Potential involvement of beta-lactamase homologous proteins in resistance to beta-lactam antibiotics in gram-negative bacteria of the ESKAPEE group.

BACKGROUND: Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher's exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.

Sensibilidad disminuida a los antibóticos Beta-Lactamicos en estreptococos Beta-Hemolíticos / Decreased sensitivity to beta-lactam antibiotics in beta-hemolytic streptococci

Últimamente, se están detectando mutaciones en las proteínas ligadoras de penicilina (PBP) de los estreptococos beta-hemolíticos que corresponden a sitios que en Streptococcus pneumoniae han determinado sensibilidad disminuida a los antibióticos beta-lactámicos. Primero, se describieron cepas con sensibilidad intermedia a penicilina en Streptococcus agalactiae (estreptococos del grupo B), luego en Streptococcus dysgalactiae subsp. equisimilis (mayormente grupos C y G) y, más recientemente, cepas con sensibilidad disminuida a aminopenicilinas y cefalosporinas de tercera generación en Streptococcus pyogenes (grupo A). El costo biológico de estas modificaciones nos permite pensar que los niveles de resistencia no han de ser tan elevados como para comprometer por ahora la efectividad clínica de los beta-lactámicos (AU)

Recently, mutations in penicillin-binding proteins (PBPs) of beta-hemolytic streptococci have been detected corresponding to sites that in Streptococcus pneumoniae have been determined to have decreased sensitivity to beta-lactam antibiotics. First, strains with intermediate sensitivity to penicillin were described in Streptococcus agalactiae (group B streptococci), subsequently in Streptococcus dysgalactiae subsp. equisimilis (mainly groups C and G) and, more recently, strains with decreased sensitivity to third-generation aminopenicillins and cephalosporins were found in Streptococcus pyogenes (group A). The biological cost of these modifications suggests that, for now, resistance levels are not high enough to compromise the clinical effectiveness of beta-lactams (AU)

Escherichia coli y Klebsiella pneumoniae productoras de betalactamasas de espectro extendido en un hospital de La Habana

RESUMEN Introducción: En microorganismos gramnegativos la producción de enzimas betalactamasas es el mecanismo más común de resistencia. Las de espectro extendido constituyen un grupo importante por su capacidad de inactivar las cefalosporinas de tercera y cuarta generación y el aztreonam. Su detección es vital para indicar el tratamiento óptimo y las medidas de aislamiento que eviten la dispersión de los microorganismos que las portan. Objetivos: Determinar la incidencia y principales características de los aislados de Escherichiacoli y Klebsiellapneumoniae productores de betalactamasas de espectro extendido en muestras no urogenitales. Métodos: Estudio transversal realizado en el hospital "Salvador Allende" durante el año 2017. Se determinó la frecuencia de Escherichia coli y Klebsiella pneumoniae productoras de betalactamasas de espectro extendido, su procedencia según servicio del hospital, tipo de muestra clínica, y su sensibilidad antimicrobiana. La identificación de betalactamasas de espectro extendido se hizo por el método de doble disco de Jarlier. Resultados: Fueron productores de betalactamasas de espectro extendido 46 y 50 % de aislados de Escherichia coli y Klebsiella pneumoniae, respectivamente. La mayoría provenían de muestras de las salas del Instituto de Angiología, el antimicrobiano con mayor efectividad fue el meropenem, la sensibilidad al resto de los antimicrobianos estuvo por debajo de 80 % y no hubo aislados sensibles a las cefalosporinas de tercera generación. Conclusiones: Se demuestra una alta incidencia de aislados de Escherichia coli y Klebsiella pneumoniae productores de betalactamasas de espectro extendido en el Hospital "Salvador Allende" de La Habana, más marcada en las salas del Instituto de Angiología y en muestras de piel.

ABSTRACT Introduction: Beta-lactamase production is the most common resistance mechanism in gram-negative microorganisms. Extended-spectrum beta-lactamases are an important group of enzymes capable of inactivating third- and fourth-generation cephalosporins and aztreonam. Their detection is important to indicate the optimum treatment as well as isolation measures aimed at preventing the spread of carrier microorganisms. Objectives: Determine the incidence and main characteristics of isolates of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae from non-urogenital samples. Methods: A cross-sectional study was conducted at Salvador Allende hospital during the year 2017. Determination was made of the frequency of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae, their origin by hospital service, the type of clinical sample and their antimicrobial sensitivity. Identification of extended-spectrum beta-lactamases was based on the Jarlier double disc method. Results: Of the total Escherichia coli and Klebsiella pneumoniae isolates studied, 46% and 50%, respectively, were extended-spectrum beta-lactamase producers. Most had been obtained from samples taken in wards of the Institute of Angiology; the most effective antimicrobial was meropenem; sensitivity to the remaining antimicrobials was below 80%; no isolates were sensitive to third-generation cephalosporins. Conclusions: A high incidence was found of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates at Salvador Allende Hospital in Havana, more noticeably in Institute of Angiology wards and skin samples.

Prevalencia de Escherichia coli, productora de BLEE en pacientes ambulatorios de la ciudad de Cuenca / Prevalence of Escherichia coli, ESBL producer in outpatients of the city of Cuenca

Se analizó un total de 274 muestras de orina de pacientes ambulatorios que acudieron a los centros de salud 1, 2 y 3 de la ciudad de Cuenca durante el periodo comprendido entre mayo y junio del año 2013, con el fin de obtener al menos 100 muestras de orina con presencia de Escherichia coli. Se recuperaron 103 cepas de Escherichia coli y se continuó el estudio con la identificación de la producción de Betalactamasas de Espectro Extendido (BLEE), mediante la técnica descrita en el manual CLSI. Se realizaron las pruebas presuntivas y confirmatorias, aplicando el método convencional de difusión en agar, en placas de agar Mueller-Hinton, con un inóculo Mac Farland 0,5 y ensayando los discos de antimicrobiano. Para la prueba presuntiva se emplearon los discos de aztreonam, cefotaxime, ceftazidime y ceftriaxona; para la prueba confirmatoria se utilizaron los discos de ceftazidime y cefotaxime en combinación con ácido clavulánico; la producción de BLEE se determinó mediante la diferencia del diámetro de los halos según se indica en la técnica. Los resultados mostraron que de 103 cepas de E. coli se recuperaron siete (6,8 %) cepas productoras de BLEE y si se considera que la población con la que se trabajó fue de pacientes ambulatorios resulta muy importante la realización de los métodos de identificación de BLEE como apoyo para la correcta terapia antimicrobiana, previniendo de esta manera la diseminación de cepas de E. coli productoras de BLEE.

A total of 274 urine samples were analyzed from outpatients presenting to Health Centers 1, 2 and 3 of the city of Cuenca during the period between May and June 2013, with the purpose to get at least 100 samples positive urine with Escherichia coli. Were retrieved 103 strains of Escherichia coli and the study was continued with the identification of the production of Extended Spectrum Beta Lactamases (ESBL), using the technique described in the CLSI manual. Were performed presumptive and confirmatory tests, applying the conventional method of agar diffusion plates in Mueller-Hinton agar, with a 0.5 McFarland inoculum and tested antimicrobial discs. For the presumptive test was used discs aztreonam, cefotaxime, ceftazidime and ceftriaxone, for the confirmatory test were used discs of ceftazidime and cefotaxime in combination with clavulanic acid. ESBL production was determined by the difference in the diameter of the halos as indicated in the technique. The results showed that of 103 strains of E. coli recovered seven (6,8 %) ESBL producing strains, when considering that the population which was worked was of outpatient, is very important the implementation of identification methods as ESBL for correct support of antimicrobial therapy, preventing this way the spread of strains of E. coli ESBL producing.

Surface restructuring of red mud to produce FeO x (OH) y sites and mesopores for the efficient complexation/adsorption of ß-lactam antibiotics.

In this work, iron oxide in the red mud (RM) waste was restructured to produce mesopores with surface [FeO x (OH) y ] sites for the efficient complexation/adsorption of ß-lactam antibiotics. Red mud composed mainly by hematite was restructured by an acid/base process followed by a thermal treatment at 150-450 °C (MRM150, MRM200, MRM300, and MRM450) and fully characterized by Mössbauer, XRD, FTIR, BET, SEM, CHN, and thermogravimetric analyses. The characterization data showed a highly dispersed Fe3+ oxyhydroxy phase, which was thermally dehydrated to a mesoporous α-Fe2O3 with surface areas in the range of 141-206 m2 g-1. These materials showed high efficiencies (21-29 mg g-1) for the adsorption of ß-lactam antibiotics, amoxicillin, cephalexin, and ceftriaxone, and the data was better fitted by the Langmuir model isotherm (R 2 = 0.9993) with monolayer adsorption capacity of ca. 39 mg g-1 for amoxicillin. Experiments such as competitive adsorption in the presence of phosphate and H2O2 decomposition suggested that the ß-lactamic antibiotics might be interacting with surface [FeO x (OH) y ] species by a complexation process. Moreover, the OH/Fe ratio, BET surface area and porosity indicated that this complexation is occurring especially on [FeO x (OH) y ]surf sites contained in the mesopore space.

Dissociation of cephamycin C and clavulanic acid biosynthesis by 1,3-diaminopropane in Streptomyces clavuligerus.

Streptomyces clavuligerus produces simultaneously cephamycin C (CephC) and clavulanic acid (CA). Adding 1,3-diaminopropane to culture medium stimulates production of beta-lactam antibiotics. However, there are no studies on the influence of this diamine on coordinated production of CephC and CA. This study indicates that 1,3-diaminopropane can dissociate CephC and CA productions. Results indicated that low diamine concentrations (below 1.25 g l(-1)) in culture medium increased CA production by 200%, but not that of CephC. Conversely, CephC production increased by 300% when 10 g l(-1) 1,3-diaminopropane was added to culture medium. Addition of just L-lysine (18.3 g l(-1)) to culture medium increased both biocompounds. On the other hand, while L-lysine plus 7.5 g l(-1) 1,3-diaminopropane increased volumetric production of CephC by 1100%, its impact on CA production was insignificant. The combined results suggest that extracellular concentration of 1,3-diaminopropane may trigger the dissociation of CephC and CA biosynthesis in S. clavuligerus.

Actividad comparativa in vitro de doripenem y otros carbapenemicos frente a pseudomonas aeruginosa aisladas en el laboratorio de microbiología del hospital Vargas de Caracas / Comparative in vitro activity of dorypenem and other carbapenems in pseudomonas aeruginosa isolates on Vargas Caracas Hospital´s Microbiology

Los ensayos de cuantificación de ARN plasmáticos de VIH-1 son importantes para el control de pacientes infectados, así como el monitoreo de la respuesta a la terapia antirretroviral. Por lo tanto, los ensayos comerciales empleados para este propósito deben presentar buena correlación entre si, para dar lugar al manejo terapéutico apropiado. El objetivo del estudio consistió en correlacionar los resultados obtenidos mediante el ensayo de amplificación de señal (bDNA) y PCR en tiempo real (RT-PCR), ambas casas comerciales aprobadas por la FDA y con diferente diana de detección del VIH-1. La validación se realizó con 180 muestras clínicas de pacientes referidos al INHRR. Los resultados fueron comparados con la subpoblación de linfocitos TCD4+ determinados mediante citometría de flujo. El análisis estadístico se realizó empleando el coeficiente de regresión lineal de Pearson (R2) y el valor de contraste de hipótesis con una significancia del 95 %, usando el programa SPSS Statistics v10.0. Se observó una buena correlación entre los ensayos (R2=0.961, p<0.05), siendo la RTPCR más sensible. Las diferencias cuantitativas de carga viral entre las técnicas ensayadas fue menor de 0.5 log10 copias/ml para el 89% de las muestras, y >1 log10 copias/ml solo en dos pacientes, no indicando necesariamente cambio terapéutico. Adicionalmente, se encontró una correlación inversa entre los linfocitos TCD4+ y carga viral del VIH-1 medida por bDNA (R2= 0.20, p<0.05) y RT-PCR (R2= 0.15, p<0.05). Los ensayos evaluados mostraron que ambas técnicas puedes ser empleadas indistintamente para el control de los pacientes VIH positivo.

The assay for quantification of plasma HIV-1 RNA are important for the control of patients infected, as well as the monitoring of the response to antiretroviral therapy. Therefore, the commercial assays used for this purpose must submit good correlation between to give place to the appropriate therapeutic management. In this study, we correlate the results obtained through the testing of signal amplification (bDNA) and real-time PCR (RT-PCR), two comercial technical approved by the FDA and with different targets of detection HIV-1. The validation was carried out with 180 clinical samples of patients referred to the INHRR. The results were compared with the subpopulation of lymphocytes TCD4+ determined by flow cytometry. The statistical analysis was performed using the program SPSS Statistics v10. It was observed good correlation between the tests studied (R2=0.961, p<0.05), with RT-PCR more sensitive. The quantitative differences in viral load between the techniques tested was less than 0.5 log10 copies/ml for the 89% of the samples, and >1 log10 copies/ml in only two patients. Additionally, it was found an inverse correlation between lymphocytes TCD4+ and viral load of HIV-1, measured by bDNA (R2= 0.20, p<0.05) and RT-PCR (R2= 0.15, p<0.05). Therefore, these assays can be employed for the patient control HIV.

Determinación de la calidad composicional y de residuos antibióticos betalactámicos en leche cruda expendida en el sector urbano del municipio de ipiales

El estudio se realizó con el fin de determinar la calidad composicional e higiénica de la leche cruda que consumen los habitantes de la zona urbana del municipio de Ipiales. Se determinó el porcentaje de muestras que no cumplen con las características óptimas para su comercialización y el porcentaje de muestras positivas a residuos de antibióticos betalactámicos. En la zona urbana del municipio de Ipiales, existen 180 expendedores ambulantes reportados, de leche cruda, Para calcular el tamaño de la muestra se aplicó la fórmula estadística de Blaha, con un nivel de confianza del 95%, con un error máximo admitido del 10% lo cual dio un total de 96 muestras, las cuales se procesaron en el laboratorio de análisis lácteo de la empresa Alquería de la ciudad de Ipiales. Los resultados que se obtuvieron en este estudio determinaron que ninguna de las muestras tomadas a los diferentes expendedores, cumplen totalmente con los requisitos en calidad composicional (decreto 616 de 2006 del Ministerio de la protección Social) e higiénica (decreto 2838 del 2006 sobre las características óptimas de la leche cruda) necesarios para asegurar un consumo inocuo de este producto. Para la presencia de antibióticos betalactámicos con SNAP test, el 5,2% de las muestras resultaron positivas y el 94,8% fueron negativas. La adición de neutralizantes en este estudio fue del 0%. En cuanto a la detección de adulterantes se encontró un 2% que corresponde a la adición de harinas.

The study was conducted to determine the compositional and hygienic quality of raw milk consumed by inhabitants of the urban area in the town of Ipiales. The percentage of samples that do not meet the optimal characteristics for marketing and the percentage of positive samples for beta-lactam antibiotic residues were determined. There are 180 reported raw milk sellers in the urban area of the municipality of Ipiales. To calculate the size of the sample the statistical formula Blaha was applied with a 95% of reliability and a maximum error of 10%. 96 not homogenized samples of raw milk, sold in the area were collected and later processed in the milk laboratory of “Alqueria” in Ipiales. The results obtained in this study determined that none of the samples of raw collected from different vendors complies with all quality (decree 616 of 2006 from the Ministry of Social protection) and hygienic requirements (decree 2838, 2006 concerned with the best characteristics of raw milk) necessary to ensure that the human consumption of this product is harmless. 5, 2% of the samples were positive for the presence of beta-lactam antibiotics with Snap test, while 94, 8% tested negative. The addition of neutralizers in this study was 0%. Additionally, it was found that regarding the detection of adulterants 2% corresponded to the addition of flour.