In-vitro Antibacterial activity of Rhus javanica Against Multidrug-Resistant Uropathogens

Introduction

Antimicrobials are medicines used to prevent and treat infections in human, animals and plants. Haphazard use or misuse of antimicrobials in daily life leads to evolutionof alteration in microbial response and become antibiotic resistant. Globally, Antimicrobials resistance is rising rampantly which leads to increased mortality and morbidity [1]. In developed country like United State, 2 million peoples are infected and 23,000 peoples die due to antibiotic-resistant bacteria every year [2]. In Nepal the mortality rate is high due to diarrheal diseases, respiratory tract infection, blood stream infection, urinary tract infection, tuberculosis and sexually transmitted diseases [3]. The evidence of multidrug resistant pathogens is reported from the various studies conducted in different part of Nepal [4, 5]. The major culprits include pathogens like extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumonia, carbapenem-resistant Enterobacteriaceae, Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococcus (VRE), Pseudomonas aeruginosa and Acinetobacter baumannii.

A total of 75% of the population of developed and developing countries have been using plant extracts for medicinal purpose and it is believed that plants provide variety and best source of drug [6]. In Nepal, about 70-80% population from mountain and hilly regions is dependent on traditional medicine for primary health care [7]. Globally, various studies revealed that natural herbs are effective against human pathogenic bacteria in both in-vitro and in-vivo conditions [8, 9]. Rhus javanica, a member of Anacardiaceae is traditionally used to treat dysentery and diarrhea [7]. In phytochemical screening it is evident that, Rhus javanica contains phenolics, glycosides, flavonoids, terpenoids and organic acids. The antibacterial activity might be due to the presence of phenolic compound [10]. However, there is limited evidence regarding the effect of Rhus javanicaon Multi Drug Resistant (MDR) bacterial isolates from Urinary Tract Infection (UTI) patients. In this study, we aimed to investigate the prevalence of UTI causing bacterial pathogens and its Antibiotic Sensitivity Testing (AST) pattern, and evaluation of antibacterial effect of Rhus javanica against MDR bacterial isolates in standard in-vitrocondition.

Materials and Methods

Cross-sectional study was designed from August to October 2019 urine samples were collected from 133 UTI suspected participants visiting clinic in Kathmandu. Further processing was done in the Department of Medical Laboratory Technology, JF Institute of Health Sciences, Hattiban, Lalitipur. Samples were inoculated into MacConkey Agar (Hi-media,India) and Blood Agar(Hi-media, India) by standard loop inoculation method (1μl volume) and incubated in controlled in-vitro condition. Standard biochemical tests were performed for further identification.

Antibiotic susceptibility testing for the pathogenic organism isolated in culture was done by Kirby-Bauer Disk Diffusion method as per the recommendation of Clinical and Laboratory Standard Institute (CLSI) using Mueller Hinton agar (MHA) (Hi-media, India). McFarland turbidity standard 0.5 was used and Hi-media, India antibiotic disk were used for AST, which can be enlisted as follows: Amoxycillin (10 μg) Nalidixic acid (30 μg), Nitrofurantoin (300 μg), Norfloxacin (10 μg), Ofloxacin (5 μg), Amikacin (30 μg), Cefixime (30 μg), Ampicillin (10 μg) and Ceftriaxone (30 μg) [11].

Rhus javanica,selected in this study was collected from Swoyambhu area of Kathmandu valley. The identification of the specimen was confirmed by Departmentof Plant Resources, Godawari, Nepal and voucher specimen was deposited at the Herbarium of the Department. Dried leaves, exactly, 600 g was soaked in 3000 mL of 50.0% methanol for 72 hour at room temperature. The extracted solution was filtered and evaporated under reduced pressure to yield methanol extract of 42.4 g (7.1%). After the extract was thoroughly dried to facilitate complete removal of the solvent, the dry extract was dissolved in Dimethyl sulfoxide (DMSO) to give the desired stock solution. Antibacterial testing was performed in MHA media by cork borer method in different concentrations of extract (i.e. 25, 50, 75 and 100 mg/ml. and 0.1% DMSO as control) prepared in 0.1 % DMSO. All experiments were performed in duplicate. Data were entered and analyzed by using the Statistical Package for Social Sciences (SPSS) version 20.

Results

Out of total 133 urine samples, 35.33% (47/133) showed bacterial growth and 64.67% (86/133) did not show any bacterial growth. Among them, female to male ratio was 2:1. Among the total positive cases of 47, 65.95% (31/47) of the females were reported with urinary tract infection whereas 34.05% (16/47) of the males were reported with urine infection. Highest number of positive cases were observed from the age of 60-70 years (27.7%; 13/47), followed by age group 10-20 and 40-50 years (17.0%; 8/47). The least number ofpositive cases was obtained from the age group of less than 10 years (0.0%; 0/47). During the study period, 6 different species of bacteria were isolated.Escherichia coli 23 (17.3%) was the predominant organism among the total organism followed by Enterococcus faecalis 9(6.8%), Klebsiella pneumoniae 5(3.8%), Pseudomonas aeruginosa 4(3%), Proteus vulgaris 3(2.3%), and Staphylococcus aureus 3(2.3%) as shown in figure 1.

Figure 1: Bacterial isolates from UTI suspected patients urine samples

Among the antibiotics used against the isolated organism, the most effective antibiotic was Nitrofuration (70.2%) followed by Levofloxacin (68.0%) and Amikacin (53.2%). Most of the organisms were resistance to Amoxycillin(95.7%) followed by Ampicillin(85.1%), Nalidixic acid (80.9%) shown in figure 2.

Figure 2: Antibiotic susceptibility pattern of bacterial isolates

Most of the bacteria isolated (53.2%)(n=25) were found to be resistant to antibiotics and were considered MDR and the remaining 46.8% (n=22)were non-MDR. It was found that 17(36.1%) isolates were sensitive to all antibiotic and (5.5%) (n=4) were resistant to 1 drug. Among the total isolates, 25 were MDR strains, where MDR strains in individual bacteria was 66.6% (n=2) in Proteus vulgaris and Staphylococcus aureus, 55.5% (n=5) in Enterococcus faecalis, 52.2% (n=12) in Escherichia coli, 50.0% (n=2) in Pseudomonas aeruginosa and 40% (n=2) Klebsiella pneumoniae, as shown in Table 1

                                                     Table 1: Resistance pattern and distribution of MDR bacterial isolates

Methanolic extract of Rhus javanica showed antibacterial effect against all MDR bacterial isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginos, Proteus vulgaris and Staphylococcus aureu. Zone of Inhibition (ZOI) was seen to increase with increasing concentration of extract. Diameter of inhibition was found to range from12 mm.to 28 mm, as shown in table 2.

                                        Table 2: Effect of methanolic extract of Rhus javanica against MDR bacterial isolates

Discussion

Development of antimicrobial resistant in bacteria, virus and fungi is become major issue for human, animal and plants. Both developed and developing countries are equally affected by this burden. In present study, the prevalence of bacterial infection among study subjects is found to be 35.3%, in which Escherichia coli is the predominant organism and followed by Enterococcus faecalis and Klebsiella pneumoniae. AST showed that, out of total isolates, 53.2% were MDR.Similar finding was reported by Shakya et al.,where 52.3% of the total isolates were MDR, in which the main pathogens are Escherichia coli 79.7%, Providencia species 5.1%,and Klebsiella pneumoniae 3.4% [4]. A study from eastern Nepal also showed that, 43.98 % of urine samples showed significant growth and pathogens were resistant to amoxycillin, co-trimoxazole, flouroquinolones and third-generation cephalosporins [12]. In addition, Chaudhary et al. reported that 39.2% of the total isolates were MDR main pathogens are Staphylococcus aureus and Escherichia coli in various surgery sites [13].

In the present study, we examined the antimicrobial activity of Rhus javanica extract against MDR urinary tract bacterial pathogens. Our study showed that, 25 to 100 mg/mL of the methanol extract inhibitedgrowth of MDR isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginos, Proteus vulgaris and Staphylococcus aureus in standard in-vitro condition. The diameter of zone of inhibition is high with increased concentration of extract where the diameter of inhibition was found range from12 to 28 mm in MHA. A study conducted in MRSA, revealed that extract of Rhus javanica inhibited the bacterial growth by significantly decreasing the expression of mecA, sea, agrA and sarA gene [10]. Another study showed that, extract showed inhibitory effect against shigellosis causing pathogens,Shigella flexneri and Shigella dysenteriae, like other bacterial pathogens [14].

Rhus javanica has been used to treat dysentery and diarrhea in different Asian countries including Korea, China, and Japan [15]. Several studies showed that, Rhus javanica extract was effective against animal husbandry disease-related bacteria like Listeria monocytogenes and Staphylococcus epidermidis, fish pathogens like Vibrio ichthyoenteri and Streptococcus iniae [16, 17]. Leaves of Rhus javanica primarily contains gallic methy lester, syringic acid, protocatechuic acid, and 1, 2, 3, 4, 6-penta-O-galloyl--D-glucose [18]. You et.al., suggested that phenolics may have been responsible for the antibacterial activity and Leaves of Rhus javanica contains phenolics, glycosides, flavonoids, steroids and organic acids [10]. Various factors like lack of knowledge and awareness regarding rational use of antibiotics in human and animals, lack of sophisticated laboratory for diagnosis and practice of medicine that usually overlooks standard microbiology protocol, are responsible for emergence of antibiotic resistance in Nepal [3, 19]. Our study revealed that crude extract of Rhus javanica shows antimicrobial activity against all MDR uropathogens in standard in-vitro condition.

Conclusion

Crude extract of Rhus javanica shows antibacterial activity in all the MDR isolates. Further study is needed for purification and isolation of the active compound responsible for such activity in vitro.

Authors Contribution

BS, GP &TS- Study design and laboratory test. BS, GP & MG - data analysis and prepare manuscript, MG, RKS &NB- plant collection and processing and GD & VS- critical comment on manuscript.

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