VETERINARSKI ARHIV 92 (4), 525-530, 2022

DOI: 10.24099/vet. arhiv.1346

Detection of virulence genes and determination

of the antimicrobial susceptibility of Escherichia coli isolates
with mastitis in Mashhad, Iran – a short communication

Fateme Aflakian1, Mehrnaz Rad1, Himen Salimizand2,3, Ali Nemati1,

and Abolfazl Rafati Zomorodi4,5*

1
Department of Pathobiology, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
2
Vaccine and Infectious Disease Organization Saskatoon, SK, Canada
3
Department of Vaccinology and Immunotherapeutics, School of Public Health,
University of Saskatchewan, Saskatoon, SK, Canada
4
Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Science, Shiraz, Iran
5
Student Research Committee, Shiraz University of Medical Science, Shiraz, Iran

Aflakian, F., M. Rad, H. Salimizand, A. Nemati, A. R. Zomorodi: Detection of virulence genes

and determination of the antimicrobial susceptibility of Escherichia coli isolates with mastitis in Mashhad, Iran
– a short communication. Vet. arhiv 92, 525-530 2022.

ABSTRACT
The purpose of this study was to determine the virulence genes and antimicrobial resistance patterns of Escherichia
coli isolated from milk samples of cows with bovine mastitis. Forty-seven E. coli isolates from clinical mastitis milk
samples, from five dairy farms in Northeast of Iran, were subjected to multiplex PCR to determine virulence genes
stx1, stx2, eaeA, hlyA, sta, F4, F17, fliC, and rfbE. In addition, antimicrobial susceptibility was assessed by applying
disk diffusion methods. The eaeA and stx1 genes were most frequently detected in 42 (89.3%) and 34 (72.3%) isolates,
respectively. However, the least frequent gene was F41 as it was found in only one isolate (2.1%). Furthermore,
9 out of 47 isolates were hlyA positive, and four isolates harbored the sta gene. The antimicrobial susceptibility
demonstrated the highest resistance against lincomycin (100%) and neomycin (91.4%). Since these bacteria represent
a high-risk pathogen on farms, the emergence of multiple antibiotic-resistant and pathogenic E. coli strains should be
of great concern for public health.
Key words: E. coli, virulence genes; antimicrobial resistance; mastitis

Introduction
Mastitis causes a large amount of economic loss negative, opportunistic bacterium, which is
since it causes medical and veterinary expenses commonly found in the environment of dairy
and through declining milk production, disposal cows, that infects the mammary glands through
of milk from cows treated with antibiotics, and environmental contact (TODOROVIĆ et al., 2018).
ultimately the extirpation of dairy cattle in herds However, non-pathogenic E. coli strains are used
(ZAFARANE et al., 2017). E. coli is a Gram- in dairy products for several metabolic activities

∗
Corresponding author:
Abolfazl Rafati Zomorodi, Department of Bacteriology and Virology, Shiraz Medical School, Shiraz University Medical Science, Shiraz, Iran

ISSN 0372-5480
Printed in Croatia 525
 F. Aflakian et al.: Virulence genes and the antimicrobial susceptibility of Escherichia coli

(MLADENOVIĆ et al., 2018). Although therapy Materials and methods

with antibiotics can significantly reduce E. coli Bacterial identification. In the present study, 47
mastitis, antimicrobial resistance has arisen due to E. coli isolates were collected from October 2015 to
the use of broad-spectrum antimicrobial agents for December 2016, from clinical mastitis milk samples
coliform mastitis treatment (FERNANDES et al., from five different farms in Mashhad. Standard
2011). Antibiotic monitoring should be undertake biochemical tests, as published by MLADENOVIĆ
to detect and control microbial resistance. This et al. (2018) were carried out in order to identify
study was carried out to assess the major virulence E. coli isolates. The isolates were then preserved at
genes and antibiotic susceptibility profile of E. coli -20 °C in nutrient broth with 15% glycerol in the
isolates from milk samples from dairy cattle with microbiology laboratory of the Veterinary Faculty,
clinical mastitis in Mashhad, North-East Iran. Ferdowsi University of Mashhad.

Fig. 1. The detection of stx1, stx2, eaeA and hlyA genes by multiplex PCR. stx1 amplicon size, 210bp;
stx2 amplicon size, 484bp; eaeA amplicon size, 397 bp; hlyA amplicon size 166 bp.

Fig. 2. The detection of F17 gene by monoplex PCR.

526 Vet. arhiv 92 (4), 525-530, 2022

 F. Aflakian et al.: Virulence genes and the antimicrobial susceptibility of Escherichia coli

Antimicrobial Susceptibility Testing. The Detection of the Virulence Genes. The multiplex
evaluation of antimicrobial susceptibility was PCR assay described by FRATAMICO et al. (2000)
carried out with the Kirby-Bauer disk diffusion was performed to assess nine virulence genes,
method (HUDZICKI, 2009) for 10 antibiotic disks including: stx1 and stx2, eaeA, hlyA, sta, F4, F17,
(MAST, United Kingdom) as follows: linco-spectin fliC and rfbE. In this order, genomic DNA was
(15/200 μg), gentamicin (10 μg), lincomycin (2 extracted by the boiling method (AHMED et al.,
μg), ampicillin (10 μg), oxytetracycline (30 μg), 2014). Further information regarding primers is
enrofloxacin (5 μg), trimethoprim/sulfamethoxazole given in Table 1 (BERTIN et al., 1996; DONG et
(1.25/23.75 μg), tetracycline (30 μg), streptomycin al., 2015; FRANCK et al., 1998).
(10 μg), and neomycin (30 μg).

Table 1. Sequences of the oligonucleotides used as primers in multiplex PCR and

monoplex PCR

Primer Product size Reference

PCR name Direction Primer sequence (5´-3´)
name (bp)
8

Forward GCATCAGCGGCAGTATCT
F41 380
Reverse GTCCCTAGCTCAGTATTATCACCT
PCR1

Forward GCTAATGTTGGCAATTTTTATTTCTGTA
Sta 190
Reverse AGGATTACAACAAAGTTCACAGCAGTAA 8
Forward TGTAACTGGAAAGGTGGAGTATACA 9
Stx1 210
Reverse GCTATTCTGAGTCAACGAAAATAAC
Forward GTTTTTCTTCGGTATCCTATTCC 9
Stx2 484
Reverse GATGCATCTCTGGTCATTGTATTAC
PCR2 Forward ATTACCATCCACACAGACGGT 9
eaeA 397
Reverse ACAGCGTGGTTGGATCAACCT
9
Forward ACGATGTGGTTTATTCTGGA
hlyA 166
Reverse CTTCACGTCACCATACATAT
Forward GCAGAAAATTCAATTTATCCTTGG 10
PCR3 F17 537
Reverse CTGATAAGCGATGGTGTAATTAAC
Forward GGATGACAAATATCTGCGCTGC 9
rfbE 213
Reverse GGTGATTCCTTAATTCCTCTCTTTCC
PCR4
Forward GCGCTGTCGAGTTCTATCGAGC 9
fliC-H7 625
Reverse CAACGGTGACTTTATCGCCATTCC

Results
Antimicrobial susceptibility testing. The in 47 (100%) and 43 (91.4%) isolates, respectively.
interpretation of the antimicrobial susceptibility Also, the least resistance was demonstrated for
test was performed according to EUCAST 2018 gentamycin and ampicillin in zero and one (2.13%)
guidelines. The disc diffusion method revealed the isolate, respectively. The results of resistotyping
highest resistance against lincomycin and neomycin are listed in Table 2.

Vet. arhiv 92 (4), 525-530, 2022 527

 F. Aflakian et al.: Virulence genes and the antimicrobial susceptibility of Escherichia coli

Table 2. Susceptibility testing of E. coli isolates (n=47). Results presented as percentages.

Antibiotic disks LSP NFX AMP STR TET GEN OTE TMP NEO LIN
Susceptible 76.6 87.2 82.9 76.5 68 100 80.8 85.1 0 0
Resistant 23.4 12.8 17.1 23.4 31.9 0 19.1 14.8 100 100
Abbreviations: LSP, linco-spectin; NFX, enrofloxacin; AMP, ampicillin; STR, streptomycin; TET, tetracycline; GEN, gentamycin;
OTE, oxytetracycline; TMP, Trimethoprim/sulfamethoxazole; NEO, neomycin; LIN, lincomycin.

The frequency of virulence genes. The isolates from fecal samples of diarrheic children,
most frequent genes, among all the isolates, as sheep, and cattle (BADOUEI et al., 2016). The
determined by PCR, were eaeA 42 (89.3%) and fimbriae F41 and F17-related adhesins were the
stx1 34 (72.3%). The least frequent gene was F41, most common in E. coli isolates from diarrheic
in one isolate (2.1%). The frequencies of stx2, calves (KIM et al., 2016). The incidences of F41
hlyA, F17 and sta genes in E. coli isolates were and F17 were 2.1% and 12.7% in the current study,
found to be in 26 (55.3%), 9 (19.1%), 6 (12.7%) and these results were close to previous studies
and 4 isolates (8.5%), respectively. In addition, 12 (GÜLER and GÜNDÜZ, 2007; MEMON et al.,
(25.5%) and four (8.5%) isolates carried only rfbE 2016). According to these reports, it seems that
and fliC, respectively. two F17 and F47 genes do not play any important
role in mastitis infections. In this study, fliC was
Discussion detected in four isolates (8.5%), while in another
In the present paper we investigated the presence survey this gene was found in 49% E. coli isolates
of virulence genes among pathogenic E. coli strains from raw milk samples (CAINE et al., 2014). The
that were collected from dairy cattle with clinical differences in the presence of virulence genes
mastitis. There were non-pathogenic E. coli strains in E. coli depend on sampling strategy, seasonal
used in the processing of dairy products, so these variations, geographic area, hygiene on farms and
commensal strains are capable to turn into the the number of cows included for sampling.
wide strains by transferring virulence genes (BOK The resistance rate to enrofloxacin and
et al., 2015). In the current study, the prevalence ceftriaxone was similar to the report by
of stx1 and stx2 genes were (72.3%) and (55.3%), IWERIEBOR et al. (2015), but the observation
respectively. The results of our study were in of resistance to ampicillin in E. coli isolates was
accordance with previous research that reported the dissimilar to another study since those authors
prevalence of stx1 at the rate of 35.7% (CENGIZ observed a high resistance rate in samples from
et al., 2014). However, ZAFARANE et al. (2017) raw milk in mastitis cases (FERNANDES et al.,
and IWERIEBOR et al. (2015) observed greaater 2011; ZAFARANE et al., 2017). Resistance to
distribution of stx2 than stx1 in their studies. tetracycline and oxytetracycline was detected in
Intimin was encoded by the eaeA gene which 31.9% and 19.1% of isolates, respectively, but
produces a bacterial outer-membrane protein it was determined that the resistance rate was
associated with the intimate connection of the not as high as in previous studies since there 80-
bacteria to the gut mucosa of the host (BEAN et 90% isolates were resistance to these antibiotics
al., 2004). In our study, 42 isolates (89.3%) were (IWERIEBOR et al., 2015; KEANE, 2016). Also,
eaeA positive, in contrast to previous studies in earlier investigations a higher resistance rate to
that recorded the eaeA gene in 3.8% - 66% of cotrimoxazole was recorded (FERNANDES et al.,
isolates (TAVAKOLI and POURTAGHI, 2017; 2011; ZAFARANE et al., 2017).
ZAFARANE et al., 2017). In this study, nine The differences between studies might be
isolates (19.1%) were observed as hlyA positive. related to variations in geographic region, the
This finding was in accordance with another survey ages of the cows, different serotypes, and the use
in Iran, which reported 87.3 % of hlyA positive of different antibiotics for therapy on the farms.

528 Vet. arhiv 92 (4), 525-530, 2022

 F. Aflakian et al.: Virulence genes and the antimicrobial susceptibility of Escherichia coli

Thus, organizing an orderly monitoring system for Determinants and Antimicrobial Resistance among
recognition of cases of clinical mastitis, restriction Commensal Escherichia coli Derived from Dairy and Beef
Cattle. Int J Environ Res Public Health 12, 970-985.
of the widespread use of common antibiotics, and
DOI: 10.3390/ijerph120100970.
assessing antibiotic susceptibility are suggested
CAINE, L.-A., U. U. NWODO, A. I. OKOH, R. N. NDIP, E.
to decrease the prevalence of resistant strains in
GREEN (2014): Occurrence of virulence genes associated
industrial dairy herds. with diarrheagenic Escherichia coli isolated from raw
There is a possibility that virulence genes cow’s milk from two commercial dairy farms in the
and antibiotic-resistance genes exist on the same Eastern Cape Province, South Africa. Int J Environ Res
plasmids, so transfer of these genes could happen Public Health 11, 11950-11963.
together. Therefore, the strains which had a DOI: 10.3390/ijerph111111950.
resistant gene, had more than one virulence gene, CENGIZ, S., G. DINÇ, M. Ü. SÖGÜT (2014): Detection of
although in this survey, antibiotic resistance was not several virulence properties, antibiotic resistance and
phylogenetic relationship in E.coli isolates originated from
assessed at the genomic level, genomic resistance
cow mastitis. Acta Vet 64, 413-425.
determinants should be analyzed in the future.
DOI: 10.2478/acve-2014-0039.
Acknowledgments DONG, P., L. ZHU, Y. MAO, R. LIANG, L. NIU, Y. ZHANG,
The authors thank Mrs. Bagherzadeh for her technical X. LUO (2015): Prevalence and characterization of
assistance. Escherichia coli O157: H7 from samples along the
production line in Chinese beef-processing plants. Food
Conflict of interest Control 54, 39-46.
None to declare. DOI: 10.1016/j.foodcont.2015.01.038.
FERNANDES, J. B. C., L. G. ZANARDO, N. N. GALVAO,
Funding I. A. CARVALHO, L. A. NERO, M. A. S. MOREIRA
Authors declare that there was no funding for this work. (2011): Escherichia coli from clinical mastitis: serotypes
and virulence factors. J Vet Diagn Invest 23, 1146-1152.
DOI: 10.1177%2F1040638711425581.
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Received: 20 October 2020

Accepted: 29 December 2020

Aflakian, F., M. Rad, H. Salimizand, A. Nemati, A. R. Zomorodi: Geni virulencije i antimikrobna

osjetljivost izolata bakterije Escherichia coli dobijenih od krava s mastitisom u pokrajini Mashhad, Iran –
kratko priopćenje. Vet. arhiv 92, 525-530 2022.

SAŽETAK
Cilj je istraživanja bio odrediti gene virulencije i antimikrobnu rezistencije bakterije Escherichia coli izolirane
iz uzoraka mlijeka krava s mastitisom. Ukupno 47 izolata bakterije E. coli iz uzoraka mlijeka krava s kliničkim
mastitisom, s pet mliječnih farmi u sjeveroistočnom Iranu, podvrgnuto je protokolu multipleks PCR-a kako bi se
odredili geni virulencije stx1, stx2, eaeA, hlyA, sta, F4, F17, fliC i rfbE. Antimikrobna je osjetljivost procijenjena
primjenom disk-difuzijske metode. Najčešće određeni geni jesu gen eaeA, u 42 izolata (89,3 %) i gen stx1, u 34 izolata
(72,3 %). Najrjeđi gen bio je F41, koji je pronađen u jednom izolatu (2,1 %). Nadalje, 9 od 47 izolata bilo je hlyA
pozitivno, a četiri su izolata sadržavala gen sta. Procjena antimikrobne je osjetljivosti pokazala je najveću rezistenciju
na linkomicin (100 %) i neomicin (91,4 %). Nalazi upućuju da se radi o visokorizičnim patogenima na farmama krava,
stoga bi pojava višestruko rezistentnih i patogenih sojeva E. coli trebala izazvati veliku javnozdravstvenu zabrinutost.
Ključne riječi: E. coli; virulencija gena; antimikrobna rezistancija; mastitis

530 Vet. arhiv 92 (4), 525-530, 2022