In looking for affordable and alternative therapy in dairy mastitis, the present study investigated the in-vitro antimicrobial activity of four selected medicinal plants against S. aureus and E. coli clinical isolates of bovine mastitis. Our study showed that Brucea antidysenterica was effective at MIC of 100mg/mL on E. coli and 50mg/mL on S. aureus isolates and mean inhibition zone (mm) of 19.28 ± 1.302 and 13.48 ± 0.36 was also recorded at this MIC respectively. Compared to the standard cut-off value for the susceptibility range for the many antibiotics [33], this plant can be considered as effective. As its effect on test organisms, this finding is consistent with one of the previous findings which reported inhibition of S. aureus at 400mg/mL, 200mg/mL, 100mg/mL, 50mg/mL and 25mg/mL concentration levels [34]. Additionally this finding was in line with the previous report of 8mm mean inhibition zone at 20% MIC against the S. aureus isolated from clinical bovine mastitis [16]. A relatively higher degree of wound healing percent of this plant extract at 50mg/mL- 100mg/mL MIC was also reported [35].
The methanolic extracts of Crotona macrostachyus showed minimum inhibition zones at the concentration of 100mg/mL against both E. coli and S. aureus using broth dilution method and 20.2 ± 1.48 and 11.64 ± 0.86 mean zone inhibition (mm) at this concentration respectively. Compared to the standard cut-off value for the susceptibility range for the conventional antibiotics [33], this plant can be considered effective. This result agrees with the finding of Biruhalem et al., [36], who reported the MIC of 12.5% against S. aureus and E. coli isolated from wound specimens at Aklilu Lemma Institute of Pathobiology, Ethiopia. Sendeku et al.,[37] also reported efficacy of this plant extract with 1.5% MIC for both isolates and 8.33 ± 1.53 and 24.00 ± 1.73 mean zone of inhibition (mm) on both S. aureus and E. col isolated from patient specimens visiting Hospital respectively.
The methanolic extract of Datura Stamonium had recorded a higher MIC against E. coli. Accordingly, MIC up to 200mg/mL and 100mg/mL were recorded on E. coli and S. aureus isolates respectively while 15.04 ± 0.58 and 13.8 ± 0.84 mean zone inhibition (mm) at this MIC was also recorded using Muller Hinton agar diffusion method. The current study agrees with 20mg/mL MIC of methanol extract Datura Stamonium on E. coli procured from the microbial type culture collection [38]. However, a study conducted on S. aureus and E. coli clinical isolates of hospital patients at MIC of inhibition (mm) of 12.5mg/mL and 6. 25mg/mL concentrations was 14.6 ± 2.3 and 9.2 ± 1.6 respectively[39]. Alemu et al.,[40] also reported at MIC 12.5mg/mL the mean inhibition zone (mm) 13 and 15 on clinical isolates of pathogenic bacteria of S. aureus and E. coli, obtained from Hospital. The current finding is nearly in agreement with this finding concerning the concentration levels and the mean zone inhibition at a concentration of MIC against S. aureus.
The extract of Prunes africanas was also tested for its effect as an antimicrobial against E. coli and S. aureus isolates of mastitic cow milks. During this study 50mg/mL and 100mg/mL MIC of Prunes africanas against S. aurues and E. coli respectively. Likewise, 12.9 ± 0.7416198 and 13.2 ± 1.643168 visible mean zone of inhibition (mm) at this MIC was recorded on E. coli and S. aureus respectively. The variation of efficacy among the above-mentioned phytopreparations could be attributed to the way of plant preparation, the season of collection, stage of the plant, place of collection, way of extract drying, means of extraction, the solvent used, preservation or storage of the extract till evaporation and other unnoticed factors.
The result from comparison of MIC of plant extract with conventional antimicrobial discs showed that the efficacy of all tested plant extract against S. aureus at their respective MIC were comparable except with Chloramphenicol. The least mean inhibitory zone recorded by Prunes Africana against E. coli might be due to the capacity of this plant extract penetrating through the media [41]. The mean inhibition zone obtained by Oxytetracycline was almost comparable to that obtained by Prunes Africana and Datura Stamonium against E. coli while Crotona macrostachis and Brucea antidysenterica was greater than the drugs. The mean inhibition zone of Erythromycin, Ampicillin, Amoxicillin was less than that of all tested plant extracts, however, chloramphenicol and cefotaxime were higher than that of all tested plant extract against E. coli. Similarly, the mean inhibition zone obtained by Kanamycin was almost comparable to that obtained by Datura Stamonium while less than that of Crotona macrostachis and Brucea antidysenterica and a little higher than that of Prunes Africana against E. coli as reported by previous study[42].
In case of S. aurues the mean zone inhibition of Datura Stamonium, Brucea antidysenterica and Prunes Africana was comparable with that of Amoxicillin, Streptomycin and Cetaximine. Almost nearly equivalent mean zone of inhibition were noted by Crotona macrostachis, to that of Ampicillin resistant isolates of S. aureus, however a bit lower than the mean inhibition zone of Brucea antidysenterica, Datura Stamonium and Prunes Africana. The mean inhibition zone obtained by Penicillin G against resistant isolates of S. aureus was also higher than that obtained by four tested plant extract. Although there is no established standard formula to judge the level of the zone of inhibition to say resistant, intermediate, and susceptible for plant extract, the mean inhibition zone of Penicillin G against resistant isolates of S. aureus in this study was below the established standard of resistance formulated by CLSI [33].
Interestingly, all tested plant extracts were showed a greater inhibitory zone than Ampicillin, Amoxaccillin and Streptomycin resistant isolates of E. coli. The mean inhibition zone obtained by Oxytetracycline against both E. coli and S. aureus was smaller than that obtained by Brucea antidysenterica, Datura Stamonium and Prunes Africana and still it was less than the result obtained by Crotona macrostachis. The comparison among these test materials suggests that the herbal preparations do have a capacity to inhibit the growth of test organisms and thus can be used to treat an emerging antibiotic-resistant pathogens that has implications for not only veterinary patients, but also humans [43].
The overall finding showed that medicinal plant extract has highest inhibition zone against E. coli organism at the highest MIC (100-200mg/mL) as compared to S. aureus in which the lowest inhibition zone was seen at the lowest MIC (50-100mg/mL). The observed difference in antibacterial activities of extracts between E. coli and S. aureus clinical isolate may be attributed to the difference in the outer membrane of both isolates. Gram-negative (E. coli) bacteria possess a high permeability barrier for numerous antibiotic molecules similarly for these extracts. Their periplasmic space also contains enzymes, which are capable of breaking down foreign molecules and appears to be less susceptible to plant extracts than gram-positive bacteria [44]. Despite the limitation of infrastructures and facilities to conduct HPLC-based purify the active ingredient from the plant and genetic based identification of drug resistant gene, this study delineated the possibility that phytopreparations could play an important role in the treatment of resistant isolates of mastitis causal agents and it could be the alternative solution to prevent the problem of ever-emerging resistant isolates in any disease situation.