CC BY
2© 2015, Scienceline Publication
World's Veterinary Journal
World Vet J, 5(4): 46-50, December 25, 2015
Antibiotic Resistance Profile of Coagulase Positive Staphylococcal infection in Dairy Buffaloe
Alsagher Omran Ali1*, Hassan Youssef Mahmoud1, Arwa Sameh2, Sayed Zaky2 and Adel Elsayed3
1Division of Infectious Diseases, Animal Medicine Department, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt 2National Research Center, Qena, Egypt
3Division of clinical laboratory diagnosis, Animal Medicine Department, Faculty of Veterinary Medicine, South Valley university, Qena, Egypt
Corresponding author's email: [email protected] ABSTRACT
For isolation and identification of the antibiotic resistance profile of Staphylococcal infection derived from buffalo (Bubalus bubalis) milk, a total of 100 milk samples have been collected from different villages in Qena governorate, Egypt, these animals were owned by small scale breeding farmers. S. aureus formed 23%, 58% other Staphylococci and other pathogens contained19% of the total number of samples. The S. aureus isolates have been tested for antibiotic susceptibility against 14 different widely used antibiotics, all S. aureus strains were resistant to Nalidixic acid by 100% and the lower percentage of resistance was to Ampicillin by 4.8%. The Multiple antibiotic resistances have been calculated for all S. aureus strains, the highest value was 1 and the least one was 0.143. The Buffaloe is considered one of the most important dairy animals in Egypt especially in the small scale breeding and the presence of S. aureus and other Staphylococci causing a great risk on the productive level of these animals due to mastitis as well as for the human beings, from here comes the importance of the detection of the antibiotic resistance profile.
Key words: S. aureus, Antibiotic susceptibility test, Mastitis INTRODUCTION
Subclinical mastitis has no gross pathological changes in the udder and the milk looks apparently normal. The main bacterial agents inducing mastitis are Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae and streptococcus agalactiae, which account for around 80% of all diagnostic cases (Bradely, 2002).
The prevalence of S. aureus mastitis varies, depends on many factors from country to another, such as the top 17 dairy producing states, USA which accounted for 79.5 % of dairy operations and 82.5 % of U.S. milk cows were infected 43% at the herd level (Lombard et al., 2008), 30% in 112 Holstein herds in Mexico (Miranda-Morales et al., 2008), 52% in bulk tank milk on Prince Edward Island (Riekerink et al., 2006), in Finland decreased from 11.1% in 1995 to 10.1% in 2001 (Pitkala et al., 2001), mastitis and teat canal infections in South African dairy herds increased from 8.1% and 24.1% in 2002, to 15.4% and 30.0% in 2006, respectively (Petazer et al., 2009) and in a similar study was done in the department of animal medicine, South Valley University revealed that the S. aureus constitutes only 24 (14.7%) isolates out of 163 staphylococci isolates grown on Baird-Parker media and a percentage of (9.7%), which was similar to another study that showed a percentage of 24.8% (El-jakee et al., 2008) and 20.7% (Jakeen et al., 2010) and 21% (Malahat et al., 2010) respectively.
The penicillin significantly decreased the S. aureus produces by four major penicillin binding proteins (PBPs), PBP 1 to 4, which catalyze the transpeptidation reaction that cross-links the peptidoglycan of the bacterial cell wall which is essential for the cell wall synthesis, but beta lactam antimicrobials can bind to the original PBP active site and thereby inhibit the cell wall synthesis. However, PBP-2a, which has a reduced affinity for binding with beta-lactam antimicrobials (Hartman and Tomasz, 1984).
The aim of the present study was to estimate the antibiotic resistance profile of coagulase positive Staphylococci in dairy Buffaloe which is considered a very important step to compare the phenotypic and genotypic characterizations of S. aureus isolates in further investigations.
ISSN 2322-4568
> R
£
OA1
» g O 22 2 0
2 i 5 5
0
S
1
2 A L
A R
S
C L E
To cite this paper:| Ali AO, Mahmoud HYA.H., Dairy Buffaloe. World's Vet. J. 5(4): 46-50. Journal homepage: http://wvj.science-line.com/
46
MATERIAL AND METHODS
Sampling
A total of 100 raw milk samples were collected from dairy buffaloes in Qena Governorate, Egypt. The animals are apparently healthy with no local or systemic infection. Samples were immediately subjected to analysis within 24 hours, the milk samples were collected and kept in an ice in sterile single plastic tube (Pamela, 2005).
Isolation and culturing of Staphylococcus aureus
Samples were mixed thoroughly with vortex till the complete homogenization of the samples and the preparation of the Baired-Parker media according to Vanderzant and Splittstoesser (1992). 3-4 Typical and large colonies were harvested and picked up by a sterile metal bacteriological loop and then immersed in the glycerol stock in Eppendorf tube and kept immediately at -70 to -80 °C for further studies (Jones et al.,1991).
Biochemical tests
The coagulase test was performed by two different methods; the slide coagulase test and tube coagulase test (Wichelhaus et al., 1999) and thermostable nuclease test "deoxyribonuclease activity" (Lachia et al., 1971)
Antibiotic susceptibility test
Antimicrobial susceptibility was tested by the single diffusion method according to (Deresse et al., 2012) for S. aureus. The antibiotic discs used with variable concentrations were Nalidixic acid (30 mg), Kanamycin (30 mg), Neomycin (30 mg), Sulfamethoxazole (25 mg), Gentamicin (10 mg), Oxytetracycline (30 mg), Penicillin (10 IU), Chloramphenicol (30mg), Norfloxacin (10mg), Streptomycin (10mg), Amoxicillin (30mg), Ciprofloxacin (5mg), Erythromycin (15mg) and Ampicillin (10mg). They were used to determine the susceptibility of the isolated S. aureus strains (Oxoid Limited, Basingstoke, and Hampshire, UK). Therefore, the antimicrobial susceptibility testing was applied according to the guidelines stipulated by National Committee for Clinical Laboratory Standards "NCCLS" (2001). The tested strains were evaluated as susceptible, intermediate and resistant. MAR index for each strain was determined according to the formula stipulated by Singh et al. (2010) as following:
MAR index= Number of resistance (Isolates classified as intermediate were considered sensitive for MAR index) / Total Number of tested antibiotics
RESULTS
Prevalence of staphylococci
The total prevalence of different staphylococci and S. aureus which isolated from a total of 100 raw milk samples collected from dairy buffaloes at farmers' houses which isolated on Baird-Parker agar media were S. aureus 23 %, other Staphylococci 58% and other pathogens 19% (Table1).
Biochemical examination
All S.aureus isolated from examined raw milk samples showed coagulase positive with a percentage of 100% and D-Nase positive with a percentage of 86.9 % and D-Nase negative with a percentage of 13.04% (Table 2).
Antibiotic sensitivity tests
The frequency of different levels of resistance to various antibiotics among S. aureus isolates are shown in Table 3, the highest resistance level was recorded for the Nalidixic acid (100%) and the lower one was for Ampicillin (4.8%) and the antimicrobial resistance profile of different S. aureus isolates are shown in Table 4.
Table1. Incidence of S. aureus and other Staphylococci isolated from Buffaloe's milk in Qena, Egypt
Number of milk samples S. aureus Other Staphylococci Other Pathogens
100 23(23%) 58(58%) 19(19%)
Table 2. Biochemical reactions of S.aureus isolated from Buffaloe'smilk in Qena, Egypt
Number of S. aureus Coagulase Test D-Nase Test
Twenty three Positive Negative Positive Negative
23 (100%) 0 (0%) 20 (86.9%) 3 (13.4%)
To cite this paper:| All AO, Mahmoud HYA.H., Dairy Buffaloe. World's Vet. J. 5(4): 46-50. Journal homepage: http://wvj.science-line.com/
47
Table 3. Antimicrobial susceptibility of S. aureus isolated from Buffaloes' milk in Qena, Egypt
Antimicrobial agent SIR
Nalidixic acid (30 mg) - - 21(100%)
Kanamycin (30mg) - 1(4.8%) 20(95.2%)
Neomycin (30mg) 1(4.8%) - 20(95.2%)
Sulfamethoxazole (25mg) 1(4.8%) 2(9.5%) 18(85.7%)
Gentamicin (10mg) 2(9.5%) 2(9.5%) 17(80.9%)
Oxytetracycline (30mg) 3(14.3%) 1(4.8%) 17(80.9%)
Penicillin (10 IU) 3(14.3%) 4(19.0%) 14(66.7%)
Chloramphenicol (30mg) 5(23.8%) 3(14.3%) 13(61.9%)
Norfloxacin (10mg) 6(28.6%) 4(19.0%) 11(52.4%)
Streptomycin (10mg) 8(38.1%) 2(9.5%) 10(47.6%)
Amoxicillin(30mg) 9(42.9%) 6(28.6%) 6(28.6%)
Ciprofloxacin (5mg) 14(66.7%) 2(9.5%) 5(23.8%)
Erythromycin (15mg) 16(76.2%) 2(9.5%) 3(14.3%)
Ampicillin (10mg) 19(90.5%) 1(4.8%) 1(4.8%)
S: susceptible, I: intermediate, R: resistant
Table 4. Antimicrobial resistance profile of S. aureus strains isolated from Buffaloes' milk in Qena, Egypt
No. Id. Strain Antimicrobial resistance profile MAR index
1 S. aureus NA,K,N, SXT, G, T, P, C, NOR,S, AMX, CP, E, AM 1
2 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S, AMX, CP, E 1
3 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S, AMX, CP, E 0.928
4 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S, AMX, CP 0.928
5 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S, AMX, CP 0.857
6 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S, AMX, 0.857
7 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S 0.786
8 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S 0.714
9 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S 0.714
10 S. aureus NA,K,N, SXT, G, T, P, C, NOR, S 0.714
11 S. aureus NA,K,N, SXT, G, T, P, C, NOR 0.643
12 S. aureus NA,K,N, SXT, G, T, P, C 0.571
13 S. aureus NA,K,N, SXT, G, T, P, C 0.571
14 S. aureus NA,K,N, SXT, G, T, P 0.500
15 S. aureus NA,K,N, SXT, G, T 0.500
16 S. aureus NA,K,N, SXT, G, T 0.500
17 S. aureus NA,K,N, SXT, G, T 0.500
18 S. aureus NA,K,N, SXT 0.428
19 S. aureus NA,K,N 0.286
20 S. aureus NA,K,N 0.286
21 S. aureus NA 0.143
Average 0.598
DISCUSSION
Using culture method identified about 23% of examined samples infected with S. aureus. These results were higher than 14% (Abdel et al., 2004), while were in accordance with 22.9% (Turkyilmaz et al., 2010) and 24.8% (El-Jakee et al., 2008) but were lower than 44.4% (Mahmoud et al., 2008).
Identification of these isolates was performed using biochemical (phenotypic) methods, 100% of these isolates showed positive for coagulase test and 86.9% for D-Nase test. Present results were higher than the report of Abdel et al. (2010), that recorded 33.55% of isolates were positive for coagulase test and 32.06 % were positive for D-Nase test while the present study results were some extent close to Mohamed (2012) as recorded 98.5% of isolates were positive for coagulase test. El-Jakee et al. (2008) reported that the production of coagulases and thermonuclease were not unique features of S. aureus but were shared by S. intermedius and S. hyicus. The prevalence of subclinical mastitis caused by S. aureus were studied by many investigators, the obtained result came in coincides with the results of Fox and Gay (1993) as they stated that S. aureus caused mastitis in cattle ranged from 7-40 % of total number of animals, while it was lower
48
To cite this papery Ali AO, Mahmoud HYA.H., Sameh A, Zaky S and Elsayed A. 2015. Antibiotic Resistance Profile of Coagulase Positive Staphylococcal infection in Dairy Buffaloe. World's Vet. J. 5(4): 46-50. Journal homepage: http://wvj.science-line.com/
for high extent than that recorded by Janosi and Balty (2004) which was 80%. The antibiotic sensitivity testing of the isolated strains (n= 21) to various antibiotics shown in Table 3 and 4. The results of penicillin (66.7%) were similar to Moronie et al. (2006) 69.1% and higher than Gentilini et al. (2002) 40% but less than Abera et al. (2010) 94.4%. The results of erythromycin 14.3% were similar to 15.7% (Mohamed, 2012), but lower than 40.9% (Ghaleb, 2006). The results of amoxicillin 28.6% were similar to 34.2% (Mohamed, 2012) but less than 81.3% (Klimien et al., 2011).
CONCLUSION
Subclinical mastitis of S. aureus was common in dairy buffaloes in Qena Governorate affecting milk yield (quality and quantity) and consumer safety. Culturing method and biochemical tests were not enough for detection of S. aureus subclinical mastitis but it is considered an important preliminary step but it needs further confirmation for detection of MRSA strain which acquire the mecA gene that responsible for the beta-lactam resistance.
Acknowledgements
We appreciate from all members of Animal Medicine Department, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt for their support and help.
REFERENCES
Abdel AAA, Bashandy MM, Yasin MH and Ibrahim AK (2010). Assessment of conventional and molecular features of Staphylococcus aureus isolated from bovine raw milk samples and contact dairy workers, Global Veterinaria, 4: 168-175.
Abdel HK, Sender G, Prusak B and Ryniewicz Z (2004). Multiplex PCR protocol for the diagnosis of cow udder
infection with Staphylococcus aureus, Animal Science Papers and Reports, 22: 679-685. Abera M, Demie B, Aragaw K, Regassa F and Regassa A (2010). Isolation and identification of Staphylococcus aureus from bovine mastitic milk and their drug resistance patterns in adama town, Ethiopia. Journal of Veterinary Medicine and Animal Health, 2: 29-34. Bradely AJ (2002). Bovine mastitis: an evolving diseases. Veterinary Journal, 163: 1-13.
Chambers HF (2001). The changing epidemiology of Staphylococcus aureus. Emerging Infectious Diseases, 7:178-182. Deresse D, Solomon G and Dawit Y (2012). Antibiotic-resistance Staphylococcus aureus isolated from cow's milk in the
Hawassa area, South Ethiopia. Annals Clinical Microbiology, 11: 26-34. El-Jakee J, Nagwa AS, Bakry M, Zouelfakar SA, Elgabry E and Gad El-Sead WA (2008). Characteristics of Staphylococcus aureus Strains Isolated from Human and Animal Source. American-Eurasian Journal of Agriculture and Environmental Sciences, 2: 221-229. Fox LK and Gay JM (1993). Contagious mastitis. Veterinary Clinics of North America, 9: 475-488. Gentilini E, Denamiel G, Betancor A, Rebuelto M, Rodrigues- Fermepin M and De Torrest RA (2002). Antimicrobial susceptibility of coagulase negative Staphylococci isolated from bovine mastitis in Argentina. Journal of Dairy Science, 85: 1913-1917.
Ghaleb M (2006). Antibiotic resistance against Staphylococcal isolates recovered from subclinical mastitis in the North
of Palestine. Islamic University Journal, 14: 1-9. Hartman BJ and Tomasz A (1984). Low-affinity penicillin-binding protein associated with beta-lactam resistance in
Staphylococcus aureus. Journal of Bacteriology, 12:513-516. Janosis and Baltay (2004). Correlation among the somatic cell count of individual bulk milk, result of California Mastitis
Test and bacteriological status of the udder in dairy cows. Acta Veterinaria Hungarica, 52: 173-183. Jones D (1991). Maintenance of bacteria on glass beads at -60°C to -76°C. In: Kirsop and Doyle (Eds.) Maintenance of
microorganisms and cultures cells, (2nd Edn.) PP. 45-50. Lachia R, Genigeogis C and Hoeprich P (1971).Meta chromatie agar-diffusion methods for detecting Staphylococcal
nuclease activity. Applied Microbiology, 21: 585:587. Lombard J, Van Slyke T, Welcome F,Schukken Y and Kopral C (2008). Prevalence of contagious mastitis pathogens on
US dairy operations NMC 47th Annual Meeting Proceedings, New Orleans LA. 170-171. Mahmoued MM, Salama ME and Galal H (2008). Rapid diagnosis of subclinical Staphylococcus aureus-mastitis in
dairy buffaloes using PCR technigue. Assiut Veterinary Medicine Journal, 54: 116. Malahat A, Sayed M, Razavi R and Nooshin A (2010). Detection of staph.aureus by PCR . comparative clinical Pathology, 19: 91-94
Miranda-Morales RE, Rojas-Trejo V, Segura-Candelas R, Carrillo-Casas EM, Sanchez-Gonzalez MG, Castor RS and Trigo-Tavera FJ (2008). Prevalence of pathogens associated with bovine mastitis in bulk tank milk in Mexico. Annals of the New York Academy of Sciences, 1: 300-302. Mohamed MH (2012). Penicillin resistance against staphylococcal isolates recovered from subclinical mastitis is Sohag Governorate, Master Veterinary Science in Microbiology Department of Veterinary Medicine, South Valley University.
Moronie P, Pisoni G, Antonini M, Villa R, Boettcher P and Carli S(2006). Antimicrobial drug susceptibility of Staphylococcus aureus from subclinical bovine mastitis in Italy. Journal of Dairy Science, 89: 2973-2976.
TQSEEBB Ali AO, Mahmoud HYA.H., Dairy Buffaloe. World's Vet. J. 5(4): 46-50. jounSQEmpSgSB http://wvj.science-line.com/
49
National Committee for Clinical Laboratory Standards "NCCLS" (2001). Performance standards for antimicrobial susceptibility testing. Supplement M100-S11. Villanova PA, USA.
Petazer I M, Karzis J, Watermeyer J, Van der Schans T and Van Reenen R (2009). Trends in udder health and emerging mastitogenic pathogens in South African dairy herds. Journal of the South African Veterinary Association, 1: 1722.
Pitkala A, Haveri M, Pyorala S, Myllys V and Honkanen-Buzalski T (2001).Bovine mastitis in Finland 2001-prevalence, distribution of bacteria, and antimicrobial resistance. Journal of Dairy Science, 8: 2433-2441.
Riekerink MO, Barkema HW, Veenstra S, Poole DE, Dingwell RT and Keefe GP (2006). Prevalence of contagious mastitis pathogens in bulk tank milk in prince Edward island. The Canadian Veterinary Journal, 6: 567.
Turkyilmaz S, YildizO, Orya§inE,Kaynarca S and Bozdogan B (2010). Molecular identification of bacteria isolated from dairy herds with mastitis, The Journal of the Faculty of Veterinary Medicine, University of Kafkas, 16: 10251032.
Vanderzant C and Splittstoesser DF (1992). Compendium of methods for the microbiological examination of food. (3rd Edn.) American Public Health Association, Washington, D.C. PP. 20-35.
Wichelhaus TA, Kern S, SchaferBrade V and Hunfeld KP (1999). Evaluation of modern agglutination tests for identification of methicillin-suspected and methicillin-resistant Staphylococcus aureus. European Journal of Clinical Microbiology and Infectious Diseases: Official Publication of the Europea. Society of Clinical Microbiology, 18: 756-758.
TQSEEBB Ali AO, Mahmoud HYA.H., Dairy Buffaloe. World's Vet. J. 5(4): 46-50. jouSaQSmpSgSB http://wvj.science-line.com/
50
