Bacteriological Analysis of Raw Beef Retailed in Selected Open Markets in Accra, Ghana

Beef is an important and popular source of protein and nutrients. Constant monitoring and evaluation of the microbiological contamination is pertinent for its safety and quality. This study aimed to assess the bacteriological proﬁles and the bacterial loads in beef, as well as investigate food safety practices among beef retailers in selected open markets in Accra, Ghana. This was a cross-sectional study with beef samples randomly purchased from six selected open markets in Accra metropolis. Beef samples were examined for total aerobic count (TAC) and total coliform count (TCC). Bacterial identiﬁcation was performed by standard bacteriological methods and a well-structured questionnaire was administered to assess food safety and hygienic practices of beef retailers. Overall, mean TACs ranged between 2.2 × 10 3 and 1.3 × 10 4 CFU/g, while mean TCCs ranged between 1.9 × 10 2 and 7.4 × 10 2 CFU/g. Beef samples were mostly contaminated with Staphylococcus spp. (34%), Klebsiella oxytoca (17%), Enterobacter spp. (15%), and Proteus vulgaris (3%). This study showed varying levels of bacterial contamination of retailed beef sold in diﬀerent Accra markets. According to the Health Protection Agency guidelines for assessing microbiological safety of ready-to-eat foods, our mean TACs were satisfactory, while mean TCCs were within borderline quality. The results of this study revealed that the majority of beef vended in open markets in Accra is contaminated with coliform and pathogenic bacteria. Therefore, more training in safe food handling and good hygiene practices is required for beef retailers. Conclusively, eﬀective monitoring of the bacteriological proﬁles and bacterial loads of meat and meat products in abattoirs and points of sale should be established, since such a continuous monitoring system could help prevent potential health problems for consumers and provide information on possible sources of meat borne intoxication and infection.


Introduction
Beef (meat) is a nutritive-rich food that contributes vital proteins, vitamins, and minerals to higher bioavailability than other food sources [1,2]. In most developed countries, beef constitutes a significant part of the normal diet and contributes more than 15% energy, 40% protein, and 20% fat.
e demand for meat in developing countries has continuously grown as the production and consumption pattern of meat increases with the availability of income [3]. However, meat has been found to be a prime vehicle for the dissemination of foodborne pathogens to humans. e water content activity of fresh meat and its neutral pH play a vital role in the growth of microbes. Beef may be susceptible to contamination at different stages of processing, from personnel and during transportation [4,5]. e incidence of beef contamination and related illnesses worldwide has led to an increase in public health concern related to meat safety in both developed and developing countries [6]. In 2015, a report by the World Health Organization (WHO) documented that 600 million foodborne illnesses were responsible for 420,000 deaths in 2010 [7]. Furthermore, in 2016, the Centers for Disease Control and Prevention [8] reported that a total of 839 cases of foodborne disease outbreaks cumulated in 14,972 illnesses, 794 hospitalizations, and 17 deaths with 18 food products. In Pakistan, the microbial load of raw meat at abattoirs and retail outlets in different areas of Lahore had been reported as a mean aerobic count of 7.15, 6.92, and 6.62 log CFU/cm 2 and 5. 35, 5.42, and 4.84 log CFU/cm 2 for beef, sheep, and goat meat at abattoirs and retail outlets, respectively, with a prevalence of 45%, 51%, and 18% for Escherichia coli, Staphylococcus aureus, and Salmonella spp., respectively [9]. In Egypt, a study monitored the bacterial contamination during different stages of beef carcass preparation at an abattoir and found surface counts to exceed 10 6 CFU/cm 2 [10]. In India, a study determined the microbiological quality of meat collected from municipal slaughterhouses and retail meat shops revealed 29.7% of the samples had exceeded the legislative limit of 10 4 CFU/g, with varying prevalence for Escherichia coli (10%), Staphylococcus aureus (8.3%), and Salmonella spp. (5%) [11]. In Ghana, a study determined the microbiological quality of fresh meat from retail outlets in the Northern Region of Ghana and revealed mean total aerobic counts of 5.74 × 10 6 CFU/cm 2 , 7.58 × 10 6 CFU/cm 2 , and 8.85 × 10 6 CFU/cm 2 for morning, afternoon, and late afternoon samples, respectively [12]. However, there is a lack of periodic monitoring of raw meats and limited information on the bacteriological nature of meat and meat products in open markets. is study aimed to assess the bacteriological profiles and loads of raw beef sold in open markets in the Accra metropolis and to investigate food safety practices among beef retailers in order to provide additional information to inform policies to protect public health.

Study Area.
e study was conducted in the Greater Accra Region which is the smallest among the 16 administrative regions in Ghana. It occupies a total land surface of 3,245 km 2 or 1.4% of the total land area of Ghana. However, in terms of population, it is the second only to the Ashanti Region, with an estimated urban population of 2.5 million, accounting for 15.4% of Ghana's total population [13]. Accra metropolitan markets used for this study included Makola, Kaneshie, Agbogbloshie, Malata, Maamobi, and Nima markets.

Study Design and Sample Collection.
is was a descriptive cross-sectional study of retailed beef in some open markets in the Accra metropolis ( Figure 1 (A)). One hundred (100) beef samples were randomly purchased from each of the six selected open markets within the Accra metropolis, Makola (10), Kaneshie (15), Agbogbloshie (20), Malata (25), Maamobi (10), and Nima (20) markets. Purchased beef samples were aseptically collected in sterile zip loc bags (Kolysen, China), sealed, and transported in an ice chest within 2 hours to the University of Ghana School of Biomedical and Allied Health Sciences Microbiology Laboratory for bacteriological analysis.

Survey
Questionnaire. Additionally, a well-structured questionnaire was used to obtain data relevant to food safety awareness from retailers. e main areas of assessment were processing, storage, selling conditions, personal and environmental hygiene, and waste disposal as well as hygienic handling of used materials such as knives and water.

Bacteriological Analysis of Samples.
Approximately 10 g of raw beef sample was added to 90 ml of 0.1% buffered peptone water (Oxoid, UK) to prepare homogenate suspension in a stomacher bag (Seward, UK), followed by tenfold serial dilutions (10 −2 to 10 −4 ) as described by Clarence et al. [14]. An aliquot of 0.1 ml from each serial dilution was inoculated onto plate count agar (Oxoid, UK), blood agar (Oxoid, UK), and MacConkey agar plates (Oxoid, UK), employing the spread plate method for total aerobic count (TAC) and total coliform count (TCC). Inoculation was done in triplicate and the plates were incubated at 37°C for 24-48 hours. Enumeration of colonies took place on plate count agar (TAC) and Mac-Conkey agar (TCC). Furthermore, dilutions that resulted in 30-300 colonies were considered countable and were included in the calculations. For bacteria identification purpose, 3-4 well-isolated colonies were obtained from primary cultures by subculturing and checking for purity onto MacConkey agar (Oxoid, UK) and blood agar (Oxoid, UK). e identification of pure culture colonies was based on colonial morphology, Gram staining, and biochemical reactions of triple sugar iron (TSI), oxidase, coagulase, citrate utilization, urease, and indole tests [15]. API 20E identification system (bioMerieux SA, Marcyl'Etoile, France) was also used to confirm the Gramnegative isolates.

Statistical Analysis.
Bacterial counts were calculated as colony forming units per gram (CFU/g) and then converted into log10 values. Descriptive statistics were computed using the Microsoft Excel data analysis Fischer's exact chisquare and one-way ANOVA was analyzed using Graph-Pad Prism software, version 6, to find any statistical differences. Statistical significance was set at a P value of < 0.05.

Demographics and Food Safety Awareness of Beef
Retailers. Overall demographics, educational status, and food safety awareness of beef retailers are summarized in Table 3. With respect to what causes diseases, 80% were aware that microorganisms are responsible for diseases, while 18% said they were not aware of what causes diseases. In terms of inspection, sampling, and control by health authorities, 85% of the respondents claimed to have regular experience in the matters, while 8% rarely confront that issue. With regard to handwashing practices, 96% said they washed their hands after using the urinal and toilet, while 4% responded that such measures were not necessary.

Discussion
In this study, 60% of beef retailers were male and 40% were female, and while 65% of the retailers were within the age range of 16-35 years, 35% were above 35 years. ese findings are in agreement with a previous study by Adzitey et al. [16], where it was reported that butchering and retailing of beef were mostly practiced by the youth and middle-aged men, since the beef retailing business may be physically exhaustive. With regard to the education level of the beef retailers, primary education accounted for 50%, followed by secondary (35%), and tertiary (15%) education, respectively. ese findings are in agreement with a previous study by Adzitey et al. [17] conducted in the Northern Region of Ghana, which reported that 62% of respondents possessed an elementary form of education. e standard of education is pertinent to the awareness of beef safety practices, since it may influence retailers' acceptability of modern and more effective ways of safety practices as well as their adherence to strict hygienic and standard slaughtering practices. Bhandare et al. [18] have highlighted that most abattoir and retail beef workers in developing nations are mostly untrained and ignorant of hygienic standards that may alleviate bacterial contamination of beef products.
In the present study, 70% of beef retailers were licensed, whereas 25% did not possess any formal proof of evidence (e.g., professional license) or other legal binding state documents for doing business. Contrary to this study findings, Mcarthur-floyd et al. [19] in Madina, Accra, evaluated the practice of food safety among fast-food operators and reported the possession of health license by all fast-food operators surveyed. Furthermore, their findings revealed that the food inspection agencies are more stringent on ready-to-eat food sellers as compared to retailers of raw and uncooked food commodities such as beef retailers. Moreover, in our study, 87% of beef retailers admitted refrigerating beef products as a form of storage; however, 10% used freezers to store their meats. Most of the beef retailers admitted employing traditional or other nonconforming means such as salting and usage of antibiotics in order to preserve meat. With regard to the usage of water for cleaning purposes, 25% of beef retailers made use of well water for washing of meat, whereas this practice may be considered worrisome in terms of cross-transmission of meat with pathogenic bacteria. A study conducted by Ngwa and Chrysanthus [20] on the usage of well water revealed that this type of water is highly contaminated with bacteria of fecal origin such as Klebsiella spp. (95%) and Escherichia coli (52%), as well as pathogenic bacteria like Salmonella typhi (32.5%). Additionally, salting as a means of preservation for meat is not only inadequate but also could predispose consumers to health risks such as high blood pressure and other cardiovascular diseases [21]. e combined retailing of beef along with other meats like chevron, chicken, and mutton may facilitate cross-contamination with microorganisms, due to the fact that contamination of meats may occur through different meat sources [22]. In this study, some retailers were not alarmed by the presence of increased bacterial loads in meat, since they believed that cooking would eradicate the organisms present. However, Prescott and Klein [23] have reported that most strains of Staphylococcus aureus implicated in staphylococcal enteritis are responsible for the synthesis of extracellular toxins, which are actually heat-resistant proteins that remain pathogenic when consumed even in cooked foods [23]. e general indicators of the microbiological food quality are TAC under favorable conditions that may enhance the multiplication of microorganisms [24]. In this study, an overall, mean TAC of beef in the surveyed markets in Accra ranged between 2.19 × 10 3 and 1.26 × 10 4 CFU/g (3.34-4.1 log CFU/g). ese findings are comparable to another Ghanaian study by Soyiri et al. [25] which revealed a TAC range of 1.9 × 10 2 -2.3 × 10 4 CFU/g in retailed beef. In contrast, a higher mean TAC of 2.5 × 10 5 -2.25 × 10 8 CFU/g and 2.7 × 10 4 -2.0 × 10 8 CFU/g was reported by Jahan and Siddique [26] in Bangladesh and Erdem et al. [27] in Turkey,

Journal of Food Quality
respectively. e International Commission on Microbiological Specification for Foods (ICMSF) has proposed that ready-to-eat foods with TAC between 0 and 10 3 CFU/g are considered acceptable, within 10 4 -10 5 CFU/g are considered tolerable, and ≥10 6 CFU/g are considered unacceptable [28]. erefore, the level of contamination in this study could be considered tolerable based on the recommended standards. Our mean TCC of beef surveyed ranged between 1.9 × 10 2 and 7.4 × 10 2 CFU/g. In a similar study by Koffi-Nevr et al. [29], a TCC of 1.6 × 10 2 -4.0 × 10 2 CFU/g was reported.
According to Health Protection Agency guidelines for assessing the microbiological safety of ready-to-eat foods [30], our mean TAC was satisfactory, while the mean TCC findings were within borderline quality and signals that the hygiene of practices of meat handling and processing along the meat production chain must be urgently improved. erefore, considering the rather limited number of samples analyzed from selected locations and the varying environmental conditions for some retail joints, there is a certain degree of possibility that the bacterial loads could escalate to  e occurrence of bacteria in this study may have originated from the usage of contaminated well water used for washing the beef before retailing. In addition, bacteria may have gained access from the soil, since in some abattoir settings postslaughter processing is performed on the bare floor [27]. Bacterial isolates of the genus Klebsiella spp. and Enterobacter spp. are of clinical significance and may act as opportunistic pathogens that could cause infections of the lower respiratory and urinary tract system [33]. e occurrence of coagulase-positive Staphylococcus aureus may be attributed to insufficient hygiene practices and cross-contamination by handling meat and money with the same unwashed hands [34]. Staphylococcus aureus is known to cause food poisoning, which is often characterized by a sudden start of nausea, vomiting, stomach cramps, and diarrhea. Endotoxins that are produced when Staphylococcus aureus loads exceed 10 5 CFU/g are the Journal of Food Quality primary cause of food poisoning [35,36]. Globally, Staphylococcus aureus is one of the common causes of foodborne disease and staphylococcal intoxication could be attributed to improper handling of foods (meat) [37]. A previous study by Essayagh et al. [38] have reported an outbreak of food poisoning in Morocco at a family dinner, which resulted in 9 individuals being hospitalized [38] with clinical signs suggestive of Staphylococcus aureus foodborne poisoning. Finally, in the present study, some markets were more contaminated in terms of the number and bacterial diversity. e difference in the level of beef contamination could be attributed to varying levels of hygienic practices of retailers in the various markets [39]. is encompasses the need to adhere to safety processing and handling practices, as well as strict sanitary standard operating procedures along the beef processing and production chain. us, Nima market was the market with the lowest mean TAC of 2.19 × 10 3 CFU/g and a mean TCC of 3.7 × 10 2 CFU/g. Our observations in this market during the survey revealed that the beef retailers were relatively concentrated on one particular area of the market, which increased the likelihood that their meat product may have originated from the same abattoir. Moreover, most of the retailers in this market were largescale retailers with good storage facilities for the preservation of meat products.

Conclusion
is study showed varying levels of bacterial contamination of retailed beef sold in selected open markets within Accra metropolis. According to the Health Protection Agency guidelines for assessing microbiological safety of ready-toeat foods, our mean TACs were satisfactory, while mean TCCs were within borderline quality. e presence of different bacteriological profiles and loads still represents a public health hazard and signals the possible occurrence of foodborne intoxication and infection. erefore, strict hygiene conditions, proper handling, and proper storage of meat products by retailers should be adhered to. Also, continuous monitoring of bacteriological profiles and loads in abattoirs and sales points should be implemented.

Data Availability
e datasets used are available upon reasonable demand.

Conflicts of Interest
e authors declare that no conflicts of interest exist.