RESUMEN
Regulatory RNAs, present in many bacterial genomes and particularly in pathogenic bacteria such as Staphylococcus aureus, control the expression of genes encoding virulence factors or metabolic proteins. They are extremely diverse and include noncoding RNAs (sRNA), antisense RNAs, and some 5' or 3' untranslated regions of messenger RNAs that act as sensors for metabolites, tRNAs, or environmental conditions (e.g., temperature, pH). In this review we focus on specific examples of sRNAs of S. aureus that illustrate how numerous sRNAs and associated proteins are embedded in complex networks of regulation. In addition, we discuss the CRISPR-Cas systems defined as an RNA-interference-like mechanism, which also exist in staphylococcal strains.
Asunto(s)
ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Staphylococcus aureus/genética , Animales , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Humanos , ARN Bacteriano/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismoRESUMEN
Escherichia coli strains MC4100 (parent) and a mutant strain derived from this (IC007) were evaluated for their ability to produce H(2) and organic acids (OAs) via fermentation. Following growth, each strain was coated with Pd(0) via bioreduction of Pd(II). Dried, sintered Pd-biomaterials ('Bio-Pd') were tested as anodes in a proton exchange membrane (PEM) fuel cell for their ability to generate electricity from H(2). Both strains produced hydrogen and OAs but 'palladised' cells of strain IC007 (Bio-Pd(IC007)) produced ~threefold more power as compared to Bio-Pd(MC4100) (56 and 18 mW respectively). The power output used, for comparison, commercial Pd(0) powder and Bio-Pd made from Desulfovibrio desulfuricans, was ~100 mW. The implications of these findings for an integrated energy generating process are discussed.
Asunto(s)
Fuentes de Energía Bioeléctrica , Electricidad , Escherichia coli/metabolismo , Hidrógeno/metabolismo , Paladio/metabolismo , Ácidos Carboxílicos/metabolismo , Desulfovibrio desulfuricans/metabolismo , FermentaciónRESUMEN
Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.
Asunto(s)
Antibacterianos/farmacología , Luciferasas/biosíntesis , Streptococcus/efectos de los fármacos , Streptococcus/genética , Antiinfecciosos/farmacología , Western Blotting , Cloranfenicol/farmacología , Ciprofloxacina/farmacología , Recuento de Colonia Microbiana , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Genes Bacterianos/genética , Genes Reporteros/genética , Mediciones Luminiscentes , Pruebas de Sensibilidad Microbiana , Resistencia a las Penicilinas/genética , Penicilinas/farmacología , Streptococcus/enzimología , Transcripción GenéticaRESUMEN
In 1875, 7 years prior to the description of the Koch bacillus, Klebs visualized the first Streptococcus pneumoniae in a pleural fluid. Since then, this organism has played a determinant role in biomedical science. From a biological point of view, it was largely implicated in the development of passive and active immunization by serotherapy and vaccination, respectively. Genetic transformation was also first observed in S. pneumoniae, leading to the discovery of DNA. From a clinical point of view, S. pneumoniae is still today a prime cause of otitis media in children and of pneumonia in all age groups, as well as a predominant cause of meningitis and bacteremia. In adults, bacteremia is still entailed with a mortality of over 25%. Although S. pneumoniae remained very sensitive to penicillin for many years, penicillin-resistance has emerged and increased dramatically over the last 15 years. During this period of time, the frequency of penicillin-resistant isolates has increased from < or = 1% to frequencies varying from 20 to 60% in geographic areas as diverse as South Africa, Spain, France, Hungary, Iceland, Alaska, and numerous regions of the United States and South America. In Switzerland, the current frequency of penicillin-resistant pneumococci ranges between 5 and > or = 10%. The increase in penicillin-resistant pneumococci correlates with the intensive use of beta-lactam antibiotics. The mechanism of resistance is not due to bacterial production of penicillinase, but to an alteration of the bacterial target of penicillin, the so-called penicillin-binding proteins. Resistance is subdivided into (i) inter mediate level resistance (minimal inhibitory concentration [MIC] of penicillin of 0.1-1 mg/L) and (ii) high level resistance (MCI > or = 2 mg/L). The clinical significance of intermediate resistance remains poorly defined. On the other hand, highly resistant strains were responsible for numerous therapeutical failures, especially in cases of meningitis. Antibiotics recommended against penicillin-resistant pneumococci include cefotaxime, ceftriaxone, imipenem and in some instances vancomycin. However, penicillin-resistant pneumococci tend to present cross-resistances to all the antibotics of the beta-lactam family and could even become resistant to the last resort drugs mentioned above. Thus, in conclusion, the explosion of resistance to penicillin in pneumococci is a ubiquitous phenomenon which must be fought against by (i) a strict utilization of antibiotics, (ii) the practice of microbiological sampling of infected foci before treatment, (iii) the systematic surveillance of resistance profiles of pneumococci against antibiotics and (iv) the adequate vaccination of populations at risk.
Asunto(s)
Resistencia a las Penicilinas/fisiología , Infecciones Neumocócicas/tratamiento farmacológico , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/fisiología , Adulto , Antibacterianos/uso terapéutico , Niño , Humanos , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Selección de Paciente , Infecciones Neumocócicas/epidemiología , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas , Prevalencia , Serotipificación , Streptococcus pneumoniae/clasificaciónRESUMEN
Penicillin tolerance is an incompletely understood phenomenon that allows bacteria to resist drug-induced killing. Tolerance was studied with independent Streptococcus gordonii mutants generated by cyclic exposure to 500 times the MIC of penicillin. Parent cultures lost 4 to 5 log(10) CFU/ml of viable counts/24 h. In contrast, each of four independent mutant cultures lost < or =2 log(10) CFU/ml/24 h. The mutants had unchanged penicillin-binding proteins but contained increased amounts of two proteins with respective masses of ca. 50 and 45 kDa. One mutant (Tol1) was further characterized. The two proteins showing increased levels were homologous to the arginine deiminase and ornithine carbamoyl transferase of other gram-positive bacteria and were encoded by an operon that was >80% similar to the arginine-deiminase (arc) operon of these organisms. Partial nucleotide sequencing and insertion inactivation of the S. gordonii arc locus indicated that tolerance was not a direct consequence of arc alteration. On the other hand, genetic transformation of tolerance by Tol1 DNA always conferred arc deregulation. In nontolerant recipients, arc was repressed during exponential growth and up-regulated during postexponential growth. In tolerant transformants, arc was constitutively expressed. Tol1 DNA transformed tolerance at the same rate as transformation of a point mutation (10(-2) to 10(-3)). The tolerance mutation mapped on a specific chromosomal fragment but was physically distant from arc. Importantly, arc deregulation was observed in most (6 of 10) of additional independent penicillin-tolerant mutants. Thus, although not exclusive, the association between arc deregulation and tolerance was not fortuitous. Since penicillin selection mimicked the antibiotic pressure operating in the clinical environment, arc deregulation might be an important correlate of naturally occurring tolerance and help in understanding the mechanism(s) underlying this clinically problematic phenotype.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Hidrolasas/genética , Proteínas de la Membrana/genética , Operón/genética , Resistencia a las Penicilinas/genética , Proteínas Quinasas/genética , Proteínas Represoras , Streptococcus/enzimología , Streptococcus/genética , Aminoácidos/análisis , Mapeo Cromosómico , Cromosomas Bacterianos/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Luciferasas/genética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación/genética , Factores de Tiempo , Transformación Genética/genéticaRESUMEN
Levofloxacin was investigated against viridans group streptococci in vitro and in rats with experimental aortic endocarditis. The MIC(90)s of levofloxacin and ciprofloxacin for 20 independent isolates of such bacteria were 1 and 8 mg/L, respectively. Rats were infected with two types of organism: either fully susceptible to levofloxacin MIC < or = 0.5 mg/L) or borderline susceptible (MIC 1-2 mg/L). Fully levofloxacin-susceptible bacteria comprised one penicillin-susceptible (MIC 0.004 mg/L) Streptococcus gordonii, and one penicillin-tolerant as well as one intermediate penicillin-resistant (MIC 0.125 mg/L) isogenic strains. Borderline levofloxacin-susceptible bacteria comprised one penicillin-susceptible Streptococcus sanguis and one highly penicillin-resistant Streptococcus mitis (MIC 2 mg/L). Rats were treated for 5 days with drug dosages simulating the following treatments in humans: (i) levofloxacin 500 mg orally once a day (q24 h), (ii) levofloxacin 500 mg orally twice a day (q12 h), (iii) levofloxacin 1 g orally q24 h, (iv) ciprofloxacin 750 mg orally q12 h, and (v) ceftriaxone 2 g iv q24 h. Levofloxacin was equivalent or superior to ceftriaxone, and was successful in treating experimental endocarditis irrespective of penicillin resistance. Nevertheless, standard levofloxacin treatment equivalent to 500 mg q24 h in human was less effective than twice daily 500 mg or once daily 1 g doses against borderline-susceptible organisms. Ciprofloxacin, used as a negative control, was ineffective and selected for resistant isolates. This underlines the importance of MIC determinations when treating severe streptococcal infection with quinolones. In the case of borderline-susceptible pathogens, total daily doses of 1 g of levofloxacin should be considered.
Asunto(s)
Antiinfecciosos/uso terapéutico , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/microbiología , Levofloxacino , Ofloxacino/uso terapéutico , Infecciones Estreptocócicas/tratamiento farmacológico , Animales , Antiinfecciosos/farmacocinética , Antiinfecciosos/farmacología , Cateterismo , Ceftriaxona/farmacocinética , Ceftriaxona/farmacología , Ceftriaxona/uso terapéutico , Ciprofloxacina/farmacocinética , Ciprofloxacina/farmacología , Ciprofloxacina/uso terapéutico , Pruebas de Sensibilidad Microbiana , Ofloxacino/farmacocinética , Ofloxacino/farmacología , Resistencia a las Penicilinas , Ratas , Infecciones Estreptocócicas/microbiología , Streptococcus/efectos de los fármacos , Streptococcus/crecimiento & desarrollo , Streptococcus sanguis/efectos de los fármacos , Streptococcus sanguis/crecimiento & desarrollo , Resultado del TratamientoRESUMEN
Genotypic and phenotypic tolerance was studied in penicillin treatment of experimental endocarditis due to nontolerant and tolerant Streptococcus gordonii and to their backcross transformants. The organisms were matched for in vitro and in vivo growth rates. Rats with aortic endocarditis were treated for 3 or 5 days, starting 12, 24, or 48 h after inoculation. When started at 12 h, during fast intravegetation growth, 3 days of treatment cured 80% of the nontolerant parent compared with <30% of the tolerant derivative (P < .005). When started at 24 or 48 h and if intravegetation growth had reached a plateau, 3 days of treatment failed against both bacteria. However, a significant difference between the 2 organisms was restored when treatment was extended to 5 days. Thus, genotypic tolerance conferred a survival advantage in both fast- and slow-growing bacteria, demonstrating that the in vitro-defined tolerant phenotype also carried the risk of treatment failure in vivo.