Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Effect of Atorvastatin and Vitamin D against Multi-drug Resistant Staphylococcus spp and Escherichia coli Isolated from Bovine Mastitis Cases

K.S. Sriraam1, T. Ramasamy1,*, K. Porteen1, A. Elamaran1, M. Pavithra1, T. Jagadeesh1, S. Rames1
1Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
Background: In the current scenario, the emergence of antimicrobial resistance has made it difficult to treat bacterial infections alone using antibacterial agents.

Methods: Milk samples collected from 100 mastitis affected cows from August 2018 to June 2019 and were stored at 4°C. After species confirmation, isolates were subjected to ABST and screened for resistance genes - mecA, blaTEM using PCR. Minimum Inhibitory Concentration (MIC) of tetracycline and ampicillin were determined against both Staphylococcus spp and E. coli, alone and in combination with atorvastatin, vitamin D by using a modified microdilution method. 

Result: Staphylococcus and E. coli isolates showed 80.65% and 85.71%, 100% and 85.71% resistance against tetracycline and ampicillin, respectively. Atorvastatin and vitamin D did not display antibacterial effects as sole agents against both bacterial species. However, there was a significant decrease in the MIC of ampicillin against E. coli and Staphylococcus spp when combined with atorvastatin and vitamin D but not for tetracycline.
Mastitis is an inflammation of the udder caused by a various microorganisms, is a widespread and economically devastating disease in dairy cattle reported across the world (Abebe et al., 2016). A wide variety of organisms are implicated in the occurrence of mastitis, chief among them being Staphylococcus aureus and E. coli (Aksoy, 2021). The principal treatment for mastitis is administration of antibacterial agents, namely ampicillin, cloxacillin, penicillin G, streptomycin and tetracycline (Bhosale et al., 2014). However, the effective treatment of bovine mastitis mainly depends on the antimicrobial susceptibility of the organisms, the type of mastitis, the type of cattle breed and the treatment regimen (Barkema et al., 2006).

As the udder quarters were infected with penicillin-sensitive pathogens, penicillin G to be used as first-line antibiotic agents (Grave et al., 1999). However, the appearance of penicillin G resistant staphylococci causing bovine mastitis has been reported (Ramasamy et al., 2021). Tetracycline is the most intensively used antibiotics due to its relative safety, low cost and broad-spectrum activity against Gram-positive, Gram-negative and Mycoplasma (Al-Nazawi, 2006). Antibiotics like ampicillin and tetracycline, which were hitherto effective in the treatment of mastitis, have reported significantly reduced efficacy (Mirzaagha et al., 2011). Treatment of mastitis has become increasingly difficult and challenging owing to the rapid emergence of antimicrobial resistance (Beuron et al., 2014).

Recently, the non-antibiotic drugs such as atorvastatin and vitamin D possess pleiotropic properties, the anti- bacterial effect being one such quality that has received a lot of attention lately (Masadeh et al., 2012; Golpour et al., 2019). The increasing problem of antibiotic resistance, the discovery of newer antibiotics having almost come to a standstill, has prompted the scientific community to look for ways to strengthen the efficacy of antibiotics through synergistic interaction with non-antibiotic drugs. Because of the above, the purpose of this study was to determine the in-vitro antibacterial effect of atorvastatin and vitamin D alone and in combination with antibiotics against multi-drug resistant Staphylococcus spp and E. coli isolated from bovine mastitis cases.
Sample collection
The study was conducted at the Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Chennai, Tamil Nadu, India from August 2018 to June 2019. A total of 100 milk samples were collected from different cows suspected for mastitis from the Department of Clinics, Madras Veterinary College Teaching Hospital, Chennai. About 15 mL of milk was aseptically transferred to a sterile universal sampler tube and were refrigerated at 4°C.
Isolation and characterization
Selective isolation of Staphylococcus spp and E. coli was performed using Mannitol Salt Agar (MSA) and Eosin Methylene Blue agar (EMB), respectively. The individual colonies obtained from selective media were stored as glycerol stocks at -20°C. Biochemical kits were used for the characterization of the isolates (HiMediaTM). Polymerase Chain Reaction (PCR) was performed for genotypic characterization of the isolates (Fang and Hedin, 2003) including tuf gene for Staphylococcus spp, uspA gene for E. coli and nuc gene for S. aureus.
Resistance genes
PCR was carried out for detection of resistance genes blaTEM for ampicillin, tetM and tetB for tetracycline. The PCR products were separated using electrophoresis and the gel were visualized on Mega Capt Gel Doc. A 100bp DNA ladder (ThermoscientificTM) was employed to determine the size of the PCR products. The list of primers, cycling conditions for identifying genes and resistance genes are given in Table 1-3, respectively.

Table 1: List of primers used in this study.

Table 2: PCR cycling conditions for identification of genes.

Table 3: Cycling conditions for resistance genes.

Antibacterial sensitivity patterns
An antibiotic sensitivity test (ABST) was performed using the modified Kirby-Bauer disc diffusion method. An overnight inoculum of culture was diluted to 0.5 McFarland standards and used for ABST. All the antibiotic discs were purchased from HiMediaTM. For evaluation of combinations, 1mg of atorvastatin, vitamin D (separately) was dissolved in 1ml of Dimethyl Sulfoxide (DMSO) and 8 µl (8 µg) and 15 µl (15 µg) of the same solution was added to the tetracycline and ampicillin discs. The discs were allowed to be set for 20 minutes at room temperature and then incubated at 35-37°C for 24 hours. A sterile disc inoculated with the same volume of DMSO served as a control (Haeri et al., 2015).
Minimum inhibitory concentration (MIC)
MIC was evaluated using the modified micro-dilution method using the resazurin indicator. A 96 welled plate was used for testing of samples and the resulting colour change was noted. The concentration of the drug in the well was calculated to find MIC (Elshikh et al., 2016). The drug solutions used were ampicillin (300 mg/ml), tetracycline (100 mg/ml), atorvastatin (100 mg/ml) and vitamin D (100 mg/ml) in DMSO.
Statistical analysis
Using one way ANOVA, followed by a Post-hoc Duncan test was performed with SPSS software for statistical analysis of the data generated. A p-value <0.005 was considered significant.
100 milk samples were collected from cows with mastitis, 60 samples (60%) showed growth on MSA and/or EMB agar. In that, 40 samples were positive for Staphylococcus spp. (66.67%) (Fig 1) and other 20 samples streaked onto EMB agar produced metallic green sheen (33.33%) positive for E. coli (Fig 2). Six samples (10%) were produced colonies in both MSA and EMB agar, indicative of polymicrobial infection.The isolates were characterized as Staphylococcus spp (72.5%) and E. coli (35%) by using commercial biochemical test kits.

Fig 1: MSA - golden yellow colonies.

Fig 2: EMB plates - metallic green colonies.

Genotypic confirmation of Staphylococci spp was carried out through tuf gene (Fig 3) and S. aureus through nuc gene using PCR (Fig 4). Based on results, 77.5% (31/40) isolates were Staphylococci spp and among these 41.94% (13/31) isolates were S. aureus. The presence of E. coli was identified in 35% of isolates (Fig 5).

Fig 3: Genotypic confirmation of tuf gene.

Fig 4: PCR assay for nuc gene.

Fig 5: PCR assay for E. coli targeting uspA gene.

The ABST targeted for Staphylococcus spp isolates revealed 100% resistance against penicillin and ampicillin, 83.87% samples were observed resistant to methicillin and 80.65% were found to be resistant to tetracycline, gentamicin, vancomycin and amikacin. The co-trimoxazole showed 74.19% resistance and 61.29% samples resistant to ciprofloxacin and ceftriaxone. 58.06% and 51.61% noticed resistance to cefotaxime and enrofloxacin respectively.

Based on the sensitivity test, E. coli isolates revealed that 85.71% were found to be resistant to tetracycline, gentamicin, cefotaxime, ceftriaxone, co-trimoxazole, ampicillin, amikacin and ciprofloxacin and 75.43% samples resistant to enrofloxacin. Complete resistance (100%) was observed against penicillin, methicillin and vancomycin. Methicillin resistance was found that 38.46% in S. aureus (Fig 6) and 16.67% in other Staphylococcus spp. The blaTEM gene were expressed in 16.13% of Staphylococcus spp and 14.29% of E. coli isolates.

Fig 6: Staphylococcus aureus isolates containing mecA gene.

The antibacterial effect of atorvastatin, vitamin D revealed no significant antibacterial activity against both Staphylococcus spp and E. coli isolates (Fig 7). However, the combination of atorvastatin and vitamin D with ampicillin indicated wider zones of inhibition against both Staphylococcus spp and E. coli (Fig  8 and 9). 

Fig 7: Zone of inhibition of atorvastatin and vitamin D alone.

Fig 8: Zone of inhibition of atorvastatin with ampicillin and tetracycline.

Fig 9: Zone of inhibition of vitamin D with ampicillin and tetracycline.

There was increased MIC of tetracycline for E. coli (2.53 µg/ml) and Staphylococcus spp (2.45 µg/ml) observed. The combination of tetracycline with atorvastatin and vitamin D decreased the MIC to 1.73 µg/ml and 1.51 µg/ml for Staphylococcus spp respectively. Similarly, for E. coli, the MIC was reduced 1.89 µg/ml with tetracycline-atorvastatin and 1.56 µg/ml with tetracycline-vitamin D combination, which was non significant (Table 4).

Table 4: MICs of tetracycline.

The MIC of ampicillin for E. coli isolates was 22.62µg/ml and the recommended CLSI (2016) MIC breakpoint for ampicillin against E. coli is £8 µg/ml. The increase in MIC of ampicillin for Staphylococcus spp isolates was 17.20 µg/ml where as the recommended MIC for ampicillin against Staphylococcus spp by CLSI (2016) is £4 µg/ml. The MIC of ampicillin in combination with atorvastatin against Staphylococcus spp and E. coli was 2.49 µg/ml and 2.61 µg/ml, respectively. The combination of ampicillin with vitamin D against both Staphylococcus spp and E. coli produced an average MIC of 2.742 µg/ml and 1.092 µg/ml, respectively, which was statistically significant (Table 5).

Table 5: MICs of Ampicillin and its combinations.

In the present study, Staphylococcus spp (77.5%) was the predominant bacteria isolated from the bovine mastitis, followed by E. coli (35%). Previous studies in our laboratory have also shown that Staphylococcus spp (94%) and E. coli (50%) are the most common bacteria usually in mastitis affected milk (Ramasamy et al., 2021). Similar findings were also reported by Jeykumar et al., (2013) and Nalband et al., (2020). Among these Staphylococcus spp, 41.94% of samples were carried the S. aureus (nuc) gene, which is the highest prevalence compared to our previous work (12.9%) (Ramasamy et al., 2021). This may be due to increased resistance S. aureus organism in years progress.

ABST results revealed complete resistance of penicillin against both bacterias. The isolates of Staphylococcus spp and E. coli were showed 80.65% and 85.71% resistance to tetracycline, respectively. Recent study also reported the Staphylococcus spp and E. coli were resistant to penicillin and tetracycline with high MIC (Anurag et al., 2021; Ramasamy et al., 2021). The increased resistance pattern observed in present study shows that β- lactams and tetracyclines are leading in front for the treatment of mastitis.

The lowest prevalence of beta lactam resistance genes were found in the both Staphylococcus spp and E. coli isolates from bovine mastitis.  None of the tested isolates was positive for tet M and tet B genes. There was increased resistance reported for tet M (67.70%) and tet B (75%) in our previous study (Ramasamy et al., 2021). This variation in the resistance genes expression may be due to changes in the study period.

In the current study, the sole antibacterial effect of atorvastatin was not observed. This result was supported by Manalo et al., (2017), who observed no antibacterial activity in in vitro. This may be due to resistant bacterial isolates from clinical samples used in this study, whereas previous workers were used the laboratory standard bacterial culture. The combination of ampicillin and atorvastatin reduced the MIC of ampicillin against both Staphylococcus spp and E. coli. In vitro and in vivo studies of statins have shown antimicrobial effects against both organisms (Ko et al., 2018; Choudhary et al., 2015; Thangamalai et al., 2014).

The ampicillin-vitamin D displayed a synergistic antibacterial effect against both study organisms, with a significant decrease in MIC. Also, there is ample evidence to prove that vitamin D possesses antimicrobial properties (Saputo et al., 2018; Youssef et al., 2011). However, it is still unclear how it is responsible for potentiating the antibacterial effect of another antibiotic.

Though the combination of tetracycline with the non-antibiotic drugs viz., atorvastatin and vitamin D produced non-significant reduction in MIC against previously resistant clinical isolates. This difference between tetracycline and ampicillin might be because of tetracycline is primarily inhibiting the protein synthesis and ampicillin is a cell wall synthesis inhibitor.
The atorvastatin and vitamin D improves the effect of ampicillin against resistant bacteria indicating their role in bacterial cell wall synthesis. However, further studies are required to ascertain the exact mechanism of action insofar as its antibacterial property is concerned.
This study was funded by Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India.

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