PREVALENCE OF ENTEROBACTER AEROGENES IN RAW MILK AND SOME MILK PRODUCTS

Introduction: Enterobacter aerogenes is known as Aerobacter aerogenes and belongs to the family Enterobacteriace . It normally habitats the intestine of the animals so it is widely found in soil, sewage and water. It causes a wide variety of health problems for humans and animals. Objective: The following study was to detect Enterobacter aerogenes in milk, yoghurt and ice cream to assess their hygienic quality. Methods: The number of samples was 300 as follows: raw milk samples, 100 yoghurt samples and 100 ice cream samples. Samples were collected in Assiut Governorate and examined for the presence of E. aerogenes. Results: E. aerogenes were found in 13% of raw milk samples, and 5% of examined yoghurt samples, while detected in 6% of examined ice cream samples. Conclusion: E. aerogenes in milk is an index of direct fecal contamination of milk and milk products which is considered a public health hazard. Consumers and emphasizes must improve their hygienic standards to avoid E. aerogenes .


INTRODUCTION
E. aerogenes known as Aerobacter aerogenes belong to the family Enterobacteriace.About its morphological characters, E. aerogenes is a Gram-negative, facultatively anaerobic, rod-shaped bacillus, and it is motile by peritrichous flagella (Anne Davin et al., 2019).It normally habitats the intestines of large animals so it

1-Collection of samples:
Samples were collected through one year in the period between April 2021 to May 2022.The number of samples was 300 samples as follows: 100 raw milk samples, 100 yoghurt samples and 100 ice cream samples.Samples were collected in Assuit Governate and examined for the presence of E. aerogenes.Samples were collected from farmer's houses and supermarkets in Assuit Governate.The samples were preserved in clean and sterile containers and transferred to the laboratory within 1-2 hours to be examined.

2-Preparation of samples: A) Milk samples:
Ten ml of milk samples were centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded.A loopful from the processed samples was incubated onto the MacConkey's agar for 24 hrs at 37˚C (S.E.Duncan et al., 2012).

B) Yogurt samples:
The samples of yogurt were shaken vigorously to suspend the microbial content.A loopful from these samples was separately inoculated on MacConkey's agar and then incubated at 37˚C for 24 hrs (Salisu M.D et al., 2016)

C) Ice cream Samples:
Ice cream samples were kept in a 45°C water bath, after thawing, 10 ml of ice cream was pipetted out and transferred into a glass bottle.One ml of previously prepared was pipetted out aseptically and transferred into a sterile test tube.This test tube contained 9 ml of distilled water to give a 1:10 dilution.Then the processed samples were inoculated on MacConkey's agar for 24 hrs at 37˚C (Abo El-Makarem et al., 2017).

3-Isolation and identification of E. aerogenes: a-Colony morphology: 1-On MacConkey's Agar:
The presumptive colonies of E. aerogenes appeared on MacConkey's agar as pink, round lactose fermenting colonies.

C. Biochemical reactions of isolates E. aerogenes: 1-Oxidase test: (Baron et al., 1994).
Bacteriological differential discs for oxidase testing were used.It was performed by adding an isolated colony on an oxidase disc.The reaction was observed within seconds as a positive reaction detected by purple-blue color change while a negative reaction which is presumptive for E. aerogenes remained colorless.

2-Catalase activity test: (Baron et al., 1994).
Pure growth was transferred with a sterile loop onto the surface of the glass slide then a drop of 3% H2O2 was placed onto the colony on the glass slide.The evolution of bubbles of gas indicates a positive result which is presumptive for E. aerogenes.

3-Citrate utilization test: (Winn et al, 2005)
Simon's Citrate agar tubes were tightly streaked with a pure culture of the organism.The tubes were inoculated for 24 hrs at 37˚C.blue coloration of the medium indicated a positive result which is presumptive for E. aerogenes.

4-Urease test (Benita Brink et al., 2010)
We used an inoculum from a 20-24 hrs culture to inoculate the broth.The tube was shaken and then incubated at 35˚C.Color change of the broth was observed at 24 hrs.
Urease production was indicated by a bright pink color throughout the broth while the negative result appeared yellow color which is presumptive for E. aerogenes.

5-Motility test (Sagar Aryal et al., 2022).
Bacterial motility was seen by a diffuse zone of bacterial growth which extended out from the line of inoculation.A needle touched a colony of 18-24 hrs then was grown on agar medium and stabbed it into the center of a semisolid agar.Incubate at 37˚C and examine after 24 hrs.A diffuse zone of growth is grown out from the line of inoculation causing cloudiness indicating positive motility which is presumptive for E. aerogenes.

6-Ornithine decarboxylase (MacFaddin J.F et al., 2000)
Pure culture was inoculated for 18-24 hrs in an Ornithine decarboxylase broth tube and overlaid the tubes with 2 ml mineral oil.The inoculated tubes were inoculated at 37˚C for 24 hrs.Changing the color of the indicator in Ornithine decarboxylase broth from yellow to purple or violet is presumptive for E. aerogenes.

b) Yogurt samples:
The total number of positive samples for E. aerogenes in yogurt samples was 5 samples.Table (1).

c) Ice Cream samples:
The total number of positive samples for E. aerogenes in Ice cream samples was 6 samples.Table (1).et al., 2001et al., Song et al., 2010)).E. aerogenes is a notorious hospitalacquired infection, some infections, result from E. aerogenes result from venous catheter insertions.Healthcare workers considered subjects that could transmit E. aerogenes infection.Bacteremia due to E. aerogenes was reported in cases associated with catheters and may cause sepsis and septic shock.(Bilevicius et al., 2001).E. aerogenes causes respiratory tract infections and is implicated in urinary tract infections (Piagnerelli et al., 2002).Also, associated with meningitis (Désinor et al., 2004), especially in people who had neurosurgery and medical devices in surgery.(Fooster et al., 2005;Hamid et al., 2007).The presence of E. aerogenes in milk indicates unfit for human consumption.(Nyein et al., 2002).E. aerogenes are killed by pasteurization, there for it is recommended to pasteurized milk used for human consumption (Branciari et al., 2004).A high count of E. aerogenes in ice cream and yogurt samples may attributed to a lack of attention to personal hygiene, so sanitation routines during production and handling are required.Ingredients must be of high quality especially when it added after heat treatment to ensure that it is free from pathogens.Attention should be given to the equipment that is in contact with yogurt and ice cream in order to be clean and dry (Omnia M. El-Tayeb et al., 2023).The presence of E. aerogenes indicates postpasteurization contamination, unsanitary manufacturing methods, and unhygienic pasteurization.E. aerogenes was reported to be mastitic in cows (Jain et al., 1971).Healthy cows recovered within 14 days of infection but neutropenic cows had necrosis of the mammary gland (Wenz et al., 2001).In the present study, the prevalence of E. aerogenes in raw milk was 13% which was similar to the results obtained by (Vivian Hoffmann et al., 2022) and (Jaime Olivares-Pérez et al., 2015) who reported 12% and 12.5% E. aerogenes in raw milk respectively.On the other hand, high results 17.5% were reported by (Eman A. El-Mokadem et al., 2023), this may be due to a lack of routine sanitation of equipment particularly during handling of milk.In contrast, lower results of 6.54% were reported for E. aerogenes in raw milk by (Azza M.K. Sobeiha et al., 2020), this may be due to good sanitary measures they used.Regarding yogurt and ice cream, the incidence of E. aerogenes was 5% and 6% respectively, similar results were reported by (Azza M.K. Sobeiha et al., 2020) who reported 5.06% in yogurt and 7.27% in ice cream samples.In contrast, higher results 34% were reported for E. aerogenes in Ice cream by (T Masud et al., 1989).On the other hand, lower results 2.6 were obtained by (S. Mathews et al., 2023).

CONCLUSION
In the present study, the results concluded that the sanitary measures during the handling, production and distribution of the examined milk and milk products (yoghurt and ice cream) are neglected.Also, the presence of E. aerogenes is considered an index for fecal contamination of milk and milk products which is a public health hazard.So, Good hygienic measures, sanitation all operating procedures, standardized pasteurization and (HACCP) Hazard analysis and critical control points are effective methods to prevent food-borne infection caused by E. aerogenes.

Table 1 :
E. aerogenes in the examined samples.