Characterization and AST pattern of bacterial pathogens of bovine mastitis milk

Introduction

Bovine mastitis is one of the most prevalent and economically significant diseases affecting dairy cows worldwide. It is characterized by inflammation of the parenchyma of mammary gland. Mastitis occurs in both clinical and subclinical forms. SCM often goes unnoticed but contributes to treatment and substantial hidden losses (Parasana et al., 2021) [14] . It is a multietiological (Hawari and Fawzi, 2008) [10] caused by a plethora of microorganisms (Vakkamaki et al., 2017) [17] and multi-factorial disease as well (Deb et al., 2013) [4] Bovine mastitis is a complicated disease that is difficult to prevent by vaccination. Global surveys indicate that Subclinical Mastitis (SCM) affects about 40-42% of dairy animals and Clinical Mastitis (CM) accounts for about 25% of cases. Pathogens like Staphylococcus aureus, coagulase-negative staphylococci (CoNS), Escherichia coli, Klebsiella and Pseudomonas spp. are the most common pathogens. So dairymen rely heavily on antibiotics for mastitis treatment. This overuse and irrational application leads to development of antibiotic resistant bacteria (Acar and Rostel, 2001) [1] . Which compromises therapeutic success and poses public health risks.

Study objective Detection of antibiotic resistance in Bacterial pathogens of bovine mastitis

Study area

A total of 153 bovine milk samples were collected from dairy herds in Chengalpattu, Chennai and Thiruvallur districts (North Eastern zone of Tamil Nadu). Briefly, milk samples were collected, from every udder quarter (1sample per quarter) and diagnosed on the basis of visual abnormality and inflammation of the udder. Samples were collected in sterile bottles (5-10 ml) and transported on ice to the laboratory within 6 hours.

Materials and Methods

Mastitis diagnosis was performed on bovine milk for the Somatic Cell Count (SCC) using TANUCHECK SCC kit based on an enzymatic reaction, which is directly related to the number of somatic cell present. This is a cow side kit that helps to screen and grade the quality of milk as good, mild, moderate, severe for mastitis followed by poor or bad quality milk. Electrical Conductivity (EC) was also done for all the milk samples.

Sample processing for phenotypic characterization

Bovine milk was divided as sub-clinical (mild to moderate) and clinical mastitis as per screening by TANUCHECK SCC kit and Electrical conductivity (Rana et al., 2023) [15] . For Bacterial isolation, conventional microbiological methods are considered as gold standard and are routinely employed for confirmation of the causative agents (Javia et al., 2018) [11] . About 20µl of mastitis milk was inoculated into 3ml of Tryptic soya broth and incubated overnight at 37°C for 16-18 hr.

Cultural characterization

Bacterial cultures were stained by Gram's method and then streaked on various selective media like, Mannitol Salt, Baird Parker, MacConkey, Nutrient and 5% Sheep blood agar. Biochemical test were done for differential identification of Bacterial isolates of Enterobacteriaceae family (IMViC).

Antimicrobial Sensitivity Test was done for all Bacterial isolates using 16 different antibiotics and the pattern was recorded (Forbes et al., 2007) [7] .

Antimicrobial Susceptibility Test (AST)

Single pure colony from selective agar plate of each Bacterial isolate was inoculated in 5ml of Tryptic soya broth and incubated at 37°C for 6 hours till a turbidity of 10 5-6 cfu per ml was obtained. Culture was centrifuged and the pellet was suspended in Phosphate buffer saline and adjusted to 0.5 scale McFarland turbidity. About 20µl of re-suspended culture was added on Muller Hinton agar and swabbed to form a lawn. AST was done as per the guidelines of CLSI (Wayne, 2013) [18] . All cultures were tested against sixteen antibiotics as listed in Table 1 Himedia (Mumbai). The zone of inhibitions were measured and interpreted.

Results

Out of the total bovine milk (N=153) screened, 33% (N=32) were of good quality milk and 79.08 % were found to be Mastitis positive (N=121) with 71 subclinical (59%), 50 (41.3%) clinical by TANUCHECK kit as detailed in Table .2.

The SCC values ranged from 1-7 lakh cells/ml, correlating with increasing EC scores. SCC and EC are used as indicators for detecting bovine mastitis and show a positive correlation. An increase in SCC causes damage to udder tissue leading to changes in milk composition, like elevated sodium and chloride ion concentrations and increasing EC of the milk (Tommasoni et al., 2024) [16] . The milk samples identified as SCM using the TANUCHECK SCC kit revealed 62% of the samples had SCC in the range of 1-1.5 lakhs graded as Good quality and 72% samples had a SCC range of 2-3.5 lakhs graded as Mild. In CM milk, 42% had a SCC range of 3.5-4.2 lakhs graded as Moderate and 58% had a SCC range of 5.5-6.5 lakh and was graded as Poor quality. Further, 8% milk was unfit with SCC range of 7 to 8lakhs and was discarded. [14] . The antibiotic susceptibility for the pathogens involved in mastitis milk revealed resistance to multiple antibiotics tested. Multidrug resistance (MDR) is defined as resistance to ≥ 3 antimicrobials (Magiorakos et al., 2012) [12] . The number of samples showing sensitivity or resistance was expressed in percentage. Milk of cows in the third or higher lactation stages showed resistance to more number of antimicrobial classes than mastitis isolates from younger cows.

The Bacterial isolates obtained from the milk samples were found to be highly sensitive to Fosfomycin, cefotaxime and Ampicillin sulbactum followed by gentamicin, ciprofloxacin and intermediate or resistant to the other tested antibiotics as given in Table 4.

Table 3: Bacterial strains obtained by cultural isolation (Harini and Sumathi 2011) [9] No [8] of the Gram positive family are as indicated in Table 5. For Staphylococcus spp the most sensitive drug was Fosfomycin (42%) and Cefotaxime (50%),while Ampicillin (34%) was sensitive .The drugs with intermediary action were Bacitracin (31%), Gentamicin (19.3%) Ceftrioxne (15%).The resistant drugs were Penicillin G (85%) Streptomycin (31%) and Ampicillin (24%).They were resistant to Cloxacillin (53%).

The Streptococcus spp isolates were most sensitive to Ampicillin (100%); Ampicillin sulbactam (86%) and Ceftrioxne (71%) while sensitive to Fosfomycin (57%) and Norfloxacin (43%) whereas Intermediary reaction against Cotrimaxazole (43%) Streptomycin and Erythromycin (29% each) and resistant to Penicillin-G (100%) Erythromycin (71%) and Oxacillin (43%).

The Bacillus spp showed 100% sensitivity to Ampicillin and Enrofloxacin, 80% sensitivity to Streptomycin and 60% to Bacitracin whereas completely resistant to Penicillin G (100%) and 80% to Ampicillin. Clostridium spp could be successfully isolated from gangrenous cases which were sensitive to Strep to or Benzyl penicillin and to Ciprofloxacin (100%) and Cefotaxime (80%). The gram negative Bacterial isolates belonging to E.coli (79%) and Klebsiella spp (38%) were most sensitive to Fosfomycin and followed by Ciprofloxacin (56%) and Ampicillin (29%) respectively. Sensitive drugs were Doxycycline (58%) and Neomycin (46%) for E. coli whereas for Klebsiela it was Ampicillin (38%) and Oxytetracycline (21%). Intermediary drug was Ampicillin (63%) for E. coli along with Gentamicin (56%) and (71%) for Klebsiella spp.

Discussion

In India, the burden of bovine mastitis is much higher, with SCM reported at 65% and CM at 35% (Dua K 2001) [5] making it a persistent challenge to milk production and herd health. Most of the bovine intra-mammary infections are Bacterial in origin (Bradley AJ, 2002) [2] . It can effectively be cured by early detection and judicial use of antimicrobials. This study used antimicrobial susceptibility test to screen and select effective drugs for early treatment of mastitis. Multiple drug resistance in mastitis bacteria has been reported by several researchers (Das et al., 2017) [3] and can be attributed to the injudicious use of antibiotics or selection pressure of pathogens to antimicrobials. Isolates in this study, showed resistance to third generation cephalosporin's viz. cefotaxime and ceftriaxone which is similar with the recent reports of drug resistance pattern conducted on mastitis pathogens (Fair and Tor 2014) [6] .

Conclusion

The study highlights a high prevalence of mastitis and significant antimicrobial resistance (Acar and Rostel, 2001) [1] among the pathogens causing bovine mastitis in the study region. This increase in antimicrobial resistance is a new hurdle-in treatment options.Bacterial species, managemental practices, region and preferences of antibiotics used in the study gave variation in anti-biogram patterns between the three districts (Deb et al., 2013) [4] . So screening mastitis milk by AST will be useful for selecting an appropriate antibiotic and also to know the changing trends of antibiotic resistance. This may limit the indiscriminate use of many types of antibiotics.Proper hygiene and milking methods helps to prevent and control mastitis.

Conflict of Interest Not available

Financial Support

Acknowledgement

18. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: 21 st Informational supplement. CLSI document M100-S23. Wayne (PA): CLSI; 2013;33:1.

References

1. Acar J, Rostel B. Antimicrobial resistance: An overview. Rev Sci Tech. 2001;20:797-810.
2. Bradley AJ. Bovine mastitis: an evolving disease. Vet J. 2002;164:116-128.
3. Das A, Guha C, Biswas U, Jana PS, Chatterjee A, Samanta I. Detection of emerging antibiotic resistance in bacteria isolated from subclinical mastitis in cattle in West Bengal. Vet World. 2017;10:517-520.
4. Deb R, Kumar A, Chakraborty S. Trends in diagnosis and control of bovine mastitis: A review. Pak J Biol Sci. 2013;16(23):1653-1661.
5. Dua K. Incidence, aetiology and estimated economic losses due to mastitis in Punjab and in India-an update. Indian Dairyman. 2001;53(10):41-48.
6. Fair RJ, Tor Y. Antibiotics and bacterial resistance in the 21 st century. Perspect Medicin Chem. 2014;6:25-64.
7. Forbes BA, Sahm DF, Weissfeld AS. Laboratory methods and strategies for antimicrobial susceptibility testing. In: Bailey & Scott's Diagnostic Microbiology. St. Louis: Mosby; 2007, p. 187-214.
8. Gao J, Wu H, Barkema HW, Zhang G, Liu Z, Deng L, et al. Incidence of clinical mastitis and distribution of pathogens on large Chinese dairy farms. J Dairy Sci. 2017;100:4797-4806.
9. Harini H, Sumathi BR. Screening of bovine milk samples for subclinical mastitis and antibiogram of bacterial isolates. Vet World. 2011;4(8):358-359.
10. Hawari AD, Fawzi A. Prevalence and distribution of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Jordan. Am J Anim Vet Sci. 2008;3:36-39.
11. Javia B, Purohit J, Mathapati B, Barad D, Savsani H, Kalariya V, et al. Molecular detection and antimicrobial resistance pattern of staphylococci isolated from clinical and subclinical bovine mastitis. Indian J Vet Sci Biotechnol. 2018;14(1):13-16.
12. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268-281.
13. Nathawat P, Bhati T, Sharma SK. Prevalence and antimicrobial profile of bacterial isolates from clinical mastitis. Vet World. 2013;6:237-238.
14. Prasanna KPR, Rizvi SH, Singh SK, Kumar A, Singh M. The hidden cost of disease: I. Impact of the first incidence of mastitis on production and economic indicators of primiparous dairy cows. J Dairy Sci. 2021;104(7):7487-7501.
15. Rana MS. Electrical conductivity and total dissolved solids of raw milk for the detection of bovine subclinical mastitis. Vet World. 2023;16(12):2521-2525.
16. Tommasoni L. Exploring the sources of variation of electrical conductivity and differential somatic cell count in Italian Mediterranean buffalo milk. J Dairy Sci. 2024;107(1):329-342.
17. Vakkamäki J, Taponen S, Heikkilä AM, Pyörälä S. Bacteriological etiology and treatment of mastitis in Finnish dairy herds. Acta Vet Scand. 2017;59:33.