Assessment of Pathogenic Contamination and Antimicrobial Activity of Selected Herbal Medicinal Remedies in Mbarara City, South Western Uganda

Background: Herbal formulations in Mbarara have been used in the treatment and management of several disease conditions extensively overtime due to low cost compared to empirical synthetic medicine, however evidenced that they can be contaminated with dangerous pathogenic organisms which are all tailored to handling practices, storage, and other environmental conditions thus, the need to further asses these herbs for safety to the consumers. Materials and methods: Forty-ve (45) liquid herbal formulations for the treatment and management of communicable infections were purchased on the open market. All Samples were cultured on plate count agar for colony counts and then subcultured on different laboratory media and then analyzed for antimicrobial activity using the agar diffusion method. Results: Out of the 45 herbal formulations, 32(71.1 %) were contaminated while 13 (28.9 %) were not. Out of the organisms isolated from individual formulations, 19 (59.4 %) had Bacillus subtillis and S. aureus, 4(12.5%) had C. freundi and Proteus mirabilis, 2(6.3%), C.divergens, 1(3.1 %) Rhodotorula, 5(15.6%) Aspergillus spp, had E. cloace. 1 (3.1%) had Klebsiella spp. Of these, 29(87.9%) had contaminants within acceptable limits of less than 10 3 CFU/mL, while 12(36.4%) beyond 10 3 CFU/m. Out of the 45 formulations, not even one could qualify for pharmaceutical use, all MICs all were >1000mcg/mL64.4%) were active while 16(35.6%) had no activity. Conclusion: Herbal formulations in Mbarara are contaminated with various microbes and have very limited antimicrobial activity, herbalists therefore should be trained on good harvesting, safe handling, storage, and good manufacturing practices of these medicinal raw materials and their products, responsible authorities should enact policies and regulations to guide the herbalists and protect the public from adverse effects of consuming these unveried herbal medicinal remedies.


Background
Herbal medicines' use is the exploration of utilising homes' grown solutions for treating various forms of health disorders including but not limited to gastrointestinal, eye defects and respiratory [1]. Nearly 80% of the people in the world depend on these local medicines for primary health care needs [2][3][4]. In developing countries, almost hundreds of people utilise these mostly plant-exploited herbal products for microbial infections [5][6][7]. However, these can either be a source of great medical value or complications such as drug resistance [1,[8][9][10].
In East Africa and Uganda, the use of herbal medicines is a common habit with about 80% of the population accustomed to such a tradition, especially in rural settings [2,11]. To some extent, this practice has been backed by a demonstration of antimicrobial bioactivity against microbiological agents by some of the commercially available herbal remedies [12][13][14].
In this regard, the Ugandan government, for instance, is encouraging the use of such formulations and an integration of their applications into the orthodox medicine framework [15,16]. However, there are no systems in Uganda to monitor and control the herbal medicine chain of production, their hygiene conditions during production, and bioactivity spectrum, thus these products could get contaminated with pathogens leading to secondary infections [17]. Contaminants such as Staphylococcus, Escherichia coli, Shigella, and bread moulds have already been reported in some of these products. Thus, consumers of these products are at risk of exposure to such pathogens [3,18]. This is further complicated by their unchecked administration and prescription guidelines, for instance, under or over dosing with herbal products could lead to clinical complications such as development of drug resistance and death. Therefore, this study aimed at determining the contamination levels and antimicrobial activity of randomly selected locally branded antimicrobial herbal medicinal remedies on the open market in Mbarara city, South Western Uganda.

Ethical approval
The proposal was submitted to the Department of Microbiology, Faculty of Medicine Review Committee (FRC) and approved by the Institutional Review Committee (IRC) of Mbarara University of Science and Technology.

Results
Common microbiological contaminants of herbal formulations around Mbarara municipality.
Our study found that Out of the 45 herbal formulations, 29 (64.4%) were active while 16(35.6%) had no activity against all standard organisms, of the 29 active herbal formulations, 26(89.6%) of the herbal formulations were active against staphylococcus aureus, 11(37.9%) against Candida albicans. 9(31.03%) against E.coli .The least activity was noted against Pseudomonas aeruginosa where 6(20.7%) of the herbal formulations acted against these microorganisms as shown in Tables 4 and 5 below.

Discussion
In this study, herbal preparations contained varying degrees of both bacteria and fungi as common contaminants .This has also been reported in similar studies by Archibong and colleagues [20][21][22]. Of concern is the high levels of contaminants such as B. subtillis, C. freundi, C. divergens, E. cloacae and P. mirabilis although some had acceptable values for Enterobacter cloacae, Rhodotorula and Aspergillus spp, Staphylococcus Aureus, Klebsiella spp according to WHO standards. This is similar to studies done in Mwanza with the exception of Shigella and Salmonella that we didn't obtain [23,24].
Contamination of these herbal preparations could be resulting from the methods involved in the production processes as previously reported by [20,25,26]. Storage conditions, methods of drying herbal raw materials such as roots, leaves and stems, bark, improper handling, and the soil from which raw materials are obtained may contribute to the presence of these contaminants in the herbal products.
Presence of microorganisms in therapeutic herbal preparations incapacitates the pharmacological activity of the herbal products and contributes to secondary infections to the consumers. Previous studies have reported that the majority of these isolates are implicated in causing various disease conditions including gastritis. The cultivation of high numbers of Bacillus subtilis could be because of inadequate heat processing methods, improper handling of the products, and contaminated processing equipment.
Our results are in agreement with that obtained by Oluwatoyin and colleagues [24,27] which found that most herbal remedies were contaminated because of improper handling of the products, and contaminated processing equipment used in the production process.

Bacillus subtilis is not considered a human pathogen nor is it toxigenic like some other members of the genus, however in immune-suppressed individuals, it's implicated in various systemic infections such as endocarditis, meningitis [26-28].
On the other hand, the presence of members of the Enterobacteriaceae family such as C. freundi , C. divergens , E. cloacae , P. mirabilis, Enterobacter cloacae and Klebsiella spp is an indication of fecal contamination. This revealed poor hygienic practices of the herbalists in the process of production and handling of these products [24,25,29]. These results are similar to those done in Croatia and Tanzania, .They are known food borne pathogens and thus implicated in food poisoning and diarrhea diseases [25,30,31].
Not only did we obtain the above but also Staphylococcus aureus was also identi ed in this study, S. aureus has also been reported in similar study done in Brazil and Kenya [24,25] except that its prevalence was higher compared to our study and its implicated in scalded skin syndrome, wound abscess, and gastroenteritis [25,32].
Microbial analysis of these samples also indicated the presence of molds, especially Aspergillus and Rhodotorula species. These have also been reported in similar studies done in Brazil [25,33]. Since fungi are ubiquitous in nature, their presence reveals that it could be because of improper storage and handling practices of raw materials during pre-and post-harvesting and processing periods. Rhodotorula spp is known to have high a nity for plastics, therefore its presence indicates poor packaging of the products since the majority of the herbal preparations were sold in unauthentic plastic bottles which appeared to have been used for other products such as commercial mineral water [34,35].
With all negative impacts of the contaminants mentioned above, Rhodotorula was't left out. Previously it was considered nonpathogenic but has emerged as pathogenic opportunists that may colonize susceptible patients, and it causes bloodstream infections, meningeal and skin infections [36][37][38].
Aspergillus spp causes a range of diseases referred to as áspergillosis with a variety of clinical syndromes, the spectrum of these illness depending on host pathogen interactions. These include invasive pulmonary aspergillosis, chronic pulmonary aspergillosis, and allergic Broncho pulmonary aspergillosis. [39][40][41].
Herbal extracts have proven antibacterial activity in studies reported previously [26, [42][43][44]. This is similar to what we obtained in this study. We established that majority of the extracts were active against S .aureus, followed by E.coli, C .albicans, and least against P.aeruginosa. This is in agreement with other studies by Shu and collegues [26,43].
However, effectiveness was established by determining Minimum Inhibitory Concentrations, in this context, the most effective crude extract regarded as sensitive should have an MIC of <100mcg/mL, and that >1000mcg/mL is regarded as resistant [45,46] .In our study, none of the samples had an MIC <100mcg/mL, all were >1000mcg/mL. This could be because of the inadequate and improper puri cation methods involved in extraction.
Our results are similar to those obtained in Northern Peru [47,48] but contrally from those obtained by Wong and colleagues [49,50].

Conclusion
Herbal formulations in Mbarara are contaminated with various microbes and have very limited antimicrobial activity, herbalists therefore should be trained on good harvesting, safe handling, storage, and good manufacturing practices of these medicinal raw materials and their products, responsible authorities should enact policies and regulations to guide the herbalists and protect the public from adverse effects of consuming these unveri ed herbal medicinal remedies.

Materials And Methods
Study site.
Study design.
Herbal products were randomly sampled at market price and the choice of herbal formulations was obtained by asking the vendors the herb treating a particular infection in local language and in this case skin infections such as boils, diarrhoea, stomach pain, respiratory tract, genital, urogenital and gastrointestinal tract infections were considered. Five out of the six divisions of Mbarara Municipality were randomly selected for sampling of herbal products.
Sample size determination.
A pilot study was done in two divisions of Mbarara Municipality to identify the common herbal remedies on the market and it was found out that Kakoba division had 11 different types of herbs treating different infections and Nyamitanga division had 7 different herbal remedies for different ailments giving a total of 18 herbal remedies for each division in the municipality with average of 9 therefore a total of 45 samples was used for the study.
Inclusion criteria.
Liquid formulations branded to treat cultivatable etiological agents and not expired at the time of conducting the study.
Herbal remedies used to treat non-communicable diseases.
Data collection instruments/methods: Data collection commenced following a thorough explanation of the study objectives.
Preparation of culture media.
All dehydrated media were prepared according to the manufacturer's instructions. They were mixed with distilled water and dissolved by gentle heat to boil. The media were sterilized in an autoclave at 121˚C for 15 min at 15 pounds of pressure. The sterile media were then dispensed into sterilized petri dishes and allowed to cool. The sterility of the prepared media was checked by incubation of blindly selected plates at 37˚C for 24 hrs.
Determination of the presence of bacterial contaminants.
Microbial parameters of signi cance such as colony forming units were determined by dispensing 1mL of the herbal formulation into the middle of a sterile culture plate using a microliter pipette followed by addition of 15-20 mL of plate count agar. The mixture was gently shaken back and forth then round to allow uniform mixing of the agar and herbal extracts. It was left to stand for 30 minutes to solidify, and each of the samples were incubated at 37o, C for 48 hrs. CFUs were then determined by manual counting of distinct colonies and expressed in colony forming units per/mL. properties, and biochemical reactions. Lactophenol blue stain was used to identify moulds with aid of microscope [19].
Determination of antimicrobial activity of selected herbal product samples using agar diffusion assay.
The herbal samples were further examined for antimicrobial activity against selected bacterial and fungal isolates, respectively.
Individual colonies of standard organisms were inoculated on Mueller Hinton agar for bacteria using sterile swabs, two holes of 8 mm diameter and equidistant from each other were bored into the plates using a sterile glass cork borer. A volume of 1 mL of herbal formulation and 1 mL of control drug cipro oxacin for bacterial activity was dispensed into the holes and left to stand for 5 minutes to allow adequate diffusion of the samples. The plates were then taken for incubation at 37°C for 24hrs.
The diameter of the zones of inhibition around each hole in the plate were measured in millimetres. The same procedure was repeated for the test with C. albicans ATCC 10231 but using Sabouraud Dextrose Agar and Fluconazole as control drugs and incubation at 25-30°C for 48hrs.
Determination of minimum inhibitory concentrations (MICs) of the herbal formulations using broth dilution assay.
To determine the herbal MICs, 1 mL of the original herbal formulation was serially diluted in the ratio of 1/2, 1/4, 1/8, 1/16, 1/32 using sterile water in bijou bottles, these were then seeded on to MHA for bacteria and SDA for fungal standards. The dilutions which inhibited growth of the test organisms were read as the MICs for each of the herbal formulations.

Declarations
Ethical consideration.
Ethical approval was sought from Research Ethics Committee (REC) of Mbarara University of Science and Technology (reference number MUREC 1/7).
We blinded the products during practical procedures, analysis of samples, and dissemination of results of the study to ensure anonymity of the manufacturer and source of the product using letter codes followed by gures representing the sample number according to the sample size and this was W01-W45.

Availability of data and materials
Data and materials are readily available from the corresponding author upon request.
GW, BM contributed in study conception and design, CO and KM Collected data, MC, HZ, IM, and GM participated in the laboratory analysis, KK and CO carried out data cleaning and analysis. MB and FT wrote the rst draft of the manuscript while HI reviewed the manuscript and supervised the whole research process.

Con ict of Interest
The authors declare that they have no Con ict of interest.