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EFFICACY OF ANTI-BACTERIAL COMPOUNDS AND PLANT EXTRACTS AGAINST METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS ISOLATES FROM NOSOCOMIAL INFECTIONS.

Byline: M. Q. Zeshan, M. Ashraf, M. O. Omer, H. M. Khan, M. A. Ali and A. A. Anjum

ABSTRACT

Sensitivity of Methicillin Resistant Staphylococcus aureus (MRSA) isolates purified from post operative wounds (n=100) from patients in surgical wards of Mayo, SIMS and Jinnah Hospital in Lahore-Pakistan was evaluated for antibiotics and plant extracts. The minimum inhibitory concentrations of ethanol extracts of medicinal plants (Opuntia delinii, Acacia nilotica and Aloe vera) and antibiotics (Moxifloxacillin, Cefipime, Imipenem/Cilastatin and Ampicillin+Cloxacillin) were determined by micro broth dilution method. Staphylococcus aureus isolates were identified on the basis of colonial morphology, microscopic morphology and biochemical characteristics. Resistance to Methicillin recorded was 72, 63 and 66 percent in isolates recovered from Mayo, SIMS and Jinnah hospital patients, respectively. Methanol extracts of Aloe vera, Opuntia delinii leaves, bark and leaves of Acacia nilotica were dried.

Activity of Aloe vera against MRSA was highest with MIC value 32.248 g/mL followed by Acacia nilotica leaves (62.5 g/mL) and then of Acacia nilotica bark (84 g/mL). The extracts of Opuntia dillenii leaves had very low anti- MRSA activity having highest MIC value (1228 g/mL). The MIC of Moxifloxacillin and Imipenem, Cefipime and Ampicillin+Cloxacillin calculated against MRSA isolates was 2.68, 2.85, 57.81 and 11.32 g/mL, respectively. Results of few selected anti-bacterial compounds were in close association with Linezolid (1.61 g/mL) and Vancomycin (2.43 g/mL).Among selected anti-bacterial agents efficacy of Moxifloxacillin, Imipenem, Ampicillin+Cloxacillin and Cefipime was comparable with standards. Both anti-bacterial compounds and extracts of studied plants may efficiently be used as alternate therapeutics against MRSA isolates.

Key words: Methicillin resistant Staphylococcus aureus, Plant extracts, Anti-bacterial, Minimum inhibitory concentration and Nosocomial infections

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) is a public health hazard since its inception and has spread across the world as an indicator agent of Nosocomial infections. Post surgical infections of patients in orthopedic units are mainly (48%) caused by S. aureus and 68% by MRSA (Eseonu et al., 2011). Prolonged hospital stays, indiscriminate and irrational use of anti-bacterial compounds prior to visit physicians are main factors of MRSA emergence (Huttner et al. 2012; Kaleem et al., 2010). Estimated frequency of MRSA is much higher (61%) in areas with thick human population in different cities of the Pakistan (Hafiz et al., 2002).

S. aureus is normal inhabitant on skin and mucous membranes in human beings. In certain instances, bacteria enter through abraded skin or mucous membrane and can cause abscesses, pneumonia, meningitis, endocarditis and septicaemia (You et al., 2011). It is established fact that MRSA isolates resist to numerous available beta-lactam antimicrobials. This resistance is mediated by the mecA gene which codes for a penicillin binding protein (PBP)2a with low affinity for beta-lactams (Akliluet al., 2013).

MRSA emerged in1970s and its number increased dramatically by 1990s throughout the world. In recent years a major change in epidemiology of MRSA has been observed, with the appearance of cases in the community affecting people having no epidemiological connection with hospitals. The strains isolated from such cases are called Community-acquired or Community-associated MRSA (CA-MRSA). Isolates from these cases have both phenotypic and genotypic differences with classically health-care associated MRSAs (Roberts 2013).

Alkaloids, tannins and flavonoids extracted from different medicinal plants exhibit antimicrobial potential (Fernandez et al., 2012). The Acacia nilotica, Aloe vera and Opuntia dillenii have been tried successfully for therapy of gram positive bacterial infections including those caused by MRSAs (Abd-El-Nabi, 1992).

Infections caused by MRSA isolates are treated by Vancomycin (glycopeptide antibiotic) routinely. It has been reported that MRSA is resistant to most of the antibiotics with the exception of Vancomycin and Linzolid (Kaleem et al., 2011). Some reports on resistance of MRSA to Vancomycin have been documented (MACopyrightlard et al., 2013). Cefepime, Moxifloxacin, Imipenem and combination of Ampicillin with Cloxacillin are being practiced to limit the post operative infections (Jacobsen et al., 2011). In view of these facts present work was planned to evaluate the efficacy of selected antibacterial agents and medicinal plant extracts against MRSA isolated from indoor patients of selected tertiary care hospitals.

MATERIALS AND METHODS

In-vitro efficacy of selected anti-bacterial agents (Moxifloxacin, Imipenem, Cefepime and Ampicillin- Cloxacillin) and medicinal plant extracts (Opuntia dillenii, Acacia nilotica and Aloe vera) was determined against Methicillin Resistant Staphylococcus aureus (MRSA) isolates. Tested MRSAs were isolated from post-operative wounds of indoor patients.

Isolation of MRSA: Pus samples (n=100) were collected aseptically from surgical wards of Mayo hospital (n=40), SIMS (n=30) and Jinnah Hospital (n=30), Lahore- Pakistan from post-operative wounds of indoor patients admitted not less than five days. The samples were processed for identification of S. aureus using cultural, morphological and biochemical characteristics. Resistance of S. aureus isolates to methicillin was determined by disc diffusion method following the protocol described by Anupurba et al. (2003). Briefly, Staph.110 medium was used to check the resistance of isolates against methicillin. Bacterial suspension (01 mL) having 5x105 colony forming units was spread over the surface of solidified agar under sterilized conditions. Methicillin (05 g) impregnated discs were placed over the inoculated culture plates and incubated for 24 hours. The isolates showing no zone of inhibition around methicillin discs were declared as MRSA.

Plant extracts: Leaves of three medicinal plants (Opuntia dillenii, Acacia nilotica and Aloe vera) were collected and got identified from Herbarium, Government College University, Lahore. Ethanol extracts of medicinal plants (leaves of Opuntia dilenii, Acacia nilotica, Aloe vera and bark of Acacia nilotica) were prepared using Soxhlet apparatus. All the extracts were filtered through membrane filter of 0.22 m pore size in a safety cabinet to avoid bacterial contamination (Wittschier et al., 2009). Ethanol extracts were solidified by evaporating ethanol in a hot air oven.

Minimum Inhibitory Concentrations: Minimum inhibitory concentrations of plant extracts and antibiotics were determined by broth micro dilution method in 96 wells microtitre plates. Stock solutions (2%) of powdered plant extracts were prepared in normal saline (0.89%).

Concentrations of medicinal plant extracts and antibiotics used were 4-2048g/mL and 0.9-200 g/mL. To prepare the inoculums, fresh colonies of MRSA were re- suspended in phosphate buffer saline. Optical density was adjusted to 0.5 McFarland standards and further diluted by 1:100 times in LB broth. Each dilution of antibiotic solutions (50 l) was added in 96 well micro-titration plate wells and inoculated with previously prepared MRSA inoculums (100 l). Positive controls contained only the media and inoculums without antibiotic, while negative control had media only. Plates were incubated aerobically at 37C for 24 hours. Minimum inhibitory concentration values for each of the antibiotics and medicinal plant extracts were recorded (Modarresi- Chahardehi et al., 2012).

Chi-Square test was used for the statistical analysis of the data.

RESULTS AND DISCUSSION

Staphylococcus aureus causes infections of skin, respiratory system, blood, soft tissues, intestinal and urinary tracts (Verhagen et al., 2013).Therapy of bacterial infections fails due to acquisition of resistance against anti-bacterial agents (Martins et al., 2013). Percent of septic wounds by methicillin resistant S. aureus (MRSA) infection was recorded 66% on the basis of microscopic studies, biochemical profile and resistance to methicillin. Infection of wounds by MRSA recorded was little higher (27/40=72%) in patients of Mayo hospital than Jinnah (20/30=66%) and SIMS (19/30=63%). However, the difference observed was statistically non significant (p=0.932). Difference among patients admitted in different surgical wards of the hospitals was non- significant (p=0.936). Butt et al. (2013) recorded 34.6% prevalence of MRSA infection in patients admitted in a tertiary care hospital Karachi.

Hafiz et al. (2002) recorded different MRSA infection frequency in different parts of the country; 61%, 57%, 46%, 36%, 32%, 26% and 2% in Lahore, Karachi, Rawalpindi, Peshawar, Azad Kashmir, Quetta and Sukkur, respectively. Therapeutic use of antibiotics in post operative wounds was non- significantly associated with MRSA isolation (p= 0.652). Between genders, non-significant association of MSRA was observed (p= 0.461). Higher prevalence rate of MRSA infection in males as compared to females has been reported by Halablab et al. (2010); Tacconelli et al. (2004) and Selvey et al. (2000). Continuous use of methicillin resulted in the emergence of MRSA strains. Most strains of S. aureus are resistant to penicillin (Dezfulian et al., 2012). Forty two percent prevalence of MRSA was reported in Lahore-Pakistan by Hafiz et al. (2002). Percent prevalence of 70 and 37 was reported in different cities of India by Anupurba et al. (2003) and Rajaduraipandi et al. (2006).

Age of the patients was found insignificantly related to MSRA isolation (p= 0.427). Out of 66, bacterial isolates 10, 20, 18, 7, 5 and 6 were obtained from patients having age of 11-20, 21-30, 31-40, 41-50, 51-60 and 61-70 years, respectively. Statistically non- significant difference was observed in relation to span of stay in hospitals (p= 0.218). Same findings have been reported by Davis et al. (2014). In contrast McClelland et al. (1999) reported higher percentage (14.5) of MRSA infection among elderly people than young i.e. 6.3.

The patients suffering from appendicitis had highest infection with MRSA (14/14) followed by bullet fire wound (3/3), gynecological surgery (10/12), diabetic foot wound (9/11), bed sour (5/8), burn case (6/11), throat surgery (4/8), chest surgery (5/8) Colostomy (6/15), and hernia (4/10).Different parameters are presented at table (01).

Table 1: Prevalence of methicillin resistant Staphylococcus aureus in post operative hospitalized patients of tertiary care hospitals.

###MRSA

###Positive###Negative###Chi-Square###p-value

N###66###34

###Male###39###19

Gender###0.095###0.461

###Female###27###15

###11-20###10###19

###21-30###20###7

###31-40###18###9

Age (years)###41-50###7###3###5.965###0.427

###51-60###5###4

###61-70###6###1

###71-80###0###1

###1-5###10###12

###6-10###15###9

Hospital###11-15###13###6

Stays###16-20###7###3###8.291###0.218

(days)###21-25###13###3

###26-30###6###1

###31-35###2###0

###Mayo###27###13

Hospitals###Jinnah###19###11###0.141###0.932

###SIMS###20###10

###Colostomy###6###9

###Hernia###4###6

###Bed Sour###5###3

###Appendicitis###14###0

###Burn case###6###5

Diseases###20.765###0.014

###Chest surgery###5###3

###Diabetic foot wound###9###2

###Gynecological surgery###10###2

###Throat surgery###4###4

###Bullet fire wound###3###0

###South surgical Mayo Hospital###6###4

###North surgical Mayo Hospital###8###2

###East surgical Mayo Hospital###6###3

###West surgical Mayo Hospital###7###4

###Surgical ward 1 Jinnah Hospital###5###2

Wards###Surgical ward 2 Jinnah Hospital###5###2###4.906###0.936

###Surgical ward 3 Jinnah Hospital###5###2

###Surgical ward 4 Jinnah Hospital###4###5

###Surgical unit 1 SIMS###4###3

###Surgical unit 2 SIMS###4###3

###Surgical unit 3 SIMS###6###1

###Surgical unit 4 SIMS###6###3

###Ciprofloxacin###10###8

###Augmentin###15###7

###Imepenem###9###4

###Linzolid###7###5

###Ampicillin###3###2

Antibiotics###6.858###0.652

###Amoxicillin###3###3

###Metronidazole###1###1

###Vancomycin###12###1

###Moxifloxcin###5###3

###Levofloxacin###1###0

Sensitivity of plant extracts against MRSA isolates was checked for four selected antibiotics (Moxifloxacillin, Cefipime, Imipenem and Ampicillin plus Cloxacillin) in comparison with Linezolid and Vancomycin. The minimum inhibitory concentration (MIC) of Moxifloxacillin and Imipenem, Cefipime and Ampicillin+Cloxacillin calculated against MRSA isolates was 2.68, 2.85, 57.81 and 11.32 g/mL, respectively. The MICs of Moxifloxacillin and Imipenem were close to Linezolid (1.61 g/mL) and Vancomycin (2.43 g/mL). Anti-MRSA activity of Moxifloxacillin, Imipenem, Ampicillin+Cloxacillin and Cefipime was comparable with standards.

Vancomycin is extensively used in MRSA infected wounds (Allen and Nicas, 2003). Most of the selected MRSA isolates were sensitive to vancomycin (99%) and strengthened the observations of Kaleem et al. (2011). In contrast, intermediate level of resistance against the vancomycin was recorded by MACopyrightlard et al. (2013). Similar pattern of intermediate resistance of MRSA to vancomycin was analyzed by Vidaillac et al. (2013) and Assadullah et al. (2003) in India (6%). Calculated values of MIC (vancomycin) against MRSA ranged from 0.5 - 4 g/mL and drug may be the choice agent against MRSA.

Linezolid, a standard drug, showed 100 percent effectiveness against the MRSA as no isolate found to be resistant. In corroborations findings for efficacy of linezolid were recorded by Fatholahzadeh et al. (2008) in Iran and Kaleem et al. (2010) in Pakistan. In agreement results were declared by Hannan et al. (2009) for effectiveness of linezolid against MRSA. The MIC values of linezolid calculated ranged from 0.37 to 3 g/mL. Tunger et al. (2004) recorded 2 g/ml MIC of linezolid against MRSA isolates. MRSA isolates resistant to vancomycin were susceptible to linezolid and may be a better choice for treatment of infections (Kohno et al., 2007).

Minimum inhibitory concentration of moxifloxacin against MRSA was 2.68 g/mL that fall within the range (MIC 0.25-4 g/mL) reported by Farrell et al. (2011). Resistance of MRSA to the fluroquinolones has been observed by Biedenbach et al. (2010). Reported Moxifloxacin inhibitory concentrations (0.5 to 2 g/mL) by Morrow et al. (2011) at New Jersey were in accord with present work. Production of alternate target enzymes and drug efflux by the microorganisms were possible resistant mechanism (Bolon, 2009). Moxifloxacin was found to be effective as an alternative to vancomycin and linezolid against MRSA. Variations in the MIC values of imipenem against MRSA isolates were recorded. Observed lowest and highest values were 0.39 and 6.25 g/mL. Comparable results were reported by Fan et al. (1986) with activity range of 0.049-50 g/ml. Few MRSA isolates showed resistance against imipenem so will not be a better choice for therapy.

Cefepime MIC value (4 g/mL) was much higher than reported by Huang et al. (2004). Variation may be due to resistance developed by MRSA against this antibiotic. However, higher MIC (256 g/mL) was observed by Queenan (2007) for cefipime and is in agreement with present findings. All isolates were sensitive to different concentrations of cefipime (25-100 g/mL) as compared to standard drugs.

Combination of amoxicillin and cloxacillin exhibits better activity against the multi drug resistant bacteria (Cantoni et al., 1989). Combination was checked against MRSA isolates and results were in line. However, activity was much lower. Antibacterial activity of Acacia nilotica and Aloe vera plant extracts against MRSA isolates was comparable with vancomycin and linezolid. It is suggested that extracts can be used as alternate therapeutic agents against MRSA.

In another experiment sixteen MRSA isolates were selected to assess the sensitivity pattern of bacteria against ethanol extracts of Opuntia dillenii, Acacia nilotica and Aloe vera. All tested isolates showed sensitivity to these extracts. The MICs of plant extracts against MRSA isolates were also calculated. Activity of Aloe vera against MRSA was highest (32.248 g/mL) followed by Acacia nilotica leaves (62.5 g/mL) and Acacia nilotica bark (84 g/mL). Extracts of Opuntia dillenii leaves had very low anti-MRSA activity with highest MIC value (1228 g/mL).

Active ingredients (terpenoids, saponins, alkaloids and glycosides) present in plant extracts have antibacterial activity against different bacterial strains. Most MRSA strains showed susceptibility to extracts of Acacia nilotica leaves at MIC (62 g/mL). Concentration of leaves extract reported by Arias et al. (2004) was higher (125 g/mL). In accord MIC of Acacia leaves (39-78 g/mL) was recorded by Khan et al. (2009). Average value of MIC for Acacia nilotica bark was found to be less than leaf extracts. It was observed that few strains showed the susceptibility at half concentration of bark extracts as compared to leaves. Results for Acacia bark extracts against MRSA are in agreement with findings of Eldeen et al. (2010). Opuntia dillenii exhibited low activity as compared toAcacia nilotica and Aloe vera against MRSA.

Aloe vera is used in cosmetics and nutraceuticals for skin infections, burns and cuts. It stimulates the cell growth and helps in repairing the damaged cells (Lu et al., 2013). Ethanol extracts of Aloe vera showed highest antibacterial activity against MRSA isolates (MIC: 32.25 g/mL). Comparable results for Aloe vera were recorded by Pandey et al. (2010) (MIC: 1-20 g/ml) against MRSA.

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Publication:Journal of Animal and Plant Sciences
Date:Aug 31, 2015
Words:4294
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