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INTRODUCTION
Plants had been used for medicinal purposes long before recorded history (1). World Health Organization (WHO) survey indicates that about 70~80% of the world population particularly in the developing countries rely mainly on herbal medicines for their primary healthcare (2). The growth of herbal markets has increased substantially in South Korea, but the worldwide market share remains small despite significant governmental efforts (3) However, Chinese herbal medicines have successfully entered the international market, and their use is increasing worldwide (3).
Microbes might contaminate herbal medicines during storage and improper handling. Although herbal remedies are often perceived as natural and therefore safe, they are not free from adverse effect (4). With increasing popularity, the safety, efficacy and quality of these medicines have become an important issue for health authorities and health professionals (4). Counting bacterial colonies on agar plates is a simple and effective method for determining the number of viable bacteria in a sample (5). In this study, 194 agricultural herbal products were collected from Gyungdong wholesale market in Seoul. The purpose was to determine the microbial load in agricultural herbal products and ascertain the sanitation control of herb products in the market.
MATERIALS AND METHODS
Random sampling
Agricultural herbal samples were collected by random sampling from Gyungdong herb products wholesale market of Dongdaemun-gu, Seoul, Korea, from November, 2015 to October, 2016. One-hundred and ninety-four agricultural herb products were collected, and each agricultural herb products had been identified by the number of each species. The quantities of the sample distribution were as follows: Seven were Acanthopanax Root Bark, eight were Achyranthis Radix, six were Acori Gramineri Rhizoma, four were Alismatis Rhizoma, eight were Angelica Gigantis Radix, four were Armeniacae Semen, fourteen were Astragali Radix, three were Atractylodis Rhizoma Alba, four were Carthami Tinctoriin Fructus, three were Cassiae Semen, three were Chrysanthemum, four were Cinnamon Bark, four were Cinnamoni Cortex Spissus, nine were Citri Unshii pericarpium, sixteen were Cnidii Rhizoma, three were Corni Fructus, eight were Crataegi Fructus, three were Eleutherococcus senticosus, five were Eucommiae Cortex, nine were Gardeniae Fructus, three were Gastrodiae Rhizoma, four were Glycyrrhzae Radix et Rhizoma, six were Liriopis Tuber, seven were Lycii Fructus, five were Menthae Herba, three were Nelumbinis Semen, thirteen were Paeoniae Radix, five were Platycodi Radix, ten were Puerariae Radix, four were Rubi Fructus, three were Schisandrae Fructus, three were Zingiberis Rhizoma, three were Zizyphi Semen.
Determination of microbial quality by aerobic plate count method
Microbial quality was determined by the aerobic plate count method using the standard protocol established by Lee et al. (6). Ten grams of samples were added in 90 ml 0.85% saline solution to achieve a ten-fold dilution of the original sample by BagMixer 400 (Interscience, St. Nom la Bretèche, France). Ten-fold serial dilutions were carried out up to the concentration 10−5 of the original sample. One ml from each dilution was poured into the petri-plate. Standard plate count agar (Oxoid, Hampshire, England) having a temperature of around 45℃ was poured and the petri-plates were rotated to distribute the sample uniformly in the media. Media on the petri-plates were allowed to solidify and then the plates were incubated at 37℃ for 24 h (4).
Determination of microbial quality by coliforms plate count
The number of coliforms was determined by a pouring method of desoxycholate agar (Difco, Sparks, MD, USA) using the same protocol as in the aerobic plate count assay.
Identification of bacterial isolates
Bacterial isolates were identified based on cultural, morphological and biochemical tests. All types of colonies obtained on tryptic soy agar plate (Oxoid) after incubating in a 37℃ incubator for 24 h were subcultured on separate tryptic soy agar plate to obtain pure cultures under the same condition. Colony characteristics on tryptic soy agar plate were studied (4). Gram staining was performed for morphological study. Different biochemical tests were performed by API 20E kit, API CHB kit, API strep kit, API staph kit, and API listeria kit (bioMerieux, Marcy l'Etoile, France).
Antibiotics susceptibility test of Bacillus cereus (B. cereus) isolates
One to two isolated colonies from the fresh culture of test bacteria were transferred into test tubes containing 1 ml of nutrient broth, and incubated at 37℃ for 3~4 h until the development of turbidity equivalent to 0.5 McFarland Nephelometer Standard. This standard is estimated to have the bacterial suspension of 1.5×108 CFU/ml (4). The antibacterial study was conducted by the antibiotics disc diffusion method as described previously (789). Thirty-six B. cereus isolates were tested by 36 kinds of antimicrobial drugs discs.
RESULTS
Based on the results from each samples, the highest average aerobic plate counts were revealed in six agricultural herb products [Acanthopanax Root Bark (1.8×105), Acori Gramineri Rhizoma (7.9×105), Atractylodis Rhizoma Alba (3.4×106), Cinnamoni Cortex Spissus (1.6×105), Lyriopis Tuber (1.8×105), and Lycii Fructus (5.9×106)]. Moreover, the highest concentration of coliforms was revealed in two agricultural herb products [Angelica Gigantis Radix (1.6×104) and Atractylodis Rhizoma Alba (4.4×104)] (Table 1). The highest incidence (100%) in aerobic plate counts was revealed in six agricultural herb products [Acori Gramineri Rhizoma, Atractylodis Rhizoma Alba, Cassiae Semen, Cinnamoni Cortex Spissus, Eleutherococcus senticosus, and Rubi Fructus], and the highest incidence (100%) in coliforms was revealed in Atractylodis Rhizoma Alba (Table 1).
Bacterial growth was observed in tryptic soy agar. Out of 194 agricultural herbal products, 491 bacteria were isolated based on colony morphology and different biochemical tests at 36.5℃. Out of the numbers, there were 252 coliforms, 148 Bacillus spp., 75 Enterococcus spp., 10 Staphylococcus spp., and 6 Listeria spp. respectively (Table 2 and Table 3). The incidences rates were as follows: coliforms 51.32% (252/491), Bacillus spp. 30.14% (148/491), Enterococcus spp. 15.27% (75/491), Staphylococcus spp. 2.04% (10/491), and Listeria spp. 1.22% (6/491) respectively (Table 2 and Table 3). Of them, the incidence rates of bacteria related to human diseases were as follows: Escherichia coli isolates was 1.2% (3/252), Enterococcus faecalis isolate was 1.3% (1/75), Enterococcus faecium isolate was 1.3% (1/75), and B. cereus isolates were 23.0% (34/148). Based on these observations, B. cereus was the most prevalent pathogenic species in the genus Bacillus (Table 2 and Table 3).
Out of coliforms, the most prevalent species were Enterobacter cloacae, Pantoea spp., Cronobacter spp., and Serratia ficaria. Out of Bacillus spp. isolates, the most prevalent species were B. mycoides, B. cereus, B. circulans, B. coagulans, and B. megatorium. Out of Enterococcus spp. isolates, the most prevalent species were Leuconostoc spp., Aerococcus viridans, and Globicatella sanguinis (Table 2 and Table 3).
Antibiotic susceptibility test of B. cereus was performed against 34 B. cereus isolates. B. cereus isolate was resistant against 20 kinds of antibiotics including Amoxicillin/clavulanic acid (AMC30), Ampicillin (AMP10), Ampicillin/sulbactam (SAM20), Bacitracin (B10), Carbenicillin (CAR100), Cefamandole (MA30), Cefoxcitin (FOX30), Ceftriazone (CRO30), Cephalothin (KF30), Colistine sulfates (CT10), Compound sulfonamides (S3-300), Nitrofrantoir (F300), Novobiocin (NV30), Penicillin G (P10), Polymyxin B (PB300), Rifampicin (RD5), Sulphomethoxazole/trimethoprim (SXT25), Tetracycline (TE30), Ticarcillin (TIC75), and Trimethoprim (W5) (Table 4), and was susceptible to 14 kinds of antibiotics including Amikacin (AK30), Chloramphenicol (C30), Ciprofloxacin (CIP5), Clindamycin (DA2), Doxycycline (DO30), Erythromycin (E15), Gentamicin (CN10), Kanamycin (K30), Levofloxacin (LEV5), Oleandomycin (OL15), Oxolinic acid (OA2), Streptomycin (S10), Tobramycin (TOB10), and Vancomycin (VA30) (Table 4).
DISCUSSIONS
Based on the results from aerobic plate counts, samples with higher average aerobic bacteria counts were Acanthopanax Root Bark, Acori Gramineri Rhizoma, Atractylodis Rhizoma Alba, Cinnamoni Cortex Spissus, Lyriopis Tuber, and Lycii Fructus. Coliforms were prevalent in Angelica Gigantis Radix and Atractylodis Rhizoma Alba (Table 1). These results were similar to the findings from Lee et al.'s report (6), in which the following was observed: the total aerobic plate counts were smaller in Astragali Radix, Eucommiae Cortex, and Zizyphi Semen, but much was measured in Acori Gramineri Rhizoma, Atractylodis Rhizoma Alba, Achyranthis Radix, and Liriopis Tuber.
Out of 194 agricultural herbal products, 491 bacteria were isolated. Out of these number, there was 51.3% (252/491) coliforms, 30.1% (148/491) Bacillus spp., 15.3% (75/491) Enterococcus spp., 2.0% (10/491) Staphylococcus spp., and 1.2% (6/491) Listeria spp. (Table 2, Table 3). Of them, B. cereus was the most prevalent pathogenic species in the genus Bacillus, but the most prevalent species in the genus Bacillus was B. mycoides (39.9%, 59/148) (Table 2, Table 3). This result is comparable with the results obtained by Oyetayo (10). He assessed Nigerian herbal medicines and reported that almost all of the herbal medicines tested were contaminated with Bacillus spp. which is commonly found in soil, air, dust, etc. (10). Many aerobic species of Bacillus spp. produce endospore that helps them not only resist environmental stress but also ensure their long-term survival under adverse conditions (10). In his study, all agricultural herbal products in which microorganisms were not isolated in aerobic bacteria count showed an antibacterial property (10). The antibacterial property of agricultural herbal products might be one of the reasons that they are not contaminated by bacteria, with the exception of Bacillus spp. (10). Among the Bacillus spp., the most predominant species was B. cereus (23.0%, 34/148) (Table 2). Esimone et al. (11) had also reported Bacillus spp. (28.4%) as the most common organism in herbal products. The presence of B. subtilis and B. cereus observed in agricultural herbal products is comparable to a similar study conducted by Adeleye et al. (12). Microbial contamination usually occurs due to improper drying or storage of the plant material which eventually results in degradation of the plant constituents (4). Microbial contamination can also render plant material toxic, either by transforming the chemicals in the plant material or from the production of toxic compounds by the microbes (4). Therefore, microbial quality tests should be conducted as necessary throughout the lifestyle of the plant materials, from the raw material to the finished product stages (4). The result is comparable with the result obtained by Okunlola et al. (13). They had also reported the presence of Escherichia coli, Salmonella spp. and Staphylococcus aureus in agricultural herbal products. Similarly, the same microorganisms were also detected in this research. During the quality analysis, precautions must be taken to ensure that conditions do not adversely affect any microorganisms that are to be measured (4).
Lee et al. (6). reported that B. cereus was isolated to 50.4% (59/117) of all isolated aerobic bacteria in medicinal herb products. B. cereus was isolated to many numbers, possibly because most Bacillus spp. was originated from soil and that isolate's spore.
Antibiotic susceptibility tests were performed on 34 B. cereus isolates. Over 50% of B. cereus isolates were resistant to antibiotics.
Most of the agricultural herbal products had a large number of coliforms and Bacillus spp. and B. cereus isolates from agricultural herbal products showed significant resistant patterns to the antibiotics tested.
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