Occurrence of Potential Bacterial Pathogens and Their Antimicrobial Susceptibility Patterns Isolated from Herbal Medicinal Products Sold in Different Markets of Gondar Town, Northwest Ethiopia.

Background. The World Health Organization estimates that about 80% of the world's population uses herbal medicine to treat various illnesses as means of primary healthcare. However, during preparation, herbal plants may be exposed to contamination by potential pathogens, and this may lead to infections. The aim of this study was to determine bacterial contamination of herbal medicinal products and to assess the antibiotic susceptibility pattern of the isolated bacteria. Methods. A cross-sectional study was conducted from January 1 to May 25, 2013, at Gondar Town. A total of 55 samples used as oral, local, and intranasal routes of administration were collected from the herbalists. Results. In the present study the total aerobic bacterial count ranges from zero to 2.41 × 10(9) CFU/g with mean count of 1.99 × 10(8) CFU/g or mL while the total coliform count showed an average of 1.05 × 10(8) CFU/g or mL with a range of zero to 2.1 × 10(9) CFU/g. The most common bacteria isolated were Bacillus spp. followed by Enterobacter spp., Shigella dysenteriae, and Salmonella spp. Multiple drug resistance was not uncommon and it was found that 125 (83.4%) of the isolates were resistant to two or more antibiotics. Conclusion. Herbal medicinal preparations were highly contaminated with pathogenic microorganisms with high microbial load. Most of the isolates have multiple drug resistance. Using those contaminated herbal medicines may lead to infection of other health related risks. Therefore, this warrants urgent training of herbalists and management scale-up for quality and safety of medicinal plants.

1. Introduction

The use of herbal medicine is generally increasing worldwide [1]. In many developed countries, 70–80% of the population still depends upon herbal drugs for their health care [1-3]. Similarly in developing countries such as Africa, up to 80% of the population relies on herbal medicine as a major source of therapy [1, 3, 4].

Although the World Health Organization (WHO) has advocated the integration of herbal medicinal products (HMPs) into the primary health care system of developing countries, safety issues related to herbal drug preparations continue to be ignored by the herbalist [5]. So the safety of herbal products became a major concern in public health [6]. This is because microorganisms of various kinds are normally adherent to leaves, stem, flowers, seeds, and roots from which herbal medicine can be prepared and potential pathogens may also be introduced during harvesting, handling [6, 7], open-air drying, preserving, manufacturing [6], and use of contaminated materials for storage [7, 8]. According to some reports, the consumers may possibly fall into illness because of taking herbs incriminated with pathogenic microorganisms [8] and sometimes the presence of antibiotic resistant microbial isolates in the HMPs will lead to transfer of antibiotic resistance strains to consumers [9]. Different studies that have been performed on herbal medicinal products revealed the presence of bacterial pathogens with multiple drug resistance [10].

In Ethiopia, due to the cultural acceptability of healers, the relatively low cost of traditional medicine, and limited access to modern health facilities [11] up to 80% of the population uses traditional medicine [11, 12]. Ethiopia has high diversity of plant species most of which are used in the preparation of herbal medicinal products and it is one of the six African countries where about 60% of the plants are said to be indigenous with their healing potential [13]. The growing and unhygienic use of herbal medicinal products has become a major concern in public health [6]. However, in Ethiopia particularly in the study area, there is no sufficient data which addresses the bacteriological quality of herbal medicinal products. Hence the aim of this study was to assess the occurrence of potential bacterial pathogens and their antimicrobial susceptibility patterns isolated from herbal medicinal products sold in different markets of Gondar Town, Northwest Ethiopia.

2. Materials and Methods

2.1. Study Area, Design, and Period

A cross-sectional study was conducted from January 1 to May 25, 2013, at Gondar Town. Gondar is located in the Northwestern part of Ethiopia, about 740 km far from Addis Ababa, the capital city of Ethiopia. The town has an estimated area of 41.27 square Km, with altitude of 2200 meters above sea level, having a total population of more than 252,537 [14].

2.2. Source of Population

All types of herbal medicinal products with oral, local, and intranasal route of administration have been sold at different markets of Gondar Town.

2.3. Study Subjects

All types of herbal medicinal products prepared by the 20 herbalists of Gondar Town who have been enrolled in association of traditional medicine named “Zeria-Biruk Association of Traditional Medical Practitioner” were the study subjects.

2.4. Sample Size and Sampling Technique

In the present study, a total of 55 (13 liquid and 42 solid) samples were taken from all herbalists. From each herbalist herbal medicinal products prepared for oral, local (body lotion), and inhalation use were considered. All orally, locally, and intranasal consumed powder form preparations and all liquid samples were purchased and included in the study. Route of administration of the herbal traditional medicine can be mainly in oral, body lotion, and inhalation type and the present study gives more attention to those herbal medicinal products.

Inclusion Criteria. Those powder and liquid preparations of herbal medicinal products administered orally, locally, and intranasally without further processing were included in the study.

Exclusion Criteria. Those powder form herbal medicinal products with further processing and other routes of administration were excluded in the study.

2.5. Data Collection, Handling, and Transporting of Specimens

A total of 55 different herbal (42 powder and 13 liquid) preparations were purchased randomly from identified herbalists in Gondar Town. Using aseptic techniques, from each liquid herbal preparation about 5 mL of sample was collected in a sterile screw-capped test tube and about 3 to 10 g of solid (powder) sample was also collected by using labeled wide-necked test tubes. All the samples were transported to the Medical Microbiology Laboratory of Gondar University Hospital with cold box within one hour of collection. Liquid specimens were refrigerated at 4°C till processing.

2.6. Isolation and Identification of Bacteria

Liquid samples were homogenized by mixing vigorously and transferred 1 mL sample to 9 mL sterile saline. At the same time, the liquid solution was prepared by mixing 1-gram powder with 9 mL of tryptone soy broth as a stock solution and then serial dilution was made to get appropriate dilution. All microbiological analyses were carried out in triplicate according to the standard [15-17]. By using a calibrated micropipette, 100 μL volume of 10−3, 10−5, and 10−7 was measured and dispensed by glass rod spreader to standard plate count and violet red bile agar media. Liquid preparations were also cultured on blood agar, MacConkey agar, and Salmonella Shigella agars and then incubated at 35–37°C for 18 to 24 hours.

At the end of the incubation period, the colonies on standard plate count media and violet red bile agar media were enumerated by using surface plate agar method using colony counter for total aerobic count and total coliform bacteria count, respectively. By multiplying the average number of colonies with dilution factor, it was calculated and reported as a colony forming unit per gram (CFU/g) of sample [16-19]. The obtained CFU/g from 1-gram sample was compared with WHO standard [5, 20]. Pure isolate of bacterial pathogen was preliminarily characterized by colony morphology and gram stain [19]. Bacterial identification was made using biochemical tests, namely, indole, citrate, oxidase, H2S production, lysine decarboxylase, lactose fermentation, urea hydrolysis, gas production catalase, the coagulase, mannitol fermentation, and novobiocin susceptibility test. A standard biochemical procedure was also used for full identification of gram positive and gram negative bacteria [21].

2.7. Antimicrobial Susceptibility Testing

The antibiotic susceptibility test was performed by using disc diffusion method recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines [22] on Mueller-Hinton agar plate (Oxoid Ltd., Basingstoke, Hampshire, UK). The antibiotic discs and their concentration were amoxicillin (AML, 10 μg), chloramphenicol (C, 10 μg), cloxacillin (CXL, 5 μg), cotrimoxazole (SXT, 50 μg), ampicillin (AMP, 10 μg), ceftriaxone (CRO, 30 μg), gentamicin (CN, 10 μg), penicillin (PEN, 5 μg), nitrofurantoin (F, 300 μg), norfloxacin (N), and erythromycin (E, 5 μg). The inoculum was standardized by transferring pure colonies of the test organism into 5 mL of tryptone soy broth. The broth then incubates for three hours at 35–37°C to allow the growth of test organism and their inoculums sizes were compared with the turbidity of 0.5 McFarland standard [10]. A sterile cotton swab was dipped into the suspension and excess suspension was removed by gentle rotation of the swab against the surface of the tube. Before placing the antimicrobial disc, the swab with the bacterial suspension was distributed evenly over the entire surface of Mueller-Hinton plates [10]. The plates were incubated at 37°C for 18–24 hours. The diameter of the zone of inhibition was measured and interpreted using standard chart as sensitive, intermediate, and resistant [22]. The reference strains used as control were Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923).

2.8. Data Analysis

Data was checked for completeness, cleaned manually, entered, and analyzed using SPSS version 20 statistical package. Analysis was made using frequency tables. Pearson's test and odds ratio with 95% CI were used for measures of association and P value less than 0.05 was considered as statistically significant.

2.9. Ethical Considerations

This research project was conducted after obtaining institutional ethical clearance from School of Biomedical and Laboratory Sciences Ethical Committee. A supporting letter was obtained from Zonal Health Bureau and the chairman of the Association for Herbal Medicinal Plants. Informed consent was also obtained from each study participant. All the information obtained from the study was kept confidential.

3. Results

3.1. Sociodemographic Characteristics

Among the total of 22 herbalists, 20 (16 males and 4 females) of them were enrolled during the study while the other 2 (both males) refused to participate in the study. The mean age of the herbalists was 41.6 years with minimum and maximum age of 27 and 70 years, respectively. Of the study participants 7/20 (35%) of them were illiterate and 1/20 (5%) of them can only read and write but the rest 12/20 (60%) of the herbalists were literate. 16/20 (80%) of the herbalists were Orthodox and 4/20 (20%) of them were Muslims. Regarding their experience, 13 (65%) of the herbalists have more than 20 years, 2 (10%) have 16–20 years, 4 have (20%) 6–10 years, and 1 (5%) herbalist has 5 years.

3.2. Total Aerobic and Total Coliform Count

Among 55 samples analyzed, 5 samples (3 liquid and 2 powder) obtained from 5 different herbalists have no growth and 13 samples (9 powder and 4 liquid) obtained from 6 herbalists have zero coliform count. The total aerobic bacterial count of 55 samples was recorded with minimum count of zero (for samples of no growth) and maximum count of 2.41 × 109 CFU/g with mean count of 1.99 × 108 CFU/g and, except one (6 × 104 CFU/mL, which was liquid), all the aerobic counts were beyond WHO tolerable limit. Total coliform count shows an average of 1.05 × 108 CFU/g or per mL with minimum of zero and maximum of 2.1 × 109 CFU/g (Table 1).

Table 1

Total aerobic and total coliform count of herbal medicinal products sold in different markets of Gondar, Northwest Ethiopia, from April 1 to May 25, 2013.

Sample ID	Total aerobic bacteria count	Total coliform count	Sample ID	Total aerobic bacteria count	Total coliform count	Sample ID	Total aerobic bacteria count	Total coliform count
01A	1.84 × 107	1.5 × 107	07A	1.81 × 108	1 × 107	16C	1.61 × 107	1.12 × 107
01B	0	0	08A	8.1 × 106	7.1 × 106	16D	5.5 × 108	5.1 × 108
01C	1.05 × 106	1 × 106	08B	4.1 × 107	5.1 × 106	16E	5.2 × 107	0
01D	4.9 × 107	3.4 × 106	09A	4.6 × 105	3.2 × 105	16F	1.2 × 106	0
01E	5 × 106	0	09B	4.11 × 107	3.1 × 107	16G	2.4 × 108	3.6 × 106
01F	1.11 × 106	0	10A	9.8 × 108	7.6 × 108	17A	0	0
01G	2.68 × 106	2.51 × 106	10B	2.84 × 108	4.2 × 107	17B	3.3 × 107	1.8 × 107
01H	4.7 × 107	0	11A	2.12 × 106	9.7 × 105	18A	5 × 107	1.2 × 107
01I	9.5 × 108	8.9 × 108	11B	0	0	18B	1 × 106	1.9 × 104
02A	5.7 × 107	4.67 × 106	12A	2.02 × 106	1.15 × 105	19A	1.05 × 105	0
02B	1.82 × 108	1.41 × 108	13A	0	0	19B	9.3 × 108	0
03A	9.7 × 107	7.5 × 107	13B	1 × 107	0	19C	1.2 × 107	5.9 × 106
03B	2.41 × 109	2.1 × 109	13C	2 × 106	0	19D	1.02 × 107	1 × 105
03C	5 × 106	0	13D	3 × 107	0	19E	9.4 × 108	8.9 × 108
03D	6 × 104	5.7 × 104	14A	1.9 × 107	4.1 × 106	19F	0	0
03E	1.82 × 107	8.4 × 106	14B	7 × 107	2.9 × 106	20A	2.89 × 108	3.3 × 106
04A	8.7 × 105	0	15A	2.6 × 108	8.2 × 107	20B	8.2 × 107	1 × 106
05A	6.1 × 106	5.5 × 107	16A	9.2 × 108	0	—	—	—
06A	9.3 × 107	4.3 × 105	16B	9.41 × 107	0	—	—	—

3.3. Correlation of Microbial Contaminants with Different Herbal Medicines

A total of 150 strains were isolated from the herbal preparations (Table 2). Of the isolates, 98/150 (65.3%) were gram negatives and 52/150 (34.7%) gram positives. 50 (90.9%) contained pathogenic microorganisms or fecal indicators such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The organism most commonly isolated from the herbal medicines was Bacillus 31 (20.7%) followed by Enterobacter cloacae 18 (12%), Shigella dysenteriae 13 (8.7%), Citrobacter spp. 12 (8%), Providencia spp. 12 (8%), Klebsiella pneumoniae 11 (7.3%), Escherichia coli 10 (6.7%), Staphylococcus aureus 10 (6.7%), Staphylococcus epidermidis 5 (3.3%), and others (Table 3).

Table 2

Correlation microbial contaminants with different herbal preparations in Gondar Town, Northwest Ethiopia, from April 1 to May 25, 2013.

Sample ID	Nature of sample/therapeutic claims	Scientific name	Isolated bacteria	Sample ID	Nature of sample/therapeutic claims	Scientific name	Isolated bacteria
01A	Liquid: antigastric & GI problem	ND	Enterobacter cloacae, Citrobacter	07A	Powder: antiabdominal problem	Cucurbita maxima	Bacillus spp. S. epidermidis, Citrobacter diversus
01B	Liquid: anticonstipation	ND	No	08A	Powder: antiliver infection	Terminalia schimperiana	Providencia stuartii
01C	Powder: antihypertension & fungal infection	Dodonaea angustifolia	Enterobacter aerogenes, Bacillus spp., P. aeuroginosa	08B	Powder: evil eye	Myrica salicifolia	E. coli, Bacillus spp. Providencia
01D	Powder: anti-heliments & sinus	Cucurbita maxima	S. aureus, S. dysenteriae, Providencia stwarti	09A	Liquid: antiwound & fungal infection	Plumbago zeylanicum	Bacillus spp. & Enterobacter cloacae
01E	Powder: antiwound infection	Plumbago zeylanicum	Bacillus spp.	09B	Powder: antiabdominal problem	ND	Citrobacter diversus, E. coli, S. dysenteriae, Bacillus spp.
01F	Powder: anti-HIV	Foeniculum vulgare	S. pyogen	10A	Powder: anti-UTI infection	Clerodendrum myricoides	Enterobacter cloacae, K. pneumonia
01G	Powder: effective for menstrual abnormality	ND	Enterobacter cloacae, Citrobacter, Acinetobacter spp., S. dysenteriae	10B	Powder: acts as pellagra	ND	Providencia,Citrobacter, K. ozaenae, Bacillus spp.
01H	Powder: effective against warts & bleeding	Calotropis procera	Bacillus spp.	11A	Powder: antigastric	ND	Bacillus spp., S. aureus, Pseudomonas aeruginosa
01I	Liquid: antiotitis media	Withania somnifera	Salmonella spp., S. dysenteriae, E. coli,  K. pneumoniae	11B	Powder: antiliver disease	ND	No growth
02A	Powder: effective for kidney and liver infections	Jestica schimperiana	Enterobacter cloacae, Serratia, Bacillus spp.	12A	Liquid: effective for otitis media	Withania somnifera	S. aureus, S. pyogen, K. pneumonia, enterococci, K. ozaenae
02B	Powder: effective for kidney infections	Plumbago zelylanicum	Enterobacter cloacae, Acinetobacter, Bacillus spp., E. coli	13A	Liquid: swollen wound	Brucea antidysenterica	No growth
03A	Powder: antiwart & wound	ND	Enterobacter cloacae & K. ozaenzae	13B	Powder: wound infection	Clerodendrum myricoides	S. aureus, Bacillus spp.
03B	Powder: antiasthma	Rumex abyssinicus	Bacillus spp., S. aureus, P. aurognosa  Acinetobacter spp.	13C	Powder: effective against liver disease	Thymus schimperi	Bacillus spp., S. epidermidis
03C	Powder: antiscar	ND	Bacillus spp.	13D	Powder: rabbis virus	ND	S. saprophyticus & Bacillus spp.
03D	Liquid: antifungal	Dodonaea amgustifolica	Serratia	14A	Powder: antiepilepsy	ND	S. aureus, Bacillus spp., K. pneumoniae, Citrobacter diversus
03E	Powder: antifungal & skin disease	Brucea antidysenterica	Providencia stuartii, E. coli, Serratia, Salmonella spp., S. dysenteriae	14B	Powder: anticancer	Plumbago zeylanicum & ND	Serratia, E. coli, S. dysenteriae, Bacillus spp.
04A	Powder: antiwart	Cucumis prophetarum	Bacillus spp.	15A	Powder: antiparalysis	ND	E. coli, K. pneumonia, Enterobacter aerogenes, Salmonella spp., S. epidermidis
05A	Powder: antiwart	Plumbago zeylanicum	E. coli, S. dysenteriae, Enterobacter	16A	Liquid: antidiarrheal	ND	S. pyogenes & Bacillus spp.
06A	Powder: acts as pellagra	Aloe vera	Enterobacter cloacae, S. aureus, S. epidermidis, K. pneumonia	16B	Liquid: anti-intestinal parasites	Oxalis semiloba	S. pyogen
16C	Powder: anti-intestinal parasites	ND	Enterobacter cloacae	18B	Powder: antiherpes zoster virus	ND	S. saprophyticus, K. pneumonia
16D	Powder: anti-intestinal parasites & food poisoning	ND	K. pneumonia, S. dysenteriae	19A	Liquid: antirash	ND	Bacillus spp.
16E	Powder: antisinus & nose asthma	Myrica salicifolia	S. epidermidis, Bacillus spp.	19B	Liquid: antiepilepsy	ND	Bacillus spp.
16F	Liquid: antiwound infection	Clerodendrum myricoides	Bacillus spp.  Enterobacter cloacae	19C	Powder: antimalaria intestinal parasites & cancer	Croton macrostachyus	S. dysenteriae, K. pneumonia
16G	Powder: antiwound infection	Clerodendrum myricoides	S. dysenteriae, Bacillus spp.	19D	Powder: antiwound	Clerodendrum myricoides	S. aureus, S. dysenteriae
17A	Powder: antiwound infection & diarrhea	ND	No growth	19E	Powder: antifungal	Aloe vera	K. pneumonia, Providencia stuarti,   Serratia
17B	Powder: antigastric	ND	S. aureus, K. pneumonia,  S. dysenteriae	19F	Liquid: antiotitis media	Withania somnifera	No growth
18A	Powder: anti-intestinal parasites & burns	ND	Shigella dysenteriae, E. coli	20A	Powder: antiepilepsy	ND	Citrobacter, Bacillus spp.
				20B	Powder: antidiabetes & MTB	ND	E. coli, Bacillus spp.

ND: not yet determined, ID: identification number, the same number or ID indicates a single herbalist and spellings in front of numbers indicate different herbal medicines of the same herbalist.

Table 3

Bacterial profiles isolated from herbal medicinal products sold in different markets in Gondar Town, Northwest Ethiopia, from April 1 to May 25, 2013.

Bacterial isolates	Frequency N (%)
Gram negatives
Enterobacter cloacae	18 (12)
Enterobacter aerogenes	4 (2.7)
Shigella dysenteriae	13 (8.7)
Klebsiella pneumoniae	11 (7.3)
Klebsiella ozaenae	4 (1.7)
Escherichia coli	10 (6.7)
Providencia spp.	12 (8.0)
Citrobacter spp.	12 (8.0)
Serratia spp.	5 (3.3)
Acinetobacter spp.	4 (2.7)
Salmonella spp.	3 (2.0)
Pseudomonas aeruginosa	2 (1.3)
Gram positives
Bacillus spp.	31 (20.7)
Staphylococcus aureus	10 (6.7)
Staphylococcus epidermidis	5 (3.3)
Staphylococcus saprophyticus	2 (1.3)
Streptococcus pyogenes	4 (2.7)
Total	150 (100%)

3.4. Antimicrobial Susceptibility Patterns of the Isolates

Antimicrobial susceptibility patterns of gram negative and gram positive bacterial isolates from herbal medicinal products in Gondar Town are presented as shown in Tables 4(a) and 4(b). Majority of the isolates were resistant to ampicillin (131) (87.3%), followed by amoxicillin clavulanic acid (95) (63.3%), amoxicillin (92) (61.3%), penicillin (30) (57.7%), and nitrofurantoin (73) (48.7%). However, the lowest rates of resistance were observed in gentamycin (4.7%) followed by vancomycin (8) (5.3%), erythromycin (9) (6.0%), ciprofloxacin (11) (7.3%), and norfloxacin (11) (7.3%) (Table 5).

Table 5

Drug resistant patterns of bacterial isolates identified from herbal medicinal products in Gondar, Northwest Ethiopia, from April 1 to May 25, 2013.

List of antibiotics	Rate of resistance among gram negatives N = 98 (%)	Rate of resistance among gram positives N = 52 (%)	Total N = 150 (%)
Cotrimoxazole	10 (10.2)	5 (9.6)	15 (10%)
Gentamycin	6 (6.1)	1 (1.9)	7 (4.7%)
Tetracycline	11 (11.2)	2 (3.8)	13 (8.7%)
Nitrofurantoin	57 (58.2)	16 (30.8)	73 (48.7%)
Amoxicillin clavulanic acid	65 (63.3)	30 (42.3)	87 (63.3%)
Ceftriaxone	22 (22.4)	16 (30.8)	38 (25.4%)
Ampicillin	88 (89.8)	43 (82.7)	131 (87.3%)
Amoxicillin	58 (59.8)	34 (65.4)	92 (61.3%)
Chloramphenicol	11 (11.2)	1 (1.9)	12 (8%)
Norfloxacin	7 (7.1)	4 (7.6)	11 (7.3%)
Ciprofloxacin	9 (9.2)	2 (3.8)	11 (7.3%)
Cloxacillin	—	23 (44.2)	23 (15.3%)
Penicillin	—	30 (57.7)	30 (20.0%)
Erythromycin	—	9 (17.3)	9 (6.0%)
Vancomycin	—	8 (15.4)	8 (5.3%)

Multiple drug resistance bacteria were among the most isolated groups. The antimicrobial susceptibility-resistance pattern of the contaminating microorganisms revealed that 9/31 Bacillus species were resistant to 8 antimicrobial agents (ciprofloxacin, tetracycline, norfloxacin, augmentin, vancomycin, ceftriaxone, gentamicin, and cotrimoxazole) and it was also found that 131 (87.3%) of the isolates were resistant to ampicillin, 95 (63.3%) to amoxicillin clavulanic acid (augmentin), 92 (61.3%) to amoxicillin, and 30 (57.7%) to penicillin. Among bacterial isolates Pseudomonas aeruginosa were found to be resistant to at least 5 antibiotics tested while half of the isolates of Klebsiella ozaenae, Providencia spp., and Streptococcus pyogenes were resistant to 5 or more antibiotics tested (Table 6).

Table 6

Multidrug resistance patterns of bacterial isolates from herbal medicinal products sold in Gondar Town, Northwest Ethiopia, 2013.

Bacterial isolates	Total (%)	Number of antibiotics resistance patterns N (%)
		R0	R1	R2	R3	R4	≥R5
Gram negatives	98 (65.3)	7 (7.1)	9 (9.2)	19 (19.4)	22 (22.5)	19 (19.4)	22 (22.4)
Enterobacter cloacae	18 (18.4)	0 (0)	0 (0)	4 (22.2)	5 (27.7)	5 (27.7)	4 (22.2)
Enterobacter aerogenes	4 (4)	0 (0)	0 (0)	0 (0)	2 (50)	1 (25)	1 (25)
Shigella dysenteriae	13 (13.3)	2 (15.4)	1 (7.7)	2 (15.4)	2 (15.4)	5 (38.5)	1 (7.7)
Klebsiella pneumonia	11 (11.2)	0 (0)	1 (9.1)	3 (27.3)	3 (27.3)	0 (0)	4 (36.4)
Klebsiella ozaenae	4 (4)	0 (0)	1 (25)	1 (25)	0 (0)	0 (0)	2 (50)
Escherichia coli	10 (10.2)	2 (20)	1 (10)	3 (30)	1 (10)	1 (10)	2 (20)
Providencia stuartii	12 (12.2)	1 (8.3)	0 (0)	1 (8.3)	3 (25)	4 (33.3)	3 (25)
Citrobacter spp.	12 (12.2)	1 (8.3)	3 (25)	2 (16.7)	2 (16.7)	3 (25)	0 (0)
Serratia	5 (5.1)	1 (20)	2 (40)	1 (20)	1 (20)	0 (0)	0 (0)
Salmonella spp.	3 (3)	0 (0)	0 (0)	1 (33.3)	2 (66.7)	0 (0)	0 (0)
Pseudomonas aeruginosa	2 (2)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (100)
Acinetobacter	4 (4)	0 (0)	0 (0)	0 (0)	1 (25)	0 (0)	3 (75)
Gram positives	52 (34.7)	7 (13.5)	2 (3.8)	4 (7.7)	12 (23.1)	9 (17.3)	18 (34.6)
Bacillus spp.	31 (59.6)	4 (12.9)	1 (3.2)	4 (12.9)	4 (12.9)	7 (22.6)	11 (35.5)
Staphylococcus aureus	10 (19.2)	1 (10)	0 (0)	0 (0)	5 (50)	1 (10)	3 (30)
Staphylococcus epidermidis	5 (9.6)	1 (20)	0 (0)	0 (0)	2 (40)	0 (0)	2 (40)
Staphylococcus saprophyticus	2 (3.8)	0 (0)	1 (50)	0 (0)	0 (0)	1 (50)	0 (0)
Streptococcus pyogenes	4 (7.7)	1 (25)	0 (0)	0 (0)	1 (25)	0 (0)	2 (50)
Total MDR (G− and G+)	150 (100)	14 (9.3)	11 (7.3)	23 (15.3)	34 (22.7)	28 (18.7)	40 (26.7)

MDR: multiple drug resistance (resistant to 2 or more antibiotics), R0: no antibiotic resistance, R1: resistance to one, R2: resistance to two, R3: resistance to three, R4: resistance to four, and ≥R5: resistance to five and more drugs.

4. Discussion

Most of the medicinal plants are prepared in open environment and unhygienic condition which gradually lead to contamination of enteric pathogens having public health importance [20]. In the present study, from 55 herbal medicinal preparations, aerobic bacterial counts were obtained from 50 samples, of which only one has permutable limit of bacterial count according to WHO standard [20]. The other 49 samples were beyond WHO limit with minimum count of 4.6 × 105 CFU/mL and maximum of 2.41 × 109 CFU/g with mean count of 2.15 × 108 CFU/g or mL. The total aerobic bacteria count in the present study is in agreement with the higher counts of aerobic bacteria found in herbal materials which have been studied by Ogunshe and Kolajo in Nigeria [23]. On the contrary, the present study has higher aerobic bacteria count than the study conducted from herbal medicinal preparation by Adeleye et al. at Lagos, Nigeria [24]. The reason why the total bacterial count in our study was higher may be due to the primitive ways of preparation of the plant products, poor environmental sanitation, and storage conditions.

In the present study, a positive coliform count was obtained from 37 samples with a minimum count of 1.9 × 104 CFU/g and maximum count of 2.1 × 109 CFU/g with a mean count of 1.56 × 108 CFU/g or mL. All of the positive coliform counts were values above WHO tolerable limit [20]. In a study conducted by Khattak in Peshawar City, Pakistan, the coliform bacteria were detected and the counts ranged from 1.5 × 102 CFU/g to 1.6 × 104 CFU/g [8]. This high coliform count in the present study may be due to unhygienic practice and most of the herbalists work with their living house which may increase human drug contact.

Among the total 55 samples 23 (41.8%) and from the positive total coliform bacteria count samples 23 (62.2%) show fecal indicator organisms like Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, or Pseudomonas aeruginosa [7, 9]. Similar studies conducted in Nigeria and Kaduna Metropolis by Esimone et al. [9] and by Danladi et al. [7] showed the presence of fecal indicators organisms in herbs due to unsafe collection, transportation, drying, preparing, storing, and dispensing processes.

The present study showed bacterial isolates like Bacillus, Citrobacter, Clostridium, Enterobacter, Escherichia, Klebsiella, Pseudomonas, Salmonella, Shigella, Staphylococcus, Serratia, and Streptococcus. This report was similar to reports in Nigeria [12, 23, 25–27]. The finding of coliforms like Escherichia coli, Salmonella spp., and Shigella dysentery is very important public health concern that needs urgent need of management of herbal medicinal products to insure their safety and quality issue.

Multiple drug resistance was common in the present study; it was found that 131 (87.3%) of the isolates were resistant to ampicillin, 95 (63.3%) resistant to amoxicillin clavulanic acid (augmentin), 92 (61.3%) to amoxicillin, and 30 (57.7%) to penicillin. Adeleye et al., from Nigeria [24], reported that most of the isolates were resistant to ampicillin, penicillin, cotrimoxazole, and gentamicin. A study conducted in Saudi Arabia showed that bacterial isolates of Shigella spp., Enterobacter spp., Escherichia coli, Staphylococcus spp., and Klebsiella spp. were sensitive to amoxicillin and gentamicin [9]. But the present study showed high level of resistance to amoxicillin. This may be due to the overprescription of amoxicillin in our setup which may act as selective pressure for the growth of amoxicillin resistant strains by suppressing the sensitive strains.

5. Conclusion and Recommendation

The present study showed that herbal medicinal preparations sold in the study area were highly contaminated with pathogenic microorganisms with very high microbial load. More than 40% of the samples contain fecal indicator organisms. Multiple drug resistance was not uncommon and it was found that 125 (83.3%) of the isolates were resistant to 2 or more antibiotics tested.

Using those contaminated herbal medicines may have very high health risk due to their biological hazard. This warrants urgent need of management of herbalists or herbal medicinal products to scale up its quality and safety issue. Furthermore, establishing a quality and safety diagnostic service and competency certificate for safe drugs may provide quality and safety initiation between herbalists and good information for the customers.

(a)

Bacterial isolate	Total number	Pattern	AMPnumber (%)	AMLnumber (%)	AMCnumber (%)	SXTnumber (%)	CNnumber (%)	Fnumber (%)	TEnumber (%)	Cnumber (%)	CROnumber (%)	Nnumber (%)	CIPnumber (%)
Enterobacter cloacae	18	S	0 (0)	7 (38.8)	5 (27.7)	15 (83.3)	17 (94.4)	8 (44.4)	15 (83.3)	15 (83.3)	12 (66.6)	16 (88.8)	15 (83.3)
		R	18 (100)	11 (61.1)	13 (72.2)	3 (16.6)	1 (5.4)	10 (55.5)	3 (16.6)	3 (16.6)	6 (33.3)	2 (11.1)	3 (16.6)
Shigella dysenteriae	13	S	2 (15.4)	3 (23)	7 (53.8)	12 (92.3)	12 (92.3)	7 (53.8)	13 (100)	13 (100)	12 (92.3)	12 (92.3)	12 (92.3)
		R	11 (84.6)	10 (76.9)	6 (46.1)	1 (7.7)	1 (7.7)	6 (46.1)	0 (0)	0 (0)	1 (7.7)	1 (7.7)	1 (7.7)
Klebsiella pneumonia	11	S	0 (0)	2 (18.2)	5 (45.5)	8 (72.7)	10 (90.9)	6 (54.5)	9 (81.8)	10 (90.9)	7 (63.6)	11 (100)	10 (90.9)
		R	11 (100)	9 (81.8)	6 (54.5)	3 (27.3)	1 (9.1)	5 (45.5)	2 (18.2)	1 (9.1)	4 (36.4)	0 (0)	1 (9.1)
Escherichia coli	10	S	2 (20)	4 (40)	7 (70)	9 (90)	10 (100)	5 (50)	10 (100)	8 (80)	10 (100)	9 (90)	10 (100)
		R	8 (80)	6 (60)	3 (30)	1 (10)	0 (0)	5 (50)	0 (0)	2 (20)	0 (0)	1 (10)	0 (0)
Citrobacter diversus	9	S	1 (11.1)	7 (77.8)	6 (66.7)	9 (100)	9 (100)	5 (55.6)	9 (100)	9 (100)	8 (88.9)	9 (100)	8 (88.9)
		R	8 (88.9)	2 (22.2)	3 (33.3)	0 (0)	0 (0)	4 (44.4)	0 (0)	0 (0)	1 (11.1)	0 (0)	1 (11.1)
Providencia stuartii	8	S	0 (0)	6 (75)	1 (12.5)	8 (100)	8 (100)	1 (12.5)	7 (87.5)	8 (100)	6 (75)	7 (87.5)	7 (87.5)
		R	8 (100)	2 (25)	7 (87.5)	0 (0)	0 (0)	7 (87.5)	1 (12.5)	0 (0)	2 (75)	1 (12.5)	1 (12.5)
Serratia	5	S	3 (60)	4 (80)	4 (80)	5 (100)	5 (100)	3 (60)	4 (80)	5 (100)	5 (100)	5 (100)	5 (100)
		R	2 (40)	1 (20)	1 (20)	0 (0)	0 (0)	2 (40)	1 (20)	0 (0)	0 (0)	0 (0)	0 (0)
Klebsiella ozaenae	4	S	1 (25)	2 (50)	2 (50)	3 (75)	3 (75)	0 (0)	2 (50)	2 (50)	3 (75)	4 (100)	4 (100)
		R	3 (75)	2 (50)	2 (50)	1 (25)	1 (25)	4 (100)	2 (50)	2 (50)	1 (75)	0 (0)	0 (0)
Enterobacter aerogenes	4	S	0 (0)	2 (50)	1 (25)	3 (75)	4 (100)	1 (25)	3 (75)	4 (100)	2 (50)	3 (75)	3 (75)
		R	4 (100)	2 (50)	3 (75)	1 (25)	0 (0)	3 (75)	1 (25)	0 (0)	2 (50)	1 (25)	1 (25)
Providencia	4	S	1 (25)	1 (25)	1 (25)	4 (100)	4 (100)	1 (25)	4 (100)	3 (75)	3 (75)	3 (75)	3 (75)
		R	3 (75)	3 (75)	3 (75)	0 (0)	0 (0)	3 (75)	0 (0)	1 (25)	1 (25)	1 (25)	1 (25)
Acinetobacter	4	S	0 (0)	1 (25)	1 (25)	4 (100)	3 (75)	0 (0)	4 (100)	2 (50)	2 (50)	4 (100)	4 (100)
		R	4 (100)	3 (75)	3 (75)	0 (0)	1 (25)	4 (100)	0 (0)	2 (50)	2 (50)	0 (0)	0 (0)
Salmonella spp.	3	S	0 (0)	0 (0)	2 (66.7)	3 (100)	3 (100)	2 (66.7)	3 (100)	3 (100)	3 (100)	3 (100)	3 (100)
		R	3 (100)	3 (100)	1 (33.3)	0 (0)	0 (0)	1 (33.3)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Citrobacter	3	S	0 (0)	2 (66.7)	0 (0)	3 (100)	3 (100)	2 (66.7)	3 (100)	3 (100)	2 (66.7)	3 (100)	3 (100)
		R	3 (100)	1 (33.3)	3 (100)	0 (0)	0 (0)	1 (33.3)	0 (0)	0 (0)	1 (33.3)	0 (0)	0 (0)
Pseudomonas aeruginosa	2	S	0 (0)	0 (0)	0 (0)	2 (100)	1 (50)	0 (0)	1 (50)	2 (100)	1 (50)	2 (100)	2 (100)
		R	2 (100)	2 (100)	2 (100)	0 (0)	1 (50)	2 (100)	1 (50)	0 (0)	1 (50)	0 (0)	0 (0)

AMP: ampicillin, AML: amoxacillin, AMC: amoxicilin + clavulanic acid, SXT: cotrimoxazole, CN: gentamicin, F: nitrofurantoin, TE: tetracycline, C: chloramphenicol, CRO: ceftriaxone, N: norfloxacin, and CIP = ciprofloxacin.

(b)

Bacterial isolate	Total number	Pattern	AMP  N (%)	AML  N (%)	AMC  N (%)	SXT  N (%)	CN  N (%)	F  N (%)	TE  N (%)	C  N (%)	CRO  N (%)	N  N (%)	CIP  N (%)	CXC  N (%)	PEN  N (%)	ERY  N (%)	V   N (%)
Bacillus spp.	31	S	3 (10)	16 (52)	5 (26)	29 (93.5)	31 (100)	21 (68)	30 (97)	30 (97)	22 (71)	16 (94)	31 (100)	14 (45.2)	12 (38.7)	24 (77.4)	23 (74.2)
		R	28 (90)	15 (48)	23 (74)	2 (6.5)	0 (0)	10 (32)	1 (3.2)	1 (3.2)	9 (29)	2 (6.5)	0 (0)	17 (54.8)	19 (61.3)	7 (22.6)	8 (25.8)
Staphylococcus aureus	10	S	2 (20)	6 (60)	6 (60)	10 (100)	10 (100)	6 (60)	9 (90)	10 (100)	9 (90)	10 (100)	10 (100)	7 (70)	4 (40)	10 (100)	10 (100)
		R	8 (80)	4 (40)	4 (40)	0 (0)	0 (0)	4 (40)	1 (10)	0 (0)	1 (10)	0 (0)	0 (0)	3 (30)	6 (60)	0 (0)	0 (0)
Staphylococcus epidermidis	5	S	1 (20)	3 (60)	2 (40)	3 (60)	5 (100)	5 (100)	5 (100)	5 (100)	3 (60)	4 (80)	4 (80)	5 (100)	2 (40)	3 (60)	5 (100)
		R	4 (80)	2 (40)	3 (60)	2 (40)	0 (0)	0 (0)	0 (0)	0 (0)	2 (40)	1 (20)	1 (20)	0 (0)	3 (60)	2 (40)	0 (0)
Staphylococcus saprophyticus	2	S	0 (0)	2 (100)	2 (100)	2 (100)	2 (100)	1 (50)	2 (100)	2 (100)	1 (50)	2 (100)	2 (100)	0 (0)	2 (100)	2 (100)	2 (100)
		R	2 (100)	0 (0)	0 (0)	0 (0)	0 (0)	1 (50)	0 (0)	0 (0)	1 (50)	0 (0)	0 (0)	2 (100)	1 (50)	0 (0)	0 (0)
Streptococcus pyogenes	4	S	3 (75)	3 (75)	4 (100)	3 (75)	3 (75)	3 (75)	4 (100)	4 (100)	2 (50)	3 (75)	3 (75)	3 (75)	1 (50)	0 (0)	4 (100)
		R	1 (25)	1 (25)	0 (0)	1 (25)	1 (25)	1 (25)	0 (0)	0 (0)	2 (50)	1 (25)	1 (25)	1 (25)	1 (25)	0 (0)	0 (0)

AMP: ampicillin, CAF: chloramphenicol, N: norfloxacin, AML: amoxacillin, CIP: ciprofloxacin, SXT: cotrimoxazole, CRO: ceftriaxone, CN: gentamicin, TTC: tetracycline, AMC: amoxicilin + clavulanic acid, CXC: cloxacillin, PEN: penicillin, ERY: erythromycin, and V: vancomycin.