EJBR2017v7i4art366-373 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2017; 7 (4): 366-373 Virulence genes and antibiotic resistance of Yersinia enterocolitica strains isolated from children Barbara Kot*, Małgorzata Piechota, Kinga Jakubiak Department of Microbiology, Institute of Biology, Siedlce University of Natural Sciences and Humanities, 12 Bolesława Prusa Str., 08-110 Siedlce, Poland *Corresponding author: Barbara Kot; Phone: +48 256431339, E-mail: barbara.kot@uph.edu.pl ABSTRACT Yersinia enterocolitica is a foodborne pathogen which is primarily responsible for gastrointestinal infections. The presence of the virulence genes in Y. enterocolitica strains isolated from children and antimicrobial resistance was studied in this work. The PCR, biotyping and disc diffusion method were used for analysis of Y. enterocolitica strains. Most of Y. enterocolitica strains belonged to biotype 4 and all carried ail, myfA and ystaA genes. Most of them also had the plasmid yadA gene. These genes were also detected in the strains of biotype 2, while in the two strains of biotype 1A only myfA gene was found. The blaA gene was present in all the strains of biotype 4 and 2, while blaB in the strains of biotype 2 and in some of biotype 4 strains. The presence of β-lactamase genes in Y. enterocolitica was not detected in biotype 1A. All strains were resistant to ampicillin, 76.2% and 47.6% were resistant to ticarcillin and piperacillin, respectively. Two strains (9.5%) were resistant to amoxicillin/ clavulanic acid and aztreonam, three (14.3%) to chloramphenicol, four (19%) to amikacin and trimethoprim/sulfamethoxazole, six (28.6%) to gentamicin. A few strains of Y. enterocolitica were multidrug resistant. The Y. enterocolitica strains isolated from the faeces of children suffering from diarrhea carried virulence genes and some of them were resistant to antibiotics used in extra-intestinal yersiniosis treatment. Keywords: Yersinia enterocolitica; Virulence genes; Antibiotic resistance; PCR; Yersiniosis. 1. INTRODUCTION Yersinia enterocolitica is an important human pathogen with the global distribution and a variety of clinical disorders such as enteritidis, enteroco- litis, gastroeneritidis, mesenteric lymphadenitis and others [1]. Yersiniosis is a zoonotic foodborne bacterial disease with high public health relevance. In Europe it is the third most common bacterial enteric disease after campylobacteriosis and salmo- nellosis [2]. Animals such as pigs, rodents, sheep, goats, cattle, horses are reservoirs of Y. entero- colitica. Pigs are a major reservoir for human pathogenic strains, especially for bioserotype 4/O3 [3]. This microorganism is considered an important foodborne pathogen including strains of diverse pathogenicity. Infections are most often acquired through ingestion of contaminated pork, milk, dairy foods, vegetables and contaminated drinking water or pet animal contact [4, 5]. The pathogenic Y. enterocolitica strains were also isolated from waste water samples in Turkey [6] or from river water in Poland [7]. Y. enterocolitica is rarely transmitted Received: 03 October 2017; Revised submission: 14 November 2017; Accepted: 22 November 2017 Copyright: © The Author(s) 2017. European Journal of Biological Research © T.M.Karpiński 2017. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. DOI: http://dx.doi.org/10.5281/zenodo.1064835 367 | Kot et al. Virulence of Yersinia enterocolitica from children European Journal of Biological Research 2017; 7 (4): 366-373 through contaminated blood during transfusion [8]. The species Y. enterocolitica is divided into six biotypes. Strains of biotype 1A are generally regar- ded as nonpathogenic, whereas strains of biotypes 1B, 2, 3, 4, and 5 carry a virulence plasmid pYV. This plasmid encodes type III secretion system and the outer membrane protein YadA (Yersinia adhesin A). YadA was found to play multiple functions in pathogenesis because it protects bacterial cells against antibacterial activity of complement and mediates specific binding of Y. enterocolitica to laminin, collagen and cellular fibronectin [9]. The chromosomal Y. enterocolitica virulence markers are ail, ystA and myfA genes. The ail gene encodes a small outer membrane protein (Ail adhesin), which promotes adhesion of Y. enterocolitica and invasion of epithelial cells. The ystA gene encodes entero- toxin YstA, which activates the guanylate cyclase that leads to the increased cGMP level. High level of cGMP causes fluid accumulation in the intestine [10]. The major subunit of antigen Myf is encoded by the myfA gene. This fibrillar structure promotes the colonization of the intestine by yersiniae [11]. Biotyping is used for clinical and epidemiological classification of Y. enterocolitica, but the hetero- genous nature of Y. enterocolitica, including diffe- rences in virulence, requires genotyping methods and this may be a novel way of pathogenic characterization of this microorganism. The aim of this study was the description of Y. enterocolitica strains isolated from the faeces of children suffering from diarrhea by using PCR assays for the detection of some virulence genes and in vitro evaluation of antibiotic sensitivity of this pathogen. The presence of genes coding β-lactamases was also detected in the genome of Y. enterocolitica strains. 2. MATERIALS AND METHODS 2.1. Strains Twenty one Y. enterocolitica strains were isolated from the faeces of children suffering from diarrhea. The strains were isolated from children treated in different hospitals and outpatients in Warsaw (Poland) over the period 2009-2015. The identification of the strains was performed with the VITEK GNI card system (VITEK 2 instrument, version 4.01, bioMérieux). Biotyping of Y. entero- colitica strains was performed according to Wauters et al. [12]. The strains were stored at -70°C in Brain Heart Infusion (BHI) Broth (BHI; BBL, Becton Dickinson) containing 15% glycerol. 2.2. Antibiotic susceptibility testing The susceptibility of the strains was tested with a disc diffusion method using the following antibiotic discs (Oxoid, Basingstoke, UK): ampi- cillin (25 µ g), amoxicillin/clavulanic acid (20/10 µ g), cefepime (30 µ g), cefotaxime (30 µ g), cefuro- xime (30 µ g), ceftazidime (30 µ g), ceftriaxone (30 µ g), gentamicin (10 µ g), imipenem (10 µ g), norflo- xacin (10 µ g), piperacillin (100 µ g), ticarcillin (75 µ g), tobramycin (10 µ g), aztreonam (30 µ g), cipro- floxacin (5 µ g), amikacin (30 µ g), chloramphenicol (30 µ g) and trimethoprim/sulfamethoxazole (1.25/ 23.75 µ g). The results were recorded by measuring the inhibition zones and scored as susceptible, intermediately susceptible, and resistant, according to the Clinical and Laboratory Standards Institute [13]. 2.3. DNA isolation Genomic DNA was isolated from Y. entero- colitica strains by using the Genomic DNA PrepPlus (A&A Biotechnology, Poland), according to the manufacturer’s protocol. 2.5 µ l of the total extracted material from each test sample was used as a template DNA for PCR application. 2.4. Primers and PCR conditions The primers specific for the ail, ystA, myfA, yadA, blaA, blaB and 16S rRNA genes of Y. entero- colitica, synthesized at DNA-Gdańsk (Gdańsk, Poland), are listed in Table 1. The duplex PCR for ail and ystaA genes was performed in a 25-µ l volume containing 2.5 µ l of DNA template, 1×PCR buffer, 0.2 mM each dATP, dCTP, dGTP, and dTTP (Fermentas, Lithuania), the ail-specific primers and ystA-specific primers at 50 nM, with 1 U of RedTag Genomic DNA polymerase (Sigma-Aldrich, Ger- many). The amplification was carried out under the following conditions: initial denaturation (94°C, 3 min), followed by 30 subsequent cycles consisting 368 | Kot et al. Virulence of Yersinia enterocolitica from children European Journal of Biological Research 2017; 7 (4): 366-373 of denaturation (94°C, 1 min), primer annealing (52°C, 1.5 min), extension (72°C, 1.5 min), and final extension (72°C, 10 min). The duplex PCR for blaA and blaB genes was also performed in a 25 µl volume containing 2.5 µl of DNA template, 1 x PCR buffer, 200 µ M of each: dATP, dCTP, dGTP, and dTTP (Fermentas, Lithuania), 100 nM of the blaA and the blaB pair of specific primers, and 1U of RedTag Genomic DNA polymerase (Sigma-Aldrich, Germany). The ampli- fication was carried out under the following conditions: initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 0.5 min, primer annealing at 50°C for 0.5 min and extension at 72°C for 1 min. A 5 min extension at 72°C was performed at the end of the final cycle. The monoplex PCR for myfA gene and yadA gene as described earlier [19] and monoplex PCR for the 16S rRNA gene for species identification as described by Wannet et al. [18] were also performed. The amplifications were carried out in the Multi Gene II thermal cycler (Labnet International, Inc., USA). The PCR products were analysed by electrophoresis in 1.5% agarose gels stained with ethidium bromide. Molecular size markers (Sigma- Aldrich) were also run for product size verification. The gel was electrophoresed in 2 × Tris-borate buffer at 70 V for 1.5 h. Table 1. Oligonucleotide primers used in the study. Primers Sequence (5' → 3') Amplicon length (bp) References ail-a (F) TGGTTATGCGCAAAGCCATGT 356 [14] ail-b (R) TGGAAGTGGGTTGAATTGCA ystA-a (F) GTCTTCATTTGGAGGATTCGGC 134 [14] ystA-b (R) AATCACTACTGACTTCGGCTGG myfA-1 (F) CAGATA CAC CTG CCT TCC ATCT 272 [15] myfA-2 (R) CTCGACATATTCCTCAACACGC yadA-1 (F) TAAGATCAGTGTCTCTGCGGCA 747 [16] yadA-2 (R) TAGTTATTTGCGATCCCTAGCAC blaA-1 (F) blaA-2 (R) AAATGCGCTACCGGCTTCAG AGTGGTGGTATCACGTGGGT 439 [17] blaB-1 (F) blaB-2 (R) CCCACTTTATACCTTGGCACAAA GAACATATCTCCTGCCTGGAAAT 781 [17] 16S rRNA-Y1 (F) 16S rRNA-Y2 (R) AATACCGCATAACGTCTTCG CTTCTTCTGCGAGTAACGTC 330 [18] 3. RESULTS Biotype 4 was most numerously represented by 71.4% of Y. enterocolitica strains. A small group included strains of biotype 2 and biotype 1A (Table 2). The 330 bp fragment, specific amplification product for the Y. enterocolitica 16S rRNA gene, was obtained in case of all the strains (Fig. 1A). A duplex PCR was used for the detection of the ystA- specific PCR product of 134 bp and the ail-specific product of 356 bp (Fig. 1B). These genes were present in all the strains of 4 and 2 biotype (Table 2). The yadA-specific amplification product of 747 bp was detected in all the strains of biotype 2 and the majority of strains belonging to biotype 4 (86.6%) (Fig. 1C). The myfA-specific PCR product of 272 bp (Fig. 1D) was detected in all the strains which belonged to different biotypes. Using multiplex PCR, 439 bp fragment for blaA gene in all the strains of biotype 4 and 2 was obtained (Fig. 1E). The amplification products for blaB (827 bp) were detected in all strains of biotype 2, and only in eight strains of biotype 4. The presence of β-lactamase genes in Y. enterocolitica was not detected in biotype 1A. 369 | Kot et al. Virulence of Yersinia enterocolitica from children European Journal of Biological Research 2017; 7 (4): 366-373 Table 2. Virulence genes and resistance profiles of Y. enterocolitica strains from the faeces of children with intestinal yersiniosis. Bt - biotype, AMP - ampicillin, TIC - ticarcillin, AMC - amoxicillin plus clavulanic acid, PIP - piperacillin, GM - gentamicin, AN - amicacin, C - chloramphenicol, SXT - trimethoprim/sulfamethoxazole, ATM - aztreonam, * - multidrug resistance strains, „-„ no amplification. Strains Year Bt Results of PCR for: Resistance profile ail yadA myfA ystA blaA blaB 9996 2009 2 + + + + + + AMP/TIC/AMC 6068 2010 1A - - + - - - AMP/PIP 10743 2010 4 + + + + + + AMP/TIC/GM 15869 2010 4 + + + + + + AMP/PIP/TIC 6528 2010 4 + - + + + + AMP/AN/C/SXT* 6701 2010 4 + - + + + + AMP/TIC 7217 2012 2 + + + + + + AMP/PIP/TIC/SXT 20179 2013 2 + + + + + + AMP/TIC/C/SXT* 10510 2013 1A - - + - - - AMP/PIP/TIC/SXT 15395 2013 4 + + + + + + AMP/GM 26530 2014 4 + + + + + + AMP/PIP/AN/GN 13004 2015 4 + + + + + - AMP/PIP/TIC 2 2015 4 + + + + + - AMP/TIC 13571 2015 4 + + + + + - AMP/TIC/ATM/AMC 601 2015 4 + + + + + - AMP/TIC 1 2015 4 + + + + + - AMP, TIC 158 2015 4 + + + + + - AMP/TIC 450/6 2015 2 + + + + + + AMP/PIP/TIC/GM 448/7 2015 4 + + + + + + AMP/PIP/ATM/AN/GN/C* 511/8 2015 4 + + + + + + AMP/PIP/TIC/AN/GM 301/3 2015 4 + + + + + - AMP/TIC The Y. enterocolitica strains showed high resistance to antibiotics belonging to penicillin group because all the strains were resistant to ampicillin, above 76% of the strains were resistant to ticarcillin and about 48% were resistant to piperacillin. Additionally, two strains (9.5%) were resistant to amoxicillin/clavulanic acid. About 29% and 19% of the strains were resistant to gentamicin and amikacin, respectively. Moreover, two strains (9.5%) were resistant to aztreonam. In case of chloramphenicol, 14.3% of the strains showed resistance and 19% of the strains were resistant to trimethoprim/sulfamethoxazole. All the strains were sensitive to cephalosporins, fluoroquinolones, imipenem and tobramycin (Fig. 2). Among the tested Y. enterocolitica, three strains were multidrug resistant. Two strains of biotype 4 showed resistance to antimicrobial agents from four various chemical groups and one strain of biotype 2 was resistant to antimicrobial agents belonging to three different chemical groups (Table 2). 4. DISCUSSION Y. enterocolitica is an important foodborne pathogen which is primarily responsible for gastrointestinal infections in young children. The incidence of Y. enterocolitica infection is highest among children under 5 years of age [20]. 370 | Kot et al. Virulence of Yersinia enterocolitica from children European Journal of Biological Research 2017; 7 (4): 366-373 A B M 1 2 3 4 5 M 1 2 3 4 5 C D M 1 2 3 4 5 M 1 2 3 4 5 E M 1 2 3 4 5 Figure 1. Electrophoresis in 1.5% agarose gel PCR products obtained by using specific primers for 16S rRNA gene (A), ail and ystA genes (B), yadA gene (C), myfA (D) and blaA and blaB genes (E). Figure 2. Antimicrobial resistance of Y. enterocolitica strains isolated from the faeces of humans with intestinal yersiniosis. AMC - amoxicillin/clavulanic acid, SXT - trimethoprim/sulfamethoxazole. The high incidence of Y. enterocolitica infections in this age group, compared with other gastrointestinal infections, such as salmonellosis and campylobacteriosis, may result from eating food prepared from raw pork products, use of baby's dummy or contact with domestic animals, such as dogs and cats [21]. In addition, factors that may contribute to the high incidence of Y. enterocolitica infection in young children include an increased rate of exposure to this pathogen as a result of fecal-oral contamination, predisposition to infection due to immature immune system [22] and higher frequency of testing stool samples in case of children when affected [23]. In our research we investigated Y. enterocolitica strains isolated from the faeces of children suffering from diarrhea. Among them, strains belonging to biotype 4 carrying the ail, myfA and ystaA genes predominated. Most of them had also the plasmid gene yadA, confirming the presence of the plasmid pYV. These results demonstrated the pathogenic potential of the investigated strains to susceptible hosts. Our results are similar to those 330 bp (16SrRNA) 356 bp (ail) 134 bp (ystA) 747 bp (yadA) 272 bp (myfA) 827 bp (blaB) 439 bp (blaA) 371 | Kot et al. Virulence of Yersinia enterocolitica from children European Journal of Biological Research 2017; 7 (4): 366-373 obtained by other authors that also showed that strains belonging to biotype 4 are responsible for most infections caused by Y. enterocolitca in Europe [4, 20]. The strains of biotype 2 are rarely isola- ted from humans. The pathogenic potential of the biotype 2 strains examined in this study was highlighted by the occurrence of the virulence markers investigated. Similar results were obtained by Frazão and Falcão [24], who also studied strains of Y. enterocolitica biotype 2. Uncomplicated course of yersiniosis usually does not require the use of antibiotics. However, some cases of yersiniosis, such as sepsis, focal extra-intestinal infection or infection in immune- compromised patients require antimicrobial treat- ment. Y. enterocolitica strains are β-lactamase producers. Most Y. enterocolitica strains harbored chromosomal genes blaA and blaB encoding BlaA (a non-inducible broad-spectrum carbenicillinase) and BlaB (an AmpC-type inducible cephalospori- nase) [25]. In our study, the presence of blaA gene in all the strains of biotype 4 and 2 was detected, while blaB gene was carried by biotype 2 strains and over 50% of the biotype 4 strains. These genes were not detected in the strains of biotype 1A, although in previous studies, in which were used additional primers designed using the conserved regions of the blaA genes of Y. enterocolitica 8,081, biotype 1B, has been shown the presence of this gene in the majority of Y. enterocolitica strains of biotype 1A [26]. Heterogeneity in blaA gene of Y. enterocolitica of biotype 1A was confirmed by Sharma et al. [27]. Inability to detect blaA gene in these strains may result from a genetic variability in blaA preventing the binding of primers. The antimicrobial suscepti- bility test revealed high resistance of Y. entero- colitica to antibiotics belonging to penicillin group such as ampicillin, ticarcillin and piperacillin. This was in accordance with the results obtained by other authors [28]. Two strains (9.5%) belonging to 2 and 4 biotype were also resistant to amoxicillin with clavulanic acid, while Frazão et al. [29] showed that 19/34 of Y. enterocolitica strains isolated from different sources in Brazil were resistant to this combination. In our study, all the strains were sensitive to the second (cefuroxime), third (cefo- taxime, ceftazidime, ceftriaxone) and fourth gene- ration cephalosporins (cefepime), fluoroquinolones and imipenem. Fluoroquinolones and the third generation cephalosporins are the best therapeutic options to treat enterocolitis in compromised hosts and in patients with septicemia or invasive infection [30]. In case of extra-intestinal yersiniosis, also aminoglycosides in combination with other anti- biotics are used for treatment. In our research, four (19%) and six (28.6%) strains were resistant to amikacin and gentamicin, respectively. Rusak et al. [28] obtained one strain (2%) resistant to amikacin, while all the strains were sensitive to gentamicin. In Switzerland during 2001-2010 also no gentamicin- resistant strains were found [4]. Trimethoprim/ sulfamethoxazole are also used to treat yersiniosis. In this study, four strains (19%) were resistant to this sulfonamide. Sporadic resistance to trimetho- prim/sulfamethoxazole occurred in Switzerland [4], while in Brazil trimethoprim/sulfamethoxazole resi- stance was found in 8.8% to 10% of the strains [28, 29]. In our study, three strains were multidrug resistant. Two strains belonging to biotype 4 showed resistance to four different classes of antimicrobial agents (penicillins, aminoglycosides, chloramphe- nicol, sulfonamides and penicillins, aminoglyco- sides, chloramphenicol, monobactams) and one strain of biotype 2 was resistant to antimicrobial agents belonging to three groups (penicillins, chloramphenicol, sulfonamides). Multiple resistance phenotypes were rarely reported in Y. enterocolitica. Only one out of from 60 Y. enterocolitica strains investigated by Rusak et al. [28] showed resistance to the three classes of antimicrobial agents (cephalo- sporin, sulfonamide, and tetracycline). Fredriksson- Ahomaa et al. [4] also reported that only one out of 128 Y. enterocolitica strains isolated from human clinical samples in Switzerland showed resistance to multiple antimicrobial agents. The multiresistance of Y. enterocolitica strains (19%) was found in Finland, and these strains were significantly associated with traveling abroad [31]. Our study showed that Y. enterocolitica strains from children in Poland belonging to biotype 4 and 2 had all investigated virulence genes, including the plasmid gene yadA, except the two strains of biotype 4 in which this gene was not detected. These strains showed high resistance to penicillin, although they remain susceptible to drugs used for treating gastroenteritidis, as well as extra- intestinal infections. However, it should be stressed 372 | Kot et al. Virulence of Yersinia enterocolitica from children European Journal of Biological Research 2017; 7 (4): 366-373 that some strains were resistant to antibiotics used in extra-intestinal yersiniosis treatment and few strains were multidrug resistant. AUTHORS' CONTRIBUTION BK: study design, laboratory investigation, data interpretation, preparation of manuscript; MP and KJ: laboratory investigation, literature analysis. The final manuscript has been approved by all authors. 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