Effects of selected Taiwanese endemic plants on anti-lipid peroxidation and antibacterial activities against clinically isolated extended-spectrum-β- lactamase producing Klebsiella pneumoniae and Escherichia coli


 It has been a challenge for many clinicians to treat a complicated extended-spectrum-β-lactamase (ESBL) producing Klebsiella Pneumoniae (Kp) and Escherichia coli (E. coli) infection due to widespread antibiotic abuse with renal damage as one of its common side effects. Therefore, this study aimed to assess the antibacterial activity of extracts from several Taiwanese folk medicinal plants against ESBL- Kp and E. coli. with renal protecting ability against lipid peroxidation (LPO) on mice kidney mitochondria. Preliminary antibacterial activities of ethanol extracts from twenty (20) Taiwanese folk medicinal plants were measured by agar-dilution method against standard ESBL strains of E. coli (ATCC 25922, ATCC 35218) and Kp (ATCC 23856, ATCC 700603). Rhus semialata var. roxburghiana DC. (RSR) exerted the most inhibitory effect and then further extracted with n-hexane, ethyl acetate, acetone, ethanol, and water, respectively. Each extract also evaluated against the four standard ATCC microorganisms. Their MIC50, MIC90, and time kill assay were adapted with detecting the maximum inhibitory activities and the antibacterial spectrum range of each extract was measured against twenty-four (24) kinds of microbes. Which were used including gram-positive, gram-negative bacteria and fungus by agar dilution method. Finally, renal protective ability was detected inhibitory effect of ferrous induced lipid peroxidation on mice mitochondria. Among 20 Taiwanese folk medicinal plants tested, Rhus semialata var. roxburghiana DC. (RSR) exhibited maximum inhibition against clinical ESBL-producing Kp and E. coli strains with acetone extracts showing MIC50/MIC90 values at 1000 µg/mL, the course of antimicrobial action was bacteriostatic and with inhibitions to all 24 kinds of microbial including Gram positive and negative bacteria and fungi. Furthermore, result of thiobarbituric acid reactive substances (TBARS) assay from this extract showed high lipid peroxidative (LPO) protective capability on mice kidney mitochondria (IC50: 29.29 ± 0.35µg/mL). RSR acetone extract, with its maximum activity against clinical isolated ESBL-producing Kp and E. coli, antimicrobial effect against other wide spectral range bacteria and relatively high LPO protective ability on mice kidney mitochondria, is a potential source, albeit further studies have yet to be conducted, to develop an antimicrobial drug against ESBL-Kp and E. coli.


Introduction
Medicinal plants have been the major source of new medicines and offer an alternative to usual drugs (Tepe at al., 2004). As the primary source for human treatment, World Health Organization (WHO) reported that approximately 80% of the world population relies on plants or derived products (WHO, 1993). In case of infectious diseases, one of the reasons of unsuccessful treatment could be attributed to increasing number of drug-resistant bacteria (Al-Mariri and Sa , 2014). This indeed is associated with the potencies of existing antibiotics are decreasing steadily (Shahghasi et al., 2004). Since multidrug resistance of microorganisms is a major medical concern, screening of natural products in search for new antimicrobial agents is the need of the hour (Zgoda and Porter, 2001).

Treatment of complicated extended-spectrum-β-lactamase (ESBL) Klebsiella Pneumoniae (Kp) and
Escherichia coli infection is a challenging for clinicians due to the antibiotic abuse (Hoban et al., 2001) (Sah et al., 2019) (Tan et al., 2020). ESBL-producing bacteria is able to hydrolyze third-generation cephalosporin, aminoglycoside and uoroquinolone (Harish et al., 2007). Kp and E. coli are normal ora in human intestine. Kp is a frequent agent causing nosocomial or community-acquired bacteraemia as well as pneumonia, pyogenic brain abscess, meningitis, and pyogenic liver abscesses (Tsai et al., 2010) (Hui et al., 2007). ESBL-E. coli urinary tract infection is increasing, and in more serious cases, the bacteria enter the bloodstream and cause toxemia. (Rodríguez-Baño et al., 2004). Several studies have been reported that utilizes antibacterial properties of plant extracts against Kp and E. coli (Sharmeen et al., 2012) (Shaik et al., 2014) (Haroun and Al-Kayali, 2016). Moreover, renal damage is one of the side effects of antibiotics therapy with increasing possibility leading to acute renal failure in newborn (Fanos and Cataldi, 1999). In Taiwan, the risk of end-stage renal disease (ESRD) increased among those that reported regular use of antibiotics and has become another public health problem shouldered by the health insurances on medicinal care (Tsai et al., 2009

Extraction and sample solution preparation
Dried plants were washed continuously with tap water to remove impurities, cut into small pieces and then dried in the air circulating oven at 60℃ for 48 hr. Each 100 g of dried material was extracted twice with 1000 mL of ethanol, ltered, and combined the ltrates, then concentration and removed the solvent in vacuo by vacuum rotary evaporator (Eyela CCA-1111) to 50mL. The concentrated extract solutions were dried by lyophilizer (Eyela FDU-1200) to obtain the extract. The preliminary antimicrobial activity to standard strains of Kp and E. coli ATCC bacteria of each extract was identi ed to nd RSR the most potent extract from the 20 Taiwanese endemic plants. Therefore, RSR was further extracted by n-hexane, ethyl acetate, acetone, ethanol and water, respectively. Each crude extract (20 mg) was dissolved in 1 mL DMSO as a stock solution and serial dilution with PBS for the following assays. ( DMSO <0.05%).
Protocol of an antimicrobial agent development from natural plants source procedure Five (5) steps were carried out to determine the most potent extract with antibacterial capability against Kp and E. coli and renal damage protective effect ( Figure 1). Agar-dilution method was used to preliminary screening assess the antibacterial activity of crude extracts from 20 Taiwanese plants against standard strains of Kp and E. coli ATCC bacteria (E. coli ATCC 25922, ATCC 35218 and Kp ATCC 23856, ATCC 700603). RSR crude extract exhibited the maximum inhibitory effects and then subjected to further extract by n-hexane, ethyl acetate, acetone, ethanol and water, respectively. Each of these extracts was prepared as mentioned previously prior to antimicrobial assay against the same 4 bacteria by agardilution method.
The most potential extract of RSR (acetone extract) was chosen to evaluate the MIC 50  To assess the antibacterial spectral range of the extract, agar-dilution method was performed against 24 kinds of microbes including Gram positive, gram negative bacteria and fungi. Finally, the renal protective capability was measured by ferrous ion-induced lipid peroxidation (LPO) on mice mitochondria.

Agar-dilution method
Agar-dilution method described by Nauman and Arshad (2011) was used with some modi cation. clinical ESBL-Kp 1520914 as well as ESBL-E. coli 1526829 were diluted to 4×10 4 to 1×10 5 CUF/ mL and then exposed to RSR acetone extract (1, 2 and 4 x MIC), 1 mL DMSO, polymyxin B sulfate salt (PB) (0.5 and 1 x MIC) and 1 x MIC RSR acetone extract combined with 0.5 x MIC PB, respectively, incubated for 48 hours period in a total volume of 2 mL of MH media. Viable cell count was determined by agar plating (onto MH agar). Bactericidal activity was de ned as > 3 log decrease in cell counts. For all strains in the present report, time kill assays were performed at least twice independently with similar results.
Inhibitory capability of LPO on mice kidney mitochondria Preparation of mice kidney mitochondria and protein content quantitation Method described by Lin et al. (2013) was used with some modi cation. Male ICR mice (4~6 weeks) were sacri ced by carbon dioxide and the kidneys were removed as soon as possible and perfuse with ice-cold PBS (0.1 M, pH 7.4) prior to homogenizing in a Potter Elvehjem homogenizer. The homogenate was suspended in PBS and centrifuged at 2000 rpm for 10 min at 4°C to separate the nuclear debris. The clear suspensions were re-centrifuged at 13000 rpm for 10 min at 4°C to obtain mitochondrial fraction and then suspended in PBS. Different concentrations of mitochondrial suspended solution were pipetted into 1.5 mL eppendorf tubes, and total volume was adjusted to 50 μL with PBS. Protein reagent was added and mixed with a vortex-mixer before measurement of absorbance at 595 nm by ELISA spectrophotometer (Synergy H4 Hybrid Reader). Quantitative analysis of the protein content was determined from bovine serum albumin standard curve. More recent studies focused on extraction of phytochemical constituents using different solvents of variable polarities to analyze their pharmacological properties. Hence, extracts from RSR where further partitioned in different solvents to obtain the crude extracts with n-hexane, ethyl acetate, acetone, ethanol and water, respectively, and the same antibacterial tests were applied using the same four microorganisms from preliminary screening. Results showed that all RSR extracts partitioned in solvents except in ethanol, showed inhibitions against the two Kp (ATCC 23856 and 700603) strains with high MIC value observed in the acetone extracts (500 µg/mL) against Kp (ATCC) 23856 (Table 2). Similarly, EA and acetone extracts gave positive inhibitions against E. coli (ATCC 25922 and ATCC 35218) except n-hexane extract, which gave negative inhibitions. In addition, both ethanol and water extracts exhibited negative inhibitions against all E. coli strain used. These maybe associated with the polar constituents present in the RSR acetone extracts such as phenolics that are often related to antioxidant properties. Anokwuru et al. (2013) reported that acetone is highly effective at extracting biomolecules that are antioxidant. Although there is no direct correlations between antimicrobial and antioxidant activities, many studies on plant extracts reported that biomolecules are antioxidant and antimicrobial too but further studies have yet to be conducted on these extract to validate this relationship.
Since acetone extract from RSR gave a good inhibition against the four Kp and E. coli strains, the same extract was used to evaluate its property against seventeen (17) and sixteen (16) clinically isolated Kp and E. coli strain, respectively. Results showed that acetone extract of RSR exhibited positive inhibitions against all thirty-three (33) clinically isolated Kp and E. coli strains with MIC values ranging from 500 to 1000 µg/mL (Table 3). No inhibitions were observed in DMSO against all the strains used.
Time-kill assay was used to assess the mode of action of RSR against the tested Kp and E. coli strains. Although time consuming, the time-kill assay provides a dynamic picture of antibiotic action over time (Perez et al., 2016). In this study, the time-kill kinetics of RSR acetone extract against Kp (ATCC) 23856 and 700603 showed a gradual increased in the number of viable cells in a dose-dependent manner, as well as when combined with polymyxin B sulfate salt (PB) (0.5X MIC) at 1X MIC. However, against clinical ESBL-Kp 1520914, almost same number of viable cells was observed at 4X MIC over the rst 6 to 12 hours followed by a gradual rise up to 24 hour (Figure 2). Similar trends have been observed in the time-kill kinetics of RSR acetone extract against E. coli (ATCC) 25922 as well as when combined with polymyxin B sulfate salt (PB) (0.5X MIC) at 1X MIC. However, against (ATCC) 35218 and clinical ESBL-E. coli 1526829, almost the same number of viable cells was observed at 4X MIC over the rst 6 hours followed by a gradual rise up at 12 hours ( Figure 3). Overall, time-kill assay suggest that RSR acetone extract demonstrated a slight bacteriostatic action with strain-selectivity at relatively high MIC (4X MIC).
Disrupted balance between essentiality and toxicity of iron as an important trace element in the body is known to induce oxidative stress (OS) via Fenton reaction (Fischer et al., 2002)(Jomova and Valko, 2011) (Pari et al., 2015). Measurement of lipid peroxidase product, MDA, as the end product of poly-unsaturated fatty acid peroxidation and a marker of free radical mediated LPO injury have been applied to detect renal injury and its levels were found to signi cantly increase in kidney after treatment of ferrous ion indicating an increased LPO activity. In this study, LPO induced by ferrous ion on mice kidney mitochondria was signi cantly affected by the addition of RSR acetone extract, gallic acid and trolox in a dose-dependent manner (Table 4). However, IC 50 value of the extract (29.29±0.35 µg/mL) is only comparable to gallic acid (27.07±6.61 µg/mL) than to trolox (4.44±0.23 µg/mL).
Antimicrobial properties of RSR acetone extract were assessed over a wide range of bacterial spectrum and fungi. Results showed that RSR acetone extract has inhibitions to all the microorganisms used (Table 5). However, positive inhibition was also observed from DMSO against Samonella multivorum, Candida albicans and Candida parapsilosis suggesting that the inhibitions observed from RSR acetone extract against these microorganisms were not purely attributed from the extract alone.

Conclusions
Out of 20 Taiwanese folk medicinal plants tested, only RSR exhibited a positive inhibition in the preliminary antimicrobial screening. RSR acetone extract, with its maximum activity against clinical isolated ESBL-producing Kp and E. coli, antimicrobial effect against other wide spectral range bacteria and relatively high LPO protective ability on mice kidney mitochondria, is a potential source, albeit further studies have yet to be conducted to isolate and purify the agents or molecules from this extract which is responsible for the antimicrobial properties and may serve as potential antibiotics against ESBL-Kp and E. coli.

Abbreviations
Extended-spectrum-β-lactamase (ESBL)     Positive control agents are Gallic acid and Trolox Table 5-Antibacterial spectrum range of RSR acetone extract against other 24 kinds of microbial strains Figure 1 The ow chart of antibacterial capability against Kp and E. coli detection procedure and renal damage protective effect.