. and L. H. Schultz, 1987. Dynamics and significance of coagulase negative staphylococcal intramammary infections. J. Dairy Sci. 70: 2648-2657.

Table 1: Comparison of nitrocefin and oxacillin discs for the detection of coagulase negative, methicilline resistant staphylococci

Table 2: Antimicrobial susceptibility pattern of coagulase negative, methicilline resistant staphylococci