Microbial quality and antimicrobial evaluation of some herbal concoctions in a Rural Town in Nigeria

The use of local herbal concoctions has been on the increase for the cure of various illnesses in Iwo community and Nigeria generally. Experiment aimed at isolating major microorganisms and carrying out antibiotic resistant test on the bacterial isolates was carried out. Bacteria isolated were identified through biochemical tests and molecular characterization. Susceptibility levels of these isolates was evaluated using antibiotic rapid test multidisc containing eight different antibiotics and resistance genes were amplified from some of the isolates. Five fungal species were isolated from the samples and these included Aspergillus niger and Mucor sp which occurred the most (33.3%), while Rhizopus stolonifera and Aspergillus nidulans had the lowest occurrence (6.7%). Twentyfour bacterial species belonging to fifteen (15) genera were identified. These includes Providencia sp 17(19%) recorded the highest occurrence, followed by Pantoea sp 13(14.4%) and Citrobacter sp 10(11.1%) while Salmonella sp, Cedecea sp, Pseudomonas sp and Yersinia sp had the lowest occurrence (2.2%) each. All the isolates showed resistance to three or more antibiotics used, especially the cephalosporins. The genes found to be responsible for the cephalosporin resistance in some of the isolates were TEM-972 and CTX-M-200. Presence of the multi-antibiotic resistant microorganisms in herbal concoctions poses a serious public health threat.


Introduction
The first line of therapy known to man in the past was medicinal crops, traditional medicinal exercise continues to be a significant component of the main care scheme in most emerging countries [1]. Approximately 70-80 per cent of the world's inhabitants lives in developing nations and depends on herbal concoctions due to their simple availability and cheapness [2,3]. Recently used synthetic or standard drugs have been obtained from herbs such as Cinchona sp from where the antimalarial medicine quinine is obtained [4]. In the US, about 25% of prescribed medicinal products have a minimum of one active component extracted from crop products [5]. The majority of herbal preparations are in various forms. The existence of plant secondary metabolites has been ascribed to most plants' medicinal activity [3,4]. In general, crops represent a significant source of orthodox drugs. In Nigeria, "public awareness and use of herbal medicines in the treatment and/or prevention of diseases" appear to be increasing significantly. However, it is important that herbal preparations are of high quality and safety. WHO [6] said the quality is the foundation of herbal pharmaceutical reproductive efficacy and safety, and that the quality of the plant products or preparations is of great significance in order to guarantee that study standards in herbal medicines are maintained. The quality requirements for medicinal medicines have been shown by [7] based on a definite science concept of raw materials.
Herbal medicine has also been defined by the World Health Organization as complete label medicines containing active constituent's plants or other herbal materials or combinations in crude form, or in plant preparations [8]. Plant products include fatty oils, gums, juices and other such ingredients. Using herbal medication has always been component of human society, as some crops have significant medicinal characteristics that can be used to heal human and other animal illnesses [9]. The primary health care recognized to humans is herbal medicine, practiced throughout history by all societies, it has become an essential component of the growth of contemporary civilization [10].
Herbs and herbal products have long been commercialized and are currently being used for a variety of applications [11]. Herbal medicine has also been defined by the World Health Organization (WHO) as a fresh or dry, fractioned or pulverized plant material that can be utilized in its raw form or subsequently processed into something more refined. Herbal products such as resins, essential oils, fixed oils, gums, infusions, decoctions, tinctures and juices can be produced by subjecting the herb to processes as baking or sweetening with honey, alcohol extraction, infusing in water, decocting, roasting, steaming or even squeezing. The resulting mixture can then be used as medical supplements or as food stuffs and starting material for further processing [12]. The products may be subjected to a number of biological, chemical or physical processes, including fermentation, concentration, purification, fractionation, expression, distillation, extraction or pulverization, depending on how sophisticated the herbal preparation is. The finished product can be formulated by blending one or more plant preparations with animal products or minerals and components isolated from herbal or synthetic materials [13].
Antibiotics or antimicrobials are drugs or chemicals used to combat infections caused by bacteria [14]. They constitute one of the world's most effective types of treatment, but a growing amount of antibiotic-resistant pathogens compromise their efficacy [15]. Almost all major bacterial diseases today in the globe are caused by antibiotic resistance; it is one of the most urgent public health issues in the globe with enhanced mortality and morbidity, and increased expenses for therapy [16,17]. In this work we investigated the microbial quality of some local herbal concoctions and antibacterial sensitivity reactions of isolated bacteria.

Sample collection
Twenty-one different samples of locally prepared herbal concoctions were bought from vendors in Iwo town and designated as A to U. The concoctions were classified based on diseases they cure as claimed by the vendors. These diseases include worm, malaria, constipation, stool control, rheumatism, diabetes, typhoid fever and pile. The concoctions were collected and transferred to the laboratory for assessment.

Isolation of bacterial isolates
Isolation of bacteria was carried out by inoculating nutrient agar to aid the total viable bacterial count, Eosine methylene blue (EMB) agar was used for selective bacterial count. Routine serial dilutions of the herbal concoctions were carried out up to dilution 10 -5 . One millilitre was taken at different dilutions. Routine bacterial identification steps included Gram's staining, catalase test, MR-VP test, indole test, sugar fermentation test etc.

Antibiotic Sensitivity Test
Antibiotic sensitivity test was carried out using Mueller Hinton agar. The inoculum was prepared using the 0.5 McFarland standard. A sterile inoculating loop was used to pick the test organism and suspended into 2ml of sterile normal saline. The absorbance of this suspension was measured using the spectrophotometer (Jenway 6305) at a wavelength of 625nm. The turbidity of the suspension was adjusted by adding more organisms when the suspension was found to be too light, and diluting with sterile normal saline when the suspension was too turbid. A sterile swab stick was dipped into the inoculum suspension and allowed to soak. The dried surface of the agar was then inoculated by streaking the entire surface uniformly with the swab stick. Using sterile forceps, a Gram negative antibiotic multidisc containing 8 antibiotics was placed aseptically on the plate and pressed firmly onto the surface of the agar to allow proper diffusion of the antibiotics on the agar. The antibiotics on the disc are ceftazidime (30µg), cefuroxime (30µg), gentamicin (10 µg), cefixime (5µg), ofloxacin (5µg), augmentin (30µg), nitrofurantoin (300µg) and ciprofloxacin (5µg). This process was repeated for all test organisms, and the plates were incubated at 37 o C for 24 hours. After incubation, the zones of inhibition were measured using a transparent ruler and were recorded in millimeter [18].

Extraction of genomic DNA
The boiling technique was used to extract DNA from each organism. Cells of each organism was harvested into 1000µL of sterile water, and were vortexed until completely dissolved. Centrifugation was carried out at 10000 rpm for 5 minutes. The supernatant was discarded, and another 1000µL of sterile water was added, after which the mixture was vortexed and centrifuged again. The supernatant was discarded and 200µL of sterile water was added. The mixture was vortexed again until thoroughly mixed. The mixture was boiled at 100 0 C after which it was cooled immediately on ice. The mixture was again vortexed and centrifuge at 10000rpm for five minutes. The supernatant was transferred into fresh Eppendorf tubes, and the pellets were discarded. The DNA was then stored at 4 o C.

ESBL Multiplex PCR amplification
The PCR reaction was carried out using the Solis Biodyne 5X HOT FIREPol Blend Master mix. PCR was performed in 20 µl of the reaction mixture, and the reaction concentration was brought down from 5x concentration to 1X concentration. The concentration contains 1X Blend Master mix buffer Buffer (Solis Biodyne), 1.5 mM MgCl2, 200µM of each deoxynucleoside triphosphates (dNTP)(Solis Biodyne), 20pMol of each primer (Jena Bioscience, Germany), 2 unit of Hot FIREPol DNA polymerase (Solis Biodyne), Proofreading Enzyme, 5µl of the extracted DNA, and sterile distilled water which was used to make up the reaction mixture. Thermal cycling was conducted in an Pielter thermal cycler (MJ Research Series) for an initial denaturation of 95°C for 5 minutes followed by 30 amplification cycles of 95 0 C for 30 seconds at; 56 0 C for 1 minute and 72 0 C for 1 minute 30 Seconds. This was followed by a final extension step of 72 0 C for 10 minutes. The amplification product was separated on a 1.5% agarose gel and electrophoresis was carried out at 80V for 1 hour 30 minutes. After electrophoresis, DNA bands were visualized by ethidium bromide staining and 100bp DNA ladder (Solis Biodyne) was used as DNA molecular weight marker [19].

Discussion
Herbal concoctions used in the present study are orally taken in the form of water extracted remedies, fermentedsteeped milled corn water ('omikan'), soft drink or alcoholic spirits. More than 60% of the herbal specimens used in this research, however, were water-based. Food and drinks based study were not carried out by producers on any of the analyzed herbal samples which most likely accounted for the elevated rates of regeneration of herbal coliforms. Coliforms are members of the Enterobacteriaceae family and are the most reliable indicators of faecal pollution in drinks. The test of their presence is therefore a pollution level index that can indicate potential presence of hazardous disease causing organisms [20,21,22].
The predominant fungal isolates obtained in the present study are the members of Aspergillus sp. The presence of these fungi in herbal medicines has been previously reported by [23,24,25]. The predominance of Aspergillus sp in herbal drugs have been reported by Elshafie et al. [26] and Mandeel [27]. The contamination of herbal preparations by fungi has been attributed to neutral extraneous contamination by dust following storage in humid conditions [28]. Some of the identified contaminants have been reported to have the ability to produce mycotoxins [29].
The microbiological status of herbal mixtures relies on many environmental variables and this could have major effects on the general quality of herbal products [30]. Results from this study have shown that the herbal concoctions being consumed by some or most residents of the Iwo Local Government Area of Osun State, Nigeria are contaminated with microorganisms (both bacterial and fungal), which might have detrimental health implications on consumers.
In order to enhance consumer acceptability, most herbalists in Nigeria have adopted the use of bottles and plastic containers as packaging vessels for their preparations. Unfortunately, these vessels are not subjected to any form of sterilization after mere rinsing them. Contamination of the preparations coupled with the humid tropical environments may result in the proliferation of microbial contaminants in the herbal concoctions; this probably explains the high microbial counts recorded in this study. Such elevated concentrations of microbial contamination have proven to spoil and degrade products or pose a health risk to the consumer [31,32,33]. Most herbal preparations are made up of different components of various plant species and these preparations are not standardized with respect to colour, taste, consistency etc. Unlike the orthodox drugs, changes in the appearance, odour, taste etc of herbal concoctions due to spoilage are hardly readily detected by the patients.
Some of the bacteria isolated in this research were Proteus sp, Pseudomonas sp, Salmonella sp which were in agreement with studies by Kolajo [34] and Mukhtar et al. [35]; all of which are naturally occurring in soil and water habitats. Their occurrence, well stated in the results, was probably due to their presence on plant parts and in the soil as their natural habitat. Kolajo [34] had reported that the presence of these microorganisms in the herbal mixtures signifies the substandard processing steps in the production of the herbs. Some other bacterial isolates like Providencia sp, Citrobacter sp, Klebsiella sp and Eschrichia coli in this study were also reported in the work of Esimone et al. [36]. Poor hygiene conditions during the production of the herbal concoctions could introduce these microorganisms into the preparations. Bacterial contaminants could also be associated with bad quality of water used for the processing of these herbal concentrations, as reported by Mukhtar et al. [35].
Some of the genera that cause enterotoxigenic gastroenteritis such as Pseudomonas sp, Enterobacter sp, Klebsiella sp, Proteus sp [37] isolated from herbal concoctions in this research were also earlier associated with infant gastroenteritis by Back et al. [38] and Jiva et al. [39]. In infantile gastroenteritis transmitted by water and food, Pseudomonas aeruginosa was likewise implicated [40]. Klipstein and Engert [41] also identified Enterobacter sp as an opportunistic pathogen in children's diarrhoea-related extra-intestinal diseases. Also acknowledged as opportunistic pathogens, Klebsiella sp, has become increasingly important. Klebsiella pneumoniae, was also involved as a cause of diarrhea [42], while Citrobacter sp. was likewise implicated as an opportunistic pathogen [43]. Guarino et al. [44] proposed its role in diarrhoeal disease. It was also revealed that Proteus mirabilis was connected with diarrhoeal illness [45].
Some fungal isolates were also obtained from some of the herbal concoctions. The fungal strains were distributed into the following genera: Mucor sp, Penicilliun sp, Aspergillus sp, Candida sp which were also in agreement with the work of Esimone et al. [36]. The presence of yeasts, Aspergillus spps, Rhizopus spps, Mucor spps, and Penicillium spps, were not so surprising as they are part of the mycoflora of plants and the soil and they are common spoilage organisms of carbohydrate containing foods [46]. The isolation of Aspergillus niger was similar to the results obtained in the isolation of Aspergillus sp. by Odedara and Muletiwon [47]. The isolation of Candida sp. corroborated the results of Adeleye et al. [24] and Anyawu [48], who in their studies isolated Candida sp. from various Nigerian herbal preparations.
Fungal species such as Rhizopus, Mucor, Penicillium and Aspergillus may endanger consumer health as they are involved in human pathogenicity. They produce potent mycotoxins that are involved in cancer, dermatitis, hepatotoxicity and nephrotoxicity [21,49,50].
Most of the bacteria isolated were mostly resistant to ceftazidime, cefuroxime, and cefixime which belong to the class of beta-lactam antibiotics known as cephalosporin, used to treat most bacterial infections [51].
As earlier mentioned contamination could get into the concoctions through the type of water used as solvent. According to Samie et al. [52] who studied the diversity of bacteria isolated from samples of household drinking water (which happens to be the solvent used for most of the herbal concoctions), ciprofloxacin is one of the most active antibiotics used, and is proving to be more effective than the cephalosporins due to the plasmid-mediated genes, and indiscriminate use of the cephalosporins. In a similar study by Ben-Ami et al. [53], enterobacteria have been found to increasingly produce extended-spectrum beta-lactamase, thereby increasing their resistance to the beta-lactam antibiotics such as the cephalosporins.
Among the antibiotics studied in this work, the third class cephalosporins such as cefuroxime, ceftazidime and cefizime were found to be the least active agents against all the organisms isolated, while gentamicin was one of the most active agent. Moosavian & Deiham [54] corroborated this finding in their study, indicating the increasing resistance of Gram negative bacteria to antibiotics, especially the third class cephalosporins. Dou et al. [55] and Ee et al. [56] suggested that the use of cefuroxime, ceftazidime and other cephalosporins should be used cautiously in treating infections to counter the related increase in resistance levels. Most of the isolates were found to be resistant to cefixime, cefuroxime and ceftazidime which are members of the class of cephalosporins.
Molecular analysis was carried out on few of the isolates which included DNA Extraction, ESBL Multiplex PCR analysis and amplification of three genes, the TEM, CTX-M and SHV genes. TEM gene was detected in three isolates and CTX-M gene was detected in only one isolate. However, SHV gene was not detected in any of the isolates. The TEM gene are found to be among those found responsible for the extended-spectrum beta-lactamase (ESBL) phenomenon [57]. While the CTX-M genes are usually the most frequent ESBL-producing genes for Gram negative bacteria as reported by Ben-Ami et al. [53], it was found in only one isolate in this study, and this corroborated the result of Champs et al. [58] who showed that among Enterobacteriaceae isolates, CTX-M genes were the least isolated. Extended spectrum betalactamase (ESBL) is a class of beta-lactamase enzyme responsible for the hydrolysis and inactivation of a wide range of beta-lactam antibiotics [59]. The emergence of the TEM gene could be due to selective pressure from the incorrect use of the cephalosporin class of antibiotics. The transfer of resistant genes by plasmids and extra chromosomal elements may also be responsible for the resistance of isolates who were previously susceptible to the beta-lactam antibiotics, according to Moosavian & Deiham [54].

Conclusion
In conclusion in spite of the benefits of using traditional medicine (e.g. herbal concoctions). The results of the present study reveal that the herbal concoctions were all contaminated by potentially pathogenic microbes to an unacceptable level, thereby making them un-safe for consumption. The herbal concoctions sold in Iwo town were, therefore, not sterile and could be a vehicle for transmission of pathogens to the end users. The ability of most of the isolated bacteria to resist more than one antibiotic which leads to multi-drug and multi-antibiotic resistance is of public health concern because the consumers of these concoctions run a risk of getting infected while trying to cure other illnesses, hence, most of the herbal concoctions used in this study cannot be recommended for use as drugs. It is therefore necessary to monitor and regulate the quality of herbal medicines sold in local communities continuously.