Biofilm formation and biocide susceptibility testing of Mycobacterium fortuitum and Mycobacterium marinum

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC trade mark assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state.     

Biofilm Formation and Biocide Susceptibility Testing of Mycobacterium fortuitum and Mycobacterium marinum

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC™ assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state. Received: 15 February 2002 / Accepted: 28 March 2002     

Rapidly growing mycobacteria in TB/HIV co-infection: a report of two cases focusing on difficulties in diagnosis and management

Recent reports indicate an increase in rates of infection and disease due to rapidly growing mycobacteria (RGM) in patients with pre-existing chronic lung disease. Studies have described difficulties in correctly identifying closely related species, even when proper methodologies are adopted, and several different gene targets have been proposed. We describe two cases of RGM infection in a 29-year-old HIV-1 positive Congolese man and a 19-year-old HIV-1 positive Liberian woman, respectively, both with bronchiectasis due to previous Mycobacterium tuberculosis (MTB) infection. Mycobacterium porcinum and Mycobacterium bolletii were identified in bronchoalveolar lavage fluid and sputum, respectively. After starting the patients on antiretroviral treatment and primary prophylaxis against non-tuberculous mycobacteria (NTM), and ensuring that they adhered to their prescribed regimen, we observed an improvement in their clinical condition and mycobacteria cleared from their respiratory specimens. Management of RGM respiratory infection in immunocompromised patients has to be evaluated on a case-by-case basis, taking into account the patient's pulmonary sequelae, adherence to multiple treatments and immune profile.     

Mycobacterial biofilms: a greasy way to hold it together

Microorganisms growing on surfaces can form biofilms under certain conditions. In this issue of Cell, Ojha et al. (2005) investigate biofilm formation in mycobacteria. They identify new cell-wall components that are required for the formation of architecturally complex mature biofilms in these bacteria and the surprising involvement of a chaperone protein in this process.     

Biofilm formation and dispersal and the transmission of human pathogens

Several pathogenic bacterial species that are found in the environment can form complex multicellular structures on surfaces known as biofilms. Pseudomonas aeruginosa, Vibrio cholerae and certain species of nontuberculous mycobacteria are examples of human pathogens that form biofilms in natural aquatic environments. We suggest that the dynamics of biofilm formation facilitates the transmission of pathogens by providing a stable protective environment and acting as a nidus for the dissemination of large numbers of microorganisms; both as detached biofilm clumps and by the fluid-driven dispersal of biofilm clusters along surfaces. We also suggest that emerging evidence indicates that biofilm formation conveys a selective advantage to certain pathogens by increasing their ability to persist under diverse environmental conditions.     

Isolation and automated ribotyping of Mycobacterium lentiflavum from drinking water distribution system and clinical specimens

Automated ribotyping as a tool for identifying of nontuberculous mycobacteria was evaluated. We created a database comprising of riboprints of 60 strains, representing 32 species of nontuberculous mycobacteria. It was shown that combined ribopatterns generated after digestion with EcoRI and PvuII were distinguishable between species of both slow-growing and rapid-growing mycobacteria. The findings were in good agreement with the 16S rRNA gene sequencing results, allowing correct identification of Mycobacterium lentiflavum isolated from clinical specimens and from biofilms growing in public water distribution system. The automated ribotyping was powerful in discriminating between M. lentiflavum and closely related species M. simiae and M. palustre. Mycobacterium lentiflavum strains from drinking water biofilms were resistant to two to four antimycobacterial drugs. The drinking water distribution system may, thus, be a source of nontuberculous mycobacteria resistant to multiple drugs.     

Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter(-1). The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r(2) = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm(2)). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.     

Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation,Structural Integrity,and Tolerance to Antibiotics

Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.     

Mycobacterium marinum biofilm formation reveals cording morphology

Abstract The emergence of the nontuberculosis mycobacteria (NTM) as clinically relevant pathogens has warranted the study of these ubiquitous organisms in the context of their likely environmental niche, the biofilm. We assayed the NTM bacterium Mycobacterium marinum strain 1218R, a fish outbreak isolate, for biofilm formation on different surfaces over time using three different methods. Using the MBEC system, biofilm development occurred continually over the 14-day culture period reaching a mature or stable biofilm state after 7 days postinoculation. Quantification of M. marinum biofilm formation on high-density polyethylene (HDPE), polycarbonate (PC) and silicon (Si) coupons over a 14-day period was evaluated using a continuous flow reactor system. M. marinum developed biofilms on all of the surfaces tested. However, substantially more biofilm accumulated on the silicon than on the other substrates (Si>HDPE>PC) under the same growth conditions indicating that silicon was the most effective substratum studied for the generation of M. marinum biofilms and suggesting a correlation between surface hydrophobicity and attachment. Finally, confocal laser scanning microscopy was used to visualize M. marinum biofilm development in situ over time and revealed an unusual biofilm ultrastructure. Large cell clusters attached to the surface grew in parallel sinuous arrays of cells that formed large cords.     

The Effect of Long-Term Storage on Mycobacterium bovis

It was established that when stored for many years (10–13 years) in low-temperature conditions (3°C), without sub-culture on a nutrient medium, Mycobacterium bovis grew as visible colonies along the line of inoculation. However, due to long-term storage in conditions of low temperature (3°C) morphology of mycobacteria differed significantly from initial cultures formed by rod-shaped bacteria. Some of them became pigment-forming and smooth on the surface. Unlike the initial strain of mycobacteria, a perennial bacteria stored under hard conditions did not cause the death of guinea pigs or their sensitization to a purified protein derivative for mammals. Morphological forms of the perennial mycobacteria had the following changes: pigment forming, L-forms of the vesicular type, non-acid-fast thread-like (filamentous) bacillary forms, and elementary bodies when compared to the initial strain. There were also some genetic changes in the target DNA due to the long-term storage of M. bovis. It may indicate a mutation in the pathogen’s DNA. These mycobacteria had altered biochemical activity during storage. The number of passages on the solid nutrient medium did not affect their fermentative activity. However, the low cultivation temperature increases mycobacterial catalase activity and the ability to hydrolyze Tween-80.     

Impacts of hydrodynamic conditions and microscale surface roughness on the critical shear stress to develop and thickness of early-stage Pseudomonas putida biofilms

Biofilms can increase pathogenic contamination of drinking water, cause biofilm-related diseases, alter the sediment erosion rate, and degrade contaminants in wastewater. Compared with mature biofilms, biofilms in the early-stage have been shown to be more susceptible to antimicrobials and easier to remove. Mechanistic understanding of physical factors controlling early-stage biofilm growth is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and microscale surface roughness on the development of early-stage Pseudomonas putida biofilm through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that early-stage biofilm growth is suppressed under high flow conditions and that the local velocity for early-stage P. putida biofilms (growth time < 14 h) to develop is about 50 μm/s, which is similar to P. putida's swimming speed. We further illustrate that microscale surface roughness promotes the growth of early-stage biofilms by increasing the area of the low-flow region. Furthermore, we show that the critical average shear stress, above which early-stage biofilms cease to form, is 0.9 Pa for rough surfaces, three times as large as the value for flat or smooth surfaces (0.3 Pa). The important control of flow conditions and microscale surface roughness on early-stage biofilm development, characterized in this study, will facilitate future predictions and managements of early-stage P. putida biofilm development on the surfaces of drinking water pipelines, bioreactors, and sediments in aquatic environments.     

Specific antibodies and mycobacterial antigens in patient sera with pulmonary tuberculous and nontuberculous diseases. Note II

"Two-assay" tests (TAT), immunoenzymatic determination of both specific antibodies and mycobacterial antigens in sera of tuberculous and non-tuberculous subjects, was undertaken in our territorial conditions, where BCG vaccination is systematically applied and the prevalence of tuberculous infection is relatively high. The sensitivity of the method, calculated on 42 patients with active pulmonary tuberculosis and on 39 patients with post-tuberculosis syndromes is high, i.e. 0.952. The specificity of the method separately calculated for 44 young subjects (under 21 years old), for 78 healthy adults and for 201 lung diseased patients, bacteriologically not ascertained as tuberculosis at the moment of sera prelevation, varied between 0.830 and 0.489. "TAT", performed with crude immunologic reagents, produces false-positive reactions in early BCG vaccinated subjects. Method specificity low values in pulmonary non-tuberculous patients group may be partially explained by the difficulty in establishing the real relationships, in time, between host and mycobacteria, by the bacteriological method imperfections or sample prelevating methods. Our results certainly underestimate the diagnosis value of "TAT".     

Glycopeptidolipid Acetylation Affects Sliding Motility and Biofilm Formation in Mycobacterium smegmatis

The absence of glycopeptidolipids (GPLs) abolishes the ability of mycobacteria both to slide over the surface of motility plates and to form biofilms on polyvinyl chloride. In a screen for biofilm-defective mutants of Mycobacterium smegmatis mc(2)155, a new mutant was obtained that resulted in partial inhibition of both processes and also showed an intermediate rough colony morphology. The mariner transposon insertion mapped to a GPL biosynthesis gene (atf1) which encodes a putative acetyltranferase involved in the transfer of acetyl groups to the glycopeptide core. Physical characterization of the GPLs from the atf1 mutant demonstrated that they were not acetylated.     

Factors Influencing Numbers of Mycobacterium avium, Mycobacterium intracellulare, and Other Mycobacteria in Drinking Water Distribution Systems

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter⁻¹. The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r² = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm²). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.     

Mycobacteria in water and loose deposits of drinking water distribution systems in Finland

Drinking water distribution systems were analyzed for viable counts of mycobacteria by sampling water from waterworks and in different parts of the systems. In addition, loose deposits collected during mechanical cleaning of the main pipelines were similarly analyzed. The study covered 16 systems at eight localities in Finland. In an experimental study, mycobacterial colonization of biofilms on polyvinyl chloride tubes in a system was studied. The isolation frequency of mycobacteria increased from 35% at the waterworks to 80% in the system, and the number of mycobacteria in the positive samples increased from 15 to 140 CFU/liter, respectively. Mycobacteria were isolated from all 11 deposits with an accumulation time of tens of years and from all 4 deposits which had accumulated during a 1-year follow-up time. The numbers of mycobacteria were high in both old and young deposits (medians, 1.8 x 10(5) and 3.9 x 10(5) CFU/g [dry weight], respectively). Both water and deposit samples yielded the highest numbers of mycobacteria in the systems using surface water and applying ozonation as an intermediate treatment or posttreatment. The number and growth of mycobacteria in system waters correlated strongly with the concentration of assimilable organic carbon in the water leaving the waterworks. The densities of mycobacteria in the developing biofilms were highest at the distal sites of the systems. Over 90% of the mycobacteria isolated from water and deposits belonged to Mycobacterium lentiflavum, M. tusciae, M. gordonae, and a previously unclassified group of mycobacteria. Our results indicate that drinking water systems may be a source for recently discovered new mycobacterial species.     

Nutrient dynamics and successional changes in a lentic freshwater biofilm

SUMMARY 1. Colonisation, species composition, succession of microalgae and nutrient dynamics in biofilms grown under light and dark conditions were examined during the initial phases of biofilm development in a lentic freshwater environment.
2. Biofilms were developed on inert (perspex) panels under natural illuminated and experimental dark conditions and the panels were retrieved for analysis after different incubation periods. Analysed parameters included biofilm thickness, algal density, biomass, chlorophyll a , species composition, total bacterial density and nutrients such as nitrite, nitrate, phosphate and silicate.
3. Biofilm thickness, algal density, biomass, chlorophyll a and species richness were significantly higher in light-grown biofilms, compared with dark-grown biofilms. The light-grown biofilms showed a three-phased succession pattern, with an initial domination of Chlorophyceae followed by diatoms (Bacillariophyceae) and finally by cyanobacteria. Dark-grown biofilms were mostly dominated by diatoms.
4. Nutrients were invariably more concentrated in biofilms than in ambient water. Nutrient concentrations were generally higher in dark-grown biofilms except in the case of phosphate, which was more concentrated in light-grown biofilms. Significant correlations between nutrients and biofilm parameters were observed only in light-grown biofilms.
5. The N : P ratio in the biofilm matrix decreased sharply in the initial 4 days of biofilm growth; ensuing N-limitation status seemed to influence biofilm community structure. The N : P ratios showed significant positive correlations with the chlorophycean fraction in both light and dark-grown biofilms, and low N : P ratio in the older biofilms favoured cyanobacteria. Our data indicate that nutrient chemistry of biofilm matrix shapes community structure in microalgal biofilms.     

Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks,and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.     

Biofilms: new ideas for an old problem

Microbial cells, under moist conditions, are able to adhere to surfaces and to form structured communities embedded in a matrix of extracellular polymeric substances (EPS). In industrial environments, biofilms can cause heat and mass transfer limitations whilst in medical facilities they can be a source of contamination and proliferation of infections. Biofilm formation is related to the pathogenicity of some bacterial strains and cells in biofilms are usually resistant to antimicrobials agents, which increases the interest in new and sound methods for their prevention and destruction.