RESUMEN
The algorithm of the identification of the bla(CTX-M) genes coding CTX-M-type beta-lactamases providing resistance to cephalosporins III-IV was developed. This algorithm provides identification of 49 genes of 96 genes presented in the GenBank database so far. Remaining 47 genes can be identified as consisting of small sub-groups composed of 2-6 genes with the exception of sub-group of the bla(CTX-M-14)-like genes composed of 13 genes. The identification of the bla(CTX-M) genes is based on two-step restriction fragment length polymorphism analysis of 544 bp PCR-product (PCR-RFLP). In the first step, determination of subtype (cluster) of the bla(CTX-M) gene occurred using the restriction nuclease Alu I: cluster 1, -2, -8, -9 or -25. Moreover, four genes can be identified just at this step: bla(CTX-M)-59, (cluster 2); bla(CTX-M-63) (cluster 8), bla(CTX-M-45) (cluster 9), and bla(CTX-M-78) (hybrid gene between cluster 2 and cluster 25). At the second step gene identification goes on inside of each cluster separately using a set of 26 restriction nucleases. As a result of the PCR-RFLP-analysis, 23 bla(CTX-M) genes can be identified at the cluster 1, 11 genes--at the cluster 2, 4 genes--at the cluster 8, 9 genes--at the cluster 9, 1 gene--at the cluster 25, and 2 hybrid genes: bla(CTX-M-78) (between clusters 2 and 25), and bla(CTX-M-64) (between clusters 1 and 9). The described algorithm was used for identification of the blac(CTX-M) genes (n = 585) detected in Enterobacteriaceae nosocomial isolates (n = 877), collected from Russial hospitals in 2003-2007. It was shown that major genes belonged to cluster 1 (n = 543), namely--bla(CTX-M-15) gene (n = 515), bla(CTX-M-3) (n = 25), bla(CTX-M-22) (n = 1), bla(CTX-M-23) (n = 1), and bla(CTM-34) (n = 1). Moreover, the genes atributed to cluster 2 were identified: bla(CTX-M-2) (n = 1), and bla(CTX-M-5) (n = 4); and genes belonged to cluster 9: bla(CTX-M-9) (n = 2), and bla(CTX-M-14) (n = 35).
Asunto(s)
Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , Farmacorresistencia Microbiana/genética , Polimorfismo de Longitud del Fragmento de Restricción , beta-Lactamasas/clasificación , beta-Lactamasas/genética , Algoritmos , Cefalosporinas/farmacología , Infección Hospitalaria/microbiología , Enterobacteriaceae/genética , Técnicas de Genotipaje , Humanos , Familia de Multigenes , Estándares de Referencia , Venenos de Escorpión/farmacologíaRESUMEN
Nosocomial bacterial isolates collected within 2003-2004 (n=411) and 2005-2007 (n=422) were highly resistant to cephalosporins III-IV and antibacterials of other groups (aminoglycosides, fluoroquinolons, chloramphenicol, and co-trimoxazole). Genes encoding TEM, SHV, CTX-M, OXA-2, and AmpC types of beta-lactamases (BLs) in the E. coli, Klebsiella spp., and Enterobacter spp. isolates were detected using polymerase chain reaction (PCR). Prevalent CTX-M-type BLs were detected in 85% of the E. coli, 87% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the first strain collection and in 94% of the E. coli, 91% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the second one. Genes belonging to three subtypes of blacTx-M genes were identified: bla(CTX-M-1) (228 bla(CTX-M-15) and six bla(CTX-M-3) of the first strain collection; 275 bla(CTX-M-15), three bla(CTX-M-3), and one bla(CTX-M-22) of the second one), bla(CTX-M-2) (one bla(CTX-M-5) of the first strain collection and one bla(CTX-M-2) of the second one), bla(CTX-M-9) (17 bla(CTX-M-14) and one bla(CTX-M-9) of the first strain collection; seven bla(CTX-M-14) and one bla(CTX-M-9) of the second one). Three isolates of the first strain collection and one isolate of the second one carried two genes belonging to two different subtypes, i.e., bla(CTX-M-15) and bla(CTX-M-14) simultaneously. The bacterial isolates had high levels of associative resistance to ciprofloxacin, co-trimoxazole, gentamicin, amikacin, and chloramphenicol associated with the resistance gene cassettes aadA1, aadA2, aadA5, aadB, aacA4, aac(6')Ib; dfrA1, dfrA5, dfrA12, dfrA17, cmlA1, ereA2, and catB8 in the class 1 integrons and the resistance gene cassettes dfrA1, sat1, and aadA1 in the class 2 integrons.
Asunto(s)
Antiinfecciosos/farmacología , Farmacorresistencia Bacteriana/genética , Enterobacter/genética , Escherichia coli/genética , Klebsiella/genética , Infección Hospitalaria/microbiología , Enterobacter/efectos de los fármacos , Enterobacter/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Frecuencia de los Genes , Genes Bacterianos/genética , Humanos , Integrones/genética , Klebsiella/efectos de los fármacos , Klebsiella/aislamiento & purificación , Federación de Rusia , Resistencia betalactámica/genética , beta-Lactamasas/genéticaRESUMEN
The addition of 3-methyl benzoate to the culture of the recombinant strain Pseudomonas putida IPM-36 (bearing the cryIIIA gene of B. thuringiensis subsp, tenebrionis under the control of the Pm promoter and the regulator gene xylS) slowed down the growth rate of the recombinant strain and increased, under non-selective conditions, the number of plasmid-free cells. Intense synthesis of the Coleoptera-specific delta-endotoxin encoded by the cryIIIA gene began 6-8 h after the addition of the inducer 3-methyl benzoate, no matter whether it was added in the early or late logarithmic phase. Maximal production of endotoxin (0.5-0.6 g/l) was observed when the inducer was added in the early logarithmic phase (3 h of growth). Overproduction of delta-endotoxin impaired cell division, so that filamentous cells became predominant in the culture. delta-Endotoxin accumulated in overproducing cells as irregular crystalloid inclusions.