Comparison of methods for the isolation of mycobacteria from water treatment plant sludge.

Nontuberculous mycobacteria (NTM) are ubiquitous organisms in all natural ecosystems, including water environments. Several of these species are potential pathogens which affect human health. NTM most commonly cause pulmonary, skin or soft tissue infections. Primary sludge obtained from the water treatment plants of four drinking water reservoirs were subjected to analysis for mycobacteria. Five decontamination methods (5% oxalic acid, modified Petroff, HCl-NaOH, N-acetyl-L-cysteine-sodium hydroxide and 0.05% cetylpyridinium chloride), three growth media (Herrold's egg yolk medium with and without the antibiotic cocktail PANTA and Löwenstein-Jensen medium with sodium pyruvate) and three incubation temperatures (25, 30 and 37 °C) for isolation of mycobacteria were compared in the analysis of 18 sludge samples. To evaluate examined methods, the overall positive, negative, and contamination rate, and these rates in respect to localities are taken into account. Statistical analysis demonstrated that the best combination for the recovery of mycobacteria with the minimum number of contaminating microorganisms is 5% oxalic acid decontamination cultured on Herrold's egg yolk medium with the antibiotic cocktail PANTA at 25 °C. The least suitable is N-acetyl-L-cysteine-sodium hydroxide decontamination cultured on Löwenstein-Jensen medium with sodium pyruvate at 25 °C. From 18 sludge samples we isolated 27 mycobacterial species or groups; Mycobacterium algericum, M. arabiense, M. heraklionense, M. minnesotense, M. moriokaense, M. salmoniphilum and M. vulneris were isolated from the natural water environment for the first time. Because the natural water environment is the main source of potentially pathogenic mycobacteria for humans, it is important to direct particular focus to newly described mycobacterial species.

The water environment as a source of potentially pathogenic mycobacteria.

Nontuberculous mycobacteria (NTM) are ubiquitous organisms of a wide variety of environmental reservoirs, including natural and municipal water, soil, aerosols, protozoans, animals and humans. Several of these species are potential pathogens which affect human health. The aim of this study was to determine the occurrence of NTM in the water environment. Samples were taken from 13 water-related facilities including fish ponds, storage ponds, drinking water reservoirs and an experimental recirculation system. Altogether, 396 samples of water, sediment and aquatic plants were collected and analysed. All samples were examined using conventional culture methods. Suspected microbial isolates were subjected to polymerase chain reaction analysis and identified using partial sequence analysis of the 16S rDNA gene. The culture revealed 94/396 samples (23.7%) that contained mycobacteria. Among known NTM we identified potentially pathogenic mycobacteria isolated from the fresh water environment for the first time: Mycobacterium asiaticum, M. chimaera, M. interjectum, M. kumamotonense, M. lentiflavum, M. montefiorense, M. nebraskense, M. paraffinicum and M. simiae. Epidemiologic studies suggest that the natural water environment is the principal source of human exposure. Our results indicate that besides the well-known potentially pathogenic mycobacteria it is important to observe occurrence, proliferation and persistence of newly discovered mycobacterial species.

Nontuberculous mycobacteria in freshwater fish and fish products intended for human consumption.

Nontuberculous mycobacteria (NTM) are potentially pathogenic agents commonly found in natural ecosystems, while food is considered to be another source of NTM for humans. We investigated a total of 92 tissue samples of freshwater fish and fish products: fish directly obtained from ponds (n=25), retail fresh (n=23) and frozen fish (n=23) and smoked fish products (n=21). Culture examination for the presence of mycobacteria was positive in 11 (11.9%) from all the examined samples. The 15 obtained isolates were identified as Mycobacterium fortuitum (n=5), M. immunogenum (n=2), M. phocaicum/ mucogenicum (n=1), M. neoaurum (n=2), M. peregrinum (n=2), M. porcinum (n=1) and M. senegalense/houstonense/conceptionense (n=2). NTM DNA was found in one (4.0%) sample of fresh fish from ponds and in 60.9% and 91.3% of retail fresh and frozen fish, respectively. None of the smoked fish products contained NTM DNA. The results of our study suggest that freshwater fish and fish products, especially retail frozen fish, might be a reservoir of NTM for humans, and proper handling and treatment before consumption of such products is recommended.

Dynamics of mono- and dual-species biofilm formation and interactions between Staphylococcus aureus and Gram-negative bacteria.

Microorganisms are not commonly found in the planktonic state but predominantly form dual- and multispecies biofilms in almost all natural environments. Bacteria in multispecies biofilms cooperate, compete or have neutral interactions according to the involved species. Here, the development of mono- and dual-species biofilms formed by Staphylococcus aureus and other foodborne pathogens such as Salmonella enterica subsp. enterica serovar Enteritidis, potentially pathogenic Raoultella planticola and non-pathogenic Escherichia coli over the course of 24, 48 and 72 h was studied. Biofilm formation was evaluated by the crystal violet assay (CV), enumeration of colony-forming units (CFU cm-2 ) and visualization using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). In general, Gram-negative bacterial species and S. aureus interacted in a competitive manner. The tested Gram-negative bacteria grew better in mixed dual-species biofilms than in their mono-species biofilms as determined using the CV assay, CFU ml-2 enumeration, and CLSM and SEM visualization. In contrast, the growth of S. aureus biofilms was reduced when cultured in dual-species biofilms. CLSM images revealed grape-like clusters of S. aureus and monolayers of Gram-negative bacteria in both mono- and dual-species biofilms. S. aureus clusters in dual-species biofilms were significantly smaller than clusters in S. aureus mono-species biofilms.

Nontuberculous Mycobacteria on Ready-to-Eat, Raw and Frozen Fruits and Vegetables.

The consumption of fruits and vegetables is increasing worldwide because of the positive impact of these foods on human health. Ready-to-eat, raw whole, and frozen fruits and vegetables were purchased from markets and examined for the presence of nontuberculous mycobacteria (NTM) using culture, real-time PCR (qPCR), and sequencing. Using qPCR, Mycobacterium sp. at 10(0) to 10(4) ge/g (genome equivalents per gram) was found in almost all of the 178 samples; members of the M. avium complex were found only sporadically. Culture and sequencing revealed the presence of 22 viable NTM isolates in 17 samples. In addition to NTM commonly found in the environment, several rarely described isolates of viable NTM were recovered. The presence of Mycobacterium shigaense, which has been previously isolated only from human patients, was found in lettuce, the first time that this species has been found in an environmental sample. Mycobacterium parmense, Mycobacterium palustre, and Mycobacterium llatzerense, which have been previously isolated from human patients and occasionally from soil and water, were recovered from leafy green vegetables. Strawberries and cut salad mixes contained Mycobacterium algericum, Mycobacterium fallax, and Mycobacterium minnesotense. NTM are primarily nonpathogenic. However, consumption of fruits or vegetables contaminated with NTM could represent a health risk for immunocompromised people, children, and the elderly.

Presence of Mycobacterium avium subspecies and hepatitis E virus in raw meat products.

Meat and meat products may be the source of various pathogenic and potentially pathogenic agents for humans. We ascertained the occurrence of Mycobacterium avium subsp. paratuberculosis, subsp. avium, and subsp. hominissuis, and hepatitis E virus in retail raw meat products. The DNA of at least one of the target M. avium subspecies was detected in 26 (29.2%) of 89 analyzed samples of meat products. Fourteen (15.7%), 1 (1.1%), and 17 (19.1%) samples contained the DNA of Mycobacterium avium subsp. paratuberculosis, subsp. avium, and subsp. hominissuis, respectively. The number of mycobacterial cells per gram of meat products determined by real-time quantitative PCR ranged from 1.15 × 10(2) to 6.97 × 10(3). Mycobacterium chitae and Mycobacterium nonchromogenicum were isolated from three (3.4%) samples. Culture examination was not positive for any M. avium subspecies. Hepatitis E virus RNA was not detected in any of the samples.

The influence of culture conditions on the identification of Mycobacterium species by MALDI-TOF MS profiling.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) represents a simple reliable approach for rapid bacterial identification based on specific peptide/protein fingerprints. However, cell-wall characteristics of mycobacterial species, and their well known stability, complicate MALDI-TOF MS profiling analysis. In this study, we tested two recently published protocols for inactivation and disruption of mycobacteria, and we also examined the influence of different culture conditions (four culture media and five cultivation times) on mass spectral quality and the discriminatory power of the method. We found a significant influence of sample pretreatment method and culture medium on species identification and differentiation for a total of 10 strains belonging to Mycobacterium phlei and Mycobacterium smegmatis. Optimum culture conditions yielding the highest identification success rate against the BioTyper database (Bruker Daltonics) and permitting the possibility of automatic acquisition of mass spectra were found to be distinct for the two mycobacterial species examined. Similarly, individual changes in growth conditions had diverse effects on the two species. For these reasons, thorough control over cultivation conditions should always be employed to maximize the performance and discriminatory power of MALDI-TOF MS profiling, and cultivation conditions must be optimized separately for individual groups of mycobacterial species/strains.