Natural Occurrence of Horizontal Transfer of Mycobacterium avium-Specific Insertion Sequence IS1245 to Mycobacterium kansasii

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. the insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.Foundation for Research Support of the State of São PauloConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilInst Adolfo Lutz Registro, Setor Micobacterias, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilFoundation for Research Support of the State of São Paulo: FAPESP-06/01533-9Web of Scienc

An experimental model of mycobacterial infection under corneal flaps

In order to develop a new experimental animal model of infection with Mycobacterium chelonae in keratomileusis, we conducted a double-blind prospective study on 24 adult male New Zealand rabbits. One eye of each rabbit was submitted to automatic lamellar keratotomy with the automatic corneal shaper under general anesthesia. Eyes were immunosuppressed by a single local injection of methyl prednisolone. Twelve animals were inoculated into the keratomileusis interface with 1 µl of 10(6) heat-inactivated bacteria (heat-inactivated inoculum controls) and 12 with 1 µl of 10(6) live bacteria. Trimethoprim drops (0.1%, w/v) were used as prophylaxis for the surgical procedure every 4 h (50 µl, qid). Animals were examined by 2 observers under a slit lamp on the 1st, 3rd, 5th, 7th, 11th, 16th, and 23rd postoperative days. Slit lamp photographs were taken to document clinical signs. Animals were sacrificed when corneal disease was detected and corneal samples were taken for microbiological analysis. Eleven of 12 experimental rabbits developed corneal disease, and M. chelonae could be isolated from nine rabbits. Eleven of the 12 controls receiving a heat-inactivated inoculum did not develop corneal disease. M. chelonae was not isolated from any of the control rabbits receiving a heat-inactivated inoculum, or from the healthy cornea of control rabbits. Corneal infection by M. chelonae was successfully induced in rabbits submitted to keratomileusis. To our knowledge, this is the first animal model of M. chelonae infection following corneal flaps for refractive surgery to be described in the literature and can be used for the analysis of therapeutic responses.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de OftalmologiaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de Microbiologia, Imunologia e ParasitologiaUNIFESP, EPM, Depto. de OftalmologiaUNIFESP, EPM, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Distribución de patrones PRA en aislamientos clínicos del complejo Mycobacterium avium procedentes de España y Suramérica.

Mycobacterium avium complex (MAC) infections are the most frequent systemic infections associated with advanced AIDS. DNA probes for accurate identification of mycobacteria are available but are very expensive in many Latin American settings. Consequently, most Latin American diagnostic laboratories employ inaccurate and outdated tests for mycobacteria identification. Therefore, PCR restriction analysis (PRA) of the hsp65 gene was evaluated for the identification of 163 MAC human isolates originated from Spain and South America. The predominant PRA type in each country was: M. avium type I in Argentina (23/42, 55%) and Brazil (48/72, 67%), M. avium type II in Spain (18/26, 69%) and M. avium type III in Colombia (10/23, 43%). The Colombia frequency is noteworthy, since the PRA type III was quite infrequent in the other three countries. Furthermore, its presence has not been reported outside the Americas. The advantages and disadvantages of PRA in diagnostic mycobacteriology are discussed.La infección por el complejo Mycobacterium avium (MAC) es la infección sistémica más frecuente en la fase terminal del SIDA. Las sondas de ADN disponibles en el mercado para la identificación de micobacterias son muy precisas pero extremadamente costosas. Por eso, la mayoría de los laboratorios clínicos de Latinoamérica aún tipifican micobacterias mediante pruebas fenotípicas que son lentas, laboriosas y poco precisas. En este trabajo se aplicó el análisis del polimorfismo de los fragmentos de restricción del gen hsp65 (PRA) a la identificación de MAC en 163 aislamientos clínicos procedentes de España y Suramérica. El genotipo PRA predominante en cada país fue: M. avium tipo I en Argentina (23/42, 55%) y Brasil (48/72, 67%), M. avium tipo II en España (18/26, 69%) y M. avium tipo III en Colombia (10/23, 43%). Este último genotipo, que aún no fue descrito fuera del continente americano, resultó muy infrecuente en los otros tres países del estudio. Se discuten ventajas e inconvenientes de la aplicación del PRA al diagnóstico micobacteriológico

First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

Background: in a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. the transfer mechanism of this insertion sequence to M. kansasii was investigated here.Methodology/Principal Findings: A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. the linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau.Conclusions/Significance: Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Cooperacion Interuniversitaria UAM-Banco Santander con America Latina (CEAL), UAM, SpainConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilLab Nacl Comp Cient, Petropolis, BrazilUniv Autonoma Madrid, Fac Med, Dept Prevent Med, Madrid, SpainInst Adolfo Lutz Registro, Nucleo TB & Micobacterioses, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilFAPESP: FAPESP - 06/01533-9Web of Scienc

Comparison of genetic patterns of Mycobacterium avium strains isolated from humans and animals

BV UNIFESP: Teses e dissertaçõe

Analysis of the behavior of phenotypic variants of Mycobacterium avium strains in animals and cells

Mycobacterium avium é uma bactéria ambiental e na classificação de patogenicidade está incluída entre as micobactérias potencialmente patogênicas, pois se trata de um patógeno oportunista em animais e humanos. O interesse em estudar fatores de virulência e patogenicidade destas bactérias aumentou após o isolamento de M. avium em amostras de pacientes portadores do vírus da Imunodeficiência Humana (HIV). O objetivo deste estudo foi isolar variantes de colônias de sete cepas de M. avium isoladas de fontes humanas e animais, caracterizadas molecularmente em nosso laboratório e avaliar o comportamento e a capacidade de multiplicação das variantes fenotípicas em experimentos com animais (hamster) e cultura de células (macrófagos). Nos cultivos iniciais, cinco das sete cepas (71,4 por cento) apresentaram variantes de colônias OP e TL e duas cepas (28,6 por cento) não apresentaram variações no fenótipo das colônias. As colônias OP recuperadas dos baços dos animais inoculados mantiveram a mesma morfologia, branca opaca e lisa, enquanto que houve alteração na mortologia nas variantes TL, de lisa transparente para rugosa transparente (TL-Rg). As variantes mantiveram a mesma identificação original por PRA-hsp65 e a mesma tipificação por RFLP-IS1245 após a passagem por animais. Com todas as cepas houve maior recuperação de UFC por grama de baço e maior índice de multiplicação intracelular com a variante TL quando comparada à variante OP. Foi avaliado o percentual de células infectadas nos dias O e 7. Houve aumento no percentual de macrófagos infectados no dia 7 com todas as cepas, com diferença estatisticamente significante em 5 das 12 variantes das cepas estudadas. Quanto ao número de bacilos por macrófago infectado, foi observado que no dia O a maioria dos macrófagos infectados com as variantes OP e TL albergaram de 1 a 15 bacilos enquanto que no dia 7 a quantidade de bacilos que os macrófagos albergaram foi distribuída em freqüências de 1 a mais que 50. Com todas as cepas, a variante TL apresentou uma tendência de distribuição nas freqüências mais elevadas quando comparada à distribuição da variante OP no dia 7. A variante TL das cepas do estudo apresentou maior capacidade de sobrevivência e multiplicação em experimentos "in vivo" e "in vitro".BV UNIFESP: Teses e dissertaçõe

Natural Occurrence of Horizontal Transfer of Mycobacterium avium- Specific Insertion Sequence IS1245 to Mycobacterium kansasii▿

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. The insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome

Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting

Abstract Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting