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Revista Brasileira de Saúde e Produção Animal

On-line version ISSN 1519-9940

Rev. bras. saúde prod. anim. vol.16 no.2 Salvador Apr./June 2015

https://doi.org/10.1590/S1519-99402015000200006 

Medicina Veterinária Preventiva

Antimicrobials resistance patterns and the presence of stx1, stx2 and eae in Escherichia coli

Padrões de resistência antimicrobianos e da presença de stx1, stx2 e "eae" em "Escherichia coli"

Gustavo Lacerda Homem ASSUMPÇÃO 1  

Marita Vendovelli CARDOZO 1  

Lívia Gerbase BERALDO 1  

Renato Pariz MALUTA 2  

Joviany Talita SILVA 3  

Fernando Antonio de AVILA 4  

Douglas McINTOSH 5  

Everlon Cid RIGOBELO 6   *  

1Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Programa de Pós-Graduação em Microbiologia Veterinária, Jaboticabal, São Paulo, Brasil.

2Universidade Estadual de Campinas, Departamento de Genética, Campinas, São Paulo, Brasil.

3Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Programa de Pós-Graduação em Microbiologia Agropecuária, Jaboticabal, São Paulo, Brasil.

4Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Patologia Veterinária, Jaboticabal, São Paulo, Brasil.

5Universidade Federal do Rio de Janeiro, Instituto de Veterinária, Departamento de Parasitologia Animal, Seropédica, Rio de Janeiro, Brasil.

6Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Produção Vegetal, Jaboticabal, São Paulo, Brasil.


ABSTRACT

The objectives of this study were to investigate whether antimicrobial resistance (AMR) or the presence of resistance genes was associated with the occurrence of the virulence genes, stx1, stx2 andeae. Three virulence genes and 11 AMR phenotypes were examined using polymerase chain reaction (PCR) and antimicrobial susceptibility tests. From 800 samples collected in this study, 561 samples were isolatesE. coli strains , being: 90 (16.0%) carriers ofstx1, 97 (17.3%) of stx2 and 45 (8.0%) ofeae genes singly. Thirty seven (6.6%) isolates were carriers of stx1 and stx2, 110 (19.6%) were carriers of stx1 and eae and 67 (11.9%) were carriers of stx2 and eae. The most common virulence gene detected was stx1 followed bystx2. The findings showed no relationship between presence of virulence factors and antimicrobial resistance. Also was not found relationship between serogroup and virulence factors.

Key words: antimicrobial resistance; dairy cow; Escherichia coli; serogroups; STEC

RESUMO

Objetivou-se com este estudo investigar se a resistência antimicrobiana (RAM) ou a presença de genes de resistência foi associada com a ocorrência dos genes de virulência, stx1, stx2 e eae e os sorogrupos deEscherichia coli isoladas a partir de vacas-vacas leiteiras. Três genes de virulência e 11 fenótipos RAM foram examinados através de PCR e testes de susceptibilidade aos antimicrobianos. Das 800 amostras colhidas neste estudo, 561 amostras foram isoladas cepas de E. coli, sendo: 90 (16,0%) portadoras de stx1, 97 (17,3%) de stx2 e 45 (8,0%) dos genes eae, separadamente. Trinta e sete (6,6%) isolados foram portadores de stx1 e stx2, 110 (19,6%), foram portadores destx1 e eae e 67 (11,9%) foram portadores de stx2 e eae. O gene de virulência mais comum detectado foi stx1 seguido por stx2. O sorogrupo predominante entre os isolados portadores de stx1 foi O119. Entre as cepas portadoras de stx2, eae e também estirpes com nenhum fator de virulência, o sorogrupo predominante foi O9. O RAM dos isolados, medido fenotipicamente, não foi associado com a presença ou ausência de genes de virulência na população saudável de vaca leiteira-nem a sua ocorrência a qualquer um dos sorogrupos foi associada com genesstx1 e stx2 e eae.

Palavras-Chave: Escherichia coli; resistência antimicrobiana; sorogrupos; STEC; vaca leiteira

INTRODUCTION

Currently, Shiga toxigenic Escherichia coli (STEC) strains are the most important emerging groups of foodborne pathogens (BEUTIN et al., 2002). These strains are producer of one or two cytotoxins called Shiga toxins (Stx1 and Stx2) (PATON & PATON, 1998). Intimin is another virulence factor responsible for intimate attachment of STEC and it is encoded by the chromosomal gene eae, which is part of a large cluster of virulence genes on a pathogenicity island termed the locus for enterocyte effacement (LEE) (KAPER et al., 1998).

There have been recent reports suggesting that antimicrobial resistance (AMR) levels in STEC is increasing (GALLAND et al., 2001;MENG et al., 1998). The natural evolution of bacteria has been changed by antibiotics use. In specific situations, there is a great probability of association between the presence of virulence factors and antimicrobial resistance (NAGASHINTA et al., 2008). It occurs because both AMR and virulence genes are carried in a similar fashion, may be linked and then co-selected (MARTINEZ & BAQUEIRO, 2002). When the association between the presence of virulence factors and antimicrobial resistance happens, the antimicrobial use may potentially enhance the selection of bacteria carrying virulence genes, accelerating the spread of virulence genes within bacterial populations (BOERLIN et al., 2005). Contrary, some studies have reported that the acquisition of antimicrobial resistance by some bacterial strains may have a fitness cost which leads to decreased virulence (CALHAU et al., 2013; CLARK et al., 2012).

Gow & Waldner (2009) demonstrated that antimicrobial resistance is not substantially more likely to be identified instx positive than in stx-negative E. coli isolates from healthy beef calves. However, there are dearth information in the literature if this association occurs at dairy cow herds and also if there is an association between serogroups of E. coli with bothstx and eae genes. Therefore, the aims of this study were to investigate if the antimicrobial resistance phenotype was associated with the occurrence of virulence genes, and also investigate if the presence or absence of virulence genes is associated with the serogroups of E. coli isolated from dairy cows.

MATERIAL AND METHODS

Fecal samples were collected in 10 different farms located in São Paulo State, Brazil, from January 2012 to February 2013. From each farm, faeces from the rectum of 80 animals were sampled using sterile swabs. In each farm, the samples were collected in a single day and represented more than 50% of each population at the time of sampling. Isolates were obtained by directly spreading the samples on MacConkey agar and sorbitol MacConkey agar. After incubation, a minimum of three colonies from each plate were analyzed biochemically (KONEMAN et al., 2001).

Up to two E. coli isolates from each fecal sample were characterized using PCR. To perform PCR, the DNA template was obtained using a thermal cell lysis procedure (KESKIMAKI et al., 2001) andstx1, stx2 and eae were collected using primers and PCR conditions describe by China et al. (1996).

Antimicrobial disk susceptibility tests were performed using the disk diffusion method recommended by Clinical Laboratory Standard Institute (WATTS et al., 2002). Drug-impregnated disks (CEFAR, São Paulo, Brazil, accessed in 2014) were placed on the surface of Muller-Hinton agar using a disk dispenser.

The serogroup was determined by standard agglutination methods (EDWARDS & EWING, 1972). The serogroups were either commercially prepared (Probac, Brazil). The following "O" antigens, which include most of the serogroups reported in cow or associated with severe enteric-disease in humans, were used: O5, O6, O8, O9, O20, O26, O55, O75, O86, O91, O101, O146, O149, O153, O157, O158 and O172.

Chi-square and Duncan exact tests were performed for the analysis of associations using software R version 2.12.0 (http://www.r-project.org/) accessed in 2014. A P value of p<0.01 was considered to be statistically significant.

RESULTS AND DISCUSSION

From 800 samples collected of dairy cow herds, it was isolated 561 E. coli strains. From these strains, 90 (16.0%) harbored onlystx1, 97 (17.3%) harbored only stx2 and 45 (8.0%) harbored only eae. Thirty seven E. colistrains (6.6%) harbored both stx1 and stx2; 110 (19.6%) harbored stx1 and eae; and 67 (11.9%) harbored stx2 and eae. Interestingly no strain was harbored to three genes, stx1, stx2 andeae together. The number of E. coli strains harboring at least one virulence gene was 446 (79.5%) and the number of strains presenting no gene was 115 (20.5%) (Table1).

Table 1 Frequency of Escherichia coli isolates harbored at least one virulence factor for Shiga like toxin (STEC) followed by total of strains harbored of genes for STEC, total of strains without genes for STEC, total of E. coli isolates, total of samples without E. coli and total of samples collected 

Virulence geneEschericia coli Number of isolates Frequency isolates (%)
stx1 90 16.0
stx2 97 17.3
eae 45 8.0
stx1+stx2 37 6.6
stx1+eae 110 19.6
stx2+eae 67 11.9
Number of isolates with genes 446 79.5
Number of isolates without genes 115 20.5

Total of isolates 561 100.0

Total of samples without gene 239 29.9

Total of samples isolates of the total collected 800 70.13

The frequencies of resistance to antimicrobial agents were verified with allE. coli strains carrying a single virulence factor and also with all strains carrying no virulence factor. The 90 E. coliisolates that carried stx1 only showed highest antimicrobial resistance to streptomycin (85.6%), followed by kanamycin (72.2%) and nalidixic acid (70.0%). For this same group, the lowest antimicrobial resistances levels were for cephalotin (20.0%), ampicillin (30.0%) and cefoxitin (35.6%) (Table2).

Table 2 Frequency of resistance to antimicrobial agents among E. coli Shiga like toxin (STEC) isolates harboringstx1 from dairy-cow herds (n=90) 

Antimicrobial agent Percentage of resistance - % (-number of resistant/total number of isolates)
Ampicillin 30.0 (27/90)
Amoxicillin – clavulanic acid 40.0 (36/90)
Cefoxitin 35.6 (32/90)
Ceftriaxone 45.6 (41/90)
Cephalothin 20.0 (18/90)
Streptomycin 85.6 (77/90)
Kanamycin 72.2 (65/90)
Gentamicin 55.6 (50/90)
Amikacin 67.8 (61/90)
Tetracycline 38.9 (35/90)
Nalidixic acid 70.0 (63/90)

For E. coli strains carrying stx2 only, highest antimicrobial resistance levels were related to streptomycin (73.2%), followed by kanamycin and nalidixic acid (70.1%) and the lowest antimicrobial resistance levels were due to cephalotin (14.4%), ampicillin (29.9%) and amoxicillin – clavulanic acid (33.0%) (Table 3).

Table 3 Frequency of resistance to antimicrobial agents among E. coli Shiga like toxin (STEC) isolates harboringstx2 from dairy-cow herds (n=97) 

Antimicrobial agent Percentage of resistance (nº of resistant/total nº of isolates)
Ampicillin 29.9 (29/97)
Amoxicillin – clavulanic acid 33.0 (32/97)
Cefoxitin 40.2 (39/97)
Ceftriaxone 46.2 (45/97)
Cephalothin 14.4 (14/97)
Streptomycin 73.2 (71/97)
Kanamycin 70.1 (68/97)
Gentamicin 60.8 (59/97)
Amikacin 67.0 (65/97)
Tetracycline 32.0 (31/97)
Nalidixic acid 70.1 (68/97)

For E. coli strains harboring only eae, amikacin (77.8%), nalidixic acid (75.6%) and streptomycin (73.3%) were the antimicrobials with the highest resistance levels. For this group, the lowest antimicrobial resistance levels were due to cephalotin (15.6%), tetracycline (24.4%) and ampicillin (26.7%) (Table 4).

Table 4 Frequency of resistance to antimicrobial agents among E. coli Shiga like toxin (STEC isolates harboring eaefrom dairy-cow herds (n=45). 

Antimicrobial agent Percentage of resistance (nº of resistant/total nº of isolates)
Ampicillin 26.7 (12/45)
Amoxicillin – clavulanic acid 28.9 (13/45)
Cefoxitin 44.4 (20/45)
Ceftriaxone 46.7 (21/45)
Cephalothin 15.6 (7/45)
Streptomycin 73.3 (33/45)
kanamycin 64.4 (29/45)
Gentamicin 48.9 (22/45)
Amikacin 77.8 (35/45)
Tetracycline 24.4 (11/45)
Nalidixic acid 75.6 (34/45)

For the E. coli strains with no virulence factor, the highest antimicrobial resistance levels were to streptomycin (78.2%), followed by kanamycin and nalixic acid (74.1%), amikacin (64%). For the same group, the lowest antimicrobial resistance levels were to cephalotin (17.4%), ampicillin (32.9%) and amoxicillin – clavulanic acid (36.0%) (Table 5).

Table 5 Frequency of resistance to antimicrobial agents among ordinaryE. coli isolates without stx1,stx2 or eae from dairy-cow herds (n=115) 

Antimicrobial agent Percentage of resistance (nº of resistant/total nº of isolates)
Ampicillin 32.9 (38/115)
Amoxicillin – clavulanic acid 36.0 (41/115)
Cefoxitin 45.5 (52/115)
Ceftriaxone 41.2 (47/115)
Cephalothin 17.4 (20/115)
Streptomycin 78.2 (90/115)
Kanamycin 74.1 (85/115)
Gentamicin 68.9 (79/115)
Amikacin 64.0 (74/115)
Tetracycline 38.0 (44/115)
Nalidixic acid 74.1 (85/115)

Among the groups presenting only one virulence factor and the group presenting no virulence factor, there was no statistical difference (p>0.01) when comparing their antimicrobial resistance patterns.

Table 6 shows the frequency of serogroups ofE. coli strains that harbour stx1. The serogroups detected were O119 (58.0%), O114 (17%), O111 (9.0%), O26, O126, O127, O55 (4.0%). For E. coli strains that carry stx2, the serogroups and frequencies were O9 (57.0%), O8 (32.0%), O101 (9.0%), O20 (2.0%). Foreae+ E. coli strains, the serogroups and frequencies were O9 (34.0%), O8, O127 (22.0%), O119, O20 (11.0%) and for E. coli strains negative for the virulence genes, the serogroups and frequencies presented were O9 (19.0%), O119 (16.0%), O114 (17.0%), O86 (6.0%), O125 (5.0%), O126 (3.0%), O101, O26, O127, O128 (2.0%) and O20 (1.0%). The comparison between the presence of serogroups with the presence or absence of virulence genes did not show correlation.

Table 6 Frequency of serogroups of E. coli strains carryingstx1, stx2, eaegenes or none of these genes 

E. colistrains carriers of stx1 gene (n=90)
Serogroup Frequency (%)
O119 58.0
O114 17.0
O111 9.0
O26 O126, O127, O55 4.0

E. coli strains carriers of stx2gene (n=97)
O9 57.0
O8 32.0
O101 9.0
O20 2.0

E. coli strains carriers of eaegene (n=45)
O9 34.0
O8, O127 22.0
O119, O20 11.0

E. coli strains with none of the virulence genes (n=115)
O9 19.0
O119 16.0
O114 17.0
O86 6.0
O125 5.0
O126 3.0
O101, O26, O127, O128 2.0
O20 1.0

No statistically significant association was detected between any AMR phenotype and virulence genes and neither between virulence genes and serogroups in this E coli population isolated of dairy-cow herds. The antimicrobial resistance patterns between the isolates carriers of at least one virulence gene or lacking any virulence gene were not different. The results showed that, the presence or absence, and the type of virulence gene did not interfere with the antimicrobial resistance level and also did not favor the selection of resistance genes (Tables1-6). Gow & Waldner (2009)verified no association between antimicrobial resistance measured phenotypically or the presence of AMR genes and virulence genes (stx1,stx2 and eae) in STEC.

The lack of association between AMR and stx virulence genes may be explained, at least in part, by the requirement for a specific receptor for phage attachment and also the limited amount of DNA incorporated into a phage head and by specific mechanism adopted by bacteriophages to incorporate the bacterial DNA in its genome (SCHWARTS et al., 2006). The mechanisms by which these virulence and resistance genes are transmitted from bacteria to bacteria are determinants for occurrence of these associations (ACHESON et al., 1998; MUNIESA, et al., 2004; NEELY et al., 1998).

Gow & Waldner (2006) analyzed 106 fecal generic E. coli isolates from calves in cow-calf herds and the most common virulence gene detected was stx2 followed byeae. In our study the most common virulence gene detected wasstx1 followed by stx2 (Table 1). Also, in our study, all antimicrobial resistance levels were measured via phenotype rather than genotype. This measurement can lead to some differences. On this account, the presence of unexpressed genes would not be measured. However, Gow & Waldner (2009) measured the antimicrobial resistance level of their isolates phenotypically and also genotypically and did not find difference between both methods.

When the association between antimicrobial resistance and virulence factor occurs, as demonstrated with other genes in previous works (BOERLIN et al., 2005, NAGACHINTA et al., 2008), it becomes more beneficial for the spread of pathogenic bacteria. The association between these factors depends on four main determinants: the bacterial species involved, virulence and resistance mechanisms, the ecological niche, and the host (BECEIRO et al., 2013).

Pereira et al. (2011) analyzed 117 fecal samples from dairy calves divided into two groups, one group received growth promoting antimicrobials (GPA) through milk and another group (NGPA) did not. The results of this study showed that NGPA group had statistically significantly lower levels of antimicrobial resistance for most of the antimicrobials tested than GPA group. Also, there was no association between virulence factors and NGPA or NGA and neither between antimicrobial resistance and virulence factors. Likely, the use of antimicrobials selects some resistance genes, but not virulence genes. Even though, in this and previous studies, there was no association between virulence factors and antimicrobial resistance. This association could take place when the virulence factors and antimicrobial resistance genes are carried in a similar fashion. In this case, it is possible that they could be linked and then co-selected (MARTINEZ & BAQUEIRO, 2002). Calhau et al. (2013) found a profile of virulence and resistance more prevalent in E. coli. They suggest that these features may be responsible for making them concomitantly virulent and extremely resistant. Nagachinta et al. (2008)found an association between virulence factors and antimicrobial resistance inE. coli strains isolated from dairy cows. However, it is important to point out that these studies have analyzed others genes different thanstx1,stx2 and eae.

Concerning the presence of serogroups, even though there had been the prevalence of O9 between E. coli strains carriers of stx2 andeae, there was also the prevalence of the same serogroup between the strains with no virulence factor, suggesting that there was no association between serogroups and virulence factors in this study.

The results did not show relationship between virulence factor and antimicrobial resistance when this association occurs the antimicrobial resistance might be selected through the antimicrobial use and thereby the virulence factor would be selected as well. Probably the mechanism of transfer from one bacterium to another does not permit the association with antimicrobial resistance genes.

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Received: July 10, 2014; Accepted: June 18, 2015

*Endereço para correspondência:everlonnsms@fcav.unesp.br

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