Caracterización molecular de carbapenemasas en el Perú durante el 2019 / Molecular characterization of carbapenemases in Peru during 2019

RESUMEN La resistencia a los carbapenémicos es un problema de salud pública. Este estudio presenta la identificación de enzimas carbapenemasas en Enterobacteriaceae, Pseudomonas spp. y Acinetobacter spp. presentes en cepas de 30 instituciones prestadoras de servicios de salud del Perú como parte del proceso de control de calidad en diagnósticos. La confirmación fenotípica e identificación enzimática se realizó utilizando la prueba de Blue CARBA y la prueba de sinergia con discos de ácido fenilborónico y ácido etilendiaminotetraacético/ácido mercaptoacético de sodio. Se identificaron 185 cepas con carbapenemasas: 78 en Enterobacteriaceae, 61 en P. aeruginosa y 46 en Acinetobacter spp. Los tipos de carbapenemasas identificadas fueron: blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-23, blaOXA-24, blaOXA-51 y la coproducción de blaVIM/IMP. Es importante reforzar la promoción del uso racional de antimicrobianos y la vigilancia epidemiológica en los nosocomios del país.

ABSTRACT Resistance to carbapenems is a public health problem. This study presents the identification of carbapenemase enzymes in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. present in strains from 30 institutions that provide health services in Peru as part of the quality control process in diagnoses. Phenotypic confirmation and enzymatic identification were performed using the Blue CARBA test and the synergy test with phenylboronic acid and ethylenediaminetetraacetic acid/sodium mercaptoacetic acid discs. 185 strains with carbapenemases were identified: 78 in Enterobacteriaceae, 61 in P. aeruginosa and 46 in Acinetobacter spp. The types of carbapenemases identified were: blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-23, blaOXA-24, blaOXA-51 and the blaVIM/IMP co-production. It is important to strengthen the promotion of the rational use of antimicrobials and epidemiological surveillance in the country's hospitals.

Caracterización molecular de carbapenemasas en el Perú durante el 2019 / Molecular characterization of carbapenemases in Peru during 2019

RESUMEN La resistencia a los carbapenémicos es un problema de salud pública. Este estudio presenta la identificación de enzimas carbapenemasas en Enterobacteriaceae, Pseudomonas spp. y Acinetobacter spp. presentes en cepas de 30 instituciones prestadoras de servicios de salud del Perú como parte del proceso de control de calidad en diagnósticos. La confirmación fenotípica e identificación enzimática se realizó utilizando la prueba de Blue CARBA y la prueba de sinergia con discos de ácido fenilborónico y ácido etilendiaminotetraacético/ácido mercaptoacético de sodio. Se identificaron 185 cepas con carbapenemasas: 78 en Enterobacteriaceae, 61 en P. aeruginosa y 46 en Acinetobacter spp. Los tipos de carbapenemasas identificadas fueron: blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-23, blaOXA-24, blaOXA-51 y la coproducción de blaVIM/IMP. Es importante reforzar la promoción del uso racional de antimicrobianos y la vigilancia epidemiológica en los nosocomios del país.

ABSTRACT Resistance to carbapenems is a public health problem. This study presents the identification of carbapenemase enzymes in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. present in strains from 30 institutions that provide health services in Peru as part of the quality control process in diagnoses. Phenotypic confirmation and enzymatic identification were performed using the Blue CARBA test and the synergy test with phenylboronic acid and ethylenediaminetetraacetic acid/sodium mercaptoacetic acid discs. 185 strains with carbapenemases were identified: 78 in Enterobacteriaceae, 61 in P. aeruginosa and 46 in Acinetobacter spp. The types of carbapenemases identified were: blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-23, blaOXA-24, blaOXA-51 and the blaVIM/IMP co-production. It is important to strengthen the promotion of the rational use of antimicrobials and epidemiological surveillance in the country's hospitals.

Contaminación ambiental por microorganismos multirresistentes y el efecto de la limpieza y desinfección en una unidad de cuidados intensivos / Environmental contamination by multi-resistant microorganisms and the effect of cleaning and disinfection in an intensive care unit / Contaminação ambiental por micro-organismos multirresistentes e o efeito de limpeza e desinfecção em unidade de terapia intensiva

Conocer el rol del medio ambiente es fundamental para evitar las infecciones intra-hospitalarias. Con ese objetivo, se planteó evaluar la prevalencia de contaminación ambiental por microorganismos multirresistentes (MMR) antes y después de la limpieza terminal de habitaciones de pacientes colonizados y establecer si la aparatología de uso común actuaba como reservorio de estos en la unidad de cuidados intensivos (UTI). Se obtuvieron muestras ambientales de las habitaciones, 48 h posteriores a la detección de colonización y luego de las limpiezas. Los resultados mostraron que luego de ambos procedimientos de limpieza se logró reducir de 28,2% a 2,6% la contaminación por Acinetobacter spp. multirresistente (AMR). También, se tomaron muestras de aparatología de uso común encontrándose entre 1,8 y 5,4% de contaminación por MMR. La limpieza y desinfección reducen significativamente la contaminación ambiental. Sin embargo, la colonización de equipos por MMR y el incumplimiento de precauciones universales representan una posibilidad de transmisión cruzada.

It is essential to understand the role of the environment in order to avoid intrahospital infections. To achieve this objective, this research proposes to assess the prevalence of the environmental contamination caused by multi-resistant microorganisms (MRM) before and after terminal disinfection in rooms with colonized patients, but also to establish whether the commonly used device acts as a reservoir of those micro-organisms in an intensive care unit (ICU). Environmental samples were obtained from the rooms, 48 hours after detecting colonization and also after the first and second final cleaning. The results showed that after both procedures, there was a reduction from 28.2% to 2.6% of contamination caused by multi-resistant Acinetobacter spp. (AMR). Samples from appliances and supplies were taken as well, in which case, between 1.8 and 5.4% of contamination levels induced by MMR were found. Cleaning and disinfecting significantly reduce environmental contamination. However, both MMR bacterial colonization and the lack of universal precautions enforcement represent a possibility of cross-transmission.

É essencial conhecer o papel do meio ambiente para evitar as infecções intra-hospitalares. Com esse objetivo, planejou-se avaliar a prevalência de contaminação ambiental por microorganismos multirresistentes (MMR) antes e depois da limpeza final dos quartos de pacientes colonizados e estabelecer se os aparelhos de uso comum atuavam como um reservatório deles na unidade de terapia intensiva (UTI). Obtiveram-se amostras ambientais dos quartos 48 horas após a detecção da colonização e logo após as limpezas finais. Os resultados mostraram que depois dos dois procedimentos de limpeza se obteve uma redução de 28,2% para 2,6% da contaminação por Acinetobacter spp. multirresistente (AMR). Foram obtidas também amostras de aparelhos de uso comum onde se encontraram entre 1,8% e 5,4% de contaminação por MMR. A limpeza e a desinfecção reduzem significativamente a contaminação ambiental. Contudo, a colonização de equipamentos por MMR e o não cumprimento de providências universais representam uma possibilidade de transmissão cruzada.

Effects of topical azithromycin, moxifloxacin, and povidone iodine on conjunctival bacterial flora in patients undergoing intravitreal injection / Os efeitos da azitromicina tópica, moxifloxacina e iodopovidona na flora bacteriana conjuntival em injeções intravítreas

ABSTRACT Purpose: To compare effects of 5% topical povidone iodine with prophylactic topical azithromycin and moxifloxacin on bacterial flora in patients undergoing intravitreal injection. Methods: A total of 132 patients were randomly assigned to receive treatment with azithromycin or moxifloxacin, or no treatment (control group). In total, 528 specimens were obtained at the time of admission, 4 days before intravitreal injection, 4 days after intravitreal injection, and 8 days after intravitreal injection. Samples were immediately sent to the microbiology laboratory for incubation. Results: The microorganism observed most frequently was coagulasenegative Staphylococcus (23.8%). When the results of samples obtained on Day 4 before injection were assessed, growth of coagulase-negative Staphylococcus was significantly lower in the moxifloxacin group, compared with controls (p=0.049). Acinetobacter baumannii continued to grow after administration of azithromycin (p=0.033). When the results of four days after intravitreal injection were evaluated, growth of coagulase-ne gative Staphylococcus was higher in controls, compared with patients who received azithromycin or moxifloxacin (p=0.004). Eradication rate was significantly higher in the moxifloxacin group than in the control group (p=0.001). Samples obtained on Day 8 after intravitreal injection showed similar levels of bacterial growth in all groups (p=0.217). Conclusion: Moxifloxacin was more effective than 5% povidone iodine in controlling the growth of conjunctival bacterial flora. Use of moxifloxacin in combination with 5% povidone iodine resulted in a synergistic effect.

RESUMO Objetivo: Comparar os efeitos de iodopovidona tópico a 5% com azitromicina e moxifloxacina profiláticas sobre a flora bacteriana em pacientes submetidos à injeção intravítrea. Métodos: Um total de 132 pacientes foram aleatoriamente designados para receber tratamento com azitromicina ou moxifloxacina ou nenhum tratamento (grupo controle). No total, 528 amostras foram obtidas no momento na admissão, 4 dias antes da injeção intravítrea, 4 dias após a injeção intravítrea e 8 dias após a injeção intravítrea. As amostras foram imediatamente enviadas para o laboratório de microbiologia para incubação. Resultados: O microorganismo mais frequentemente observado foi o Staphylococcus coagulase-negativo (23,8%). Quando os resultados das amostras obtidas no dia 4 antes da injeção foram avaliados, o crescimento do Staphylococcus coagulase-negativo foi significativamente menor no grupo mo xifloxacina, em comparação com os controles (p=0,049). Acinetobacter baumannii continuou a crescer após a administração de azitromicina (p=0,033). Quando os resultados de 4 dias após a injeção intravítrea foram avaliados, o crescimento do Staphylococcus coagulase-negativo foi maior no controle, em comparação com pacientes que receberam azitromicina ou moxifloxacina (p=0,004). A taxa de erradicação também foi significativamente maior no grupo moxifloxacina do que no grupo controle (p=0,001). As amostras obtidas no dia 8 após injeção intravítrea mostraram níveis semelhantes de crescimento bacteriano em todos os grupos (p=0,217). Conclusão: A moxifloxacina foi mais eficaz do que 5% de iodopovidona no controle do crescimento da flora bacteriana conjuntival. O uso de moxifloxacina em combinação com 5% de iodopovidona resultou em um efeito sinérgico.

Avaliação da contaminação por Acinetobacter spp. em uma unidade de terapia intensiva / Evaluation of contamination by Acinetobacter spp. in an intensive care unit / Evaluación de la contaminación por Acinetobacter spp. en una unidad de cuidados intensivos

Justificativa e Objetivos: A relevância clínica das Infecções Relacionadas à Assistência à Saúde (Iras) ocasionadas pelo Acinetobacter spp. e a confirmação da existência de cepas com multirresistência no meio hospitalar mostram a necessidade de se conhecer melhor a epidemiologia dessas infecções, a fim de auxiliar a implantação de medidas mais efetivas de prevenção e controle deste patógeno. Objetivou-se avaliar a diversidade fenotípica e o perfil de sensibilidade de Acinetobacter spp. isolados de pacientes internados, de mãos de profissionais e de superfícies inanimadas em uma Unidade de Terapia Intensiva de um hospital público da região sudeste do estado do Pará. Métodos: As coletas das superfícies e das mãos dos profissionais foram realizadas utilizando swabs umedecidos em soro fisiológico estéril e friccionados por meio de rolamento. Para análise dos dados, foram utilizadas técnicas de estatística descritiva por meio de distribuições absolutas e percentuais. Resultados: Das 163 amostras coletadas, 87 (53,4%) foram das superfícies, 47 (28,8%) dos pacientes e 29 (17,8%) das mãos dos profissionais. Em 28% observou-se o crescimento de bactérias Gram-negativas, sendo o Acinetobacter baumannii a cepa mais prevalente, estando presente nos isolados clínicos de pacientes e nas superfícies após o processo de limpeza. O A. baumannii apresentou-se resistente a todos os antimicrobianos testados. Conclusão: O A. baumannii foi a única espécie do gênero Acinetobacter a ser encontrada, sendo tais cepas resistentes a todos os antibióticos testados.(AU)

Background and Objectives: The clinical relevance of Healthcare Associated Infections caused by Acinetobacter spp. and the confirmation of the existence of strains with multi-resistance in the hospital environment show the need to know better the epidemiology of these infections, in order to help the implantation of more effective measures of prevention and control of this pathogen. The objective of this study was to evaluate the phenotypic diversity and the sensitivity profile of Acinetobacter spp. isolated from inpatients, hands of professionals and inanimate surfaces in an Intensive Care Unit of a public hospital in the southeast region of the state of Pará. Methods: The professionals' hands and surfaces were collected using swabs moistened in sterile saline and rubbed by rolling. Data analysis was performed using descriptive statistics techniques using absolute and percentage distributions. Results: Of the 163 samples collected, 87 (53.4%) came from the surfaces, 47 (28.8%) from the patients and 29 (17.8%) from the hands of professionals. Growth of Gram-negative bacteria was observed in 28%, being Acinetobacter baumannii the most prevalent strain, present in the clinical isolates of patients and on the surfaces after the cleaning process. A. baumannii was resistant to all antimicrobials tested. Conclusion: A. baumannii was the only species of the genus Acinetobacter to be found, being such strains resistant to all antibiotics tested.(AU)

Justificación y Objetivos: La relevancia clínica de las infecciones relacionadas con el cuidado de la salud ocasionadas por el Acinetobacter spp. y la confirmación de la existencia de cepas con multirresistencia en el medio hospitalario implican la necesidad de conocer mejor la epidemiología de esas infecciones, a fin de auxiliar en la implantación de medidas más efectivas de prevención y control de este patógeno. El objetivo de este estudio fue evaluar la diversidad fenotípica y el perfil de sensibilidad de Acinetobacter spp. aislados de pacientes hospitalizados, de las manos de profesionales y de superficies inanimadas en una Unidad de Cuidados Intensivos de un hospital público en la región sureste del estado de Pará. Métodos: Las colectas de las superficies y de las manos de los profesionales se realizaron utilizando hisopos humedecidos en suero fisiológico estéril y frotados por medio de rodamiento. El análisis de los datos se realizó mediante técnicas de estadística descriptiva utilizando distribuciones absolutas y porcentuales. Resultados: De las 163 muestras recogidas, 87 (53,4%) fueron de las superficies, 47 (28,8%) de los pacientes y 29 (17,8%) de las manos de los profesionales. El crecimiento de bacterias Gram-negativas se observó en el 28,0%, siendo Acinetobacter baumannii la cepa más prevalente en los aislados clínicos de los pacientes y en las superficies después del proceso de limpieza. A. baumannii fue resistente a todos los antimicrobianos probados. Conclusiones: El A. baumannii fue la única especie del género Acinetobacter encontrada, siendo tales cepas resistentes a todos los antibióticos probados.(AU)

Earliest identification of New Delhi metallo-ß-lactamase 1 (NDM-1) in Acinetobacter pittii in Brazil

Abstract We report the occurrence in Brazil of the bla NDM-1 gene in Acinetobacter pittii, prior to the previously described first reports regarding the species Providencia rettgeri and Enterobacter hormaechei. Clinical isolates were investigated by polymerase chain reaction followed by bidirectional sequencing, and species was confirmed by 16S rDNA sequencing and matrix-assisted laser desorption-ionization time-of-flight spectrometry. A. pittii carrying bla NDM-1 was confirmed in a patient with no national or international travel history, or transfer from another hospital. The findings warn of the possibility of silent spread of bla NDM-1 to the community.

High rate of detection of OXA-23-producing Acinetobacter from two general hospitals in Brazil

Abstract INTRODUCTION In recent decades, the prevalence of carbapenem-resistant Acinetobacter isolates has increased, and the production of oxacillinase (OXA)-type carbapenemases is the main mechanism underlying resistance. We evaluated OXA production from 114 Acinetobacter isolates collected between March and December 2013 from different clinical specimens of patients in two hospitals (Hospital 1 [n = 61] and Hospital 2 [n = 53]) located in Niterói, Rio de Janeiro, Brazil. We also evaluated the genetic diversity of OXA-producing isolates. METHODS All the isolates were identified through the automated system Vitek II and matrix-assisted laser desorption ionization-time of flight mass spectrometry MALDI-TOF MS as belonging to the A. baumannii-A. calcoaceticuscomplex. Antimicrobial susceptibility profiles were verified through agar diffusion tests. The presence of OXA-encoding genes was confirmed by PCR. The genetic diversity of isolates positive for carbapenemase production was analyzed through pulsed-field gel electrophoresis. RESULTS There was a high rate of resistance to carbapenems in the isolates (imipenem: 96%; meropenem: 92%) from both hospitals. Moreover, a high percentage (95.6%) of OXA-23-positive isolates was observed for both hospitals, indicating that this was the main mechanism of carbapenem-resistance among the studied population. In addition, most isolates (96.5%) were positive for bla OXA-51. A high genetic diversity and a few major genotypes were found among the OXA-23-positive isolates analyzed. Only intra-hospital dissemination was observed. CONCLUSIONS The elevated dissemination of bla OXA-23-like observed among Acinetobacter isolates from both the studied hospitals highlights the need for continuous epidemiological surveillance in these institutions.

Diversity of metallo-b-lactamase-encoding genes found in distinct species of Acinetobacter isolated from the Brazilian Amazon Region

BACKGROUND The multidrug resistance (MDR) phenotype is frequently observed in Acinetobacter baumannii, the most clinically relevant pathogenic species of its genus; recently, other species belonging to the A. calcoaceticus-A. baumannii complex have emerged as important MDR nosocomial pathogens. OBJECTIVES The present study aimed to verify the occurrence of metallo-β-lactamase genes among distinct Acinetobacter species in a hospital located in the Brazilian Amazon Region. METHODS Antimicrobial susceptibility profiles were determined by broth microdilution. The genetic relationships among these isolates were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Pyrosequencing reads of plasmids carrying the bla NDM-1 gene were generated using the Ion Torrent™ platform sequencing. FINDINGS A total of six isolates carried bla NDM-1: A. baumannii (n = 2), A. nosocomialis (n = 3), and A. pittii (n = 1); three carried bla IMP-1: A. baumannii, A. nosocomialis, and A. bereziniae. Resistance to colistin was observed for an NDM-1-producing A. nosocomialis isolate. Diverse PFGE patterns and sequence types were found among A. nosocomialis and A. baumannii isolates. The bla NDM-1 sequence was inserted in a Tn125 transposon, while the bla IMP-1 was found as a gene cassette of the class 1 integron In86. MAIN CONCLUSIONS To the best of our knowledge, this is the first report describing the dissemination of bla NDM-1 among distinct Acinetobacter species recovered from the same hospital in South America.

The antimicrobial activity of ephedrine and admixture of ephedrine and propofol: an in vitro study / A atividade antimicrobiana de efedrina e da combinação de efedrina e propofol: um estudo in vitro

Abstract Introduction Propofol and Ephedrine are commonly used during anesthesia maintenance, the former as a hypnotic agent and the later as a vasopressor. The addition of propofol to ephedrine or administration of ephedrine before propofol injection is useful for decreasing or preventing propofol related hemodynamic changes and vascular pain. This in vitro study evaluated the antibacterial effect on common hospital-acquired infection pathogens of ephedrine alone or combined with propofol. Material and method The study was performed in two stages. In the first, the Minimum Inhibitory Concentration of propofol and ephedrine alone and combined was calculated for Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Pseudomonas aeruginosa, and a clinical isolate of Acinetobacter spp. at 0, 6, 12 and 24 h, using the microdilution method. In the second stage, the same drugs and combination were used to determine their effect on bacterial growth. Bacterial solutions were prepared at 0.5 MacFarland in sterile 0.9% physiological saline and diluted at 1/100 concentration. Colony numbers were measured as colony forming units.mL-1 at 0, 2, 4, 6, 8, 10 and 12th hours. Results Ephedrine either alone or combined with propofol did not have an antimicrobial effect on Escherichia coli, Enterococcus faecium, or Pseudomonas aeruginosa and this was similar to propofol. However, ephedrine alone and combined with propofol was found to have an antimicrobial effect on Staphylococcus aureus and Acinetobacter species at 512 mcg.mL-1 concentration and significantly decreased bacterial growth rate. Conclusion Ephedrine has an antimicrobial activity on Staphylococcus aureus and Acinetobacter species which were frequently encountered pathogens as a cause of nosocomial infections.

Resumo Introdução Propofol e efedrina são fármacos comumente usados durante a manutenção da anestesia, o primeiro como agente hipnótico e o segundo como vasopressor. A adição de propofol à efedrina ou a administração de efedrina antes da injeção de propofol é útil para diminuir ou prevenir alterações hemodinâmicas e dor vascular relacionadas ao propofol. Este estudo in vitro avaliou o efeito antibacteriano de efedrina, isolada ou em combinação com propofol, em patógenos comuns implicados em infecção hospitalar. Material e método O estudo foi feito em duas etapas. Na primeira, a concentração inibitória mínima (CIM) de propofol e de efedrina isolada e em combinação foi calculada para Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Pseudomonas aeruginosa e um isolado clínico de Acinetobacter spp às 0, 6, 12 e 24 horas, com o método de microdiluição. Na segunda etapa, o mesmo fármaco e sua combinação foram usados para determinar seus efeitos no crescimento bacteriano. As soluções bacterianas foram preparadas em soro fisiológico a 0,9% em 0,5 McFarland e diluídas a uma concentração de 1/100. Os números das colônias foram medidos como ufc.mL-1 às 0, 2, 4, 6, 8, 10 e 12 horas. Resultados Efedrina isolada ou em combinação com propofol não apresentou efeito antimicrobiano sobre E. coli, E. faecium ou P. aeruginosa, um resultado semelhante ao de propofol. Porém, efedrina isolada e em combinação com propofol apresentou efeito antimicrobiano sobre Staphylococcus aureus e Acinetobacter spp, em concentração de 512 mcg.mL-1, e redução significativa da taxa de crescimento bacteriano. Conclusão Efedrina tem atividade antimicrobiana em S. aureus e Acinetobacter spp, patógenos frequentemente identificados como causa de infecções nosocomiais.

Perfil de resistencia antimicrobiana en bacilos Gram negativos no fermentadores aislados en fuentes hídricas / Antimicrobial resistance profile in non-fermenting gram negative bacilli isolated in water resources

La amplia distribución de los bacilos gram negativos no fermentadores en medios ambientales como el agua y especies vegetales cobra importancia al ser reconocidos como agentes causales de enfermedades en pacientes inmunocomprometidos, de allí la relevancia del porque debemos conocer la prevalencia y perfil de susceptibilidad de estos microorganismos en ambientes no hospitalarios. Materiales y Métodos: Estudio transversal, realizado en muestras hídricas de fuentes naturales y artificiales de almacenamiento para el consumo humano en la ciudad de Bogotá y municipios aledaños. La identificación se realizó a través de pruebas IMVIC y el perfil de resistencia a través del método de kirby bauer o E-TEST®. Resultados: Se obtuvieron 42 muestras, 7 (16,6%) con aislamientos de interés: 3 (60%) Pseudomonas spp, 2 (20%) Acinetobacter spp, 1 (10%) Sphingomonas paucimobilis y 1 (10%) Pantoea spp. El 70% presento resistencia a la ceftriaxona, el 30% a cefoxitina, 20% a gentamicina, 10% a ciprofloxacina y 10% a piperacilina-tazobactam. No se presentó resistencia a imipenem. Conclusión: 5 de 7 aislamientos revelaron un BGNNF de importancia en infección en humanos, siendo importante la resistencia encontrada a la ceftriaxona.

The wide distribution of non-fermenting gram negative bacilli in environmental media such as water and plants becomes important as they are recognized a cause of diseases in immunocompromised patients, that’s the reason why we should to know the prevalence and the susceptibility profile of these microorganisms in non-hospital environments. Materials and Methods: Cross-sectional study done with samples of natural and artificial water storage for human consumption in the city of Bogotá and surrounding municipalities. The identification was made through IMVIC tests and the resistance profile through the kirby bauer or E-TEST® method. Results: 42 samples were obtained, 7 (16.6%) with isolates of interest: 3 (60%) Pseudomonas spp, 2 (20%) Acinetobacter spp, 1 (10%) Sphingomonas paucimobilis and 1 (10%) Pantoea spp. The 70% had resistance to ceftriaxone, 30% to cefoxitin, 20% to gentamicin, 10% to ciprofloxacin and 10% to piperacillin-tazobactam. No resistance to imipenem was shown. Conclusion: 5 of 7 isolates revealed a BGNNF of importance in infection in humans, with an important resistance to ceftriaxone.

Characterization of multidrug-resistant Acinetobacter ssp. strains isolated from medical intensive care units in Cali - Colombia / Caracterización de cepas de Acinetobacter spp. resistentes a múltiples fármacos aisladas de unidades de cuidados intensivos en Cali - Colombia

Abstract Introduction: The extensive use of antibiotics has led to the emergence of multi-resistant strains in some species of the genus Acinetobacter. Objective: To investigate the molecular characteristics of multidrug-resistant of Acinetobacter ssp. strains isolated from 52 patients collected between March 2009 and July 2010 in medical intensive care units in Cali - Colombia. Methods: The susceptibility to various classes of antibiotics was determined by disc diffusion method, and the determination of the genomic species was carried out using amplified ribosomal DNA restriction analysis (ARDRA) and by sequencing of the 16s rDNA gene. Also, the genes of beta-lactamases as well as, integrases IntI1 and IntI2 were analyzed by PCR method. Results: The phenotypic identification showed that the isolates belong mainly to A. calcoaceticus- A. baumannii complex. All of them were multi-resistant to almost the whole antibiotics except to tigecycline and sulperazon, and they were grouped into five (I to V) different antibiotypes, being the antibiotype I the most common (50.0%). The percent of beta-lactamases detected was: blaTEM (17.3%), blaCTX-M (9.6%), blaVIM (21.2%), blaIMP (7.7%), blaOXA-58 (21.2%), and blaOXA-51 (21.2%). The phylogenetic tree analysis showed that the isolates were clustering to A. baumannii (74.1%), A. nosocomialis (11.1%) and A. calcoaceticus (7.4 %). Besides, the integron class 1 and class 2 were detected in 23.1% and 17.3% respectively. Conclusion: The isolates were identified to species A. baumanii mainly, and they were multiresistant. The resistance to beta-lactams may be by for presence of beta-lactamases in the majority of the isolates.

Resumen Introducción: El uso extensivo de antibióticos ha llevado a la emergencia de cepas multirresistentes en algunas especies del género Acinetobacter. Objetivo: Investigar las características moleculares de resistencia a múltiples fármacos de cepas aisladas de Acinetobacter spp. colectadas entre marzo de 2009 y julio de 2010 en 52 pacientes de unidades de cuidados intensivos en Cali - Colombia. Métodos: La susceptibilidad a diversas clases de antibióticos se determinó mediante el método de difusión de disco, y la determinación de la especie genómica se llevó a cabo usando un análisis de restricción de ADN ribosómico amplificado (ARDRA) y mediante la secuenciación del gen 16s de ADNr. Además, se analizaron por el método de PCR los genes de las beta-lactamasas, como también, las integrasas IntI1 e IntI2. Resultados: La identificación fenotípica mostró que los aislamientos pertenecen principalmente al complejo A. calcoaceticus - A. baumannii. Todos ellos eran multirresistentes a casi todos los antibióticos excepto tigeciclina y sulperazón, y se agruparon en cinco (I a V) antibitipos diferentes, siendo el antibiotipo I el más común (50%). El porcentaje de betalactamasas detectadas fue: blaTEM (17,3%), blaCTX-M (9,6%), blaVIM (21,2%), blaIMP (7,7%), blaOXA-58 (21,2%), blaOXA- 51 (21.2%). El análisis del árbol filogenético mostró que los aislados se agrupaban en A. baumannii (74.1%), A. nosocomialis (11.1%) y A. calcoaceticus (7.4%). Además, el integrón clase 1 y clase 2 se detectaron en 23.1% y 17.3% respectivamente. Conclusión: los aislamientos se identificaron principalmente como la especie A. baumanii, y fueron multirresistentes. La resistencia a los betalactámicos puede deberse a la presencia de betalactamasas en la mayoría de los aislamientos.

Glimpse into the genome sequence of a multidrug-resistant Acinetobacter pittii ST950 clinical isolate carrying the blaOXA-72 and blaOXA-533 genes in China

The development of carbapenem-resistant Acinetobacter species is of serious concern in the hospital settings and naturally occurring oxacillinase genes (blaOXA) have been identified in several Acinetobacter species. In this study, we report the genome sequence of A. pittii TCM178 belongs to ST950, a multidrug-resistant isolate that harbored the blaOXA-72 and blaOXA-533 genes simultaneous. The genome size was estimated to be 3,789,564 bp with 3,501 predicted coding regions, and G+C content is 37.60%. Our findings have raised awareness of the possible constitution of a reservoir for peculiar carbapenemase genes in A. pittii that may spread among other Acinetobacter species in China.

Draft genome sequence of a multidrug-resistant beta-lactamase OXA-357-producing Acinetobacter pittii ST865 clinical isolate from China

Abstract Worldwide increasing emergence of carbapenem-resistant Acinetobacter spp. has rendered the limited availability of effective antimicrobial agents and has become a major public health concern. In this study, we report the draft genome sequence of A. pittii TCM156, a multidrug-resistant isolate that harbored the blaOXA-357 gene. The genome sequence was further analyzed by various bioinformatics methods. The genome size was estimated to be 3,807,313 bp with 3508 predicted coding regions and G + C content is 38.7%. These findings have raised awareness of the possible emergence of OXA-type enzyme-producing A. pittii isolate in China.

Phenol degradation and genotypic analysis of dioxygenase genes in bacteria isolated from sediments

Abstract The aerobic degradation of aromatic compounds by bacteria is performed by dioxygenases. To show some characteristic patterns of the dioxygenase genotype and its degradation specificities, twenty-nine gram-negative bacterial cultures were obtained from sediment contaminated with phenolic compounds in Wuhan, China. The isolates were phylogenetically diverse and belonged to 10 genera. All 29 gram-negative bacteria were able to utilize phenol, m-dihydroxybenzene and 2-hydroxybenzoic acid as the sole carbon sources, and members of the three primary genera Pseudomonas, Acinetobacter and Alcaligenes were able to grow in the presence of multiple monoaromatic compounds. PCR and DNA sequence analysis were used to detect dioxygenase genes coding for catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and protocatechuate 3,4-dioxygenase. The results showed that there are 4 genotypes; most strains are either PNP (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is positive) or PNN (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is negative). The strains with two dioxygenase genes can usually grow on many more aromatic compounds than strains with one dioxygenase gene. Degradation experiments using a mixed culture representing four bacterial genotypes resulted in the rapid degradation of phenol. Determinations of substrate utilization and phenol degradation revealed their affiliations through dioxygenase genotype data.

Draft genome sequence of phenol degrading Acinetobacter sp. Strain V2, isolated from oil contaminated soil

Abstract We report here the draft genome sequence of Acinetobacter sp. Strain V2 isolated from the oil contaminated soil collected from ENGEN, Amanzimtoti, South Africa. Degradation of phenolic compounds such as phenol, toluene, aniline etc. at 400 ppm in 24 h and oil degrading capability makes this organism an efficient multifunctional bioremediator. Genome sequencing of Acinetobacter spp. V2 was carried out on Illumina HiSeq 2000 platform (performed by the Beijing Genomics Institute [BGI], Shenzhen, China). The data obtained revealed 643 contigs with genome size of 4.0 Mb and G + C content of 38.59%.

Emergence of colistin resistance in the largest university hospital complex of São Paulo, Brazil, over five years

Abstract Colistin resistance involving Gram-negative bacilli infections is a challenge for health institutions around of the world. Carbapenem-resistance among these isolates makes colistin the last therapeutic option for this treatment. Colistin resistance among Enterobacteriaceae, Acinetobacter spp., and Pseudomonas spp. was evaluated between 2010 and 2014 years, at Hospital das Clínicas, São Paulo, Brazil. Over five years 1346 (4.0%) colistin resistant Gram-negative bacilli were evaluated. Enterobacteriaceae was the most frequent (86.1%) pathogen isolated, followed by Acinetobacter spp. (7.6%), and Pseudomonas spp. (6.3%). By temporal analysis there was a trend for an increase of colistin resistance among Enterobacteriaceae, but not among non-fermentative isolates. Among 1346 colistin resistant isolates, carbapenem susceptibility was observed in 21.5%. Colistin resistance in our hospital has been alarmingly increased among Klebsiella pneumoniae isolates in both KPC positive and negative, thus becoming a therapeutic problem.

ß-lactamase-producing Gram-negative bacteria in an intensive care unit in southern Brazil

ABSTRACT The present study evaluated the antimicrobial susceptibility profile, ß-lactamase production, and genetic diversity of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. using phenotypic identification, antimicrobial susceptibility testing, and ß-lactamase phenotypic detection. Isolates were obtained from patients in an intensive care unit in a hospital in southern Brazil. Bacterial genomic DNA was extracted, followed by the genotypic detection of carbapenemases and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Fifty-six isolates (26 Klebsiella pneumoniae, five Escherichia coli, three Enterobacter aerogenes, nine P. aeruginosa, and 13 Acinetobacter spp.) were evaluated. The phenotypic extended spectrum ß-lactamase (ESBL) test was positive in 53.8% of the K. pneumoniae isolates, 100.0% of the E. coli isolates, and 100.0% of the E. aerogenes isolates. Phenotypic and genotypic testing of K. pneumoniae carbapenemase (KPC) was positive in 50.0% of the K. pneumoniae isolates. Phenotypic and genotypic testing showed that none of the P. aeruginosa or Acinetobacter spp. isolates were positive for metallo- ß-lactamase (MBL). The bla OXA gene was detected only in Acinetobacter spp. The lowest genetic diversity, determined by ERIC-PCR, was observed among the KPC-producing K. pneumoniae isolates and OXA-producing Acinetobacter spp. isolates, indicating the inadequate dissemination control of multidrug-resistant bacteria in this hospital environment.

Caracterização fenotípica e genotípica de Acinetobacter spp. e Pseudomonas aeruginosa produtores de carbapenemases / Phenotypic and genotypic characterization of carbapenemase-producing Acinetobacter spp. e Pseudomonas aeruginosa

A resistência bacteriana a antibióticos é um grave e crescente problema de saúde pública de âmbito mundial. O principal, e mais eficiente, mecanismo de resistência aos ß-lactâmicos em bacilos Gram-negativos é a produção de ß-lactamases, que possuem a capacidade de hidrolisar o anel ß-lactâmicos e consequentemente inativar essa classe de antibióticos. Vale ressaltar, que atualmente os antibióticos ß-lactâmicos são os mais utilizados clinicamente, particularmente em infecções graves. Dentre as ß-lactamases existentes destacam-se as carbapenemases, enzimas capazes de inativar a maioria dos antibióticos ß-lactâmicos. Uma grande preocupação é o fato dessas enzimas, em sua maioria, serem codificadas por plasmídeos, o que propicia a disseminação desses genes de resistência; portanto, é de extrema importância a realização de um rápido e efetivo monitoramento da presença de patógenos portadores desses genes de resistência, para que assim se possa prevenir a disseminação desses determinantes. Foram incluídos neste estudo 230 amostras únicas de Acinetobacter e Pseudomonas aeruginosa resistentes a imipenem detectados em pacientes internados em hospitais privados da cidade de São Paulo durante o período de fevereiro a outubro de 2013. As amostras foram avaliadas quanto à hidrólise de imipenem por espectrofotometria, quanto à presença de genes de carbapenemases por PCR e sequenciamento, e quanto à clonalidade por eletroforese em campos pulsados (PFGE) ou ERIC-PCR. Foram realizados ensaios de conjugação, transformação e sequenciamento completo de plasmídeos. Dentre as amostras de Acinetobacter spp. 80% (88) foram capazes de hidrolisar o imipenem. Dentre esses 76,1% (67) foram positivos para blaOXA-51-like, 19,3% (17) foram positivos para blaOXA-72. blaOXA-23, blaOXA-482 e blaIMP-1 foram detectados isoladamente em isolados distintos. O gene blaIMP-1 foi detectado em A. ursingii inserido em integron de classe 1 e representa a primeira descrição no Brasil. Uma nova carbapenemase OXA-482-like foi detectada em A. baumanii. Utilizando-se ERIC-PCR, observou-se uma grande diversidade de grupos clonais, com o máximo de quatro isolados por grupo. Dentre as amostras de P. aeruginosa, apenas 35,3% foram capazes de hidrolisar o imipenem. Dessas amostras, 14 possuíam o gene blaSPM-1, e isolados únicos possuíam, individualmente, os genes blaIMP, blaVIM, blaKPC-2 ou blaGES-23. O gene blaKPC-2 foi detectado inserido em contexto genético diferente dos descritos anteriormente, em plasmídeo IncU de 32 Kb, mobilizável, mas não conjugativo. Esta é a primeira descrição da sequencia completa de plasmídeo albergando o gene blaKPC-2 em P. aeruginosa no Brasil. Nas demais amostras (20) com atividade hidrolítica, não foram detectados genes de carbapenemase conhecidos, o que sugere a presença de genes de carbapenemase ainda não descritos. Em três amostras foi possível obter transformantes com plasmídeos, resistentes a carbapenêmicos. As amostras com blaSPM-1 apresentaram perfis de PFGE estreitamente relacionados. Em contraste, os perfis de PFGE das amostras com potenciais novas carbapenemases apresentaram índice de similaridade de Dice inferior ix a 80%, evidenciando grande diversidade clonal. Nossos achados evidenciam que a carbapenemase não intrínseca predominante em Acinetobacterem hospitais privados da cidade de São Paulo é OXA-72, e em hospitais privados há uma grande diversidade clonal. Em P. aeruginosa, a carbapenemase predominante é SPM-1, cuja disseminação é mediada por um único clone. Há potencialmente um número significativo de novas carbapenemases em Acinetobacter e P. aeruginosa, algumas delas mediadas por plasmídeos

Bacterial resistance to antibiotics is a serious and growing public health problem worldwide. The main and most efficient mechanism of resistance to ß-lactams in Gram-negative bacilli is the production of ß-lactamases, which have the ability to hydrolyze the ß-lactam ring and consequently inactivate this class of antibiotics. It is worth mentioning that currently ß-lactam antibiotics are the most used clinically, particularly in severe infections. Among the existing ß-lactamases, carbapenemases are capable of inactivating most ß-lactam antibiotics. A major concern is that these enzymes are mostly encoded by plasmids, which facilitates the spread of these resistance genes; therefore, it is of extreme importance to carry out a rapid and effective monitoring of the presence of pathogens bearing these resistance genes, in order to prevent the dissemination of these determinants. This study included 230 unique samples of imipenem-resistant Acinetobacterand Pseudomonas aeruginosa detected in patients hospitalized in private hospitals in the city of São Paulo during the period from February to October 2013. The samples were evaluated for the imipenem hydrolysis by spectrophotometry, the presence of carbapenemase genes by PCR and sequencing, and concerning clonality by pulsed field electrophoresis (PFGE) or ERIC-PCR. Conjugation, transformation and complete sequencing of plasmids were performed. Among Acinetobacter spp. samples, 80% (88) were able to hydrolyze imipenem. Among these, 76.1% (67) were positive for blaOXA-51-like genes and 19.3% (17) were positive for blaOXA-72. The blaOXA-23, blaOXA-482 and blaIMP-1 genes were detected alone in distinct isolates. The blaIMP-1 gene was detected in A. ursingii inserted in class 1 integron and represents the first description in Brazil. A novel OXA-482-like carbapenemase was detected in A. baumanii. Using ERIC-PCR, a great diversity of clonal groups was observed, with a maximum of four isolates per group. Among P. aeruginosa samples, only 35.3% were able to hydrolyze imipenem. Of these samples, 14 had the blaSPM-1 gene, and single isolates individually possessed the blaIMP, blaVIM, blaKPC-2 or blaGES-23 genes. The blaKPC-2 gene was found inserted in a genetic context different from those described previously, in a mobilizable, but not conjugative, 32 Kb IncU plasmid. This is the first description of the complete nucleotide sequence of a plasmid harboring the blaKPC-2 gene in P. aeruginosa in Brazil. In the remaining samples (20) with hydrolytic activity, no known carbapenemase genes were detected, suggesting the presence of carbapenemase genes not yet described. In three samples it was possible to obtain transformants with plasmids, resistant to carbapenems. Samples with blaSPM-1 showed closely related PFGE profiles. In contrast, the PFGE profiles of the samples with potential new carbapenemases showed Dice similarity index lower than 80%, evidencing a great clonal diversity. Our findings show that the predominant non-intrinsic carbapenemase in Acinetobacter in the city of São Paulo is OXA-72, and in private hospitals there is great clonal diversity. In P. aeruginosa, the predominant carbapenemase is SPM-1, the spread of this enzyme is mediated by a single clone. There are potentially a significant number of new carbapenemases in Acinetobacter and P. aeruginosa, some of them plasmid mediated

Draft genome sequence of Acinetobacter pittii ST643 shared by cystic fibrosis patients

Acinetobacter pittii has emerged as an important hospital pathogen that is associated with outbreaks and drug resistance. In cystic fibrosis (CF) patients, the detection of Acinetobacter spp. is rare; however, we isolated the A. pittii sequence type ST643 in several Brazilian CF patients treated in the same centre. The current study describes the draft genome of A. pittii ST643.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.