RESUMEN
In recent work, we described excision of a large genomic region from Enterococcus faecium D344R resulting from the interaction of Tn916 and a related transposon designated Tn5386. In the present study, we present and analyze the complete sequence of Tn5386. Tn5386 is 29,451 bp in length. Fifteen of its 30 open reading frames are analogous to ORFs found in Tn916. Significant differences include a series of ORFs with homology to lantibiotic immunity genes in the same location where tetM is found in Tn916, insertion of a Group II intron and an ORF with similarities to previously described surface exposed collagen adhesion proteins. Our results indicate that Tn5386 falls within the Tn916 family of transposons, and in place of tetM encodes a novel region that may confer resistance to lantibiotics.
Asunto(s)
Elementos Transponibles de ADN/genética , Enterococcus faecium/genética , Antibacterianos/farmacología , Bacteriocinas/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Enterococcus faecium/efectos de los fármacosRESUMEN
In recent work, we described the excision of a large genomic region from Enterococcus faecium D344R in which the sequence from "joint" regions suggested that excision resulted from the interaction of conjugative transposon Tn916 and the related mobile element Tn5386. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn916 and Tn5386 to promote the excision of constructs consisting of the termini of Tn916, Tn5386, and the VanB mobile element Tn5382. Integrases alone from either Tn916 or Tn5386 promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn916 and Tn5382 found in all joints were consistent with previously identified Tn916 and Tn5382 termini. Substantial variation was seen in the integrase terminus of Tn5386 used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn5386 constructs, in contrast to joints observed with the termini of Tn916 or Tn5382. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn916-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn916-like integrases, which bind to the ends and facilitate strand exchange.
Asunto(s)
Elementos Transponibles de ADN/genética , Enterococcus faecium/enzimología , Enterococcus faecium/genética , Integrasas/genética , Secuencia de Bases , Biología Molecular , Plásmidos/genética , Especificidad por SustratoRESUMEN
To test the hypothesis that establishing gastrointestinal colonization with multiresistant Enterococcus faecium (VRE) C68 results from expansion of the enterococcal population in the upper small bowel, we compared VRE quantities recovered from the proximal, middle, and distal segments of the small bowel from mice treated with different antimicrobial agents. Antibiotics associated with high-level VRE fecal colonization (cefotetan, ceftriaxone, clindamycin, and ticarcillin-clavulanic acid) increased VRE quantities in all small-bowel segments, whereas cefepime and piperacillin-tazobactam did not. Enterococcal expansion did not correlate with reductions in numbers of native gram-negative or anaerobic flora. Green fluorescence protein-expressing E. faecium bacteria were found adjacent to the small bowel epithelial lining in colonized mice. These data indicate that enterococcal bowel colonization begins within the proximal small bowel and does not correlate with inhibition of other cultivable flora. Host or enterococcal factors induced by exposures to certain antibiotics may play a role in facilitating E. faecium colonization of the mammalian gastrointestinal tract.
Asunto(s)
Antibacterianos/farmacología , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/crecimiento & desarrollo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/microbiología , Animales , Cefalosporinas/farmacología , Ácidos Clavulánicos/farmacología , Clindamicina/farmacología , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Metronidazol/farmacología , Ratones , Ácido Penicilánico/análogos & derivados , Ácido Penicilánico/farmacología , Piperacilina/farmacología , Combinación Piperacilina y Tazobactam , Ticarcilina/farmacologíaRESUMEN
Using 15 unrelated Enterococcus faecium isolates as donors, we demonstrated that ampicillin resistance was transferable to an E. faecium recipient containing a pbp5 deletion for all but four strains. The transfers occurred at low frequencies (generally ca. 10(-9) transconjugants/recipient CFU), consistent with chromosome-to-chromosome transfer. pbp5 transfer occurred within large genetic regions, and insertion into the recipient genome occurred most commonly into the recipient SmaI restriction fragment that had been created by the previous pbp5 deletion. Restriction mapping of the region upstream of pbp5 revealed a commonality of fragment sizes among the clinical isolates from the United States which differed significantly from those of three strains that were isolated from turkey feces. These data prove conclusively that E. faecium pbp5 is a transferable determinant, even in the absence of a coresiding vancomycin resistance mobile element. They also suggest that the spread of high-level ampicillin resistance among U.S. E. faecium strains is due in part to the transfer of low-affinity pbp5 between clinical isolates.
Asunto(s)
Conjugación Genética , Enterococcus faecium/genética , Proteínas de Unión a las Penicilinas/genética , Farmacorresistencia Microbiana/genética , Enterococcus faecium/efectos de los fármacos , Vancomicina/farmacologíaRESUMEN
We describe Tn5386, a novel ca.-29-kb Tn916-like mobile element discovered to occur in ampicillin-resistant, Tn916-containing Enterococcus faecium D344R. PCR amplification experiments after overnight growth with or without tetracycline revealed "joint" regions of circularized Tn5386 composed of 6-bp sequences linking different transposon termini. In one case (no tetracycline), the termini were consistent with those derived by target site analysis of the integrated element. In the other case, the termini were virtually identical in distance from the integrase binding regions, as seen with Tn916. These data are consistent with a model in which one PCR product results from the action of Tn5386 integrase, whereas the other results from the action of the Tn916 integrase on Tn5386. Spontaneous conversion of D344R to an ampicillin-susceptible phenotype (D344SRF) was associated with a 178-kb deletion extending from the left end of Tn5386 to the left end of Tn916. Examination of the Tn5386 junction after the large deletion event suggests that the deletion resulted from an interaction between the nonintegrase ends of Tn5386 and Tn916. The terminus of Tn5386 identified in this reaction suggested that it may have resulted from the activity of the Tn916 integrase (Int(Tn916)). The "joint" of the circular element resulting from this excision was amplifiable from D344R, the sequence of which revealed a heteroduplex consistent with Int(Tn916)-mediated excision. In contrast, Tn5386 joints amplified from ampicillin-susceptible D344SRF revealed ends consistent with Tn5386 integrase activity, reflecting the absence of Tn916 from this strain. Tn5386 represents a new member of the Tn916 transposon family. Our data suggest that excision of Tn5386 can be catalyzed by the Tn916 integrase and that large genomic deletions may result from the interaction between these heterologous elements.
Asunto(s)
Elementos Transponibles de ADN/genética , Enterococcus faecium/genética , Eliminación de Gen , Genoma Bacteriano , Resistencia a la Ampicilina/genética , Secuencia de Bases , Cromosomas Bacterianos/genética , Enterococcus faecium/efectos de los fármacos , Evolución Molecular , Genotipo , Datos de Secuencia Molecular , Fenotipo , Inhibidores de la Síntesis de la Proteína , Tetraciclina , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
We compared ceftriaxone and piperacillin-tazobactam at doses ranging from 0.1 to 2 times the human equivalent daily dose (HEDD), to determine their impact on gastrointestinal colonization by ampicillin- and vancomycin-resistant Enterococcus faecium C68 in a mouse model. Ceftriaxone failed to promote colonization at doses up to 0.25 times the HEDD, whereas piperacillin-tazobactam promoted colonization at doses up to 0.5 times the HEDD. Ceftriaxone promoted colonization at doses at least 0.5 times the HEDD, whereas piperacillin-tazobactam inhibited colonization at doses at least 0.75 times the HEDD. Both piperacillin-tazobactam and ceftriaxone inhibited colonization by an enterococcal strain devoid of low-affinity penicillin-binding protein-5 (significantly increasing its susceptibility to these agents), at doses that promoted colonization by E. faecium C68. These results support a model in which the impact that different beta -lactam agents have on colonization by VRE is related to the level of the beta -lactam agent's intrinsic antienterococcal activity against the colonizing strain.
Asunto(s)
Antibacterianos/farmacología , Portador Sano/prevención & control , Ceftriaxona/farmacología , Enterococcus faecium/efectos de los fármacos , Ácido Penicilánico/farmacología , Piperacilina/farmacología , Animales , Ceftriaxona/administración & dosificación , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana Múltiple , Heces/microbiología , Femenino , Tracto Gastrointestinal/microbiología , Infecciones por Bacterias Grampositivas/prevención & control , Ratones , Ácido Penicilánico/administración & dosificación , Ácido Penicilánico/análogos & derivados , Piperacilina/administración & dosificación , Combinación Piperacilina y TazobactamRESUMEN
We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all beta-lactams. Affinity for penicillin generally correlated with beta-lactam MICs for the mutants, but these associations were not strictly proportional.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Hexosiltransferasas/genética , Hexosiltransferasas/fisiología , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutación/genética , Mutación/fisiología , Peptidil Transferasas/genética , Peptidil Transferasas/fisiología , Resistencia betalactámica/genética , Resistencia a la Ampicilina/genética , Cristalografía por Rayos X , Vectores Genéticos/genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Proteínas de Unión a las Penicilinas , Penicilinas/metabolismo , Plásmidos/genética , Unión ProteicaRESUMEN
We studied the effect of different subcutaneously administered beta-lactam antibiotics on the establishment of gastrointestinal colonization by vancomycin-resistant Enterococcus faecium C68 in a mouse model. Aztreonam, cefazolin, cefepime, and, to a lesser extent, ceftazidime, which neither have significant antienterococcal activity nor are secreted into human bile at high concentrations, did not promote significant vancomycin-resistant enterococci (VRE) colonization. Piperacillin-tazobactam, which has antienterococcal activity and is secreted in human bile at high concentrations, inhibited colonization after limited exposure to the inoculum but was associated with progressively increased VRE colony counts in stool samples after repeated exposure to the VRE inoculum. Ceftriaxone and cefotetan, which lack antienterococcal activity but are secreted into human bile at high concentrations, were associated with rapid and high-level colonization. These data suggest that the risk of VRE colonization varies during exposure to different beta-lactam antimicrobial agents and that the risk is related to biliary concentration and antienterococcal activity of the specific beta-lactam.
Asunto(s)
Antibacterianos/farmacología , Sistema Digestivo/microbiología , Enterococcus faecium/efectos de los fármacos , Resistencia a la Vancomicina , beta-Lactamas/farmacología , Animales , Antibacterianos/farmacocinética , Bilis/metabolismo , Femenino , RatonesRESUMEN
In vitro linezolid resistance was selected at a higher frequency in Enterococcus faecalis JH2-2 than in recombination-deficient E. faecalis UV202. Resistance in JH2-2 was related to accumulated G2576T mutations in 23S rRNA genes, with the least resistance conferred by mutations in two of four copies. UV202 resistance was associated with a G2505A mutation present in a single copy in mutants with different MICs.