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

Authors: Abdela Yesuf; Yitayih Wondimeneh; Teklay Gebrecherkos; Feleke Moges
Journal: International Journal of Bacteriology
 (2016)

---

## Abstract

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 wereBacillus spp. followed byEnterobacter spp.,Shigella dysenteriae, andSalmonella 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.


---

## Body

## 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 wereEscherichia coli (ATCC 25922) andStaphylococcus 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 andP 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.


## 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 wereEscherichia coli (ATCC 25922) andStaphylococcus 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 andP 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 (Table2). 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 asEscherichia coli,Staphylococcus aureus, andPseudomonas aeruginosa. The organism most commonly isolated from the herbal medicines wasBacillus 31 (20.7%) followed byEnterobacter 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
Bacillusspp. 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, Bacillusspp., P. aeuroginosa
08B
Powder: evil eye
Myrica salicifolia
E. coli, Bacillusspp.
Providencia
01D
Powder: anti-heliments & sinus
Cucurbita maxima
S. aureus, S. dysenteriae, Providencia stwarti
09A
Liquid: antiwound & fungal infection
Plumbago zeylanicum
Bacillusspp.&Enterobacter cloacae
01E
Powder: antiwound infection
Plumbago zeylanicum
Bacillusspp.
09B
Powder: antiabdominal problem
ND
Citrobacter diversus, E. coli, S. dysenteriae, Bacillusspp.
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, Bacillusspp.
01H
Powder: effective against warts & bleeding
Calotropis procera
Bacillusspp.
11A
Powder: antigastric
ND
Bacillusspp., S. aureus, Pseudomonas aeruginosa
01I
Liquid: antiotitis media
Withania somnifera
Salmonellaspp., 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, Bacillusspp.
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, Bacillusspp., 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, Bacillusspp.
03B
Powder: antiasthma
Rumex abyssinicus
Bacillusspp., S. aureus, P. aurognosa
Acinetobacterspp.
13C
Powder: effective against liver disease
Thymus schimperi
Bacillusspp., S. epidermidis
03C
Powder: antiscar
ND
Bacillusspp.
13D
Powder: rabbis virus
ND
S. saprophyticus&Bacillusspp.
03D
Liquid: antifungal
Dodonaea amgustifolica
Serratia
14A
Powder: antiepilepsy
ND
S. aureus, Bacillusspp., K. pneumoniae, Citrobacter diversus
03E
Powder: antifungal & skin disease
Brucea antidysenterica
Providencia stuartii, E. coli, Serratia, Salmonellaspp., S. dysenteriae
14B
Powder: anticancer
Plumbago zeylanicum & ND
Serratia, E. coli, S. dysenteriae, Bacillusspp.
04A
Powder: antiwart
Cucumis prophetarum
Bacillusspp.
15A
Powder: antiparalysis
ND
E. coli, K. pneumonia, Enterobacter aerogenes, Salmonellaspp., S. epidermidis
05A
Powder: antiwart
Plumbago zeylanicum
E. coli, S. dysenteriae, Enterobacter
16A
Liquid: antidiarrheal
ND
S. pyogenes &Bacillusspp.
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
Bacillusspp.
16E
Powder: antisinus & nose asthma
Myrica salicifolia
S. epidermidis, Bacillusspp.
19B
Liquid: antiepilepsy
ND
Bacillusspp.
16F
Liquid: antiwound infection
Clerodendrum myricoides
Bacillusspp.
Enterobacter cloacae
19C
Powder: antimalaria intestinal parasites & cancer
Croton macrostachyus
S. dysenteriae, K. pneumonia
16G
Powder: antiwound infection
Clerodendrum myricoides
S. dysenteriae, Bacillusspp.
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, Bacillusspp.
20B
Powder: antidiabetes & MTB
ND
E. coli, Bacillusspp.
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
FrequencyN (%)
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)
Providenciaspp.
12 (8.0)
Citrobacterspp.
12 (8.0)
Serratiaspp.
5 (3.3)
Acinetobacter spp.
4 (2.7)
Salmonellaspp.
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 Tables4(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 4
(a) Antimicrobial susceptibility pattern of gram negative bacterial isolates from herbal medicinal products in Gondar Town, Northwest Ethiopia, from April 1 to May 25, 2013. (b) Antimicrobial susceptibility pattern of gram positive bacterial isolates from herbal medicinal products in Gondar Town, Northwest Ethiopia, from April 1 to May 25, 2013.
(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 (%)
Bacillusspp.
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.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 9/31Bacillus 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 isolatesPseudomonas aeruginosa were found to be resistant to at least 5 antibiotics tested while half of the isolates ofKlebsiella ozaenae, Providencia spp., andStreptococcus 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 patternsN (%)
R
0
R
1
R
2
R
3
R
4
≥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)
Citrobacterspp.
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)
Salmonellaspp.
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)
Bacillusspp.
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),R
0: no antibiotic resistance, R
1: resistance to one, R
2: resistance to two, R
3: resistance to three, R
4: resistance to four, and ≥R5: resistance to five and more drugs.


## 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 (Table2). 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 asEscherichia coli,Staphylococcus aureus, andPseudomonas aeruginosa. The organism most commonly isolated from the herbal medicines wasBacillus 31 (20.7%) followed byEnterobacter 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
Bacillusspp. 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, Bacillusspp., P. aeuroginosa
08B
Powder: evil eye
Myrica salicifolia
E. coli, Bacillusspp.
Providencia
01D
Powder: anti-heliments & sinus
Cucurbita maxima
S. aureus, S. dysenteriae, Providencia stwarti
09A
Liquid: antiwound & fungal infection
Plumbago zeylanicum
Bacillusspp.&Enterobacter cloacae
01E
Powder: antiwound infection
Plumbago zeylanicum
Bacillusspp.
09B
Powder: antiabdominal problem
ND
Citrobacter diversus, E. coli, S. dysenteriae, Bacillusspp.
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, Bacillusspp.
01H
Powder: effective against warts & bleeding
Calotropis procera
Bacillusspp.
11A
Powder: antigastric
ND
Bacillusspp., S. aureus, Pseudomonas aeruginosa
01I
Liquid: antiotitis media
Withania somnifera
Salmonellaspp., 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, Bacillusspp.
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, Bacillusspp., 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, Bacillusspp.
03B
Powder: antiasthma
Rumex abyssinicus
Bacillusspp., S. aureus, P. aurognosa
Acinetobacterspp.
13C
Powder: effective against liver disease
Thymus schimperi
Bacillusspp., S. epidermidis
03C
Powder: antiscar
ND
Bacillusspp.
13D
Powder: rabbis virus
ND
S. saprophyticus&Bacillusspp.
03D
Liquid: antifungal
Dodonaea amgustifolica
Serratia
14A
Powder: antiepilepsy
ND
S. aureus, Bacillusspp., K. pneumoniae, Citrobacter diversus
03E
Powder: antifungal & skin disease
Brucea antidysenterica
Providencia stuartii, E. coli, Serratia, Salmonellaspp., S. dysenteriae
14B
Powder: anticancer
Plumbago zeylanicum & ND
Serratia, E. coli, S. dysenteriae, Bacillusspp.
04A
Powder: antiwart
Cucumis prophetarum
Bacillusspp.
15A
Powder: antiparalysis
ND
E. coli, K. pneumonia, Enterobacter aerogenes, Salmonellaspp., S. epidermidis
05A
Powder: antiwart
Plumbago zeylanicum
E. coli, S. dysenteriae, Enterobacter
16A
Liquid: antidiarrheal
ND
S. pyogenes &Bacillusspp.
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
Bacillusspp.
16E
Powder: antisinus & nose asthma
Myrica salicifolia
S. epidermidis, Bacillusspp.
19B
Liquid: antiepilepsy
ND
Bacillusspp.
16F
Liquid: antiwound infection
Clerodendrum myricoides
Bacillusspp.
Enterobacter cloacae
19C
Powder: antimalaria intestinal parasites & cancer
Croton macrostachyus
S. dysenteriae, K. pneumonia
16G
Powder: antiwound infection
Clerodendrum myricoides
S. dysenteriae, Bacillusspp.
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, Bacillusspp.
20B
Powder: antidiabetes & MTB
ND
E. coli, Bacillusspp.
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
FrequencyN (%)
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)
Providenciaspp.
12 (8.0)
Citrobacterspp.
12 (8.0)
Serratiaspp.
5 (3.3)
Acinetobacter spp.
4 (2.7)
Salmonellaspp.
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 Tables4(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 4
(a) Antimicrobial susceptibility pattern of gram negative bacterial isolates from herbal medicinal products in Gondar Town, Northwest Ethiopia, from April 1 to May 25, 2013. (b) Antimicrobial susceptibility pattern of gram positive bacterial isolates from herbal medicinal products in Gondar Town, Northwest Ethiopia, from April 1 to May 25, 2013.
(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 (%)
Bacillusspp.
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.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 9/31Bacillus 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 isolatesPseudomonas aeruginosa were found to be resistant to at least 5 antibiotics tested while half of the isolates ofKlebsiella ozaenae, Providencia spp., andStreptococcus 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 patternsN (%)
R
0
R
1
R
2
R
3
R
4
≥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)
Citrobacterspp.
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)
Salmonellaspp.
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)
Bacillusspp.
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),R
0: no antibiotic resistance, R
1: resistance to one, R
2: resistance to two, R
3: resistance to three, R
4: 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 likeEscherichia coli,Salmonella,Shigella,Staphylococcus aureus, orPseudomonas 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 likeBacillus,Citrobacter,Clostridium,Enterobacter,Escherichia,Klebsiella,Pseudomonas,Salmonella,Shigella,Staphylococcus,Serratia, andStreptococcus. This report was similar to reports in Nigeria [12, 23, 25–27]. The finding of coliforms likeEscherichia coli,Salmonella spp., andShigella 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 ofShigella spp.,Enterobacter spp.,Escherichia coli,Staphylococcus spp., andKlebsiella 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.



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*Source: 1959418-2016-05-19.xml*