ABSTRACT Introduction: Cholera is an acute intestinal infection caused by toxigenic strains of Vibrio choleare. The rapid dissemination and emergence of the multiresistance that characterizes this pathogen could interfere with the success of antimicrobial therapy, so it is a priority to monitor changes in susceptibility patterns, as a transcendental part of the resistance control policy antimicrobial. Objective: To determine the behavior of antimicrobial resistance against the antimicrobials of interest used in the treatment, the presence of enzymatic virulence factors and whether there is a relationship between them. Methods: A descriptive cross-sectional study was conducted during July 2012 to December 2015. Where 500 isolates belonging to the cepary of the National Reference Laboratory for Acute Diarrheal Diseases of the Institute of Tropical Medicine "Pedro Kourí", from outbreaks of EDA of the national network of Microbiology laboratories in Cuba. Conventional phenotypic methods were applied to determine the behavior of antimicrobial resistance, the presence of enzymatic factors and their relationship with antimicrobial resistance. Results: The highest percentages of sensitivity were obtained against azithromycin (98%), doxycycline (96%) and ciprofloxacin (93%) and resistance to ampicillin (100%) and trimethoprim-sulfamethoxazole (99.4%). 44 isolated (8.8%) multi-resistant were found. All isolates had at least two extracellular enzymes as virulence factors, the most frequent: gelatinase (96%) and lecithinase (95%). Conclusions: There is a direct and proportional relationship between the presence of virulence factors and antimicrobial resistance, synergism that arises greater pathogenicity of the isolates studied from epidemic outbreaks.

RESUMEN Introducción: El cólera es una infección intestinal aguda causada por cepas toxigénicas de Vibrio choleare. La rápida diseminación y emergencia de la multirresistencia que caracteriza a este patógeno, podría interferir en el éxito de la terapia antimicrobiana, por lo que constituye una prioridad monitorear los cambios en los patrones de susceptibilidad, como parte trascendental de la política de control de la resistencia antimicrobiana. Objetivo: Determinar el comportamiento de la resistencia antimicrobiana frente a los antimicrobianos de interés empleados en el tratamiento, la presencia de factores de virulencia enzimáticos y si existe relación entre ambos. Métodos: Se realizó un estudio descriptivo de corte transversal durante julio de 2012 a diciembre de 2015. Se estudiaron 500 aislamientos pertenecientes al cepario del Laboratorio Nacional de Referencia de Enfermedades Diarreicas Agudas del Instituto de Medicina Tropical “Pedro Kourí”, procedentes de brotes de enfermedades diarreicas agudas de la red nacional de laboratorios de Microbiología de Cuba. Se aplicaron métodos convencionales fenotípicos para determinar el comportamiento de la resistencia antimicrobiana, la presencia de factores enzimáticos y la relación de estos con la resistencia antimicrobiana. Resultados: Los mayores porcentajes de sensibilidad se obtuvieron frente a azitromicina (98 %), doxiciclina (96 %) y ciprofloxacina (93%) y de resistencia frente a ampicilina (100 %) y trimetoprim-sulfametoxazol (99,4 %). Se encontraron 44 aislados (8,8 %) multirresistente. Todos los aislamientos poseían al menos dos enzimas extracelulares como factores de virulencia, las más frecuentes: gelatinasa (96 %) y lecitinasa (95 %). Conclusiones: Se evidencia una relación directa y proporcional entre la presencia de los factores de virulencia y resistencia antimicrobiana, sinergismo que surgiere mayor patogenicidad de los aislados estudiados procedentes de brotes epidémicos.

ABSTRACT Introduction: Acute diarrheal disease is a frequent cause of morbidity and mortality in developing countries. One of the leading causes is diarrheagenic Escherichia coli, which affects patients at extreme ages and with immunodeficiencies. Objective: Identify the most active pathotypes of diarrheagenic Escherichia coli and the antimicrobial resistance phenotypes expressed by the prevailing pathotype. Methods: A study was conducted from July 2012 to February 2014 of 184 isolates obtained from 15 provincial Hygiene, Epidemiology and Microbiology Centers in Cuba and the Isle of Youth. Identification of the genus, species and pathotypes was based on conventional and molecular diagnostic methods. Determination of antimicrobial susceptibility was performed by the Bauer-Kirby method in compliance with guidelines from the Clinical and Laboratory Standards Institute of 2013. Results: A total 108 (58%) diarrheagenic Escherichia coli were identified. The following pathotypes were confirmed: Multiplex PCR 1 revealed 5 (6%) enteropathogenic, 4 (4%) enterotoxigenic and 0 (0%) enterohemorrhagic Escherichia coli. Multiplex PCR 2 found 72 (82%) enteroaggregative Escherichia coli, which was the prevailing type in the study. PCR 3 (simple) detected 7 (8%) enteroinvasive Escherichia coli. 100% of the prevailing pathotype displayed resistance to at least one of the antimicrobials tested, a single resistance pattern to two antimicrobials, and nine multiresistance patterns. Conclusions: The study showed the importance of the use of rapid molecular tests to confirm diarrheagenic E. coli pathotypes, which cause mild dehydration, serious complications and death. Identification could be done of the four most common pathotypes and enteroaggregative E. coli, the one with the highest incidence in the study population. The prevailing pathotype displayed high percentages of antimicrobial resistance to beta-lactams and good antimicrobial sensitivity to third-generation cephalosporins and aminoglycosides. The study contributed knowledge not revealed by previous research about Cuban isolates. Such information is considered to be highly valuable for clinicians, pediatricians and epidemiologists in the country.

RESUMEN Introducción: En los países en vías de desarrollo las enfermedades diarreicas agudas son causa frecuente de morbilidad y mortalidad. Entre las primeras causas se encuentra Escherichia coli diarrogénicos, que afecta a pacientes en edades extremas de la vida y con inmunodeficiencias. Objetivo: Identificar los patotipos de Escherichia coli diarrogénicos que más inciden y los fenotipos de resistencia antimicrobiana expresados por el patotipo predominante. Métodos: Se estudiaron 184 aislamientos procedentes de 15 centros provinciales de Higiene, Epidemiología y Microbiología de Cuba e Isla de la Juventud. La investigación se realizó desde julio de 2012 hasta febrero de 2014. La identificación de género, especie y patotipos fue realizada por métodos de diagnóstico convencional y molecular. La determinación de la susceptibilidad antimicrobiana se realizó por el método de Bauer y Kirby, y las normas del Clinical and Laboratory Institute Standards de 2013. Resultados: Se identificaron 108 (58 %) Escherichia coli diarrogénicos. Los patotipos confirmados fueron: en la PCR múltiple 1, 5 (6 %) de Escherichia coli enteropatogénico, 4 (4 %) de enterotoxigénico y 0 (0 %) de enterohemorrágico. La PCR múltiple 2 reveló 72 (82 %) Escherichia coli enteroagregativo, que resultó el predominante en el estudio. La PCR 3 (simple) detectó 7 (8 %) de enteroinvasivo. El 100 % del patotipo predominante mostró resistencia, al menos a un antimicrobiano de los probados, un solo patron de resistencia a dos antimicrobianos, y nueve patrones de multirresistencia. Conclusiones: El estudio demuestra la importancia del uso de pruebas moleculares rápidas para la confirmación de los patotipos de E. coli diarrogénicos, los que provocan deshidratación ligera, complicaciones graves y la muerte. Se logra identificar los cuatro patotipos más frecuentes y E. coli enteroagregativo, el de mayor incidencia en la población estudiada. El patotipo predominante mostró altos porcentaje de resistencia antimicrobiana a betalactámicos y buena sensibilidad antimicrobiana a los aminoglucósidos y cefalosporinas de tercera generación. La investigación aporta conocimientos, no revelados en estudios anteriores con aislados cubanos, lo que es considerado de alto valor para los clínicos, pediatras y epidemiólogos del país.

ABSTRACT Vibrio cholerae non-O1, non-O139 is the third bacterium group from the genus Vibrio most commonly causing diarrhea. It survives in aquatic environments, using the formation of biofilm as a survival mechanism facilitating the transmission of diarrheal disease. Multi-drug resistant V. cholerae isolates have been characterized since the year 1977, and some of the mechanisms involved include the production of extended-spectrum β-lactamases (ESBLs). The purpose of the study was to determine the formation of biofilm in Cuban isolates of V. cholerae non-O1, non-O139 causing acute diarrheal disease (ADD), and detect the production of ESBLs in those with total or intermediate resistance to ampicillin. A descriptive cross-sectional study was conducted from January 2014 to June 2015. The study sample was 55 previously characterized isolates obtained from the strain collection at the ADD National Reference Laboratory of Pedro Kourí Institute. For phenotypic determination of ESBLs, 43 were studied which were known to be susceptible to ampicillin. 54.5 % of the isolates tested positive for biofilm formation, with a predominance of those classified as moderate (46.6 %) and weak (36.6 %) biofilm producers. Of the 34 isolates resistant to ampicillin, 26.5 % were positive for ESBL production. Of the 9 with intermediate ampicillin resistance, 44.4 % were positive. The results of the present study contribute knowledge about their ability to persist in the environment, and provide insight into antimicrobial resistance mechanisms.

RESUMEN Vibrio cholerae no-O1, no-O139 es el tercer grupo de bacterias del género Vibrio que con más frecuencia producen diarreas. Sobrevive en los ambientes acuáticos, utilizando la formación de biopelícula como mecanismo de supervivencia que propicia la transmisión de la enfermedad diarreica. Desde 1977 se caracterizan aislados de V. cholerae con resistencia múltiple, y algunos de los mecanismos involucrados incluyen la producción de β-lactamasas de espectro extendido (BLEE). Este trabajo tuvo como objetivo determinar la formación de biopelícula en los aislados cubanos de V. cholerae no-O1, no-O139, causantes de enfermedad diarreica aguda (EDA), y detectar la producción de BLEE en aquellos con resistencia total e intermedia a ampicilina. Se realizó un estudio descriptivo de corte transversal, entre enero 2014 y junio 2015. Se estudiaron 55 aislados caracterizados previamente, que formaban parte del cepario del Laboratorio Nacional de Referencia de EDA del Instituto “Pedro Kourí”. Para la determinación fenotípica de BLEE se estudiaron 43, de los que ya se conocía su susceptibilidad a ampicilina. El 54,5 % de los aislados resultaron positivos a la formación de biopelícula, y predominaron los clasificados como formadores moderados (46,6 %) y débiles (36,6 %). De los 34 resistentes a ampicilina, 26,5 % resultaron positivos a la producción de BLEE. En el caso de los nueve aislados con resistencia intermedia a ampicilina, 44,4 % resultaron positivos. Los resultados del presente estudio contribuyen al conocimiento sobre la capacidad que tienen de persistir en el ambiente y permiten profundizar sobre los mecanismos de resistencia a los antimicrobianos.

ABSTRACT INTRODUCTION In the Caribbean region, acute diarrheal diseases caused by the Vibrio genus have increased in recent years, following the 2010 earthquake in Haiti. Based on its capsular lipopolysaccharide, Vibrio cholerae is classified into more than 200 serogroups, divided into O1, O139 and non-O1, non-O139. Non-O1 serogroups produce clinical features ranging from mild diarrhea to severe dehydration. In Cuba, circulation of non-O1, non-O139 V. cholerae has been reported both in outbreaks and sporadic cases. OBJECTIVE Describe the antimicrobial susceptibility of V. cholerae to the drugs of interest used in its treatment and verify the presence of enzymatic virulence factors. METHODS A descriptive, cross-sectional study was conducted in January through November 2014, based on 125 non-O1, non-O139 V. cholerae isolates obtained during 2013 and 2014 from patients with acute diarrheal disease (isolates from the National Reference Laboratory for Acute Diarrheal Diseases of the Pedro Kouri Tropical Medicine Institute, Havana). Bacteriological identification was performed according to conventional methods. Antimicrobial susceptibility was determined by the Bauer-Kirby agar diffusion method. The technique described by Robinson in 1986 was used to determine virulence factors, hemolytic activity and enzyme factors (DNase, elastase, gelatinase), and Karagozova’s method for the enzyme lecithinase. RESULTS Highest percentages of sensitivity were obtained for azithromycin (98.4%), doxycycline (97.6%) and ciprofloxacin (96.8%), and highest resistance values for ampicillin (60%), sulfonamide (46.4%) and trimethoprim–sulfamethoxazole (32%). Six resistance patterns were detected, four found in Cuba for the first time, as well as six patterns of multidrug resistance (4.8%). All isolates had at least two extracellular enzymes as virulence factors. The most frequent were gelatinase (90.4%) and lecithinase (77.6%). The percentage of virulence factors was lower in the group of isolates resistant to ≥2 antimicrobials. An inverse relationship was found between presence of enzymatic virulence factors and resistance in the isolates studied. CONCLUSIONS The results suggest that azithromycin, doxycycline and ciprofloxacin should continue to be used for treatment of V. cholerae-caused infections, and confirmed the presence of four new resistance patterns in isolates circulating in Cuba.

In vitro antifungal susceptibility testing is really necessary at present in medical mycology laboratories. Minimal inhibitory concentration of 210 Candida yeast strains isolated from HIV/AIDS patients with oral lesions was determined to find out susceptibility to fluconazole and amphotericin B. A CLSI´s M27A2 protocol-based dilution micromethod was used. C. albicans was predominant (62.4 %)followed by C. glabrata, C. parapsilosis, and C. tropicalis. Regarding amphotericin B, the mode value and MIC50 were 0,125 µg/mL, which indicated the effectiveness of this drug in vitro conditions. Only C. krusei strain showed a minimal inhibitory concentration over 1 µg/mL. 8.1 % of strains were resistent to fluconazole whereas 8.1 % was dose- depending susceptible. The less susceptible species were C. krusei and C. glabrata. The achieved results laid the foundations for wider studies to be made in the Cuban context and indicated the need of keeping surveillance on the behaviour of clinical yeast isolates to antifungal drugs.

Las pruebas de sensibilidad in vitro a los agentes antifúngicos se han convertido en una necesidad actual de los laboratorios de micología médica. Se determinó la concentración mínima inhibitoria de 210 cepas de levaduras del género Candida aisladas de pacientes VIH/SIDA que presentaban lesiones orales, con el objetivo de conocer la sensibilidad a fluconazol y anfotericina B. Fue utilizado un micrométodo de dilución según el protocolo M27A2 del CLSI. C. albicans fue la especie predominante (62,4 %), seguida, en orden de frecuencia, de C. glabrata, C. parapsilosis, C. krusei y C. tropicalis. Frente a la anfotericina B el valor de la moda y la CMI50 fue de 0,125 µg/mL, lo que indica la efectividad in vitro de este fármaco. Solo una cepa (C. krusei) mostró una concentración mínima inhibitoria superior a 1 µg/mL. De las cepas, 8,1 % resultó resistente al fluconazol y 8,1 % sensible dependiente de la dosis. Las especies que se comportaron menos sensibles fueron C. krusei y C. glabrata. Los resultados obtenidos sentaron las bases para estudios más amplios en el medio cubano y sugirieron la necesidad de continuar la vigilancia del comportamiento de los aislamientos clínicos de levaduras frente a los agentes antifúngicos.