STUDIES ON ENTEROHAEMORRHAGIC ESCHERICHIA COLI (EHCE) STRAINS NON O157:H7 IN CHICKEN WITH REGARD TO ANTIBIOTIC RESISTANCE GENE ON PLASMID

Assiut University web-site: www.aun.edu.eg

STUDIES ON ENTEROHAEMORRHAGIC ESCHERICHIA COLI (EHCE) STRAINS NON O157:H7 IN CHICKEN WITH REGARD TO ANTIBIOTIC RESISTANCE GENE ON PLASMID

SOAD A. NASEF ¹; AMAL S. EL OKSH ² and GHADA A. IBRAHIM ³

¹ Chief Researcher, RLQP, Dokki, Poultry Diseases

² Researcher, Bacteriology Department, RLQP, Zagazig Branch, Bacteriology

³ Researcher, Bacteriology Department, AHRI, Ismailia Branch, Bacteriology

Received: 8 March 2017;       Accepted: 30 March 2017

INTRODUCTION

Chicken meat is one of the most popular foods among population. However, the epidemiology and prevalence of EHEC strains in chicken meat is essentially unknown. Enterohemorrhagic Escherichia coli (EHEC) is considered recently as one of important emerged groups of food-borne pathogens. Although E. coli O157:H7 is currently the most common EHEC strains in many regions of the world (Armstrong et al., 1996) but serotypes O103, O26, O104,O111, O 113,O 128, O5 and O145 (which are now usually referred to as non-O157 EHEC) constituted also  a serious threat to public health (Bettelheim,1996). They characterized by their specific virulence factors and its ability to produce potent cytotoxins (verotoxins). All strains produce hemolysin (which is encoded by hlyA) (Schmidt et al., 1994) and at least one Shiga-like toxin (encoded by stx1 or stx2) (O’Brien et al., 1987). Shiga – like toxins have similar biological activities including cytotoxicity to Vero and HeLa cells, but they are different immunologically (Law, 2000).

Corresponding author: Dr. AMAL S. EL OKSH

E-mail address: saidamal19@yahoo.com

Present address: Researcher, Bacteriology Department, RLQP, Zagazig Branch, Bacteriology

Antimicrobials are routinely used for disease prevention and growth promotion in poultry production however, they are not recommended for treating EHEC O157:H7 infection because antimicrobials may lyse bacterial cell walls, thereby liberating Shiga toxins (Wong et al., 2000 and Hedican et al., 2009), and/or cause increased expression of Shiga toxin genes in vivo (Zhang et al., 2000).

Although EHEC infections are not aggressively treated with antimicrobial therapy and many isolates are susceptible to numerous antimicrobials, recent reports indicate that antimicrobial resistance of EHEC is on the rise (Farina et al., 1996; Galland et al. 2001 and Schroeder et al., 2002).Moreover, it is proven that repeated sublethal exposure to antibacterial agents not only promotes adaptative resistance but also confers decreased sensitivity to antibiotics (Braoudaki and Hilton, 2003). This practice leads to a selection of antimicrobial resistance among commensals in the intestinal tracts of chicken, which poses a public health threat (Witte, 1998).

Ideally, PCR-based detection methods are rapid and sensitive without need for extensive sample preparations. Large plasmids resembling carried the genetic information for most non-O157 EHEC strains which is responsible for the so-called enterohaemolytic phenotype. (Schmidt et al., 1995).

Due to the fact of definite zoonotic origin and pathogenic STEC/EHEC group. This paper will review the recentfinding on the most virulence factors of EHEC, trying to seek out what makes a STEC, an EHEC highly pathogenic to poultry production and evaluation of antibiotic resistance properties in EHEC serogroups isolated from chicken meat (Momtaz and Jamshidi, 2013).

MATERIALS AND METHODS

1. Bacterial strains

A total of 50 EHEC strains that were recovered from chicken and were collected from different sources in Sharkia province, Egypt. The majority of strains included in the present study had been obtained in previously published studies (Blanco et al., 2001; Blanco et al., 2003 and Blanco et al., 2004) and the procedures for their isolation were described in detail in those studies.

2. Serotyping

EHEC strains were serotyped by slide agglutination test with commercially available polyvalent and monovalent anti E coli O and K sera (Test Sera Enteroclon, Anti –Coli, SIFIN Berlin, Germany).

3. Antimicrobial susceptibility testing

The susceptibility of identified EHEC strains to a panel of ten commonly used antimicrobial agents was performed by the standard Kirby–Bauer disc diffusion method (Bauer et al., 1966) and the results were interpreted according to the criteria recommended by the Clinical and Laboratory Standards Institute for antimicrobial susceptibility testing (CLSI, 2011) Isolates resistant to three or more antibiotics were classified as MDR strains.

4. Molecular detection of antibiotic resistance and virulence genes

Plasmid DNA from 10 MDR EHEC strains was isolated using QIAprep Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany). Screening for the presence of some antibiotic resistance and virulence plasmid-associated genes was carried out by PCR amplifications using specific primers and different cycling conditions as previously described by (Clark et al., 1999; Colom et al., 2003; Ewers et al., 2007 and Santos et al., 2014). The PCR products were tested for positive amplification by agarose gel electrophoresis (Sambrook et al., 1989). For each PCR experiment, appropriate positive and negative controls were included. The primer sequence from Metabion (Germany) of virulent and resistance genes and the amplification cycling conditions are listed in tables (1 & 2).

Table1: Oligonucleotide primer sequences of virulence and antibiotic resistance genes of E.coli.

Table 2: Cycling conditions and predicted sizes of PCR products for virulence and antibiotic resistance genes.

RESULTS

This study was done on fifty chicken E.coli strains isolated from avian origin as shown in Table 3, thirty five (70%) strains were confirmed to be EHEC positive, also there were significant differences between the presence of attaching and effacing E.coli EPEC and EHEC subgroups in strains. Among the 35 EHEC included in this study, 5 (10%) strains were serotyped as O157:H7 and 30 (60%) non-O157. Non-O157 EHEC strains were belonged to 8 different O:H serotypes (O26:H-, O111:H-,O 103:H3,O104, O145,O5,O113,O128). Based on the results, O26 (16%) and O111(14%) were of high incidence ratio among serogroups whereas O128 had the lowest incidence in EHEC strains 2%.

Table 4 shows the distribution of virulence factors of non-O157EHEC strains. All of the EHEC-positive samples had stx1, stx2, and hly virulence genes with significant differences as shown in Table 5.

The antibiotic resistance profiles of the thirty non-O157 strains were significantly different with respect to the levels of resistance of E. coli to the tested antibiotics. Non-O157 EHEC serogroups demon- strated high rates (100%) for sulfamethoxazole, trimethoprim, chlorumpheneol, colistin, gentamycin and tetracycline while 85% for streptomycin, doxycycline, 80% for cefotraxone and 75% for amoxicillin clavulanic acid.

Phenotypic resistance of non-O157 to amoxicillin- clavulanic acid, sulfamethoxazole- trimethoprim and streptomycin antibiotics could be explained by the presence of blaTEM, sulI and aadA resistance genes among the tested strains (60%,85% and 75%), respectively.

The frequencies and combinations of virulence genes carried on plasmid (stx1, stx2 and hly) of twenty non-O157 EHEC strains were assessed. Among the detected virulence genes, stx1 and stx2 were the most prevalent gene (45% and 65%), followed by hlygene (80%) fig. (1).

Finally, characterization of the selected non-O157 EHEC according to their serotypes, virulence potential and corresponding antibiotic resistance pattern were shown in table (5). It was demonstrated that MDR E. Coli serovars possessed at least two virulence genes accompanied by attendance of antibiotic resistance genes. The co-occurrence of these concerning trends confirmed that the acquisition of antimicrobial resistance by E. coli has been accompanied by increased virulence.

Table 3: Prevalence of detected serotypes based on total number of E.coli isolates.

Table 4: The prevalence of virulence genes and antibiotic resistance genes among examained EHEC.

Fig. (1): Shows agarose gel electrophoresis of PCR amplified products of (A,B): sulI, (C,D):aadA1, (E,F) blaTEM resistant genes, (G,H):stx1,(I,J):stx2 and (K,L): hly virulence genes. Lane M: DNA molecular size marker (100 bp), lanes 1-20: E.coli isolates, lane (+ve): positive control and lane (-ve): negative control. The size in base pairs (bp) of each PCR product is indicated on the right of the bands

Table 5: Resistance profiles and genotypic characterization of some virulence and antibiotic resistance genes of EHEC strains on their plasmid.

DISCUSSION

E.coli strains that cause entric infections are generally called diarrheagenic E.coli strains, and their pathogenesis is associated with a number of virulence attributes which vary according to pathotypes (Vidal et al., 2005). Currently, Diarrheagenic E.coli strains are classified into 6 pathotypes based on their distinct virulence determinants and pathogenic features, including Enterohemorrhagic E.coli (EHEC) that produceshiga-like toxins (Xia et al., 2010).

In this recent investigation, high recovered rate of EHEC strains (70%) was recorded. Similar results were reported by (Lyhs et al., 2012; Eid and Erfan, 2013 and Peer et al., 2013) where E.coli was isolated in percentage of 94.5%, 80% and 84%, respectively. Serotyping of EHEC strains confirmed the identification of O157 (10%) and non O157 serotypes (60%). The non-O157 STEC serotypes were: O26 (16%), O103 (6%), O111 (14%), O145 (6%), O5(4%), O113(4%), O128(2%) and O104 (8%). This result was in agreement with that recorded by (Hedican et al., 2009). However, another study in Korea showed that (41 / 900) from poultry samples were E. coli positive and non O157 serogroups were included in a percentage of 4.6% (Lee et al., 2009).

Because of the high indiscriminate use of antibiotics, especially in veterinary medicine, antibiotic resistance against most effective antibiotics was recorded. Several recent studies have documented antibiotic resistance especially among non-O157 EHEC (Farina et al., 1996; Horii et al., 1998; Galland et al., 2001 and Khan et al., 2002). This study is matching with our study which recorded high levels of resistance for some antimicrobial agents (sulfamethoxazole- trimethoprim, cholorumphenicol, colistin, gentamycin, tetracycline). Likewise, higher resistance rates have been reported for avian E. coli isolates to tetracycline (100%) in Brazil (Lima-filho et al., 2013) and 84% in Korea (Kim et al., 2007). Meanwhile, streptomycin, doxycycline and amoxicillin clavulanic acid antibiotics rates were (70%, 70% and 75%), respectively, while doxycycline was recorded in another study in Egypt (51%) (Eloksh, 2014).

The increased prevalence of resistance of E.coli isolates to these antibiotics is due to their regular usage in poultry industry for control of pathogenic avain colibacillosis in many districts in Egypt because of their low cost and availability. Also, it would seem that the discrepancies in the rate of E.coli resistance among different countries are due to differences in the level of dependence on antimicrobial usage and management practices in poultry production as well as variations in legislation guiding the use of antimicrobials from region to region. Therefore, non-O157 EHEC strains could be a potential reservoir of antimicrobial resistance genes.

As shown in figure (1), the highest incidence of antimicrobial resistance genes was for sul1 (100%), followed by aadA1 (85%), blaTEM (75%), however some researchers in Iran detected these genes in percentage of 64.63%and 62.19%, respectively. (Momtaz and Jamshidi, 2013).

While the serogroups are important for determining potential pathogens, the presence of virulence attributes, such as stx1, stx2 and hly, are important parameters for pathogenicity of the strains. In addition, the virulence factors associated with these strains are linked to plasmids which detected by PCR. Consequently, they are likely to be subjected to horizontal gene transfer between the species as exhibited by dissemination of plasmids (Magwedere et al., 2013).

In prevalence of virulence genes (stx1 and stx2) in the present study were 45% and 65%, respectively whereas nine only out of 987from poultry samples were positive for the same genes (Lukásová et al., 2004).

Boerlin et al. (1999) found non-O157 EHEC serotypes O26, O103, O111, and O145 expressing stx2 gene concluding that this makes the organism significantly more likely to cause serious disease, while Friedrich et al. (2002) found that of 87 isolates of non-O157 (including O26, O103, O121, and O145) harbored stx2 gene.

Hemolysin (hly) gene is also one of important virulence factors (Gyles, 2006) which is more common in EHEC strains and in this study recorded in 80% of total non-O157 EHEC strains. It is widely distributed among non-O157 strains and could cause lysis of red blood cells in vitro. Approximately 90% of all EHEC strains possess genes encoding hemolysin (Kilic et al., 2007)

Plasmid profiles is one of the several useful methods  for determining the relatedness or unrelatedness of bacterial strains that contain plasmid DNA. A large number of plasmids had been detected in E.coli as well as a large number of other bacterial species and their role in antibiotic resistance and other variable characters has been well established. The occurrence of multiple antibiotic resistances has also been shown to be due to a greater genetic mobilization of the antibiotic resistance genes carried by the plasmids (Gaddad and Shivannavar, 2011). In fact, plasmid profiles of the isolates are generally a useful tool for obtaining knowledge about resistance of the isolates to the antimicrobial substances and transfer of a plasmid among closely related isolates from different sources. Our results showed the high presence of virulence factors and multiple antibiotic-resistant properties among E.coli serotypes that were isolated from chicken meat samples (Momtaz and amshidi, 2013).

In conclusion there is an increase in detection of EHEC non O157. As well as there is an increase in antimicrobial resistance among the examined EHEC non O 157 so paying attention to this group of E.coli is required to avoid the risk to human. To the authors´ knowledge, excessive prescribing, crowding and poor sanitation are the primary factors responsible for the high antibiotic resistance in E.coli isolated from chicken meat. The sanitation conditions, especially in poultry slaughter houses and supermarkets could help to reduce the contamination rate of poultry meat. Finally, to prevent antibiotic resistance in bacteria, we have to prescribe antibiotics more cautiously in animals and periodically use different antibiotics.

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