In vitro antibacterial activity of crude extracts of 9 selected medicinal plants against UTI causing MDR bacteria

1 Introduction

Urinary tract infection (UTI) was mostly caused by Gram negative (GN) bacteria, predominately by Escherichia coli and by mixed infections of (Gram positive, GP) Staphylococcus aureus, Enterococcus faecalis, including other GNs, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter aerogenes, Proteus mirabilis, Citrobacter freundii, Proteus vulgaris and Klebsiella oxytoca in the decreasing order of prevalence, when monitored in the Institute of Medical Sciences and Sum Hospital, for example (Mishra et al., 2013). The infecting bacteria normally constitute the faecal flora, and the UTI episode is initiated, when the urine flow in an individual is obstructed by one of several reasons such as, strictures, calculi, tumours, prostatic hypertrophy, vesicourethral reflux, diabetes, anal disease, pregnancy, catheterization, some surgical procedure at the urinogenital region and cystoscopy (Saint et al., 2002). The infecting bacteria invade urethra and bladder with a compromised body defense mechanism and decreased urine flow. In these conditions, the bacteria ascend via urethra move to the bladder mucosa, colonize, multiply and cause inflammation; this causes intolerable pain, burning, frequency and urgency of urination, nocturia, foul smelling, cloudy urine and haematuria. Indeed, at the onset of the problem, the patient reports to the physician and an empiric therapy is started before the culture report of the urine sample is obtained.

If any infection in a patient is not controlled, infecting microbes get resistance to the applied antibiotics intrinsically and a drug resistant cell survives and predominates with concomitant bacterial genetic exchanges mechanisms (McMurry and Levy, 2011). In short, there are several factors of antibiotic resistance in pathogenic bacteria and this situation has become a clinical consternation. A physician often prescribes some higher generation antibiotics in empiric therapy to avoid the debacle from treatment failure arising from the possible presence of MDR bacteria. And the UTI being a graver problem than imagined because of frequent attacks in females mainly, some complementary/adjuvant/synergistic therapeutic strategy is warranted.

Furthermore, information from ethnobotany/traditional medicine has been seen useful for several health complications other than infectious diseases. Nine flowering plants (Anogeissus acuminata, Azadirachta indica, Bauhinia variegata, Boerhaavia diffusa, Punica granatum, Soymida febrifuga, Terminalia chebula, Tinospora cordifolia and Tribulus terrestris) (Fig. 1) were used. These plants are in use traditionally by local ethnic tribes against infectious diseases; and these were examined in a systematic screening with UTI causing bacteria for use as source of non-microbial antimicrobials, so that these could serve as complementary medicine, along with mainstream drugs, the antibiotics, as it takes 3–4 days of time of arrival of the culture report of the urine during which period, infecting bacteria cause further problems. UTI episodes need to be controlled with an iron hand. And to avoid the use of any antibiotic of higher generation during empiric therapy, complementary or synergistic therapy using phyto-drugs with any ongoing antibiotic could be prudently used against UTI.

Close

Figure 1

Photographs of plants: a, Anogeissus acuminata; b, Azadirachta indica; c, Bauhinia variegata; d, Boerhaavia diffusa; e, Punica granatum; f, Soymida febrifuga; g, Terminalia chebula; h, Tinospora cordifolia; i, Tribulus terrestris.

Export to PPT

In continuation to previous work on scientific verification of ethnomedicinal information of a group of plants from Kalahandi (Odisha) forest (Mishra and Padhy, 2013; Rath and Padhy, 2014), the cited 9 plants were selected. These plants were too recorded in Indian pharmacopeia (Anonymous, 2014), as plants frequently used against general infectious diseases: A. acuminata is in use for UTI and skin diseases; whole plant of A. indica is used as an antiseptic; B. variegata is used against diarrhoea and throat infections; B. diffusa is used for UTI and dysentery; P. granatum is used for treating diarrhoea, dysentery and throat problems; S. febrifuga is used against diarrhoea, dysentery and UTI; T. chebula is used for diarrhoea and dysentery; T. cordifolia has anti-tubercular activity; and T. terrestris is used against UTI. Several other plants, Terminalia alata, Lantana camara, Combretum albidum and Woodfordia fruticosa had comparable antibacterial activities, which had been used for further pharmacognostical studies (Rath and Padhy, 2012; Rath and Padhy, 2013; Dubey et al., 2014; Sahu et al., 2014), but these 9 cited plants were not used for the isolation of individual compounds against any bacteria. Detailed antibacterial work with pathogenic bacteria isolated from clinical samples of patients in the hospital, with 9 plants is described.

2 Materials and methods

2.4

2.4 Determinations of MIC and MBC of plant extracts

Original stock solutions of leaf extracts were prepared with methanol, at 44.44 mg plant extract/ml 10% DMSO solution, with distilled water. Each stock solution was diluted to obtain final concentrations of 0.29, 0.67, 1.51, 3.41, 4.27, 9.63, 21.67 and 44.44 mg/ml with the DMSO solution. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the methanol leaf extracts were determined in a well on a 96-welled (12 × 8) micro-titre plate (Fig. 2), as described elsewhere (Mishra and Padhy, 2013; Rath and Padhy, 2014).

Close

Figure 2

Determination of minimum inhibitory concentrations (MICs), in a 96-well microtiter plate, of 44.44 mg/ml of methanol leaf extract of P. granatum against 8 MDR UTI causing pathogenic bacteria (1 = S. aureus, 2 = E. faecalis, 3 = A. baumannii, 4 = E. aerogenes, 5 = K. pneumoniae, 6 = K. oxytoca, 7 = P. mirabilis, 8 = P. vulgaris). M = MIC at numbers that signifies the lowest concentration of leaf extract. C = control without plant leaf extract; A = Gentamicin (30 μg/ml) as control without any plant leaf extract.

Export to PPT

3 Results

3.5

3.5 Qualitative phytochemical analyses

Qualitative phytochemical analyses were done for methanol leaf extracts. Phytochemicals, alkaloids, flavonoids, carbohydrates, terpenoids, steroids, tannins, resins, saponins and anthraquinones, which could be attributed to the recorded significant antibacterial activities in most extracts (Table 5). From PubChem database, structure and related information of one leading antimicrobial compound of each plant was presented (Table 6). From previous studies, the antibacterial nature of these cited 9 compounds were ascertained (Table 6). The Molsoft tool (http://molsoft.com/mprop/) was used to find out the drug likeness scores of each compound, according to their structure canonical ‘simplified molecular-input line-entry system’ (SMILES). Thus theoretically, based on the available data on drug likeness scores, phytochemicals could be arranged in the decreasing order of drug-likeness scores mentioned against: quercetin (0.93) > berberin (0.91) > luteolin-7-O-glugoside (0.86) > kaempferol (0.77) > ursolic acid (0.65) > argungenin (0.61) and mahmoodin (0.61) > conocarpan (0.13) and dihydrodedrodehydrodiconiferylalcohol (0.13) > anolignan B (−0.78). It could be inferred here that S. febrifuga among the 3 best plants is the most leading plant with luteolin-7-O-glugoside, which has a good score of drug-likeness (Table 6).

Table 5 Qualitative phytochemical analyses of methanol extracts of 9 medicinal plants.

Sl.No.	Plants	Alkaloids	Resins	Glycosides	Terpenoids	Carbohydrates	Saponins	Tannins	Flavonoids	Steroids	Anthraquinones
1	A. acuminata	+	−	+	+	+	+	+	+	+	+
2	A. indica	−	+	+	+	+	+	−	−	+	−
3	B. variegata	+	+	+	−	+	+	+	−	+	+
4	B. diffusa	+	−	−	−	−	−	+	+	+	+
5	P. granatum	+	−	+	+	−	+	+	+	+	+
6	S. febrifuga	+	+	+	+	+	−	+	+	+	+
7	T. chebula	+	+	+	+	+	−	+	+	−	+
8	T. cordifolia	+	−	+	−	+	+	−	−	+	−
9	T. terrestris	−	−	−	+	−	−	−	−	+	−

Note: “+” sign denotes presence, and “− “sign denotes absence of the compound in a plant.

Table 6 Leading antimicrobial phytochemicals structure, information with properties.

Plant name	Leading antimicrobial phytochemical structure	Information and properties of leading phytochemical	Drug-likeness score	References
A. acuminata		Anolignan B (oc) Molecular weight: 266.33432 [g/mol] Molecular formula: C18H18O2 XLogP3-AA: 5.5 H-Bond donor: 2 H-Bond acceptor: 2	−0.78	Rimando et al. (1994a,b), Eldeen et al. (2006)
		Conocarpan (oc) Compound ID: 6474521 Molecular Weight: 266.33432 [g/mol] Molecular Formula: C18H18O2 XLogP3-AA: 4.4 H-Bond donor: 1 H-Bond acceptor: 2	0.13
		Dihydrodehydrodiconiferylalcohol (oc) Compound ID: 5274623 Molecular weight: 360.40096 [g/mol] Molecular formula: C20H24O6 XLogP3-AA: 2.1 H-Bond donor: 3 H-Bond acceptor: 6	0.13
A. indica		Mahmoodin (l) Compound ID: 126566 Molecular weight: 526.61792 [g/mol] Molecular formula: C30H38O8 XLogP3-AA: 3.7 H-Bond donor: 1 H-Bond acceptor: 8	0.61	Siddiqui et al. (1992)
B. variegata		Kaempferol (f) Compound ID: 5280863 Molecular weight: 286.2363 [g/mol] Molecular formula: C15H10O6 XLogP3: 1.9 H-Bond donor: 4 H-Bond acceptor: 6	0.77	Holler et al. (2012)
B. diffusa		Ursolic acid (t) Compound ID: 64945 Molecular weight: 456.70032 [g/mol] Molecular formula: C30H48O3 XLogP3-AA: 7.3 H-Bond donor: 2 H-Bond acceptor: 3	0.65	Jiménez-Arellanes et al. (2013)
P. granatum		Stigmasterol (s) Compound ID: 5280794 Molecular weight: 412.69082 [g/mol] Molecular formula: C29H48O XLogP3-AA: 8.6 H-Bond donor: 1 H-Bond acceptor: 1	0.73	Awouafack et al. (2013)
S. febrifuga		Luteolin-7-O-glucoside (f) Compound ID: 5291488 Molecular weight: 448.3769 [g/mol] Molecular formula: C21H20O11 XLogP3-AA: 0.5 H-Bond donor: 7 H-Bond acceptor: 11	0.86	Khatkar et al. (2014)
T. chebula		Arjungenin (t) Compound ID: 12444386 Molecular weight: 504.69852 [g/mol] Molecular formula: C30H48O6 XLogP3-AA: 4.5 H-Bond donor: 5 H-Bond acceptor: 6	0.61	Manosroi et al. (2013)
T. cordifolia		Berberin (a) Compound ID: 2353 Molecular weight: 336.36122 [g/mol] Molecular formula: C20H18NO4+ XLogP3-AA: 3.6 H-Bond donor: 0 H-Bond acceptor: 4	0.91	Choudhary et al. (2013)
T. terrestris		Quercetin (f) Compound ID: 5280343 Molecular weight: 302.2357 [g/mol] Molecular formula: C15H10O7 XLogP3: 1.5 H-Bond donor: 5 H-Bond acceptor: 7	0.93	Rashed and Butnariu (2014)

Note: a, alkaloid; f, flavonoid; l, limonoid; oc, organic compound; s, sterol; t, terpene.

4 Discussion

All of these 9 plants were used by an ethnic tribe, the Kandha tribe of Kalahandi district, since time immemorial for primary healthcare needs specifically for infectious diseases. It was seen that, 11 bacteria isolated from urine samples were resistant to the following: aminoglycosides, β-lactams (amoxyclav and ampicillin), two cephalosporins (ceftriaxone and cefpodoxime) as well as, chloramphenicol, signifying most bacterial strains as resistant to most antibiotics. Additionally, plants, A. acuminata, A. indica, B. variegata, P. granatum and S. febrifuga had control capacity on all the 11 strains of MDR bacteria. Moreover, among these 3 best plants in the control of MDR bacteria in vitro were A. acuminata, P. granatum and S. febrifuga. P. granatum has stigmasterol, a sterol with the drug-likeness score, 0.73 while, S. febrifuga has luteolin-7-O-glucoside, a flavonoid with the drug-likeness score, 0.86. Further work on antibacterial bioactive compounds of A. acuminata is in progress, as it had significant microbial activity. However, the plant, T. terrestris has the famous antimicrobial agent, quercetin, which has an effective drug likeness score of 0.93; but this plant did not have the best antibacterial activity, in this study or with these bacteria.

Antibiotic sensitive pathogens have a limited capacity of virulence as the employed antibiotic controls them in vivo. At a particular density, the host defense system too helps control pathogens, when the later are in a limiting number. As known, for the internal protection, antibiotic producing organisms harbour antibiotic resistant genes in plasmids and chromosomes, as well as the transfer mechanisms remain active (Mamelli et al., 2009). Therefore, such genes and/or transposon are taken up, horizontally by the susceptible group of bacteria, through bacterial transformation and/or conjugation (Pages et al., 2008; Warnes et al., 2012).

Moreover, bacteria having simple genomes undergo intrinsic (mutations) or acquired genetic (conjugations and transformation) changes in the presence of an antibiotic, as a stress factor (Groisman and Ochman, 1996). As a result, accrual antibiotic resistance mechanisms are the clinical determinants of the pathogenesis. It had been known that in areas, where a particular group of antibiotics are used bacteria resistance to same antibiotics were in higher numbers (Shrestha et al., 2002). Indeed, the horizontal transfer of genetic materials from one organism to another appears faster than mutational changes, a phenomenon popularly called as ‘evolution of quantum leaps’ (Groisman and Ochman, 1996). Progressively, the use of more antibiotics even of higher generations for the control of infectious diseases have led to multiple resistances, i.e., too many antibiotics are ineffective to progressively increasing resistant strains of pathogens, as if growth and momentum gained by a descending snow-ball, during the passage of time by mutation and acquisition of genes from related/unrelated bacteria, ending in shockingly repellant multidrug bacterial resistance. Older antibiotics slowly become moribund, even the resistant mechanism against those are found in certain bacteria for which, those antibiotics were never applied. Drug resistant bacteria gain the capability of surviving and multiplying under antibiotic-stress conditions, confirming the biological rule, ‘any limiting condition for the majority would be an excellent opportunity for the minority’. In the presence of a drug in a body in vivo, the progeny of a drug sensitive strain is eliminated and the resistant strain survives, multiplies as if, developing from a doppelgänger, and predominates ultimately in causing a characteristic pathogenesis. It is because a suitable emulating agent for the control is absent, and if plant-based antimicrobial would be present in parallel along with the employed antibiotic, there would be the coveted blithesome result, since no bacterium how much genetically well-equipped be it may as in a cohort of MDR bacteria, can never over-ride complexities of phytochemicals for survival. This fact is repeatedly seen in vitro with several plant extracts (Dubey and Padhy, 2013; Mishra and Padhy, 2013; Rath and Padhy, 2013; Sahu et al., 2015). Thus, those in a coalesced manner, as in a crude extract, have a combined controlling effect.

It has been demonstrated with Salmonella enterica serotype typhimurium (Alekshun and Levy, 1999). Moreover, MDR Neisseria gonorrheae had been known to acquire ‘MTR and SAP A MDR’ systems of genes, from S. enterica serotype typhimurium (Hagman and Shaferm, 1995). Discovery and development of antibiotics in the last century have not only saved countless human lives, but have provided assurances in clinical management all over. But, concomitant development of antibiotic-resistance mainly in bacteria has dismayed both preventive and therapeutic potencies of antibiotics today. In the odyssey of drug development, antibiotics are introduced continually and a few of them are modified suiting to the need to overcome bacterial resistance. Eventually, today there are a large number of antibiotics in use. The demand for newer antibiotics for MDR bacteria in colossal scale has arisen, which has become difficult to meet, as these small molecules are extremely complex in functionality linked to chemical structure. Secondly, an antibiotic ensconced for a typical set of infections cannot ordinarily be abandoned as an obsolete drug, due to reports of dogmatic/realistic resistance in a geographical zone; rather, along with the same antibiotic the introduction of complementary or adjuvant drug could be aimed, when considered with contemplation the problem of morbidity/mortality from infections due to MDR bacteria (Davies and Davies, 2010).

Applied antibiotics, being of microbial origin, are readily won over by pathogenic microbes in vivo. The cell producing an antibiotic has the characters of the self-protective mechanism as characters/genes, which direct the modes of resistance such as, alteration in the cell membrane by efflux mechanism or production of external enzymes like, β-Lactamases are intrinsically transmitted to similar bacteria, as discussed (Rout et al., 2014). In short, suitable antibiotics are required in colossal scale globally, which would give a way to the chance of development of resistant strain(s) of pathogenic bacteria in a Darwinian way, further. Indeed, the present methodology of methanol-extraction of phytochemicals is a unique approach, as this solvent helps extraction of most polar to non-polar phytocompounds (Rezaie et al., 2015). The β-Lactam group of antibiotics consisting of penicillins, cephalosporins, monobactams, glycopeptides and penems target the peptidoglycan biosynthesis of bacteria. Tetracyclines, aminoglycosides, macrolides, lincosamides, streptogramins, oxazolidinone (linezolid) and phenicols cause a thwart to the translation process in the parasitic cell; quinolones inhibit the DNA replication. Pyrimidines and sulphonamides alter carbon metabolism during inhibition of parasitic growth; and the most recent ones such as, daptomycin and colistin inhibit cell membrane functions (see, Davies and Davies, 2010). However, the first effective antimicrobials were the sulphonamides, which have been amply lent themselves for further uses in the drug development process as antibacterials in the last several decades. Thus, the concept of complementary use of phytocompounds along with main stream drugs, the antibiotics has immersed when the myriad mechanisms of drug resistance is considered in the face of success of phytocompounds as non-microbial antimicrobials (Sahu et al., 2015).

Our school has screened out about 250 plants in the last 4 years (Rath and Padhy, 2012; Rath et al., 2012; Dubey and Padhy, 2012; Mishra and Padhy, 2013; Rath and Padhy, 2014; Sahu et al., 2015), using mainly ethanol and water as extracting solvents. Among them for 47 plants methanol was used as the solvent (Mishra and Padhy, 2013; Rath and Padhy, 2014). A comparative account of MIC values of the best plants among 47 against MDR strains of 8 gruesome bacteria is considered, along with the present 3 best plants (Table 7). It is discernible that methanol extracts of most plants had MIC values as 3.41 or more, except those of Cinnamomum tamala, A. acuminata, P. granatum and S. febrifuga, which had comparatively lower MIC values, as 1.51 or 0.67 or 0.29 mg/ml.

5 Conclusion

Antibiograms of 11 isolated pathogenic bacteria with 17 antibiotics of the day ascertained that all were amply MDR. The work on individual 9 plants in controlling all MDR strains of bacteria was evident, mostly with lower MIC and MBC values. All these used plants have ethnomedicinal uses and 3 best plants could be promoted as complementary medicine. The recorded data of 3 best plants, A. acuminata, P. granatum and S. febrifuga, are anticipated to trigger work on the isolation of pure compounds for further scientific use in the crusade of the control of MDR bacteria. Phytocompounds, stigmasterol and luteolin-7-O-glucoside already isolated from the second and the third best antibacterial plant, respectively have significant drug-likeness scores. Thus, the presently used three best plants could be regarded as the most effective plants studied for further consideration for complementary medicine as sources of non-microbial anti-microbials against most MDR UTI causing bacteria.