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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 232-238

Antibacterial potential of neem (Azadirachta indica) against uropathogens producing beta-lactamase enzymes: A clue to future antibacterial agent?


1 Department of Microbiology, Santosh Medical College and Hospital, Ghaziabad, Uttar Pradesh, India
2 Department of Microbiology, Maharishi Markandeshwar Medical College and Hospital, Solan, Himachal Pradesh, India

Date of Submission18-Mar-2020
Date of Acceptance02-May-2020
Date of Web Publication12-Sep-2020

Correspondence Address:
Prof. Dakshina Bisht
Department of Microbiology, Santosh Medical College and Hospital, Ghaziabad - 201 009, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_38_20

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  Abstract 


Background: Emergence of drug resistance in Gram-negative bacilli due to production of extended-spectrum beta-lactamases (ESBL), metallo-beta-lactamases (MBL), and AmpC beta-lactamase is very common nowadays; therefore, we are left with less choice for antibiotics that is why we are in the need for the new alternatives. Hence, the current study was done to demonstrate antibacterial properties of neem (Azadirachta indica) leaves against ESBL, MBL, and AmpC beta-lactamase-producing Gram-negative uropathogens. Methods: Gram-negative uropathogens (221) were isolated and further tested for beta-lactamase (ESBL, MBL, and AmpC) production. Neem (A. indica) was tested for its antibacterial activity against all uropathogens. Results: Ethanolic extract of neem leaves showed good antibacterial activity against all isolates. Maximum zone of inhibitions and lowest minimum inhibitory concentration and minimum bactericidal concentration values were revealed by Acinetobacter baumannii. Conclusion: Extract of neem leaves at different concentrations showed potential antibacterial activity against both beta-lactamase-producing and nonproducing Gram-negative bacilli.

Keywords: Ethanolic extract, extended-spectrum beta-lactamases, metallo-beta-lactamases, and AmpC beta-lactamase, neem (Azadirachta indica), urinary tract infection, uropathogens


How to cite this article:
Faujdar SS, Bisht D, Sharma A. Antibacterial potential of neem (Azadirachta indica) against uropathogens producing beta-lactamase enzymes: A clue to future antibacterial agent?. Biomed Biotechnol Res J 2020;4:232-8

How to cite this URL:
Faujdar SS, Bisht D, Sharma A. Antibacterial potential of neem (Azadirachta indica) against uropathogens producing beta-lactamase enzymes: A clue to future antibacterial agent?. Biomed Biotechnol Res J [serial online] 2020 [cited 2020 Sep 22];4:232-8. Available from: http://www.bmbtrj.org/text.asp?2020/4/3/232/294853




  Introduction Top


Millions of people get affected by urinary tract infections (UTIs) annually, which can lead to serious health issues.[1] Bacteria are the most common cause of both nosocomial and community acquired UTIs therefore antibiotics are commonly prescribed against UTIs. Indiscriminate use of antibiotics has led to antimicrobial resistance occurring globally, leading to serious complications.[2],[3] Difficulty in treating these infections has caused higher mortality and morbidity. Beta-lactamases such as extended-spectrum beta-lactamases (ESBL), metallo-beta-lactamases (MBL), and AmpC production are one of the common reasons of antibiotics resistance, especially in Gram-negative bacilli. Therefore, the urge to discover new antimicrobials has increased nowadays.[4],[5] Natural bioproducts such as medicinal plant extracts are gaining limelight for the production of new antimicrobials because of their known antimicrobial properties.[6] Neem (Azadirachta indica) is one of the most popular medicinal plants and well known for its medicinal uses. It is distributed in tropical and subtropical areas of different countries, but is a native plant of India.[7] Leaves of neem plant have been used in treating acne, eczema, and dermatophytic infections. It has well documented antihyperglycemic, anti-inflammatory, and antimicrobial properties. Neem ethanolic extracts have showed goodin vitro antibacterial activity.[8],[9] Hence, considering the importance of neem as an antibacterial agent, the present study has been designed to analyze the antibacterial potential of neem against ESBL, MBL, and AmpC beta-lactamase-producing uropathogens.


  Materials and Methods Top


This study was conducted in the Department of Microbiology, Santosh Medical College and Hospital, Ghaziabad, and Department of Microbiology, M. M. Medical College and Hospital, Solan. Approval was taken from the Institutional Ethical Committee F. No. SU/2017/683 (16) on 26/05/2017. All the urine specimens of clinically suspected patients of UTI sent to the microbiology laboratory from the different departments were accepted and processed further. All samples were cultured on blood agar, cystine–lactose–electrolyte-deficient agar, and MacConkey agar and were incubated further at 37°C for 18 h.[7] More than 105 cfu/mL colony count for urine specimens was considered as significant bacteriuria for UTI. Bacterial identification for positive urine cultures were performed using standard microbiological tests and were further processed for antibiotic susceptibility testing.[10],[11]

ESBL, MBL, and AmpC beta-lactamase detection of all Gram-negative uropathogens were performed using phenotypic methods in accordance with the Clinical and Laboratory Standards Institute guidelines.

Screening for extended-spectrum beta-lactamases

ESBL-producing isolates were screened in accordance with the zone of inhibition of ≤25 mm for ceftriaxone and ≤22 mm for ceftazidime using disk diffusion method, which was further confirmed by cephalosporin/clavulanate combination disk method.[12]

Phenotypic confirmatory test for ESBL production was done using cephalosporin/clavulanate combination disk method.

All isolates were inoculated in peptone water and adjusted to 0.5 McFarland unit. Isolates were then swabbed on to Mueller-Hinton agar (MHA). A 30 μg disk of ceftazidime and 30/10 μg disk of ceftazidime-clavulanic acid were placed on the same plate by keeping a minimum distance of 30 mm between them. Plates were further incubated for overnight at 37°C. Zone size of more than 5 mm around ceftazidime-clavulanic disk compared to ceftazidime disk alone was considered positive for ESBL production. Control strains  Escherichia More Details coli ATCC 25,922 and Klebsiella pneumoniae ATCC 700,603 were used for the procedure.[12]

Screening for AmpC beta-lactamases

AmpC production by the isolates was screened by disk diffusion method using cefoxitin disk and zone size of <18 mm was considered possible producer. It was further confirmed using cefoxitin-cloxacillin double disk synergy test.[13]

Phenotypic AmpC confirmatory test by cefoxitin-cloxacillin double disk synergy test:

Thirty micrograms disk of cefoxitin and combination of cefoxitin and 200 μg of cloxacillin were used for this study. All strains of 0.5 McFarland unit were inoculated on Mueller-Hinton (MH) agar and further kept for overnight incubation at 37°C. Equal or more than 4 mm zone size difference between both the disks were indicative of AmpC production.[13]

Screening for metallo-beta-lactamases

All the clinical isolates showing resistance to imipenem disk were considered positive for MBL screening and were further subjected to confirmation by combined disk test.[14]

Phenotypic confirmatory test by imipenem-ethylenediaminetetraacetic acid (EDTA) combined disk test:

MBL-screened isolates were swabbed on MH agar and two disks of imipenem (10 μg) and imipenem with 10 μL of an EDTA solution were placed on the same plate, further incubated for overnight at 37°C. A zone of inhibition ≥7 mm around with the imipenem-EDTA disk compared to imipenem disk alone indicated MBL production.[14]

Collection and certification of medicinal plant

Neem (A. indica) was obtained and certified (University of Horticulture and Forestry (UHF) herbarium No. 13,588) from the Department of Forestry, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India.

Preparation of plant extract

The leaves of A. indica were separated, washed, and dried in shade. Plant extract was prepared with 800 g of dry plant powder soaked in 2.5 l of 70% ethanol, for 10 days and stirred after every 10 h using a sterilized glass rod. At the end of extraction, it was passed through Whatman filter paper No. 1. This ethanoic filtrate was concentrated using water bath at 40°C till the sticky semisolid mass was obtained and then stored at 4°C for further use. The crude extract was prepared by dissolving known amount of the extract in dimethylsulfoxide, to have a stock solution of 100 mg/ml concentration.[15]

Antibacterial activity of plant extract

Antibacterial activity of ethanolic neem extract was carried out by disk diffusion method. Turbidity of the culture was adjusted to 0.5 McFarland standards. Culture suspensions were inoculated on MHA using lawn culture. Sterile paper disks (6 mm, HiMedia, Mumbai, Maharashtra, India) were impregnated with 20 μl of the different concentration (100, 50, 25, 12.5, and 6.25 mg/ml) of plant extracts and were placed on the inoculated agar. For negative control, disks impregnated with 20 μL of 70% ethanol were placed at the center of inoculated MHA. Culture plates were incubated at 37°C for 24 h. After incubation period, the zones of inhibition was measured in mm with the help of antibiotic susceptibly scale (HiMedia Laboratories, Mumbai, Maharashtra, India).[16]

Determination of minimum inhibitory concentration and minimum bactericidal concentration

Broth microdilution method was used for estimation of minimum inhibitory concentration (MIC), which is the lowest concentration of the antibacterial agent at which there is no visible growth seen. Microplate (96 polystyrene well) was used for the preparation of different concentration of neem extract (100-6.25 mg/ml) by serial dilution. The final concentration of each strain suspension was adjusted to 5 × 105 CFU/ml with 10 μL aliquot of bacterial suspension in supplemented MH broth, in a final volume of 100 μL. Positive growth control (well with overnight culture in MH broth), negative controls (MH broth medium alone), and color control (wells containing only extracts) were also prepared. The plates were covered with a sterile plate sealer, carefully mixed, and incubated at 37°C for 24 h. Bacterial growth was indicated by the turbidity, relative to the negative and positive controls. The minimum bactericidal concentration (MBC) was obtained by subculturing from each well of microplate onto a nutrient agar plate. The well containing the lowest concentration of the extract that failed to show growth on subculture was considered as MBC for that test strain.[17],[18]


  Results Top


A. indica was tested for antibacterial properties against 221 Gram negative uropathogens causing urinary tract infection [Table 1].
Table 1: Distribution of uropathogens

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Antibacterial activity of neem (Azadirachta indica) against Escherichia coli

Neem was tested against 100 E. coli in which 32 (32%) were ESBL, 18 (18%) were AmpC producers, and 50 (50%) were non beta-lactamase-producing strains. Maximum zone of inhibition (13 mm) was obtained at the highest concentration and minimum zone of inhibition (07 mm) was obtained at the lowest concentration. MIC and MBC of neem against E. coli was turned out to be 1.56 and 3.12 mg/ml, respectively [Table 2].
Table 2: Antibacterial activity of A.indica against E. coli and its distribution

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Antibacterial activity of neem (Azadirachta indica) against Klebsiella pneumoniae

Of the total 37 K. pneumoniae isolates, 14 (38%) were ESBL producers, 7 (19%) were AmpC producers, and 16 (43%) were non-beta-lactamase-producing strains. Neem was tested against all isolates for zone of inhibition, MIC, and MBC. Maximum zone of inhibition (14 mm) was obtained at 100 mg/ml and minimum zone of inhibition (07 mm) was obtained at 6.25 mg/ml. MIC was 1.56 and MBC was 3.12 mg/ml [Table 3].
Table 3: Antibacterial activity of A. indica against K. pneumoniae and its distribution

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Antibacterial activity of neem (Azadirachta indica) against Enterobacter species

Of the 20 Enterobacter species isolated, 4 (20%) were ESBL producers and 16 (80%) were non-beta-lactamase-producing strains. Maximum zone of inhibition (14 mm) was obtained at 100 mg/ml and minimum zone of inhibition (7 mm) was obtained at 6.25 mg/ml. MIC was 3.1 mg/ml and MBC was 6.25 mg/ml [Table 4].
Table 4: Antibacterial activity of A. indica against Enterobacter species and its distribution

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Antibacterial activity of neem (Azadirachta indica) against Citrobacter species

Of the 17 Citrobacter species, 5 (29%) were ESBL producers and 12 (71%) were non-beta-lactamase-producing strains. Citrobacter species showed maximum zone (14 mm) at 100 mg/ml and minimum zone (07 mm) at 6.25 mg/ml concentration of neem. MIC and MBC of neem was 1.56 and 3.12 mg/ml, respectively, against all Citrobacter isolates [Table 5].
Table 5: Antibacterial activity of A. indica against Citrobacter species and its distribution

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Antibacterial activity of neem (Azadirachta indica) against Proteus species

Of the 10 Proteus species, 3 (30%) were ESBL producers and 7 (70%) were non-beta-lactamase-producing strains. Maximum zones (15 mm) were obtained at 100 mg/ml and minimum zones (07 mm) obtained at 6.25 mg/ml concentration. Proteus species showed 3.12 mg/ml MIC and 6.25 mg/ml MBC of neem [Table 6].
Table 6: Antibacterial activity of A. indica against Proteus species and its distribution

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Antibacterial activity of neem (Azadirachta indica) against Pseudomonas aeruginosa

Of the 25 Pseudomonas aeruginosa isolates, 7 (28%) isolates were MBL producers and the rest 18 (72%) were non-beta-lactamase-producing strains. P. aeruginosa did not show any zone of inhibition at 6.25 mg/ml concentration, whereas at 100 mg/ml concentration, it showed 12 mm zone of inhibition. MIC was 6.25 mg/ml and MBC was 12.5 mg/ml for all P. aeruginosa isolates [Table 7].
Table 7: Antibacterial activity of A. indica against P. aeruginosa and its distribution

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Antibacterial activity of neem (Azadirachta indica) against Acinetobacter baumannii

Of the 12 Acinetobacter baumannii isolates, 4 (33%) were MBL producers and 8 (67%) were non-beta-lactamase-producing strains. Maximum zone of inhibition (15 mm) was found at 100 mg/ml and minimum (8 mm) inhibitory zone was found at 6.25 mg/ml concentration of neem.

MIC was 1.56 mg/ml and MBC was 3.12 mg/ml for all A. baumannii isolates [Table 8].
Table 8: Antibacterial activity of A. indica against A. baumannii and its distribution

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  Discussion Top


Neem (A. indica) is a tree in the mahogany family Meliaceae and is used in traditional medicine as a source of therapeutic agent in the Indian culture and grows well in tropical countries. Neem ingredients are applied in Ayurveda, Unani, Homeopathy, and modern medicine for the treatment of many infectious, metabolic, or cancer diseases. It has a complex of various constituents including nimbin, nimbidin, nimbolide, and limonoids and such types of ingredients play a role in disease management through modulation of various genetic pathways and other activities. Quercetin and beta-sitosterol were first polyphenolic flavonoids purified from fresh leaves of neem and were known to have antifungal and antibacterial activities.[19],[20]

Ethanolic extract of neem leaves was effective in all concentrations (100-6.25 mg/ml) against all seven clinical isolates; however, neem revealed best antibacterial activity against A. baumannii as it showed maximum zone of inhibitions (15 mm) and minimum values of MBC (3.12 mg/ml) and MIC (1.5 mg/ml) [Figure 1] and [Figure 2].
Figure 1: Maximum zone of inhibition (mm) of neem against uropathogens

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Figure 2: Average minimum inhibitory concentration and minimum bactericidal concentration values of neem against uropathogens

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Our study showed a range of 13-7 mm zone of inhibitions against E. coli at different concentrations of neem. Krishnan et al. reported 9–13 mm inhibitory zones at different concentration (10–100 mg/ml) of leaves extracts against E. coli, which was almost similar to the present study.[21] Raut et al. also demonstrated 11 mm zone of inhibition at 100 mg/ml concentration against E. coli.[22] Other authors also stated antibacterial activity of neem against E. coli at various concentrations.[23],[24],[25] MBC and MIC value of neem against E. coli was 3.12 and 1.56 mg/ml, respectively, similar MIC value being reported by Yehia,[24] whereas in a study done by Krishnan et al.,[21] MIC was 10 mg/ml.

In the present study, neem showed 14-7 mm inhibitory zones against K. pneumoniae at various concentrations. Similar antibacterial activity (14.5-7.8 mm) was demonstrated by Mohammed and Omer who also used the same concentrations as our study.[26] Another study done by Kalita et al. revealed 8.33 and 15.1 mm zone of inhibitions at 10% of aqueous and acetone extracts of neem respectively against K. pneumonia.[27] Chaturvedi et al. too reported significant activity against K. pneumoniae at different concentrations of neem aqueous extracts.[28]

Citrobacter species showed 14-7 mm zone of inhibitions against neem. Thanigaivel et al.[29] also reported good anti Citrobacter freundii activity by neem. In contrast to our study Sakha et al.[30] and Jadhav et al.[31] did not find any antibacterial activity against C. freundii.

There is no literature available of antibacterial activity of neem against Enterobacter species, but studies suggest that neem has good antibacterial potential against members of Enterobacteriaceae family[32] and our study also showed moderate antibacterial activity (14-7 mm, MBC 6.25 and MIC 3.1 mg/ml) of neem against Enterobacter species.

Researchers from Sudan had screened neem with similar concentrations (100-6.25 mg/ml) against 21 isolates of Proteus mirabilis and revealed 14.9-8.4 mm inhibitory zones which were in concordance to our study.[26] Another study from India mentioned good antibacterial activity (15-13 mm) by aqueous extract of neem at lower concentrations (2000-500 μg/ml).[33] Singaravelu et al. also reported 25-11 mm zone of inhibition against P. mirabilis at 2000-500 μg/ml concentrations and MIC was 0.5 mg/ml which was lower compared to our study.[34]

Our study demonstrated 13-07 mm inhibitory zones for P. aeruginosa. In a study done in Malaysia, chloroform extract of neem leaf was used and it showed 14.33 ± 0.58-8.00 ± 0.00 zones against P. aeruginosa at different concentrations (100-10 mg/ml).[21] Another author used same concentrations on 12 strains of P. aeruginosa and observed 15.8-10.6 mm zones of inhibitions.[26] Raja Ratna Reddy Y et al. also observed good anti-P. aeruginosa activity (15-13 mm) at lower concentrations.[33] In contrast to the present study, Singaravelu et al. found out 450 μg/ml MIC of methanolic neem extract against P. aeruginosa which was much lower.

A. baumannii showed 15-08 mm zones. Kavitha M et al. stated 12 mm inhibitory zones at 250 μg/ml against Acinetobacter.[35] Another author also demonstrated 14.56 mm zone with methonolic of neem which is also in concordance to our study.[36] On the other hand R. H. Autade et al. did not observe any antibacterial activity against A. baumannii.[37]


  Conclusion Top


Our study concluded that ethanolic extract of neem was over all effective against all beta-lactamase and non-beta-lactamase-producing clinical isolates. On the basis of these evidences, our study suggests that neem leaves contain several bioactive compounds which has potential anti-bacterial activity against wide range of bacterial pathogens. However, the mechanism behind its antibacterial property remains unclear. Further, well-designedin vivo clinical trials are needed to identify and characterize newer bio molecules which can be used in alternative to antibiotics.

Acknowledgment

I humbly thank the Department of Forestry, Dr. Y. S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, for neem plant authentication and certification.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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