Non-tuberculous mycobacteria (NTM) are studied not only for their importance as emerging opportunistic pathogens but also for their applications in biotechnology and bioremediation. Our aim was to determine the occurrence and diversity of mycobacteria in different aquatic habitats of General Pico city, Province of La Pampa. The percentage of samples with positive cultures for mycobacteria were the following: 37.5% recovered from the water supply distribution system; 32.6% from the aquifer that supplies water to the distribution system; 36.8% from rain water; 53.1% from the two wetlands in the area of influence; 80% from indoor swimming pools; and 33.3% from water fountains in downtown public squares. Of the 90 NTM isolates, 8.9% could not be identified at the species level with any of the used methods, phenotypic tests and molecular methods. Mycobacterium fortuitum and Mycobacterium gordonae were the most frequently isolated species. Some of the identified species such as, M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum and M. nonchromogenicum, have been reported in cases of mycobacteriosis in Argentina. Mycobacteria with values higher than 0.8mg/ml of residual active chlorine were not recovered from the drinking water supply network, whereas in the swimming pools the presence of up to 1.5 mg/l was not a constraint. Based on our results, the presence of mycobacteria in aquatic environments is close to 35% and their occurrence and diversity is affected both by contact with man and his activities as well as by the existence of animal life.

Las micobacterias no tuberculosas (MNT) no solo se estudian por su importancia como patógenos oportunistas, sino también por sus aplicaciones en biotecnología y biorremediación. Nuestro objetivo fue determinar la presencia de micobacterias en los distintos hábitats acuáticos de la ciudad de General Pico (provincia de La Pampa), así como su diversidad. Los porcentajes de muestras positivas a micobacterias fueron los siguientes: 37,5% en el sistema de distribución de agua de red, 32,6% en el acuífero que abastece dicho sistema, 36,8% en el agua proveniente de las precipitaciones, 53,1% en los humedales del área de influencia, 80% en los natatorios cubiertos y 33,3% en las fuentes decorativas ubicadas en plazas públicas. De los 90 aislamientos de MNT obtenidos el 8,9% no logró ser identificado a nivel de especie con los métodos utilizados, que incluyeron pruebas fenotípicas y métodos moleculares. Las especies más frecuentemente aisladas fueron Mycobacterium fortuitum y Mycobacterium gordonae. Algunas especies identificadas han sido reportadas en casos de micobacteriosis en nuestro país, entre ellas M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum y M. nonchromogenicum. No se aislaron MNT en muestras de agua de red con concentraciones de cloro activo residual mayores de 0,8mg/l, mientras que en los natatorios la presencia de hasta 1,5mg/l de cloro activo residual no fue una limitante para la proliferación de estos microorganismos. Se puede considerar que la incidencia de micobacterias en los ambientes acuáticos de General Pico es cercana al 35%, y que la presencia de estos microorganismos y su diversidad se ve afectada por el contacto con el hombre y sus actividades, como así también por la existencia de vida animal.

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

Neste estudo, realizou-se genotipagem de isolados de Mycobacterium bovis, provenientes de amostras de tecidos de bovinos positivos no teste cervical comparativo (TCC) para tuberculose em Mato Grosso do Sul, por meio da técnica de spoligotyping. Tecidos de 13 bovinos positivos, oriundos de diferentes municípios do estado, foram cultivados em meio de Stonebrink. As colônias resultantes foram submetidas à coloração de Ziehl-Neelsen e todos os isolados apresentaram características tintoriais de BAAR. Os 13 isolados de BAAR foram identificados por PCR multiplex (mPCR). O gene hsp65 foi alvo para identificação de Mycobacterium spp, a sequência de inserção IS6110 foi alvo para identificação de complexo Mycobacterium tuberculosis (CMT) e a região rvd1rv2031c foi explorada para detecção de M. bovis. Os isolados micobacterianos foram genotipados pela técnica de spoligotyping. Dos 13 bovinos, sete tinham pelo menos uma lesão sugestiva de tuberculose em linfonodos retrofaríngeos, parotídeos e pulmonares ou no pulmão, e em seis não foram encontradas lesões visíveis sugestivas da doença. Na mPCR, 11/13 (84,6%) isolados foram positivos para Mycobacterium spp; 8/13 (61,5%) positivos para CMT e 7/13 (53,8%) positivos para M. bovis. Com base no spoligotyping, oito isolados de BAAR foram agrupados dentro de três diferentes agrupamentos de genótipos e uma amostra remanescente apresentou perfil único, sendo quatro isolados com padrão de espoligotipo SB0121, dois SB1145, dois SB0881 e um SB0140. A técnica de spoligotyping demonstrou que há diversidade genética entre os espoligotipos presentes no estado de Mato Grosso do Sul, embora predomine o perfil SB0121

Spoligotyping was performed in the present study to genotype Mycobacterium bovis isolates obtained from tissues of cattle that were positive in the comparative intradermal tuberculin test (CITT) in the state of Mato Grosso do Sul (Brazil). Tissue samples from 13 positive cattle from different municipalities of the state were cultured using a Stonebrink medium. The resulting colonies were subjected to Ziehl-Neelsen staining and all isolates exhibited the staining characteristics of AFB. The 13 isolates of AFB were identified by means of a multiplex PCR (mPCR) assay. The hsp65 gene was targeted for the identification of Mycobacterium spp., whereas the IS6110 insertion sequence was targeted for the identification of the Mycobacterium tuberculosis complex (MTC) and the rvd1rv2031c region was explored for the detection of Mycobacterium bovis. The spoligotyping assay was performed to genotype mycobacterial isolates. Of the 13 cattle, seven had at least one lesion suggestive of tuberculosis in the retropharyngeal, parotid and lung lymph nodes or lung. The remaining six exhibited no lesions suggestive of the disease. In the mPCR, 11 of the 13 isolates (84.6%) were positive for Mycobacterium spp., 8/13 (61.5%) were positive for the MTC and 7/13 (53.8%) were positive for M. bovis. Based on the spoligotyping, eight isolates were grouped into three different groups of genotypes and one isolate exhibited an orphan type. Four isolates exhibited spoligotype pattern SB0121, while two isolates were associated with the pattern SB1145, another two were associated with pattern SB0881 and one was associated with pattern SB0140. Spoligotyping confirmed the genetic diversity present among isolates found in the state of Mato Grosso do Sul. In addition, SB0121 was confirmed as the predominant profile.

Tuberculosis (TB) and multidrug and extensively drug-resistant (DR) TB are important public health problems that are spreading worldwide. The aims of this study were to determine the sensitivity and specificity of the GenoType® MT BDRplus assay from smear-positive clinical specimens and isolates and to explore its possible application in routine work. Clinical samples were previously decontaminated using NaOH-N-acetyl-L-cystein or NaOH-ClNa hypertonic solution for Ziehl-Neelsen staining and cultures. The leftover sediments of smear-positive samples were stored at -20 ºC, 70 of which were selected to be included in this study according to their DR profile. Thirty DR Mycobacterium tuberculosis isolates were also assessed. Sequencing was used as gold standard to detect mutations conferring isoniazid (INH) and rifampicin (RIF) resistance. Valid results were obtained in 94.0 % of the samples and 85.5 % (53/62) of the INH-R samples were properly identified. Mutations in the katGS315T gene and inhA C-15T gene promoter region were present in 59.7 % (37/62) and 25.8% (16/62) of the INH-R samples, respectively. The system could also identify 97.7 % (41/42) of the RIF-R samples; the mutations found were rpoBS531L (66.7 %, 28/42), D516V (19.0 %, 8/42), H526Y and S531P/W (4.8 %, 2/42 each one), and S522L/Q (2.4 %, 1/42). A 98.8 % concordance between the GenoType assay and sequencing was obtained. GenoType® MT BDRplus has demonstrated to be easy to implement and to perform in clinical laboratories and useful for a rapid detection of DR M. tuberculosis from decontaminated sputa and clinical isolates. Therefore, this assay could be applied as a rapid tool to predict INH-R and/or RIF-R in DR risk cases.

La tuberculosis (TBC), y la TBC multi y extensivamente drogo-resistentes (DR) son importantes problemas de salud pública mundial. El objetivo de este estudio fue determinar la sensibilidad y especificidad del sistema GenoType® MT BDRplus a partir de esputos (baciloscopía positiva) y aislamientos clínicos de Mycobacterium tuberculosis, explorando su aplicación clínica. Previo a la tinción de Ziehl-Neelsen y al cultivo, las muestras fueron descontaminadas mediante NaOH-N-acetyl-L-cisteina o la solución hipertónica NaOH-NaCl. Los sedimentos remanentes se conservaron a -20 ºC, y 70 de ellos fueron incluidos en este estudio según su perfil de DR. Treinta cepas de M. tuberculosis DR fueron también evaluadas. La secuenciación fue utilizada como método de referencia para la detección de mutaciones que confieren resistencia a isoniacida (INH) y rifampicina (RIF). Se obtuvieron resultados válidos en el 94,0 % de las muestras, identificándose al 85,5 % (53/62) de las INH-R. La mutación katG S315T estuvo presente en 59,7 % (37/62), y la mutación C-15T del promotor del gen inhA en 25,8 % (16/62) de las mismas. El sistema identificó el 97,7 % (41/42) de las muestras RIF-R. Las mutaciones encontradas fueron rpoB S531L (66,7 %, 28/42), D516V (19,0 %, 8/42), H526Y y S531P/W (4,8 %, 2/42 cada una de ellas) y S522L/Q (2,4 %, 1/42). La concordancia entre el GenoType y la secuenciación fue del 98,8 %. El sistema GenoType® MT BDRplus resultó ser útil, fácil de realizar e implementar para la detección rápida de M. tuberculosis DR. Por lo tanto, este ensayo podría ser aplicado como una herramienta rápida para el diagnostico de TBC DR, principalmente en aquellos casos asociados a factores de riesgo.