Microscopy, culture, and quantitative real-time PCR examination confirm internalization of mycobacteria in plants.

The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10(8) to 10(10) cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10(4) cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.

Mycobacterium avium subsp. avium and Mycobacterium neoaurum detection in an immunocompromised patient.

Non-tuberculous mycobacteria are increasingly described as infectious agents in immunocompromised patients. A 17-year-old male patient suffering from secondary non-Hodgkin's lymphoma and treated with chemotherapeutic agents was admitted to hospital due to pleuropneumonia. Mycobacterium neoaurum was cultured repeatedly from his sputum and, Mycobacterium avium subsp. avium (M. a. avium) was detected by IS901 qPCR from detached fragments of his intestinal mucosa. We attempted to determine the possible sources of infection by analysing environmental samples from the closed oncology unit and conventional unit in the hospital, and from the patient's home residence and places which he frequented. The environment of the patient harboured mycobacteria (41 isolates in total); however, M. neoaurum was not recovered. M. a. avium was detected by qPCR in the environmental samples from a small flock of hens kept by his neighbour. Although it was not confirmed by DNA fingerprinting methods, the M. a. avium infection could have been acquired through the eating of incompletely cooked eggs.

Real-time quantitative PCR detection of Mycobacterium avium subspecies in meat products.

The aim of this work was to examine various purchased meat products and to find out if any traces of Mycobacterium avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis could be detected in these samples. Analysis of the meat products (raw, cooked, and fermented) was performed using a real-time quantitative PCR (qPCR) method for the detection of specific insertion sequences: duplex qPCR for the detection of IS900 specific for M. avium subsp. paratuberculosis, and triplex qPCR for the detection of IS901 specific for Mycobacterium avium subsp. avium and IS 1245 specific for M. avium subsp. hominissuis. Of the 77 analyzed meat samples, 17 (22%) were found to contain M. avium subsp. paratuberculosis DNA, 4 (5%) samples contained Mycobacterium avium subsp. avium DNA, and in 12 (16%) samples M. avium subsp. hominissuis DNA was detected. The concentration of M. avium subsp. paratuberculosis and M. avium subsp. hominissuis DNA in some meat products exceeded 10(4) genomes per g. Culture examination of these mycobacterial subspecies was negative. By analyzing a range of meat products, we have provided evidence to support the hypothesis that M. avium is present in everyday commodities sold to the general public.

Distribution of Mycobacterium avium subsp. avium and M. a. hominissuis in artificially infected pigs studied by culture and IS901 and IS1245 quantitative real time PCR.

Mycobacterium avium subsp. avium (MAA) and M. a. hominissuis (MAH) belong to the Mycobacterium avium complex (MAC) and are frequently associated with diseases in animals and humans. The aim of this study was to develop a system for rapid and accurate real time quantitative PCR (qPCR) identification and quantification of MAA and MAH. This study included 22 per os infected pigs, of which 10 were infected with MAA, 10 with MAH and 2 were present as a negative control group. From each animal, 21 different tissue samples as well as blood were tested by microscopy, culture and triplex qPCR. The developed triplex qPCR reaction was based on the simultaneous detection of specific insertion sequences, IS901 and IS1245, and also contained an internal amplification control. In both groups of experimentally infected animals, the newly developed triplex qPCR assay proved to be more specific and sensitive in comparison with the other methods used. Contrary to culture examination, triplex qPCR confirmed the infection in all animals infected with MAA, and in eight animals infected with MAH. In conclusion, we developed a quick and sufficiently sensitive triplex qPCR for MAA and MAH detection in tissue and blood samples. From the food safety point of view the presence of MAH in muscles should be considered as a possible threat to human health.