# Microbial Load and Antibiotic Resistance ofEscherichia coli and Staphylococcus aureus Isolated from Ready-to-Eat (RTE) Khebab Sold on a University Campus and Its Environs in Ghana

Authors: Azumah Ayamah; Augustina Angelina Sylverken; Linda Aurelia Ofori
Journal: Journal of Food Quality
 (2021)

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## Abstract

The demand for ready-to-eat (RTE) foods is handy to busy urban dwellers. Handling, processing, and selling are known to contaminate these foods and cause foodborne outbreaks. This study assessed a load ofS. aureus and E. coli in khebabs (beef, chevon, and gizzard) sold on the KNUST campus and its environs and how resistant they are to clinically relevant antimicrobial agents. Thirty-six (36) khebab samples were purchased from vendors at Kotei, Ayeduase, Kentinkrono, Boadi, KNUST campus, and Ayigya. They were analyzed for S. aureus and E. coli and their resistance to clinically relevant antimicrobial agents checked using standard methods. S. aureus and E. coli load ranged from 4.09 to 5.96 CFU/g and 1.79 to 6.12 MPN/g in beef, 4.02 to 6.01 CFU/g and 1.99 to 4.44 MPN/g in chevon, and 5.37 to 6.18 CFU/g and 1.79 to 6.10 MPN/g in gizzard khebabs in the different locations. E. coli (n = 27) were multiresistant to ampicillin, tetracycline, gentamicin, cefuroxime, ceftriaxone, cefotaxime, and cotrimoxazole (51.85%) and susceptible to chloramphenicol (100%). S. aureus (n = 36) isolates were multiresistant to penicillin, tetracycline, flucloxacillin, cefuroxime, ampicillin (97.22%), erythromycin (75%), cotrimoxazole (86.11%), and gentamicin (69.44%). It can therefore be concluded that the majority of khebabs from the KNUST campus and its environs were contaminated with S. aureus and E. coli above the acceptable standard limits (≤4 log10CFU/g and ˂2 log10MPN/g, respectively). Also, the S. aureus and E. coli isolated were multiresistant to the antibiotics tested and could be a medium for the transmission of antibiotic-resistant bacteria and therefore expose consumers to a high risk of contracting foodborne infections with drug-resistant strains.


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## Body

## 1. Introduction

Ready-to-eat (RTE) foods have been defined as foods that do not require further preparation with exception of reheating [1]. These RTE foods are usually consumed raw or cooked, or hot or cold without further heat treatment [2, 3]. Khebab is a popular ready-to-eat street-vended meat in Ghana, which is easily accessible to most Ghanaians. The khebab is usually prepared using meat from cow, goat, sheep, chicken, etc. The khebab may contain high protein or carbohydrate, high moisture content, or low acidity, which provides a suitable medium for the rapid growth of microorganism, therefore making it potentially hazardous food. Because it provides a medium for rapid microorganism’s growth, microbial pathogens or toxins can affect consumer’s health and may even lead to death when they are contaminated [4].S. aureus and E. coli have been found to contaminate ready-to-eat foods [5–9]. They have the ability to survive on hands and other surfaces and are therefore transferred to foods [10]. Feglo and Sakyi [11] recorded different levels of S. aureus, E. coli, and other bacteria in different ready-to-eat foods in the Kumasi metropolis of Ghana. S. aureus is a major foodborne pathogen in ready-to-eat foods responsible for foodborne infections across the globe [12, 13]. It grows rapidly under room temperature and produces toxins that cause the infections. Every year, about 241,000 foodborne diseases of S. aureus are reported in the United States [14, 15]. Also, China recorded 12.5% foodborne diseases associated with S. aureus in 2013 [14]. E. coli on the other hand is one of the most dangerous foodborne pathogens [16, 17]. It produces one or more types of cytotoxins such as shiga toxin (Stx) or vero-cytotoxin (VT). Those producing Stx and VT are referred to as shiga toxin-producing E. coli (STEC) and vero-cytoxigenic E. coli (VTEC), respectively [18]. According to Monaghan et al. [19], STEC is one of the most important foodborne pathogens to emerge within the past two decades. It is a frequent cause of foodborne diarrhea outbreak [20–22]. In North America, Europe, Asia, and Africa, E. coli, particularly serotype O157 : H7, is an important food-borne pathogen responsible for gastroenteritis epidemics [23, 24]. In 2011, the CDC reported that shiga toxin-producing Escherichia coli (STEC) O157 infections cause more than 63,000 illnesses, 2,100 hospitalizations, and 20 deaths in the United States [15]. In the world, STEC O157 is the most common serotype associated with human gastrointestinal illness and ranks third in the cause of bacterial foodborne-related hospitalizations in the United States [15, 25]. Individuals infected with STEC usually develop hemolytic uremic syndrome, hemolytic anemia, and thrombocytopenia that can be fatal [26].The increased contaminations of RTE foods in the society could be linked to the increased mobility of people due to urbanization, larger number of itinerant workers, and less home activities have resulted in large percentage of the population depending on RTE foods for employment and food. This situation, however, resulted that food sanitary measures and proper handling have been transferred from individual and families to the food vendors who rarely enforce such practices [27, 28]. Majority of these vendors are unaware of improper handling of food and their role in the transmission of infections [29, 30]. Most RTE foods are also sold under insanitary conditions that make them prone to contamination [4, 31–33]. Additionally, most RTE foods are sold openly, with little or no covering, and are therefore easily contaminated by flies and dust in the environment [34].Besides foodborne contamination, the emergence of antimicrobial-resistant bacteria is a great challenge to public health. The indiscriminate uses of antimicrobials in animals and humans have led to the development of antimicrobial-resistant microorganisms [35]. In recent times, more and more multidrug-resistant (MDR) S. aureus food poisoning outbreaks have been reported [36–39]. There are limited data on antimicrobial resistance of S. aureus and E. coli foods in Ghana. It is with this background that this study was conducted to assess the S. aureus and E. coli loads in khebabs sold on the KNUST campus and its environs and how resistant they are to clinically relevant antimicrobial agents.


## 2. Materials and Methods

### 2.1. Study Design

A cross-sectional study was conducted in 2018 to assess theS. aureus and E. coli loads in khebabs sold on the KNUST campus and its environs, and how resistant they are to clinically relevant antimicrobial agents [40].


### 2.2. Study Location

The study was conducted in the KNUST campus and its environs in the Oforikrom Municipal Assembly, Kumasi, Ghana (Figure1). According to Ababio and Adi [41], the KNUST campus and its environs are one of the communities in Kumasi with high population of food vendors because of the socioeconomic activities that take place in this area. Majority of students, staff, and indigenous people residing on the KNUST campus and its environs patronize these ready-to-eat foods.Figure 1
Map showing sampling locations.


### 2.3. Collection of Samples

Samples of khebab made from beef, chevon, and gizzard were purchased from two vendors in each location. A purposive sampling technique was employed in the selection of the vendors. About one hundred grams of the khebabs were purchased from each vendor into a sterile Ziploc bag. The purchased khebab samples were kept in an ice chest with ice packs and transported immediately to the laboratory and stored at 4°C and analyzed the following day [40].


## 2.1. Study Design

A cross-sectional study was conducted in 2018 to assess theS. aureus and E. coli loads in khebabs sold on the KNUST campus and its environs, and how resistant they are to clinically relevant antimicrobial agents [40].


## 2.2. Study Location

The study was conducted in the KNUST campus and its environs in the Oforikrom Municipal Assembly, Kumasi, Ghana (Figure1). According to Ababio and Adi [41], the KNUST campus and its environs are one of the communities in Kumasi with high population of food vendors because of the socioeconomic activities that take place in this area. Majority of students, staff, and indigenous people residing on the KNUST campus and its environs patronize these ready-to-eat foods.Figure 1
Map showing sampling locations.


## 2.3. Collection of Samples

Samples of khebab made from beef, chevon, and gizzard were purchased from two vendors in each location. A purposive sampling technique was employed in the selection of the vendors. About one hundred grams of the khebabs were purchased from each vendor into a sterile Ziploc bag. The purchased khebab samples were kept in an ice chest with ice packs and transported immediately to the laboratory and stored at 4°C and analyzed the following day [40].


## 3. Microbiological Analysis

### 3.1. Sample Analysis

Serial dilutions of 10−1 to 10−5 of the sample were carried out by placing ten grams of each khebab sample into ninety milliliters of sterilized buffered peptone water (Oxoid CM 0009; Oxoid Ltd Basingstoke, Hampshire, England) and pulsified for 15 seconds [40].


### 3.2. Total Viable Count (TVC)

Pour plate technique was employed to estimate the TVC ofS. aureus using mannitol salt agar. Aliquot of 1 ml from each of the dilutions for the khebab samples was placed into labeled petri dishes, and about 10 ml of molten (45°C) mannitol salt agar (Oxoid CM 0085; Oxoid Ltd Basingstoke, Hampshire, England) was poured into the labeled petri dishes. The plates were swirled slowly for uniform mixing, allowed to solidify, sealed with parafilm, and then incubated at 35°C for 24 hours. Colonies with yellow and pink zones observed indicated the presence of S. aureus, which were counted using colony counter (Stuart colony counter, UK). Colonies between 30 and 300 were used in their estimation by using the following formula:(1)CFU=average colonies×dilution factoraliquot platedand expressed as CFU/g.


### 3.3. Isolation ofS. aureus

Yellow and pink zone colonies on the mannitol salt agar were isolated onto nutrient agar as presumptiveS. aureus [42].


### 3.4. Enumeration, Isolation, and Characterization ofEscherichia coli

The most probable number (MPN) method was employed to enumerate the totalE. coli in the khebab samples. One milliliter aliquot from each of the dilutions was inoculated into a 5 ml sterile MacConkey Broth (Oxoid CM 0085; Oxoid Ltd Basingstoke, Hampshire, England) and incubated at 44°C for 24 hours. Tubes showing colour change from purple to yellow after incubation were identified as presumptive positive for thermotolerant coliforms. From each of the positive tubes, 1 ml was transferred into a test tube containing 5 ml of sterile tryptophan broth (Scharlau 02-418; Scharlau chemie S.A, Barcelona, Spain) and incubated at 44°C for 24 hours. A drop of Kovacs’ reagent was added to the overnight (ON) culture tubes of tryptophan broth. All tubes showing a red ring development after gentle agitation denoted the presence of indole and were recorded as presumptive positive for thermotolerant coliforms (E. coli). Counts per gram were calculated from the most probable number (MPN) tables.The tubes that showed ring colour development were further streaked on eosin methylene blue (EMB) agar media (Oxoid CM 0069; Oxoid Ltd Basingstoke, Hampshire, England) and incubated at 37°C for 24 hours and colonies with green metallic sheen indicated E. coli. The colonies were then subcultured into nutrient agar to obtain pure isolates.Bacteria were characterized using an API 20E Biochemical Test Strip following manufacturer’s manual (bioMérieux, France). All codes obtained were referenced to the API database for organism identification.


### 3.5. Antimicrobial Susceptibility ofE. coli and S. aureus

Antimicrobial susceptibility ofE. coli and S. aureus was determined by the Kirby Bauer agar disk diffusion method on Mueller–Hinton agar according to the protocol and guidelines of the European Committee on Antibiotic Susceptibility Testing [43].Broth cultures of the isolates and Muller–Hinton agar (Oxoid CM 0337; Oxoid Ltd Basingstoke, Hants, United Kingdom) plates were prepared. The Muller–Hinton agar plates were streaked with the broth cultures with the help of a sterile swap and allowed to dry for 2–5 minutes. Sterile forceps were used to aseptically pick each of the antibiotic disks (Abtek Biologicals Ltd) and placed on each plate. The plates were labeled and incubated at 37°C for 24 hrs. The zone of inhibition (ZOI) was measured using a ruler, and data were recorded and interpreted according to the EUCAST breakpoints [44].


### 3.6. Statistical Analysis

The data collected were subjected to the analysis of variance (ANOVA) using Genstat statistical software version 12. Significant differences were assessed at 5% level of significance (p = 0.05). Where there was a significant difference, means were separated using the Fishers protected least significant difference (LSD) procedure. The data were logarithmically (log10) transformed to minimize the variations associated with the enumeration techniques.


## 3.1. Sample Analysis

Serial dilutions of 10−1 to 10−5 of the sample were carried out by placing ten grams of each khebab sample into ninety milliliters of sterilized buffered peptone water (Oxoid CM 0009; Oxoid Ltd Basingstoke, Hampshire, England) and pulsified for 15 seconds [40].


## 3.2. Total Viable Count (TVC)

Pour plate technique was employed to estimate the TVC ofS. aureus using mannitol salt agar. Aliquot of 1 ml from each of the dilutions for the khebab samples was placed into labeled petri dishes, and about 10 ml of molten (45°C) mannitol salt agar (Oxoid CM 0085; Oxoid Ltd Basingstoke, Hampshire, England) was poured into the labeled petri dishes. The plates were swirled slowly for uniform mixing, allowed to solidify, sealed with parafilm, and then incubated at 35°C for 24 hours. Colonies with yellow and pink zones observed indicated the presence of S. aureus, which were counted using colony counter (Stuart colony counter, UK). Colonies between 30 and 300 were used in their estimation by using the following formula:(1)CFU=average colonies×dilution factoraliquot platedand expressed as CFU/g.


## 3.3. Isolation ofS. aureus

Yellow and pink zone colonies on the mannitol salt agar were isolated onto nutrient agar as presumptiveS. aureus [42].


## 3.4. Enumeration, Isolation, and Characterization ofEscherichia coli

The most probable number (MPN) method was employed to enumerate the totalE. coli in the khebab samples. One milliliter aliquot from each of the dilutions was inoculated into a 5 ml sterile MacConkey Broth (Oxoid CM 0085; Oxoid Ltd Basingstoke, Hampshire, England) and incubated at 44°C for 24 hours. Tubes showing colour change from purple to yellow after incubation were identified as presumptive positive for thermotolerant coliforms. From each of the positive tubes, 1 ml was transferred into a test tube containing 5 ml of sterile tryptophan broth (Scharlau 02-418; Scharlau chemie S.A, Barcelona, Spain) and incubated at 44°C for 24 hours. A drop of Kovacs’ reagent was added to the overnight (ON) culture tubes of tryptophan broth. All tubes showing a red ring development after gentle agitation denoted the presence of indole and were recorded as presumptive positive for thermotolerant coliforms (E. coli). Counts per gram were calculated from the most probable number (MPN) tables.The tubes that showed ring colour development were further streaked on eosin methylene blue (EMB) agar media (Oxoid CM 0069; Oxoid Ltd Basingstoke, Hampshire, England) and incubated at 37°C for 24 hours and colonies with green metallic sheen indicated E. coli. The colonies were then subcultured into nutrient agar to obtain pure isolates.Bacteria were characterized using an API 20E Biochemical Test Strip following manufacturer’s manual (bioMérieux, France). All codes obtained were referenced to the API database for organism identification.


## 3.5. Antimicrobial Susceptibility ofE. coli and S. aureus

Antimicrobial susceptibility ofE. coli and S. aureus was determined by the Kirby Bauer agar disk diffusion method on Mueller–Hinton agar according to the protocol and guidelines of the European Committee on Antibiotic Susceptibility Testing [43].Broth cultures of the isolates and Muller–Hinton agar (Oxoid CM 0337; Oxoid Ltd Basingstoke, Hants, United Kingdom) plates were prepared. The Muller–Hinton agar plates were streaked with the broth cultures with the help of a sterile swap and allowed to dry for 2–5 minutes. Sterile forceps were used to aseptically pick each of the antibiotic disks (Abtek Biologicals Ltd) and placed on each plate. The plates were labeled and incubated at 37°C for 24 hrs. The zone of inhibition (ZOI) was measured using a ruler, and data were recorded and interpreted according to the EUCAST breakpoints [44].


## 3.6. Statistical Analysis

The data collected were subjected to the analysis of variance (ANOVA) using Genstat statistical software version 12. Significant differences were assessed at 5% level of significance (p = 0.05). Where there was a significant difference, means were separated using the Fishers protected least significant difference (LSD) procedure. The data were logarithmically (log10) transformed to minimize the variations associated with the enumeration techniques.


## 4. Results

### 4.1. Mean Load ofE. coli and S. aureus in Beef Khebab

The beef khebab from the different locations recorded varying mean loads ofE. coli ranging from 0.00 to 9.30 × 106 MPN/g with those from Ayigya recording the highest load (6.12 log10 MPN/g) and Boadi the lowest (1.79 log10 MPN/g) (Table 1). E. coli load was significantly different (p = 0.019) and was above the Food Standards of Australia New Zealand (FSANZ) guidelines of ˂100 MPN/g (˂2 log10 MPN/g). Nonetheless, the E. coli load in beef at Kentinkrono and Boadi was within the acceptable limit.Table 1
Mean loads ofE. coli and S. aureus in beef khebab sold on the KNUST campus and its environs.
LocationRange ofE. coli load (MPN/g)Mean load ofE. coli (log10 MPN/g)Range ofS. aureus load (CFU/g)Mean load ofS. aureus (log10 CFU/g)Kotei0.00–9.30 × 1053.77 ± 2.94ab3.10 × 102–1.44 × 1064.09 ± 1.47Ayeduase9.00 × 104–2.10 × 1065.61 ± 0.57b6.40 × 103–2.96 × 1075.12 ± 1.81Kentinkrono0.00–9.30 × 1051.99 ± 3.08a8.80 × 102–9.60 × 1065.46 ± 1.52Boadi0.00–2.30 × 1051.79 ± 2.77a3.70 × 103–2.94 × 1065.43 ± 1.40KNUST campus0.00–2.40 × 1063.91 ± 3.06ab6.40 × 102–4.40 × 1064.80 ± 1.56Ayigya2.30 × 105–9.30 × 1066.12 ± 0.60b4.40 × 105–1.94 × 1065.96 ± 0.24CV (%)63.227.8p value0.0190.323MPN = most probable number; CFU = colony-forming unit. Mean loads with the same letters are not significantly different (p > 0.05).The mean load ofS. aureus ranged from 3.10 × 102 to 2.96 × 107 CFU/g with no significant difference among them. Samples from Ayigya recorded the highest load of 5.96 log10 CFU/g, whereas those from Kotei recorded the least load of 4.09 log10 CFU/g (Table 1). S. aureus load was, however, above the FSANZ acceptable limit of ≤104 CFU/g (≤4 log CFU/g) for safe foods.


### 4.2. Mean Load ofE. coli and S. aureus in Chevon Khebab

E. coli load in chevon khebab ranged from 0.00 to 9.30 × 106 MPN/g with Ayeduase recording the highest mean E. coli load of 4.44 log10 MPN/g and Boadi the least (1.99 log MPN/g) (Table 2). The differences were not significant (p= 0.779) but were above the FSANZ guidelines of ˂100 MPN/g (˂2 log10 MPN/g) with the exception of Boadi, which was within the guidelines limit. Staphylococcus aureus ranged from 3.60 × 103 to 2.88 × 107 CFU/g in chevon khebab irrespective of the location. Samples from Kentinkrono recorded the highest mean load of 6.53 log10 CFU/g, whereas those from Kotei the least (4.02 log10 CFU/g) (Table 2). The mean loads of S. aureus were significantly different (p = 0.013) and above the FSANZ acceptable limit of ≤104 CFU/g (≤4 log CFU/g).Table 2
Mean loads ofE. coli and S. aureus in chevon khebab sold on the KNUST campus and its environs.
LocationRange ofE. coli load (MPN/g)Mean load ofE. coli (log10 MPN/g)Range ofS. aureus load (CFU/g)Mean load ofS. aureus (log10 CFU/g)Kotei0.00–2.40 × 1063.91 ± 3.063.60 × 103-3.70 × 1044.02 ± 0.39aAyeduase0.00–9.30 × 1064.44 ± 3.452.40 × 104-2.88 × 1076.01 ± 1.31bcKentinkrono0.00–9.30 × 1064.43 ± 3.441.80 × 105-1.64 × 1076.53 ± 0.71cBoadi0.00–9.30 × 1051.99 ± 3.083.60 × 103-1.40 × 1064.82 ± 1.13abKNUST campus0.00–4.30 × 1063.99 ± 3.144.50 × 103-1.28 × 1075.71 ± 1.62bcAyigya0.00–2.40 × 1064.25 ± 3.294.60 × 103-1.52 × 1075.81 ± 1.49bcCV (%)84.721.7p value0.7790.013MPN = most probable number; CFU = colony-forming unit. Mean loads with same letters are not significantly different (p > 0.05).


### 4.3. Mean Load ofE. coli and S. aureus in Gizzard Khebab

For the gizzard khebab, the mean load ofE. coli for the locations ranged from 0.00 to 2.90 × 107 MPN/g with Kotei recording the highest mean load (6.10 log10 MPN/g) and the least observed in Boadi (1.79 log10 MPN/g). Although the mean load of E. coli in the gizzard khebab from the various locations was not significantly different (p = 0.207), it was above the FSANZ guidelines of ˂ 100 MPN/g (˂2 log10 MPN/g), with the exception of Boadi, which had a mean E. coli load within the acceptable limit. S. aureus load ranged from 4.20 × 103 to 1.64 × 107 CFU/g. Samples from Kentinkrono recorded the highest mean load (6.18 log CFU/g), whereas those from Kotei recorded the least (5.37 log10 CFU/g) (Table 3). The mean load differences of S. aureus were statistically not significant (p = 0.788); however, they were above the FSANZ acceptable standard limit of ≤ 104 CFU/g (≤4 log10 CFU/g).Table 3
Mean load ofE. coli and S. aureus in gizzard khebab sold on the KNUST campus and its environs.
LocationRange ofE. coli load (MPN/g)Mean load ofE. coli (log10 MPN/g)Range (CFU/g) ofS. aureus loadMean load ofS. aureus (log10 CFU/g)Kotei2.30×105-4.30 × 1066.10 ± 0.521.52 × 104-3.50 × 1065.37 ± 0.98Ayeduase0.00–2.40 × 1063.85 ± 3.034.20 × 103-2.96 × 1065.43 ± 1.36Kentinkrono0.00–2.90 × 1072.94 ± 3.853.00 × 105-1.64 × 1076.18 ± 0.81Boadi0.00–2.40 × 1051.79 ± 2.781.24 × 105-1.76 × 1065.80 ± 0.54KNUST campus0.00–1.50 × 1064.05 ± 3.141.48 × 105-9.60 × 1065.82 ± 0.65Ayigya0.00–9.30 × 1064.38 ± 3.417.40 × 103-1.64 × 1075.69 ± 1.50CV (%)79.118.1p value0.2070.788MPN = most probable number; CFU = colony-forming unit.


### 4.4. Antimicrobial Susceptibility ofE. coli and S. aureus from Khebab on KNUST Campus and Its Environs

#### 4.4.1.E. coli

Based on the British Society for Antimicrobial Chemotherapy (BSAC) Methods for Antimicrobial Susceptibility Testing, version 14, May 2015,E. coli (n = 27) were all resistant to the antibiotics tested with the exception of chloramphenicol, which was susceptible (Figure 2).Figure 2
A graph showing percentage resistance and susceptibility ofE. coli.


#### 4.4.2.S. aureus

S. aureus (n = 36) was resistant to the antibiotics tested (ampicillin, tetracycline, gentamicin, cefuroxime, ceftriaxone and cefotaxime = 100%, cotrimoxazole = 52%) with the exception of few which were susceptible (chloramphenicole = 100% and cotrimoxazole = 48%).


## 4.1. Mean Load ofE. coli and S. aureus in Beef Khebab

The beef khebab from the different locations recorded varying mean loads ofE. coli ranging from 0.00 to 9.30 × 106 MPN/g with those from Ayigya recording the highest load (6.12 log10 MPN/g) and Boadi the lowest (1.79 log10 MPN/g) (Table 1). E. coli load was significantly different (p = 0.019) and was above the Food Standards of Australia New Zealand (FSANZ) guidelines of ˂100 MPN/g (˂2 log10 MPN/g). Nonetheless, the E. coli load in beef at Kentinkrono and Boadi was within the acceptable limit.Table 1
Mean loads ofE. coli and S. aureus in beef khebab sold on the KNUST campus and its environs.
LocationRange ofE. coli load (MPN/g)Mean load ofE. coli (log10 MPN/g)Range ofS. aureus load (CFU/g)Mean load ofS. aureus (log10 CFU/g)Kotei0.00–9.30 × 1053.77 ± 2.94ab3.10 × 102–1.44 × 1064.09 ± 1.47Ayeduase9.00 × 104–2.10 × 1065.61 ± 0.57b6.40 × 103–2.96 × 1075.12 ± 1.81Kentinkrono0.00–9.30 × 1051.99 ± 3.08a8.80 × 102–9.60 × 1065.46 ± 1.52Boadi0.00–2.30 × 1051.79 ± 2.77a3.70 × 103–2.94 × 1065.43 ± 1.40KNUST campus0.00–2.40 × 1063.91 ± 3.06ab6.40 × 102–4.40 × 1064.80 ± 1.56Ayigya2.30 × 105–9.30 × 1066.12 ± 0.60b4.40 × 105–1.94 × 1065.96 ± 0.24CV (%)63.227.8p value0.0190.323MPN = most probable number; CFU = colony-forming unit. Mean loads with the same letters are not significantly different (p > 0.05).The mean load ofS. aureus ranged from 3.10 × 102 to 2.96 × 107 CFU/g with no significant difference among them. Samples from Ayigya recorded the highest load of 5.96 log10 CFU/g, whereas those from Kotei recorded the least load of 4.09 log10 CFU/g (Table 1). S. aureus load was, however, above the FSANZ acceptable limit of ≤104 CFU/g (≤4 log CFU/g) for safe foods.


## 4.2. Mean Load ofE. coli and S. aureus in Chevon Khebab

E. coli load in chevon khebab ranged from 0.00 to 9.30 × 106 MPN/g with Ayeduase recording the highest mean E. coli load of 4.44 log10 MPN/g and Boadi the least (1.99 log MPN/g) (Table 2). The differences were not significant (p= 0.779) but were above the FSANZ guidelines of ˂100 MPN/g (˂2 log10 MPN/g) with the exception of Boadi, which was within the guidelines limit. Staphylococcus aureus ranged from 3.60 × 103 to 2.88 × 107 CFU/g in chevon khebab irrespective of the location. Samples from Kentinkrono recorded the highest mean load of 6.53 log10 CFU/g, whereas those from Kotei the least (4.02 log10 CFU/g) (Table 2). The mean loads of S. aureus were significantly different (p = 0.013) and above the FSANZ acceptable limit of ≤104 CFU/g (≤4 log CFU/g).Table 2
Mean loads ofE. coli and S. aureus in chevon khebab sold on the KNUST campus and its environs.
LocationRange ofE. coli load (MPN/g)Mean load ofE. coli (log10 MPN/g)Range ofS. aureus load (CFU/g)Mean load ofS. aureus (log10 CFU/g)Kotei0.00–2.40 × 1063.91 ± 3.063.60 × 103-3.70 × 1044.02 ± 0.39aAyeduase0.00–9.30 × 1064.44 ± 3.452.40 × 104-2.88 × 1076.01 ± 1.31bcKentinkrono0.00–9.30 × 1064.43 ± 3.441.80 × 105-1.64 × 1076.53 ± 0.71cBoadi0.00–9.30 × 1051.99 ± 3.083.60 × 103-1.40 × 1064.82 ± 1.13abKNUST campus0.00–4.30 × 1063.99 ± 3.144.50 × 103-1.28 × 1075.71 ± 1.62bcAyigya0.00–2.40 × 1064.25 ± 3.294.60 × 103-1.52 × 1075.81 ± 1.49bcCV (%)84.721.7p value0.7790.013MPN = most probable number; CFU = colony-forming unit. Mean loads with same letters are not significantly different (p > 0.05).


## 4.3. Mean Load ofE. coli and S. aureus in Gizzard Khebab

For the gizzard khebab, the mean load ofE. coli for the locations ranged from 0.00 to 2.90 × 107 MPN/g with Kotei recording the highest mean load (6.10 log10 MPN/g) and the least observed in Boadi (1.79 log10 MPN/g). Although the mean load of E. coli in the gizzard khebab from the various locations was not significantly different (p = 0.207), it was above the FSANZ guidelines of ˂ 100 MPN/g (˂2 log10 MPN/g), with the exception of Boadi, which had a mean E. coli load within the acceptable limit. S. aureus load ranged from 4.20 × 103 to 1.64 × 107 CFU/g. Samples from Kentinkrono recorded the highest mean load (6.18 log CFU/g), whereas those from Kotei recorded the least (5.37 log10 CFU/g) (Table 3). The mean load differences of S. aureus were statistically not significant (p = 0.788); however, they were above the FSANZ acceptable standard limit of ≤ 104 CFU/g (≤4 log10 CFU/g).Table 3
Mean load ofE. coli and S. aureus in gizzard khebab sold on the KNUST campus and its environs.
LocationRange ofE. coli load (MPN/g)Mean load ofE. coli (log10 MPN/g)Range (CFU/g) ofS. aureus loadMean load ofS. aureus (log10 CFU/g)Kotei2.30×105-4.30 × 1066.10 ± 0.521.52 × 104-3.50 × 1065.37 ± 0.98Ayeduase0.00–2.40 × 1063.85 ± 3.034.20 × 103-2.96 × 1065.43 ± 1.36Kentinkrono0.00–2.90 × 1072.94 ± 3.853.00 × 105-1.64 × 1076.18 ± 0.81Boadi0.00–2.40 × 1051.79 ± 2.781.24 × 105-1.76 × 1065.80 ± 0.54KNUST campus0.00–1.50 × 1064.05 ± 3.141.48 × 105-9.60 × 1065.82 ± 0.65Ayigya0.00–9.30 × 1064.38 ± 3.417.40 × 103-1.64 × 1075.69 ± 1.50CV (%)79.118.1p value0.2070.788MPN = most probable number; CFU = colony-forming unit.


## 4.4. Antimicrobial Susceptibility ofE. coli and S. aureus from Khebab on KNUST Campus and Its Environs

### 4.4.1.E. coli

Based on the British Society for Antimicrobial Chemotherapy (BSAC) Methods for Antimicrobial Susceptibility Testing, version 14, May 2015,E. coli (n = 27) were all resistant to the antibiotics tested with the exception of chloramphenicol, which was susceptible (Figure 2).Figure 2
A graph showing percentage resistance and susceptibility ofE. coli.


### 4.4.2.S. aureus

S. aureus (n = 36) was resistant to the antibiotics tested (ampicillin, tetracycline, gentamicin, cefuroxime, ceftriaxone and cefotaxime = 100%, cotrimoxazole = 52%) with the exception of few which were susceptible (chloramphenicole = 100% and cotrimoxazole = 48%).


## 4.4.1.E. coli

Based on the British Society for Antimicrobial Chemotherapy (BSAC) Methods for Antimicrobial Susceptibility Testing, version 14, May 2015,E. coli (n = 27) were all resistant to the antibiotics tested with the exception of chloramphenicol, which was susceptible (Figure 2).Figure 2
A graph showing percentage resistance and susceptibility ofE. coli.


## 4.4.2.S. aureus

S. aureus (n = 36) was resistant to the antibiotics tested (ampicillin, tetracycline, gentamicin, cefuroxime, ceftriaxone and cefotaxime = 100%, cotrimoxazole = 52%) with the exception of few which were susceptible (chloramphenicole = 100% and cotrimoxazole = 48%).


## 5. Discussion

### 5.1. Microbiological Quality

The meanE. coli count in the beef, chevon, and gizzard khebab samples from the various locations ranged from 1.79 to 6.12 log10 MPN/g, 1.99 to 4.44 log10 MPN/g, and 1.79 to 6.10 log10 MPN/g, respectively. The mean E. coli count for the khebab samples (beef, chevon, and gizzard) in the locations was above the standard acceptable limit, except Boadi, which was within the acceptable limit. The high count of E. coli in the khebab samples suggests a possible faecal contamination. This may be due to poor hygienic practices of the vendors. Hygienic practices such as protection of food from flies and dust, cleaning of fingernails, use of aprons, and head cups by street food vendors are one of the key measures that helps to prevent foodborne contamination [40]. But majority of street food vendors are uneducated and therefore lack the knowledge in safe food preparation and handling. As a result, they rarely adhere to hygienic practices and this might have accounted the high level of E. coli count in the khebab samples. Additionally, meat generally contains the necessary nutrients for microbial growth as well as their metabolism, making it prone to microbial contaminations. Thus, if the vendor does not practice proper hygiene, it facilitates the growth of the organism resulting in their high numbers [45]. According to Oladipo-Adekeye and Tabit [46], Carrasco et al. [47], and Wei et al. [48], poor sanitary practices by street food vendors results in food contaminations. The use of nonportable water by vendors during preparation or processing of the khebab could also contaminate the khebab with E. coli. E. coli is destroyed at a temperature above 70°C, so their presence in the khebab samples could be that the khebabs were insufficiently heated (roasted) or grilled [49]. Bryan et al. [50] have reported similar high E. coli count of 6 log10 CFU/g in street-vended foods. Djoulde et al. [51] also reported E. coli counts of 3.3 log10 CFU/g and 2.8 log10 CFU/g in mobile and stationary food sellers, respectively, which are lower than the current findings but were above the permissible limit. Ologhobo et al. [52] also reported unsatisfactory levels of E. coli counts in–RTE beef (3.3 × 104 CFU/g) and chicken (3.4 × 105 CFU/g) “suya,” which is lower than the current findings but above the permissible limit. Similar results of high E. coli count above the permissible limit have also been reported by Salihu et al. [53] in traditionally prepared ground beef.The mean total viable count (TVC) ofS. aureus in the beef, chevon, and gizzard khebabs in the locations was all above the standard acceptable limit of ≤104 CFU/g (≤4 log10 CFU/g). According to Bishop and Onyowoicho [54] and Gilbert and Harrison [55], S. aureus is a normal flora of the human skin. Therefore, the high levels of S. aureus in the khebab samples might be a cross-contamination from the vendor. Gay [56] also indicated that the presence of S. aureus in food is mainly a result of human contamination, which is normally from the human respiratory passages and the skin, as well as the superficial wounds on the human skin surfaces. But according to Levine [57], the TVC of microbes present in any food is not indicative of their safety for consumption, but it is a supreme importance of judging the hygienic conditions under which the foods had been prepared, handled, and stored. The current results are consistent with the findings of Hassan et al. [58], who reported microbial load of S. aureus ranging from 0.125 to 2.6 × 106 CFU/g in barbecue meat (suya). Bagumire and Karamuna [1] and Selvan et al. [59] also reported higher S. aureus count of 4.37 and 4.88 log10 CFU/g in ready-to-eat beef and chicken samples, respectively. However, the current results are in contrast to the findings of Uzeh et al. [60] and Djoulde et al. [51] who reported relatively lower S. aureus count of 1.0×102-12.0 × 102 CFU/g and 1.10 log10 CFU/g in tsire-suya and roasted chicken, respectively.


### 5.2. Antimicrobial Susceptibility ofS. aureus and E. coli Isolated from Khebab Samples

The antibiotic resistance pattern observed in theE. coli (n = 27) and S. aureus (n = 36) revealed a multiresistance to the antibiotics tested, although some of them were susceptible to few of the antibiotics tested (Figures 2 and 3). E. coli (n = 27) were 100% resistant to ampicillin, tetracycline, gentamicin, cefuroxime, ceftriaxone, and cefotaxime, and 100% susceptible to chloramphenicol. The current findings are in line with work carried out by Ahmadi et al. [61] who reported that E. coli isolates from meat and meat product were resistant to ampicillin, tetracycline, and other antibiotics. Barua [62] also reported a higher percentage of VTEC isolates resistant to ampicillin. Additionally, Somda et al. [63] reported E. coli isolates from fumed, grilled, and flamed chicken resistant to cefotaxime (7.14%), ceftriaxone (10.71%), ampicillin (42.86%), tetracycline (64.3%), and other antibiotics that support the current results. The current findings also agree with work carried out by Apun et al. [64] who reported E. coli isolates from Malaysian broiler chickens resistant to ampicillin, tetracycline, and gentamicin ranging from 11 to 95%. Tricia et al. [65] reported 43% of E. coli isolates resistant to ampicillin but were not resistant to gentamicin, and this partly agrees with the current findings.Figure 3
A graph showing percentage resistance and susceptibility ofS. aureus.In the case of theS. aureus (n = 36), majority of them were multiresistant to all the antibiotics tested and only few were susceptible to four of the antibiotics namely ampicillin (2.78%), erythromycin (25%), cotrimoxazole (13.89%), and gentamicin (30.56%). Since the S. aureus been multiresistant to the antibiotics tested, the khebabs could pose a health risk to the consumer. The current findings agree with work carried out by Temesgen et al. [66], who reported S. aureus isolates from ready-to-eat foods resistant to ampicillin, ceftriaxone, cloxacillin, and other antimicrobial drugs. This therefore makes treatment of S. aureus infection difficult.TheE. coli and S. aureus exhibited a similar antibiotic resistance pattern even though the khebab samples were taken from different locations. This agrees with work carried out by Achi and Madubuike [67], who reported similar antibiotic resistance patterns of S. aureus isolates from different vendors (hawkers and retail outlets) in food samples. These antibiotic-resistant bacterial isolates from the khebab samples give an indication that street foods might pose serious health problems to the public who patronize them. According to Umoh et al. [68] and Chigbu and Ezeronye [69], the level of antimicrobial resistance is because of the result of indiscriminate use and abuse of antibiotics in the environment. Additionally, the practice of administering antimicrobials agents to domestic livestock as a way of preventing and treating diseases as well as serving as promoting growth could result in the emergence of antimicrobial-resistant bacteria.In recent years, antimicrobial resistance in foodborne pathogens has been on the rise. According to Allison and Gilbert [70], the increase could be attributed to the selection pressure created by using antimicrobials in food-producing animals and unregulated use of antibiotics by humans in developing countries. De Briyne et al. [71] also indicated the prevalence of resistance to these antibiotics is expected as the result of extensive use of these substances in veterinary medicine.


## 5.1. Microbiological Quality

The meanE. coli count in the beef, chevon, and gizzard khebab samples from the various locations ranged from 1.79 to 6.12 log10 MPN/g, 1.99 to 4.44 log10 MPN/g, and 1.79 to 6.10 log10 MPN/g, respectively. The mean E. coli count for the khebab samples (beef, chevon, and gizzard) in the locations was above the standard acceptable limit, except Boadi, which was within the acceptable limit. The high count of E. coli in the khebab samples suggests a possible faecal contamination. This may be due to poor hygienic practices of the vendors. Hygienic practices such as protection of food from flies and dust, cleaning of fingernails, use of aprons, and head cups by street food vendors are one of the key measures that helps to prevent foodborne contamination [40]. But majority of street food vendors are uneducated and therefore lack the knowledge in safe food preparation and handling. As a result, they rarely adhere to hygienic practices and this might have accounted the high level of E. coli count in the khebab samples. Additionally, meat generally contains the necessary nutrients for microbial growth as well as their metabolism, making it prone to microbial contaminations. Thus, if the vendor does not practice proper hygiene, it facilitates the growth of the organism resulting in their high numbers [45]. According to Oladipo-Adekeye and Tabit [46], Carrasco et al. [47], and Wei et al. [48], poor sanitary practices by street food vendors results in food contaminations. The use of nonportable water by vendors during preparation or processing of the khebab could also contaminate the khebab with E. coli. E. coli is destroyed at a temperature above 70°C, so their presence in the khebab samples could be that the khebabs were insufficiently heated (roasted) or grilled [49]. Bryan et al. [50] have reported similar high E. coli count of 6 log10 CFU/g in street-vended foods. Djoulde et al. [51] also reported E. coli counts of 3.3 log10 CFU/g and 2.8 log10 CFU/g in mobile and stationary food sellers, respectively, which are lower than the current findings but were above the permissible limit. Ologhobo et al. [52] also reported unsatisfactory levels of E. coli counts in–RTE beef (3.3 × 104 CFU/g) and chicken (3.4 × 105 CFU/g) “suya,” which is lower than the current findings but above the permissible limit. Similar results of high E. coli count above the permissible limit have also been reported by Salihu et al. [53] in traditionally prepared ground beef.The mean total viable count (TVC) ofS. aureus in the beef, chevon, and gizzard khebabs in the locations was all above the standard acceptable limit of ≤104 CFU/g (≤4 log10 CFU/g). According to Bishop and Onyowoicho [54] and Gilbert and Harrison [55], S. aureus is a normal flora of the human skin. Therefore, the high levels of S. aureus in the khebab samples might be a cross-contamination from the vendor. Gay [56] also indicated that the presence of S. aureus in food is mainly a result of human contamination, which is normally from the human respiratory passages and the skin, as well as the superficial wounds on the human skin surfaces. But according to Levine [57], the TVC of microbes present in any food is not indicative of their safety for consumption, but it is a supreme importance of judging the hygienic conditions under which the foods had been prepared, handled, and stored. The current results are consistent with the findings of Hassan et al. [58], who reported microbial load of S. aureus ranging from 0.125 to 2.6 × 106 CFU/g in barbecue meat (suya). Bagumire and Karamuna [1] and Selvan et al. [59] also reported higher S. aureus count of 4.37 and 4.88 log10 CFU/g in ready-to-eat beef and chicken samples, respectively. However, the current results are in contrast to the findings of Uzeh et al. [60] and Djoulde et al. [51] who reported relatively lower S. aureus count of 1.0×102-12.0 × 102 CFU/g and 1.10 log10 CFU/g in tsire-suya and roasted chicken, respectively.


## 5.2. Antimicrobial Susceptibility ofS. aureus and E. coli Isolated from Khebab Samples

The antibiotic resistance pattern observed in theE. coli (n = 27) and S. aureus (n = 36) revealed a multiresistance to the antibiotics tested, although some of them were susceptible to few of the antibiotics tested (Figures 2 and 3). E. coli (n = 27) were 100% resistant to ampicillin, tetracycline, gentamicin, cefuroxime, ceftriaxone, and cefotaxime, and 100% susceptible to chloramphenicol. The current findings are in line with work carried out by Ahmadi et al. [61] who reported that E. coli isolates from meat and meat product were resistant to ampicillin, tetracycline, and other antibiotics. Barua [62] also reported a higher percentage of VTEC isolates resistant to ampicillin. Additionally, Somda et al. [63] reported E. coli isolates from fumed, grilled, and flamed chicken resistant to cefotaxime (7.14%), ceftriaxone (10.71%), ampicillin (42.86%), tetracycline (64.3%), and other antibiotics that support the current results. The current findings also agree with work carried out by Apun et al. [64] who reported E. coli isolates from Malaysian broiler chickens resistant to ampicillin, tetracycline, and gentamicin ranging from 11 to 95%. Tricia et al. [65] reported 43% of E. coli isolates resistant to ampicillin but were not resistant to gentamicin, and this partly agrees with the current findings.Figure 3
A graph showing percentage resistance and susceptibility ofS. aureus.In the case of theS. aureus (n = 36), majority of them were multiresistant to all the antibiotics tested and only few were susceptible to four of the antibiotics namely ampicillin (2.78%), erythromycin (25%), cotrimoxazole (13.89%), and gentamicin (30.56%). Since the S. aureus been multiresistant to the antibiotics tested, the khebabs could pose a health risk to the consumer. The current findings agree with work carried out by Temesgen et al. [66], who reported S. aureus isolates from ready-to-eat foods resistant to ampicillin, ceftriaxone, cloxacillin, and other antimicrobial drugs. This therefore makes treatment of S. aureus infection difficult.TheE. coli and S. aureus exhibited a similar antibiotic resistance pattern even though the khebab samples were taken from different locations. This agrees with work carried out by Achi and Madubuike [67], who reported similar antibiotic resistance patterns of S. aureus isolates from different vendors (hawkers and retail outlets) in food samples. These antibiotic-resistant bacterial isolates from the khebab samples give an indication that street foods might pose serious health problems to the public who patronize them. According to Umoh et al. [68] and Chigbu and Ezeronye [69], the level of antimicrobial resistance is because of the result of indiscriminate use and abuse of antibiotics in the environment. Additionally, the practice of administering antimicrobials agents to domestic livestock as a way of preventing and treating diseases as well as serving as promoting growth could result in the emergence of antimicrobial-resistant bacteria.In recent years, antimicrobial resistance in foodborne pathogens has been on the rise. According to Allison and Gilbert [70], the increase could be attributed to the selection pressure created by using antimicrobials in food-producing animals and unregulated use of antibiotics by humans in developing countries. De Briyne et al. [71] also indicated the prevalence of resistance to these antibiotics is expected as the result of extensive use of these substances in veterinary medicine.


## 6. Conclusion

The prevalence of foodborne illness in Ghana is rising each day, which is a major threat to public health. The current research sought to determine the quality of khebabs on the KNUST campus and its environs as well as the antibiotic resistance ofE. coli and S. aureus isolated from the khebabs. The findings of the study generally suggest that street-vended khebabs on the KNUST campus and its environs were contaminated. Isolated E. coli and S. aureus were multiresistant to the antibiotics tested and therefore expose consumers to risk of contracting multiresistant E. coli and S. aureus illness.



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*Source: 8622903-2021-12-29.xml*