Joseph O. Falkinham

Health Impacts of Environmental Mycobacteria

SUMMARY Environmental mycobacteria are emerging pathogens causing opportunistic infections in humans and animals. The health impacts of human-mycobacterial interactions are complex and likely much broader than currently recognized. Environmental mycobacteria preferentially survive chlorination in municipal water, using it as a vector to infect humans. Widespread chlorination of water has likely selected more resistant environmental mycobacteria species and potentially explains the shift from M. scrofulaceum to M. avium as a cause of cervical lymphadenitis in children. Thus, human activities have affected mycobacterial ecology. While the slow growth and hydrophobicity of environmental mycobacteria appear to be disadvantages, the unique cell wall architecture also grants high biocide and antibiotic resistance, while hydrophobicity facilitates nutrient acquisition, biofilm formation, and spread by aerosolization. The remarkable stress tolerance of environmental mycobacteria is the major reason they are human pathogens. Environmental mycobacteria invade protozoans, exhibiting parasitic and symbiotic relationships. The molecular mechanisms of mycobacterial intracellular pathogenesis in animals likely evolved from similar mechanisms facilitating survival in protozoans. In addition to outright infection, environmental mycobacteria may also play a role in chronic bowl diseases, allergies, immunity to other pulmonary infections, and the efficacy of bacillus Calmette-Guerin vaccination.

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

ABSTRACT 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.

Persistent colonisation of potable water as a source of Mycobacterium avium infection in AIDS.

The source of Mycobacterium avium infection in AIDS has not been identified and it is not known whether most patients with AIDS acquire the organism from recent infection or by reactivation of previous infection. As part of a prospective epidemiological study, we isolated multiple colonies of M avium from patients with AIDS and from potable water to which they had been exposed. All isolates were analysed with pulsed field gel electrophoresis (PFGE). As judged by PFGE, 29 (81%) of 36 patients were infected with one or more unique clinical strains of M avium. 7 patients (19%) were infected with three groups of common strains. Group 1 included 3 patients who lived in separate rural areas and had no common exposures apart from treatment at hospital A. The same strain was isolated repeatedly during 41 months from a recirculating hot water system at hospital A; residential water cultures were negative. Group 2 included 2 patients with no common exposures apart from treatment at hospital B; the same strain was isolated repeatedly over a period of 24 months from a recirculating hot water system at hospital B. Patients in groups 1 and 2 had numerous possible exposures to hospital hot water. Group 3 included 2 patients treated at the same methadone treatment facility. In an institution the hot water system may be persistently colonised with a particular strain of M avium. HIV-infected patients exposed to these water sources can develop disseminated M avium infection.

Nontuberculous mycobacteria in the environment

It is likely that the incidence of infection by environmental opportunistic mycobacteria will continue to rise. Part of the rise will be caused by the increased awareness of these microbes as human pathogens and improvements in methods of detection and culture. Clinicians and microbiologists will continue to be challenged by the introduction of new species to the already long list of mycobacterial opportunists (see Table 3). The incidence of infection will also rise because an increasing proportion of the population is aging or subject to some type of immunosuppression. A second reason for an increase in the incidence of environmental mycobacterial infection is that these microbes are everywhere. They are present in water, biofilms, soil, and aerosols. They are natural inhabitants of the human environment, especially drinking water distribution systems. Thus, it is likely that everyone is exposed on a daily basis. It is likely that certain human activities can lead to selection of mycobacteria. Important lessons have been taught by study of cases of hypersensitivity pneumonitis associated with exposure to metalworking fluid. First, the implicated metalworking fluids contained water, the likely source of the mycobacteria. Second, the metalworking fluids contain hydrocarbons (e.g., pine oils) and biocides (e.g., morpholine) both of which are substrates for the growth of mycobacteria [53,193]. Third, outbreak of disease followed disinfection of the metalworking fluid [136,137]. Although the metalworking fluid was contaminated with microorganisms, it was only after disinfection that symptoms developed in the workers. Because mycobacteria are resistant to disinfectants, it is likely that the recovery of the mycobacteria from the metalworking fluid [137] was caused by their selection. Disinfection may also contribute, in part, to the persistence of M avium and M intracellulare in drinking water distribution systems [33,89,240]. M avium and M intracellulare are many times more resistant to chlorine, chloramine, chlorine dioxide, and ozone than are other water-borne microorganisms [141,236]. Consequently, disinfection of drinking water results in selection of mycobacteria. In the absence of competitors, even the slowly growing mycobacteria can grow in the distribution system [33]. It is likely that hypersensitivity pneumonitis in lifeguards and therapy pool attendants [139] is caused by a similar scenario.

Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment

A majority of the Mycobacterium species, called the nontuberculous mycobacteria (NTM), are natural inhabitants of natural waters, engineered water systems, and soils. As a consequence of their ubiquitous distribution, humans are surrounded by these opportunistic pathogens. A cardinal feature of mycobacterial cells is the presence of a hydrophobic, lipid-rich outer membrane. The hydrophobicity of NTM is a major determinant of aerosolization, surface adherence, biofilm-formation, and disinfectant- and antibiotic resistance. The NTM are oligotrophs, able to grow at low carbon levels [>50 microg assimilable organic carbon (AOC) l(-1)], making them effective competitors in low nutrient, and disinfected environments (drinking water). Biofilm formation and oligotrophy lead to survival, persistence, and growth in drinking water distribution systems. In addition to their role as human and animal pathogens, the widespread distribution of NTM in the environment, coupled with their ability to degrade and metabolize a variety of complex hydrocarbons including pollutants, suggests that NTM may be agents of nutrient cycling.A majority of the Mycobacterium species, called the nontuberculous mycobacteria (NTM), are natural inhabitants of natural waters, engineered water systems, and soils. As a consequence of their ubiquitous distribution, humans are surrounded by these opportunistic pathogens. A cardinal feature of mycobacterial cells is the presence of a hydrophobic, lipid‐rich outer membrane. The hydrophobicity of NTM is a major determinant of aerosolization, surface adherence, biofilm‐formation, and disinfectant‐ and antibiotic resistance. The NTM are oligotrophs, able to grow at low carbon levels [>50 μg assimilable organic carbon (AOC) l−1], making them effective competitors in low nutrient, and disinfected environments (drinking water). Biofilm formation and oligotrophy lead to survival, persistence, and growth in drinking water distribution systems. In addition to their role as human and animal pathogens, the widespread distribution of NTM in the environment, coupled with their ability to degrade and metabolize a variety of complex hydrocarbons including pollutants, suggests that NTM may be agents of nutrient cycling.

Chlorine, Chloramine, Chlorine Dioxide, and Ozone Susceptibility of Mycobacterium avium

ABSTRACT Environmental and patient isolates of Mycobacterium avium were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine, the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five M. avium strains ranged from 51 to 204. Chlorine susceptibility of cells was the same in washed cultures containing aggregates and in reduced aggregate fractions lacking aggregates. Cells of the more slowly growing strains were more resistant to chlorine than were cells of the more rapidly growing strains. Water-grown cells were 10-fold more resistant than medium-grown cells. Disinfectant resistance may be one factor promoting the persistence of M. avium in drinking water.

Nontuberculous Mycobacteria from Household Plumbing of Patients with Nontuberculous Mycobacteria Disease

To determine whether plumbing could be a source of nontuberculous mycobacteria (NTM) infection, during 2007–2009 I isolated NTM from samples from household water systems of NTM patients. Samples from 22/37 (59%) households and 109/394 (28%) total samples yielded NTM. Seventeen (46%) of the 37 households yielded >1 Mycobacterium spp. isolate of the same species as that found in the patient; in 7 of those households, the patient isolate and 1 plumbing isolate exhibited the same repetitive sequence-based PCR DNA fingerprint. Households with water heater temperatures <125°C (<50°C) were significantly more likely to harbor NTM compared with households with hot water temperatures >130°F (>55°C) (p = 0.0107). Although households with water from public or private water systems serving multiple households were more likely to have NTM (19/27, 70%) compared with households with a well providing water to only 1 household (5/12, 42%), that difference was not significant (p = 0.1532).

Molecular Survey of the Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems

ABSTRACT The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems.

The international epidemiology of disseminated Mycobacterium avium complex infection in AIDS

Objective:To determine rates of disseminated Mycobacterium avium complex (MAC) infection among AIDS patients in developed and developing countries, and to determine whether different rates reflect differences in exposure or immunity, or both. Design:Prospective cohort study. Setting:University hospitals and outpatient AIDS programs. Methods:HIV-infected subjects with CD4 counts < 200×106/l were interviewed and had CD4 lymphocyte counts, blood cultures for mycobacteria (baseline and at 6 months), and skin tests with purified protein derivative (PPD) and M. avium sensitin. Results:Among 566 study patients rates of disseminated MAC were 10.5–21.6% in New Hampshire, Boston and Finland compared to 2.4–2.6% in Trinidad and Kenya (P < 0.001). PPD skin test reactions ≥ 5 mm were present in 20% of patients from Kenya compared to 1% at other sites (P < 0.001). Among patients from the United States and Finland, multiple logistic regression indicated that occupational exposure to soil and water was associated with a decreased risk of disseminated MAC, whereas the following were associated with an increased risk of disseminated MAC: low CD4 count, swimming in an indoor pool, history of bronchoscopy, regular consumption of raw or partially cooked fish/shellfish and treatment with granulocyte colony-stimulating factor. Conclusions:Rates of disseminated MAC in AIDS are higher in developed than developing countries and are due to both differences in exposure and differences in immunity. These data provide a rationale for prevention of MAC through both active immunization and reduction in exposure to the organism.

Effect of growth in biofilms on chlorine susceptibility of Mycobacterium avium and Mycobacterium intracellulare.

ABSTRACT Mycobacterium avium and Mycobacterium intracellulare were grown in suspension and in biofilms, and their susceptibilities to chlorine were measured. M. avium and M. intracellulare readily adhered within 2 h, and numbers increased 10-fold in 30 days at room temperature in biofilms on both polystyrene flasks and glass beads. The chlorine resistance of M. avium and M. intracellulare cells grown and exposed to chlorine in biofilms was significantly higher than that of cells grown in suspension. Survival curves showed no evidence of a resistant, persisting population after 6 h of exposure to 1 μg chlorine/ml. The chlorine susceptibility of cells grown in biofilms and exposed in suspension (cells detached from bead surfaces) was also significantly higher than that of cells grown and exposed in suspension (planktonic cells), although it was lower than that of cells grown and exposed in biofilms. The higher resistance of the detached biofilm-grown cells was reversed upon their growth in suspension. There was a strong correlation between the chlorine susceptibility of cells of both M. avium and M. intracellulare and cell surface hydrophobicity measured by contact angle for both biofilm- and suspension-grown cells.