Abstract: Multiple antibiotic resistance S. aureus and coagulase negative Staphylococci (CoNS) constitute a major healthcare problem but with the resuscitation of traditional and complementary alternative medicine, these infectious diseases have been reduced drastically. Two hundred swab samples were obtained from various clinical sites of patients in surgical unit. Grounded plants were soxhlet extracted using ethanol and n-hexane and purified. Primary characterization, antibiotic sensitivity testing of commercial antibiotics and impregnated disk of plant extracts were analyzed according to standards. Fifty three isolates were characterized as S. aureus (47) and CoNS (6) 15 isolates were β-lactamase producing and 39 were DNase positive. Multiple antibiotic resistances were observed against the isolates with 17 S. aureus and 4 CoNS strains demonstrating multiple resistance to all the 9 antibiotics tested. High resistance pattern were seen in rifampicin (76.6), streptomycin (70.2), ampiclox (68.1), erythromycin (63.8) and the fluoroquinolones (51.1-61.7). The multiple antibiotic resistance index ranges between 0.11-1.0. These results therefore call for an urgent attention in addressing drugs misuse, specific clinical diagnosis and treatment to the emerging staphylococcal infection especially the CoNS and incorporating herbal drugs prescription in orthodox medicine.
INTRODUCTION
Staphylococci have again emerged as the predominant organisms causing infections in the hospital setting and are the leading cause of nosocomial infections and community-acquired infections including impetigo, folliculitis, furuncles, sycosis barbe, cellulitis, abscesses, osteomyelitis and bacteraemia (Al-Hamdani and Hamad, 2012; Gad et al., 2010; Yameen et al., 2010; Bashir et al., 2007). Most developed countries have reported an increase in colonization and infection in hospitalized patients by coagulase negative Staphylococci (CoNS) while there are scanty data in developing countries.
Since the introduction of antimicrobials, bacteria have developed mechanism for resisting the effects of antibiotics and the levels of antibiotic resistant infections in the developing world have increased steadily in the last few decades as a result of combination of microbial characteristics and the selective pressure of antimicrobial use (Bashir et al., 2007; Blondeau and Tillotson, 2000). Several mechanisms by microorganisms in overcoming the activities of antimicrobial agents include the production of structure-altering or inactivating enzymes (e.g., beta-lactamase or aminoglycoside-modifying enzymes), alteration of penicillin-binding proteins or other cell wall target sites, altered DNA gyrase targets, permeability mutations, active efflux systems and ribosomal modification (Akinjogunla and Enabulele, 2010; Gad et al., 2010). Multi-drug resistance bacteria in both the hospital and community environment are of importance to the clinician, as it is the major cause of failure in the treatment of infectious diseases, increased morbidity and mortality and the evolution of new pathogens (Akinjogunla and Enabulele, 2010).
However, multi-drug resistant strains of Staphylococci have been reported with increasing prevalence worldwide among macrolides, aminoglycosides, fluoroquinolones or combination of these antibiotics. Thus, making infection caused by Staphylococci practically difficult to treat especially among the catheter in-dwelling patients and patients with prolonged hospital stay.
Recently, the discovery and development of medicinal plants as drugs (especially from China, India and Nigeria and some African countries) has proven effective in the treatment of multi-drug resistance pattern among clinical and environmental isolates, which has plagued the healthcare system especially in developing and underdeveloped countries. The primary benefits of using plant-derived medicines are that they are safer than synthetic alternatives, offering profound therapeutic benefits and more affordable treatment (Ojo et al., 2013a). They are effective with minimal or no side effects in the treatment of infectious diseases, while simultaneously mitigating many of the side effects that are often associated with synthetic drugs (Ojo et al., 2013b). This study, however tends to present the effectiveness of Diodia scandens and Phyllanthus amarus on the multi-drug resistant Staphylococci from surgical unit in Delta State Tertiary Hospitals, Nigeria.
MATERIALS AND METHODS
Collection of samples: Two hundred (200) clinical swab samples were obtained from septic wounds and burns patients undergoing injury dressing on various body sites at the General Hospital, Ekpan, Warri, Delta State, Nigeria. Wound exudates were obtained from the infected sites of each patient with a sterile cotton swab and applied to freshly prepared slant of nutrient agar and mannitol salt agar (oxoid) and were incubated at 37°C for 24 h.
Isolation and identification: Colonies growing on slants were streaked on top of freshly prepared plates of mannitol salt agar and incubated again at 35°C. Primary characterization of isolates was based on Gram stain, morphological and cultural characteristics, growth on nutrient agar and DNase agar and fermentation on mannitol salt agar, catalase and coagulase tests. β-lactamase assay was performed using the method as described by Ako-Nai et al. (2005).
Antibiotic sensitivity testing: The isolates were subjected to antibiotic screening by disk diffusion method using CLSI (2008) standard with a reference S. aureus strain type 25923. The inocula were prepared by diluting overnight cultures in sterile sodium chloride (0.9%) suspension and then matched with the McFarland turbidity index. Bacterial suspensions were then plated onto Mueller Hinton agar and the commercially available antibiotic disc were placed on the lawn of culture and inoculated overnight at 35°C. The following disks were used: Ciprofloxacin (10 μg), Levofloxacin (5 μg), Norfloxacin (20 μg), Gentamycin (10 μg), Ampiclox (30 μg), Streptomycin (30 μg), Erythromycin (10 μg), Rifampicin (20 μg) and Chloramphenicol (25 μg).
Determination of multiple antibiotic resistance index (MAR): This was determined using the equation:
where, x was the number of antibiotics to which test isolate displayed resistance and y is the total number of antibiotics to which the test organism has been evaluated for sensitivity.
Determination of antimicrobial effect of plant extracts on multi-drug resistant Staphylococci: The multi-drug resistant Staphylococci obtained were cultured on Mueller Hinton Agar and incubated at 35°C for pure isolates. The extracts were prepared as earlier described by Ojo et al. (2013a). The antimicrobial susceptibility profiles of the isolates were determined using agar diffusion method as described by the Clinical Laboratory Standards Institute CLSI (2008) Wayne, PA, USA. Paper disks of 6 mm diameter in size were impregnated for 6 h in various concentrations of ethanolic and n-hexane extracts of D. scandens and P. amarus. Reference type strains of S. aureus ATCC 25923 were included for quality control.
RESULTS
Various sample sources in this study reveal the isolation rate of Staphylococci with the highest from lap (23.4), hand (17.0) and leg and feet (14.9) respectively for S. aureus while for CoNS, the leg (33.3) showed higher isolation rate (Table 1). Table 2 showed the biochemical analysis of the clinically isolated Staphylococci from various sites of the body, thus indicating presence of β-lactamase producing and DNase positive Staphylococci. The antibiotic sensitivity pattern on S. aureus and CoNS revealed high resistance pattern among the rifampicin (76.6), streptomycin (70.2), ampiclox (68.1), erythromycin (63.8), gentamycin (59.6) and the fluoroquinolones (51.1-61.7) (Table 3). Seventeen isolates of the S. aureus and 4 CoNS isolates showed multiple antibiotic resistance on the 9 antibiotics tested with at least 1 isolate showing multiple antibiotic resistance on 2 antibiotics. Thus, indicating high level of multiple antibiotic resistance with the multiple antibiotic index ranging from 0.11-1.0 in the Staphylococci isolates (Table 4, 5).
| Table 1: | Sample source composition of Staphylococci from wounds and burns patients |
| Table 2: | Biochemical analysis of clinically isolated Staphylococci from wounds and burns patients |
| W: Wound, B: Burns, +ve: Positive, -ve: Negative | |
| Table 3: | Profile of antibiotic sensitivity testing on Staphylococci isolated from wounds and burns patients |
| ND: Not determined | |
Antimicrobial effect of D. scandens and P. amarus were demonstrated on the multi-drug resistance Staphylococci using the absolute ethanol and n-hexane extracts of the two plants at different concentrations. However, results showed the zone sizes of inhibition of P. amarus ethanolic extracts (7-16 mm), P. amarus n-hexane extracts (6-24 mm), D. scandens ethanolic extracts (6-20 mm) and D. scandens n-hexane extracts (7-13 mm) (Table 6).
DISCUSSION
Staphylococcal infections have become a significant clinical problem in medical practice (Gad et al., 2010) posing great health threat both to the immunocompetent and immunosuppressed individuals. The exceedingly increases and emergence of multidrug resistance pathogens (especially among S. aureus and CoNS) in the developing countries can be attributed to the indiscriminate use of antibiotics, complex socio-economic, behavioural antecedents and dissemination of drug-resistant pathogens in human medicine (Yang et al., 2008). Thus, the multi-drug resistant strains among the S. aureus and the more emerging CoNS is complicating the management of wounds and burns infection and increasing the risk for treatment failure.
| Table 4: | Antibiogram pattern of resistant antibiotics |
| Table 5: | Multiple antibiotic resistance (MAR) index of Staphylococci from wounds and burns |
It was obvious from our study and similar studies that Staphylococci are becoming more resistance to various antibiotics commonly administered by clinicians or as over-the-counter (OTC) use especially among gentamicin, erythromycin, streptomycin, chloramphenicol and ampiclox. S. aureus tested against fluoroquinolones used in this study showed a high resistance pattern as compared with an earlier report (Yameen et al., 2010), whereas other studies indicated a low level or no resistance pattern (Akinjogunla and Enabulele, 2010; Al-Hamdani and Hamad, 2012). The result of CoNS resistance to all antibiotics tested especially the fluoroquinolones in this study is alarming, corroborating the reports from Akinkunmi and Lamikanra (2010), Akinjogunla and Enabulele (2010) and Bashir et al. (2007). Akinjogunla and Enabulele (2010) reported the multiple antibiotic resistance index to range between 0.25-1.00 while Al-Hamdani and Hamad (2012) reported a range between 0.2-0.75. These earlier reports were in agreement with the result of this study.
| Table 6: | Zone sizes (mm) on antimicrobial effect of Diodia scandens and Phyllanthus amarus on clinically isolated Staphylococci |
| PE1-P. amarus ethanolic extract (100 μg mL-1), PE2-P. amarus ethanolic extract (200 μg mL-1), P. amarus ethanolic extract (400 μg mL-1), DN1-D. scandens n-hexane extract (100 μg mL-1), DN2-D. scandens n-hexane extract (200 μg mL-1), DN4-D. scandens n-hexane extract (400 μg mL-1) | |
Most studies on medicinal plants have shown results only on MIC and MBC determination. However, this study has revealed the antimicrobial effect of the two plants extracts at various concentrations from ethanol and n-hexane using disc diffusion method according to CLSI (2008) standards. The antimicrobial potency of P. amarus ethanol extracts was high at the 100 μg mL-1 (6-16 mm) and 400 μg mL-1 (11-22 mm) than 200 μg mL-1 (8-10 mm), which was synonymous with Oluwafemi and Debiri (2008) who reported antimicrobial activity at 100 mg mL-1 with zone size of 8.3±0.12 mm while those of P. amarus n-hexane extracts showed low potency from the diameter of zone sizes. Conversely, the antimicrobial potency of D. scandens ethanol extract had a good antimicrobial effect at the 400 μg mL-1 (11-20 mm), 200 μg mL-1 (6-13 mm) and 100 μg mL-1 (6-12 mm), respectively as compared with the n-hexane extract which had a low antimicrobial effect on the isolates. This however elucidates the medicinal use of these plants in treating antibiotic resistance Staphylococci (MRSA and MRCoNS) and possibly other organisms. It can be deduced from the result that ethanol extraction tends to extract most of the active ingredients than n-hexane, which is reflected in the extraction yield. Hence, n-hexane is not a good solvent for extraction.
CONCLUSION
This study showed the prevalence of multi-drug resistant strains of S. aureus and CoNS as a major cause of wound infection among patients in surgical unit of the hospital which can be transferred to both in-patients and out-patients. It is therefore recommended that since Staphylococci causes nosocomial infection, overcrowding in healthcare institution should be avoided to reduce the spread of the infection within the hospital and strict policy on OTC use of antibiotics be implemented among the pharmaceutical outlets and patent-medicine dealers vis-à-vis the introduction and prescription of purified traditional medicine in patients’ treatment. We know that our result will enhance clinicians’ capability to better manage this infection and reduce possible squealer.