Food Control 34 (2013) 121e125

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Food Control
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Prevalence, characterisation, and antimicrobial resistance of Listeria

species and Listeria monocytogenes isolates from raw milk in farm bulk
tanks
Hossein Jamali a, b, Behrad Radmehr c, Kwai Lin Thong a, b, *
a
Microbiology Division, Institute of Biological Science, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia
b
Laboratory of Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya, 50603, Kuala Lumpur, Malaysia
c
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Islamic Azad University-Karaj Branch, Karaj, Iran

a r t i c l e i n f o a b s t r a c t

Article history: A total of 446 various raw milk samples were collected from numerous farm bulk milk tanks to examine
Received 19 January 2013 the presence of Listeria species. These isolates were further characterised by biochemical tests and
Received in revised form Polymerase Chain Reaction (PCR). Listeria spp. was isolated in 83 of the 446 samples (18.6%). The highest
8 April 2013
prevalence of Listeria spp. was detected in raw cow milk samples (22.5%), followed by raw sheep milk
Accepted 16 April 2013
(16.4%) and raw goat milk (4.9%). The most common species isolated was Listeria innocua (57.8%); the
remaining Listeria isolates were Listeria monocytogenes (21.7%), Listeria welshimeri (12%), and Listeria
Keywords:
seeligeri (8.4%). Based on PCR serotyping, the 18 L. monocytogenes isolates were distributed into three
Listeria spp.
Listeria monocytogenes
serogroups “1/2a, 3a” (n ¼ 11), “1/2c, 3c” (n ¼ 5), and “4b, 4d, 4e” (n ¼ 2). All the examined
Raw milk L. monocytogenes were positive for internalin genes (inlA, inlC, and inlJ). The Listeria spp. isolates were
PCR serogrouping resistant to tetracycline (49.4%) and penicillin G (43.4%) but remained susceptible to gentamicin,
Internalin genes vancomycin and rifampicin. The findings of this study show that consumption of raw milk could be a
Antimicrobial resistance potential risk of human listeriosis.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
The genus Listeria is a Gram-positive, non-spore forming, and
facultatively anaerobic bacteria. It comprises ten species: Listeria
monocytogenes, Listeria ivanovii, Listeria seeligeri, Listeria innocua,
Listeria welshimeri, Listeria grayi, Listeria marthii, Listeria rocourtiae,
Listeria fleischmannii, and Listeria weihenstephanensis (Halter,
Neuhaus, & Scherer, 2013; Zhang et al., 2007). L. monocytogenes is
an important foodborne pathogen in humans and animals (Nadon,
Woodward, Young, Rodgers, & Wiedmann, 2001), however L iva-
novii is considered a pathogenic species for animals (Guillet et al.,
2010). Pregnant women, new-born babies, elderly people and
immunocompromised individuals are grouped as high risk for
listeriosis (McLauchlin, Mitchell, Smerdon, & Jewell, 2004). Also,
members of this group are considered the main consumers of dairy
products. L. monocytogenes has also been detected in raw and ready
to eat (RTE) foods (Jamali, Chai, & Thong, 2013) and in raw milk and
* Corresponding author. Microbiology Division, Institute of Biological Science,
Faculty of Science, University of Malaya, Jalan Pantai Dalam, 50603, Kuala Lumpur,
Malaysia. Tel.: þ60 3 79674437; fax: þ60 3 7967 5908.
E-mail addresses: thongkl@um.edu.my, mordabenilofari@yahoo.com (K.L. Thong).
dairy products (Rahimi, Ameri, & Momtaz, 2010). The high preva-
lence of Listeria spp. in foods and the high mortality rate associated
with its infection make listeriosis a public health concern (Farber &
Peterkin, 1991).
Recently, several foodborne outbreaks of listeriosis due to the
consumption of contaminated dairy products have been docu-
mented (Fretz et al., 2010; Oliver, Boor, Murphy, & Murinda, 2009).
Increased consumption of raw milk has contributed to the increase
in listeriosis outbreaks (Rahimi et al., 2010). In some countries, raw
milk is sold as an RTE food after packaging (Vanegas, Vasquez,
Martinez, & Rueda, 2009). Dairy products which are produced
from unpasteurised milk such as cheese and ice cream (Brooks
et al., 2012) are frequently contaminated with the pathogen
(Swaminathan & Gerner-Smidt, 2007).
L. monocytogenes contains 13 serotypes (1/2a,1/2b, 1/2c, 3a, 3b, 3c,
4a, 4b, 4ab, 4c, 4d, 4e, and 7). Ninety-five percent of L. monocytogenes
strains associated with human listeriosis and food samples belong to
serotypes 1/2a, 1/2b, and 4b (Kathariou, 2002). Serotype 1/2a has
frequently been detected in different food matrices (Martins & Leal
Germano, 2011).
Various Listeria determinants have been reported to mediate
bacterial adherence into target cells. Internalin genes can increase
the invasion or virulence of the pathogen in animal models or in

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.04.023
122 H. Jamali et al. / Food Control 34 (2013) 121e125
tissue cultures (Bierne & Cossart, 2002; Orsi, Ripoll, Yeung,
Nightingale, & Wiedmann, 2007; Sant’Ana, Igarashi, Landgraf,
Destro, & Franco, 2012). L. monocytogenes needs inlA for internal-
isation of the intestinal epithelial cells (Hamon, Bierne, & Cossart,
2006). On the other hand, the presence of inlC and inlJ in
L. monocytogenes could be a rapid method to differentiate virulent
from avirulent strains of the pathogen (Liu, Lawrence, Austin, &
Ainsworth, 2007).
Antimicrobials are increasingly used in feeds for the control and
treatment of diseases in animals. However, the European Union
(1998) prohibits the use of antimicrobials which are consumed as
human medicine in animals’ foods. Increased antimicrobials con-
sumption by animals could be the cause of antimicrobial resistance
in L. monocytogenes (Jansen, Koknen, Obst, & Schwartz, 2003). The
pathogen usually shows susceptibility to a variety of antimicrobials
while some multidrug resistant L. monocytogenes strains have been
isolated from foods and human listeriosis cases (Marian et al., 2012;
Safdar & Armstrong, 2003). Due to the high mortality rate and the
increased multidrug resistance in the foodborne pathogen, anti-
microbial susceptibility testing is done to understand changes in
the resistance patterns of L. monocytogenes strains (Harakeh et al.,
2009).
In Iran, there is limited data on the prevalence and resistance
patterns of Listeria spp. in foods. Hence, this study was carried out
to determine the prevalence, serogroups, virulotypes and antimi-
crobial resistance status of Listeria spp. and L. monocytogenes iso-
lated from raw milk in Tehran province, Iran.
2. Materials and methods
2.1. Samples
Between September 2008 and August 2010, 446 samples of raw
milk (cow milk, n ¼ 240; sheep milk, n ¼ 165; goat milk, n ¼ 41)
were aseptically collected from farm bulk milk tanks in Tehran
province, Iran. The samples were transferred into sterile plastic
bags and transported in ice box to the laboratory within 4 h of
sampling.
2.2. Isolation and detection
The United States Department of Agriculture (USDA) method
was used in the isolation and detection of Listeria spp. in this study
(McClain & Lee, 1988). Briefly, 25 ml of each raw milk sample was
added to 225 mL of Listeria enrichment broth (Oxoid, Basingstoke,
UK), blended for 2 min and incubated for 24 h at 37  C. Then, 1 mL of
Listeria enrichment broth was added to 9 ml of Fraser broth (Oxoid,
Basingstoke, UK) as a second enrichment culture and incubated at
37  C for 24 h. Finally, enriched Fraser broth-culture was streaked
onto Palcam agar (Oxoid, Basingstoke, UK) and Oxford agar (Oxoid,
Basingstoke, UK) and incubated for 48 h at 37  C. Three to five
presumptive Listeria colonies were sub-cultured onto Tryptic Soy
agar (Oxoid, Basingstoke, UK) with 0.6% yeast extract (Oxoid,
Basingstoke, UK) (TSAYE) and were then incubated for 24 h at 37  C.
Table 1
Prevalence of Listeria spp. in raw milk samples.
No. Sample Listeria spp.
Cow milk 240 54 (22.5%)
Sheep milk 165 27 (16.4%)
Goat milk 41 2 (4.9%)
Total 446 83 (18.6%)
The colonies from TSAYE were then subjected to biochemical tests
(Aygun & Pehlivanlar, 2006).
2.3. Confirmation of Listeria spp. by Polymerase Chain Reaction
Further confirmation of Listeria spp. isolates was performed by
Polymerase Chain Reaction (PCR). Crude DNA was prepared by
direct boiling of a suspension of the cell lysates (Thong, Lai, Teh, &
Chua, 2011). A duplex-PCR was used to detect the presence of
Listeria spp. (U1/LI1) and L. monocytogenes (LM1/LM2) simulta-
neously (Rossmanith, Krassnig, Wagner, & Hein, 2006). Primers
U1/LI1 and LM1/LM2 amplified the 16S rRNA (938 bp) and Lister-
iolysin O gene (702 bp), respectively.
2.4. Multiplex-PCR serogrouping
A multiplex-PCR to distinguish four major serotypes of
L. monocytogenes (1/2a, 1/2b, 1/2c, and 4b) developed by Doumith,
Buchrieser, Glaser, Jacquet, and Martin (2004) was adopted. Five
pairs of primers, ORF2819, ORF2120, lmo0737, lmo1118, and prs
were used.
Serogroup “1/2a, 3a” displays a 691 bp fragment with the
primers lmo0737. Serogroup “1/2c, 3c” shows two fragments, 691
bp and 906 bp when amplified with the primers lmo0737 and
lmo1118, respectively. Serogroup “1/2b, 3b, 7” displays a 471 bp
fragment with the primer ORF2819. Serogroup “4b, 4d, 4e” shows
two fragments with the primers ORF2819 (471 bp) and ORF2120
(597 bp). All members of the Listeria genus display a fragment of
370 bp with the prs primers.
2.5. Virulence genes detection using a multiplex-PCR
Detection of inlA, inlC, and inlJ genes was performed by a
multiplex-PCR using primers and cycling conditions as described
by Liu et al. (2007). Representative amplicons of inlA, inlC and inlJ
were purified and sequenced to validate their identities.
2.6. Antimicrobial susceptibility testing
The KirbyeBauer disc diffusion method was applied to deter-
mine the antimicrobial susceptibility of the isolates. Mueller Hinton
agar (Oxoid, Basingstoke, UK) supplemented with 5% defibrinated
sheep blood was used according to the Clinical and Laboratory
Standards Institute (CLSI, 2006). The following panel of antimi-
crobial agents and concentrations were used; amoxicillin-
clavulanic acid (20/10 mg), chloramphenicol (30 mg), clindamycin
(2 mg), erythromycin (15 mg), gentamycin (10 mg), kanamycin
(30 mg), penicillin G (10 unit), rifampicin (5 mg), streptomycin
(10 mg), tetracycline (30 mg), trimethoprim-sulfamethoxazole
(1.25/23.75 mg), and vancomycin (30 mg).
2.7. Statistical analysis
The relationship between the contaminated samples and the
different kinds of raw milk was analysed using Chi-square analysis.
L. monocytogenes L. welshimeri L. innocua L. seeligeri
13 (5.4%) 6 (2.5%) 33 (13.8%) 2 (0.8%)
4 (2.4%) 4 (2.4%) 14 (8.5%) 5 (3%)
1 (2.4%) e 1 (2.4%) e
18 (4%) 10 (2.2%) 48 (10.8%) 7 (1.6%)
 H. Jamali et al. / Food Control 34 (2013) 121e125 123

Table 2 (8.4%). The isolates were resistant to tetracycline (49.4%), penicillin

Prevalence of L. monocytogenes serogroups in raw milk samples. G (43.4%), and amoxicillin-clavulanic acid (14.5%). All isolates of
Serogroups of L. monocytogenes Listeria spp. were susceptible to gentamicin, rifampicin and
“1/2a, 3a” “1/2c, 3c” “4b, 4d, 4e”
vancomycin.

Raw cow milk 8 (61.5%) 3 (23.1%) 2 (15.4%)

Raw sheep milk 2 (50%) 2 (50%)
Raw goat milk 1 (100%) e e
Total 11 (61.1%) 5 (27.8%)
All statistical and chi-square analyses were performed using SPSS
18.0 (SPSS Inc., Chicago, IL, USA).
3. Results
In this study, a total of 446 raw milk samples were examined for
Listeria spp. (Table 1). Out of 446 samples, 83 (18.6%) were
contaminated with Listeria spp. More than half (57.8%) of isolated
Listeria spp. was L. innocua. The remaining isolates were
L. monocytogenes (21.7%), L. welshimeri (12%), and L. seeligeri (8.4%).
A total of 18 isolates of L. monocytogenes which were identified by
using biochemical tests were also confirmed using a duplex-PCR.
Among the 446 samples, 54 raw cow milk (22.5%), 27 raw sheep
milk (16.4%), and two raw goat milk (4.9%) were naturally
contaminated with Listeria species. There was a significant differ-
ence between contaminated raw cow milk samples from other
kinds of raw milk (P < 0.05).
The 18 L. monocytogenes isolated from raw milk samples were
distributed into serogroups “1/2a, 3a” (61.1%); “1/2c, 3c” (27.8%);
and “4b, 4d, 4e” (11.1%) by PCR-serogrouping (Table 2, Fig. 1).
L. monocytogenes of serogroup “4b, 4d, 4e” was isolated only from
raw cow milk samples. However, serogroup “1/2a, 3a” was detected
in all kinds of raw milk tested.
All the 18 L. monocytogenes isolates showed the presence of
internalin genes, inlA, inlC, and inlJ (Fig. 2).
The resistance patterns of Listeria spp. to 10 antimicrobial agents
examined is shown in Table 3. On the whole, 44 of 83 Listeria spp.
isolates (53%) showed resistance to at least one antimicrobial agent.
Twenty (24.1%) and 17 (20.5%) of Listeria spp. isolates showed
resistance to one and two antimicrobial agents, respectively.
Multidrug resistance was observed in 7 isolates of Listeria spp.
e 4. Discussion
2 (11.1%) Among the different kinds of raw milk tested in this study, cow
milk had the highest percentage rate of Listeria spp., particularly
L. innocua and L. monocytogenes. L. monocytogenes was isolated
from raw cow milk (1.1%), raw sheep milk (6.5%), and raw goat milk
(1.7%) in Isfahan province, Iran (Rahimi et al., 2010). In another
study, Mahmoodi (2010) isolated L. monocytogenes from 2.5% of raw
milk samples collected from two traditional dairy manufactures in
Noorabad, located in Southern Iran. In other studies,
L. monocytogenes was isolated in 3.2% of raw cow milk (Gelbícová &
Karpísková, 2012a) and 3.8% of raw goat milk samples (Abou-
Eleinin, Ryser, & Donnelly, 2000). Furthermore, 29% of soft cheese
samples made from raw sheep milk were also contaminated with
L. monocytogenes (Pintado, Oliveira, Pampulha, & Ferreira, 2005).
Both L. ivanovii and L. seeligeri were detected from 1.4% of raw cow
milk samples in Egypt (El-Sherbini, Al-Agili, & Garbaj, 1998). In this
study, L. innocua was the main Listeria spp. isolated from raw milk
which is in agreement with the earlier findings reported by
Dhanshree, Otta, Karunasagar, Goebel, and Karunasagar (2003),
Jalali and Abedi (2008), and Rahimi et al. (2010).
Infected animals and poor silage quality in dairy farms have
been considered the source of Listeria spp. in raw milk, as well as
environmental contamination which could occur during milking
and storage (Bemrah, Sanaa, Cassin, Grifiths, & Cerf, 1998; Sagun,
Sancak, Isleyici, & Ekic, 2001). Therefore, in this study insufficient
hygiene and as well as poor silage quality in dairy farms are likely
the most common causes of Listeria spp. in raw milk.
In this study, serogroup “1/2a, 3a” was the predominant
L. monocytogenes serogroup. The serotype distribution of the raw
milk isolates was predictable simply because the 1/2a has been
reported dominant among strains isolated from food samples
(Gilbreth et al., 2005; Wieczorek, Dmowska, & Osek, 2012). The
high percentage rate of serogroup “1/2a, 3a” in the current study is
in agreement with earlier findings (Gelbícová & Karpísková, 2012a;
Waak, Tham, & Danielsson-Tham, 2002). Furthermore, Muraoka,
Gay, Knowles, and Borucki (2003) had detected 89.1% and 10.9%

Fig. 1. A representative gel of DNA amplicons generated by multiplex PCR for identification of L. monocytogenes serogroups. Serogroup “1/2a, 3a” is indicated by the amplicon of 691
bp, serogroup “1/2c, 3c” by two amplicons 691 bp and 906 bp, serogroup “1/2b, 3b, 7” by amplicon 471 bp and serogroup “4b, 4d, 4e” by two amplicons 471 bp and 597 bp. All
members of the Listeria genus give an amplicon of 370 bp with prs primers. Lanes 1 and 11, 100 bp molecular size marker; lanes 2 to 4, and 9, serogroup “1/2c, 3c”; lanes 5, 7, and 8,
serogroup “4b, 4d, 4e”; lane 6, serogroup “1/2a, 3a”; lane 10, negative control.
124 H. Jamali et al. / Food Control 34 (2013) 121e125

Fig. 2. Representative PCR-amplified products of three Listeria monocytogenes virulence genes: inlA, inlC, and inlJ. inlA, inlC, and inlJ are indicated by the amplicons of 800 bp, 517 bp
and 238 bp, respectively. Lanes 1 and 12, 100 bp molecular size marker; lanes 2 to 10, L. monocytogenes; lane 11; negative control.

serotypes 1/2a and 4b, respectively from bulk milk in the Pacific
Northwest.
The human foodborne listeriosis is commonly caused by sero-
types 1/2a, 1/2b and 4b (Martins & Leal Germano, 2011). Although
serotype 1/2a is the most isolated serotype from food samples, the
majority of listeriosis in humans is linked to serotype 4b. The dif-
ference in presence of serotype 4b in food samples and human
listeriosis cases could be due to the fact that serotype 4b is more
virulent. Serotypes 3a, 3b, 4d and 4e were seldom detected from
food matrices (Zhang et al., 2007). In this study, three serogroups
“1/2a, 3a”; “1/2c, 3c”; and “4b, 4d, 4e” were identified which were
probably serotypes 1/2a, 1/2c, and 4b, respectively.
The internalin genes (inlA, inlC and inlJ) tested in this study were
present in all 18 isolates of L. monocytogenes. Our finding is in
agreement with earlier findings, whereby the internalin genes
were present in almost all of the examined isolates of
L. monocytogenes isolated from different types of food categories
(Indrawattana et al., 2011; Lomonaco, Patti, Knabel, & Civer, 2012;
Sant’Ana et al., 2012), animal (Marien et al., 2007), human listeriosis
(Mammina et al., 2009), as well as environmental samples
(Gelbícová & Karpísková, 2012b). The presence of inlA, inlC and inlJ
genes showed the virulence potential of foods L. monocytogenes
isolates.
In the current study, Listeria species were resistant to tetracy-
cline and penicillin G, and to a lesser extent, to amoxicillin-
clavulanic acid, chloramphenicol, erythromycin, clindamycin, and
kanamycin. Resistance of food Listeria spp. to tetracycline and
penicillin G was reported by Rahimi et al. (2010), Marian et al.
(2012) and Ayaz and Erol (2010). About half (53%) of all Listeria
spp. isolates showed resistance to at least one antimicrobial agent
however, 7 (8.4%) showed multidrug resistance. The multidrug
resistant isolates were separated into L. monocytogenes (71.4%) and
L. innocua (28.6%). Our findings showed that food L. monocytogenes
isolates are sensitive to gentamicin, vancomycin and rifampicin
which are in agreement to previous studies (Arslan & Özdemir,
2008; Conter et al., 2009; Hof & Emmerling, 1984; Srinivasan
et al., 2005). Recently Harakeh et al. (2009) also reported food
L. monocytogenes isolates which showed high sensitivity to van-
comycin and gentamicin.
A high prevalence of resistance to tetracycline was found by
Harakeh et al. (2009) and Conter et al. (2009), while in the earlier
study a low number of Listeria showed resistance to tetracycline
(Arslan & Özdemir, 2008). Since the previous decade, tetracycline
has been applied in animals’ food to treat infection diseases
particularly in dairy farms of Iran. The high resistance rate of Lis-
teria spp. to tetracycline could be due to widespread use of the
antimicrobial. The presence of antimicrobial resistance Listeria in
different food matrices and transmission of Listeria with contami-
nated food can be an important public health concern.
In summary, the presence of L. monocytogenes in raw milk sam-
ples showed that consumption of raw and unpasteurised milk could
be a potential risk of foodborne listeriosis especially at the high risk
groups. Continued surveillance on prevalence of L. monocytogenes
and also on emerging antimicrobial resistance is needed to enable
the recognition of foods that may represent risks and also ensure the
effective treatment of listeriosis.

Table 3
Resistance profiles of Listeria spp. isolated from raw milk samples.

Listeria spp. L. monocytogenes L. welshimeri L. innocua L. seeligeri

Penicillin G 36 (43.4%) 11 (61.1%) 4 (40%) 18 (37.5%) 3 (42.9%)

Amoxicillin-clavulanic acid 12 (14.5%) 4 (22.2%) e 7 (14.6%) 1 (14.3%)
Chloramphenicol 9 (10.8%) 5 (27.8%) e 3 (6.3%) 1 (14.3%)
Erythromycin 6 (7.2%) 2 (11.1%) e 4 (8.3%) e
Gentamicin e e e e e
Tetracycline 41 (49.4%) 13 (72.2%) 3 (30%) 24 (50%) 1 (14.3%)
Vancomycin e e e e e
Rifampicin e e e e e
Clindamycin 4 (4.8%) 1 (5.6%) e 3 (6.3%) e
Kanamycin 1 (1.2%) e e 1 (2.1%) e
Resistance to 1 Antimicrobial 44 (53%) 10 (22.7%) 5 (11.4%) 25 (56.8%) 4 (9.1%)
Resistance to 2 Antimicrobial 17 (20.5%) 2 (11.8%) 1 (5.9%) 15 (88.2%) 1 (5.9%)
Resistance to  3 Antimicrobial 7 (8.4%) 5 (71.4%) e 2 (28.6%) e
 H. Jamali et al. / Food Control 34 (2013) 121e125
Acknowledgement
This study was supported by PPP grant (PV123/2012A) and High
Impact Research Grant e Molecular Genetics (UM.C/625/1HIR/
MOHE/ -02 [A000002-5000 1]) from the University of Malaya.
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