1. Prevalence of biofilm producing
Staphylococcus aureus of dairy origin,
and its response against antibiotics
Muhammad Aamir Naseer
2012-ag-2609
M.Phil. CMS
Supervisor Dr. M. Ijaz Saleem
2. Why Staphylococcus aureus
• Potent pathogenic spectrum
including
Skin infections
Respiratory infections
Digestive disorders
Cardiovascular ailments
Common isolate in mastitis
• Ranked 4th most potent nosocomial
pathogen
3. Introduction
• Cattle 46.1 million and buffalo 38.8 million milk ~45 million tons
• Mastitis – decline in milk production, altered milk composition and
compromised udder health
• S. aureus – one of the most common isolate of mastitis
• Resistance against antibiotics – most important factor “ Biofilm production”
5. Objectives
• To estimate prevalence and risk factors of biofilm producing Staphylococcus
aureus of dairy origin
• To evaluate antibiotics efficacy against biofilm establishing and biofilm
established Staphylococcus aureus
6. Materials and Methods
Sampling SFMT screening microbiological and biochemical
analysis
Biofilm detection S. aureus isolation & identification
8. Tube method
• Culture in tryptic soy broth- over
night incubation at 37° C
• Decant Eppendorf- PBS washings
• Crystal violet stain- washing
• Clearly lined walls and bottom-
Biofilm present
(Christenson et al. 1988)
Observations Biofilm score
No biofilm 0
Weak biofilm 1
Moderate biofilm 2
Strong biofilm 3
9. Congo red agar method
• Congo red agar- swabbing
• 24 h incubation at 37°C
• Check growth
characteristics
• Interpret the results
(Freeman et al., 1989)
Colony characteristics Results
Reddish to Pink No Biofilm
Blackish Weak Biofilm
Black Dry Moderate Biofilm
Jet Black Crystalline sheet Strong Biofilm
10. Microtitration Plate method
• Overnight broth culture – 50µl to wells
• Negative control – only Nutrient broth
• Incubation – overnight
• Washing & stain
• OD value at 570nm
(Christenson et al. 1988)
OD value Adherence Biofilm
formation
< 0.12 None None/ Weak
0.12-0.24 Moderate Moderate
> 0.24 Strong High
12. Statistical Analysis
• Prevalence calculation by
Prevalence % =
No. of infected Animal (n)
Total no. of sampled Animals (N)
× 100
• Risk factors association – chi-square (p value 5%)
13. Results
• Sub-clinical mastitis 64%
• 58.75% samples harboring S. aureus
• Most of isolates are biofilm positive along
with resistant of commonly used antibiotics
14. Biofilm identification
Sr.
No
.
Method Total Weak Biofilm
% age
Moderate
Biofilm
% age
Strong
Biofilm
% age
No Biofilm
%age
1 Tube
Method 154
25.58 6.98 2.33 65.11
2 Congo
red agar
Method 154
23.25 30.23 2.33 44.19
3 Plate
Method 154
48.83 20.93 2.33 27.91
16. Risk factors
• Body condition
• Teat dipping practice
• Professional handling
• Udder and teat abnormalities
• Knowledge about mastitis
17. Conclusion
• 64% subclinical mastitis shows higher occurrence rate of mastitis
• 58.75% of positive samples harboring S. aureus indicating potential threat for
community health.
• High prevalence of biofilm producing isolates – indicative of evolving
resistive behavior in S. aureus.
• Narrowing antibiogram of isolates indicates the need of revision in
antibiotic selection criteria at clinics.
18. References
• Economic Survey Pakistan 2017-18
• Thiran E, Di Ciccio PA, Graber HU, et al., 2018. Biofilm formation of Staphylococcus aureus dairy isolates representing
different genotypes. J Dairy Sci 101:1000–1012.
• Merritt JH, Kadouri DE and O’Toole GA, 2005. Growing and Analyzing Static Biofilms. Curr Protoc Microbiol 0 1:Unit-
1B.1.
• Mathur T, Singhal S, Khan S, et al., 2006. Detection of biofilm formation among the clinical isolates of Staphylococci: an
evaluation of three different screening methods. Indian J Med Microbiol 24:25–29.
• Hassan A, Usman J, Kaleem F, et al., 2011. Evaluation of different detection methods of biofilm formation
in the clinical isolates. Braz J Infect Dis 15:305–311.
• Hiramatsu K, Katayama Y, Matsuo M, et al., 2014. Multi-drug-resistant Staphylococcus aureus and
future chemotherapy. J Infect Chemother 20:593–601.
