Bacteria from bovine clinical mastitis showed multiple drug resistance

Mastitis, which often manifests as udder infection in dairy animals, is of great concern as it affects public health and results in heavy economic losses to the dairy industry. A hospital-based cross-sectional study was conducted to determine the cultivable bacterial species associated with bovine clinical mastitis and their resistance patterns towards different antimicrobials. The milk samples from cows suffering from clinical mastitis during monsoon season were investigated. The prevalence of clinical mastitis was significantly high in Holstein–Friesian crossbred cows, followed by in Jersey crossbred, Red Kandhari and Deoni. Significantly high prevalence was observed during 4th to 6th months of lactation. A total of 110 bacterial isolates belonging to 14 different genera were isolated and identified. Aminoglycosides and quinolones were found to be the most effective antibiotics. Vancomycin resistant penicillinase producing Gram positive bacteria were demonstrated. Gram negative bacteria resistant to extended spectrum β lactamases, cephalosporins, tetracyclines, vancomycin and chloramphenicol as well as vancomycin resistant enterococci, multiple drug resistant (MDR) gram negative rods, MDR Pseudomonas and MDR Acinetobacter were found. Widespread resistance of Streptococcus uberis towards cephalosporins was documented. Variable MDR patterns were recorded within a single species. MDR transfer from non-pathogens to emerging foodborne and established mastitis pathogens could be a potential problem to the dairy industry as well as to public health.


Introduction
Bovine mastitis is professed to be the leading cause of impaired productivity and health issues in dairy animals (Hogeveen et al. 2011). Mastitis is a complex, multi-factorial illness caused by a variety of pathogens, including bacteria, viruses and fungi. The occurrence of the disease may vary depending upon the animals affected, pathogens involved and the environment (Constable et al. 2017). Several bacterial agents associated with bovine mastitis are commonly isolated from the dairy environment (Bradley 2012).
Streptococcus, Staphylococcus and Corynebacterium spp. are potential bacteria capable of causing clinical as well as subclinical mastitis. In addition, bacteria of environmental aetiology, including Escherichia, Acinetobacter, Pasteurella, Pseudomonas, Klebsiella, Arthrobacter, Bacillus, Enterobacter, Enterococcus and Serratia are involved in aggravated conditions. These organisms often carry antimicrobial resistance (AMR) genes and are developing multiple drug resistance (MDR) to popular antimicrobial agents used in treating mastitis (Weisblum 1995;McMurry and Levy 2000;Botrel et al. 2010). Not responsible use of antibiotics leads to mutations in bacteria, allowing them to survive and making treatment a complicated and costly affair. However, bacterial isolates associated with bovine mastitis are dramatically susceptible to gentamicin (Awandkar et al. 2009;Salih and Gibreel 2019). Only sporadic literature is available on MDR in Indian dairy animals. Therefore, this study was planned to decipher the antimicrobial resistance profile of bacterial pathogens linked to clinical bovine mastitis in dairy cows.

Study design, milk sampling and screening
A hospital-based cross-sectional study was performed on lactating cows that reported to the Veterinary Clinical

Bacterial isolation
Each positive sample (100 μL) was inoculated by streaking on 5% sheep blood agar and incubated aerobically for 24-48 h at 37 °C. The colonies of the suspected pathogens were further subcultured on brain heart infusion agar for lysate preparation and antimicrobial susceptibility testing.

Bacterial identification
The bacterial isolates were identified by observing the nature of growth, cultural characteristics, Gram staining reaction and matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS) analysis (Bruker Daltonics, Bremen, Germany).
The protein was extracted from the bacterial cultures using formic acid and analysed by MALDI-TOF MS as per the procedure recommended by Bruker Daltonics. The bacterial growth was pelleted by centrifugation, mixed with 1 mL of 70% ethanol, and centrifuged at 13,000×g for 2 min. The supernatant was discarded. The pellet was dissolved in 25 µL of 70% formic acid and 25 µL of acetonitrile. The mixture was centrifuged at 13,000×g for 2 min. The supernatant (1 µL) containing the bacterial protein extract was transferred onto the MALDI target plate and allowed to dry at room temperature. After drying, the sample spot was overlaid with 1 µL of MALDI matrix (a saturated solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) and air dried. Mass spectra were then acquired using the Microflex MALDI-TOF MS. MALDI BioTyper 2.0 software was employed for spectral analysis and comparison with the MALDI BioTyper database. A score of ≥ 2 was considered valid for species-level identification, while that between 1.7 and 2 was considered valid for genus-level identification. Score ≤ 1.7 was considered invalid. The staphylococci were further subjected to coagulase test as per the standard protocol.
The bacterial isolates were inoculated in brain heart infusion broth and incubated at 37 °C for 4-10 h until the turbidity reached 0.5 McFarland's standard. The inoculum was spread onto the entire surface of a sterile MHA medium with sterile cotton swabs. The plate was incubated at room temperature for 10 min before placing the antibiotic discs. The plates were then incubated at 37 °C for 16 h. The average of at least two perpendicular diameters of growth inhibition zones was recorded. Resistance of Staphylococcus aureus towards β lactam antibiotics was determined by including penicillin (10 units) discs. Resistance of Staphylococcus aureus and Enterococcus faecalis to glycopeptides was determined by including vancomycin (30 mcg) discs. Cefotaxime (30 mcg) and ceftriaxone-tazobactam (30/10 mcg) discs were used to determine extended spectrum β lactamase (ESBL) producing Escherichia coli.

Statistical analysis
The prevalence of bovine clinical mastitis and antibiotic resistance was expressed in percentage. The breed and lactation stage wise prevalence of clinical mastitis was analysed by using binary logistic regression model in IBM SPSS-20 software package. The results of antibiotic resistance were analysed for statistical significance by applying χ 2 test. Nonsusceptibility to at least one antibiotic in three or more antibiotic groups was considered as MDR.

Results
Totally, 272 milk samples (19.68%) were found to be positive upon clinical inspection as per CMT. The overall logistic regression model was significant indicating effect of breed and lactation stage on the clinical mastitis. The predicted odds of having clinical mastitis was 0.245 which was not very high. The breed (odds ratio 1.193, 95% confidence interval 1.074 to 1.327) and lactation stage (odds ratio 1.246, 95% confidence interval 1.052 to 1.475) were highly significant (p < 0.01) for clinical mastitis.

Breed-wise prevalence of clinical mastitis
The results revealed that there was highly significant (p = 0.001) change in the breed-wise prevalence of clinical mastitis. Within breed effect was significant only for HF cross bred cows, however, for others, the within breed differences were not observed. Between the breeds, the odds of clinical mastitis were not much different when compared to HF cross bred cows. The highest prevalence was recorded in HF crossbred cows, followed by Jersey crossbred, Red Kandhari and Deoni cows (Table 1).

Lactation stage-wise prevalence of clinical mastitis
The lactation stage wise occurrence of clinical mastitis was highly significant (p = 0.011). When the prevalence of clinical mastitis during 7th to 9th months of lactation was compared with the prevalence during 0th to 3rd months and 4th to 6th months, the odds of occurrence of clinical mastitis was higher in 4th to 6th months of lactation as compared to other lactation stages (Table 2).

Antibiotic sensitivity
The species-wise results of the antibiotic sensitivity test for Staphylococci aureus, NAS, Streptococcus uberis, Enterococcus faecalis, Bacillus spp., Escherichia coli, Enterobacter spp., Acinetobacter baumannii and Pseudomonas aeruginosa are presented in Fig. 2.
Acinetobacter baumannii showed almost complete resistance towards all the β lactam antibiotics and oxytetracycline. Non-significant resistance was found against amikacin. The isolates were significantly less resistant (p < 0.001) towards doxycycline, gentamicin, enrofloxacin and ciprofloxacin (16.67% each).
Pseudomonas aeruginosa demonstrated significantly high resistance (p < 0.001) towards oxytetracycline and doxycycline (90.91% each) and significant resistance (p < 0.001) towards amikacin (27.27%). These isolates were found to be completely sensitive towards gentamicin. Nonsignificant less resistance was noted for enrofloxacin and ciprofloxacin (9.09% each). The isolates were significantly sensitive towards aminoglycosides and quinolones.

Multiple drug resistance
The MDR patterns of Gram positive and Gram-negative bacteria associated with bovine clinical mastitis were evaluated (Tables 4 and 5).
At least one Enterococcus faecalis isolate presented MDR towards glycopeptides-tetracyclinequinolones-cephalosporin-β lactam, glycopeptidest e t r a c y c l i n e -c e p h a l o s p o r i n -β l a c t a m a n d glycopeptides-aminoglycosides-quinolones-cephalosporin-β lactam. Glycopeptides-cephalosporin-β lactam and tetracycline-aminoglycosides-cephalosporin-β lactam resistance patterns were recorded in two isolates each. Similarly, three isolates revealed tetracycline/macrolides/glycopeptidecephalosporin-β lactam resistance pattern.
Bacillus cereus isolates (one each) showed MDR towards eight antibiotics distributed over four and five antibiotic groups. Most of the isolates showed resistance towards chloramphenicol-glycopeptide-tetracyclinecephalosporins-β lactams.

Discussion
Our findings revealed the significant prevalence of clinical mastitis in HF crossbred followed by Jersey crossbred cows. The Red Kandhari and Deoni cows were comparatively less susceptible to clinical mastitis. Significantly higher prevalence of clinical mastitis in cross bred cows could be due to higher milk productivity and the associated stress.
The significantly high prevalence of clinical mastitis was observed during 4 th to 6 th months of lactation. This lactation stage was more prone for occurrence of clinical mastitis. The higher prevalence during this stage could be ascribed to the physiological stress of high milk production and alterations in homeostasis (Joshi and Gokhale 2006).
In clinical mastitis, a comparable distribution of Staphylococcus spp., Pseudomonas spp., Streptococcus spp., Pasteurella spp., Enterobacter spp., Klebsiella spp. and Corynebacterium spp. has been reported earlier (Das et al. 2017). When compared with certain previous reports, we have reported higher distribution of Pseudomonas spp. and Enterococcus spp. and less but variable distribution of Staphylococcus spp., Escherichia spp. and Klebsiella spp. (Gundogan and Avci 2014;Jahan et al. 2015;Gao et al. 2017;Salauddin et al. 2020). Other than Staphylococcus spp., isolates such as Pseudomonas spp., Enterococcus spp., Escherichia spp., Streptococcus spp. and Bacillus spp. emerged as the major bacteria associated with bovine mastitis in the study area. We further isolated Pseudomonas aeruginosa and Acinetobacter baumannii, which are of public health significance. Various researchers have reported low isolation rates for Acinetobacter baumannii in milk samples, which is in accordance with the findings of the present study (Gurung et al. 1993;Nam et al. 2010). Variations in the distribution patterns of mastitis bacteria may indicate their geographical dissemination, health status and biosecurity practices of the study area (Salauddin et al. 2019). Jahan et al. (2015) and Salauddin et al. (2020) documented antibiotic resistance in Staphylococcus aureus comparable to the results of the present investigation. Past studies have reported the isolation of antibiotic resistant Bacillus spp. with 91.40% resistance towards penicillin, followed by ampicillin (50.67%), ceftriaxone (35.29%), erythromycin (20.36%) and azithromycin (5.42%) from animals with bovine mastitis. These findings agree with the results of the present investigation (Sadashiv and Kaliwal 2014). All the Escherichia coli isolates were sensitive to gentamicin. On the contrary, earlier reports have suggested that majority of the Escherichia coli isolates from animals with bovine mastitis were resistant to gentamicin and susceptible to amoxicillin and ciprofloxacin (Yang et al. 2018;Lan et al. 2020). Low preference to gentamicin in veterinary therapy may be the reason behind this high sensitivity. Complete resistance to β lactams and the MDR exhibited by Staphylococcus aureus involved in bovine mastitis could be attributed to the indiscriminate use of these antibiotics in therapy (Pitkala et al. 2004;Hendriksen et al. 2008;Kalmus et al 2011). The MDR Staphylococcus aureus reported in this study demonstrated resistance towards vancomycin as reported earlier (Kateete et al. 2013;Nobrega et al. 2018). Use of vancomycin therapy for infections of Gram-positive cocci is critical as the organisms have acquired complete resistance towards β lactam antibiotics. Increasing resistance to vancomycin may have implications on animal and human health.
Earlier reports have divergently suggested the common resistance of Streptococcus uberis towards tetracyclines, followed by gentamicin, and the absence of resistance towards penicillin and ampicillin (Minst et al. 2012). Other studies have reported Streptococci with relatively high MDR as well as complete resistance towards penicillin (Schukken et al. 2012) and intermediate resistance towards vancomycin (Kateete et al. 2013;Nobrega et al. 2018). This report suggested that β lactam-resistant Streptococcus uberis has acquired considerable resistance towards vancomycin too, which may lead to health concerns in the near future.
Bacillus cereus is considered a common contaminant of milk as it is ubiquitously present in the environment of cows and is not viewed as a primary pathogen causing mastitis (Sadashiv and Kaliwal 2014). Its abundant presence in soil and teats and accidental introduction into the udder via the teat canal may explain the high isolation rate in the present study. The results are indicative of lack of hygienic practices in dairy farming operations in the study area.
Previous reports have also hinted the complete resistance exhibited by Pseudomonas aeruginosa against many antibiotics, which is similar to the findings of the current study (Bernal-Rosas et al. 2015). β lactam antibiotics and cephalosporins have been suggested to be essential components in the treatment of Pseudomonas aeruginosa infection (Mesaros et al. 2007). However, the development of high resistance towards these antibiotic groups uncovered in the present report is alarming. Ciprofloxacin remains to be effective, along with gentamicin and enrofloxacin. Similar findings were stated by Hirakawa et al. 2010;Ranjan et al. 2010;Akhoon et al. 2012;Biswal et al. 2014;Kotwal et al. 2016;Awad et al. 2017).
Quinolones are widely used in veterinary and medical healthcare. However, high quinolone resistance in Escherichia coli and other Gram-negative bacteria has been identified in the present study. Similar findings have been documented earlier by Machuca et al. (2017). A few reports on the involvement of ESBL producing and tetracycline resistant Escherichia coli in bovine mastitis (Ghatak et al. 2013;Bandyopadhyay et al. 2015) support the present findings. Indiscriminate antibiotic use may have contributed to the increased  (Chandrasekaran et al. 2014;Hinthong et al. 2017). Moreover, the presence of antimicrobials in the environment of the dairy animals may be the reason for MDR in Escherichia coli, which is ubiquitous in the cow's environment. The ability to produce β lactamase and penicillin binding proteins might have led to resistance towards the penicillin group of antibiotics . Symptomatic therapy may be helpful in alleviating the clinical symptoms instead of resorting to antimicrobial therapy against MDR Escherichia coli (Schukken et al. 2012). The resistance of Escherichia coli to β lactam and cephalosporin group of antibiotics may be due to the production of ESBL. However, resistance towards cephalosporins owing to the production of AmpC cephalosporinase needs to be explored. Our results allude the complete resistance of Klebsiella pneumoniae towards third generation cephalosporins, which reinforces the views of earlier investigators that these drugs will become ineffective against stabilized populations of Klebsiella pneumoniae in most parts of the world by 2030 (Alvarez-Uria et al. 2018). Our findings imply the high prevalence of MDR in Klebsiella pneumoniae as reported earlier (Schukken et al. 2012;Alvarez-Uria et al. 2018). In view of complete resistance to multiple antibiotic groups, therapy should be focussed on reducing the clinical symptoms and saving the cows that suffer from clinical mastitis caused by MDR Klebsiella pneumoniae (Schukken et al. 2012).
The overuse of antibiotics has been suggested to be positively associated with antibiotic resistance in mastitiscausing bacteria. Therefore, their irrational use may not be the ideal solution for mastitis in dairy animals (Saini et al., 2012;Barkema et al. 2015;Kayitsinga et al. 2017). Antibiotic sensitivity test-based selection and prudent use of antibiotics are thus the need of the hour.

Conclusion
This study has determined the wide distribution of penicillinase producing Staphylococcus aureus and ESBL producing Escherichia coli in cows with clinical bovine mastitis. Cephalosporins, tetracyclines, vancomycin and chloramphenicol resistant bacteria were found to be associated with bovine mastitis. Besides, this work has demonstrated the presence of vancomycin resistant enterococci and Staphylococcus aureus, MDR gram negative rods, MDR Pseudomonas and MDR Acinetobacter, which is an issue of public health concern. Furthermore, the widespread resistance of Streptococcus uberis towards cephalosporins, which are commonly used in treating bovine mastitis, has been recorded. MDR transfer from non-pathogens to emerging foodborne and established mastitis pathogens could be a potential problem to the dairy industry and pose public health concerns.