Antimicrobial activity of Ricinus comunis, Swietenia mahogani and Crusentia cujete ethanol extracts against multidrug resistant pathogens, recovered from a hospital environment

Medicinal plants have been used as effective approaches to manage multidrug resistant pathogens, including infectious agents that cause nosocomial infections. This study aimed to investigate the antimicrobial potentials of ethanolic extracts of Ricinus comunis, Swietenia mahogani and Crusentia cujete against five multidrug resistant nosocomial pathogens namely; Staphylococcus aureus, Pseudomonas sp., Klebsiella sp., Escherichia coli and Candida sp., which were isolated from hospital fomites. Using standard microbiological methods, fomite swab samples from ward beddings and door handles from the casualty, women, and children ward of Dalhatu-Araf Specialist Hospital, Lafia, Nigeria, were assessed. A total of 251 microbial isolates, consisting of 8 bacterial and 6 fungal genera were recovered. The highest frequency of microbial pathogens was recorded in the casualty unit (98[39%]), followed by the women’s unit (90[36%]), while the children’s ward (63[25%]) was the least contaminated. S. aureus (25[42%]) and Aspergillus sp. (43[72%]) were the most isolated bacteria and fungi; respectively, while Salmonella sp. (7[12%]) and Trichoderma sp. (9[15%]) were the least isolated. However, there were no significantly statistical differences across wards and microbial isolates. The five selected isolates were tested for in vitro susceptibility against several standard antibiotics to check their multiple drug resistance. The tested microorganisms exhibited various levels of multidrug resistance patterns except for Candida sp. which was resistant to two classes of antibiotics (azole group and griseofulvin). On the other hand, Klebsiella sp. was resistant to eight antibiotics of four classes. The ethanolic leaf extract of C. cujete was more effective against all the selected microbial pathogens, while the bark extract of S. mahogani was substantially effective. R. comunis exhibited no inhibitory potential against any of the tested pathogens. All the plant extracts were not as effective against the tested microorganisms as the conventional antibiotics that were used as positive controls. Results obtained indicate the risk of nosocomial infections caused by multidrug resistant pathogens originating from the hospital environment. Good hygienic practices, public awareness on nosocomial infections and further research into ethnomedicine are hereby recommended.


Introduction
Hospital environment and fomites including; doorknobs, rails, beds, among others, serve as reservoirs of bacterial pathogens causing nosocomial infections in patients attending to the hospitals for treatment (Suleyman et al., 2018). A recent study of Peters et al., (2019) revealed that the constant increase in cases of nosocomial infections is in line with the increase in bacterial resistance to antibiotics, and constitutes a considerable risk to the human health. Hence, it is vital to search for future strategies to combat these microbial infections and antibiotic resistance.
A previous study conducted by Iwu et al., (2009) revealed that in developing countries, infectious diseases have accounted for approximately one-half of all deaths. In addition, incidence of epidemics in the industrialized nations due to multidrug-resistant microorganisms, poses enormous public health concerns. Accordingly, upon increase of the problem of antibiotic resistance, ethnomedicine is gaining popularity as an alternative to orthodox medicine.
In 2003, the World Health Organization (WHO) observed that up to 80% of the rural areas in the developing countries depend on herbal medicine, and requested the member countries to explore safe indigenous medicine for their national health care (Ogbonnia et al., 2008). The high cost of synthetic drugs has made herbs the most common means for treatment of diseases in rural areas. A high percentage of the population in developing countries, including Nigeria, do not have adequate means to purchase synthetic drugs; thus, herbs become an alternative sources for treatment, since they are readily available and abundant.
Several recent studies conducted by Abdul et al., (2018); Olaniyi et al., (2018) reported that, in many African countries; extracts from plants are currently used for treatment of several infectious diseases, varying from minor skin infections to gastrointestinal disturbances, in addition to serious infectious diseases such as typhoid fever. A previous study of Ogbonnia et al., (2008) highlighted that antimicrobial compounds from plants may inhibit bacterial infections by a mechanism that differs from that of the used antibiotics, and may have clinical values in treatment of the antibiotic resistant bacterial strains.
Therefore, it is important to ascertain the antimicrobial efficacies of more herbs, as they could provide cost-effective drug delivery strategies, to combat the multidrug resistant microorganisms. The objective of this study was to investigate the antimicrobial potentialities of R. comunis, S. mahogani and C. cujete ethanolic extracts against selected multidrug resistant nosocomial pathogens isolated from hospital fomites.

Collection and ethanolic extraction of plant materials
Fresh leaves and fruits of Crusentia cujete and Ricinus comunis, and fresh leaves and stem bark of Swietenia mahogani were collected from different sites in Lafia Local Government, Nasarawa State, Nigeria. The plants were properly air dried at room temperature under shade. Ethanol extraction of plant samples was carried out according to Akinyemi et al., (2005). Plant materials were shredded and ground into fine powder. About 100 g of each powdered plant material was transferred into Erlenmeyer flasks containing 1000 ml of 80 % ethanol and then soaked for 48 h. The mixture was separated by filtration using Whatman no. 1 filter paper; the resulting filtrate was retained and then air-dried to remove excess ethanol, in reference to Doughari et al., (2012). Air-dried extracts were collected in air-tight containers and stored at 4 o C till further analysis.
Novel Research in Microbiology Journal, 2021

Microbial samples collection and processing of hospital fomites
Using sterile swab sticks, a total of 60 samples were collected from ward beddings and door handles of the children ward, women ward, and casualty unit of Dalhatu-Araf Specialist Hospital Lafia, Nasarawa state, and then transported immediately to the Microbiology laboratory, for analysis. The door handles were those found on the entrance doors of the wards and the lavatories.
The swab sticks were dipped and gently vortexed in 1 ml 0.85% physiological saline. An aliquot of 0.1 ml of stock solution was streaked on blood agar, MacConkey agar, Mannitol salt agar and Eosin methylene blue agar media for bacterial isolation, and Potato dextrose agar (PDA) for fungal isolation. Bacterial plates were incubated at 37 °C for 24 h while fungal plates were incubated for 72 h at 27± 2 o C. After incubation, the bacterial isolates were subcultured on nutrient agar to obtain pure distinct colonies (Cheesbrough, 2006;Nwankwo, 2012). The assay plates were used in triplicates for each culture medium.

Biochemical identification of the microbial isolates
The bacterial and fungal isolates were identified as described by Cheesbrough, (2006);Crous et al., (2009), respectively. In addition to Gram staining, the used biochemical assays included; catalase test, indole test, coagulase test and citrate utilization test, to identify the bacterial isolates (Lincy et al., 2016). On the other hand, the fungal isolates were identified through staining the cultures with lactophenol blue, and then examining the spores' arrangement under a light microscope.

Determination of antimicrobial potency of the plants extracts
Antimicrobial efficacy of the plants extracts against the multidrug resistant microbial isolates was tested using the agar well diffusion method (Debalke et al., 2018). A 0.1 ml aliquot of 24 h old culture of each bacterial and fungal isolate was spread on plates containing 20 ml of Muller Hinton agar using a sterile glass spreader. About 6 mm well was made individually in each of the seeded media using sterile cork borer, and then 20 µl of each ethanolic plant extract was dispensed separately into each well. The plates were allowed to stand for 1 h and subsequently incubated at 37°C for 24 h. The antimicrobial potential was assayed by measuring the diameters (in mm) of the zones of inhibition formed around each well using a calibrated ruler, and results were interpreted using CLSI. (2017) guidelines. Ciprofloxacin disc was used as a positive control for Gram-negative bacteria, Levofloxacin for Gram-positive bacteria and Fluconazole for fungi, based on CLSI. (2017) recommendations.

Statistical analysis
Data were analyzed using one-way Analysis of variance (ANOVA) to calculate the significant difference in the frequency of occurrence of microbial species in each ward and across the wards. Statistical significance differences in antimicrobial activities of the plants extracts were also analyzed. The Statistical Package for Social Sciences (SPSS) version 22 was used for the analysis.

Prevalence of nosocomial pathogens isolated from hospital fomites
A total of 251 microbial contaminants, consisting of 8 bacterial and 6 fungal genera, were isolated from the 60 collected samples, as shown in Table (  Where; the isolates were tested at p ≤ 0.05.
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Antibiotic susceptibility patterns of the microbial species
Five microbial species including; Pseudomonas sp., E. coli, Klebsiella sp., S. aureus and Candida sp., were tested for their susceptibility against a panel of antibiotics. Results of the antibiotic susceptibility of the bacterial and fungal spp. are presented in Table (6). These microorganisms were selected based on previous reports of their involvement in nosocomial infections in the hospital environment. Multidrug resistance is considered as resistance to a single antibiotic in three or more antibiotic groups. Klebsiella sp., E. coli and Pseudomonas sp. showed resistance to 8, 7 and 6 antibiotics, respectively. Klebsiella sp. demonstrated resistance to all of the tested antibiotics, except for cefprozil and gentamicin. Pseudomonas sp., and E. coli expressed resistance to several antibiotics in four classes of antibiotics including; Quinolones (ofloxacin, reflacin, ciprofloxacin and nalidixic acid), Penicillin (augmentin, streptomycin), Aminoglycosides (gentamicin, streptomycin), and Novel Research in Microbiology Journal, 2021 Trimethoprim. S. aureus is resistant to 5 antibiotics in four classes of antibiotics including; Fluoroquinolones (norfloxacin), Aminopenicillins (amoxicillin), Macrolides (erythromycin) and Penicillin (ampicillin). On the other hand, Candida sp. is resistant to 3 antibiotics in the Azole group (miconazole, itraconazole and voriconazole) and to griseofulvin. It showed intermediate reaction to ketoconazole and econazole; however, is susceptible to fluconazole and terbinafine.

Antimicrobial potential of the plants extracts against the antibiotic-resistant microbial species
The antimicrobial efficacy of the ethanolic extracts of R. comunis, C. cujete and S. mahogani plants tested against the antibiotic-resistant bacterial and fungal spp. are presented in Table (7). Both the beans and leaf extracts of R. comunis, and leaf extract of S. mahogani showed no inhibitory efficacy against all the tested isolates. Conversely, the leaf extract of C. cujete expressed significant inhibitory potential against all the selected microbial isolates. The fruit extract of C. cujete exhibited no inhibitory activity against all the Gram-negative bacteria, but is active against S. aureus and Candida sp., giving inhibition zone diameters of 2.00 ±2.82 and 6.00 ±2.82 mm, respectively. mm. However, the inhibitory efficacy of all the tested plant extracts against the selected microbial isolates is low, compared to the conventional antibiotics used as positive controls. Values are expressed as Mean ±SD (n=2). Where; * demonstrates a significant difference in the inhibitory potential among the tested plant extracts against the selected microbial isolates at p< 0.05: Positive control: Ciprofloxacin for Gram-negative bacteria, Rifampicin for Gram-positive bacteria, Fluconazole for fungal species. SD = standard deviation

Discussion
In the current study, the most frequently isolated microbial pathogens included; S. aureus, Streptococcus sp., E. coli, Pseudomonas sp., Aspergillus sp., Penicillium sp., Rhizopus and Candida spp. The highest microbial count recorded in the casualty unit was not surprising, as the unit is frequented with accident emergencies, a high population flow, and high patient turnover, compared to the other wards. Similarly, a recent study conducted by Awanye and Amrasawore, (2020) reported the high prevalence of microbial pathogens including; S. aureus, Pseudomonas sp., Klebsiella sp., Salmonella sp. and Candida sp. from fomites in the Accident and Emergency Unit, and Plastic and Burn Unit of the University of Port Harcourt Teaching Hospital, Nigeria.
Recovery of microbial pathogens from fomites supports the earlier reports of isolating similar microbes from toilet door handles in various settings including; hospitals in India (Lincy et al., 2016), beds and door handles in Kiwoko Hospital in Uganda (Segujja et al., 2016) and hospital surface samples in Ethiopia (Getachew et al., 2018). Recovery of methicillin-resistant S. aureus from different surfaces including; glass, vinyl floor tile, countertop and stethoscopes have been documented by Williams and Davis, (2009). In Mexico, Klebsiella sp., E. coli, Enterobacter sp., Pseudomonas sp. and Aspergillus sp. were isolated from the hospital environment, as documented by Garcia-Cruz et al., (2012). The risk of infection is markedly exhibited in these hospital wards sampled in this study, as these microorganisms are known to cause various forms of infections.
The highest bacterial isolates recovered from the casualty unit and children's ward was S. aureus, in accordance with Saka et al., (2016), who reported the predominance of S. aureus from the pediatric ward of the University of Ilorin Teaching Hospital, Nigeria. A previous study of Bereket et al., (2012) also documented that S. aureus is the foremost source of host infections, including surgical sites infections and lower respiratory site infections.
Aspergillus sp. was the predominant fungus recovered from fomites in all the wards, and showed statistically significant difference from all other isolates across all the wards. A total of 80 isolates of Aspergillus spp. were recovered from hospital wards (bone marrow transplant, hematology-oncology wards) in a hospital in Lisbon, Portugal (Sabino et al., 2014).
The beddings were more contaminated than the door handles. The level of contamination in the ward beddings causes a concern to the patients and medical practitioners, as it increases the possibility of transfer of infection and prolonged stay in the hospital.
Multidrug resistance is a leading factor that predisposes the patients to nosocomial infections. S. aureus was resistant to four classes of antibiotics and is known to be resistant to β -lactamases. The resistance to β-lactams, including ampicillin is due to the production of the enzyme carbapenemase, as reported by Iroha et al., (2009). As such, the presence and high frequency of S. aureus in the casualty and children's ward exposes the patient to high risk of infections.
Klebsiella sp. exhibited the highest multidrug resistance to 80 % of all the tested antibiotics. This agrees with the recent study conducted by Sserwadda et al., (2018), which reported the prevalence of high multidrug resistance in K. pneumonia recovered from fomites and work surfaces in a general hospital in Uganda. Moreover, K. pneumonia has been implicated in urinary and respiratory tract infections as reported by Chung, (2016), and its presence is indicative of poor hygienic practices and fecal contamination (Sserwadda et al., 2018).
Escherichia coli showed the second-highest resistance to five classes of antibiotics in this study. This supports the findings of Basak et al., (2016), which reported that the most common multidrugresistant microorganism isolated from the clinical specimens was E. coli. An early study of Bereket et al., (2012) revealed that E. coli carries a large number of virulence factors and is common in wound infections, urinary tract infections and causes pneumonia in immunocompromised patients. Recently, Mojica et al., (2020) highlighted that Pseudomonas, Klebsiella and E. coli are common microorganisms exhibiting multidrug resistance, due to production of β-lactamases, including extended βlactamases (ESBLs).
Candida sp. was resistant to drugs in the azole group and griseofulvin. The Triazole drugs include fluconazoles, which are the first line of antibiotics used for treatment of fungal infections, while voriconazole is a second-generation drug known for its strong activity against Candida spp. (Maroszynska et al., 2013;Mandras et al., 2009). However, resistance of Candida spp. to voriconazole (Maroszynska et al., 2013) and itraconazole (Zaidi et al., 2018) has been reported. Moreover, resistance of Candida spp. including C. albicans, C. glabrata, C. krusei, C. tropicalis to several triazole drugs have also been reported by several previous studies conducted by Maroszynska et al., (2013); Li et al., (2013); Bhattacharjee, (2016). Sensitivity to fluconazole in this study contrasts the report of Mahmoudabadi et al., (2013) and Li et al., (2013), where Candida spp. were resistant to fluconazole. Sensitivity to fluconazole has been reported to vary among the Candida spp. as revealed by Maroszynska et al., (2013); Dagi et al., (2016). Previous studies of Dagi et al., (2016); Tercas et al., (2017) have indicated the variability in response of Candida spp. to the antifungal agents; however, this was not reflected in the current study, as we did not delineate Candida spp. into several species, or test their response to the different antifungal drugs. Intermediate reaction of Candida sp. to econazole has been recorded in our study, which contradicts the sensitivity of Candida sp. to econazole reported in the early study of Mahmoudabadi et al., (2013). The ethanolic seed and leaves extracts of R. comunis demonstrated no activity against all the tested microorganisms. This agrees with the study conducted by Oloyede et al., (2012), but contrasts with the report of Jeyaseelan and Jashothan, (2012), which found that ethanolic extracts of R. comunis were the most effective against the various microbial pathogens. The study of Jeyaseelan and Jashothan, (2012) demonstrated the highest activity of ethanolic extract of R. comunis against S. aureus with an MIC of 5 mg/ ml, and was highly effective against C. albicans, A. niger and A. fumigatus. The lack of inhibitory effects of the seed and leaf extracts of R. comunis may be attributed to using ethanol as an extracting solvent. Nour et al., (2012) reported that methanol is a better extractant than ethanol in the extraction of phytochemicals from plants materials. The leaf extract of C. cujete had the highest inhibitory activity against all the microbial isolates tested, in accordance with the report of Khandaker et al., (2011). On the other hand, the fruit extract of C. cujete demonstrated no inhibitory potency against the Gram-negative bacteria which contradicts the results of Khandaker et al., (2011). The effectiveness of the leaf extract of C. cujete against the microbial isolates may be attributed to the high phytochemical residues present in its leaves.
Overall, the activities of all the plant extracts tested in this study were low, compared to the activities of the conventional antibiotics. Various reasons may account for these results, including the use of crude forms of these extracts.

Conclusion
This study evaluated the antimicrobial potential of ethanolic extracts of some plants against multidrug resistant nosocomial bacterial and fungal pathogens. The casualty unit and the children ward had the highest and lowest frequency of these pathogens across the ward beddings and door handles, respectively. The presence of multidrug resistant microorganisms responsible for nosocomial infections especially in the casualty ward is worrisome as it poses a health risk to the patients on admission to the hospital. The antimicrobial potency of the crude plants extracts tested in this study was not as effective against the microbial isolates as the conventional antibiotics. It is therefore recommended to carry out further research using different plant parts and different extractors/extraction methods. In addition, the bioactive compounds should be further isolated and then examined for their antimicrobial potential.