Sobeih et al.,2020, KVMJ, 18 (2): 9-13, DOI: 10.21608/kvmj.2020.39992.

1009

Kafrelsheikh Veterinary Medical Journal

(https://kvmj.journals.ekb.eg/)

Received Aug 21, 2020

Original Article Accepted Sep 16, 2020
Published Oct 09, 2020

Prevalence of Enterobacteriaceae in raw milk and some dairy products

Azza M.K. Sobeiha, Ibrahim I. Al-Hawaryb, Elsayed M. Khalifac and Nahla A. Ebiedc

a
Department of Food Control, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt
b
Department of Fish Processing and Biotechnology, Dean of Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University,
Egypt
c
Animal Health Research Institute, Kafr El-Sheikh Branch, Egypt
*Corresponding author: [email protected]

Abstract
Objective: This study was performed to detect and identify members of Enterobacteriaceae in raw milk, yoghurt and ice cream to assess their
hygienic quality.
Methods: A total number of 200 samples distributed as follows:100 raw milk, 25 small and 25 large scale plain yoghurt, and 25 small and 25
large scale ice cream samples collected from different localities at Kafr El-Sheikh Governorate and examined for Enterobacteriaceae count and
identification of members.
Results: Enterobacteriaceae were detected in 84% of examined raw milk samples (with mean count of 1.02x106 + 1.98x105 cfu/ml), 40% and
32% of examined small and large scale plain yoghurt samples (with mean value of 6.95x103 + 4x103 and 1.57x103 + 8.95x102 cfu/g), while
detected in 64% and 20% of examined small and large scale ice cream samples (with mean count of 1.02x104 + 4.13x103 and 1.4x103 + 8.26x102
cfu/g). The most frequent members isolated were Hafnia alive (30.95%), E. coil (44.44%), and Serratia marcescens (57.14%) from positive raw
milk, yoghurt, and ice cream samples, respectively. Other members of Enterobacteriaceae were isolated from positive samples at different
percentages.
Conclusion: Presence of members of Enterobacteriaceae is a reliable index of fecal contamination and the possibility of the presence of
associated enteric pathogenic organisms. So, it is recommended to specify Enterobacteriaceae as a sanitary index of milk and dairy products in
the Egyptian food acts and regulations.
Keywords: Enterobacteriaceae, raw milk, yoghurt, ice cream

1. Introduction
Enterobacteriaceae is a large, heterogeneous group of Gram-negative and dairy products made from raw milk may be caused by Shiga toxin-
rods, which naturally inhabit the intestinal tract of both humans and producing Escherichia coli (STEC), Salmonella spp., and Yersinia
animals. They include many genera (such as Escherichia coli, enterocolitica (Proctor and Davis, 2000; Mazurek et. al.2004; Jayarao et.
Shigella, Salmonella, Enterobacter, Klebsiella, Serratia, and al.,2006 and Cancino-Padilla et. al. 2017). The primary condition
Proteus). These genera are widespread in the environment and can associated with cases of foodborne illness caused by STEC and
contaminate milk through feces; bedding, improperly cleaned teats, Salmonella spp. is gastroenteritis which is usually self-limiting, while
milk handling, and equipment contaminated with soil or polluted immuno-compromised individuals are at a higher risk of serious illness.
water (Cohen et al., 2017). The presence of Enterobacteriaceae in The Yersinia enterocolitica disease is typically characterized by
dairy products induces undesirable changes that render the product of gastroenteritis and enterocolitis. However, debilitating post-infection
inferior quality, unmarketable, and unfit for human consumption. immunological sequel, including Guillian-Barré syndrome and reactive
Moreover, their presence is frequently considered as a reliable index arthritis are known to develop in some individuals following an episode
of fecal contamination. Therefore, the presence of Enterobacteriaceae of foodborne illness with these pathogens (Altekruse et. al., 1999 and
and Coliform are routinely assessed to determine the hygienic quality Oliver et. al., 2005).
of foods, particularly dairy products (Martin et. al., 2010). Yoghurt is the best known and popular cultured milk product
Food-borne microorganisms are major pathogens affecting food concerning nutrition as it contains a high level of protein, calcium,
safety and cause human illness worldwide as a result of the phosphorus, and vitamin B2 and B12 (Piaia, 2001). Yoghurt helps to
consumption of foodstuff, mainly animal products contaminated with maintain food and water intake, which are often reduced in older people,
vegetative pathogens or their toxins (Abebe et. al., 2020). The moreover, it is a part of the diet designed to promote successful growth.
outbreak of foodborne illnesses following consumption of raw milk Ice cream is a highly delicious and nutritionally rich frozen milk product

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 Sobeih et al.,2020, KVMJ, 18 (2): 9-13, DOI: 10.21608/kvmj.2020.39992.1009

that is widely consumed in Egypt. Microorganisms may gain entrance 3. Results and discussion
to such products during processing, handling and distribution also the Enterobacteriaceae were detected in 84 (84%) of the examined raw milk
ingredients used in the manufacture contribute to contamination of the samples with a mean count of 1.02 x 106 + 1.98 x 105cfu/ml. The highest
product and consequently lead to public health hazards as frequency distribution (48.81%) of the positive samples lies within the
gastroenteritis in humans (Yadav et.al. 1993 and Holban and range of 105: <106cfu/ml followed by 22.62% in the range of 106 : <107
Grumezescu, 2018). cfu/ml (Tables 1 and 2).
Although pasteurization can destroy most of the specific Enterobacteriaceae members were detected in 40% and 32% of the
pathogens that pose risk to public health, a potential microbial hazard examined small scale and large scale plain yoghurt samples with a mean
may be still introduced after pasteurization through adding value of 6.95 x 103 + 4 x103 and 1.57x103 + 8.95 x102 cfu/g (Table 1).
contaminated ingredients and improper handling process (Marshall, The highest frequency distribution of positive small scale plain yoghurt
1998 and Oliver, 2005). Raw milk that consumed raw or used in the samples (70%) lies within the range of 1 x 103:< 5 x 103 cfu/g while that
manufacturing of yoghurt and ice cream may be produced under poor of positive large scale plain yoghurt samples (62.5%) lies within the
hygienic status using primitive procedures. Under such range of >10:<103cfu/g (Table 3).
circumstances, the finished products would express to harbor a great On the other hand Enterobacteriaceae members were detected in
number and different types of contaminants, particularly those 64% and 20% of the examined small scale and large scale ice cream
belonging to enteric microorganisms. Therefore, this work was samples with a mean count of 1.02 x 104 +4.13 x 103 and 1.4 x 103 +8.26
planned to detect and identify members of Enterobacteriaceae in raw x 102 cfu/g (Table 1). The highest frequency distribution of both small
milk, yoghurt, and ice cream to assess their quality and sanitation. scale ice cream samples (43.75%) and large scale ice cream samples
(60%) lie within the range of >10:<103 cfu/g (Table 3). According to
2. Materials and methods Egyptian Standards for yoghurt and ice cream, Enterobacteriaceae count
must not exceed 10 cell/g (E.S.2005, a and b), consequently all positive
This study was conducted after under the ethical approval from the yoghurt and ice cream samples (>10 cfu/g) (Table 3) exceed the
Experimental Animals Care Committee in compliance with guidelines permissible limit of Egyptian Standards, 2005.
of the University of Kafrelsheikh. It is evident that the most frequent members isolated from the
examined raw milk samples were Hafnia alive (30.95%), Serratia
2.1. Collection of samples
liquefaciens (25.0%), and Klebsiella pneumonia (15.48%). Klebsiella
A total of 200 random samples of raw milk and some dairy products
pneumoniae found in soil, water, sewage and constitute a part of the flora
were collected from small dairies, groceries, and supermarkets from
of the mouth and intestinal tract of human and animals. It responsible for
different localities at Kafr El-Sheikh Governorate, Egypt. Of them,
pneumonia and upper respiratory tract infection and may be responsible
100 raw milk, 50 plain yoghurt (25 small scale and 25 large scale),
for meningitis, pyaemia, and cystitis (Martin and Bachman, 2018).The
and 50 ice cream (25 small scale and 25 large scale) samples were
prevalence of Klebsiella oxytoca, Enterobacter aerogenes and Proteus
obtained in their containers as sold to the consumer or in sterile
vulgaris was 13.1% (of the positive samples). Both Serratia marcescens
containers and dispatched directly to the laboratory with a minimum
and Proteus rettgeri were isolated at percentage of 11.9%, while, E. coli,
of delay in an insulated icebox at 4oC to be examined.
Providencia rettgeri and Citrobacter diversus could be identified in 9.52,
2.2. Examination of raw milk for the detection of heat treatment 9.52, and 3.57% of the positive samples, respectively.
All collected milk samples were subjected to Storch's test according Enterobacteriaceae members which isolated at low percentage were
to A.P.H.A, 2004 to exclude samples proved to be heat treated above Proteus morganii, Providencia alcalifaciens, Citrobacter freundii,
80oC. Shigella flexneri and Shigella sonnei each at 2.38% of the positive
2.3. Preparation of serial dilution samples, while Enterobacter cloacae and Yersinia enterocolitica
All samples were prepared and serially diluted according to A.P.H.A, isolated only from 1.19% of the positive samples. Most of these groups
2004. were isolated by many investigators (Saleh, 2000; Nyein et. al. 2002 and
Jayarao et. al. 2006).
2.4. Enterobacteriaceae count The presence of a large number of Enterobacteriaceae in raw milk
According to Harrigan 1998, 2 ml from each of the previously indicate unsafe raw milk for human consumption (Nyein et. al. 2002)
prepared serial dilution milk, yoghurt, or ice cream were transferred unless it is pasteurized as Enterobacteriaceae in raw milk were
into sterile duplicate Petri dishes (1 ml each). About 10-15 ml of violet completely removed by pasteurization and not appear during the
red bile glucose agar (VRBGA) medium (melted and tempered to 45 manufacturing process of dairy product (Branciari et. al. 2004) so milk
+ 1oC) were poured into each plate and mixed well with the inoculum for human consumption must be pasteurized. The mean
and allowed to set. The set medium was overlaid by pouring another Enterobacteriaceae count in small scale plain yoghurt was significantly
5 ml of VRBGA medium. After solidification, the inoculated plates (P<0.01) higher than that of large scale plain yoghurt (Table 1). These
were incubated at 37oC for 24 h. All typical colonies, characterized by results declare neglected sanitary measures in small-scale plain yoghurt
red or purple color, >0.5 mm in diameter and surrounded by a zone of and un-heat treated milk may be used for the preparation of yoghurt. The
precipitated bile were counted and recorded as total presence of Enterobacteriaceae in yoghurt is considered as an index of
Enterobacteriaceae count. Five colonies were picked up and unsatisfactory sanitation and the possible presence of enteric pathogens.
transferred to nutrient agar slants, then incubated at 37oC/24 h. and In normal fermentation, a final pH of <4.5 is developed in cultured
used for further identification. milk products. Low pH generally prevents the growth of most spoilage
2.5. Identification of Enterobacteriaceae and pathogenic organisms despite interference with acid development
Isolated colonies were purified on plate count agar at 37oC/24h and may allow the growth of undesirable microorganisms (A.P.H.A, 1992).
identified according to Krieg and Holt, 1984. Regarding yoghurt samples, E. coil is the most frequent member
isolated (44.44% out of 18 positive samples examined) (Table 5). E. coli
is the most common aerobic bacterium of the large bowel of man and

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 Sobeih et al.,2020, KVMJ, 18 (2): 9-13, DOI: 10.21608/kvmj.2020.39992.1009

animal, thus its presence in food generally indicates direct or indirect with a percentage of 19.05%. On the other hand, Klebsiella ozaenae,
fecal pollution, so that it has been implicated in cases of Citrobacter freundii, Proteus rettgerii, and Shigella flexneri were
gastroenteritis, epidemic diarrhea in infants, sporadic cases of summer detected in 9.52% of positive samples and Serratia odorifera,
diarrhea in children and cases of food poisoning (Bennett et. al., Enterobacter cloacae, Shigella sonnei, and Proteus vulgaris were
2015). It is important to conclude that the detection of even low isolated only from one sample at a percentage of 4.76 of the positive
numbers of E. coli in foods reveals public health risk (I.C.M.S.F, samples. This study showed that Salmonella spp. could not be detected
1978). According to Egyptian Standards yogurt must be free from in any of the examined ice cream samples as reported by many previous
E.coli (E.S. 2005, a), thus confirm public health risk for yoghurt authors (Fadel and Ismail, 2009 and El-Bana, 2011).
consumers. The high Enterobacteriaceae count in the examined ice cream
Serratia liquefaciens could be isolated from 27.78% of the samples obtained in this investigation reflects unhygienic practices
positive samples examined while Shigella sonnei, Shigella flexneri, during manufacture and distribution. This may be due to poor
Klebsiella pneumoniae, and Hafnia alive isolated at 22.22%. On the ingredients, carelessness of the employer, who may lack the necessary
other hand, Serratia marcescens, Enterobacter aerogenes, knowledge of personal hygiene, product handling, and sanitation
Providencia staurtii, Edwardsiella hoshinae, and Proteus morganii routines. So, this indicates a need for more concern on the part of the
were detected in 11.11%. Citrobacter diversus, Proteus rettgerii, and dairy industry to prevent contamination of ice cream. Ensure ingredients
Enterobacter cloacae were detected in 5.56% of the positive samples. of good quality and especially those added after heat treatment should be
No salmonellae could be detected in any of the examined yoghurt free from pathogens and conform to microbiological criteria. Sufficient
samples. Similar results were recorded by many authors (El-Kasas, heat treatment to ice cream mix, prevention of post heat treatment
2004; Braun and Preuss, 2007 and Rotar et.al., 2007). contamination, and special attention should be given to utensils and
Identification of Enterobacteriaceae isolated from ice cream equipment which come in contact with ice cream also the packaging
samples as recorded in Table (6). Serratia marcescens had the highest used.
isolation percentage (57.14% out of 21 positive samples). It was Enterobacteriaceae are sometimes contaminants of pasteurized
reported that Serratia organisms have been implicated in human dairy products. Their presence is indicative of unsanitary methods of
infection, including pulmonary and urinary tract infections as well as manufacture, inadequate pasteurization, or post-pasteurization
septicemia (Mahlen, 2011). E.coli could be isolated at 33.33% which contamination. This indicates a need for more concern on the part of the
is nearly similar to results recorded by (Yaman et.al. 2006). Both dairy industry to prevent contamination with Enterobacteriaceae and thus
Klebsiella pneumoniae and Hafnia alvie were isolated from 23.81% prevent additional outbreaks of food-borne illness caused by their
while both Citrobacter diversus and Enterobacter aerogenes isolated members.

Table 1. Statistical analytical results of Enterobacteriaceae count in the examined samples

Positive
No. of examined Enterobacteriaceae count (cfu/ml or g)
Type of samples samples
samples
No. % Minimum Maximum Mean + SE
Raw milk 100 84 84 3x 102 1.1 x 107 1.02 x 106 + 1.98 x 105
Yoghurt
-Small scale 25 10 40 9x102 4.3x102 6.95x103 + 4x103
-large scale 25 8 32 5x10 6.5x103 1.57x103 +8.95x102 *
Ice cream
-Small scale 25 16 64 5x10 5x104 1.02 x 104 + 4.13 x 103*
-Large scale 25 5 20 3 x 102 4.65x103 1.4 x 103 + 8.26 x 102
cfu = colony forming unit, SE= Standard error of mean
* Small and large scale (yoghurt and ice cream) samples differed significantly at P <0.01

Table 2. Frequency distribution of positive raw milk samples based on their Enterobacteriaceae count

Interval (cfu/ml) No. of samples %

>10 : < 104 7 8.33
104 : < 105 16 19.05
105 :< 106 41 48.81
106 : < 107 19 22.62
≥ 107 1 1.19
Total 84 100

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 Sobeih et al.,2020, KVMJ, 18 (2): 9-13, DOI: 10.21608/kvmj.2020.39992.1009

Table 3. Frequency distribution of positive yoghurt and ice cream samples based on their Enterobacteriaceae count
Yoghurt samples Ice cream samples
Interval (cfu/g) Small-scale Large-scale Small-scale Large-scale
No. % No. % No % No %
>10 : < 103 1 10 5 62.5 7 43.75 3 60
103 : < 5 x 103 7 70 2 25.0 3 18.75 2 40
5 x 103 : < 104 1 10 1 12.5 1 6.25 0 0
104: < 5x 104 1 10 0 0 5 31.25 0 0
Total 10 100 8 100 16 100 5 100
Table 4. Incidence of Enterobacteriaceae members isolated from the examined raw milk samples
Positive samples
Isolates Number %*
No. % **
Hafnia alive 54 17.65 26 30.95
Serratia liquefaciens 34 11.11 21 25.00
Klebsiella pneumoniae 18 5.88 13 15.48
Klebsiella oxytoca 30 9.80 11 13.10
Enterobacter aerogenes 20 6.54 11 13.10
Proteus vulgaris 12 3.92 11 13.10
Serratia marcescens 44 14.38 10 11.90
Proteus rettgerii 28 9.15 10 11.90
Escherichia coli 20 6.54 8 9.52
Providencia rettgeri 10 3.27 8 9.52
Citrobacter diversus 4 1.31 3 3.57
Proteus morganii 3 0.98 2 2.38
Providencia alcalifaciens 4 1.31 2 2.38
Citrobacter freundii 8 2.61 2 2.38
Shigella flexneri 6 1.96 2 2.38
Shigella sonnei 6 1.96 2 2.38
Enterobacter cloacae 4 1.31 1 1.19
Yersinia enterocolitica 1 0.33 1 1.19
* Percent calculated to total No. of isolates (306) ** Percent calculated to number of positive samples (84)
Table 5. Incidence of Enterobacteriaceae members isolated from the examined yoghurt samples
Positive sample
Isolates Number %*
No. %**
E. coli 42 26.58 8 44.44
Serratia liquefaciens 20 12.66 5 27.78
Shigella flexneri 14 8.86 4 22.22
Shigella sonnei 22 13.92 4 22.22
Klebsiella pneumoniae 12 7.59 4 22.22
Hafnia alvie 10 6.33 4 22.22
Serratia marcescens 10 6.33 2 11.11
Enterobacter aerogenes 8 5.06 2 11.11
Providencia staurtii 4 2.53 2 11.11
Edwardsiella hoshinae 2 1.27 2 11.11
Proteus morganii 2 1.27 2 11.11
Proteus rettgerii 2 1.27 1 5.56
Enterobacter cloacae 8 5.06 1 5.56
Citrobacter diversus 2 1.27 1 5.56
* Percent calculated to total No. of isolates (158) ** Percent calculated to number of positive samples (18)
Table 6. Incidence of Enterobacteriaceae members isolated from the examined ice cream samples
Positive samples
Isolates Number %*
No. %**
Serratia marcescens 24 21.82 12 57.14
Escherichia coli 16 14.55 7 33.33
Klebsiella pneumoniae 14 12.73 5 23.81
Hafnia alive 12 10.91 5 23.81
Citrobacter diversus 8 7.27 4 19.05
Enterobacter aerogenes 8 7.27 4 19.05
Klebsiella ozaenae 4 3.64 2 9.52
Citrobacter freundii 4 3.64 2 9.52
Shigella flexneri 6 5.45 2 9.52
Proteus rettgerii 6 5.45 2 9.52
Serratia odorifera 2 1.82 1 4.76
Enterobacter cloacae 2 1.82 1 4.76
Shigella sonnei 2 1.82 1 4.76
Proteus vulgaris 2 1.82 1 4.76
* Percent calculated to total No. of isolates (110) ** Percent calculated to number of positive samples (21)

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4. Conclusion Jayarao BM, Donaldson SC, Straley BA, Sawant AA, Hegde NV and
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