Isolation, Characterization, and Quantification of Bacteria from African Sausages Sold in Nairobi County, Kenya

African sausages are local popular delicacies in Kenya. Demand for these sausages has resulted in this delicacy's vendors being on the increase. However, health risk posed to unsuspecting consumers of African sausages sold in informal, unhygienic make shift road-side kiosks in major cities of Kenya is largely unknown. A descriptive study was designed to isolate, characterize and quantify bacteria from African sausages sold in Nairobi County. A total of hundred (100) African sausages (62 roasted and 38 nonroasted) were conveniently collected from three meat eatery points of Westlands, Kangemi slum, and Pangani estates. Five genera of bacteria, namely, Staphylococcus spp. at 50.4%, Bacillus spp. at 19.5%, Streptococcus spp. 9.8%, Proteus spp. 2.4%, and E. coli spp. at 1.6%, were isolated from 80 African sausage samples. The total aerobic bacterial count range was between 1.0-9.9x101 and 1.0-9.9x107 log cfu/g with 37 samples having total aerobic bacterial count of between 1.0-9.9 x104 and 1.0-9.9 x107 log cfu/g. There was no significant difference (p>0.05) in distribution of isolates and total aerobic bacterial count across geographical sites studied among the roasted and nonroasted African sausages. This study has demonstrated presence of bacteria in African sausages which are potentially zoonotic to humans. Comprehensive study is needed to sample more eatery meat points in Nairobi and other areas in order to demonstrate pathogenic attributes of these isolates and establish the respective total aerobic bacterial count. There is also need to establish the sources of bacteria due to high total aerobic bacterial count determined in the current study.


Introduction
African sausages, popularly known as Kenyan sausages locally known as "Mutura" in Kikuyu dialect, is a local delicacy for low, middle income earners and beer drinkers. It is a protein rich meat snack comprising goat or cow cleaned intestines stuffed with cooked small pieces of meat and formed into long coils; sometimes blood is added. Processed African sausages are then placed in boiling water or soup for 30 to 40 minutes and then roasted over coals on outdoor grills using low to medium heat and turning frequently to dehydrate the meat and give it the sensational smoky taste. The internal temperature should be at least 160 degrees Fahrenheit [1].
Animal proteins such as meat, meat products, and even blood are regarded as high-risk perishable commodities with respect to pathogen content, natural toxins, and other possible contaminants [2]. Among the bacteria isolated from animal products in recent studies include Staphylococcus aureus, Streptococcus sp., Escherichia coli, Campylobacter jejuni, Clostridium perfringens, Shigella sp., and Salmonella sp.
Consumption of food with such microbial pathogens and toxins is estimated to result in approximately 1.5 billion episodes of diarrhea and over 3 million deaths globally each year [3]. Increase in demand for finger-licking African sausages has resulted in this delicacy's vendors being on the increase, especially in slum areas [4]. These informal businesses mostly operate in unhygienic makeshift and roadside meat points of Nairobi and major cities in Kenya, where they are unregulated and the standardized preparation of these African sausages is disregarded.
In regard to Torok et al. [5] study, food-borne disease from African sausages arises from intestinal bacteria or external contamination, which is a result of unhygienic food preparation especially if the vendor fails to adhere to hygiene practices during processing, preparation, handling, 2 International Journal of Food Science and/or storage; it may therefore pose serious health risk to unsuspecting consumers resulting in outbreaks of foodborne illnesses. Results of this study will help the relevant regulatory body in laying down food safety measures for the African sausages.

Study Design.
A descriptive study design was employed whereby a convenience sampling of retail meat outlets from Westlands, Pangani, and Kangemi was carried out. One hundred (100) African sausages samples (38 nonroasted and 62 roasted) were collected, processed, isolated, characterized, and quantified.

Study Area.
The study was carried out in Nairobi County which is one of the 47 counties of Kenya [6]. It is the smallest yet most populous county, the capital and largest city of Kenya, which has experienced one of the most rapid growths in urban centres with a population of 3,375,000 as at year 2009 census [7]. It has a total area of 696 km 2 with 17 parliamentary constituencies. Nairobi is a cosmopolitan and a multicultural city.
Economically, it can be subdivided into three main categories: (1) the high end or leafy suburbs or upper-class estates, the likes of Muthaiga, Karen, Westlands, among others, (2) the Middle-class estates of Pangani, Buruburu, among others, and (3) low class estates of Mukuru, Mathare, Kangemi slums, among others [8]. Three ready-to-eat vending sites and meat eatery points of Westlands market, Kangemi market, and Pangani estate were conveniently selected on the basis of easy access, perceived sanitation, and relative hygiene levels. The number of vendors in these areas is not known; but they tend to converge around the shopping areas.

Sample Size Calculation.
Prevalence of common meat contaminants in previous studies was used to determine the sample size required to detect the presence of the bacteria. An expected prevalence rate of between 7% was used to estimate the sample size in this study since similar studies [9][10][11][12] reported a prevalence rate of between 3 and 14%. Using the above information, the sample size was calculated using the formula: n=z 2 pq/d 2 where n is the desired sample size, Z is the standard normal deviate set at 1.96, p is the estimated prevalence, q=1-p, and d is the degree of accuracy set at 0.05 as given by Fisher et al. [13].

Homogenate Preparation.
At the laboratory, one-gram portions of the African sausages (roasted and nonroasted) were obtained aseptically from the vendors, picked separately as they are sold using sterile glass bottles, stored on ice before processing in the laboratory within 24 hours of collection, cut into small pieces on a sterile chopping board using a sterile knife, and blended (homogenized) in 4ml of 0.1% peptone water to obtain 1:5 initial dilution.

Bacterial Isolation and Characterization.
Since the researcher suspected presence of coliforms and other fastidious organisms, the homogenates of the African sausages were streaked on general purpose enriched medium (blood agar) and selective and differential medium for members of family Enterobacteriaceae (MacConkey agar) (Oxoid Ltd. Thermo Scientific, UK) and incubated aerobically at 37 ∘ C for 24 hours. The isolated bacteria were identified based on colony morphology, Gram staining reaction, and biochemical characteristics using established standardized methods according to Bergey's Manual of determinative bacteriology [14].

Quantification of Total Aerobic Bacterial Count of the African Sausages.
For the determination of bacterial load (total bacterial count), method given by Miles and Misra [15] was used. Serial dilutions of 10 −1 to 10 −10 were prepared from the African sausage homogenate stock solution that was prepared earlier. Nutrient agar plate was divided into four quadrants, and each quadrant served as one plate. Using a 25 l calibrated dropper (equivalent to 1/40 th of an ml), one drop from each dilution tube was placed per quadrant; each dilution was done in quadruplicate.
The drop was then allowed to dry and the plate incubated aerobically at 37 ∘ C for 24 hours [16], after which the number of colonies that grew per drop was counted using Quebec Dark Field colony counter taking the average count for the quadruplicate drops of each dilution. The concentration of the original bacterial suspension was then calculated and expressed as colony forming units per millilitre (cfu/ml), using the formula, a x 40 x 10 y , where a is the average number of colonies in the 4 drops of one dilution tube/diluted suspension, 40 is the number of drops that make one millilitre (the drop being equivalent to 1.40 th of a ml), and 10 y is the dilution factor of the respective dilution tube/diluted suspension. This is then multiplied by 5, the initial dilution at homogenization stage.

Statistical Analysis.
The experiment was done in triplicate. The means for the prevalence of bacterial isolates across the three geographical sites and the bacterial counts were compared by a Paired-Samples T Test. Differences were considered statistically significant when P<0.05. Statistical analysis was conducted using the software SPSS 13.0 (SPSS, Chicago, Illinois, USA) Figure 1 gives results on the prevalence of isolated and characterized bacterial isolates. Five genera of bacteria (123 isolates) were isolated and characterized from 80/100 (80%) roasted and nonroasted African sausages.     Table 1 gives the mean distribution of bacterial isolates. There was no significant difference (p>0.05) in distribution of isolates across the geographical areas under study.

Discussion
The data obtained on isolation and characterization of bacteria in the present study where Staphylococcus spp. and Bacillus spp. were the predominant isolates concurs on some aspects with reports by Oluwafemi and Simisaye [17] and Okonko et al. [18] working on beef sausages. However, differences are noted whereby in the present study Streptococci spp., Proteus spp., and E. coli were isolated while in the latter, Enterobacter, Pseudomonas, and Klebsiella species were isolated from beef sausages and seafood, respectively. The current study showed Staphylococcus species prevalence of 50.4%. This was lower than the Staphylococcus species recovery at 58.6% from hotels, restaurants, and cafes, report by Berynestad and Granums [19]. A study by Yusuf et al. [20], on percent occurrence of bacteria isolated from the "balangu" meat product in relation to all the retail outlets, reported a lower prevalence of Staphylococcus aureus at 12.5%. A study by Aycicek et al. [21] reported that processed foods were found to be more prone to Staphylococcus species contamination. This may have been attributed to contamination from aerial spores carried in the air, throat, hands, and nail of food handling persons [22].
The results of the roasted African sausages in current study contrast the findings by Orogu and Oshilim [22] who reported a higher prevalence (30%) of Bacillus from suya meat. However, similar prevalence was obtained by a study by Matos et al. [23] working with dry smoked sausages. Presence of Bacillus contamination in some of the samples examined 6 International Journal of Food Science  [24] working on grilled beef. The prevalence of E. coli reported in present study was slightly lower than that reported by Syne et al. [25] and Onuora et al. [24]. E. coli presence in African sausages has the potential to cause diarrhea.
The incidence of E. coli obtained in this study is a cause for public health concern as this bacterium has been implicated in cases of gastroenteritis [26]. The presence of Proteus isolates was remarkably higher in a study by Gwinda et al. [27] from beef meat, compared to what was found in the current study. The presence of Proteus organisms in the meat samples can obviously be attributed to unhygienic food processing.
Staphylococcus spp., Bacillus spp., Streptococcus spp., and E. coli are known to produce potent enterotoxins and the ingestion of food containing these toxins can cause a sudden onset of illness within three to four hours, with nausea, vomiting, and diarrhea as the major symptoms [28]. There was no significant difference (p>0.05) in distribution of these organisms across the three geographical sites studied. In the present study, it was observed that there was no significant difference (p>0.05) in the total aerobic bacterial count across the three geographical sites studied. Total aerobic bacterial count of between 1.0-9.9 x 10 2 and 1.0 x 9.9 10 4 log cfu/g was reported in most 54/100 (54%) of African sausage samples.
Similar studies by Oluwafemi and Simisaye [17] reported a total aerobic bacterial count level of between 1.3 × 10 4 and 4.0 × 10 8 log cfu/g in beef sausage samples. In related studies, Inyang et al. [29] reported a total viable count of between 3.7 x 10 5 to 2.4 x10 6 log cfu/g while total viable count by Onuora et al. [24] reported a plate count of between 0.9 x 10 4 log cfu/g and 1.5 x 10 4 log cfu/g.
The difference in the total bacterial counts may be attributed to the samples used, unhygienic method of transportation, handling, processing, unhygienic environment, and practices such as dirty cutting boards and knifes or utensils. Cheesbrough [28] noted that insects also contribute to contamination by mechanical transfer of microorganisms to food products since they are left uncovered and exposed to dust.
There was no significant difference (p>0.05) in total aerobic bacterial count across the three geographical sites under study. This study therefore concludes that roasted and nonroasted African sausages sold in meat outlets in Nairobi County are contaminated with Staphylococcus, Bacillus, Streptococcus, Proteus, and E. coli organisms and poses food safety risks to the consumers. The presence of these organisms in ready-to-eat African sausages is a pointer that these African sausages were either processed under poor hygienic and sanitary conditions and insufficient processing or could have been from the animal intestines. Food safety enforcement authority therefore need to scale up inspection of establishments where African sausage vendors are.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.