RESUMEN
Enterotoxigenic E. coli (ETEC) bacteria frequently cause watery diarrhoea in newborn and weaned pigs. Plasmids carrying genes of different enterotoxins and fimbrial adhesins, as well as plasmids conferring antimicrobial resistance are of prime importance in the epidemiology and pathogenesis of ETEC. Recent studies have revealed the significance of the porcine ETEC plasmid pTC, carrying tetracycline resistance gene tet(B) with enterotoxin genes. In contrast, the role of tet(A) plasmids in transferring resistance of porcine ETEC is less understood. The objective of the present study was to provide a comparative analysis of antimicrobial resistance and virulence gene profiles of porcine post-weaning ETEC strains representing pork-producing areas in Central Europe and in the USA, with special attention to plasmids carrying the tet(A) gene. Antimicrobial resistance phenotypes and genotypes of 87 porcine ETEC strains isolated from cases of post-weaning diarrhoea in Austria, the Czech Republic, Hungary and the Midwest USA was determined by disk diffusion and by PCR. Central European strains carrying tet(A) or tet(B) were further subjected to molecular characterisation of their tet plasmids. Results indicated that > 90% of the ETEC strains shared a common multidrug resistant (MDR) pattern of sulphamethoxazole (91%), tetracycline (84%) and streptomycin (80%) resistance. Tetracycline resistance was most frequently determined by the tet(B) gene (38%), while tet(A) was identified in 26% of all isolates with wide ranges for both tet gene types between some countries and with class 1 integrons and resistance genes co-transferred by conjugation. The virulence gene profiles included enterotoxin genes (lt, sta and/or stb), as well as adhesin genes (k88/f4, f18). Characterisation of two representative tet(A) plasmids of porcine F18(+) ETEC from Central Europe revealed that the IncF plasmid (pES11732) of the Czech strain (~120 kb) carried tet(A) in association with catA1 for chloramphenicol resistance. The IncI1 plasmid (pES2172) of the Hungarian strain (~138 kb) carried tet(A) gene and a class 1 integron with an unusual variable region of 2,735 bp composed by two gene cassettes: estX-aadA1 encoding for streptothricin-spectinomycin/streptomycin resistance exemplifying simultaneous recruitment, assembly and transfer of multidrug resistance genes by the tet(A) plasmid of porcine ETEC. By this we provide the first description of IncF and IncI1 type plasmids of F18(+) porcine enterotoxigenic E. coli responsible for cotransfer of the tet(A) gene with multidrug resistance. Additionally, the unusual determinant estX, encoding for streptothricin resistance, is first reported here in porcine enterotoxigenic E. coli.
RESUMEN
Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence.
Asunto(s)
Adhesión Bacteriana/fisiología , Escherichia coli Enterotoxigénica/metabolismo , Enterotoxinas/metabolismo , Células Epiteliales/microbiología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Animales , Línea Celular , Escherichia coli Enterotoxigénica/enzimología , Infecciones por Escherichia coli/microbiología , Calor , Mucosa Intestinal/microbiología , PorcinosRESUMEN
In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential.
Asunto(s)
Escherichia coli Enterotoxigénica/genética , Infecciones por Escherichia coli/microbiología , Plásmidos/genética , Enfermedades de los Porcinos/microbiología , Resistencia a la Tetraciclina/genética , Factores de Virulencia/genética , Animales , Antibacterianos/farmacología , Toxinas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli Enterotoxigénica/efectos de los fármacos , Escherichia coli Enterotoxigénica/patogenicidad , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Sitios Genéticos , Humanos , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Plásmidos/aislamiento & purificación , Análisis de Secuencia de ADN , Porcinos , Tetraciclina/farmacología , VirulenciaRESUMEN
To study the genetic characteristics of DT104 strains of Salmonella Typhimurium and the prevalence of Salmonella Genomic Island (SGI1) in Hungary, 140 recent Salmonella strains of food and animal origin were examined. For the first time in Hungary, the SGI1 was found in 17 out of 59 S. Typhimurium isolates (all proven to be DT104 phage type). These 17 strains were then subtyped by pulsed-field gel electrophoresis (PFGE) into 6 pulsotypes which were less correlated with the geographic origin than with the animal species of origin.
Asunto(s)
Microbiología de Alimentos , Islas Genómicas/genética , Salmonelosis Animal/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/aislamiento & purificación , Salmonella/genética , Animales , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos , Hungría/epidemiología , Salmonelosis Animal/epidemiologíaRESUMEN
Enterotoxigenic Escherichia coli (ETEC) infection is the most common type of colibacillosis of young animals (primarily pigs and calves), and it is a significant cause of diarrhoea among travellers and children in the developing world. The main virulence attributes of ETEC are adhesins and enterotoxins, which are mostly regulated on large plasmids. Almost all ETEC bacteria are known to adhere to receptors on the small intestinal epithelium by their proteinaceous surface appendages (fimbriae, pili) or by afimbrial proteins without inducing significant morphological changes. Furthermore, they secrete protein toxins (enterotoxins) to reduce absorption and to increase fluid and electrolyte secretion of small intestinal epithelial cells. Regarding details of epidemiology, pathogenesis, diagnosis and prevention of ETEC infections and diarrhoea in animals, readers are referred to an earlier more extensive review [Nagy and Fekete, 1999. Enterotoxigenic Escherichia coli (ETEC) in farm animals. Vet. Res. 30, 259-284]. This paper intends to summarise our basic knowledge and to highlight the new developments and most actual research topics in the area of ETEC infections in veterinary medicine. Attention is paid to recently described new virulence factors and to new genetic vectors in ETEC bacteria. Applications of our knowledge in the diagnosis and prevention of ETEC diarrhoea in animals will also be discussed.
Asunto(s)
Enfermedades de los Animales/microbiología , Enterotoxinas/biosíntesis , Infecciones por Escherichia coli/veterinaria , Vacunas contra Escherichia coli , Escherichia coli/patogenicidad , Animales , Toxinas Bacterianas/inmunología , Escherichia coli/clasificación , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/fisiopatología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/inmunología , VirulenciaRESUMEN
The enterotoxigenic Escherichia coli (ETEC) strain Ec2173, causing post weaning diarrhoea in swine, harbours six plasmids ranging from 13 to 200 kb in size. The heat stable toxin genes sta, stb and a tetracycline resistance gene were located on a self conjugative 120-kb plasmid, called pTC. In the cloned ColE1 type origin of replication of pTC a deletion was detected compared to other ColE1 replicons affecting the replication modulator gene rom. Epidemiological studies on ETEC isolates showed that pTC-like plasmids are widely distributed among porcine ETEC strains; thus representing an example of co-evolution of antibacterial resistance and virulence in pathogenic E. coli.
Asunto(s)
Diarrea/veterinaria , Enterotoxinas/genética , Infecciones por Escherichia coli/veterinaria , Escherichia coli/patogenicidad , Plásmidos/genética , Enfermedades de los Porcinos/microbiología , Animales , Conjugación Genética , Diarrea/microbiología , Escherichia coli/clasificación , Escherichia coli/genética , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Fimbrias Bacterianas/genética , Radioisótopos de Flúor , Plásmidos/clasificación , Origen de Réplica , Porcinos , Enfermedades de los Porcinos/metabolismo , Tetraciclina/farmacología , Virulencia/genética , DesteteRESUMEN
Most virulence genes of enterotoxigenic Escherichia coli (ETEC) are located on plasmids. The gene for heat-stable enterotoxin I (sta) is part of the transposon Tn1681, and the heat-stable enterotoxin II (stb) gene was described to be part of the transposon Tn4521. In the studies presented here, we describe the linkage of the sta and stb genes on an approximately 10-kb fragment designated as toxin-specific locus (TSL). The TSL has been isolated from the 120-kb virulence plasmid pTC of the porcine ETEC strain 2173 that produces F18 fimbriae. The nucleotide sequence of the TSL fragment was determined. Sequences in the flanking regions of the sta gene indicated the presence of Tn1681, but--unexpectedly--flanking sequences of the neighbouring stb gene were completely different from those of Tn4521. The 10-kb TSL is part of a 40-kb fragment that contains the replication origin of pTC. This 40-kb fragment was mobilised into plasmid pACYC177 and the nucleotide sequence of the bordering fragments was determined. The 40-kb fragment was flanked by IS10 elements at both ends, indicating the existence of a new 40-kb pathogenicity island (PAI) in strain 2173. Several F4(K88)+ ETEC and F18+ ETEC as well as F18+ E. coli strains producing enterotoxins and verotoxin-2 (ETEC/VTEC) from weaned pigs of different geographical origin were tested for the flanking regions by PCR to see if they belong to the "Tn4521-like" or the "pTC-like" stb type. It turned out that the Tn4521-like stb-type was characteristic of F4(K88)+ ETEC, while the pTC-like stb type was present in most F18+ ETEC and F18+ ETEC/VTEC, although polymorphism was observed both in the K88 and F18 groups. These results suggest the existence and worldwide spread of a new plasmid-encoded pathogenicity island in porcine post weaning ETEC and ETEC/VTEC strains producing F18 fimbriae.