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ISOLATION AND MOLECULAR IDENTIFICATION OF YERSINIA ENTEROCOLITICA

IN LOCALLY PRODUCED RAW MILK IN IRAQ

Article in Biochemical and Cellular Archives · June 2020

DOI: 10.35124/bca.2020.20.1.1105

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 DOI : 10.35124/bca.2020.20.1.1105
Biochem. Cell. Arch. Vol. 20, No. 1, pp. 1105-1111, 2020 www.connectjournals.com/bca ISSN 0972-5075

ISOLATION AND MOLECULAR IDENTIFICATION OF YERSINIA

ENTEROCOLITICA IN LOCALLY PRODUCED RAW MILK IN IRAQ
May Mohammed Ali1* and Firas Rashad Al-Samarai2
Department of Microbiology, College of Medicine, University of Kerbala, Karbala, Iraq.
1

2
Department of Veterinary Public Health, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq.
*e-mail : [email protected], [email protected]
(Received 21 September 2019, Revised 14 December 2019, Accepted 20 December 2019)

ABSTRACT : The aim of this study was to investigate the incidence of Y. enterocolitica in locally produced raw milk samples
of two different animal species. A total of two hundred and forty cow and buffalo raw milk samples were collected from several
markets in Baghdad and four provinces of middle Euphrates, Iraq (168 samples for cow milk and 72 for buffalo). All the samples
subjected to conventional cultured on CIN agar in order to obtain Yersinia species, which revealed 58(24%) positive isolates
considered as presumptive Yersinia species then all those isolates subjected to three laboratory tests for confirmation and
identification the existence of positive Yersinia enterocolitica species isolates (traditional biochemical tests, VITEK 2 and
conventional PCR).
The total rate of infected raw milk samples was 12% with Yersinia enterocolitica. The highest contaminated samples obtained
in cow milk (14.2%) followed by buffalo milk (6.9%). The results of the conventional culture method showed a higher number
of isolates. The number of isolates reduced to 53(22%) isolates after performing traditional biochemical tests, VITEK 2
compact system achieved better performance in identification of Yersinia enterocolitica isolates, only 29 (12%) of the study
isolates successfully amplified and shown single band of 16s rRNA gene of Y. entercolitica at 1485 bp. Both ail and Inv genes
were perceived in all 29 isolates while YadA gene was positive only in 24 isolates. Based on the current results, it may be
concluded that locally produced raw milk in Iraq is not always prepared under proper hygienic practices may become a public
health hazard, as it may act as a potential vehicle for the transmission of virulence bacterial pathogens. For this reason, it is
advisable to use strict conditions in producing and processing of the raw milk to reduce the hazards that may be involved with
its consumption.
Key words : Baghdad, cow, buffaloes, milk, 16s rRNA, virulence genes, Yersinia enterocolitica.

INTRODUCTION (Momtaz et al, 2013). Yersiniosis is a typical foodborne

Foodborne diseases are an important and growing disease (Zoonotic gastroenteritis) in humans has been
public health and economic problem in many countries. identified as emerging disease in children and in indigent
Many factors related to social and environmental changes populations (Omar, 2015). Yersinia enterocolitica is the
favor the spread of infectious diseases transmitted by leading cause of yersiniosis in Europe and one of the four
food (Curtis et al, 2014). Since milk and dairy products major causes of gastrointestinal disease. Yersinia
are perishable and vulnerable to bacteria beside enterocolitica is a food-borne pathogen estimated to
impracticality to create and maintain sterile food industry cause 117,000 illnesses, cases of human Yersiniosis
environments especially under adverse tropical zone annually in the United States (EFSA and ECDC, 2015).
particularly non-health conditions in our country and other The pathogen is Gram-negative pleomorphic ranging from
third world countries, microbiological safety against food small coccobacilli with rounded ends facultative anaerobic,
pathogens have always been a major concern for public the motility is temperature regulated: at 25°C. Yersinia
health. enterocolitica is peritrichously flagellated, but at 37°C
they are unflagellated and so non motile widely distributed
A high prevalence of gastrointestinal illness, including
in nature in addition to the psychotropic nature of Y.
fatal cases attributable to yersiniosis, is also observed in
enterocolitica, which enable the bacterium to grow to
many developing countries, including Iraq, Iran,
large numbers at temperatures close to 0°C characterized
Bangladesh, and Nigeria Indicating major underlying food
by temperature-dependent adaptations (Aziz and Fatima,
safety problems in low and middle-income countries
1106 May Mohammed Ali and Firas Rashad Al-Samarai
2019). The epidemiology of yersiniosis is not entirely clear. (100-250 ml). They were collected randomly from markets
The pathogenic and nonpathogenic strains of Y. of different regions of Baghdad province and four
enterocolitica are widely distributed throughout the provinces of middle Euphrates (Karbala, Babylon, Al-
environment and have been repeatedly isolated from food Najaf and Al Qadysia) during December 2018 till May
(raw milk, chocolate milk, dairy cream and ice cream, 2019. The samples were transported in a cool box to the
meat, eggs, vegetables contaminated with feces of lab. The sampleswere analyzed and processed according
infected animals or secondarily during the technologic to different standard reference foodmicrobiological which
process, sewage-contaminated water, soil, sea-food ,many most recommended and used for isolation Y.
animal species and man). Therefore, consumption of milk enterocolitica from animal food products with some
and dairy products especially raw ones give a higher modifications in which they collected aseptically in screw-
chance for infection by Y. enterocolitica in humans (Acha capped containers (250 ml) and transported in the ice
and Szyfres, 2003). In Iraq similar to other countries box to the milk hygiene laboratory as soon as possible,
locally produced raw milk and soft cheese which then warmed and homogenized at lab temperature.
produced from raw milk with insufficient hygienic Processing of Samples unit
condition. The low isolation rates of pathogenic Y.
All the samples subjected to pre-enrichment by using
enterocolitica in food samples may be due to the limited
a modified typtone soy broth (MTSB), double strength
sensitivity of the culture methods (Fredriksson et al, 2008).
power containing tryptone soy broth (TSB) plus yeast
Subsequently, the detection, isolation, serological,
extract (YE), one part of raw milk sample (25 ml) to 9
biochemical classification and enumeration of Y.
parts (225 ml) of (MTSB-YE ) to get (1:10) (dilution food
enterocolitica remained problematic and it is important
standard formula) , then homogenized with stomacher
to determine the pathogenic significance of isolates. This
for (3-5) minutes after that incubated at 25ºC for 24 hours
can be done with several phenotypic tests which are time-
for resuscitation of stressed or sub lethally damaged cells.
consuming and not always reliable (Fredriksson and
After pre enrichment the samples underwent cold
Korkeala, 2003).
enrichment in different Yersinia selective broths which
A set of three tests has been proposed to separate are recommended by ISO10723:(2003) for the detection
pathogenic from non-pathogenic Yersinia trains; of pathogenic Y. enterocolitica in food. PSB (peptone
Pyrazinamidase activity, Esculin hydrolysis and Salicin Sorbitol bile broth), ITC (Irgasan/TriclosanTicarcillin/
fermentation. The virulence of Yersinia enterocolitica Chlorate broth base) for (48-72) hours at 4°C then the
depends on possession of both chromosomal and cold enriched samples were subjected to alkali treatment
plasmidial genes because they are essential to work as (0.5) ml of each. The culture was transferred for 20
pathogen. These genes are responsible for the assembly seconds into 4.5 ml of 0.5% KOH in 0.5% NaCl) mixed
of the proteins :Inv (invasin), Ail (attachment invasion by vortexing for (2-3) seconds, then a loopful was
locus), Yst (Yersinia stable toxin) and YadA (Yersinia streaked onto MacConkey and CIN (Cefsulodin-Irgasan-
adhesion) (Hallanvuo, 2009). Many studies targeted those Novobiocin) agar plates which supplemented with
genes to verify the pathogenicity of Y. enterocolitica Cefsulodin-irgasan-novobiocin antibiotics which
isolates in which Polymerase Chain Reaction (PCR) have simultaneously and incubated at 25°C for (24-48) hours.
been used (Darwish et al, 2015; Khalid and Abbas, 2019). The plates were examined for the presence of specific
Currently, there is no comprehensive data regarding to colonies of Yersinia species morphology, Isolates showing
the prevalence of virulent Y.enterocolitica in raw milk both Non Lactose Fermenting (NLF) colonies on
in Iraq. Therefore, the present study was undertaken to MacConkey agar andbull’s eye colonies with deep red
investigate the prevalence rate of virulent Y. center and clear colorless periphery on CIN agar plates
enterocolitica in raw milk of two ruminant species that were considered presumptive Yersinia species. The
produced locally and marketed in the different areas of suspected colonies were picked and purified then stained
Baghdad province and the provinces around Baghdad in with Gram’s technique and examined microscopically
Iraq by performing both phenotypic and genotypic methods after that the isolates were subjected to multiple traditional
and to determine the pathogenicity of the isolates rapidly lab biochemical tests for preliminary detection for Y.
by molecular method (PCR). enterocolitica (oxidase, catalase, urease, triple sugar iron
MATERIALS AND METHODS test and citrate utilization test). The presumptive Yersinia
Collection and processing of samples isolates subjected to the identification at species level by
A total (240) raw milk samples included (168 cow using VITEK2 compact system and identification at
and 72 buffalo) of raw milk each sample volume was molecular lenvel was conducted by conventional PCR
 Isolation and molecular identification of Y. enterocolitica 1107
assay. The primers were lyophilized and dissolved in free
Testing for Pathogenicity markers deionized distill water (ddH2O) to get a final concentration
of 100 pmol/µl as stock solution, which was kept at -
Y. enterocolitica strains were tested for virulence
20°C to prepare10 pmol/µl concentration as work primer
phenotypic tests including Temp-Dependent
suspended, 10 µl of the stock solution in 90 µl of free
autoagglutination (25°C-35°C) in Methyl Red-
deionized distill water (ddH2O) to reach a final volume
VogesProskauer broth, Congo red absorption and
100 µl. Different primers were used in this study. Primer
occurrence of small red colonies on Congo red agar,
sequences, their references, product lengths, and specific
pyrazinamidase production were performed three times
targets are listed in Table 1. The PCR conditions for the
for all study isolates as described by Yang and Fang
primers listed in Tables 2, 3 and 4, respectively.
(2002). Pyrazinamidase production should be negative
for virulent Y. enterocolitica while the other two tests Afterwards, 10 µl of PCR amplicon were analyzed
should be positive for the presence of plasmid in virulent by electrophoresis on a 2.5% agarose gel. Gels were
Y. enterocolitica (Yang and Fang, 2003). visualized under a UV illuminator and photographed
through gel documentation system. A 100-bp DNA ladder
PCR-based detection of Y. enterocolitica
(Bioneer, Korea) was used as DNA molecule size marker.
DNA extraction
Statistical analysis
Bacterial DNA was extracted from fresh Y.
Data were subjected to analysis using SAS software.
enterocolitica colonies grown in the broth containing
The prevalence of unpaired groups was compared using
presumptive pure culture colonies of Y. enterocolitica
Chi square whereas the four matched groups were
by using GeneaidPresto™ Mini DNA Bacteria Kit
compared using Cochran’s Q test.
(Taiwan) following manufacturer’s instructions for DNA
extraction protocol extracted DNA was stored at –20°C RESULTS
until used. In Iraq, the prevalence of Y. enterocolitica in foods
Primers and its signiûcance in public health is still unknown.
Although, in some debating investigations the prevalence
The Y. enterocolitica strains were identified by PCR
rate of Y. enterocolitica as causative agent of
for presence of 16srRNA gene for confirmation of
gastroenteritis has been evaluated, no clear data of its
pathogenicity. The PCR targeted the chromosomal
quota in intestinal infections is available. Furthermore,
virulence genes comprised in the analysis: Ail (attachment
eating habits in Iraq are different from developed
invasion locus gene), Inv (Invasin gene ) and the plasmid-
countries, as most generally prefer to consume traditional
borne virulence gene YadA (Yersinia adhesin A) gene.
andhome-made foods rather than industrially-produced
Table 1 : Primers used in this study to detect the16s rRNA, ail, inv and Yad A genes in Y. enterocolitica.
Primer Size in BP Sequence (5′′-3′′ ) Reference
16s rRNA 1485 5′-AGAGTTTGATCCTGGCTCAG-3′ Hao et al (2016)
5′-GGTTACCTTGTTACGACTT-3′
Ail 351 5′-TAATGTGTACGCTGCGAG-3′ Thoerner et al (2003)
5′- GACGTCTTACTTGCACTG-3′
Inv 183 5′-CGGTACGGCTCAAGTTAATCTG-3′ Thoerner et al (2003)
5′-CCGTTCTCCAATGTACGTATCC-3′
YadA 849 5′-CTTCAGATACTGGTGTCGCTGT-3′ Thoerner et al (2003)
5′-ATGCCTGACTAGAGCGATATCC-3′

Table 2 : Amplification of 16s rRNA gene was achieved using the Table 3 : Amplification of Ail gene was achieved using the following
following condition. condition.
Phase Tm(°C) Time No. of cycle Phase Tm(°C) Time No. of cycle
Initial Denaturation 94 °C 5 min 1 cycle Initial Denaturation 94 °C 5 min 1 cycle
Denaturation 94 °C 30 sec Denaturation 94 °C 30 sec
35 cycles 35 cycles
Annealing 62°C 30 sec Annealing 57 °C 30 sec
Extension-1 72 °C 1 min Extension-1 72 °C 30 sec
1 cycle 1 cycle
Extension-2 72 °C 5 min Extension-2 72 °C 5 min
1108 May Mohammed Ali and Firas Rashad Al-Samarai
Table 4 : Amplification of: Inv gene were achieved using the following Table 5 : Amplification of: YadA gene were achieved using the
condition. following condition.
Phase Tm(°C) Time No. of cycle Phase Tm(°C) Time No. of cycle
Initial Denaturation 94 °C 5 min 1 cycle Initial Denaturation 94 °C 5 min 1 cycle
Denaturation 94 °C 30 sec Denaturation 94 °C 30 sec
35 cycles 35 cycles
Annealing 61 °C 30 sec Annealing 60 °C 30 sec
Extension-1 72 °C 30 sec Extension-1 72 °C 1 min
1 cycle 1 cycle
Extension-2 72 °C 5 min Extension-2 72 °C 5 min

Table 6 : Prevalence of Yersinia species in raw milk samples collected from different cows and buffalos using conventional culture method,
VITEK 2 and PCR confirmation.
Type sample No. Positive Positive isolates Positive isolates Positive isolates Cochran’s P
isolateson according to according toVITEK 2 according to Q value
CIN biochemical tests compact system PCR
Cow milk 168 45(26.7%) 43(25.5%) 25(14.8%) 24(14.2%)
Buffalo milk 72 13(18%) 10(13.8%) 5(6.9%) 5(6.9%)
Total 240 58(24 %.) 53(22%) 30(12.5%). 29(12%) 15.73 0.0004
Chi square value 2.09 4.01 2.90 2.55
P 0.14 0.04 0.08 0.10

types. This isespecially true about consumption of raw samples except in second procedure of traditional
milk. Therefore, investigations dealing with the prevalence biochemical test. As shown in Fig. 1 data summarized in
rate of virulent Y. enterocolitica in traditional dairy Table 6.
products seem to be of paramount importance. The Y. enterocolitica isolates were tested for
According to the results, 29 (12%) molecularly virulence by phenotypic tests including Temp-Dependent
confirmed positive isolates of total samples consisting of autoagglutination (25°C-35°C) in Methyl Red-
240 raw milk were positive for occurrence of Y. VogesProskauer broth, Congo red absorption only 19
enterocolitica, 24(14.2%) isolates, 5 (6.9%) isolates were isolates revealed positive results for both, while only 25
found in cow milk and buffalo milk, respectively. The isolates achieved negative results for pyrazinamidase
using of the conventional culture isolation procedure production .As a whole, all 29 Y. enterocolitica positive
revealed that out of 240 milk samples, 58 (24%) positive isolates that revealed positive for 16s rRNA gene were
isolates gave the typical bull’s eye character while after examined by common PCR for existence of virulence
investigation using traditional specific biochemical tests genes in chromosomes and plasmids.
for identification of Y. enterocoliticaonly 53(22%) A gene of Y. enterocolitica visualized under
isolates revealed the typical results for Y. enterocolitica. ultraviolet light (after ethidium bromide staining).
The identification of Y. enterocolitica at species level
DISCUSSION
by using VITEK 2 compact system the positive isolates
were 30 (12.5%). One of the global public health objectives is to achieve
Food safety control and prevention the food borne diseases
In identification at molecular level by conventional
that may act as threaten on human health. Therefore,
PCR the positive isolates for 16s rRNA gene were only
detection of food pathogens is important to recognize and
29 (12%) successfully amplified and shown single band
prevent the problems concerned to health and safety
of 16s rRNA gene of Y. enterocoliticaat 1485 bp.
(Fung et al, 2018). Yersinia specifically Y. enterocolitica
According to the results there is highly significance are encompassed of strains with different degrees of
variation between the four different identification pathogenicity also special attention to the issue of safety
procedures that used in our study to investigate the of dairy products on Yersinia infection is associated with
occurrence of Y. enterocolitica isolates in the total 240 ability of Y. enterocolitica to grow to large numbers at
tested raw milk samples. While there is no significance refrigeration temperatures in the raw milk and keep on
variation between the different identification procedures viable at low temperatures for a long time, so
that used in our study to investigate the occurrence of Y. contaminated milk with the organism could become a
enterocolitica in case of cow and buffalo raw milk tested
 Isolation and molecular identification of Y. enterocolitica 1109

Fig. 1 : Identification species specific of 16s rRNA gene of Y. entercolitica PCR product at the band size 1485 bp. Lane M = DNA ladder
(100 pb), lane (1-29) represent gene of 16s rRNA of Y. enterocolitica which appear in 29 isolates, lane 21 negative of 16s rRNA gene
of Y. enterocolitica.

Fig. 2 : Identification of Ail gene the PCR product at band size 351 bp (Lane M:100 bp DNA marker, Lane (1-29) represent Ail gene of Y.
enterocolitica appeared in all 29 of Y. enterocolitica isolates visualized under ultraviolet; light (after ethidium bromide staining).

Fig. 3 : Identification of Inv gene the PCR product at band size 183bp. (Lane M:100 bp DNA marker, Lane 1-29 represent Inv gene of Y.
enterocolitica appeared in all 29 of Y. enterocolitica isolates) visualized under ultraviolet light (after ethidium bromide staining).

Fig. 4 : Identification of Yad A gene the PCR product at band size 849 bp (Lane M:100 bp DNA marker, Lane (1-3, 5-7, 9-11, 13-16, 19-29)
represent Yad A gene of Y. enterocolitica appeared in 24 of Y. enterocolitica isolates lane (4, 8, 12, 17, 18) negative of Yad.

significant health risk for consumers. Thus, the milk of samples were infected with pathogenic strains of
consumption of contaminated dairy products is a high risk Y. enterocolitica at rate (12%) in the present study, while
of yersiniosis infection (Hanifian and Khani 2012; Jamali in similar study the pathogen was isolated in lower rate
et al, 2015). During 2012, there were 6776 reported (4%) from of raw milk in southern Iraq (Khalid and Abbas,
yersiniosis cases in humans. Yersiniosis is listed in the 2019). Different studies have reported the contamination
third place after campylobacteriosis and salmonellosis rate of raw milk with Y. enterocolitica, for example:
(EFSA and ECDC, 2013). Results profile reflect that raw studies conducted in Iran recorded 7.6% by Hanifian and
1110 May Mohammed Ali and Firas Rashad Al-Samarai
Khani (2012) and 5.3% by Jamali et al (2019). In et al, 2001; Vishnubhatla et al, 2001) and some
Morocco, Hamama et al (1992) also reported 30%, in biochemical activities of Y. enterocolitica are
Lebanon, Harakeh et al (2012) reported 9.75%. Also in temperature dependent (above and below 30°C) so
several studies in Egypt reported variable rates (Ahmed misidentifications can occur (Manafi and Holzhammer,
et al, 2019), 22% (Darwish et al, 2015), 46% and 10% 1994). Thus, differentiation by biochemical tests is usually
by Ali et al (2015). In Turkey 25% by Yucel and Ulusoy based on a limited set of strains, especially among Y.
(2006) and 3.3% by Ozdemir and Arslan (2015). In enterocolitica. Since the Vitek2 card attributed 96% of
another study in India reported 24.1% in raw buffalo milk study presumptive isolates we conclude that Colorimetric
by Toora et al (1989), while in Egypt by Darwish et al card system may be a very high-performing tool can be
(2015) showed that buffalo contamination rate in Yersinia used effectively for screening with only one disadvantage
was 25%. Some authors failed in detection of Y. is that they do not distinguish between virulent and a
enterocolitica in milk samples (Zeinhom and Abdel-Latef, virulent strains and do not list all the Yersinia species in
2014). The variations between findings of numerous their database. Thus, identification based on a biochemical
authors and of this study would possibly be due to tests and manufactured diagnostic kit like VITEK2and is
many factors such as distinction in ways of sampling inadequate in order to avoid misidentifications. Generally,
range of analyzed samples, ways of analyzing, sources of our results record highly significance variation between
samples, season, sanitary measures and geographical the identification procedures that used in the current study
location. These factors might cause a rise or decrease thus gene analysis is the “gold standard” for bacterial
in the incidence of Yersinia spp. infection. The incidence identification PCR method is superior to the cultural and
of Yersinia spp. using conventional culture isolation biochemical tests as it is able to detect Y.enterocolitica
procedure revealed that out of 240 milk samples, 58(24%) isolates as well as their pathogenicity with more specificity
samples gave the typical bull’s eye character, after the and rapidity.
molecular identification for the presumptive Yersinia
Neubauer et al (2001) described a PCR method
isolates by PCR assay through successful amplification
targeting 16S rRNA gene to identify presumptive Y.
to 16s rRNA the incidence of Yersinia is reached to 29
enterocolitica strains, previously biochemically identified
(12%) confirmed Y. enterocolitica isolates. This can be
as belonging to the Yersinia genus. The result of 16s rRNA
due to several factors: Existence of large population of
gene PCR in which 29 (12%) isolates successfully
background flora which may interfere with correct
amplified single band of 16s rRNA specific gene of
selection of the specific colonies of Yersinia organisms
Y.enterocolitica at 1485bp and this is agreed with results
additionally, the capability of some types of
accomplished by Hao et al (2016). Our result shows the
Enterbacteriacae as to grow on CIN agar media and
importance of 16s rRNA PCR assay in differentiation of
yield similar colony morphology is another impeding factor
Y. enterocolitica since it offers a clear advantage over
(Fukushima, 1987). The complexity of the conventional
conventional biochemical tools such as the Vitek 2, as it
culturing method which include pre enrichment, cold
achieves better analytical performance, but at lower cost
enrichment, alkali treatment and plating on CIN media
and much shorter time for identification. For identifying
multiple steps method was not balanced by high recovery
the pathogenicity of the Y. enterocolitica isolates
rates of confirmed Y. enterocolitica strain. Absence of
virulence genes were targeted by PCR assay (Ail, Inv
a completely dependable method for isolation of Yersinia
and YadA) also several authors investigate the
from different type of samples can be additional factor
occurrence and distribution of those genes in Y.
(Neubauer et al, 2001). Thus the traditional cultural
enterocolitica strains in food samples by PCR (Thoerner
technique showed high false positive in the tested sample
as this compared by PCR analysis, the PCR technique et al, 2003; Falco et al, 2004, Saberianpour et al, 2014
facilitates epidemiological studies, the number of Positive and Darwish et al, 2015). This study shows that
presumptive Y. enterocolitica isolates according to prevalence rate of virulent Y. enterocoliticais relatively
biochemical tests was 53(22%). When this number high and there is a potential for the transmission of
compared to PCR confirmed isolates number we can bacterium to humans via the consumption of raw milk.
conclude there was false positive results in biochemical Our results from a random 240 sample of raw milk can
confirmation. This difference was also reported by Rahimi contribute important information on the role of raw milk
et al (2014), who identified only 24 out 28 Y. in foodborne human yersiniosis. Most importantly,
enterocolitica isolates by PCR. This difference can preventive measures must be adopted to avoid risks for
result from the fact that all biochemical tests are reported human being associated with consumption of milk
to be complex and provide unreliable results (Neubauer containing pathogenic Yersinia spp.
 Isolation and molecular identification of Y. enterocolitica 1111
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