Isolation, characterisation and confirmatory identification of enteropathogenic E. coli
A total of 135 isolates of
E. coli were recovered from 150 faecal samples from diarrhoeic neonatal goat-kids (Fig 1). Pathotype specific PCR targeting bfpA resulted in to a single amplicon of 324 bp, which indicates confirmatory identification of EPEC as illustrated in Fig 2. In the current study, EPEC was selected as target pathogen, because it is the most common cause of colibacillosis in neonatal goat-kids as reported by
Mishra et al., (2019). The pattern of antibiotic resistance shown by EPEC isolates indicated their multi-antibiotic resistant status (Table 1).
Antibacterial efficacy of the selected plants against the resistant EPEC
Among the 9 methanolic extracts evaluated for their antibacterial activity, only extracts of
Holarrhena antidysenterica (bark),
Acacia nilotica (bark),
Punica granatum (rind),
Gloriosa suberba (leaves) and
Aegle marmelos (fruit) showed antibacterial activity against antibiotic resistant clinical isolates of EPEC (Table 2). The leaves of other 4 medicinal plants
viz. Cassia tora,
Bambusa arundinacea,
Murraya koenigii and
Psidium guajava did not exhibit any antibacterial potential against the resistant EPEC isolates (Table 2). The maximum mean inhibition zone of antibacterial effect was demonstrated by the methanolic extract of
Holarrhena antidysenterica (19.16±0.76 mm) followed by
Acacia nilotica (18.00±1.00 mm) and
Punica granatum (16.67±1.53 mm) (Table 2). Standard antibiotic,
i.e., ciprofloxacin produced consistent inhibition zone of 32 mm and served as positive control, whereas discs impregnated with aqueous tween-20 solution (1%) didn’t exhibit any antibacterial activity in the form of inhibition zone and served as negative control.
The antibacterial property of plant extracts varies with type of solvents used (Rani and Khullar, 2004). Methanolic or acetone extracts of plants possessing antimicrobial potential exhibited much more pronounced activity in comparison to ethanol or aqueous extract against a variety of pathogenic microbes like
E. coli (Eloff, 1998). Taking these studies into the consideration, the methanolic extracts of the herbs were used for evaluation of their antimicrobial properties.
During
in vitro screening of plant extracts for antibacterial property,
Holarrhena antidysenterica exhibited maximum zone of inhibition and therefore, it was considered for further evaluation in terms of MIC and MBC. After 24 hr of incubation, turbidity appeared in tubes containing the extract @ 0.5 and 1.0 mg/ml, whereas no growth was observed in all the other tubes (containing
Holarrhena antidysenterica extract @ 2,3,4,5,6,7,8,9 and 10 mg/ml) on visual inspection. Lush growth was observed in the tube not containing any amount of the extract. On the basis of the above finding, the MIC of
Holarrhena antidysenterica extract was considered to be 2 mg/ml. During MBC trial, good growth was observed on nutrient agar plates streaked with the inoculum taken from MIC tubes having
Holarrhena antidysenterica extract @ 0.5 and 1.0 mg/ml, whereas significant reduction in bacterial growth was seen at 2 mg/ml (MIC value). No bacterial growth occurred at concentrations of 3-10 mg/ml. Hence, MBC of
Holarrhena antidysenterica extract was considered to be 3 mg/ml.
The antibacterial activity of plant extracts can be attributed to the presence of broad-spectrum antimicrobial compounds in them (Marjorie, 1999). Methanolic extracts of
Holarrhena antidysenterica,
Acacia nilotica,
Punica granatum and
Gloriosa suberba possess high amount of tannins and phenolic compounds (Senthilkumar, 2013; Kavitha and Niranjali, 2009;
Misar et al., 2007; Prasanth et al., 2001). Tannins possess antimicrobial potential owing to their astringent activity and capability to inactivate the microbial adhesins, enzymes, cell envelope transport proteins
etc. (Cowan, 1999). Like synthetic phenols compounds, tannins adversely affect the integrity of bacterial cell membranes. Tannins chelate the metal ions such as iron and form a complex, which results in antimicrobial activity through iron depletion (Scalbert, 1991). Alkaloids present in the methanolic extract of
Holarrhena antidysenterica interfere and reduce the initial bacterial adhesion to intact epithelial cells and it may exert an anti-adherence effect against the pathogenesis of enteropathogenic
E. coli in the host epithelial cells (Kavitha and Niranjali, 2009).
In the present study, the methanolic extract of semi-ripe fruit of
Aegle marmelos showed antibacterial activity against the multi-drug resistant EPEC. Similar results have also been reported regarding the antibacterial potential of
Aegle marmelos against a variety of enteropathogenic bacteria in previous studies
(Mazumder et al., 2006). In our study, the methanolic extract of the leaves of
Psidium guajava did not exhibit any antibacterial potential against multi-drug resisitant EPEC isolates. However, in the previous reports, the antimicrobial potential of
Psidium guajava against enteropathogenic
E. coli isolates from calves was confirmed (Srivastava and Mondal, 2016). This variation could be due to differences of genotypes amongst EPEC isolates and herbal plants.
It has been reported earlier that ethanolic extract of
Holarrhena antidysenterica bark had MIC value of 2 mg/ml and MBC value of 3 mg/ml against
E. coli (Singh et al., 2016). In the present study, methanolic extract of
Holarrhena antidysenterica bark exhibited a MIC value of 2 mg/ml and a MBC value of 3 mg/ml against the multi-drug resistant EPEC. Similarities in the results of present study and previous studies may be due to several factors. The most important one is that the methodology as well as the pathogen chosen for the study is same
viz. diarrhoeagenic
E. coli.
Similar to our results, exploratory studies carried out by
Misar et al., (2007) in mice also revealed presence of significant antidiarrheal activity in the methanolic extract of
Acacia nilotica. Its aqueous extract was found effective in treating biofilm forming and multidrug resistant uropathogenic
E. coli by
Elamary et al., (2020). Several studies support the ethnomedicinal use of
Acacia nilotica bark for the treatment of diarrhoea and other ailments
(Misar et al., 2007).
Though, various reports have linked the antimicrobial potential of plants to the presence of tannins in them (Girard and Bee, 2020) but in the present study, no antimicrobial activity was observed in the methanolic extracts of
Bambusa arudinacea and
Psidium guajava leaves, which contain significant amount of tannins. Hence, further detailed phytochemical evaluation of plant extracts exhibiting significant antibacterial potential is warranted.
It is quite obvious from the existing scientific literature, antibacterial potential of different plants tested, could not be endorsed entirely to presence of any singular chemical constituent
(Ozcelik et al., 2006; Marjorie, 1999). Differences in the efficacy and specificity exist among the different types of phytochemicals present in a plant or its extract. These differences are attributed to the concentration of phytochemicals and chemical structure. Further investigations are needed to determine the optimal combinations of phytochemicals and the most suitable and cost-effective manner to deliver them (Girard and Bee, 2020). Certain disagreements between
in vitro and
in vivo results could be explained by the metabolism of phytochemicals along the intestinal tract. Whether and which phytochemicals and their metabolites remain active in the intestine warrant further research. There is a necessity to further explore different phytochemical components of the plant extract in terms of their mode of action and possible interactions responsible for their antibacterial potential.
