Microbial Profile of Fresh and Semicooked Nile Tilapia (Oreochromis niloticus) and Hygienic Practice of Fish Handlers in Hawassa, Ethiopia

Despite its high nutritional quality, fish is a highly perishable food item. This study aimed at assessing the microbial quality and safety of fresh and semicooked Nile tilapia fish fillets and the food safety practices of fish handlers in Hawassa City. The microbial load of 40 for each of raw and semicooked fillet samples was estimated by the standard plate count method, and the dominant flora as well as common bacterial pathogens were identified following phenotypic procedures. Moreover, a survey was conducted to assess the hygienic conditions and food safety practices of 30 fish handlers. The mean microbial load of the raw fillet samples in log10CFUg−1 was 8.42 for aerobic mesophilic bacteria (AMBC), 2.52 for total coliforms (TCC), and 3.41 for a count of staphylococci (CS). On the other hand, the respective parameters for the semicooked fillets in log10CFUg−1 were 6.68 (AMBC), 2.52 (TCC), and 3.17 (CS). The mean AMBC of all the fresh raw fillet samples exceeded the recommended maximum permissible limits. The mean SC of raw fillets from three of the eight vendors and one semicooked fillet were at a potentially hazardous level (>4 log units). Moreover, Salmonella species were isolated from 30% to 25% of raw and semicooked samples, respectively. The mesophilic bacterial flora of both types of samples was dominated by Bacillus species, Salmonella species, E coli, and Staphylococcus species. Most fish handlers did not practice hygienic food handling and lacked basic sanitation amenities like clean water and soap for hand washing. Moreover, nearly all the fish handlers did not have any formal education. These findings call for public health intervention measures like the provision of training in good hygienic practices and certification for fish vendors in the chain.


Introduction
Fish is an important part of a healthy diet as it provides highquality protein, and omega-3 fatty acids [1,2].Globally, fsh contributes about 60% of the world's supply of protein [3].Owing to its digestibility, fsh protein complements diets provided by cereals and legumes that are typically consumed in many developing countries.Fish is also a valuable source of vitamins A and B and iodine [4].Moreover, a fsh diet is known to reduce the risk of heart disease, infammation, and cancer [5,6].Recognition of these nutritional and health benefts provided by the fsh diet is such that world per capita fsh consumption increased from 9.9 kg in the 1960s to 20.1 kg in 2016 [7].
However, in many African countries, including Ethiopia, fsh production and consumption are very low.Being a landlocked country, Ethiopia depends for its fshery on inland water bodies, which include an estimated surface area of 7,334 km 2 of major lakes and reservoirs, 7,185 km of rivers, and 275 km 2 of small water bodies [8].Te total annual fsh production potential of the country is about 51,481 tons [9].Tese estimates did not consider the potential of newly developed reservoirs and dams being constructed.A more realistic estimate of the fshery production potential of the Ethiopian inland water bodies puts it between 89,000 and 99,000 tons per year [10].Te inland capture fshery provides nearly all the fsh supplies in Ethiopia, with a barely budding aquaculture supply starting recently [7].Te country's overall per capita fsh production is less than 480 g per person per year [7].However, in areas where there is a regular and sufcient supply, annual fsh consumption can exceed 10 kg per person per year [11].According to Christophe and Damien [8], about 73% of the total fsh landed was marketed as fresh in nearby markets, while 26% was transported to distant areas as chilled or frozen, and only 1% of the products were processed in canned, smoked, or dried forms.Lake Hawassa is the major source of fsh supply for the residents of Hawassa City, the administrative center of the Sidama Regional State of Ethiopia (SRSE).Te lake has an estimated surface area of about 90 km 2 , with a potential for 600 metric tons of fsh production per year [8].Fishing activity in Hawassa is mainly an artisanal activity operated with basic rafts made of papyrus and wooden canoes, the use of hook lines, and some nets.Among the fsh types supplied from the lake, Nile tilapia (Oreochromis niloticus) is the most popular one.Te main customers are the many restaurants that serve the fsh in diferent recipes, either fried or raw.Fish dishes are special attractions for local and foreign tourists, particularly during lent and normal fasting days (Wednesdays and Fridays).Tese facts have led to increasing demand and created a lucrative business for mushrooming small-scale fsh vendors.Raw fsh fllets ("AsaKurt") and semicooked fsh fllets ("AsaLebleb") are among the most popular fsh dishes.
Despite all these positive vibes, there are many concerns, as fsh is one of the most perishable food items that deteriorate rapidly and may also serve as a vehicle for the transmission of food-borne pathogens [12,13].Te spread and persistence of human pathogens in fsh and fsh products and the formation of hazardous toxins caused by the growth of spoilage microorganisms are major concerns.Clostridium botulinum, pathogenic Vibrio spp., Aeromonas spp., and Plesiomonas shigelloides are examples of human pathogenic bacteria that may be inherently present as part of the fsh's normal fora [14][15][16].Moreover, contamination of fsh from external sources during processing and storage may add pathogenic bacteria like Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Shigella spp., Escherichia coli, and Yersinia enterocolitica [17].Studies have demonstrated the occurrence of potentially pathogenic and spoilage bacteria in fsh, including Pseudomonas angulluseptica and Streptococcus spp [3].
Several factors may contribute to the microbial deterioration of the quality and safety of fsh, including lack of proper transportation facilities, ignorance, poor handling, and processing, among others [18,19].Fish production and distribution in Ethiopia are carried out both formally (e.g., through organized cooperatives) and informally by private individuals [20].According to some estimates, there are collectively about 67,400 fshermen operating among the diferent inland water bodies in Ethiopia [10].Updated information about the number of people engaged in the sector for their livelihood is unavailable; nonetheless, one would expect a steady increase over the decade since.
Similar to most developing nations, Ethiopia's fsh supply chain primarily follows an informal market system with limited supply chain management for fsh quality and a lack of information for price determination along the chain [21,22].Under these conditions, the supply chain faces difculties in maintaining quality and tracing the paths of the products when spoiled or substandard products reach the consumer.
Experiences in other parts of the world have shown that fsh and fsh products have been involved in various foodborne disease outbreaks and product recalls annually [13].Te outbreak of fshborne disease could afect not only direct consumers but also have a ripple efect on the hotel and tourism industries by tainting the positive image of the country.From an economic perspective, the fsheries subsector contributes very little to the overall GDP in Ethiopia [10].Tis would defnitely change if spoilage and postharvest losses were minimized by installing good manufacturing and hygienic practices at the various steps along the production and supply chain [19].Te objective of this study was to assess the microbial quality and safety of raw and undercooked Nile tilapia fllets as well as the food safety practices of fsh handlers in Hawassa city, southern Ethiopia.

Description of the Study Area.
Te study was conducted from November 2017 to June 2018 in Hawassa City, the administrative center of the Sidama Regional State of Ethiopia (SRSE).Hawassa is located on the southeastern shores of Lake Hawassa, 275 km south of Addis Ababa, within the geographic coordinates 06 °58′-07 °14′ N latitudes and 38 °22′-38 °28′ E longitudes [23].Te city is one of the hotspots of tourist destinations for locals as well as foreigners and consequently hosts dozens of international hotels and resorts.Te samples for the study were collected from Lake Hawassa landing sites and its surrounding hotels.

Study Design.
A cross-sectional study was conducted based on the laboratory analysis of the microbiological profles of raw and semicooked Nile tilapia fllet samples collected from randomly selected hotels in Hawassa and lakeside fsh vendors.Moreover, a questionnaire survey of randomly selected fsh handlers was done to assess the current status of food safety practices.

Sample Collection and Culture Media Preparation.
A total of 80 (40 fresh raw and 40 semicooked) Nile tilapia fllets were collected in 20 trips from 10 randomly selected registered hotels and lakeside fsh vendors.From each vendor, four fresh, raw, and four semicooked fllet samples were considered.On each trip, Nile tilapia fllet (one fresh raw and one semicooked) samples were collected from one registered hotel and one lakeside fsh vendor.All samples were collected aseptically using a sterile stainless-steel box, labeled with the type of sample, place, date of sampling, 2 International Journal of Microbiology given an identifcation code, and transported to Hawassa University Microbiology Laboratory in an icebox containing ice packs.Upon arrival, the samples were stored in a refrigerator until processing within two hours of collection.All media used in this study were prepared according to the instructions of the manufacturers (Himedia Laboratories, India).

Preparation of Decimal Dilution of the Fish Fillet Samples.
Each sample was prepared following the protocol for the standard plate count method [24].Briefy, each cutlet of the fllet sample was well mixed, and 10 g pieces were aseptically transferred using sterile forceps and a dissecting scalpel into a labeled bottle containing 90 mL of sterile bufered peptone water as a diluent to make a tenfold dilution.Te bottle was vigorously agitated manually and by a vortex mixer for 5 minutes to release the attached microbes from the fllet pieces.Further, tenfold serial dilutions were done up to 10 −7 by transfer of 1 mL aliquots aseptically using a sterile micropipette to test tubes containing 9 mL bufered peptone water [25].Vortex mixing was done between each transfer into the tubes to ensure uniform homogenization.Te pH of the sample was measured from the 10 −1 dilution using a digital pH meter (digital pH, model 181).

Enumeration of the Microbial Load of the Fillet Samples.
For aerobic mesophilic bacterial count (AMBC), 0.1 mL aliquots from 10 −6 and 10 −7 dilution tubes were transferred on plate count agar (PCA, HiMedia) using a sterile micropipette and spread-plated with a sterile bent glass rod in duplicates.Ten, the inoculated plates were incubated at 37 °C for 48 hrs.For the enumeration of total coliforms (TCC) and Staphylococcus aureus, 0.1 mL aliquots from dilution factors of 10 −2 and 10 −3 were spread plated on violet red bile agar (VRBA, Hi Media) plates and manitol salt agar (MSA, HiMedia) plates in duplicates, respectively.Morphologically typical colonies (pink colonies on VRBA and golden yellow colonies on MSA) were counted from countable plates using a colony counter with a magnifying glass.Plates having between 25 and 250 colonies were considered to calculate the average load in colony-forming units per gram of fsh fllet samples (CFUg −1 ) [26].Similarly, the proportion of fecal coliforms (Escherichia coli) represented in the TCC was assessed by picking fve to ten typical colonies (pink pigmented) from countable VRBA plates and purifcation by subculturing on MacConkey agar and Eosin methylene blue agar (EMB) plates.Fecal coliform bacteria, such as type 1 E. coli formed pink colonies on MacConkey and black colonies with a green metallic sheen on EMB agar plates.Te standard IMVIC biochemical tests were used for confrmation when necessary.Isolates that were Indole positive, methyl red positive but negative for Voges Proskauer, and citrate were identifed as E. coli [27][28][29].

Identifcation of Dominant
For the detection of Salmonella species, a loop-full sample from 1 : 10 dilution was streaked onto the surface of Salmonella and Shigella agar (SSA).Streaking on the same media was also done after overnight culture of 0.1 mL of a 1 : 10 dilution aliquots into a tube containing 5 mL of sterile nutrient broth.Te latter method was done to resuscitate cells that might be in a viable but not cultivable state due to metabolic injury.At the end of the incubation, well-isolated typical colonies (colorless colonies with a black center) were picked and purifed by repeated subculturing, and their colony morphology was noted.Presumptive purifed isolates were subjected to a biochemical test for confrmation [27][28][29].

Data Analysis.
All data were recorded, and basic statistics for diferent datasets were analyzed using SPSS version 20.To calculate the average load from multiple plates, the following formula was used [24]: where  C is the sum of colonies counted on all the dishes retained; n 1 is the number of dishes retained in the frst dilution; n 2 is the number of dishes retained in the second dilution; and d is the dilution factor corresponding to the frst dilution.Final values were transformed into log 10 colony forming units per g (log 10 CFUg −1 ) for ease of manipulation and comparison.Descriptive summary values like average, range, and percentages were used to compare microbial load values within and between the sample types.Te paired T-test was used to compare the microbial loads of the raw and semicooked fllet samples, and p < 0.05 was used to decide on the statistical signifcance of the observed diferences.

Aerobic Mesophilic Bacterial Count (AMBC).
Te aerobic mesophilic bacterial counts (AMBC) of the raw/fresh fllet samples ranged from 6.68 to 9log 10 CFUg −1 with the mean value being 8.53 log 10 CFUg −1 (Table 1).Tese values are beyond the recommended acceptable criteria of 5.60-7.00log 10 CFUg −1 for raw fsh fllets [30].Mean AMBC in raw International Journal of Microbiology fsh fllets exceeding this value implies gross contamination as a result of poor hygienic conditions of the water source and mishandling during processing.Te range of AMBC for the raw fllet samples in this study was in agreement with the 3 × 10 7 to 4 × 10 9 CFUg −1 of raw fllet samples reported from Khartoum state, Sudan [31].On the other hand, it was higher than the reported 6.67log 10 CFUg −1 for samples from Arbaminch, [32], the 6.79 log 10 CFUg −1 for samples from Jimma, Ethiopia [33], and the 7.57 log 10 CFUg −1 reported for freshwater fsh fllets from Latvia [34].More recently, a similar study for fresh raw fllet samples of diferent fsh species during the hot summer season in Mauritius revealed mean AMBC ranging between 3.1 × 10 5 CFU/g and 1.4 × 10 7 CFUg −1 [35], which is also lower than the values in the present study.
Considering the semicooked fsh fllet samples, the average AMBC ranged from 6.38 to 6.84 log 10 CFUg −1 with the mean being 6.67 log 10 CFUg −1 (Table 1).Tis value was signifcantly lower (P < 0.05) than that for the raw, fresh fsh fllet samples (Table 2).Tis apparently refects the efect of cocking temperature and perhaps the lower pH of the semicooked fsh sample.Te pH is an important intrinsic factor related to fsh fesh and infuences freshness because it infuences bacterial growth [36].Te pH values of semicooked and fresh fsh samples ranged from 5.33 to 6.42 and 6.20-6.68,respectively (Table 1).Te variations in pH values may be due to physiological conditions, the degree of antemortem activity, or stress [37].Te lower the pH of the fsh fllet, the slower is the bacterial growth, and vice versa [38].Te higher pH in the raw fllets might be due to higher total volatile bases (TVB) that resulted from the decomposition of nitrogenous compounds by endogenous or microbial enzymes, and this corroborates with the increase in TVB-N [38].Such an increase in the pH indicates bacterial growth, loss of quality, and incipient spoilage [39].
According to the guidelines by the Institute of Food Science and Technology, London, UK, the average AMBC of heat-treated fsh prepared under good manufacturing practice should be less than 4 log units [40].However, all the semicooked fsh samples in the present study showed values above six log units.Tis fnding implies that the initial microbial load in the raw fsh fllets was so high that normal cooking temperatures were not sufcient to reduce the load to an acceptable level.
Te average AMBC of the semicooked fsh samples in this study was slightly greater than the 8.344 × 10 5 −3.108 × 10 6 CFUg −1 range of AMBC reported for smoked fsh samples [41].However, it was lower than the 1.5 × 10 5 −1.6 × 10 8 CFUg −1 range of AMBC reported for cooked fsh samples sold in Khartoum state, Sudan [31].Te variations among the studies are perhaps refections of diferences in the quality of the source water bodies and the concomitant microbial loads of the initial raw products and handling procedures.How long the raw product was stored before cooking also has a bearing since it is common practice to stock supplies in catering establishments.
According to the International Commission on Microbiological Specifcations for Foods [30], when the mean AMBC of fresh raw fllets exceeds the value of 6 log units, it suggests inadequate refrigeration or long storage under refrigeration [40].It is not uncommon among food caterers to stock supplies of fsh fllets in refrigerated storage during times of high production for use in times of scarcity.Te mean AMBC of both types of fllet samples in the present study exceeded this level.Likewise, according to Croatian microbiological standards, the maximum permissible AMBC limit for fresh/frozen fsh is 3 log units [42].All 4 International Journal of Microbiology the fsh samples in the present study showed AMBC levels two-fold higher than this limit.Usually, the muscles and internal organs of healthy fsh are sterile [16].However, various levels of contamination are inevitable during evisceration and processing by either the intestinal or skin fora of the fsh, as well as from the fsh handler, environment, and utensils.

Staphylococcus aureus Count (SC).
Te mean Staphylococcus aureus counts (SC) of the raw fsh fllet samples ranged from not detectable (zero) to 4.3 log 10 CFUg −1 with the mean value being 2.85 log 10 CFUg −1 (Table 1).Tis value is lower than the 6.4 log 10 CFUg −1 for raw fsh fllet samples sold in Jimma, Ethiopia [33] and the 2.7 × 10 4 CFUg −1 -1.23 × 10 5 CFUg −1 values reported for fresh fllets from Mauritius [35].In the present study, S. aureus was detected and counted in 34 (85%) of the raw fllet samples (Table 1).Tis value is much higher than the 65% prevalence reported for frozen raw fllet samples collected from Lake Abaya and Chamo in Arbaminch [32].Te diference in prevalence might be due to diferences in hygienic handling during processing as well as treatment received.Staphylococcus aureus is capable of producing enterotoxin and is noted to survive for extended periods in hostile environments [43].
According to recommended guidelines [44], when the count of S. aureus in ready-to-consume foods is in excess of 4 log units, the item should be regarded as potentially hazardous.
In general, S. aureus is not considered part of the normal fora of fsh [45], and its presence in fsh fllets therefore likely refects contamination due to unhygienic handling practices, cross-contamination from equipment, or processing the environment [46].Te isolation of enterotoxigenic S. aureus from raw fsh fllets was reported at varying rates elsewhere, including in 6.5% of samples in Egypt [47], in 17.75% in Iraq [48], and in 24.47% of samples in India [49].
In the absence of refrigeration, the initial contaminants can multiply to a sufcient number to cause food poisoning.Te presence of Staphylococcus aureus in ready-to-eat food items at high counts suggests poor handling and processing and should be considered a public health concern in populations where the consumption of raw fsh fllets is rampant [38,50].According to a recent survey in the central part of Ethiopia, more than three-fourths (77%) of the consumers preferred consuming raw fsh [51].
Concerning the semicooked fsh samples, the average SC ranged from not detectable (zero) to 4.2 log 10 CFUg −1 , with the mean being 2.63 log 10 CFUg −1 (Table 1).Tis value was slightly lower than the mean SC of the raw fllet samples.Te observed diference in mean SC between the two types of fllet samples was not statistically signifcant (Table 2).Te relatively lower SC in the semicooked fsh samples could be due to the combined efect of cooking and the lower pH value.However, it should be noted here that enterotoxinproducing strains of S. aureus can grow in a substratum with a pH range of 4 to 10 [52].As in the case of the raw fllet samples, S. aureus was detected and counted in 85% (34 of 40) of the semicooked fllet samples (Table 1).Eight (20%) of the semicooked fllet samples showed hazardous levels of mean SC that exceeded 4 log units (Table 1).

Total Coliform Count (TCC).
Te total coliform count (TCC) of raw/fresh fllet samples ranged from not detectable (zero) to 3.2log 10 CFUg −1 with the mean value being 1.51 log 10 CFUg −1 (Table 1).Coliform bacteria were not detectable in two of the semicooked samples and four of the raw/fresh fllet samples (Table 1).Te mean TCC in the present study was lower than the 6.82 log 10 CFUg −1 value reported for fresh fllets sold in Jimma, Ethiopia by Shiferaw et al. [33].Te fnding of coliform bacteria like E. coli in numbers exceeding 100 per gram in any type of ready-to-consume food is considered to indicate poor sanitary quality [30].
With regard to the semicooked fsh fllet samples, the mean TCC ranged from not detectable (zero) to 3.2 log 10 CFUg −1 with the mean value being 1.79 log 10 CFUg −1 (Table 1).Tis value is slightly lower than the mean TCC value for fresh raw fsh fllets, although the diference was not statistically signifcant (Table 2).

Incidence of Salmonella Species and Escherichia coli.
Te incidences of Salmonella species and E. coli in the fresh, raw, and semicooked fllet samples are presented in Table 3. Te overall incidences of Salmonella species and E. coli in the fresh, raw fllet samples were 30% and 35%, respectively.On the other hand, the incidences of the respective bacterial species in the semicooked fllet samples were 25% and 7.5%.Te higher incidences of the Salmonella species and E. coli in International Journal of Microbiology raw fresh fllet samples than in the semicooked one is commensurate with expectations since heat treatment, albeit partial, is likely to reduce their survival.Te widespread occurrence of members of the Enterobacteriaceae, including pathogenic E. coli and Salmonella species in tropical aquatic environments is well known [46].Te major inland water bodies in Ethiopia, including Lake Hawassa, are situated near major urban areas, where contamination from leaching pit latrines and septic tanks as well as fooding infows of municipal sewage, is quite common.It is not surprising to encounter E. coli and Salmonella species in fsh sourced from these water bodies.Te incidence of Salmonella species in the raw fllet samples in the present study was higher than the 7.5% incidence reported by Teka et al. [31] for fresh fllet samples from Arbaminch, southern Ethiopia.Tesfaye et al.
[53] also reported the fnding of salmonellae in 5.17% of the skin, gills, and intestine of fsh samples from Lake Haike in northern parts of Ethiopia, which is lower than the incidence in the present study.Likewise, Ohalete et al. [54] also reported a lower incidence (20%) of Salmonella species than that of the present study for fresh fllet samples from Nigeria.On the other hand, Hiko et al. [55] reported the fnding of salmonellae in the kidneys and oral cavities of 30 fsh samples from Lake Tinike, Eastern Ethiopia.Tough the isolation was from specifc fsh organs, the incidence rate is in concordance with the present study.Te observed differences in the incidences among the diferent studies may refect diferences in the analytical methods, the pollution level of the source lakes, and the sanitary and hygienic condition of the processing by the vendors.With regard to E. coli, the 35% incidence in the raw fresh fllet samples in the present study is lower than the 42.5% incidence reported for frozen Nile tilapia fllet samples from markets in Arbaminch, Ethiopia [31].On the other hand, Tilahun and Engidawork [56] reported the isolation of E. coli from 11 of 131 (8.4%) swab samples from the muscles of diferent species of fresh fsh samples in Hawassa, which is lower than that of the present study.Elsewhere, Rocha et al. [57] reported a 9.09% incidence of E. coli from fresh fllet samples in Fortaleza, Brazil.Elsherief et al. [58] also reported a 12% incidence of E. coli from fresh fllet samples, which is lower than that of the present study.While there existed diferences in the analytical methods used among the studies, the higher incidence of E. coli in the fresh fllet samples in this study implies gross pollution of the source lake as well as poor handling, preparation, and storage practices.
E. coli is a fecal coliform bacteria that usually originates from the guts of warm-blooded animals and is not part of the normal bacterial fora in fsh [57].Owing to this, E. coli is widely used as an indicator of sanitary conditions.Te fnding of a high incidence and above certain levels per gram is considered to refect the contamination of fsh or the source water with human and animal feces.Trivial observation of the surrounding area of Lake Hawassa, especially at some landing sites reveals open defecation, washing, bathing, and swimming as widespread daily activities along the shoreline and possible seepage of untreated sewage from nearby residential areas due to the absence of a protective bufer zone that separates the lake from drainages and food.In addition to being an indicator of sanitary quality, several pathovars of E. coli are known to cause diferent types of diseases in humans with varying degrees of severity.Among these, the food-borne pathogenic E. coli serotype O157: H7 has been recently reported to be isolated from diferent species of fsh samples from Lake Hawassa [56].Although this study does not include the determination of serotype, the existence of E. coli O157: H7 in Lake Hawassa should be of great concern as it is known to cause hemorrhagic colitis and life-threatening hemolytic uremic syndrome [59][60][61][62].
Likewise, the majority (43/55 or 78.18%) of the AMB isolates from the semicooked fsh samples were grampositive bacteria, dominated by Bacillus species (15/43 or 34.88%) and unidentifed, nonspore-forming, gram-positive rods (23/43 or 53.5%).Staphylococci and streptococci constituted the minor gram-positive genera, each represented by less than fve isolates.Te remaining nine AMB isolated from the semicooked fsh samples were gramnegative bacteria, belonging to E. coli, Salmonella, and Pseudomonas species, each represented by two isolates, and  1).Several factors afect the number and diversity of microbes found in fsh, including the geographical location, the season, and the method of harvest [63].Te bacterial load and diversity of fsh in warm tropical lakes are known to be higher and more diverse than those in temperate zones.In addition to the natural fora of the lakes, the fsh fllets pick up more bacteria as they proceed in the handling chain from evisceration to cleaning and retail market from handlers, utensils, and the environment.Te widespread open defecation of local residents and the unrestricted access that cattle have to freshwater lakes and reservoirs, as well as the exposure of the water bodies to contaminated water run-of, are all potential causes of contamination of the habitats used naturally for the production of fsh.In one recent study, it was reported that there is a dramatic worsening of pollution levels in the Lake Hawassa Watershed due to urbanization, the usage of fertilizers in agricultural lands in the suburbs, efuents from industrial facilities, excessive usage of detergents in domestic and industrial facilities, soil erosion, increased sewage pollution, the practice of open defecation and urban runof [64].
Te result of this study revealed that common pathogenic bacteria were associated with fresh and semicooked fllet samples.In a similar study carried out by Moshood and TengkuHaziyamin [65], Bacillus subtilis, Staphylococcus aureus, Proteus mirabilis, Klebsiella sp., Salmonella typhi, and Streptococcus sp. were all found to be associated with smoked and raw fsh.Te presence of Staphylococcus aureus was attributed to the contamination of the fsh samples by humans through handling and processing.Staphylococcus aureus is capable of producing heat-stable enterotoxin and is noted to survive for extended periods in hostile environments [50].Consumption of food that contains a high number of enterotoxigenic strains could cause gastroenteritis in susceptible individuals.Te presence of Salmonella species in semicooked fsh indicates poor hygiene handling practices and postprocessing contamination [66].
3.6.Food Handling and Hygienic Practices.Food handling and processing are among the main sources of food contamination through poor hygienic practices.In this regard, all food handlers have a basic responsibility to have a high degree of personal hygiene and safe handling practices.However, the result of this study showed that 63.33% of the vendors wash their hands using clean water and soap, but 36.67%wash their hands only with water.Besides this, none of the vendors use antiseptics or disinfectants (Table 4).Te nails of 43.33% of the fsh handlers in this study were not shortly trimmed and cleaned.Tis fnding is higher than the result of Teklemariam et al. [67] who found that 21.2% of food handlers in Hawassa city hotels did not trim their nails, but lower than the 46.5% reported in the study carried out in Arbaminch, southern Ethiopia [31].
Moreover, 43.34% of the vendors did not cover their hair during food preparation.Te present result is consistent with that of Kumie et al. [68] and Wendwesen et al. [31] who reported that 60% and 55.8% of the participants in Ziway and Arbaminch towns, respectively, did not cover their hair during the preparation of food.On the other hand, the proportion of fsh vendors who did not cover their hair during food handling in the present study was lower than the  International Journal of Microbiology reported 98.2% for vendors from Hawassa in a previous study [67].Remarkably, 63.33% of the respondents cleaned their utensils with water and soap, while 36.67%washed their utensils regularly with water only.In addition, the survey showed that 33.3% of the fsh handlers did not have separate work clothes for food preparation, and only 46.66% of them washed their work clothes regularly (Table 4).Tese fndings are concordant with those reported by Teka et al. [31], where only 32.6% of participants washed their clothes.Work clothes should be kept clean and stored separately from private clothes.Otherwise, there is a risk of bringing various types of contaminants, including pathogens, to the fsh handling locations, enhancing the risk of crosscontamination.Poor socioeconomic conditions, a lack of infrastructure, and ignorance are among the major challenges to fshery management problems in Ethiopia [18].
According to Adams and Moss [69], providing training for food handlers regarding the basic concepts and requirements of personal hygiene and sanitary handling of food plays an integral part in ensuring a safe product for the consumer.However, the result of this study showed that 96.66% of respondents had poor awareness of the consequences of microbially contaminated fsh, and none of the fsh vendors had professional training related to the safe handling and processing of fsh fllets.Circumstantial observations on the lakeside landing sites showed that most of the fsherman handled and processed fsh in unhygienic conditions near the lake shore.Tey were all performing the task without access to basic processing equipment, such as a clean, room or area, clean water, a facility for washing and disinfecting equipment, a place to wash one's hands, or a facility for handling products.All fsh were frequently processed with a single knife and the same cutting board, with infrequent washing and no disinfection in between.It is not uncommon to see some fshermen manually eviscerating fshes, removing the skin, and washing them with lake water.
Although there are some policies and legislation governing the fsheries sector in Ethiopia, they are poorly implemented.Te federal fsheries Proclamation No. 315/ 2003 for Fisheries Development and Utilization [70] was aimed to "conserve fsh biodiversity and its environment" as well as prevent and control overexploitation of the fsheries resource, increase the supply of safe and good-quality fsh, ensure a sustainable contribution of the fsheries toward food security, and expand the development of aquaculture."Tis most recent legal document that is specifcally related to the fsheries sector has these goals.Ethiopia has also endorsed the FAO Code of Conduct Article II 1998s which states that member states should adopt appropriate measures to ensure the right of consumers to safe, wholesome, and unadulterated fsh and fshery products.In the absence of vigilance in the enforcement of safety and quality standards in fshery production and supply chains, periodic cross-sectional studies like this may serve as early warning alarms [71].

Conclusion
Tis study has revealed that both fresh raw and semicooked fsh fllets marketed in Hawassa had a higher microbial load than recommended levels.Te mean microbial load of the fresh raw fllet samples was signifcantly higher than that of the semicooked fllet samples.Tis fnding implies that the microbial load in the raw fllets was so high that the normal cooking temperature was not sufcient to reduce the load in the ready-to-eat item to an acceptable level.Te aerobic mesophilic bacterial fora of both types of samples was dominated by genera known to be food-borne pathogenic bacteria.Staphylococcus aureus and Escherichia coli are potential toxigenic pathogens and are known to cause food 8 International Journal of Microbiology poisoning.Te detection of Salmonella and Shigella in fresh fsh samples should be of great public health concern in communities where the consumption of raw fllets is rampant since they are known to cause life-threatening infections.Several factors may be speculated to contribute to the contamination of fsh fllets, including poor sanitary quality of the water in the source lake, unhygienic processing, improper storage, transportation, poor handling during preparation, and display for sale.Te survey data on the level of awareness and hygienic practice of fsh vendors and handlers revealed a lack of training on the proper handling and processing of food, poor personal hygiene of vendors, and unhygienic surroundings could be possible factors for the observed problems.

Figure 1 :
Figure 1: Te frequency of the dominant aerobic mesophilic bacterial genera in raw fresh and semicooked Nile tilapia fllet samples marketed in Hawassa, southern Ethiopia, 2018.

Table 1 :
Te microbial load in log 10 CFUg −1 and pH of fresh raw fsh fllets and semicooked fsh fllets from diferent sites in Hawassa city, southern Ethiopia, 2018.

Table 2 :
Comparison of the mean microbial load (AMBC, TCC, SC) and pH values based on the student's T-test for the fresh raw fsh fllets (n � 40) and semicooked fsh fllets (n � 40) in Hawassa, southern Ethiopia, 2018.� indicates the observed diferences in mean microbial load of fsh fllets samples for the respective parameters between fresh raw fsh fllets and semicooked fsh fllets is statistically signifcant by an independent t-test, at p value <0.05.

Table 3 :
Frequency of detection of Salmonella and E. coli from raw and undercooked Nile tilapia fllets.

Table 4 :
Awareness level regarding microbial contaminants, hygiene in preparation, and food safety among fsh handlers (n � 30) in Hawassa city.