Comparative evaluation of the microbial safety of boiled locally vended ready-to-drink, HTST pasteurized and UHT bovine milk sold in Nakawa Division of Metropolitan Kampala

This study evaluated the microbial safety of vended boiled, pasteurized and UHT milk sold in Nakawa, Kampala-Uganda. 15 milk samples were analyzed; 2 samples had Salmonella , 5 had S. aureus with a count of 1.66+0.02 log 10 CFU/ml. E. coli was detected in 8 samples with 1.0+0.02 to 3.0+0.01log 10 CFU/ml count. A high load of 3.0+0.1log 10 CFU/ml was obtained in 3 samples with E . coli . Four E. coli positive samples had a contamination load of 2.0+0.015log 10 CFU/ml of which one was pasteurized milk. Only a pasteurized milk showed a low E. coli load at 1.0+0.02log 10 CFU/ml. All UHT milk had no microbial contamination . Both boiled and pasteurized milk had Salmonella, S. aureus and E. coli in levels above the set threshold limits . Milk consumers in Nakawa stand a potential public health risk of food poisoning reflected by presence of Salmonella, S. aureus and E. coli in some milk sold in the area.


Introduction
Uganda, the pearl of Africa is blessed with a copious produce of milk that is consumed locally either directly or further processed by an unexhaustive list of licensed milk processing companies.Street vendors locally prepare ready-to-do (RTD) milk and vend them amongst other street foods to earn a livelihood.However, inadequately prepared comestible streetvended foods, including milk, meat and their products are implicated vectors for the transit of resistant bacteria and pathogenic genes into humans [1][2][3].Superbugs, such as Staphylococcus aureus (S. aureus), Escherichia coli (E.coli), Listeria monocytogenes and Bacillus cereus in street vended foods have threatened public health and have been an unsurpassable subject of continuous obsessive research [4][5][6][7][8] .Milk is chemically an emulsion of butterfat globules entrained in an aqueous milieu with dissolved biological molecules of life [9] .Albeit its culinary and nutritional completeness, milk and its products ought to be obtained, handled and processed under good and strict sanitary conditions as it is highly ephemeral [10,11] and its contamination thereof is a cause of grave human health concern [12] .Microbial contamination of milk is exacerbated by its complex biochemical profile and high-water activity, which provides a good milieu wherein microbes thrives [1] , thereof triggering milk-borne infections [4,13,14] .
Interestingly, milk possess an inherent antimicrobial mechanism known to beneficially retard microbial growth in the first few hours of milk holding without treatment and/or preservation [15, 16] .Raw bovine milk can reportedly house up to 3.0log10 counts of bacteria per millilitres [16,17] .Etiologic agents, notorious in raw bovine milk are Salmonella (Typhimurium) and other Salmonella serovars, Enterobacter species (spp), Campylobacter spp, Listeria monocytogenes, contagious mastitis pathogens: S. aureus, Streptococcus agalactiae and Mycoplasma bovis, E. coli 0157:H7, Brucella spp, Coxiella burnetii, Proteus spp, Leptospira spp, Corynebacterium ulcerans, Clostridium spp, yeasts and moulds [3,[18][19][20][21][22][23] .Coliforms, including E. coli 057:H7 are innocuous colon inhabitants whose detection in bovine milk are implicatory of faeces-related contamination [11] .An Australian survey in 2007 on 183 raw milk samples recorded a heightened prevalence of E. coli, coagulase-positive S. aureus and Salmonella species at 8% [10] .E. coli O157:H7 peculiarly has been isolated from bovine dairy farms and bulk raw bovine milk globally at 1-33.5% prevalence [7] and reportedly implicated for outbreaks of fatal Hemolytic-uremic syndrome (HUS) and hemorrhagic colitis [18,21] .Microbial milk contamination is multifactorial originating from animal foods, exposure to faecal and environmental contamination as well as the holding containers and water sources [24] and directly from lactating dairy animals with mastitis [25] .Efforts of thermal treatment sought thus is always geared towards the reduction of this microbial loads to none-lethal or better still, safer levels [6] , thereby increasing its shelf life [26] and thus comestibility.The shelf life of pasteurized milk is affected by large numbers of psychotropic bacteria that reportedly predominates the microbial profile of raw milk and which profile positively translate into concentrations of plasmin (a heat-stable protease) and lipoprotein lipase in the milk; activities of these extracellular enzymes augment those of bacterial hydrolases, shortening the storage life of milk [27,28] .Albeit a vast array of microbial pathogens incessantly encountered in raw and processed bovine milk, there is finite published data on the prevalence and levels of pathogens in bovine milk [29] .In Uganda, studies of Wawa et al [7] and Mugampoza et al [8] reported the prevalence of bacteriological contaminants in selected processed bovine milk products vended in selected regions of Uganda.Further, Mugampoza et al [30] reported the microbial load and prevalence of E. coli and Salmonella spp. in ready-to-eat street-vended foods in Nakawa and Naguru parishes of Metropolitan Kampala.This study augmented the aforeacknowledged reports by investigating the microbial load and safety of locally vended RTD, high temperature short time (HTST) pasteurized and UHT milk sold in Nakawa division of Kampala.

Sample size and sample collection
The study was conducted in Banda parish of Nakawa division of Metropolitan Kampala, Uganda between July to October 2018 on a total of 15 representative bovine milk samples bought from local supermarkets and local people vending RTD boiled milk.They included 5 different brands of UHT milk samples (500ml each), 5 assorted brands of pasteurized milk (500ml each), and 5 locally boiled RTD milk obtained from 5 different local people vending RTD milk.All the samples were obtained according to ISO/DIS 707 [31] .Thus, the milk samples were aseptically collected and examined for microbial contamination using ISO Analytical methods at the laboratory of Uganda Industrial Research Institute, Department of Microbiology, 42A Mukabya Rd, Nakawa Industrial Area, Nakawa-Kampala.

Microbial Analysis
Microbial analysis involved isolation, detection and total colony count (TCC) of Salmonella, S.

Isolation and detection of Salmonella in the milk samples
Isolation of Salmonella was carried out using the ISO 6759 [32] standard method for detection and isolation of Salmonella species in dairy and food products.The milk samples were preenriched by inoculating 25ml of the samples into 225ml of Buffered Peptone Water (BPW) followed by serological incubation at 37 0 C for 24 hours.Following pre-enrichment, 1ml of the samples were selectively enriched in 10ml of Selenite Cysteine Broth, SCB (Sigma Aldrich) and incubated for 24hrs at 37 0 C. Selective plating on Xylose Lysine Deoxycholate, XLD (Oxoid, UK) agar was done by streaking a loop full of the selectively enriched contents and incubating at 37 0 C for 48hours.The XLD plates were observed for colonies that appeared pinkish in color with or without a dark or black center of hydrogen sulphite as a characteristic feature for Salmonella.Suspected colonies were then subjected to citrate and urease biochemical tests for identification of Salmonella spp.Citrate test was performed to determine the ability of the isolates previously obtained on XLD plates to utilize citrate as a carbon source.
The isolated suspect microorganisms were subsequently inoculated on Simmon's citrate agar (SCA) slant and incubated at 37 0 C for 48hours.After incubation, the tubes were examined for change in coloration of slant.A change in coloration from green to blue indicate positive test for citrate utilization and thus confirming Salmonella.Urease agar was inoculated with the colonies of the test isolates and incubated at 37℃ for 24 hours.The change of color of the slant from light orange to magenta within the hours of incubation was taken to show the presence of Salmonella.

Isolation and enumeration of S. aureus
ISO 6888-1 [33] laboratory test method for S. aureus enumeration and isolation by spread plate method was used.Briefly, 10ml portion of each sample was diluted with 90ml of sterile Baird Parker Medium to form 10 -1 sample homogenate.1ml of serially diluted samples were used as test portions on Mannitol Salt Agar, MSA (Oxoid, U.K).Then 1.0 ml test portion of 10 -1 sample homogenate was aseptically distributed and spread onto three poured plates of MSA.The plates were incubated aerobically for 48hours at 37 0 C.After the incubation, plates carrying sample portions, colonies of S. aureus appearing to be circular, smooth, convex, moist, 2-3 mm in diameter (on uncrowned plates), above all with unique features of gray to jet-black frequently with light-colored (off-white) margin which is surrounded by opaque zone were enumerated as S. aureus.The total count of S. aureus in each milk sample was computed as the number of organisms of colony forming units per ml (CFU/ml) of samples from equation ( 1)

Confirmation for S. aureus (Slide Agglutination and Catalase test)
Rapid Latex Slide Agglutination (Staphurex kit) and catalase tests were used for the confirmatory identification of presumptive Staphylococcus aureus colonies as per the standard ISO 6888-1&2 [33] procedure.Isolated colonies of the suspected S. aureus were emulsified in a 3.0ml of Brain Heart Infusion broth and incubated at 37 0 C for 18hours.One drop of overnight cultures was mixed with Latex coagulase reagent on the Latex Slide and rocked for 60 seconds observing for agglutination.For catalase test, exactly 0.1 ml of the overnight culture portions were transferred onto clean glass slides followed by equal parts of 3% hydrogen peroxide and observed for effervescence.

Detection of indicator organisms of fecal contamination
Chromocult® Coliform Agar (CCA) was used following Pour Plate Technique for the enumeration of Total coliforms and E. coli [34] .In this method, a 10ml portion of milk sample was aseptically diluted with 90ml of sterilized Buffered Peptone Water (BPW).Serial dilutions from 10 -1 to 10 -5 were aseptically prepared using a 10-fold serial dilution series.Then, 0.1 ml of the homogenate was spread onto prepared plates of Chromocult® Coliform Agar (Pronadisa).Plates were then incubated for 48 hours at 37°C.Blue colonies on CCA were identified and counted as E. coli, whereas purple-pink colonies as other coliforms.A sample of purple-pink colonies from CCA were subjected to indole tube test.Sterilized test tubes containing 4ml of tryptophan broth were aseptically inoculated with a colony and incubated at 37°C for 24 hours.After incubation, 0.5ml of Kovac's reagent was added to the broth culture.
Appearance of pink colored ring was taken as confirmatory presence of E. coli.Colonies were counted promptly after the incubation period.Plates with 0-300 colonies were selected and colonies counted with the use of a colony counter.For the dilution, whose average count (arithmetic mean) of the duplicate plates was near 30 CFU were computed using equation (1).

Results and statistical analysis
The experiment was done in triplicate and statistically significant differences between the microbial load of the milk samples was established using analysis of variance (ANOVA) performed using Minitab statistical software (v6, Minitab Inc., USA).

Salmonella
There was total absence of Salmonella in 13 (86.7%) of the 15 samples analyzed.One (20.0%) sample of the locally boiled RTD milk and one (20.0%)sample of the HTST pasteurized milk was positive for Salmonella.The overall point occurrence of Salmonella in milk samples under this study was 13.3%.

Staphylococcus aureus
Five samples (33.3%) of the 15 milk samples analyzed had Staphylococcus with a mean count of 1.66±0.02log10CFU/ml.Of these, were 3 samples (60.0%) of the locally boiled RTD with a mean count of 1.89±0.01log10CFU/mland 2 (40.0%) pasteurized milk samples with a mean count of 1.46±0.03log10CFU/ml.S. aureus was not detected in all the five (100%) UHT milk samples analyzed.The overall point occurrence of Staphylococcus in the assorted milk samples in this study was 33.3%.

Escherichia coli
E. coli was detected in 8 (53.3%) of the 15 samples analyzed with counts in the range of 1.0±0.02 to 3.0±0.01log10CFU/ml.A high load of 3.0 ±0.01log10 CFU/ml was obtained in 3 (37.5%) of the samples that showed positive for E. coli; all were samples of the boiled locally vended RTD milk.Four (50.0%) of the E. coli positive samples had a contamination load of 2.0±0.015log10CFU/ml of which one sample was a pasteurized milk sample.Only one (12.5%)sample showed a low load of E. coli at 1.0±0.02log10CFU/ml and this was a pasteurized milk sample.All the 5 (100%) UHT milk samples showed negative for E. coli.A significant difference (p<0.05) in occurrence of E. coli was observed between locally boiled RTD and HTST pasteurized milk.
Table 2 Occurrence and mean count of E. coli in the milk samples

Discussion
Thirteen (86.7%) milk samples did not have Salmonella.However, one (20.0%)sample of the locally boiled RTD milk and one (20.0%)sample of the HTST pasteurized milk samples analyzed were positive for Salmonella implying that they are not safe for human consumption since some strains of Salmonella are known to cause food poisoning and salmonellosis in humans [35] .
Five samples (33.3%) of the total samples tested in the study had S. aureus.Three samples (60.0%) of the locally boiled RTD milk had S. aureus with a mean count of 1.81.46 ±0.01 log10CFU/ml while two (40.0%)pasteurized milk samples had a mean count of 1.46 ±0.03log10CFU/ml.A significant difference (p<0.05) in occurrence of S. aureus was observed between locally boiled RTD and pasteurized milk.The study findings on S. aureus in the milk samples indicated a possible health risk because S. aureus produces a heat stable toxin in milk [13] that may lead to food poisoning.More so, its prevalence is of great concern to dairy milk processors as it is seemingly an innocuous microflora of the mammalian mucus, and skin and thus suggests there is deficient hygienic practices during milk processing [35] .The study showed that S. aureus is common in milk in Nakawa and may impose a public health hazard.However, the absence of S. aureus in all the five UHT milk samples analyzed is an indication that the elevated temperature used in UHT treatment is effective in killing S. aureus and such milk is very safe for human consumption.Matter-of-factly, UHT milk should be devoid of microorganisms capable of proliferation at conditions encountered before consumption in their packagings at 25-37℃ for a week or more.

Sample
The presence of E. coli and other faecal coliforms in eight (86.7%) of the total samples analyzed in this study is a clear implication of the possible faecal contamination of the milk [36] probably due to inapt cleanliness while milking, preparing, storing and handling of milk as well as inapt or no reheating and inapt hawked milk temperatures during sale [37, 38].This could be because the hawkers must move around in search of potential customers.All the five (100.0%)UHT milk samples had no detectable E. coli.A significant difference (p<0.05) in occurrence of E.
coli was observed between locally boiled vended RTD and HTST pasteurized milk.These findings imply that consumers of locally boiled RTD milk and some of the pasteurized milk might be at risk of Enterohaemorrhagic infections if the process of boiling and pasteurization are not well effected.E. coli can be destroyed by thorough cooking of foods until 70°C or higher.E. coli O157: H7 is the most important STEC serotype of daily increasing clinical importance in relation to public health.Therefore, boiling should be made effective by ensuring ample holding temperature and the milk should be allowed to cool in a sufficiently clean container to avoid post-processing contamination.Despite the existence of pathogenic strains, the observed presence of E. coli is an alarm indicator of contamination.Strategies should be laid to reduce microbiological contamination during milking and storage to minimize microbial growth and maintain quality milk products.It should be overemphasized that the bacterial contamination observed in some of the pasteurized milk samples in this study is a deleteriously serious observation as the raw milk had undergone heat treatment and is mostly preferred by consumers as UHT treatment causes milk to lose its natural flavour and the economically prohibitive purchasing costs make them not a first choice of some consumers [39].
The results observed with UHT milk in this study are comparable with the report of Nascentes and Araújo [5] who found that the UHT milk in Patos de Minas MG-Brazil had no detectable microbial contaminants.The elevated microbial load of RTD milk could supposedly be due to the entry of the pathogens into the milk through inadequate sanitation during milking, handling, and local preparation.Furthermore, the poor hygienic conditions noted at the sale points for the locally boiled vended RTD milk during sampling might have contributed to such elevated occurrence of the microorganisms in the RTD milk.

Preprints
(www.preprints.org)| NOT PEER-REVIEWED | Posted: 19 November 2018 Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 19 November 2018 doi:10.20944/preprints201811.0445.v15.0 Conclusions and recommendations Both boiled RTD and pasteurized milk in this study had S. aureus, E. coli and Salmonella in levels above the threshold limits by WHO and FDA and therefore poses a potential public health risk.The bacteriological profile of UHT milk samples in the study conformed to the safety requirements as set by FDA and WHO.E. coli is more prevalent than S. aureus and Salmonella in the milk samples investigated in this study.Local milk consumers in Nakawa Division stand a potential public health risk of food poisoning as shown by the presence of S. aureus and Salmonella encountered in the tested samples.Due to the observed presence of pathogens in the milk samples, proper handling and preservation of milk should be observed with respect to proper hygiene as recommended by WHO.Public health sensitization programs in milk handling should be launched.From a food safety point of view, food handlers at different points in the milk value chain should be educated on how to reduce contamination of milk and milk products from Staphylococcus species and other pathogens through personal protective equipment hygiene as well provision of cold-chain equipment in the milk supply chain.A comparative evaluation of the microbial safety of other milk brands on the market should be assessed using a larger sample size.

Table 1
Occurrence and mean count of S. aureus in the milk samples a Mean count are presented as Mean±S.E (Standard error) of experiments done in triplicate.Figure 1 XLD plates positive for Salmonella Figure 2 Baird Parker Agar plate positive for S. aureus Preprints (www.