Development of a multiplex PCR assay for the detection of metallo-beta-lactamase genes in Pseudomonas aeruginosa / Desarrollo de un PCR múltiple para la detección de genes metalo-beta-lactamasa en Pseudomonas aeruginosa

SUMMARY: The appearance of Pseudomonas aeruginosa strains with multi-resistance to antibiotics is a clinical problem of great relevance. The methods for detecting these resistances are laborious and slow, which is a complication when treating patients promptly. In this work, we developed a simple method for simultaneous detection of several carbapenem resistance genes using a multiplex PCR assay. The PCR assay developed, followed by electrophoretic separation of fragments, allows to simultaneously identify the presence of 6 antibiotic resistance genes: bla-VIM (261 bp), bla-IMP (587 bp), bla-SPM (648 bp), bla-GIM-1 (753 bp), bla-NDM-1 (813 bp) and bla-KPC (882 bp). We analyzed 7 clinical isolates of P. aeruginosa obtained in Chile, finding the resistance genes bla-VIM, bla-IMP, bla-SPM, bla-GIM, and bla-NDM in 5 of them. We found a perfect correlation between the detection of various resistance genes by PCR and the results obtained by antibiograms. Interestingly, 2 of the strains possessed 3 different resistance genes simultaneously. Finally, in this work, we found the presence of 3 genes never described before in clinical isolates of P. aeruginosa in Chile (bla-IMP, bla-SPM, and bla-GIM-1). We developed a rapid multiplex PCR test for the simultaneous detection of up to 6 antibiotic resistance genes of the metallo-β-lactamase family in P. aeruginosa.

La aparición de cepas de Pseudomonas aeruginosa con resistencias a diversos antibióticos es un problema clínico de gran relevancia. Los métodos de detección de dichas resistencias son laboriosos y lentos, lo que genera una complicación al momento de tratar a los pacientes oportunamente. En este trabajo desarrollamos un método simple de detección simultánea de varios genes de resistencia a carbapenem, mediante un sistema de PCR múltiple. El ensayo de PCR desarrollado, seguido de una separación electroforética de los amplicones, permite distinguir simultáneamente la presencia de 6 genes de resistencia a antibióticos: bla-VIM (261 pb), bla-IMP (587 pb), bla-SPM (648 pb), bla-GIM-1 (753 pb), bla-NDM-1 (813 pb) y bla-KPC (882 pb). Analizamos 7 aislados clínicos obtenidos en Chile, encontrando en 5 de ellos los genes de resistencia bla-VIM, bla-IMP, bla-SPM, bla-GIM y bla-NDM. Encontramos una perfecta correlación entre la detección de diversos genes de resistencia y los resultados obtenidos mediante antibiogramas. Interesantemente, 2 de las cepas mostraron poseer simultáneamente 3 genes de resistencia distintos. Por último, en este trabajo encontramos la presencia de 3 genes nunca antes descritos en aislados clínicos de P. aeruginosa en Chile (bla-IMP, bla-SPM y bla-GIM-1). Hemos desarrollado un test rápido de PCR múltiple, para la detección simultánea de hasta 6 genes de resistencia a antibióticos de la familia.a de las metallo-b-lactamases en P. aeruginosa.

Clinical distribution and drug resistance characteristics of carbapenem-resistant Klebsiella pneumoniae / 中华预防医学杂志

To explore the clinical distribution and drug resistance characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP), in order to provide reference for the prevention and treatment of CRKP infection. Retrospective analysis was performed on 510 clinical isolates of CRKP from January 2017 to December 2021, and strain identification and drug sensitivity tests were conducted by MALDI-TOF mass spectrometer and VITEK-2 Compact microbial drug sensitivity analyzer. The carbapenemase phenotype of CRKP strain was detected by carbapenemase inhibitor enhancement test. The CRKP strain was further categorized by immunochromogenic method and polymerase chain reaction (PCR) was used for gene detection. The results showed that 302 strains (59.2%) were derived from sputum, 127 strains (24.9%) from urine and 47 strains (9.2%) from blood. 231 (45.3%) were mainly distributed in intensive care, followed by 108 (21.2%) in respiratory medicine and 79 (15.5%) in neurosurgery. Drug susceptibility test result shows that the resistant rate of tigecycline increased from 1.0% in 2017 to 10.1% in 2021, the difference was statistically significant (χ2=14.444,P<0.05). The results of carbapenemase inhibitor enhancement test showed that 461 carbapenemase strains (90.4%) of 510 CRKP strains, including 450 serinase strains (88.2%), 9 metalloenzyme strains (1.8%), and 2 strains (0.4%) produced both serine and metalloenzyme. 49 strains (9.6%) did not produce enzymes. Further typing by immunochromogenic assay showed that 461 CRKP strains were KPC 450 (97.6%) and IMP 2 (0.4%). 7 NDM (1.5%); 2 strains of KPC+NDM (0.4%); PCR results were as follows: 450 strains of blaKPC (97.6%), 2 strains of blaIMP (0.4%), 7 strains of blaNDM (1.5%), and 2 strains of blaKPC+NDM (0.4%). In conclusion, CRKP strains mainly originated from sputum specimens and distributed in intensive care department, and the drug resistance characteristics were mainly KPC type in carbapenemase production. Clinical microbiology laboratory should strengthen the monitoring of CRKP strains, so as to provide reference for preventing CRKP infection and reducing the production of bacterial drug resistance.

Antibiotic susceptibility and fosfomycin resistance characterization in a cohort of children older than 6 years of age with urinary tract infection / Sensibilidad antibiótica y caracterización de la resistencia a fosfomicina en una cohorte de niños mayores de 6 años con infección urinaria

Fosfomycin tromethamol (FT) was reintroduced as an option for the treatment of low urinary tract infection (UTI) in children. In this study, we described the antibiotic sensitivity and mechanisms of resistance to fosfomycin in isolates from children older than 6 years with UTI. Urine culture and antibiotic susceptibility study were performed. In fosfomycin resistant strains, PCR for fos, blaCTX-M was performed followed by classification by phylogenetic group and sequencetyping. Escherichia coli was the most frequent etiological agent (89.2%). The susceptibility percentages were: fosfomycin 97.9%; amoxicillin-clavulanate 92.7%; cefuroxime and ceftriaxone 99%; nitrofurantoin 94.4%. An E. coli strain (ST69, phylogenetic group D) was resistant to fosfomycin (MIC 256mg/l) and carried the blaCTX-M-14 and fosA3 genes in a 45kb IncN-type plasmid.

La fosfomicina-trometamol (FT) se reintrodujo como una opción para el tratamiento de la infección del tracto urinario (ITU) baja en niños. En este estudio describimos la sensibilidad antibiótica y los mecanismos de resistencia a FT en aislamientos de niños mayores de 6 anos con ITU. Se realizaron urocultivos y estudios de sensibilidad antibiótica. En las cepas resistentes a fosfomicina se realizó la técnica de PCR para fos, blaCTX-M, y su identificación según su grupo filogenéticoy secuenciotipo. Escherichiacoli fue el agente etiológico más frecuente (89,2%). Los porcentajes de sensibilidad fueron: fosfomicina 97,9%; amoxicilina-clavulánico 92,7%; cefurox-ima y ceftriaxona 99%; nitrofurantoína 94,9%. Una cepa de E. coli (ST69, grupo filogenético D) fue resistente a fosfomicina (CIM 256mg/l) y portaba los genes blaCTX-M-14 y fosA3 en un plás-mido de 45 kb del tipo IncN. Este es el primer reporte de E. coli ST69 con blaCTX-M-14/fosA3 de origen humano.

Resistencia a colistina en aislados de Klebsiella pneumoniae resistente a carbapenémicos en un hospital pediátrico de Corrientes / Colistin resistance in carbapenem-resistant Klebsiella pneumoniae isolates from a pediatric hospital from Corrientes, Argentina

INTRODUCCIÓN: Existe un incremento de las infecciones por Klebsiella pneumoniae resistente a carbapenémicos (KPRC) en la población pediátrica y los datos epidemiológicos son limitados. OBJETIVOS: Conocer la frecuencia de KPRC en pacientes pediátricos, determinar la actividad in vitro de colistina y detectar el gen mcr-1 en dichos aislados. MATERIALES Y MÉTODOS: Se estudiaron 220 aislados de K. pneumoniae en un hospital pediátrico durante los años 2018 y 2019. La susceptibilidad antimicrobiana se determinó por microdilución en caldo según CLSI y EUCAST. Los genes blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48 y mcr-1 se analizaron mediante reacción de polimerasa en cadena (RPC). RESULTADOS: El 9,5% (n: 21) de los aislados fueron caracterizados como KPRC, donde se observó una resistencia a colistina de 47,6% (10/21) con valores de CIM50 de 2 μg/mL y CIM90 de > 4 μg/mL. En todos los aislados de KPRC se caracterizó el gen blaKPC y no se detectó el gen mcr-1. El perfil de resistencia observado en otros antimicrobianos fue el siguiente: gentamicina 100% (n: 21), ciprofloxacina 100% (n: 21), cotrimoxazol 100% (n: 21) y amikacina 19% (n: 4). Se observó 100% de sensibilidad a tigeciclina y ceftazidima/avibactam. CONCLUSIÓN: Este estudio demuestra un valor significativo de la resistencia a colistina en comparación a ceftazidima/avibactam y tigeciclina.

BACKGROUND: There is an increase of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in the pediatric population and epidemiological data are limited. Aim: To calculate the frequency of CRKP in pediatric patients, to determine the in vitro activity of colistin and to detect the presence of mcr-1 gene in said isolates. METHODS: 220 isolates of K. pneumoniae were studied in a pediatric hospital between January 2018 and December 2019. Antimicrobial susceptibility was determined by microdilution in broth according to guidelines of CLSI and EUCAST. The genes blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48 and mcr-1 were detected by polymerase chain reaction (PCR). RESULTS: 9.5% (n: 21) of the isolates were characterized as CRKP, where was observed a resistance to colistin of 47.6% (10/21) with values of MIC50 of 2 μg/mL and MIC90 of ≥ 4 μg/mL. In 100% of CRKP strains the blaKPC gene was detected and the mcr-1 gene was not found. The resistance profile to other antimicrobials was as follow: gentamicin 100% (n: 21), trimethoprim/sulfamethoxazole 100% (n: 21), ciprofloxacin 100% (n: 21), amikacin 19% (n: 4). All of the isolates were sensitive to ceftazidime/avibactam and tigecycline. CONCLUSION: This study demonstrates a significant value of resistance to colistin in pediatric patients compared to other last line antimicrobial such as ceftazidime/avibactam and tigecycline.

Tipos de carbapenemasas expresadas en Klebsiella spp, y Pseudomonas aeruginosa resistente a carbapenémicos en seis hospitales de alta complejidad de la Ciudad de Bogotá - Colombia / Carbapenemases produced in Klebsiella spp, and Pseudomonas aeruginosa in six hospitals in Bogotá - Colombia

INTRODUCCIÓN: En las últimas décadas, se ha incrementado la prevalencia de infecciones por bacilos gramnegativos resistentes a carbapenémicos. OBJETIVO: Determinar los tipos y la frecuencia de las distintas carbapenemasas en aislados de Klebsiella spp. y Pseudomonas aeruginosa, en seis hospitales de alta complejidad de Bogotá-Colombia. MÉTODOS: Estudio observacional descriptivo en seis hospitales de la ciudad de Bogotá, en el período de enero de 2017 a agosto de 2018. Se realizaron RPC para genes de KPC, GES, VIM, NDM, IMP y OXA-48 en cepas de Klebsiella spp y P aeruginosa resistentes a carbapenémicos. RESULTADOS: 52 aislados de P aeruginosa amplificaron para una carbapenemasa, de los cuales 39 (75%) fueron positivos para KPC, 11 (21%) para VIM y 2 co-producciones de KPC y VIM. En cuanto a Klebsiella spp., 165 cepas amplificaron al menos para una carbapenemasa, 98% expresaron KPC y 4 aislados tuvieron co-producciones de metalo-beta-lactamasas y KPC. DISCUSIÓN: Este estudio aporta información valiosa, como el incremento de producción de KPC en P. aeruginosa y la co-producción de KPC y metalo-beta-lactamasas, locual tiene una implicancia tanto en la selección del tratamiento, las medidas de aislamiento de contacto y el pronóstico de los pacientes.

BACKGROUND: In the last decades, the prevalence of infections by carbapenem resistant gram-negative bacilli has been increased. OBJECTIVE: To determine types and frequency of the different carbapenemases in Klebsiella spp. and Pseudomonas aeruginosa, in six hospitals in Bogotá-Colombia. METHODS: Descriptive and observational study, in six hospitals in the city of Bogotá, in the period ftom January 2017 to August 2018. PCR were performed for KPC, GES, VIM, NDM, IMP and OXA-48 genes, in carbapenem resistant Klebsiella spp. and P aeruginosa. RESULTS: 52 P aeruginosa isolates amplified a carbapenemase gene, of which 39 (75%) were positive for KPC, 11 (21%) for VIM and two co-productions of KPC and VIM. Regarding Klebsiella spp. 165 strains amplified at least one carbapenemase gene, 98% expressed KPC and four isolates had co-productions of metallo-P-lactamases and KPC. DISCUSSION: This study provides valuable information, such as the increased production of KPC in P. aeruginosa información valiosa, como el incremento de producción de KPC en P. aeruginosa and the co-production of KPC plus metallobetalactamases, which has an implication both in treatment selection, isolation precautions and patient prognosisy.

Restricción programada de cefalosporinas de tercera generación en contexto de un brote de bacilos gramnegativos productores de beta-lactamasas tipo AmpC en unidades críticas: una experiencia de la vida real / Third-generation cephalosporins programmed restriction in the context of an outbreak of AmpC beta-lactamase-producing gram-negative bacilli in critical units: a real-life experience

INTRODUCCIÓN: La restricción programada (RP) de antimicrobianos puede disminuir selectivamente la tasa de infecciones por determinados microorganismos. En este sentido, los bacilos gramnegativos productores de beta-lactamasas AmpC (BGN-blaAmpC) son seleccionados por el sobreuso de cefalosporinas de tercera generación (C3G). Estas bacterias, también adquieren genes y co-producen otras beta-lactamasas, como las de Nueva Delhi (BGN-blaNDM). OBJETIVOS: Disminuir la tasa de aislamiento de BGN-blaAmpC y BGN-blaNDM en cultivos de pacientes de la UCI luego de una RP de C3G en el marco de un brote nosocomial por estos microrganismos. MATERIALES Y MÉTODOS: Estudio cuasi-experimental, previo (P1= 12 meses) y posterior (P2= 12 meses) a una RP de C3G en un hospital de adultos, donde, en el contexto de brote mencionado, se aplicaron medidas de control de infecciones generales. El uso de antimicrobianos se expresó como "porcentaje de los días de tratamiento (%DDT)"/100 camas ocupadas al día (100-COD). Se compararon las tasas de aislamiento de BGN-blaAmpC y BGN-blaNDM en hemocultivos (HC), mini-lavados bronquio-alveolares (mB) y urocultivos (UC) en la UCI. RESULTADOS: En P2 el consumo de C3G fue 2,5% DDT/100-COD. Hubo un descenso en los aislamientos de BGN-blaAmpC en HC (RR 0,48 [0,2-0,9] p < 0,02) y mB (RR 0,52 [0,3-0,9] p < 0,02), así como también de BGN-blaNDM en HC (RR 8,1 [1,6-39,4] p < 0,00). Conclusiones: La RP de C3G se asoció con la reducción de los BGN-blaAmpC y BGN-blaNDM en HC, así como de los BGN-blaAmpC mB.

BACKGROUND: Programmed restriction (PR) of antimicrobials can selectively decrease the rate of infections by certain microorganisms. In this sense, AmpC beta-lactamase-producing gram-negative bacilli (GNB-blaAmpC) are selected for the overuse of third generation cephalosporins (3GC). These bacteria also acquire genes and co-produce other β-lactamases, such as New Delhi ones (GNB-blaNDM). AIM: To decrease the isolation rate of GNB- blaAmpC and GNB- blaNDM in cultures from ICU patients after a PR of 3GC. METHODS: Quasi-experimental study, before (P1= 12 months) and after (P2= 12 months) a PR of 3GC in an adults' hospital. The use of antibiotics was expressed as "percentage days of treatment (%DOT)" /100 beds occupied per day (100-BOD). The rates of GNB-blaAmpC and GNB-blaNDM were compared in blood cultures (BC), mini-bronchio alveolar lavages (mB) and urine cultures (UC) in the ICU. RESULTS: In P2, 3GC consumption was 2.5% DOT/100-COD. There was a decrease in GNB-blaAmpC from BC (RR 0.48 [0.2-0.9] p < 0.02) and mB (RR 0.52 [0.3-0.9] p < 0.02), as well as of GNB-blaNDM from BC (RR 8.1 [1.6-39.4] p < 0.00). Conclusions: PR of 3GC was linked to the reduction of GNB-blaAmpC and GNB-blaNDM in BC, as well as GNB-blaAmpC in mB from ICU patients.

Caracterización de bacilos gramnegativos multi-resistentes, aislados en pacientes hospitalizados en instituciones de salud de Barranquilla (Colombia) / Characterization of multiresistant gram-negative bacilli, isolated in patients hospitalized in health institutions in Barranquilla (Colombia)

Resumen Introducción: La resistencia a carbapenémicos en bacilos gramnegativos es un problema de salud pública mundial, debido a que se asocia con altas tasas de mortalidad, aumento en los niveles de resistencia a otros antimicrobianos, elevación en el potencial de diseminación e incremento en los costos de atención en salud. Objetivo: Caracterizar bacilos gramnegativos multirresistentes, aislados en pacientes hospitalizados en instituciones de salud de Barranquilla (Colombia). Material y Métodos: Estudio descriptivo acerca de la caracterización fenotípica y genotípica de la resistencia bacteriana en las infecciones asociadas a la atención en salud, mediada por carbapenemasas en aislados bacterianos enviados por los laboratorios pertenecientes a la red de laboratorios del Departamento del Atlántico. Resultados: La KPC fue la carbapenemasa más frecuente en las Enterobacterales (27,6%), predominando en Klebsiella pneumoniae (13,1%) sola y asociada a otras carbapenemasas. En Pseudomonas aeruginosa predominó la carbapenemasa VIM (32,8%) y la OXA en Acinetobacter baumannii (17,1%). Conclusión: Se encontró una amplia distribución de cepas multi-resistentes productoras de carbapenemasas en instituciones de salud de Barranquilla, las cuales expresaron los siguientes mecanismos de resistencia: KPC, VIM, NDM, OXA.

Abstract Background: The emergence of carbapenem resistant gramnegative bacilli has become a problem of public health worldwide, because it is associated with high mortality rates, increased levels of resistance to other antimicrobials, increased potential for dissemination transition and increase in health care costs. Aim: To characterize multiresistant gram-negative bacilli, isolated in patients hospitalized in health institutions of Barranquilla (Colombia). Methods: A descriptive study was conducted on the phenotypic and genotypic characterization of bacterial resistance in infections associated with health care, mediated by carbapenemases in bacterial isolates sent by laboratories belonging to the laboratory network of the Department of Atlántico. Results: KPC was the most frequent carbapenemase in Enterobacterales (27.6%), predominantly in Klebsiella pneumoniae (13.1%) alone and associated with other carbapenemases. In Pseudomonas aeruginosa, VIM carbapenemase (32.8%) predominated and OXA in Acinetobacter baumannii (17.1%). Conclusion: A wide distribution of multi-resistant strains producing carbapenemases in Atlantic health institutions was found, which expressed the following resistance mechanisms: KPC, VIM, NDM, OXA.

Detección fenotípica y genotípica de la producción de carbapenemasas tipo NDM-1 y KPC en enterobacterias aisladas en un laboratorio clínico en Maracay, Venezuela / Phenotypic and genotypic detection of the production of carbapenemases type NDM-1 and KPC in isolated Enterobacteriaceae in a clinical laboratory in Maracay, Venezuela

INTRODUCCIÓN: La producción de beta-lactamasas capaces de hidrolizar a los carbapenémicos es uno de los mecanismos de resistencia más preocupantes porque eliminan la última opción terapéutica frente a los microorganismos multi-resistentes. OBJETIVO: Determinar la producción de carbapenemasas tipo KPC y NDM-1, empleando métodos fenotípicos y genotípicos, en enterobacterias aisladas en un laboratorio clínico de la ciudad de Maracay, Venezuela. MÉTODOS: Se determinó la producción de carbapenemasas mediante métodos fenotípicos (según algoritmo de Malbrán) y genotípicos (amplificación de los genes blaNDM-1 y blaKPC por RPC) en enterobacterias aisladas en un laboratorio clínico durante el período marzo-agosto 2018. RESULTADOS: Se identificaron 605 enterobacterias de diferentes especies, siendo Escherichia coli la cepa con mayor porcentaje de aislamiento (61,3%), seguida por Klebsiella pneumoniae (14,9%). Diez y seis enterobacterias (2,64%) fueron positivas para la producción de carbapenemasas: 13 cepas de K. pneumoniae y tres del complejo Enterobacter cloacae. La RPC demostró que 14 cepas (87,5%) contienen el gen blaNDM-1 y dos (12,5%) el gen blaKPC; se observó 100% de concordancia entre la determinación fenotípica y la RPC para ambos grupos de enzimas. CONCLUSIONES: Los resultados mostraron mayor incidencia de la metalo-beta-lactamasa tipo NDM-1, reconocida como una alarma epidemiológica debido a que su rápida diseminación dificulta su control, por lo que la identificación del tipo de enzima permitiría establecer estrategias de manejo y control más certeras con la finalidad de erradicar a dichos patógenos.

BACKGROUND: The production of carbapenem-hydrolyzing beta-lactamases is one of the most concerning resistance mechanisms since it eliminates the last therapeutic option against multidrug resistant microorganisms. AIM: To determine the production of KPC and NDM-1 type carbapenemases, using phenotypic and genotypic methods, in isolated enterobacteria in a clinical laboratory in the city of Maracay, Venezuela. METHODS: The production of carbapenemases was determined by phenotypic (according to the Malbrán algorithm) and genotypic methods (amplification of the blaNDM-1 and blaKPC genes by PCR) in clinical isolates of Enterobacteriaceae during the period March-August 2018. RESULTS: 605 Enterobacteriaceae of different species were identified, being Escherichia coli the strain with the highest percentage of isolation (61.3%), followed by Klebsiella pneumoniae (14.9%). Sixteen strains (2.64%) were positive for carbapenemases production: 13 strains of K. pneumoniae and three of the Enterobacter cloacae complex. PCR showed that 14 strains (87.5%) carry the blaNDM-1 gene and two strains (12.5%) the blaKPC gene; 100% agreement was observed between phenotypic determination and PCR for both groups of enzymes. CONCLUSIONS: The results of this study showed a higher incidence of metallo-beta-lactamase type NDM-1, which rapid dissemination and consequently difficult control has been cause of epidemiological alert. The identification of the type of enzyme would allow establishing more accurate management and control strategies in order to eradicate these pathogens.

Resistencia enzimática en Pseudomonas aeruginosa, aspectos clínicos y de laboratorio / Enzymatic resistance in Pseudomonas aeruginosa, clinical and laboratory aspects

Resumen Pseudomonas aeruginosa es uno de los principales patógenos que causa infecciones asociadas a la atención en salud (IAAS). Su capacidad de adaptación, diseminación, resistencia intrínseca a los antimicrobianos y de adquirir nuevos mecanismos a través de elementos genéticos móviles, hacen que el tratamiento de las infecciones por este microorganismo sea un desafío para el médico clínico. Intrínsecamente, P. aeruginosa, presenta una reducida permeabilidad en la membrana externa, debido a la expresión de bombas de expulsión, y una cefalosporinasa tipo AmpC inducible. Además, P. aeruginosa es capaz de adquirir nuevos determinantes de resistencia por transferencia horizontal en forma de casetes situados en integrones, y a su vez, localizados en transposones o plásmidos. Dentro de la resistencia enzimática que presenta P. aeruginosa destacan las β-lactamasas, incluyendo aquellas de espectro extendido (BLEE) y las carbapenemasas. Pero también enzimas modificadoras de los aminoglucósidos, haciendo que este microorganismo pueda presentar fenotipos de multi-resistencia (MDR), resistencia extrema (XDR) y panresistencia (PDR) a los antimicrobianos denominados antipseudomonas, incluyendo a las nuevas cefalosporinas con inhibidores de beta-lactamasas.

Abstract Pseudomonas aeruginosa is one of the major pathogens causing healthcare-associated infections (HAI). Its capacity of adaptation, dissemination, intrinsic resistance to antimicrobials and of acquiring new mechanisms through mobile genetic elements, make the treatment of infections by this microorganism a challenge for the clinician. Intrinsically, P. aeruginosa, presents a reduced permeability in the external membrane, due to the expression of efflux pumps, and an inducible AmpC-type cephalosporinase. In addition, P. aeruginosa is able to acquire new resistance determinants by horizontal transfer in the form of cassettes located in integrons, and in turn located in transposons or plasmids. Within the enzymatic resistance that P. aeruginosa presents, betalactamases, including extended spectrum (ESBL) and carbapenemases. But also aminoglycoside modifying enzymes, stand out, causing this microorganism to present multi-resistance phenotypes (MDR), extreme resistance (XDR) and pan-resistance (PDR) to the called antipseudomonal antibiotics, including the new cephalosporins with betalactamase inhibitors.

Carbapenemasas en aislamientos de Pseudomonas aeruginosa resistentes a carbapenémicos aisladas en hospitales de Chile

Resumen Introducción: La resistencia a carbapenémicos mediada por carbapenemasas en Pseudomonas aeruginosa es un mecanismo importante; sin embargo, la pérdida de la porina OprD continúa siendo el mecanismo más frecuente. Objetivo: Determinar la proporción de aislados de P. aeruginosa, resistentes a imipenem y/o meropenem, productores de carbapenemasas, el tipo de enzima producida y la relación genética entre los aislados. Material y Métodos: Se incluyó 113 aislados resistentes al menos a un carbapenémico, provenientes de 12 hospitales de 9 ciudades de Chile. Adicionalmente se determinó la susceptibilidad a ceftazidima, amikacina, gentamicina, piperacilina/tazobactam, ciprofloxacina y colistina. Se realizó Carba NP y en los aislados positivos (n: 61) se detectó genes de carbapenemasas por RPC. Los aislados fueron tipificados por restricción con SpeI y PFGE. Resultados: No todos los aislados presentan carbapenemasas, y sólo en 61/113 de ellos (54%) se amplificó blaKPC (32) o blaVIM (29). En ninguno de los aislados se encontró co-portación de ambos genes. Los pulsotipos indican que no hay diseminación clonal de los aislados, evidenciando una importante diversidad genética. Conclusiones: Los aislados de P. aeruginosa productores de carbapenemasas, obtenidos en hospitales de Chile, portan genes blaKPC y blaVIM y, en su mayoría, son policlonales. Estos resultados ponen énfasis en la importancia de realizar estudios epidemiológicos con mayor número de aislados que permitan conocer mejor la epidemiología de P. aeruginosa productoras de carbapenemasas en Chile.

Abstract Background: Carbapenem resistance mediated by carbapenemases in Pseudomonas aeruginosa is an important mechanism; however, loss of porin OprD remains as the most frequent. Aim: To determine the proportion of P. aeruginosa isolates, resistant to imipenem and/or meropenem, producing carbapenemases, the type of enzyme produced and the genetic relationship between the isolates. Methods: One hundred and thirteen resistant to at least one carbapenem isolates, obtained in 12 hospitals and 9 cities in Chile were studied. Additionally, susceptibility to ceftazidime, amikacin, gentamicin, piperacillin/tazobactam, ciprofloxacin and colistin was determined. Carba NP was performed and in the positive isolates carbapenemase genes were detected by PCR. The isolates were typified by restriction with SpeI and PFGE. Results: Not all isolates produce carbapenemases, and only in 61/113 of them (54%) the blaKPC (32) or blaVIM (29) was amplified. In none of the isolates was found the coharboring of both genes. The pulsotypes indicated no clonal dissemination of the isolates, evidencing an important genetic diversity. Conclusions: P. aeruginosa isolates producing carbapenemases, obtained in Chilean hospitals carry blaKPC and blaVIM genes and, mostly, are polyclonal. These results emphasize the importance of carrying out epidemiological studies with a greater number of isolates to allow a better understanding of the epidemiology of carbapenemase-producing P. aeruginosa in Chile.

Alternative Splicing Analysis of LACTB Gene and Expression Characteristics of Different Transcripts in Leukemia Cell Lines / 中国实验血液学杂志

OBJECTIVE@#To detect the expression of different transcripts of lactamase β（LACTB) gene in leukemic cell lines.@*METHODS@#NCBI website and DNAstar software were used to detect the Bioinformatics analysis of LACTB. The expression of different transcripts of LACTB gene in leukemic cell lines (THP-1, HL60, K562, U937, Jurkat and Raji) was detected by reverse transcription PCR (RT-PCR), DNA and clone sequencing; the expression of different transcripts of LACTB gene in leukemic cell lines was detected by Quantitative Real-time PCR.@*RESULTS@#There were a variety of splicing isomers in LACTB, and it could produce a variety of protein isomers with conserved N-terminal and different C-terminal, moreover, there were many splice isoforms of LACTB in leukemia cell lines, and there were different expression patterns in different cell lines, including XR1, V1, V2 and V3. The expression of total LACTB showed high in HL60 cells, while low in Raji cells, and the difference was statistically significant (P<0.05). The V1 was high expression in U937 cells but low in Raji cells, and the difference was statistically significant (P<0.05). V2 was high expression in HL60 cells but lowly in Raji cells, and the difference was statistically significant (P<0.05). The expression of V3 was low in THP-1 cells, which was significantly different as compared with that in normal bone marrow (P<0.05).@*CONCLUSION@#The reaserch found that there are many splice isomers of LACTB in leukemic cell lines, and there are different expression patterns in different cell lines.

Molecular epidemiology and drug resistance of Acinetobacter baumannii isolated from a regional hospital in the Brazilian Amazon region

Abstract INTRODUCTION: In this study, we report a clonal dissemination of carbapenem resistant Acinetobacter baumannii isolates due to the acquisition of blaOXA-23 in a regional hospital located in Brazilian Amazon Region. METHODS: The isolates were identified by MALDI-TOF and the carbapenemase-encoding genes were detected by multiplex-PCR. The genetic similarity was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: Only 10 (55.6%) isolates harbored the gene bla OXA-23. PFGE analysis revealed that these isolates belong to a single clone. CONCLUSIONS: This dissemination strategy indicates the need for surveillance, adoption of control procedures defined in guidelines, and the careful administration of antimicrobials should be reinforced.

First report of the aac(6)-Ib-cr gene in Providencia stuartii isolates in Brazil

Abstract INTRODUCTION: The aac(6')-Ib-cr and bla KPC genes are spreading among Enterobacteriaceae species, including Providencia stuartii, in some countries of world. METHODS: These genes were investigated in 28 P. stuartii isolates from a public hospital in Recife, Pernambuco, Brazil, by PCR and sequencing. RESULTS: The aac(6')-Ib-cr gene was detected in 16 resistant isolates, and the bla KPC gene was seen in 14. CONCLUSIONS: The presence of these genes in P. stuartii multi- and extensively drug-resistant isolates indicates that the resistance arsenal of this species is increasing, thus limiting the therapeutic options.

First report of a blaNDM-resistant gene in a Klebsiella aerogenes clinical isolate from Brazil

Abstract INTRODUCTION: Carbapenemase-resistant enterobacteria that produce the bla NDM gene are found worldwide. However, this is the first report of blaNDM in Klebsiella aerogenes in Brazil. METHODS: The identification of bacterial species was performed using anautomated system and confirmed by biochemical tests, 16S rRNA gene sequencing, and detection of resistance genes. RESULTS: The clinical isolate showed minimum inhibitory concentration resistance to meropenem and polymyxin B at 8mg/L and 4mg/L, respectively. Only the blaNDM gene was detected. CONCLUSIONS: The current report of the blaNDM gene in isolated MDR enterobacteria indicates that this gene can spread silently in a hospital setting.

First report of bla GES-1 in Proteus mirabilis clinical isolates

Abstract Proteus mirabilis is one of the main pathogens causing urinary tract infections and sepsis. To our knowledge, this is the first report of a P. mirabilis hosting bla GES. The presence of these genes was determined using PCR and sequencing. We identified the presence of bla GES-1 in all three isolates. In addition, we identified the bla KPC-2 and bla NDM-1 genes in the two strains. These data emphasize the importance of monitoring and surveillance of all enterobacteria. The circulation of P. mirabilis strains carrying bla GES-1 constitutes a new scenario of resistance in this species and should be an epidemiological alert for global health.

Full characterization of an IncR plasmid harboring qnrSI recovered from a VIM-11-producing Pseudomonas aeruginosa

Abstract Metallo-p-lactamases (MBL) producing Pseudomonas aeruginosa isolates have been well characterized. Quinolones are commonly used in the treatment of carbapenem-resistant P. aeruginosa infections; however, data about PMQR in this species are scarce. The objective of this study was to report the simultaneous presence of qnrS and blaV-M-n in P. aeruginosa, and to characterize the qnrS-harboring plasmid. Thirty-eight carbapenem-resistant P. aeruginosa isolates were recovered from a hospital in Buenos Aires during 2012. Screening forMBL was assessed by the double disk synergy test using EDTA and carbapenem discs. Plasmid DNA extraction was performed by a method using phenol-chloroform. PCR followed by sequencing was carried out to determine each MBL and PMQR allele. PCR-BseGI-RFLP was performed to detect aac-(6')-Ib-cr. The gyrA-QRDR was sequenced in those PMQR-harboring isolates. Plasmid incompatibility groups and addiction systems were characterized by PCR. The PMQR-carrying plasmid was sequenced using Illumina technology, annotated using RAST and manually curated. Eleven/38 isolates were VIM producers (blaVIM-2 and blaVIM-11) while 1/38 harbored blaIMP-13. One isolate harbored blaVIM-11 and the PMQR qnrSI; however, both markers were located in different plasmids. The qnrSí-harboring plasmid (pP6qnrS1) was 117 945 bp in size, presented 154 CDS and corresponded to the IncR group. In addition to qnrSI, it harbored several aminoglycoside resis-tance markers. Although pP6qnrS1 was non-conjugative, it presented an oriT which made it possible for this plasmid to be transferable. This is the first report on P. aeruginosa carrying both blaVIM-11 and qnrSI, plus the first detection of an IncR plasmid in Argentina.

Resumen Las quinolonas son comúnmente utilizadas en el tratamiento de las infecciones producidas por Pseudomonas aeruginosa resistentes a carbapenems (PARC); aun así, la información sobre la resistencia a quinolonas mediada por plásmidos (PMQR) en esta especie es escasa. El objetivo de este trabajo fue reportar la presencia simultánea de los genes qnrS y blaVIM-11 en PARC y caracterizar el plásmido portador de qnrS. Durante 2012 se recuperaron 38 PARC en un hospital de Buenos Aires. El tamizaje para detectar producción de metalo-beta-lactamasas (MBL) se llevó a cabo mediante sinergia de doble disco utilizando EDTA y carbapenems. El ADN plasmídico fue extraído utilizando fenolcloroformo. Para determinar los alelos de los genes implicados en la síntesis de MBL y de PMQR, se llevó a cabo PCR-secuenciación. Para la detección de aac-(6')-Ib-cr se realizó PCR-BseGI-RFLP. En aquellos aislamientos portadores de PMQR se secuenció el gen gyrA. Los grupos de incompatibilidad y sistemas de adicción fueron caracterizados por PCR. El plásmido portador de PMQR fue secuenciado completamente y curado manualmente. De 38 aislamientos, 11 fueron productores de VIM (blaVIM-2 y blaVIM-11), mientras que uno contenía blaIMP-13. Si bien un aislamiento fue portador de blaVIM-11 y de qnrSI, dichos marcadores se encontraban en distintos plásmidos. El plásmido portador de qnrSI (pP6qnrS1) presentó un tamaño de 117.945 pby 154 secuencias codificantes (CDS); este correspondió al grupo de incompatibilidad IncR. Además de qnrSI, el plásmido portaba diversos marcadores de resistencia a aminoglucósidos. Aun cuando pP6qnrS1 no resultó conjugativo, presentó un oriT, de modo que posiblemente sea transferible. Este es el primer informe acerca de PARC portadora de blaVIM-11 y de qnrSI en simultáneo, además, es la primera descripción de un plásmido IncR en Argentina.

First characterization of K. pneumoniae ST11 clinical isolates harboring blaKPC-3 in Latin America / Primera caracterización de aislamientos clínicos de K. pneumoniae ST11 portadoresde blaKPC-3en América Latina

Abstract Antimicrobial resistance due to carbapenemase production in Enterobacteriaceaeclinical isolates is a global threat. Klebsiella pneumoniae harboring the blaKPCgene is one ofthe major concerns in hospital settings in Latin America.The aim of this study was to characterize the antibiotic resistance mechanisms and to typifyfour carbapenem-resistant K. pneumoniae clinical isolates from the city of Manizales, Colombia.We identified blaKPC-3in all four isolates by polymerase chain reaction and subsequentsequencing. The plasmid-mediated quinolone resistance genes qnrB19-like and aac(6)Ib-cr;fosfomycin resistance gene fosA and an insertion sequence IS5-like in mgrB (colistin resistance)were also detected. Sequence types ST11 with capsular type wzi75, and ST258 with wzi154,were characterized. The blaKPC-3gene was mobilized in a 100-kb IncFIB conjugative plasmidwith vagCD toxin-antitoxin system.This work reports multiple resistance genes in blaKPC-producing K. pneumoniae and the firstoccurrence of ST11 clinical isolates harboring blaKPC-3in Latin America.

Resumen La resistencia a antibióticos mediada por la producción de carbapenemasas en aislamientos clínicos de Enterobacteriaceae es una amenaza mundial. Klebsiella pneumoniae portador de blaKPC es uno de los mayores problemas a nivel hospitalario en Latinoamérica. El objetivo de este estudio fue caracterizar los mecanismos de resistencia antibiótica y tipificar cuatro aislamientos clínicos de K. pneumoniae resistentes a carbapenems obtenidos en la ciudad de Manizales, Colombia. Se identificó blaKPC-3 en todos los aislamientos mediante reacción en cadena de polimerasa y secuenciación. También se detectaron los genes de resistencia transferible a quinolonas qnrB19-like y aac(6')Ib-cr y a fosfomicina fosA, y la secuencia de inserción /S5-like en mgrB (asociada a la resistencia a colistina). Se caracterizaron los secuenciotipos ST11 (cápsula wzi75) y ST258 (cápsula wzi154). Se comprobó que blaKPC-3 fue movilizado por un plásmido conjugativo IncFIB-vagCD de 100kb. En este trabajo se reportan múltiples genes de resistencia en K. pneumoniae productor de blaKPC y se describen por primera vez aislamientos clínicos ST11 productores de blaKPC-3 en Latinoamérica.

Presencia de genes fimH y afa en aislamientos urinarios de Escherichia coli productora de betalactamasas de espectro extendido en Lima, Perú / Presence of fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases producing Escherichia coli in Lima, Peru

RESUMEN Con el objetivo de determinar la presencia de los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de betalactamasas de espectro extendido (BLEE), se realizó un estudio descriptivo, con aislamientos del cepario del proyecto TO-06/09 del Instituto Nacional de Salud del Niño en Lima, Perú. Se incluyeron 75 aislamientos urinarios de Escherichia coli. La identificación de genes se realizó por reacción en cadena de la polimerasa. De los 75 aislamientos, 74 (98,7%) fueron positivos para el gen fimH y 6 (8,0%) fueron positivos para el gen afa. Se evidenció la presencia de los factores de virulencia producidos por los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de BLEE.

ABSTRACT Descriptive study conducted in order to determine the presence of the fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli. Isolates from project TO-06/09 of the Instituto Nacional de Salud del Niño in Lima, Peru were used. A total of 75 urinary isolates of Escherichia coli were included. Gene identification was performed by polymerase chain reaction. From the 75 isolates, 74 (98.7%) were positive for the fimH gene and 6 (8.0%) were positive for the afa gene. Virulence factors produced by the fimH and afa genes were evident in urinary isolates of ESBL producing Escherichia coli.

Mobile genetic elements associated with carbapenemase genes in South American Enterobacterales

ABSTRACT Introduction: Carbapenem resistance in members of order Enterobacterales is a growing public health problem causing high mortality in developing and industrialized countries. Its emergence and rapid propagation worldwide was due to both intercontinental spread of pandemic strains and horizontal dissemination via mobile genetic elements (MGE) such as plasmids and transposons. Objective: To describe MGE carrying carbapenem resistance genes in Enterobacterales which have been reported in South America. Search strategy and selection criteria: A search of the literature in English or Spanish published until 2019 in PubMed, Google Scholar, LILACS and SciELO databases was performed for studies of MGE in Enterobacterales reported in South American countries. Results: Seven South American countries reported MGE related to carbapenemases. Carbapenemase-producing Klebsiella pneumoniae belonging to clonal complex 258 were the most prevalent pathogens reported; others carbapenemase-producing Enterobacterales such as Escherichia coli, Serratia marcescens, and Providencia rettgeri also have been reported. The MGE implicated in the spread of the most prevalent carbapenemase genes are Tn4401 and non-Tn4401 elements for bla KPC and ISAba125 for bla NDM, located in different plasmid incompatibility groups, i.e. L/M, A/C, FII and bacterial clones. Conclusion: This review indicates that, like in other parts of the world, the most commonly reported carbapenemases in Enterobacterales from South America are being disseminated through clones, plasmids, and transposons which have been previously reported in other parts of the world.

Investigation of carbapenemases and aminoglycoside modifying enzymes of Acinetobacter baumannii isolates recovered from patients admitted to intensive care units in a tertiary-care hospital in Brazil

Abstract INTRODUCTION: Acinetobacter baumannii are opportunistic bacteria, highly capable of acquiring antimicrobial resistance through the production of carbapenemases and aminoglycoside modifying enzymes (AMEs). METHODS: Carbapenemase and AME genes were investigated in A. baumannii recovered from inpatients of a Brazilian hospital. RESULTS: The key genes found were bla OXA-51-like, the association ISAba1- bla OXA-23-like, and the AME genes aph(3´)-VI, aac(6´)-Ib, aac(3)-Ia, and aph(3´)-Ia. Different clusters spread through the institution wards. CONCLUSIONS: The dissemination of bla OXA-23-like and AME-carrying A. baumannii through the hospital highlights the need for improved preventive measures to reduce the spread of infection.