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Food Control 20 (2009) 913–917
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Food Control
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Bacterial quality of raw milk investigated by Escherichia coli and isolates
analysis for specific virulence-gene markers
Abdullah D. Altalhi a, Sabry A. Hassan a,b,*
a Department of Biological Sciences, Faculty of Sciences, Taif University, P.O. Box 888, Taif, Saudi Arabia
b Department of Microbiology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt
a r t i c l e i n f o
Article history:
Received 10 June 2008
Received in revised form 10 January 2009
Accepted 20 January 2009
Keywords:
E. coli
PCR
Public health
STEC
NTEC
EPEC
ExPEC
MPN
Fecal coliform
Saudi Arabia
0956-7135/$ - see front matter � 2009 Elsevier Ltd. A
doi:10.1016/j.foodcont.2009.01.005
* Corresponding author. Address: Department of B
Sciences, Taif University, P.O. Box 888, Taif, Saudi A
fax: +966 27256570.
E-mail address: sabryhassan1968@yahoo.com (S.A
a b s t r a c t
The bacterial quality and safety of raw milk sources in Taif region (Western Saudi Arabia) were analyzed
for the natural contamination of fecal coliform and Escherichia coli by standard most probable number
method. The E. coli isolates were identified to species identity using API 20E and screened for markers
of STEC (Stx1,Stx2), ETEC (ST,LT), (EaeA) and ExPEC (CNF1, CNF2, SfaS, PapA, CdtB, IutA, FyuA, TraT) using
PCR assays. Thirty-three E. coli strains were recovered from raw milk sample sources, which were con-
taminated by fecal coliform. None of the investigated isolates reacted positively with the PapA, SfaS
and CdtB, ST and LT-specific primers. Markers of STEC and EPEC were each detected in three strains. NTEC
was detected in four strains. The most frequent virulence markers were TraT (17 strains), IutA (11 strains)
and FyuA (8 strains). Results suggested a possibility of potential public health threat of E. coli originating
from raw milk sources.
� 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Markets and consumers for raw milk and their products have
existed in many parts of the world. Raw unpasteurized milk is con-
sumed directly by a large number of people in rural areas and indi-
rectly by a much larger segment of the population via consumption
of several types of cheeses. Among the main reasons that people
may believe that the raw milk and their products have advantages
or value over the pasteurized one.
Being a highly nutritious medium, therefore many bacteria
including spoilage and pathogenic bacteria, can grow and propa-
gate in it. Generally, bacteria in the milk can occur through coloni-
zation of the teat canal or an infected udder (clinical and
subclinical mastitis) or gets contaminated at various stages be it
from the animal, milker (manual as well as automated), extraneous
dirt or unclean process water (Banwart, 1989; Gruetzmacher &
Bradley, 1999; Hayes et al., 2001; Philips & Griffiths, 1990; Rohde,
1985; Stewart, 1978). Mastitis, milk quality and dairy food safety
are all much interrelated. Collectively, the presence of foodborne
ll rights reserved.
iological Sciences, Faculty of
rabia. Tel.: +966 533473418;
. Hassan).
pathogens in unpasteurized raw milk either directly or indirectly
increases the risk of ingestion and transmission of foodborne
pathogens and ingestion of potentially harmful toxins.
Several studies have identified milkborne pathogens including
Campylobacter jejuni, Shiga-toxin producing Escherichia coli (STEC),
Listeria monocytogenes, Salmonella spp. and Yersinia enterocolitica in
farm bulk tank milk (BTM) (Lovett, Francis, & Hunt, 1987; McM-
anus & Lanier, 1987; Moustafa, Ahmed, & Marth, 1983; O’Donnell,
1995; Rahn et al., 1997; Rohrbach, Draughon, Davidson, & Oliver,
1992; Steele et al., 1997).
Raw milk is also used to produce cheeses and other home made
foods. Cheese-related foodborne disease outbreaks have been re-
ported (Johnson, 1990). Thus, raw milk containing a high bacterial
count, including foodborne pathogens, likely increases the proba-
bility of contamination of dairy products to be consumed by hu-
mans. The presence of these pathogenic bacteria in milk emerged
as major public health concerns, especially for those individuals
who still drink raw milk (Ryser, 1998). However, the true incidence
of milkborne disease is unknown.
Many microorganisms can get access to milk and products,
among these are E. coli. coliforms and E. coli are often used as
marker organisms. Recovery and counting of E. coli is used as reli-
able indicator of fecal contamination and indicates a possible
presence of enteropathogenic and/or toxigenic microorganisms
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914 A.D. Altalhi, S.A. Hassan / Food Control 20 (2009) 913–917
which constitute a public health hazard. E. coli is one of the main
inhabitants of the intestinal tract of most mammalian species,
including humans and birds. Most E. coli are harmless, but some
are known to be pathogenic bacteria, causing severe intestinal
and extraintestinal diseases in man (Kaper, Nataro, & Mobley,
2004). These potentially harmful E. coli are classified into catego-
ries based on the production of virulence factors and on the clin-
ical manifestations that they cause. Nationally, coliforms have
been reported in raw milk and products by several authors (Aly
& Galal, 2002; Chye, Abdullah, & Ayob, 2004; Lues, Venter, &
van der Westhuizen, 2003; Soomro, Arain, Khaskheli, & Bhutto,
2002). In Saudi Arabia, Salji, Sawaya, and Ayaz (1984) recorded
coliforms as the main contaminants of raw milk. Moreover, Al-
Kanhal, Abo-Tarboosh, Hamad, and Al-Shrawy (1996) reported
that coliforms count exceeded the American standards in most
of the dairy plant studied.
In addition to the presence of E. coli denoting fecal pollution, the
presence of virulence – related genes in E. coli strains refer to the
pathogenicity of the isolates. Previous studies documented the
aquation of some E. coli isolates from raw milk and products for
virulence markers (Holko, Bisova, Holkova, & Kmet, 2006; Jayarao
& Henning, 2001; Klie et al., 1997; Paneto, Schocken-Iturrino, Mac-
edo, Santo, & Marin, 2007).
Considering the above voiced, the present study thus aimed to
(1) assess the raw milk quality by investigating E. coli as fecal indi-
cator using MPN method and (2) investigate the E. coli isolates to
certain functioning virulence-associated genes using PCR assay.
2. Materials and methods
2.1. Sample collection
A total of 50 raw milk (30 samples) and product (20 samples)
samples were randomly collected from different localities in Taif
region (Western Saudi Arabia). Samples were collected immedi-
ately, delivered to the laboratory in a cool box and tested within
24 h.
Table 1
Sequences and predicted lengths of PCR amplification products of the oligonucleotide prim
Pathogenic factor Primers Primer sequence
Cytotoxic necrotizing factor 1 (cnf1) CNF1a atcttatactggatggg
CNF1b gcagaacgacgttctt
Cytotoxic necrotizing factor 2 (cnf2) CNF2f aatctaattaaagaga
CNF2r catgctttgtatatcta
Shiga toxin 1 (stx1) Stx1f aaatcgccattcgttga
Stx1r tgccattctggcaactc
Shiga toxin 2 (stx2) Stx2f cgatcgtcactcactg
Stx2r ggatattctccccactc
Heat labile toxin (LT) LTa tgtttccacttctcttag
LTb tattccctgttacgatg
Heat stable toxin (ST) STa tctgtattatctttcccc
ataacatccagcacac
STb ccctcaggatgctaaa
ttaatagcacccggta
Enteropathogenic attachment & effacement (eaeA) EAE1 tgcggcacaacaggc
EAE2 cggtcgccgcaccagg
Cytolethal distending factor (cdtB) Cdt 1 aaatcaccaagaatca
Cdt 2 aaatctcctgcaatca
P-Fimbriae (papA) PapA-f atggcagtggtgtcttt
PapA-r cgtcccaccatacgtg
S-Fimbriae adhesion (sfaS) SfaS-f gtggatacgacgatta
SfaS-r ccgccagcattccctg
Yersiniabactin (fyuA) fyuA-f tgattaaccccgcgac
fyuA-r cgcagtaggcacgatg
Aerobactin (iutA) AerJ-f ggctggacatcatggg
AerJ-r cgtcgggaacgggtag
Serum survival (traT) TraT-f ggtgtggtgcgatgag
TraT-r cacggttcagccatcc
2.2. Isolation and identificationof coliforms
Raw milk sample sources were taken for microbiological analy-
ses to detect the presence of coliforms. Initially, 25 ml/g of raw
milk or product sample was dispensed into a sterile flask contain-
ing 225 ml of buffered peptone water and homogenized with lab
stomacher. Subsequent serial decimal dilutions of milk or product
were prepared in buffered peptone water. Coliform bacteria were
enumerated by a most probable number (MPN) multiple-tube fer-
mentation method according to US standard method (US FDA,
2002). Fecal E. coli confirmation was achieved by monitoring the
acidification and gas production during growth in MacConky broth
(Oxoid, UK) at 44 ± 0.5 �C for 24 ± 3 h. From the fermentation
tubes, 33 E. coli isolates were included in this study.
The identification of E. coli was confirmed by colony morphol-
ogy on eosin methylene blue agar (EMB – Scharlau, Spain, EU)
and performing API 20E (bioMérieux – France). The strains were
stored at �70 �C in trypticase soy broth (TSB – Scharlau, Spain,
EU) with 20% glycerol for further characterization.
2.3. DNA isolation
Bacterial strains were overnight grown in trypticase soy agar
(TSA – Scharlau, Spain, EU) at 37 �C. One colony was suspended
in 100 ll of sterile distilled water. After boiling the suspension
for 13 min; this was followed by freezing and subsequently centri-
fuged at 14,000 rpm for 15 min to pellet the cell debris (Reischl
et al., 2002). The supernatant was used as a template for amplifica-
tion reaction.
3. Polymerase chain reaction (PCR)
The PCR assays, specific primer sequences and the predicted
size of the amplified products for the different pathogenic gene
coding regions were employed as previously described (Blanco
et al., 1996; Brian et al., 1992; Heuvelink, van de Kar, Meis, Monn-
ens, & Melchers, 1995; Johnson & Stell, 2000; Lang Lee, Tsai, Mayer,
ers used.
s (50–30) Product size (bp) Reference
atcatcttgg 1105 Johnson & Stell (personal communication)
cataagtatc
ac 543 Blanco et al. (1996)
ctacttct 366 Brian et al. (1992)
gcgatgca
gtttcatca 282 Brian et al. (1992)
tgacacc
258 Lang Lee et al. (1994)
t
tc 186 Schultz et al. (1994)
aggc
ccag 166
caagc
ggcga 629 Heuvelink et al. (1995)
attc
tccagtta 430 Johnson and Stell (2000)
tccagttta
ggtg 720 Johnson and Stell (2000)
ctcttc
ctgtg 240 Johnson and Stell (2000)
tattc
gggaa 880 Johnson and Stell (2000)
ttgta
aactgg 300 Johnson and Stell (2000)
aatcg
cacag 290 Johnson and Stell (2000)
ctgag
Table 4
Virulence factors of 33 E. coli isolates recovered from raw milk sources.
Virulence factors No. of positive strains % from total number (n = 33)
STX1 1 3
STX2 2 6.1
EaeA 3 9.1
CNF1 3 9.1
CNF2 1 3
FyuA 8 24.2
IutA 11 33.3
TraT 17 51.5
Table 5
Virulence gene profile of 33 E. coli strains screened by PCR.
Virulence gene (s) E. coli strains with indicated virulence genes
Raw milk Raw milk products Total (%)
cnf1, cnf2, traT, fyuA, iutA 1 – 1 (3)
cnf1, traT, iutA – 1 1 (3)
cnf1, fyuA, iutA 1 – 1 (3)
stx1, stx2, traT, iutA – 1 1 (3)
stx2, traT, fyuA 1 – 1 (3)
eaeA, traT 1 2 3 (9.1)
fyuA, iutA 2 – 2 (6.1)
fyuA, iutA 5 – 5 (15.1)
fyuA 3 – 3 (9.1)
traT 5 – 5 (15.1)
Fig. 1. The different PCR amplification showing 100 bp DNA ladder (lane 1), CNF1—
1105 bp (lane 2), FyuA—880 bp (lane 3), EaeA—629 bp (lane 4), CNF2 —543 bp (lane
5), Stx1—370 bp (lane 6), IutA—300 bp (lane7), TraT—290 bp (lane 8) and Stx2—
A.D. Altalhi, S.A. Hassan / Food Control 20 (2009) 913–917 915
Patton, & Palmer, 1994; Schultz et al., 1994). Details are shown in
Table 1. For cycling, a PXE-0.5 thermal cycler (THERMO, Electron
Corporation, Milford, MA, USA) was used. The amplified products
were visualized by ethidium bromide staining after gel electropho-
resis of 10 ll of the final reaction mixture in 1.5% agarose. Refer-
ence strains representing the amplified genes, J96 (papA, fimH,
hlyD, cnf1, fyuA); JP6 (cdtB); Bl-6-9 (sfaS) and Bl-6-10 (iutA, traT)
(kindly provided by Prof. Johnson and Dr. Stell, VA Medical Centre
and Department of Medicine, University of Minnesota, Minneapo-
lis, Minnesota, USA); reference strains for stx1, stx2 and eaeA (pro-
vided by Bundesst. bakt. serol. Untersuchngsanstalt, National
reference laboratory for EHEC, Innsbruck, Austria); for cnf2, LT
and ST (Prof. Awad-Masalmeh, department of 2nd medical clinic,
University of veterinary medicine, Vienna, Austria).
4. Results
4.1. Fecal coliform contamination
The raw milk and product samples displayed heavily coliform
contamination (Table 2). Most of the raw milk sources were pol-
luted with the fecal coliform according to the results of fermenta-
tive growth in MacConky broth at 44 ± 0.5 �C, with most probable
number (MPN), 93 to P240 CFU/ml or g (data not shown). Of a to-
tal of 50 samples collected from different sources, 40 samples
(80%) were contaminated by coliform bacteria, seven samples
(17.5%) with non-faecal coliform bacteria, and 10 (20%) were with
no growth (Table 3). Consequently, 33 of the 40 coliforms were de-
tected as E. coli which was included in this study.
4.2. Polymerase chain reaction
Of 33 E. coli strains recovered, 23 (69.7%) had one or more of the
genes responsible for pathogenicity of E. coli (Table 5). The traT,
iutA and fyuA genes coding for serum resistance, aerobactin and
yersiniabactin, are virulence associated markers of extraintestinal
pathogenic E. coli (ExPEC), were most frequently demonstrated
(51.5%, 33.3% and 24.2%, respectively) among E. coli isolates from
raw milk samples; followed by eaeA (9.1%) coding for enteropath-
ogenic E. coli (EPEC). The cnf1 and cnf2 genes both coding for nec-
Table 2
Coliform contamination levels in raw milk sources.
MPN (CFU/ml or
g)
No. of fermentative tubes
Raw milk
(n = 30)
Raw milk product
(n = 20)
Overall
(n = 50)
>240 5 8 13
240 22 11 33
93 3 1 4
Table 3
Isolation of fecal coliform and E. coli from various raw milk sources.
Sample Number
examined
Coliform E. coli
Raw milk
Cow 11 9 8
Sheep 10 8 6
Goat 5 2 1
Camel 4 4 3
Raw milk product
Baladi cheese 10 7 5
Modeer (home made
fermented milk food)
10 10 10
282 bp (lane 9).
rotizing E. coli (NTEC) were occurred in 9.1% and 3%, respectively.
stx1 and stx2 coding for Shiga-like-producing E. coli (STEC) were
detected in 3% and 6.1%, respectively (Table 4). No genes coding
for LT, ST, PapA, CdtB and SfaS were detected in any of the isolates
examined.
A representative gel electrophoresis profile of amplified prod-
ucts of the investigated pathogenic coding genes is shown in Fig. 1.
5. Discussion
Nowadays, public health concern associated with microbial
food safety has risen. Because E. coli is not only regarded as an indi-
cator of fecal contamination but more likely as an indicator of poor
hygiene and sanitary practices during milking and further han-
dling, generally, it can be concluded that samples were heavily
contaminated as indicated by MPN (Table 2). E. coli is, furthermore,
a known causative agent of diarrhea and other foodborne-related
illnesses through the ingestion of contaminated foodstuffs. Patho-
genic members of the coliform group as well as the Enterobacteri-
aceae family are represented by genera such as Salmonella and
Shigella and are found in the intestines of humans and animals
(Collins, Lyne, & Grange, 1995; Hayes et al., 2001; Le Minor,
1984; Rowe & Gross, 1984).
916 A.D. Altalhi, S.A. Hassan / Food Control 20 (2009) 913–917
E. coli was isolated from 33 (66%) of the 50 raw milk and prod-
uct samples tested. Although detection of E. coli in milk reflects fe-
cal contamination, environmental coliforms have also been
detected in milk (Shehu & Adesiyun, 1990). Milk can be easily con-
taminated by infected food handlers who practice poor personal
hygiene or by water containing human discharges. Globally, higher
percentage of coliform and E. coli was reported by many authors. In
Egypt, Aly and Galal (2002) showed the presence of coliform bac-
teria in raw milk and the number reduced in the heat treated
one. In India, the raw milk and products were heavily contami-
natedby E. coli (Soomro et al., 2002). In South Africa, Lues et al.
(2003) detected a higher percentage of E. coli in raw milk. In Malay-
sia, Chye et al. (2004) indicated that 90% of the examined raw milk
were contaminated by coliform bacteria and 65% were E. coli posi-
tive. In Saudi Arabia, Salji et al. (1984) recorded coliforms as the
main contaminants of raw milk. Moreover, Al-Kanhal et al.
(1996) reported that coliforms count exceeded the American stan-
dards in most of the dairy plant studied.
PCR showed that STEC were isolated from three isolates (9.1%).
STEC have been associated with human disease. Foods of animal
origin including raw milk have been implicated as important vehi-
cles for STEC infections in humans. In this study, a value was much
higher than registered in Spain (0.4%) by Quinto and Cepeda
(1997), in Ontario (0.87%) by Steele et al. (1997) and in Germany
(3.9%) by Klie et al. (1997). Meanwhile, was slightly higher than
6% reported by Paneto et al. (2007) in raw milk cheese in Brazil.
In the other hand, less than 13% reported by Vernozy-Rozand,
Montet, Beradin, Bavai, and Beutin (2005) in French cheese. The re-
sults showed that, two of the three isolates of E. coli encoded for
shiga-toxin 2 gene, while one strain encoded for shiga-toxin 1
gene. On the contrary, Montenegro et al. (1990) reported that most
of the STEC isolates of bovine origin encoded for shiga-toxin 1
gene.
The EaeA, virulence factor is correlate with EPEC strains. EPEC
are defined as Stx-negative E. coli strains able to produce A/E le-
sions on intestinal cells, detectable in vitro by positive eae-PCR
test. The ratio of EPEC (eaeA+) in our testing was 9.1% that
means one of two most frequently determined E. coli pathotypes.
The value was higher than the 3.09% reported by Holko et al.
(2006) from traditional cheese made from raw sheep milk in
Slovakia.
Cytotoxic necrotizing factors (CNF1, CNF2), of the most fre-
quently determined virulence genes in this study are encoding
cytotoxic necrotizing E. coli. CNF1 is a chromosomally encoded uro-
pathogenic E. coli (UPEC) toxin (De Rycke, Guillot, & Boivin, 1987;
Oswald, De Rycke, Guillot, & Boivin, 1989). Several groups have re-
ported an epidemiological link between the presence of cnf1+
genotype, or the production of CNF1, and E. coli strains that caused
extraintestinal disease. CNF2, is a commonly occurring virulence
factor among healthy cattle (Burns, Ball, & Finlay, 1996; Pohl
et al., 1993). Indeed, we reported a high prevalence of the CNF1
in faeces of healthy cattle (Hassan & Awad-Masalmeh – unpub-
lished data). The percentage of cnf1 in our testing was 9.1% and
cnf2 in 3% from a total of 33 isolates used in our investigation. This
is in agree with the previous study reported CNF1 in most of sam-
ples investigated (Holko et al., 2006).
Other occurring virulence-associated traits were FyuA, IutA and
TraT that encoding for ExPEC. The fyuA encoding yersinia high
pathogenicity island, iutA encoding aerobactin system and traT,
serum survival gene, are specific virulence markers associated with
exteraintestinal infection. The explanation of their occurrence
could be the possibility of udder infection.
In conclusion, Results clearly indicated that microbial quality
and safety of raw milk produced by local farmers and distributors
was unsatisfactory. The presence of fecal indicator organisms not
only indicate poor hygiene but also itself may be pathogenic. The
pathogenic bacteria such as E. coli may pass to the milk; this sug-
gests that raw milk should be considered a vehicle for the trans-
mission of potentially pathogenic bacteria. Since a lot of people
still drink raw milk, especially in rural areas, this emphasis’s the
need for educational efforts on health risks associated with con-
sumption of raw unpasteurized milk.
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http://www.cfsan.fda.gov/ebam/bam-4.htm1
http://www.cfsan.fda.gov/ebam/bam-4.htm1
	Bacterial quality of raw milk investigated by Escherichia coli and isolates analysis for specific virulence-gene markers
	Introduction
	Materials and methods
	Sample collection
	Isolation and identification of coliforms
	DNA isolation
	Polymerase chain reaction (PCR)
	Results
	Fecal coliform contamination
	Polymerase chain reaction
	Discussion
	References