Street‐vended grilled beef sausages as potential vehicles of bacterial and fungal pathogens: An exploratory survey in Ho, the capital city of the Volta Region of Ghana

Abstract Grilled beef sausage is a popular street delicacy in many countries, and Ghana is no exception. This study assessed street‐vended grilled beef sausages as a potential vehicle of microorganisms that present food safety risks to the general public in Ho City. Twenty grilled beef sausages were obtained from various vended locations within Ho municipality by convenient sampling and were analyzed by standard microbiological protocols for food safety followed by statistical analysis with a test of significant difference at p < .05. Total aerobic bacteria count of the potential foodborne pathogens ranged from 2.75 × 104 to 1.85 × 107 CFU/g. The microbial species identified included Staphylococcus aureus with a load from 6.15 × 102 to 1.67 × 105 CFU/g, Escherichia coli from 4.2 × 102 to 3.9 × 104 CFU/g, Bacillus cereus from 3.05 × 102 to 7.1 × 104 CFU/g, and Salmonella spp. from 2.8 × 102 to 5.5 × 104 CFU/g. Total fungal counts also ranged from 0.0 to 9.83 × 103 CFU/g, and the species identified included Aspergillus spp. and Rhizopus spp. all of which were within the acceptable limits of the International Commission for Microbiological Specification of Foods. However, for total viable bacteria, 75% of the samples were above the acceptable limits in the guidelines by the Ghana Standards Authority, indicating that the consumption of grilled beef sausages poses serious food safety and hygiene risks to consumers. Hygienic processing of the sausages under sanitary environments, proper handling and preservation procedures, and periodic follow‐up visits to the vended areas should be employed to reduce the risk of occurrence of potential pathogens in the products.

is greatly consumed in Ghana and dietitians or nutritionists recommend it as a major source of protein for growing children, immunocompromised patients, expectant mothers, and the aged.
Meat processing converts meat by employing physical and biochemical technologies that add value to the product (Teye, 2010), leading to the preservation or extension of shelf life, and improvement of tenderness and flavor of meat and its products (Heinz & Hautzinger, 2007).Meat and meat products, in general, have been implicated as vehicles for the spread of food-borne pathogens (Altajori & Elshrek, 2014).They provide adequate nutrients that encourage the growth of microorganisms, rendering meat perishable.Due to its nature, sausage presents an increased surface area which can easily get contaminated when not properly stored.Many interrelated factors such as holding temperatures, atmospheric oxygen, endogenous enzymes, moisture, light, and most importantly microorganisms influence the shelf life and freshness of meat (Eie et al., 2007;Zhou et al., 2010).The microorganisms that had been isolated from different sausage products and processing lines include Staphylococcus aureus, Bacillus cereus, Escherichia coli, Salmonella, Listeria monocytogenes, yeasts, and molds (Afshin et al., 2011;Altajori & Elshrek, 2014;Güngör & Gökoğlu, 2010).
The consumption of grilled beef sausage has become very popular in Ghana and beyond.Beef sausage is grilled on a glowing charcoal fire, and is stacked on wood sticks and spiced with vegetable oil to improve its flavor.It is usually served with onions and powdered pepper.The ready-to-eat beef sausages are often prepared and sold along the streets, in clubhouses, restaurants, and hotels under unhygienic conditions, and are normally packaged in nonsterile newspapers.Street foods play a key socioeconomic role in meeting the food and nutritional needs of city consumers at affordable prices for the lower and middle-income classes and are valued for their unique flavors and convenience (Khairuzzaman et al., 2014).However, serious concerns exist about the handling and safety of street-vended foods in developing countries (Imathiu, 2017).According to Rane (2011), high levels of pathogenic bacteria capable of causing food poisoning have been quantified in these foods and have been declared unacceptable for consumption.It is, therefore, imperative that attempts are made to reduce the high levels of bacterial contamination to acceptable levels.
In Ghana, most street vendors operate without regulation or monitoring by the responsible authorities on the foods they sell.
Kebab sellers often sell grilled sausages in an open space or along the streets which are exposed to the environment.Most of these vendors typically operate in the evening until night or daybreak and do not adhere to recommended meat safety practices (Adzitey et al., 2020).According to Atter et al. (2020), most processors purchase fresh sausages from the cold store during the period it is kept in a freezer, but the major issue is the applicable storage practices before grilling.Although grilling of beef sausages may kill all bacteria, yeast, and molds, rendering them harmless due to the heat applied during preparation (Figure 1), the operations of the meat vendors in an open space make it possible for contamination by physical, chemical, or biological hazards from the environment (Adzitey, 2016).

| Sample size determination
A convenience sampling technique, Hotjar sample size calculator, and Cochran's formula for smaller populations were employed to obtain 20 grilled beef sausages from kebab vendors located across the entire Ho municipality.
All samples were kept on ice immediately after being purchased and were transported to the microbiology laboratory of the University of Allied Health Sciences (UHAS) for microbiological analysis.

| Microbiological analysis
Bacterial species were enumerated and identified by the procedures described by Aboagye et al. (2020) and Dortey et al. (2020) whilst fungal species were determined as described by Kortei et al. (2018) and Moss (1998).

| Sample preparation
Ten grams of each sausage sample was homogenized in 90 mL of sterile distilled water.Six serial dilutions were prepared, and aliquots of 1 mL of the dilutions were plated on the respective selective media, and incubated at 37°C for up to 48 h and 7 days for bacterial and fungal species, respectively.The selective media used were products of OXOID and BIOMARK laboratories.They included nutrient agar (Biomark Laboratories) for the isolation of total aerobic bacteria count, MacConkey agar (Biomark Laboratories) for the isolation of Escherichia coli, Salmonella-Shigella Agar (OXOID CM0099) for the isolation of Salmonella spp., Mannitol salt agar (OXOID CM0085) for the isolation of Staphylococcus aureus, Bacillus cereus selective agar base (OXOID CM0617) for the isolation of Bacillus cereus, oxytetracycline-glucose-yeast extract agar (OXOID CM0545) for the isolation of yeast, and Sabouraud dextrose agar (OXOID CM0041) for the isolation of molds.All media were prepared using the manufacturer's instructions.

| Identification of bacterial and fungal species
Bacterial colonies that appeared on the media surface showed distinctive morphologies.The variation in bacterial colony morphology was observed both visually and with the aid of a light microscope (Olympus) at x1000 magnification.The morphological variation included shape, size, color, and surface elevation on the sensitive and specific media used, whilst Standard Gram stain technique was employed for additional elaboration of the species isolated from the sausages.Fungal species were identified using lactophenol cotton blue by employing morphological characterization including texture and color of colonies on the sensitive and specific media used.Microscopic examination was also carried out at x1000 magnification (Olympus).Slides of fungal cultures were prepared by gently lifting the mycelial mat with a sterile inoculation pin into a drop of lactophenol cotton blue, and mixed uniformly using sterile loop and the suspension laid with a coverslip followed by the microscopic examination.The morphological features of the isolated fungi were observed and identified as described by Moss (1998) in Table 1 below.
The percentage occurrence of both bacterial and fungal species was calculated using the following formula:

| Data analysis
The mean bacteria counts were determined along with the fungal counts as described by Kortei et al. (2018) and Odamtten et al. (2018).Standard Authority ( 2019) and the International Commission for Microbiological Specification of Food (ICMSF, 1998) were used to interpret the microbiological quality of the street-vended grilled beef sausage.

| Bacterial load in the grilled beef sausages
The results obtained from the microbial analysis are presented in  Also, on Sabouraud dextrose agar (SDA) (Figure 3), the highest total fungal counts of 8.55 × 10 3 CFU/g were recorded at Mirage vending area (sample Q) whilst the least total fungal counts of 2.0 × 10 1 CFU/g were recorded at Housing vending area (sample T).
Furthermore, the identified fungi in the grilled beef sausage samples are presented in Table 5, where most of the samples had more than one fungal species.
The percentage of occurrence of fungi isolated from the grilled beef sausage is presented in Figure 4.

| DISCUSS ION
Microscopic organisms are incredibly diverse and constitute complex communities with numerous species of prokaryotes such as bacteria and archaea, unicellular eukaryotes such as fungi, protists, and algae, and some small multicellular eukaryotes (Mestre & Höfer, 2021), whose presence in food could become a biological hazard (Osimani et al., 2013).The results from the microbiological quality of streetvended grilled beef sausages showed that total aerobic bacteria count (TABC) ranged from 2.75 × 10 4 CFU/g to 1.85 × 10 7 CFU/g for all samples collected.The analysis of variance revealed that there were statistically significant differences (p < .05)among the total viable bacteria count of grilled beef sausages sampled from the different vending areas.The Ghana Standard Authority (2019) specifies that the total aerobic bacteria count should be less than or equal to 10 4 CFU/g for ready-to-eat foods.The results obtained indicated that 15 samples representing 75% were above the acceptable microbiological limits whilst only five samples representing 25% were within the acceptable limits.
The microbial quality of meat products is influenced by several factors.The higher microbial loads in most of the samples could be attributed to unsanitary practices and inadequate grilling by the kebab vendors.The total aerobic bacteria count represents the total number of viable pathogenic microorganisms (bacteria, yeasts, and molds) present in a sample and it is an important aspect in determining the hygienic environment during the storage and processing of beef sausages.Upon observation, the beef sausages grilled by the kebab vendors were left uncovered or not stored in glass showcases, exposing them to dust and flies, which are carriers of microorganisms, possibly contributing to the high microbial load.As suggested by Ayamah et al. (2021), hygienic procedures such as covering food from flies and dust, cleaning fingernails, and using aprons and head cups by street food vendors are one of the key measures that help to prevent foodborne contamination.
The results on the enumeration of total Staphylococcus aureus counts (TSC) for all the samples of street-vended grilled beef sausages ranged from 6.15 × 10 2 CFU/g to 1.67 × 10 5 CFU/g.Statistically, there were significant differences (p < .05)among the TSC of grilled beef sausages sampled from different vending areas.Ayamah et al. ( 2021) also reported a similar high S. aureus load of 3.10 × 10 2 to 2.96 × 10 7 CFU/g with no significant difference among the results in the KNUST campus and its environs in Ghana.According to Ghana Standard Authority (2019), the limit for Staphylococcus aureus is satisfactory for samples <20 CFU/g, and the acceptable upper limit for samples with 20 to ≤10 3 CFU/g and >10 3 CFU/g is considered unsatisfactory for ready-to-eat foods.The results obtained from the microbial assay indicated that 55% of the sampled grilled beef sausages were within the borderlines which indicates the maximum acceptable limits.The results also revealed that 45% of the sampled grilled beef sausages were above the acceptable limit (unsatisfactory).This indicates that all the samples analyzed in this study were contaminated with S. aureus.This is almost sim- The presence of Staphylococcus aureus in the grilled beef sausage samples suggested that the street kebab vendors were not practicing good personal hygiene.In most studies, Staphylococcus aureus is one of the major known pathogens identified on hands (Rabbi et al., 2011;Shojaei et al., 2006).It has also been proven that  A total of nine fungal species (Aspergillus niger, Aspergillus fumigatus, Aspergillus ochraceus, Aspergillus terreus, Fusarium, Mucor, Penicillium, Rhodotorula, and Rhizopus species) belonging to six genera (Aspergillus, Fusarium, Penicillium, Mucor, Rhodotorula, and Rhizopus) were identified on both media.
F I G U R E 4 Frequency of occurrence (%) for Fungi isolates.The presence of yeasts and molds usually contaminate foods and subsequently produce mycotoxins which are most often not eliminated through processing.The results obtained for the mean fungal counts on OGYEA ranged from 0.0 CFU/g to 9.83 × 10 3 CFU/g.
Statistical analysis of the mean fungal counts on OGYEA showed no significant difference (p = .28)among the various vending areas.
A similar trend was also observed on SDA, the mean fungal counts ranged from 2.0 × 10 1 CFU/g to 8.55 × 10 3 CFU/g.Statistical analysis of the mean fungal counts on SDA also showed no significant difference (p = .33)among the various vending areas.Fungal counts recorded in this work were in the same range of values of 1.0 × 10 3 to 8 × 10 3 CFU/mL reported by EI-Hassan et al. (2018), as counts of processed meat products (Tsire) in Nigeria.Despite the fact that all of the samples analyzed were within the acceptable range of microbiological counts for ready-to-eat foods (<10 4 CFU/g) as prescribed by the International Commission for Microbiological Specification of Foods (ICMSF, 1998), the presence of fungal species in the grilled beef sausages constitutes a public health risk for consumers.The highest fungal counts were recorded at the Mirage vending area, this could be attributed to the sausages being exposed to dust and flies, as well as the kebab vendor's poor personal hygiene.According to Osakue et al. (2016), the presence of fungal species in ready-toeat food indicates a deplorable state of poor hygiene and sanitary practices used in the processing and sales of these food products.
Therefore, the presence of fungal species in the grilled beef sausages implies poor hygienic practices of food handlers and insufficient grilling.
Mold and yeast attacks on food are more severe since they may develop easily not only at room temperature but also in cool, chilled storage settings (Petruzzi et al., 2017).Even though molds are involved in the spoilage of many kinds of foods, according to Minj et al. (2020), yeasts play a significant role in the production of alcoholic beverages, due to the capacity to gather high levels of ethanol and the production of aromatic compounds.As stated by Noumavo et al. (2022), the presence of molds and yeasts in food should not be disregarded because many of them, in particular mycotoxinogenic molds (aflatoxins, ochratoxins, and fumonisins production), can be harmful to human health when consumed in large quantities.From the results obtained in this study, Aspergillus niger had the highest frequency of occurrence (25%).This is congruent with the results obtained by Makhlouf et al. (2019) who claimed that Aspergillus was the dominant genus in samples of spices used in meat processing that were analyzed.Also, EI-Hassan et al. (2018) recorded Aspergillus niger as the most frequent occurrence of species in processed meat products (Tsire) sold in Nigeria.Oranusi and Braide (2012) stated that the mishandling of food products could lead to the proliferation of fungi beyond acceptable limits.However, Algammal et al. (2021) found A. flavus as the most predominant mold species isolated from the processed meat samples.Furthermore, the presence of fungi such as Aspergillus niger in the food indicates danger to public health because many of these fungi are toxin-producing organisms (Osakue et al., 2016).
Even though low counts of species of Penicillium were isolated, it is still of pathological importance.Penicillium is in the Deuteromycetes made up of diverse fungal genera of the ascomycetous fungi and contains more than 350 species.More than 80 Penicillium species are documented toxin producers (Agriopoulou et al., 2020); the most important are ochratoxin A, citreoviridin, penitrem A, roquefortine, and secalonic acids.The genus has major importance in the natural environment as well as the food and drug production industry.The preponderant fungal species isolated in this study with toxigenic potential, human health, and pathological importance were A. niger, A. fumigatus, A. ochraceus, Fusarium verticillioides, F. oxysporum, and Penicillium spp.Three major genera of fungi are identified to produce mycotoxins, including Aspergillus, Fusarium, and Penicillium, although other genera also produce toxic compounds (Ahmed et al., 2013).Aspergilli and Penicilliums are linked to agricultural commodities during postharvest storage (Agriopoulou et al., 2020;Balendres et al., 2019).Zearalenone causes human uterotrophic (antireproduction) effects in animals and pigs (Agriopoulou et al., 2020).
Fusarium species isolated from beef sausages produce several mycotoxins such as biologically active trichothecenes which when ingested in high concentrations, cause vomiting and diarrhea in humans.Trichothecenes are also associated with reduced weight gain and immune dysfunction in animals (Huang et al., 2019).Another Fusarium species F. verticillioides (F.moniliforme) isolated from beef sausages in the study produces fumonisins which have neurotoxic effects in animals and are associated with esophageal cancer (Schrenk et al., 2020).

| CON CLUS IONS
Among the biological hazards are Salmonella, Escherichia coli, Bacillus cereus, Staphylococcus aureus, yeast, and molds.The consumption of grilled beef sausages, therefore, presents various risk levels to consumers based on the frequency of intake and hygienic practices executed by the vendors.Therefore, this study investigated the microbiological quality of street-vended grilled beef sausages, the first of its kind in the capital city of the Volta Region of Ghana to ascertain the risks in handling preparations and consumption to provide safety recommendations to consumers.2| ME THODS2.1 | SamplingSamples were collected randomly from 20 selected vending areas in the Ho municipality.The main reason for the selection of these areas was based on population density as well as patronage for the grilled beef sausage.The specific sampling point of the selected vending areas was recorded as follows: Dave vending area (Sample A), Trafalgar vending area (Sample B), Melcom vending area (Sample C), Civic Center vending area (Sample D), Dome vending area (Sample E), Market vending area (Sample F), Fiave vending area (Sample G), Zongo vending area (Sample H), Barracks vending area (Sample I), Guinness vending area (Sample J), Ahoe vending area (Sample K), Poly vending area (Sample L), Lokoe vending area (Sample M), SSNIT Flat vending area (Sample N), Bankoe vending area (Sample O), Mawuli Estate vending area (Sample P), Mirage vending area (Sample Q ), Alaye vending area (Sample R), Ola vending area (Sample S), and Housing vending area (Sample T).A total of 40 grilled beef sausage vendors were identified along the streets of Ho municipality.Samples of grilled beef sausage that are vended along the streets of Ho municipality were included in the study.This study was conducted using experimental design, that is, the data were based on direct observation.FI G U R E 1 A showcase of grilled beef sausages sold at a vending area in Ho municipality.
The data were subjected to one-way ANOVA and values were considered for statistical significance at p < .05using Statistical Package for Social Sciences (IBM SPSS) version 26.The guidelines of the Ghana Percentage ( % ) occurrence of bacteria ∕ fungi = Number of bacteria ∕ fungi Total number of bacteria ∕ fungi isolated × 100 TA B L E 1 Cultural and morphological characteristics of the fungi identified.

Figures 2
Figures 2 and 3 present the mean fungal counts for grilled beef sausages sampled from various vending areas.The results obtained indicated that, on oxytetracycline-glucose-yeast extract agar (OGYEA) (Figure2), Mirage vending area (sample Q) recorded the highest total fungal counts of 9.83 × 10 3 CFU/g whilst Housing vending area (sample T) recorded the least total fungal counts of 0.00 CFU/g.
ilar to the results obtained byAtter et al. (2020) from Ghana, who reported that out of 25 ready-to-eat grilled beef sausage samples purchased from different vendors in the Greater Accra Region, 22 of them were contaminated with S. aureus.Also, the current results conform with the results obtained byShaltot et al. (2015), Falegan et al. (2017), EI-Hassan et al. (2018), Chingoma et al. (2016), Karoki et al. (2018), Madoroba et al. (2021), Ananias and Roland (2017), and Unachukwu et al. (2019) who also recorded Staphylococcus aureus as the most frequently isolated bacteria and highest percentage occurrence in ready-to-eat meat products.However, these results disagree with the findings of Alabi et al. (2021) who recorded the lowest percentage of Staphylococcus aureus (15.8%) in ready-to-eat roasted beef in Ibadan, Nigeria.
= fungi presence.Sample A, Dave vending area; Sample B, Trafalgar vending area; Sample C, Melcom vending area; Sample D, Civic Center vending area; Sample E, Dome vending area; Sample F, Market vending area; Sample G, Fiave vending area; Sample H, Zongo vending area; Sample I, Barracks vending area; Sample J, Guiness; Sample K, Ahoe vending area; Sample L, Poly vending area; Sample M, Lokoe vending area; Sample N, SSNIT Flat vending area; Sample O, Bankoe vending area; Sample P, Mawuli Estate vending area; Sample Q, Mirage vending area; Sample R, Alaye vending area; Sample S, Ola vending area; Sample T, Housing vending area.
majority of street-vended food handlers are agents of S. aureus(Rabbi et al., 2011).It was observed that most of the kebab vendors handled the sausages with their bare hands while cutting them, and the use of old newspapers for wrapping the grilled beef sausages could be a contributing factor.Also, the presence of Staphylococcus aureus could be attributed to the raw ingredients used (such as raw sliced onion).Furthermore, street kebab vendors hardly wash their hands, especially when there are a lot of customers waiting to be served.According to Luu and Michiels (2021), it appears that foods that are extensively handled manually are more likely to be contaminated with S. aureus, bacteria that lives naturally on human skin, in the nose, and ears.The results on the enumeration of total Escherichia coli (TEC) for all the samples of street-vended grilled beef sausages ranged from 4.2 × 10 2 CFU/g to 3.9 × 10 4 CFU/g.There were statistically significant differences (p < .05)among the TEC of grilled beef sausages sampled from the different vending areas.According to Ghana Standard F I G U R E 5 Staphylococcus aureus colonies on Mannitol salt agar.F I G U R E 6 Bacillus cereus colonies on Bacillus Cereus selective agar.F I G U R E 7 Macroscopic view of Aspergillus spp.and Fusarium spp.F I G U R E 8 Macroscopic view of Aspergillus niger.F I G U R E 9 Microscopic view of Staphylococcus aureus (×1000).
This exploratory survey demonstrated that all the vending areas of grilled beef sausages serve as potential vehicles of foodborne pathogens that present risks of foodborne illness to humans such as Salmonella spp., Staphylococcus aureus, Bacillus cereus, and Escherichia coli and also the fungal species such as Aspergillus spp.andRhizopus spp.The survey also demonstrates that the materials such as used newspapers, the use of bare hands, and exposure to the environment constitute contamination of grilled beef sausages.For this reason, training regimes should be organized for the vendors of this product with regular inspection and appropriate supervision to ensure consumer safety in the consumption of the product.Also, periodic follow-up visits to inspect hygienic vending areas should be carried out to ensure that the vendors adhere to their training.Data curation (equal); investigation (equal); methodology (equal); resources (equal); writing -original draft (lead); writing -review and editing (lead).Priscilla Amenya: Conceptualization (equal); data curation (equal); resources (equal); supervision (equal); writing -original draft (equal).George Aboagye: Conceptualization (lead); data curation (lead); investigation (lead); methodology (lead); resources (equal); supervision (equal); validation (equal); Conidiophores arise from a foot cell.Club-shaped vesicles at top of the conidiophores.Conidia are found in chains Fusarium spp.White-pink sparse aerial mycelia becoming felty Macroconidia sparse, borne on phialides on branched conidiophores (Septate banana shaped).Sporangiospores are produced inside a spherical sporangium.The columella is present on the top of the sporangiophore.Root-like rhizoids are found Note: Sources: da Cunha et al. (2013), Madrid et al. (2014).

Table 2
Dave vending area (sample A) recorded the highest level of contamination with a load of 7.1 × 10 4 CFU/g whilst the least load of 3.05 × 10 2 CFU/g was recorded at Trafalgar (sample B).Dome, Market, SSNIT Flat, Mawuli Estate, and Housing vending areas had no observable TBC.For total Salmonella count (TSSC) also, The table identifies the bacteria from the highest to the lowest frequency of occurrence.From the results obtained in Table3, Staphy- In the case of the total Escherichia coli count (TEC), the highest contamination was recorded at the Poly vending area (sample L) with a load of 3.9 × 10 4 CFU/g whilst the least load of 4.2 × 10 2 CFU/g was recorded at Bankoe vending area (sample O).Mawuli Estate, Mirage, and Alaye vending areas had no observable TEC.For total Bacillus cereus count (TBC), lococcus aureus had the highest frequency of occurrence 20 (31.25%), followed by Escherichia coli with 17 (26.56%)andBacilluscereushad15(23.44%).Salmonella had the lowest frequency of occurrence 12 (18.75%).Also, the morphological and biochemical (gram staining) characteristics of the organisms isolated from the grilled beef sausage and their preliminary identification are presented in Table4below.TA B L E 2Mean microbial load of grilled beef sausage samples.Abbreviations: NOG, no observable growth, values with a and b subscripts in the same column represent maximum and minimum loads, respectively; TABC, Total aerobic bacteria count; TBC, total Bacillus cereus count; TEC, total Escherichia Coli count; TSC, total Staphylococcus aureus count; TSSC, total Salmonella count.

Fusarium sp. Mucor sp. Penicillium sp. Rhodotorula sp. Rhizopus sp.
Mean fungal counts for sausage samples on OGYEA.Isolated fungi from the grilled beef sausages.