ABSTRACT Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms’ role in this extreme environment, the characterization and description of new species is vital.

Genes associated to rhamnolipids production were molecularly characterized in 61 bacterial strains from LAMYBIM bacterial collection (Laboratorio de Microbiología y Biotecnología Microbiana, Universidad Nacional Mayor de San Marcos, Perú). Strains were isolated from peruvian environments hydrocarbons polluted and were classified as RL overproducers (n= 21), RL producers (n = 20) and non-producers (n = 20) producers. Molecular identification using the 16S rRNA gene was preceded by the biochemical identification of 61 strains selected with the API 20 NE system. Pseudomonas aeruginosa was the most prevalent strain of the RL overproducers and RL producers. Species such as Burkholderia cepacea, Pseudomonas fluorescens, Aeromonas hydrophila and Chryseobacterium indologenes, were found too. In the same way, non-producers microorganisms were also characterized. The PCR amplification and agarose gel electrophoresis techniques, standarized by the UNAM laboratory, showed that the selected strains had the genes: rhlA, rhlB, rhlR and rhlC. For the sequencing of the rhLABR gene region, four strains were selected: Pseudomonas aeruginosa T2K2, Pseudomonas aeruginosa III T1P2, Pseudomonas aeruginosa 6K-11 and Pseudomonas aeruginosa ATCC 9027, applying the methodology standardized by the UNAM and were compared with Pseudomonas aeruginosa PAO1. Our results show that the genes studied in the selected strains are synonymous with their homologues in Pseudomonas aeruginosa PAO1 standard strain. Therefore, genotypical differences that explain the overproduction of rhamnolipid might be found in other molecular markers not covered in this study.

Se realizó la caracterización molecular de los genes asociados a la producción de ramnolípidos (RL), en 61 cepas bacterianas de la colección del Laboratorio de Microbiología y Biotecnología Microbiana (LAMYBIM) de la Universidad Nacional Mayor de San Marcos, Perú. Las cepas provenian de entornos peruanos contaminados con hidrocarburos y fueron catalogadas como sobreproductoras de RL(n= 21), productoras de RL (n=20) y no productoras de RL (n= 20). Las 61 cepas fueron identificadas bioquímicamente con el sistema API 20 NE. La identificación molecular se realizó empleando el gen del RNAr 16S. Se encontró que Pseudomonas aeruginosa fue el microorganismo de mayor prevalencia en los estratos sobreproductores y productores de ramnolípidos. Además, se encontraron: Burkholderia cepacea, Pseudomonas fluorescens, Aeromonas hydrophila y Chryseobacterium indologenes. Los microorganismos no productores de ramnolípidos, también fueron caracterizados bioquímicamente. Mediante amplificación de PCR y electroforesis en gel de agarosa, estandarizados por la UNAM, se evidenció que las cepas seleccionadas poseen los genes: rhlA, rhlB, rhlR y rhlC. Para el secuenciamiento de la región génica rhLABR, se seleccionaron cuatros especies: Pseudomonas aeruginosa T2X-2, Pseudomonas aeruginosa IIIT1P2, Pseudomonas aeruginosa 6K-11 y Pseudomonas aeruginosa ATCC 9027, siguiendo metodología estandarizada por la UNAM y fueron comparados con Pseudomonas aeruginosa PAO1. Nuestros resultados muestran que los genes estudiados en las cepas seleccionadas son sinónimas de sus homólogos en la cepa patrón Pseudomonas aeruginosa PAO1, por lo que las diferencias genotípicas que expliquen la sobreproducción de ramnolípidos deberían hallarse en otros marcadores moleculares no cubiertos en el presente estudio.

No outono de 2011, em fazendas de ciprinídeos localizadas em Iasi, no rio Jijia, diversas infecções bacterianas e cistos externos macroscópicos na pele se desenvolveram como resultado do estresse induzido por fatores bióticos e abióticos. No exame do conteúdo dos cistos, a presença de diversos esporos foi observada, a maioria do gênero Dermocystidium sp. As amostras foram colhidas das seguintes espécies: carpa comum (Cyprinus carpio) e carpa cruciana (Carassius auratus gibelio) de fazenda piscícola, além do rio Jijia. Assim sendo, 35 peixes foram examinados, todos demonstrando cistos, fragmentação da barbatana dorsal e congestão das guelras. O exame histológico da pele mostrou um campo de múltiplos cistos dérmicos com formações circulares claras eosinofílicas (14-16µm) contendo corpo central refratado similar ao relatado para Dermocystidium sp. Amostras de guelras foram retiradas das áreas afetadas para exame MEV, com o propósito de se avaliar não apenas os aspectos da morfologia normal, mas também os aspectos de algumas modificações das áreas afetadas, além da presença da bactéria etiologicamente incriminada: Pseudomonas fluorescens. Os isolados foram identificados por meio de métodos fenotípicos. Todas as amostras que mostraram mobilidade e positividade-oxidase foram testadas usando-se fita API 20 NE. Consequentemente, estas foram taxonomicamente agrupadas na espécie Pseudomonas fluorescens. O microscópio eletrônico de varredura (MEV) foi usado pela primeira vez na caracterização de lesões bacterianas produzidas por Pseudomonas nas guelras de Cyprinus carpio e Carassius auratus gibelio. O diagnóstico de septicemia com espécies condicionais de patogênico de Pseudomonas fluorescens foi correlacionado com os resultados das investigações físico-químicas da água e de dados sobre as condições de reprodução dos animais investigados.

In autumn 2011 in cyprinid farms located in Iasi on the Jijia river, several infections with bacterial strains and macroscopical external cysts on the skin were diagnosedwhich developed as a result of the stress induced by biotic and abiotic factors. On the examination of the cyst contents the presence of numerous spores was observed, mostly of the Dermocystidium spgenusThe samples were taken from the common carp (Cyprinus carpio) and crucian carp (Carassius auratus gibelio)species from the fish farm as well as from the Jijia River. 35 fish were examined, all of them showing cysts, fragmentation of their dorsal fin and congestion of the gills. Histological examination of the skin showed a field of multiple dermal cysts with round light eosinophilic formations (14-16µm) containing a central refractable body similar to that reported for Dermocystidium sp.Gills samples were taken from the affected areas for the SEM examination with the purpose of evaluating not only aspects of normal morphology, but also aspects of some modifications of the affected areas as well as the presence of the etiologically incriminated bacteria Pseudomonas fluorescens. The isolates were identified through phenotypic methods. All the strains that showed mobility and oxidase-positivity were tested using API 20 NE strip. Consequently, they were taxonomically grouped into the species Pseudomonas fluorescens.The scanning electron microscope (SEM) was used for the first time in the characterization of the bacterial lesions produced by Pseudomonasstrains on Cyprinus carpioand Carassius auratus gibeliogills. The diagnosis of septicemia with conditional pathogen species of Pseudomonas fluorescens was correlated with the results of the physico-chemical investigations of water and the data concerning the breeding conditions of the investigated livestock.

RESUMOS. maltophilia contem um novo gene qnr cromossômico denominado Smqnr que contribui para baixa resistência intrínseca a quinolonas. Descrevemos Smqnr em 13 isolados clínicos de S. maltophilia de dois hospitais brasileiros, ao longo do período de dois anos. Os isolados foram identificados pela API 20 NE (bioMérieux, França). Susceptibilidade pelo método de microdiluição dos seguintes antibióticos trimetroprim/sulfametoxazol, ciprofloxacina, levofloxacina, minociclina, ceftazidima, cloranfenicol e ticarcilina/clavulanato foi realizada segundo o CLSI. Detecção do gene de Smqnr foi realizada por PCR. A sequência de Smqnr foi comparada com aquelas depositadas no GenBank. Foi realizada eletroforese em gel de campo pulsado (PFGE) de todos os isolados. Treze isolados contendo Smqnr foram sequenciados e identificados três variantes do gene Smqnr. Todos os 13 isolados de Smqnr apresentaram diferentes padrões por PFGE. Este é o primeiro relato de Smqnr em isolados de S. maltophilia no Brasil.

SUMMARYStenotrophomonas maltophilia contains a novel chromosomally-encoded qnr gene named Smqnr that contributes to low intrinsic resistance to quinolone. We described Smqnr in 13 clinical isolates of S. maltophilia from two Brazilian hospitals, over a 2-year period. The strains were identified by API 20 NE (bioMérieux, France). Susceptibility by microdilution method to trimetroprim/sulfamethoxazole, ciprofloxacin, levofloxacin, minocycline, ceftazidime, chloramphenicol and ticarcillin/clavulanate was performed according to CLSI. PCR detection of Smqnr gene was carried out. The sequence of Smqnr was compared with those deposited in GenBank. Pulsed-field gel electrophoresis (PFGE) of all strains was performed. Thirteen Smqnr positives isolates were sequenced and three novel variants of Smqnr were identified. All 13 Smqnr isolates had distinguishable patterns by PFGE. This is the first report of Smqnr in S. maltophilia isolated in Brazil.

We herein present the case of an adult male patient who consulted for lower extremity edema, a 2- month history of fever and oppressive chest pain radiating to the left arm. He referred neither contact with breeding animals nor consumption of unpasteurized dairy products. A diagnosis of endocarditis was confirmed by cardiac studies. Since the empirical treatment with cephalotin, ampicillin and gentamicin failed, the patient underwent aortic valve replacement. A total of four blood cultures were positive with a gram-negative rod. Bacterial identification was performed using the API 20 NE technique (bioMerieux), the Phoenix automated method (BD) and conventional biochemical tests which were unable to classify the isolate as to genus and species. The strain was sent to the INEI-ANLIS "Dr. Carlos G. Malbran" where it was identified as Brucella canis. The antimicrobial treatment was switched to doxycycline, rifampicin and trimethoprim-sulfamethoxazole with good evolution of the patient. The clinical significance of this case report lies in the possible failure of the empiric antibiotic therapy administered for endocarditis, since B. canis did not respond to the conventional antimicrobial treatment for this pathology.

Se describe el primer caso documentado de endocarditis por Brucella canis en Argentina. El paciente fue un varón adulto que consultó por edemas en miembros inferiores, registros febriles aislados de 2 meses de evolución y dolor precordial opresivo que irradiaba a brazo izquierdo. Negaba contacto con animales de cría o consumo de productos sin pasteurización. Estudios cardiológicos constataron endocarditis infecciosa. Se resuelve cirugía de recambio valvular ante fracaso terapéutico empírico con cefalotina, ampicilina y gentamicina. Los hemocultivos fueron positivos (4 de 4 muestras) con bacilos gram negativos. Se realizó la identificación con técnica API 20 NE (bioMérieux), el método automatizado Phoenix (BD) y las pruebas bioquímicas convencionales, sin concluir género ni especie. Se derivó la cepa al departamento de Bacteriología Especial INEI-ANLIS "Dr. Carlos G. Malbrán" donde se identificó al aislamiento como Brucella canis. Se rotó el esquema terapéutico a doxiciclina, rifampicina y trimetoprima-sulfametoxazol con buena evolución. La importancia del caso radica en la posible falla del tratamiento antimicrobiano empírico administrado para endocarditis, ya que B. canis no responde a los antimicrobianos convencionales para esta patología.

Non-O1, and non-O139 Vibrio cholerae is an infrequent cause of bacteremia. There are no reports of such bacteremia in chronic hemodialysis patients. This work describes the case of a chronic hemodialysis patient that had an episode of septicemia associated with dialysis. Blood cultures were obtained and treatment was begun with vancomycin and ceftazidime. After 6.5 hours of incubation in the Bact/Alert system there is evidence of gram-negative curved bacilli that were identified as Vibrio cholerae by conventional biochemical tests, API 20 NE and the VITEK 2 system. This microorganism was sent to the reference laboratory for evaluation of serogroup and virulence factors and was identified as belonging to the non-O1 and non-O139 serogroup. The cholera toxin, colonization factor and heat-stable toxin were not detected. The isolate was susceptible to ampicillin, trimethoprim-sulfamethoxazole, ciprofloxacin, tetracycline, ceftazidime and cefotaxime by the disk diffusion method and the VITEK 2 system. The patient received intravenous ceftazidime for a 14 day- period and had a favorable outcome.

Vibrio cholerae no-O1, no-O139 es un agente poco frecuente como causal de bacteriemias y no hay informes que documenten su presencia en pacientes en hemodiálisis crónica. Se describe el caso de una paciente en hemodiálisis crónica que presentó un cuadro de sepsis, por lo cual inició un tratamiento con vancomicina y ceftacidima. Al cabo de seis horas y media de incubación en el sistema BACT/ALERT de hemocultivo, se evidenció la presencia de bacilos curvos gram negativos, posteriormente identificados como Vibrio cholerae mediante pruebas bioquímicas convencionales y el uso de los kits API 20 NE y VITEK 2. La evaluación del serogrupo y de la presencia de factores de patogenicidad, realizada en el laboratorio de referencia, determinó que el microorganismo hallado pertenecía al serogrupo no-O1, no-O139. No se detectó la toxina de cólera, tampoco el factor de colonización ni la toxina termoestable. El aislamiento presentó sensibilidad frente a ampicilina, trimetoprima-sulfametoxazol, ciprofloxacina, tetraciclina, ceftacidima y cefotaxima por el método de difusión con discos y por VITEK 2. La paciente cumplió 14 días de tratamiento con ceftacidima endovenosa, con evolución favorable.

Ralstonia mannitolilytica is a glucose non-fermentative, aerobic gram-negative bacillus formerly known as Ralstonia picketti biovar3/"thomasii" of relatively low virulence. It has been isolated from a wide kind of infections like bacteremia, meningitis, endocarditis, osteomielitis and from the respiratory tract of patients with cystic fibrosis. The case presented is related to a 26-year-old male patient with diabetes insipidus, hypothyroidism and histiocytosis X with a two-day-febrile syndrome and chills associated with the presence of a porthacat catheter used for administrating medication. An episode of catheter-related bloodstream infection was documented by using Bact-Alert blood culture system and differential-time-to-positivity method for central venous catheter versus peripheral blood cultures (>120min). Once removed, it was confirmed through Maki semiquantitative technique (>15 ufc). The microorganism was identified by API 20 NE, Vitek 2C and Vitek 1 as. Ralstonia mannitolilytica.

Ralstonia mannitolilytica es un bacilo gramnegativo no fermentador de la glucosa, anteriormente conocido como Ralstonia picketti biovar3/"thomasii", de relativamente baja virulencia. Se aisló de una gran variedad de infecciones como bacteriemia, meningitis, endocarditis, osteomielitis y de la vía respiratoria de pacientes con fibrosis quística. Se presenta un caso de bacteriemia asociada a catéter por Ralstonia mannitolilytica en un hombre de 26 años con diabetes insípida, histiocitosis X e hipotiroidismo que presentaba fiebre de (39 - 40 °C) y escalofríos de dos días de evolución asociados a administración de medicación por portacath. Del cultivo de sangre tomada a través de catéter y de sangre periférica, se aisló Ralstonia mannitolilytica. Se utilizó el sistema automatizado de hemocultivos Bact-Alert y la metodología de tiempo diferencial (>120 minutos) y del catéter por la técnica semicuantitativa de Maki (>15 ufc). El microorganismo fue identificado por Vitek 1 (bioMerieux, Marcy, l'Ètoile, Francia),Vitek 2 Compact (bioMerieux, Marcy, l'Ètoile, Francia) y por Api 20 NE (bioMerieux, Marcy, l'Ètoile, Francia).

Objetivou-se neste trabalho, isolar, identificar e quantificar bactérias diazotróficas existentes nas raízes e folhas de coqueiro (híbrido PB121) cultivados na baixada litorânea de Sergipe. As populações de bactérias diazotróficas nesses órgãos foram quantificadas pela técnica do número mais provável (NMP) em meios semi-sólidos JNFb e NFb, e identificadas por meio de avaliações microscópicas, culturais e de testes bioquímicos dos sistemas API 20 - E e API 20 - NE. O conteúdo de N em meios semi-sólidos NFb e JNFb foi determinado colorimetricamente, após oito dias de incubação das bactérias, a 32ºC, para estimar a capacidade de fixação biológica dos isolados. Enterobactérias pertencentes ao gênero Enterobacter e bactérias com características semelhantes às do gênero Pseudomonas (pseudomonadas) foram predominantes entre os isolados obtidos. As enterobactérias e pseudomonadas isoladas de folhas foram predominantemente endofíticas. Quanto às diazotróficas isoladas de raízes, observou-se predominância de enterobactérias na sua superfície, ao passo que as pseudomonadas ocorreram em proporções semelhantes na superfície e no interior desse órgão. Após oito dias de incubação, os conteúdos de N nos meios com inoculação das bactérias pseudomonadas foram maiores do que nos meios com inoculação das enterobactérias.

The aim of this work was to isolate, identify and quantify diazotrophic bacteria existing in roots and leaves of coconut palms (hybrid PB121) grown in a coastal lowland of Sergipe, in Brazil. Diazotrophic populations in these organs were quantified by the most probable number (MNP) technique on NFb and JNFb semi-solid media and identified by the evaluation of microscopic, cultural and biochemical characteristics of the isolates. Biochemical characteristics were evaluated using the API 20 - E and API 20 - NE galleries. Nitrogen content in NFb and JNFb semi-solid media inoculated with each of the isolates and incubated for eight days at 32ºC was colorimetrically evaluated to estimate the biological nitrogen fixation capacity of the isolates. Bacteria of the Enterobacter genus and pseudomonads formed the predominant types of N2 - fixers. Enterobacteria and pseudomonads isolated from leaves were predominantly endophytic. Regarding to the diazotroph bacteria isolated from roots, it was observed that enterobacteria were predominant in the root-surface, while pseudomonads were found in nearly equivalent proportions in the surface and in the interior of this organ. After eight days of incubation, pseudomonads isolates presented higher N fixation capacity than enterobacteria.