Research Article

Comparative studies on the antibacterial activities of leaf extracts of Azadirachta indica and Psidium guajava and antibiotics on methicillin and vancomycin resistant Staphylococcus aureus

Eze C.1, Iroha I. R.2, Eluu S. C.3, Ejikeugwu P. C.2*, Iroha C. S.4, Ajah M. I.5, Nwakaeze E. A.2, Ugwu E. N.2

1National Agency for Food and Drug Administration and Control (NAFDAC) Owerri, Imo State, Nigeria

2Department of Applied Microbiology, 3Department of Biological Sciences (Biotechnology Programme), Faculty of Sciences, Ebonyi State University, 4Department of Pharmacy, 5Cancer Screening Unit, Federal Teaching Hospital, P.M.B 102, Abakaliki, P.M.B 053, Ebonyi State, Nigeria

 

*For correspondence

Ejikeugwu Chika,

Department of Applied Microbiology, Faculty of Sciences, Ebonyi State University, Abakaliki, P.M.B 053, Ebonyi State, Nigeria.

Email: ejikeugwu_chika @yahoo.com

 

 

 

 

 

 

 

 

 

Received: 26 February 2017

Accepted: 16 March 2017

ABSTRACT

Objective: This study investigated the antibacterial activity of the acetone, methanol, and ethanol leaf extracts of Azadirachta indica and Psidium guajava against methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Staphylococcus aureus (VRSA) strains as well as the antimicrobial activity of some conventional antibiotics on these multidrug resistant organisms.

Methods: MRSA and VRSA strains were obtained from the culture collection unit of a tertiary hospital in Ebonyi State, Nigeria. The test organisms and the plant extracts were processed using standard microbiology techniques. Kirby-Bauer disk diffusion technique was used to determine the antimicrobial susceptibility profile of the MRSA and VRSA strains to some conventionally used antibiotics; and the antibacterial activity of the plant extracts was evaluated using agar well diffusion technique with three solvents: ethanol, methanol and acetone.

Results: It was revealed in this study that the extracts of A. indica and P. guajava demonstrated some level of antimicrobial activity against the test organism at concentrations of 100 mg/ml and 50 mg/ml. Compared to the conventional antibiotics used, the antibiotics showed better antimicrobial activity against the test organisms than the plant extracts which was least active against the MRSA and VRSA strains.

Conclusions: Though the A. indica and P. guajava extracts showed some appreciable antimicrobial activity against the MRSA and VRSA strains, the conventional antibiotics produced better antimicrobial action against these multidrug resistant bacteria. The search for novel compounds with putative antimicrobial activity should be stepped up since plants holds the potential for discovering novel drugs.

Keywords: Antimicrobial resistance, Herbal plants, MRSA, VRSA, Nigeria

 

Introduction

Infectious diseases are becoming a major cause of human and animal mortality and morbidity around the world; and human pathogenic bacteria including Staphylococcus aureus are a major cause of bacteremia, associated with higher morbidity and mortality compared to other bacteremia-causing pathogens.1 Pathogenic S. aureus causes purulent skin and soft tissue infections that frequently reoccur in infected individuals.2 Antibiotics are one of our most important weapons in fighting bacterial infections and have greatly benefited the health-related quality of human life since their introduction into clinical medicine, but the emergence and spread of drug resistant bacteria has made some of these agents inefficacious for treatment.3 Despite advances in antibiotic therapy, infectious diseases still remain an important cause of mortality and morbidity among hospitalized patients.4 Even though pharmacological industries have produced a number of new antibiotics in the last three decades, resistance to these drugs by microorganisms has increased.5 Resistant bacteria are capable of withstanding attack by antibacterial agent; hence, infections persist irrespective of standard treatments, creating more chances for its spreading to other persons.6 Over the last few decades, the number and proportion of vancomycin-resistant S. aureus (VRSA) and methicillin-resistant S. aureus (MRSA) infections in different countries has increased.7,8 Several studies have shown that MRSA strains are highly resistant to antibiotics.9-11 Despite the discovery of new drugs for the treatment of infections caused by VSRA and MRSA, outbreaks of MRSA/VRSA infections often appear in hospitals, clinics, nursing and elderly homes.12 Thus, the search for effective antimicrobial agents and/or alternative approaches to the conventional use of antibiotics is urgently needed to contain the growing prevalence of antimicrobial resistant bacteria in both the community and hospital environments. More than 80% of the world's population of developing countries relies on traditional medicine for their primary healthcare needs; and antimicrobials of plant origin have enormous therapeutic potential.13,14 Herbal plants are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with some available synthetic antimicrobials; and Azadirachta indica and Psidium guajava are some well-known plant of medical importance that possess wide range of antibacterial potentials. This research work was therefore designed to investigate and compare the antibacterial potentials of leaf extracts of A. indica (Neem) and P. guajava (Guava) and those of some conventional antibiotics on methicillin and vancomycin resistant S. aureus.

Materials and Methods

Collection, identification and preparation of plant materials

The leaves of Azadirachta indica (neem) and Psidium guajava (guava) were collected from Abakaliki in Ebonyi State, and identified by Prof. S.C.C. Onyekwere of Applied Biology, Faculty of Sciences, Ebonyi State University, Abakaliki. The samples after identification were rinsed with clean water and sun dried for five (5) days, grinded into powder and stored separately in a transparent air-tight glass bottles, and kept in laboratory cupboard until required for use. Exactly 20 g each of the pulverized plant material was measured into 75 ml of each of the extraction solvents that included acetone, methanol, and ethanol in a conical flask and covered with an aluminum foil. The mixture was placed in a mechanical shaker and allowed for twenty four (24) hours, after which it was decanted and filtered using Whatmann number 1 filter paper. The extracts were allowed to air-dry. Purity test was conducted by plating the extracts on nutrient agar and incubated for 24 h at 37 oC.13

Test organisms

Methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Staphylococcus aureus (VRSA) were obtained from the culture collection unit of a tertiary hospital in Ebonyi State, Nigeria.

Antibacterial activity of the plant extracts

Twenty (20) milliliter each of sterilized molten Mueller-Hinton (MH) agar was poured aseptically into sterile petri dishes of equal sizes and then allowed to gel. The surface of the MH agar plates were then streaked with standardized inocula of the test bacteria (adjusted to 0.5 McFarland turbidity standards). Thereafter, a sterilized 6 mm cork borer was used to bore six (6) holes on the Mueller Hinton agar plates, and five (5) of the holes were filled with equal volumes of the respective plant extracts that was diluted with 75% DMSO. DMSO was used as a negative control. The plates were allowed to stand for about 30 minutes for pre-diffusion of the plant extracts, and these were incubated at 37oC for 24 h. After incubation, the inhibition zone diameters of each plant extracts were measured in milliliter using a meter rule.13

Determination of antimicrobial susceptibility patterns of VRSA and MRSA

Antimicrobial susceptibility studies were determined using a previously described methodology.15 VRSA and MRSA were subjected to antimicrobial susceptibility testing in line with CLSI guideline, using the Kirby-Bauer disc diffusion method. Single discs of ceftazidime (30 µg), erythromycin (15 µg), vancomycin (30 µg), cefotaxime (30 µg), tetracycline (30 µg), aztreonam (30 µg), piperacillin (100 µg), ceftriaxone (30 µg), ampicillin (10 µg), clindamycin (2 µg), ciprofloxacin (5 µg), sulphamethoxazole (25 µg), cefoxitin (30 µg), and cefuroxime (30 µg) were aseptically placed on the surface of MH agar plates already inoculated with the test organism (adjusted to 0.5 McFarland turbidity standards), and incubated at 37oC for 24 hrs. Zone clearance was measured and interpreted according to CLSI interpretative criteria.16

Results and Discussion

The antibacterial activity of the plant extracts of Azadirachta indica and Psidium guajava on the VRSA and MRSA organisms is shown in Table 1. The acetone extract of A. indica had no inhibitory effect on both the VRSA and MRSA strains. However, there was an appreciable inhibitory activity against the VRSA and MRSA strains as was produced by the methanol and ethanol extracts of both A. indica and P. guajava. The acetone extract of P. guajava had inhibitory activity on two strains of VRSA and two strains of MRSA (Table 1). Table 2 shows the antibacterial activity of the plant extracts of Azadirachta indica and Psidium guajava on the VRSA and MRSA organisms at a concentration of 50 mg/ml. The acetone extract of A. indica and the methanol extract of P. guajava had no inhibitory activity against the MRSA and VRSA strains. However, ethanol extract of A. indica showed inhibitory activity against the MRSA and VRSA strains. The methanol extract of A. indica only had activity on one VRSA strain. The ethanol and acetone extract of P. guajava also showed some antimicrobial activity against the MRSA and VRSA strains (Table 2).

Figure 1: Antibacterial susceptibility patterns of MRSA.

CAZ = ceftazidime, E = erythromycin, VA = vancomycin, CTX = cefotaxime, TE = tetracycline, ATM = aztreonam, PRL = piperacillin, CRO = ceftriaxone, AMP = ampicillin, DA = clindamycin, CIP = ciprofloxacin, SXT = sulphamethoxazole, CFX= cefoxitin, CXM = cefuroxime.

Table 1: Inhibition zone diameter of acetone, methanol, and ethanol leaf extracts of A. indica and P. guajava against VRSA and MRSA strains at concentrations of 100 mg/ml.

Bacterial strains

Inhibition zone diameters (IZDs)

 

Acetone

Methanol

Ethanol

Acetone

Methanol

Ethanol

 

A. indica

P. guajava

29A

NI

10

NI

15

NI

16

24A

NI

10

12

NI

NI

15

5A

NI

12

NI

18

15

12

9B

NI

10

12

17

12

15

13A

NI

NI

NI

20

NI

14

34A

NI

NI

12

NI

NI

19

Key: VRSA strains = 5A, 24A, 29A, MRSA strains = 9B, 13A, 34A, NI = no inhibition

Table 2: Inhibition zone diameter of acetone, methanol, and ethanol leaf extracts of A. indica and P. guajava against VRSA and MRSA strains at concentrations of 50 mg/ml.

Bacterial strains

Inhibition zone diameters (IZDs)

 

Acetone

Methanol

Ethanol

Acetone

Methanol

Ethanol

A. indica

P. guajava

29A

NI

NI

NI

12

NI

NI

24A

NI

NI

08

NI

NI

10

5A

NI

10

NI

10

NI

10

9B

NI

NI

NI

12

NI

10

13A

NI

NI

NI

16

NI

09

34A

NI

NI

09

11

NI

11

Key: VRSA strains = 5A, 24A, 29A, MRSA strains = 9B, 13A, 34A, NI = no inhibition.

Table 3. Inhibition zone diameter of acetone, methanol, and ethanol leaf extracts of A. indica and P. guajava against VRSA and MRSA strains at concentrations of 25 mg/ml.

Bacterial strains

Inhibition zone diameters (IZDs)

 

Acetone

Methanol

Ethanol

Acetone

Methanol

Ethanol

A. indica

P. guajava

29A

NI

NI

NI

NI

13

NI

24A

NI

NI

NI

NI

NI

NI

5A

NI

NI

NI

10

NI

NI

9B

NI

NI

NI

09

NI

NI

13A

NI

NI

NI

NI

NI

NI

34A

NI

NI

NI

13

NI

10

Key: VRSA strains = 5A, 24A, 29A, MRSA strains = 9B, 13A, 34A, NI = no inhibition.

Table 3 shows the antibacterial activity of the plant extracts of Azadirachta indica and Psidium guajava on the VRSA and MRSA organisms at a concentration of 25 mg/ml. The ethanol, acetone and methanol extract of the A. indica plant had no inhibitory activity against the VRSA and MRSA strains. This was also applicable to the acetone, ethanol and methanol extract of the P. guajava plant which also showed a reduced level of antimicrobial activity against the test organisms (Table 3).

Table 4 shows the antibacterial activity of the plant extracts of Azadirachta indica and Psidium guajava on the VRSA and MRSA organisms at a concentration of 12.5 mg/ml. The acetone, methanol and ethanol extract of A. indica had no inhibitory activity on the VRSA and MRSA strains. The acetone extract of P. guajava had antimicrobial activity on only one VRSA strain and 2 MRSA strains while the methanol extract of P. guajava had inhibitory activity on 2 VRSA strains and one MRSA strain. Ethanol extract of P. guajava showed antimicrobial activity on one VRSA strain and one MRSA strain (Table 4). The antibacterial activity of the plant extracts of Azadirachta indica and Psidium guajava on the VRSA and MRSA organisms at a concentration of 6.25 mg/ml is shown in Table 5. The acetone, methanol and ethanol extract of A. indica and P. guajava had no antimicrobial activity on the VRSA and MRSA strains. The antimicrobial susceptibility profiles of the MRSA and VRSA strains to some conventionally used antibiotics are shown in Figure 1 and 2. The MRSA strains were found to be more susceptible to erythromycin, tetracycline, piperacillin, ceftriaxone, clindamycin, ciprofloxacin and sulphamethoxazole trimethoprim (Figure 1). VRSA strains showed susceptibility to cefotaxime, piperacillin, ciprofloxacin, tetracycline, cefuroxime, ceftriaxone, ampicillin and sulphamethoxazole-trimethoprim (Figure 2).

Table 4: Inhibition zone diameter of acetone, methanol, and ethanol leaf extracts of A. indica and P. guajava against VRSA and MRSA strains at concentrations of 12.5 mg/ml.

Bacterial strains

Inhibition zone diameters (IZDs)

 

Acetone

Methanol

Ethanol

Acetone

Methanol

Ethanol

A. indica

P. guajava

29A

NI

NI

NI

NI

10

NI

24A

NI

NI

NI

NI

10

12

5A

NI

NI

NI

10

NI

NI

9B

NI

NI

NI

09

10

12

13A

NI

NI

NI

NI

NI

NI

34A

NI

NI

NI

13

NI

NI

Key: VRSA strains = 5A, 24A, 29A, MRSA strains = 9B, 13A, 34A, NI = no inhibition

Table 5: Inhibition zone diameter of acetone, methanol, and ethanol leaf extracts of A. indica and P. guajava against VRSA and MRSA strains at concentrations of 25 mg/ml.

Bacterial strains

Inhibition Zone Diameters (IZDs)

 

Acetone

Methanol

Ethanol

Acetone

Methanol

Ethanol

A. indica

P. guajava

29A

NI

NI

NI

NI

NI

NI

24A

NI

NI

NI

NI

NI

NI

5A

NI

NI

NI

NI

NI

NI

9B

NI

NI

NI

NI

NI

NI

13A

NI

NI

NI

NI

NI

NI

34A

NI

NI

NI

NI

NI

NI

       

NI

NI

NI

Key: VRSA strains = 5A, 24A, 29A, MRSA strains = 9B, 13A, 34A, NI = no inhibition.

Figure 2: Antibacterial susceptibility patterns of VRSA.

CAZ = ceftazidime, E = erythromycin, VA = vancomycin, CTX = cefotaxime, TE = tetracycline, ATM = aztreonam, PRL = piperacillin, CRO = ceftriaxone, AMP = ampicillin, DA = clindamycin, CIP = ciprofloxacin, SXT = sulphamethoxazole, CFX= cefoxitin, CXM = cefuroxime, MRSA Strains = 5A, 23A, 24A.

The alarming increase in the rate of infection by antibiotic-resistant microorganisms has urged scientists to search for compounds which have potential antimicrobial activity against these resistant organisms. Many researchers worldwide have investigated the antimicrobial properties of plants and its uses which are rooted in the antimicrobial traits of different species of plant around the world.13,17-20 In this study, the antimicrobial activity of the acetone, ethanol and methanol extracts of two plants, A. indica and P. guajava leaves were evaluated on Methicillin Resistant Staphylococcus aureus (MRSA) strains, and Vancomycin Resistant Staphylococcus aureus (VRSA) strains; and the antimicrobial activity of these plant extracts was compared to those of some conventionally used antibiotics. The results of the antimicrobial activity of the acetone, methanol, and ethanol extracts of A. indica leaves against the VRSA and MRSA strains showed that the extracts had greater antimicrobial activity against the VRSA and MRSA strains especially at a higher concentration. It was observed in this study that the acetone, methanol, and ethanol extracts of A. indica leaves showed appreciable levels of antimicrobial activity at 100 mg/ml and 50 mg/ml than at lower concentrations of 25, 12.5 and 6.25 mg/ml. P. guajava leave extracts also showed similar antimicrobial activity on the VRSA and MRSA strains used in this study. Surprisingly, it was observed in our study that the acetone, methanol and ethanol extract of both A. indica and P. guajava had no antimicrobial activity on the VRSA and MRSA strains. The antimicrobial activity of the acetone, methanol, and ethanol extracts of A. indica leaves and P. guajava as reported in this study is in line to previously reported work on the antimicrobial activity of herbal plants on antibiotic resistant bacteria.18,19,21 In comparison to the conventional antibiotics evaluated in this study, we discovered that the extracts of both A. indica and P. guajava showed lesser antimicrobial activity against the MRSA and VRSA strains. The conventional antibiotics showed better antimicrobial activity against the MRSA strains and VRSA strains when compared to the acetone, ethanol and methanol extracts of both A. indica and P. guajava. This goes to show that some of these conventional antibiotics are still efficacious, even though some antibiotic resistant bacteria such as VRSA and MRSA strains may remain viable in their presence. However, the search for novel and better antimicrobial agents especially from plants should be stepped up in order to discover and develop better antimicrobial therapy for containing the emergence and spread of drug resistant bacteria across the globe.

Funding: No funding sources

Conflict of interest: None declared

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