Mutaciones en genes gyrA y gyrB en cepas de bacilos Gram negativos aisladas en hospitales chilenos y su relación con la resistencia a fluoroquinolonas / Mutations in gyrA and gyrB genes among strains of Gram-negative bacilli isolated from Chilean hospitals and their relation with resistance to fluoroquinolones

Background: A progressive frequency of resistance to fluorquinolones is observed among Gram-negative bacilli. Aim: To investigate the mechanism of resistance to fluoroquinolones mediated by mutations affecting gyrA and gyrB genes in strains of Gram negative bacüli isolated from CMean hospitals. Material and method: Minimal inhibitory concentration of fluoroquinolones was determined in 91 randomly selected nalidixic acid-resistant strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, isolated from hospitals of 12 Chilean cities. Quinolone resistance determining region (QRDR) was amplified by PCR and mutations were determined by restriction fragment length polymorphism (RFLP) and DNA sequencing. Results: Strains with mutation in codon 83 of gyrA showed decreased susceptibility to ciprofloxacin with MICs ranging from 0.25 to 1024 fig/ml. The sequencing of PCR products for gyrA indicated amino acid changes in the QRDR region. One strain ofE. coli presented a double mutation, in codon 83 Ser to Leu as well as in codon 87 Asp to Asn. In strains ofK. pneumoniae, however, the change of codon 83 was Ser to Tyr, in A. baumannii was Ser to Leu and in P. aeruginosa was Thr to He. No strains with mutations affecting gyrB were found. Conclusions: Mutations in codon 83 of gyrA is a frequent genetic event involved in the mechanism leading to decreased susceptibility to fluoroquinolone in strains of Gram-negative bacilli.

Identificación molecular de enzimas modificantes de aminoglucósidos en cepas de Enterococcus spp. aisladas en hospitales de la Octava Región de Chile / Molecular identification of aminoglycoside-modifying enzymes among strains of Enterococcus spp. isolated in hospitals of the VIII Region of Chile

Background: Infectious diseases produced by Enterococcus spp, must be treated with a synergistic combination between a penicillin and an aminoglycoside. High level resistance to aminoglycosides is a serious therapeutic problem, since it predicts the loss of synergistic activity of this antimicrobial combination. Aim: To investigate the presence of genes encoding aminoglycoside-modifying enzymes (AMEs) among strains of Enterococcus spp with high level of resistance to aminoglycosides. Material and methods: The genes encoding some of the AMEs were investigated among 305 aminoglycoside-resistant strains of Enterococcus spp isolated in hospitals of the VIII region of Chile, by dot blot hybridization and Polymerase Chain Reaction (PCS). Results: High level resistance to some aminoglycosides was observed in 104 strains (34.1 percent) and 93 of these harbored at íeast one of the genes encoding the investigated AMEs. Three genes were detected: aac(6)Ie-aph(2")Ia (14.8 percent) encoding for the enzyme AAC(6)Ie-APH(2")Ia (resistance to all aminoglycosides, except streptomycin); aph(3)IIIa (26 percent), and ant(6)la (28.5 percent) encoding for the phosphorylating enzymes APH(3)Ilia (resistance to kanamycin, amikacin and neomycin), and ANT(6)-la (resistance only to streptomycin), respectively. None of the strains harbored the gene ant (4) which encode for the enzyme ANT (4). Conclusion: The low frequency of strains harbouring the bifunctional enzyme (<15 percent), conferring an extended resistance profile to aminoglycosides, allows us to propose the empirical use of aminoglycoside-aminocyclitols, associated to a penicillin, in the treatment of serious infections produced by species of enterococci.

Producción de ß-lactamasas de espectro extendido (BLEE) en cepas de Acinetobacter baumannii aisladas en hospitales de la VIIIª Región, Chile / Extended spectrum ß-lactamases (ESBL) production in Acinetobacter baumannii strains isolated from Chilean hospitals belonging to VIII Region

The resistance of Acinetobacter baumannii to ß-lactam antibiotics is mainly due to the synthesis of ß-lactamases. From a clinical point of view, this bacteria and others, grouped under the acronym SPACE (S: Serratia, P: Pseudomonas, A: Acinetobacter, C: Citrobacter, E: Enterobacter) are essentially Amp-C ß-lactamases producers. There is no local information about ESBL presence in Acinetobacter. We studied ESBL production using the Ho and col. technique modified by adding cloxacillin as chromosomal ß-lactamases inhibitor. From 69 isolates, with resistance to at least one third generation cephalosporin, only 7 showed positive synergy test. Four of these amplified for TEM family gene, and one of these amplified also for the OXA family. Our study found a low ESBL production percentage, which agrees with the premise of Amp-C as the main mechanism of resistance to ß-lactam antibiotics in A. baumannii. However, the ESBL description in these bacteria emphasizes the capacity of expressing multiple resistance mechanisms.

La resistencia de Acinetobacter baumannii a antimicrobianos ß-lactámicos se debe fundamentalmente a la síntesis de ß-lactamasas. Del punto de vista clínico se considera que esta bacteria, y otras agrupadas en el acrónimo SPACE (Serratia, Pseudomonas, Acinetobacter, Citrobacter, Enterobacter), son predominantemente productoras de ß-lactamasas tipo AmpC. No hay información en nuestro país sobre presencia de ß-lactamasas de espectro extendido (BLEE) en Acinetobacter. Se estudió la producción de BLEE en cepas de Acinetobacter, mediante una modificación de la técnica de Ho y col adicionando cloxacilina como inhibidor de ß-lactamasas cromosomales. De 69 cepas con resistencia al menos a una cefalosporina de tercera generación, sólo siete presentaron sinergia positiva. Cuatro cepas amplificaron por RPC un fragmento intragénico de genes de familia TEM y una de ellas amplificó, además, para el gen de la familia OXA. Se evidenció un bajo porcentaje de producción de BLEE, lo que confirma que la producción de Amp-C es el principal mecanismo de resistencia de A. baumannii a ß-lactámicos. Sin embargo, la descripción de BLEE en esta bacteria, enfatiza su capacidad de albergar múltiples mecanismos de resistencia.

Transferecia de de ß-lactamasas de espectro extendido desde cepas hospitalarias de Klebsiella pneumoniae a otras especies de enterobacterias / Transference of extended-spectrum ß-lactamases from nosocomial strains of Klebsiella pneumoniae to other species of Enterobacteriaceae

Background: Klebsiella pneumoniae is an important pathogenic bacterium, frequently isolated from nosocomial samples, that exhibits wide antimicrobial resistance profiles, including third generation cephalosporins (3GC), aminoglycosides and quinolones. The resistance to 3GC is mainly due to the synthesis of extended spectrum beta lactamases (ESBL), encoded by conjugative plasmids. Aim: To investigate the potential transference of resistance to 3GC from nosocomial strains of K. pneumoniae to other clinical strains of various species of Enterobacteriaceae. Material and methods: The mating experiments were carried out in liquid media and three nosocomial strains of K. pneumoniae were used as donors. These strains were ESBL-producers and resistant to, at least, one of the 3GC assayed. One strain of Citrobacter freundii, Salmonella typhimurium, Serratia marcescens and Escherichia coli, isolated from clinical specimens, were used as recipients. The presence of bla genes was investigated by PCR. Results: The three nosocomial strains of K. pneumoniae were able to transfer the resistance to 3GC and the genes encoding the ESBL to the susceptible recipient strains of enterobacteria. The frequency of transference was as high as 3.2 x 10-2 transconjugants/recipient cell when the strain of Citrobacter freundii was used as recipient. All transconjugants exhibited high level of resistance to the 3GC assayed. Conclusions: Strains of K. pneumoniae isolated from Chilean hospitals are able to disseminate the ESBL genes to clinical strains of others species of Enterobacteriaaceae.

Aminoglucósidos-aminociclitoles: características estructurales y nuevos aspectos sobre su resistencia / Aminoglycosides-aminocyclitols: structural characteristics and new aspects on resistance

Los antimicrobianos aminoglucósidos-aminociclitoles constituyen una de las familias de agentes antibacterianos de mayor actividad sobre bacilos gramnegativos aeróbicos. Estos compuestos están característicamente formados por la combinación de un alcohol cíclico aminado (aminociclitol) y aminoazúcares (aminoglucósidos) unidos por enlaces glucosídicos. La potente actividad bactericida de estos compuestos parece no sólo explicarse por su capacidad de inhibición de la síntesis proteica, sino que también por un efecto sumatorio y pleiotrópico que altera la permeabilidad de la membrana citoplasmática. La penetración de estos antibacterianos en células bacterianas ocurre mediante tres fases bien definidas, siendo las dos últimas dependientes de la fuerza protón motriz (FPM). Este hecho explica la ausencia de actividad de estos compuestos sobre bacterias anaeróbicas. La resistencia bacteriana a este grupo de antibacterianos se debe, principalmente, a la producción de enzimas modificantes de aminoglucósidos. Estas enzimas son normalmente codificadas por elementos extracromosomales tales como plßsmidos y transposones. Sin embargo, se han identificados nuevos mecanismos y elementos genÚticos que participarían en los fenómenos de resistencia. Recientemente se ha descrito la metilación de bases involucradas en la unión entre el ARN 16S y el aminoglucósido, lo que implicaría un nuevo mecanismo de resistencia contra esta familia de antimicrobianos. Por otra parte, los cassettes genéticos adquieren una creciente importancia ya que albergan genes de resistencia a múltiples familias de antibacterianos, entre ellos, los aminoglucósidos-aminociclitoles. Estos cassettes genÚticos se encuentran asociados a integrones, los cuales son capaces de captar estos determinantes de resistencia.

Resistencia a gentamicina, amikacina y ciprofloxacina en cepas hospitalarias de klebsiella pneumoniae subespecie pneumoniae productoras de beta-lactamasas de espectro extendido / Resistance to gentamicin, amikacin and ciprofloxacin among nosocomial isolates of klebsiella pneumoniae subspecie pneumoniae producing extended spectrum beta-lactamases

BACKGROUND: Klebsiella pneumoniae is a pathogenic bacterium frequently isolated from nosocomial samples, specially the subspecie pneunonlae, with extensive antibiolic resistance profiles, including third generation cepbhalosporiis, aminoglycosides and quinolones. This is specially true for those strains producing extended spectrum beta lactamases (ESBL). AIM: To investigate the susceptibility to gentamicin, amikacin and ciprofloxacin and the presence of some aminogloycoside modifying enzyme (AMEs) among nosocomial strains of K pneumoniae subspecie pneumoniae producing ESBL. MATERIAL AND METHODS: The antibiotic resistant patterns and the level of resistance (minimal inhibitory concentration, MIC) of 100 strains, isoklted from sel ,eal bospitals of dcifferent Chilean cities, were deterl,in,ed. Tbe presence of some aminoglycosides modifying enzyme (AMEs) was investigated by PCR. RESULTS: Sixty five percent of strains were resistant to gentamicin, 47% were resistant to amikacin, and 29% were resistant to ciprofloxacin. The most frequent AMEs genes detected were the aac(6')-Ib gene (6'N-Acetyltransferase type Ib enzyme) in 69% of strains, conferring resistance to amikacin, kanamycin, tobramycin, and nieoniycin, and the gene aac(3)-IIa (3-Acetyltransferase type 3-IIa enzyme), in 36% of strains, conferring resistance to gentamlicin. CONCLUSIONS: Among nosocomial strains of K pneumoniae subspecie pneumoniae isolaterd from Chilean hospitals, there is an association between the production of ESBL and the resistance to others antimicrobial agents, especially aminoglycosides. Nevertheless, 71% of isolates are susceptible to ciprofloxacin.

Integrones y cassettes genéticos de resistencia: estructura y rol frente a los antibacterianos / Integrons and resistance gene cassettes: structure and role against antimicrobials

Bacteria have developed sophisticated and successful genetic mechanisms to evade the action of antimicrobials. Bacterial multiresistance has caused serious problems in the treatment of nosocomial infections. Integrons and gene cassettes are considered the main genetic elements in the evolution of plasmids and transposons that actively participate in the mobilization of genes, codifying different bacterial resistance mechanisms. This article reviews the historical and structural aspects of integrons and resistance gene cassettes and the presence of these structures in Gram negative bacteria isolated from Chilean hospitals in the last ten years (Rev MÚd Chile 2004; 132: 619-26).

Metodología clásica y molecular en la identificación de especies de enterococcus spp / Classic and molecular methodologies for the identification of enterococcus species

Background: Enterococcus is a bacterial genus with low virulence. However, in the last years, the importance of some enterococcus species as nosocomial pathogens has increased, specially due to their resistance to some antimicrobial. Aim: To identify enterococcus strains using classical biochemical techniques and genomic amplification with Polymerase Chain Reaction (PCR). Material and methods: Three hundred and five enterococcus strains, isolated between 1996 and 1999, from different clinical specimens in hospitals and other centers of the VIIIth Region of Chile, were studied. The isolates were identified, to the species level, according to the scheme proposed by Carvalho et al. Identification of some strains was confirmed by PCR. Results: Eighty nine percent of isolates were identified as E fócalis, 10.2 percent as E fócium and 3.3 percent as other species. Conclusions: PCR is a fast and promising technique, useful in the identification of Enterococcus species

Aislamiento de enterococcus faecium resistente a vancomicina con genotipo vanB en el Hospital Clínico Regional de Concepción / Isolation of vancomycin resistant enterococcus faecium with genotype vanB at the Hospital Clínico Regional de Concepción

La resistencia bacteriana a los agentes antimicrobianos ha aumentado durante las últimas décadas. De particular importancia es la descripción de aislamientos de Enterococcus resistente a vancomicina (EVR), de reciente y progresiva descripción en nuestro país. Comunicamos el aislamiento de dos cepas de E. faecium resistentes a vancomicina de pacientes colonizados por este microorganismo en el Hospital Clínico Regional de Concepción. El estudio feno y genotípico fue positivo para vanB, además ambos aislamientos presentaron similitud genética en un estudio de tipificación molecular por rep-PCR. Interesantemente el aislamiento de estas cepas precedió al aislamiento de EVR según el protocolo ministerial. Esta diferencia puede explicarse por los factores de riesgo que presentaron los pacientes estudiados

Propiedades microbiológicas, clasificación y relación estructuras-actividad de cefalosporinas e importancia de las cefalosporinas de cuarta generación / Microbiological properties, classification and structure-activity relationships of cephalosporins and the importance of fourth generation cephalosporins

Las cefalosporinas son uno de los grupos de mayor importancia dentro de los ß-lactámicos. Existen diversas clasificaciones de esta moléculas, siendo la más utilizada aquella que agrupa a estos compuesto de acuerdo a propiedades estructurales, microbiológicas y desarrollo histórico: primera a cuarta generación. Las cefalosporinas de tercera generación han sido ampliamente utilizadas, pero la emergencia de resistencia bacteriana fundamentalmente derivada de la producción de ß-lactamasas tanto cromosomales como plasmidiales, ha limitado el uso de estos compuestos. Las cefalosporinas de cuarta generación se caracterizan por la presencia de un nitrógeno cuaternario en C, además de mantener el grupo metoxi-imino aminotiazolil en C. Presentan una elevada penetración intracelular a través de la membrana externa de bacilos Gram negativos y tienen una baja afinidad por enzimas que degradan cefalosporinas de tercera generación. Cefepime, una cefalosporina de cuarta generación, demostró una mayor actividad inhibitoria sobre cepas chilenas de Klebsiella pneumoniae y Escherichia coli productoras de ß- lactamasa de espectro extendido, que cefotaxima y ceftazidima

Presencia de integrones y su relación con la resistencia a cefalosporinas de tercera generación en cepas de acinetobacter baumannii de origen nosocomial / Presence of integrons and their relationship with the resistance to third generation cephalosporins among nosocomial isolates of acinetobacter baumannii

Background: Acinetobacter baumannii is an important etiological agent causing nosocomial infections. High level of resistance for different kind of antimicrobials has been observed, including ß-lactam antibiotics. This feature, chromosomal or plasmid encoded, has been associated to integrons harbouring antibiotic resistance gene cassettes. Aims: To investigate the presence of integrons among clinical isolates resistant to third generation cephalosporins (3GC). Material and methods: One hundred A. baumannii strains isolated from several Chilean hospitals were included in this study. Minimal inhibitory concentrations (MIC) of 3GC by an agar dilution method were carried out. Integrons class 1, 2 and 3 were investigated by colony blot hybridisation and confirmed by PCR. Results: High level of resistance to all assayed 3GC was observed. On the other hand, integrón class 2 was the most prevalent (77 percent of isolates) followed by integron class 1 (52 percent). Forty six percent of isolates hybridised with probes for both of them. However, no positive hybridisation was detected for integron class 3. Conclusions: Nevertheless, most isolates harboured one or both class of integron; there was no direct relationship between the presence of these genetic structures and the resistance to this kind of ß-lactam antibiotics

Evaluación de los criterios de Nugent y Amsel para el diagnóstico de vaginosis bacteriana / Evaluation of Nugent and Amsel criteria for the diagnosis of bacterial vaginosis

Background: Bacterial vaginosis (BV) is a common disease in reproductive-age women and is associated to important gynecologic and obstetric complications. Aim: To study the occurrence of BV in apparently healthy women attending family planning clinics, using Amsel and Nugent diagnostic criteria. Material and methods: Two hundred thirty nine women consulting for symptoms associated to cervicovaginitis, were studied. A sample from the lateral walls of the vagina was obtained with a sterile swab for microscopic analysis, Gram stain and amine test. Results: According to Amsel and Nugent criteria a 31.1 percent and 31.8 percent BV prevalence was observed. The sensitivity and specificity of Nugent criteria, compared with Amsel criteria were 83.3 percent and 92.1 percent, respectively. Conclusions: The high prevalence of BV found in this study suggests that this vaginal infection should be diagnosed with standardized methods. Nugent criteria are economic easy to perform and sensitive and we propose that they should be used in local health centers

Aislamiento de acinetobacter spp desde muestras clínicas en el Hospital Clínico Regional Guillermo Grant Benavente en Concepción / Isolation of acinetobacter spp from clinical specimens at Hospital Clinico Regional Guillermo Grant Benavente, Concepción

Debido a que Acinetobacter se ha transformado en un importante patógeno intrahospitalario el objetivo de este estudio fue investigar las especies de este género que se encontraban involucradas en procesos infecciosos en el principal centro hospitalario de la ciudad de Concepción, en el periodo comprendido entre agosto y diciembre de 1999. Se recolectaron 113 cepas de Acinetobacter, aisladas principalmente de secreciones del tractorespiratorio y heridas. El mayor número de aislamiento tuvo su origen en pacientes internados en la unidad de cuidado intensivo, seguido de pacientes que se encontraban en los servicios de traumatología y cirugía. La identificación de especie y la biotipificación de Acinetobacter baumannii se realizó mediante pruebas fenotípicas. A. baumannii fue la especie predominante, siendo los biotipos 9,6 y 8 los prevalentes en este centro hospitalario

Quinolonas: aspectos generales sobre su estructura y clasificación / Quinolones: general characteristics of structure and classification

Las quinolonas constituyen uno de los grupos de antimicrobianos de mayor desarrollo. Químicamente son estructuras bicíclicas heteroaromáticas, constituidas por un núcleo piridona- ß-ácido carboxílico y un anillo aromático. La relación entre la estructura química y la actividad biológica (relación estructura-actividad) de estas moléculas ha motivado la síntesis de compuestos con distintos radicales en la estructura química básica. Posición 1: el sustituyente de mayor importancia es el grupo ciclopropil que combina favorables propiedades estéricas, espaciales y de interacción electrónica exhibiendo las quinolonas que poseen este grupo una potente actividad sobre bacilos Gram negativos; otros sustituyentes de importante desarrollo son los anillos bencénico mono o difluorados, que amplían el espectro de actividad y mejoran las propiedades farmacocinéticas, pero podrían estar asociadas a fenómenos de toxicidad. Las posiciones 2, 3, 4 no presentan mayores variaciones. La posición 6 prácticamente define, por la presencia de flúor, a las modernas quinolonas, sin embargo, se han sintetizado quinolonas experimentales sin flúor que presentan una interesante actividad in vitro. Posición 5, la presencia de grupos amino o metilo favorece algunas propiedades farmacocinéticas. Posición 8: la presencia de halógenos aumenta la actividad antianaerobia, en particular Cl y F, pero se asocia a fenómenos de fototoxicidad incentivando la síntesis de compuestos sin halógenos. También estas moléculas se clasifican en generaciones de acuerdo al momento de su síntesis y los radicales utilizados

Aspectos microbiológicos de meropenem / Microbiological topics of meropenem

Meropenem es un compuesto del grupo carbapenem, con un amplio espectro antibacteriano y rápida actividad bactericida. Esta actividad es superior sobre bacilos gram negativos fermentadores como klebsiella sp y E. coli, e inferior sobre pseudomonas sp. Y A. baumannii. En cuanto a estas dos últimas bacterias, hay que hacer notar que la actividad de meropenem sobre pseudomonas sp pareciera ser bastante más rápida que la que ocurre sobre acinetobacter sp, aún cuando las CIM aparecen similares. La muerte bacteriana es más rápida sobre P. aeruginosa

Adherencia de acinetobacter baumannii al tejido de tráquea de la rata / Adherence of acinetobacter baumannii to rat trachial tissue

Background: Acinetobacter baumannii is an important nosocomial pathogen whose virulence factors have not been fully elucidated. Aim: To study the adherence and hemagglutinating capacity of several biotypes of Acinetobacter baumannii. Material and methods: Thirty nine strains of Acinetobacter baumannii isolated from hospitalized patients were studied. The adherence of these strains to small pieces of rat tracheal tissue was studied. Additionally, their ability to hemagglutinate human erythrocytes and the effect of D-mannose and D-galactose on the adherence and hemagglutinating capacity was assessed. Transmission electron microscopy of strains was performed looking for the presence of fimbriae. Results: All strains exhibited adherence to tissues. All strains had also D-mannose and D-galactose resistant hemagglutinating ability. Fimbriae were found in Acinetobacter baumannii and E coil cells. Conclusions: Adherence of Acinetobacter baumannii to rat tracheal tissue, apparently not related to the presence of fimbriae, may be a virulence mechanism of this bacterium