RESUMEN
The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background.
Asunto(s)
Antibacterianos/farmacología , Fenotipo , Selección Genética , Staphylococcus aureus/genética , Resistencia a la Vancomicina/genética , Vancomicina/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/química , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Elementos Transponibles de ADN/efectos de los fármacos , Expresión Génica , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains.
Asunto(s)
Aminoaciltransferasas/genética , Proteínas Bacterianas/genética , Daptomicina/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Aminoaciltransferasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Genotipo , Humanos , Resistencia a la Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/ultraestructura , Microscopía Electrónica , Mutación , Fenotipo , Plásmidos , Infecciones Estafilocócicas/microbiología , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Transformación BacterianaAsunto(s)
Humanos , Farmacorresistencia Bacteriana Múltiple/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Infecciones Estafilocócicas/microbiología , Argentina/epidemiología , Biopelículas , Células Clonales , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Transferencia de Gen Horizontal , Genes Bacterianos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Prevalencia , Infecciones Estafilocócicas/epidemiología , Virulencia/genéticaAsunto(s)
Antibacterianos/farmacología , Cefoxitina/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Oxacilina/farmacología , Argentina , Humanos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/microbiologíaAsunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Infecciones Estafilocócicas/microbiología , Argentina/epidemiología , Biopelículas , Células Clonales , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Transferencia de Gen Horizontal , Genes Bacterianos , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Prevalencia , Infecciones Estafilocócicas/epidemiología , Virulencia/genéticaAsunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas , Carbapenémicos/farmacología , Brotes de Enfermedades , Infecciones por Pseudomonas/epidemiología , Pseudomonas aeruginosa/efectos de los fármacos , Resistencia betalactámica , beta-Lactamasas , Argentina/epidemiología , Proteínas Bacterianas/biosíntesis , Ceftazidima/farmacología , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/aislamiento & purificación , beta-Lactamasas/biosíntesisRESUMEN
OBJECTIVES: One of the main characteristics of methicillin-resistant Staphylococcus aureus (MRSA) from both hospitals and community is their heterogeneous expression of resistance. Recently, we reported new heterogeneous MRSA isolates phenotypically susceptible to oxacillin despite being mecA positive. These low-level mecA-mediated resistance MRSA strains are very heterogeneous in expression (HeR) and are likely to be clinically relevant since exposure of such isolates to beta-lactams can result in high-level homotypic resistance (HoR). We hypothesized that HeR to HoR selection in these clinically relevant strains may be determined by the pre-existence of a hypermutable population that favours its selection in the presence of oxacillin. METHODS: Using established procedures, SA13011 HeR to HoR selection was performed by using subinhibitory concentrations of oxacillin and examined for mutability. Real-time RT-PCR and transcriptional profiling by DNA microarray were used to compare gene expression between both populations and related genetically modified SA13011 strain. RESULTS: We found that HeR/HoR selection by oxacillin was associated with increased mutation rate and oxacillin-mediated SOS response. We determined increased expression of both mecA and SOS response lexA/recA regulators. Mutational inactivation of lexA repressor resulted in a significant decrease in both mutation rate and oxacillin resistance in the HoR cells. Complementation of the lexA mutant strain restored oxacillin resistance to the high levels observed in the corresponding HoR wild-type strain. CONCLUSIONS: The present results support the notion that SOS response is mechanistically involved in generating mutations that, in addition to mecA induction, allow the selection of a highly oxacillin-resistant population.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/fisiología , Respuesta SOS en Genética , Estrés Fisiológico , beta-Lactamas/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/genética , Perfilación de la Expresión Génica , Humanos , Resistencia a la Meticilina , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Unión a las Penicilinas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Sixty-nine community-associated methicillin-resistant Staphylococcus aureus recovered in 6 healthcare centers from northeastern and eastern Argentina were genotyped by pulsed-field gel electrophoresis. The predominant pulsotype was widely distributed harbored SCCmec type IV and Panton-Valentine leukocidin genes. Representative isolates were characterized by multilocus sequence typing and spa typing, demonstrating that this clone belonged to ST5 and spa type 311.
Asunto(s)
Infecciones Comunitarias Adquiridas/clasificación , Infecciones Comunitarias Adquiridas/genética , Staphylococcus aureus Resistente a Meticilina/genética , Infecciones Estafilocócicas/clasificación , Infecciones Estafilocócicas/genética , Argentina/epidemiología , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Técnicas de Tipificación Bacteriana/métodos , Infecciones Comunitarias Adquiridas/epidemiología , Dermatoglifia del ADN/métodos , Farmacorresistencia Bacteriana/genética , Electroforesis en Gel de Campo Pulsado/métodos , Exotoxinas/genética , Genotipo , Humanos , Leucocidinas/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Proteínas de Unión a las Penicilinas , Reacción en Cadena de la Polimerasa/métodos , Infecciones Estafilocócicas/epidemiologíaRESUMEN
The mechanisms by which there is differential expression of resistance to oxacillin within the populations of a single strain remains to be fully understood. The purpose of this study was to evaluate and characterize 25 GOA48 methicillin-resistant Staphylococcus aureus (MRSA) oxacillin-susceptible mecA-positive strains, which were obtained by screening consecutively 832 S. aureus isolates. These 25 isolates (3% of the total strains investigated) were uniformly detected by extending the 24-h oxacillin agar screen plate to 48 h (namely, GOA48-MRSA). Twenty-two isolates tested positive for penicillin-binding protein 2a, whereas the remaining 3 isolates were inconsistently mecA positive. Inconsistent detection of mecA by polymerase chain reaction (PCR) in the mentioned 3 isolates was investigated by colony hybridization using a mecA probe (> or = 80% of colonies hybridized poorly to the probe). A PCR product that amplified the empty SCCmec insertion site (attB), present only if the element was excised, resulted positive in all 3 isolates before oxacillin exposure, whereas integrated elements were positive only for oxacillin-grown isolates. The remaining 22 strains did not reveal excision demonstrating stable mecA. We concluded that resistance to beta-lactams in MRSA-positive mecA strains susceptible to oxacillin is associated to an extreme heterogeneous expression of resistance combined in some cases to oxacillin SCCmec excision.
Asunto(s)
Antibacterianos/farmacología , Resistencia a la Meticilina , Oxacilina/farmacología , Staphylococcus aureus/efectos de los fármacos , Resistencia betalactámica , Sitios de Ligazón Microbiológica , Proteínas Bacterianas/genética , Recuento de Colonia Microbiana , ADN Bacteriano/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Viabilidad Microbiana , Hibridación de Ácido Nucleico/métodos , Sondas de Oligonucleótidos/genética , Proteínas de Unión a las Penicilinas/genética , Reacción en Cadena de la Polimerasa , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificaciónAsunto(s)
Infecciones por Pseudomonas/microbiología , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/enzimología , beta-Lactamasas/biosíntesis , Anciano , Antibacterianos/farmacología , Argentina , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Pseudomonas putida/clasificación , Resistencia betalactámica , beta-Lactamasas/genéticaRESUMEN
Methicillin resistance in Staphylococcus aureus is primarily mediated by the acquired penicillin-binding protein PBP 2a, which is encoded by mecA. PBP 2a acts together with native PBP 2 to mediate oxacillin resistance by contributing complementary transpeptidase and transglycosylase activities, respectively. In this study, we have investigated a phenotype of beta-lactam dependence in a clinical methicillin-resistant S. aureus strain (strain 2884D) obtained by in vitro selection with ceftobiprole. 28884D, which grew very poorly in blood agar, required the presence of the beta-lactam antibiotics to grow. On the basis of this observation, we hypothesized that a gene or genes essential for growth were dependent on oxacillin induction. Identification and analysis of genes regulated by oxacillin were performed by both real-time reverse transcription-PCR and spotted microarray analysis. We found that mecA was constitutively expressed in strain 2884D and that the constitutive expression resulted from perturbations in the two systems involved in its regulation, i.e., MecI/MecR1 (staphylococcal chromosome cassette mec type I) and BlaI/BlaR1 (nonfunctional penicillinase operon). PBP 2 appeared to be poorly induced by oxacillin in 2884D. Further analysis of the PBP 2 two-component VraSR regulatory system showed that it was nonfunctional, accounting for the lack of response to oxacillin. Together, these results support the notion that limited PBP 2 availability may have led 2884D to become dependent on oxacillin-mediated mecA induction as a required survival mechanism.