Antimicrobial activity of six different plant extracts against clinical isolates of Staphylococcus spp. from milk origin

Introduction

Since the dawn of time, thousands of years ago, nature has been a source of medicinal agents (Tyler et al., 1988; Chopra and Doiphode, 2002) [28,9] . Nearly all plants in India have therapeutic properties, and use of medicinal plants, particularly in traditional medicine, are now widely accepted (Clark, 1996) [6] . The presence of secondary metabolites such as alkaloids, tannins, saponins, flavonoids, anthraquinones, glycosides, volatile oils, terpenes, essential oils, and resins may account for the therapeutic benefit of the plants (Subramani et al., 2017) [25] . Around 70 to 80% populations in the developing countries use medicinal plants to treat diseases (Palhares et al., 2015) [21] . The World Health Organization states that one of the best sources for antibacterial drug is medicinal plants (Manandhar et al., 2019) [19] . In the last 20 years, research into various extracts made from conventionally used medicinal plants as possible sources of novel antimicrobial agents has gained more and more attention to combat the drug resistance (Charlandy et al., 1999) [7] . Antibacterial resistance to the medications has started to emerge as a result of the abuse and overuse of antibiotics in the treatment of diseases, and it has gotten worse due to selective pressure (Bhalodia and Shukla, 2011) [4] . Staphylococcus spp. one of the most common genus of bacteria frequently implicated in cases of bovine mastitis, has been documented multiple times as an antibioticresistant organism (Costa et al., 1992; Taponen et al., 2006) [5,30] . Because of the emergence of resistant bacterial strains and limited discovery of novel antimicrobial compound (Yadav and Agarwal, 2011) [31] , for the global management of highly contagious and recurrent infectious diseases necessitates the development of novel medicines, especially derived from naturally occurring plant metabolites. This study aimed to evaluate antibacterial activities of aqueous and methanolic extracts from six different plants viz., Piper nigrum (seed), Kigellia pinnata DC. (fruit), Azadirachta indica (leaves), Cymbopogon citrate (leaves),

Preparation of the inoculums

The cultures were grown on Mueller Hinton broth (MHB) (Himedia, India) at 37 °C for 18 hours. The turbidity was adjusted to 0.5 McFarland standards.

Antibacterial activity of plants extract 2.2.3.1 Disk diffusion method

MHB was inoculated with isolates of Staphylococcus spp. and incubated at 37 °C overnight. Bacterial suspension was adjusted to 0.5 McFarland standards before use. As per Imran et al., (2021) [14] the inoculum was spread uniformly over the entire surface of the MHA plates. These plates were allowed to dry for few minutes and then a sterile filter paper disc of 6 mm diameter, to which different extracts at a concentration of 100 mg/ml were absorbed, and were placed, with the help of forceps. Commercially available antibiotic discs were used as positive control while methanol was used as vehicle control. The plates were incubated at 37 °C for 18-24 hrs. Inhibitory zone diameters (mm) were measured with the help of scale. Each assay was repeated thrice, and the mean diameters were recorded.

Determination of Minimum Inhibitory Concentrations (MIC) of plants extract by 2.2.3.2.1 Micro Broth dilution method

The antimicrobial activity of extracts against all isolates was investigated at various concentrations in a 96-well microtiter plate. As per Sultana et al. (2016) [24] , each extract was serially diluted (two-fold) upto 9 th well using a sterile MHB medium, and the 10 th , 11 th and 12 th wells serving as vehicle, media and culture controls, respectively. Each plant extract dilution was inoculated with 100 μl of log phase microorganism (0.5 McFarland). The 96-well microtiter plate was then kept at 37 °Celsius for 18 hours. The MIC value of the extract was recorded after 18 hours. Each extract was tested for antibacterial activity in triplicate Zihadi et al., 2019 [32] .

Minimum Bactericidal Concentration (MBC)

The MBC was determined by collecting a loop full of broth from those wells which did not show any growth in MIC assay, two wells above and two wells below the MIC value are inoculated on sterile MHA plate by streaking and incubated at37 °C for 18-24 hours. The highest dilution that did not yield a colony on a solid medium was considered as MBC Zihadi et al., 2019 [32] .

Antibacterial activity with commercially available antibiotics 2.2.4.1 Disk diffusion test

A disc diffusion test was conducted in accordance with National Committee for Clinical Laboratory Standards (CLSI) guideline 2023 to determine the antibiotic susceptibility against all clinical isolates. Commercially available 6mm diameter antibiotic discs procured from Himedia were used in this study viz., Gentamicin (10 mcg), Enrofloxacin (5 mcg), Tetracycline (30 mcg), Erythromycin (15 mcg) and Chloramphenicol (30 mcg). 0.5 McFarland bacterial suspension was seeded over the MHA plate followed by placement of the disc with the help of forceps. The plates were incubated at 37 °C for 18-24 hrs. Zone of Inhibition (ZOI) in mm were measured with the help of scale. Each assay was repeated thrice, and the mean diameters were recorded.

MIC of Antibiotics

As per CLSI (2023) guideline the MIC of antibiotics was determined by first adding 100 μl of MHB to a 96-well sterile microtiter plate. Twofold dilution of antibiotics, from the first to the tenth well was done. The 11 th and 12 th wells are used for media and culture control respectively. Next, the microorganism suspension was diluted in MHB broth by a ratio of 1:100. Subsequently, the adjusted bacterial suspension was introduced into each well that had varying antibiotic concentrations. The microtiter plate was then incubated at 37 °C for 18 hours. MIC was defined as the concentration at which the organism did not appear to be growing. The bactericidal activity of each drug was examined in triplicate.

Results and discussion

Disk diffusion assay of plants extract

Six plant extracts from Piper nigrum (P), Kigellia pinnata DC. (K), Azadirachta indica (A), Cymbopogon citrates (Cy), Tagetes erecta (T) and Emblica officinalis Gaertn. (E) are investigated to evaluate their antibacterial activity against clinical isolates of Staphylococcus spp. using disk diffusion method. Evaluation of antibacterial activity of these plant extracts are presented in Table 1 and graphically represented in Fig 1 . The results revealed that all plant extracts were potentially effective in suppressing microbial growth with 100 mg/ml concentration. The highest zone of inhibition was observed with the T (12.02±0.14 mm) followed by Cy (10.60±0.15 mm), K (10.02±0.19 mm), A (9.98±0.21 mm), P (8.10±0.40 mm) and E (7.71±0.12 mm), respectively. Very less growth inhibition zone was seen with the extracts of E (7.71±0.12 mm). T (12.02±0.14 mm) was the most effective extract retarding microbial growth at concentration of 100 mg/ml. Similar studies was reported by Aldaly, 2010 [2] ; Ewansiha et al., 2012 [12] ; Latifian et al., 2021 [17] with extracts of Piper nigrum; Cymbopogon citrates; Tagetes erecta showing almost similar ZOI. On the other hand, Agyare et al., 2013 [3] studied the susceptibility of methanolic extracts of Kigellia Africana (leaf and stem) and found 14.50±0.50 mm ZOI which is slightly higher than our findings. This may be due to different range of activity with different parts of the plants as per Latifian et al., 2021 [17] or may be due to extraction media (Gupta et al., 2019) [13] . Similarly, for Emblica officinalis extracts our finds for antimicrobial susceptibility is poor when compared to findings of Gupta et al., 2019 [13] . In contrast to our findings, Kumar et al., 2018 [16] observed methanolic extracts of Azadirachta indica that had lower ZOI.

MIC and MBC of the plant extracts

The MIC of six plant extracts was employed by micro broth dilution method. The mean MIC values of all plant extracts were reported in Table 2 and the graphical representation was shown in Fig 2 . The lowest MIC was observed with the extract of T (16.96±0.00 mg/ml) followed by Cy (68.57±0.00 mg/ml), K (108.57±0.00 mg/ml), A (112.50±0.00 mg/ml), P (126.79±0.00 mg/ml), where as E, showed the highest MIC of 200.00±0.00 mg/ml, respectively. Out of all prepared plant extracts, Tagetes erecta shows a good antibacterial activity with least MIC value among all the prepared plant extracts against all the isolates followed by Cymbopogon citratus, Kigellia pinnata DC., Azadirachta indica, Piper nigrum and Emblica officinalis Gaertn., respectively. The MBC of the six plant extracts was determined by sub-culturing the well that exhibits no growth on a fresh media, two wells above and two wells below the MIC value. The MBC values of the plant extracts T, Cy, K, A, P, and E are 22.32±0.00 mg/ml, 90.00±0.00 mg/ml, 120.00±0.00 mg/ml, 143.75±0.00 mg/ml, 152.14±0.00 mg/ml, and 257.14±0.00 mg/ml, respectively. The MBC values are presented in Table 3 and graphically represented in Fig 3 . The plant extracts of Piper nigrum, Cymbopogon citrates and Emblica officinalis exhibit lower antibacterial activity in terms of MIC and MBC value when compared to study of antibacterial activity of those of Aldaly 2010 [2] , Ewansiha et al., (2013) [12] and Sharma and Pundir et al., 2018, respectively. This could be the result of the extraction medium (Gupta et al., 2019) [13] or the plant extracts themselves, which might still include impurities that would inhibit their function (Tereschuk et al., 2004) [29] . As a result, extraction in various media is necessary to determine which media performs best. Purification is also crucial since it concentrates the active component and increases its activity (Ewansiha et al., 2012) [12] . Extracts of Kigellia pinnata DC., Azadirachta indica, and Tagetes erecta were reported to have good antibacterial activity by Kimutai 2014 [15] , Muhammad et al., 2019 [20] , and Motamedi et al., 2015, respectively. However, our findings show that the plant extracts have very good antimicrobial activity with lower minimum inhibitory concentrations than their reporting. Several studies were done on P, K, A Cy, T and E plant extracts to determine their antimicrobial activity against Staphylococcus aureus; nevertheless, very few studies have been conducted on clinical isolates of Staphylococcus spp. from milk origin. In general, the prepared plant extracts exhibit a good antibacterial activity against the clinical isolates of Staphylococcus spp. However, studying a greater number of isolates as well isolation and purification are required in future to give a good antimicrobial picture by the plant extracts.

Susceptibility Test with antibiotics

To determination the susceptibility of microorganisms to antimicrobials, disk diffusion test and MIC test was performed. Disk diffusion test also commonly known as Kirby Bauer test was used to determine the susceptibility of the clinical isolates to different commercially available antibiotics. The results of disk diffusion test performed against 5 antibiotics viz., Gentamicin (Gen), Enrofloxacin (EX), Tetracycline (TE), Erythromycin (E) and Chloramphenicol (C) in freshly prepared MHA plates are given in Table 4. The mean growth inhibited zone is given in mm±standard error. Generally, all the tested isolates were found to be susceptible to extracts with variable zones of inhibition. Studies revealed ZOI for Gen, EX, TE, E and C were 14.69±0.27, 21.36±0.26, 20.64±0.33, 14.52±0.33 and 14.62±0.40, respectively against all the isolates of Staphylococcus spp. MIC is defined as the lowest concentration at which antimicrobial inhibits the visible growth of a microorganism after overnight incubation. MIC of 5 antibiotics was performed against all the isolates by micro broth dilution method in a 96 well microtiter plate. The mean MIC (µg/ml) value against all the isolates was presented in Table 5. Studies revealed that TE shows 78.57 % sensitivity, which is highest among all the antibiotics followed by Gen (57.14%), C (28.57%), whereas EX and E shows only 7.14% sensitivity. The intermediate sensitivity was observed against EX and C (28.57%), Gen and TE (7.14%). The isolates show highest resistance against E (92.86%), followed by EX (64.26%), C (42.86%), Gen (35.71%) and TE (14.29%), respectively. The percent susceptibility of the antibiotics against all isolates is graphically represented in Fig [27,22,23,10,8] and Archer and Climo, 1994 [1] . They studied the antibiotic resistance pattern in Staphylococcus spp. of milk origin and found that most of the isolates were resistant to Erythromycin and sensitive to Tetracycline and Gentamicin. 00±0.00 60.00±0.00 30.00±0.00 60.00±0.00 60.00±0.00 60.00±0.00 120.00±0.00 60.00±0.00 60.00±0.00 60.00±0.00 60.00±0.00 120.00±0.00 60.00±0.00 120.00±0.00 68.57±0.00 T 6.25±0.00 12.50±0.00 6.25±0.00 12.50±0.00 12.50±0.00 12.50±0.00 12.50±0.00 12.50±0.00 12.50±0.00 12.50±0.00 12.50±0.00 50.00±0.00 12.50±0.00 50.00±0. 00±0.00 120.00±0.00 30.00±0.00 120.00±0.00 60.00±0.00 120.00±0.00 120.00±0.00 120.00±0.00 120.00±0.00 60.00±0.00 60.00±0.00 120.00±0.00 60.00±0.00 120.00±0.00 90.00±0.00 T 6.25±0.00 25.00±0.00 6.25±0.00 25.00±0.00 12.50±0.00 25.00±0.00 25.00±0.00 25.00±0.00 25.00±0.00 12.50±0.00 12.50±0.00 50.00±0.00 12.50±0.00 50.00±0.00 22.32±0.00 E 200.00±0.00 200.00±0.00 200.00±0.00 400.00±0.00 200.00±0.00 200.00±0.00 400.00±0.00 200.00±0.00 200.00±0.00 200.00±0.00 200.00±0.00 400.00±0.00 200.00±0.00 400.00±0.00 257.14±0.00 Values are expressed as Mean±Standard Error. Experiments were performed in triplicates. P = Piper nigrum, K = Kigellia pinnata DC., A = Azadirachta indica, Cy = Cymbopogon citrates,T = Tagetes erecta and E = Emblica officinalis Gaertn. 0.50±0.00 1.00±0.00 0.50±0.00 0.50±0.00 64.00±0.00 2.00±0.00 64.00±0.00 0.25±0.00 0.25±0.00 64.00±0.00 64.00±0.00 8.00±0.00 1.00±0.00 64.00±0.00 Enrofloxacin (EX) 0.50±0.00 32.00±0.00 2.00±0.00 2.00±0.00 64.00±0.00 4.00±0.00 16.00±0.00 2.00±0.00 2.00±0.00 64.00±0.00 64.00±0.00 64.00±0.00 8.00±0.00 64.00±0.00 Tetracycline (TE) 2.00±0.00 4.00±0.00 4.00±0.00 2.00±0.00 4.00±0.00 16.00±0.00 4.00±0.00 4.00±0.00 4.00±0.00 8.00±0.00 2.00±0.00 1.00±0.00 4.00±0.00 64.00±0.00 Erythromycin (E) 32.00±0.00 64.00±0.00 64.00±0.00 64.00±0.00 64.00±0.00 16.00±0.00 16.00±0.00 16.00±0.00 16.00±0.00 32.00±0.00 32.00±0.00 64.00±0.00 0.50±0.00 64.00±0.00 Chloramphenicol (C) 1.00±0.00 32.00±0.00 16.00±0.00 16.00±0.00 64.00±0.00 32.00±0.00 4.00±0.00 16.00±0.00 16.00±0.00 8.00±0.00 32.00±0.00 32.00±0.00 4.00±0.00 32.00±0.00 Values are expressed as Mean±Standard Error. Experiments were performed in triplicates.

Conclusion

The study's findings show that the prepared plant extracts exhibit strong antibacterial activity against Staphylococcus spp. clinical isolates from milk origin. They can therefore be used to exploit for novel drugs in near future. More isolates, as well as the isolation and purification of the phytochemical compound using different extraction media, may be studied in future research.

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