Applied and Environmental Microbiology, September 2003, p . 5685-5689, Vol . 69, No . 9

One reason for the persistence of M . avium in drinking water is its resistance to agents commonly used to disinfect water . Expressed as CT_99.9 values (concentration of chlorine in parts per million multiplied by the time [in minutes] required for 99.9% inactivation), strains of M . avium were more than 500 times more resistant to chlorine than was Escherichia coli, the standard for disinfection (35) . In addition, the M . avium strains were also resistant to chloramine, chlorine dioxide, and ozone (35) . Other opportunistic environmental mycobacteria, including Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium phlei, and Mycobacterium chelonae, have been shown to be relatively resistant to chlorine at concentrations used in municipal water systems for disinfection (4, 26, 27) .

Not only is M . avium resistant to chlorine, but it and other MAIS organisms are also resistant to heavy metals and oxyanions (12), antibiotics (30), and disinfectants such as quaternary ammonium compounds, phenols, and glutaraldehyde (3, 6) . Because broad-spectrum antimicrobial resistance is expected to be a major determinant of the epidemiology of opportunistic environmental mycobacteria, I have initiated a long-term study whose objective is to identify the cellular and molecular bases for resistance . As a first step, I herein report the effect of colony type, growth phase, growth rate, temperature, and growth in 6% oxygen upon the susceptibility to chlorine . These studies grew out of evidence of wide variations in chlorine susceptibility of the same strains tested under apparently the same conditions (35), which suggested the influence of unknown factors . Further, evidence that water-grown cells of M . avium were significantly more resistant to chlorine than medium-grown cells (35) suggested that one factor influencing mycobacterial susceptibility to disinfectants could be the metabolic state of the cells . In addition, here we report the physiological characteristics of MAIS strains that appear to correlate with resistance to chlorine .

The following mycobacterial strains were used in the study . M . avium and M . intracellulare strains were identified with a DNA probe (Gen-Probe, San Diego, Calif.), and M . scrofulaceum strains were identified by cultural and biochemical tests . M . avium strain A5 is a plasmid-free, transformable AIDS patient isolate (1) . The unpigmented colonial variant of M . avium strain 2812 [2812 (U)] was isolated from the pigmented strain 2812 [2812 (P)], recovered from an AIDS patient (32) . The pairs of M . avium strains 1508 (water) and 1060 (AIDS patient) and 5502 (water) and 5002 (AIDS patient) share identical pulsed-field gel electrophoresis fingerprints (37) . The opaque colonial variant of Va14 [Va14 (O)] was isolated from the transparent colonial strain Va14 [Va14 (T)] isolated from a non-AIDS pulmonary patient (14) . M . intracellulare strains TMC 1406^T, TMC 1403, and TMC 1405 and M . scrofulaceum strains TMC 1323^T, TMC 1312, and TMC 1306 are from the Trudeau Memorial Collection (21) .

Strains were grown in Middlebrook 7H9 broth (M7H9) (BBL Microbiology Systems, Cockeysville, Md.) containing 0.5% (vol/vol) glycerol and 10% (vol/vol) oleic acid-albumin (OAA) (28) . Cells were also grown in M7H9-OAA containing either 0.1% (vol/vol) glycerol or 0.1, 0.2, or 0.5% (wt/vol) sodium acetate and in M7H9-glycerol-OAA containing 0.1 or 0.5% (vol/vol) Tween 80 . Cells were grown at 30 and 40°C and in 6% oxygen at 37°C . Turbidity (absorbance at 580 nm) was plotted against incubation period (in days) to identify when the cultures reached early log, log, and stationary phases . The cultures were streaked to confirm purity and colony morphology . Cell suspensions lacking aggregates were prepared and exposed to chlorine, and the surviving cells were enumerated as described previously (35) . All colony counts were obtained by spreading suspensions on the surfaces of M7H10 agar medium plates that were dried to the same extent (3 days) and spread to ensure the disruption of aggregates . The data are the averages of the results of a minimum of three replicates, and CFU counts were used to calculate the logarithm of the percent survival with time (in minutes) for each strain or condition and subsequently used to calculate concentration multiplied by time to 3 logs of the cell inactivation (CT_99.9) values . Chlorine concentrations were measured by the N,N-diethyl-p-phenylenediamine colorimetric method according to the manufacturer's directions (Hach Co., Loveland, Colo.) . Statistical analysis (e.g., linear regression, correlation, and t tests) was performed using InStat version 3.0 (GraphPad Software, Inc., San Diego, Calif.) .

MICs of Tween 80, para-hydroxybenzoate, hydroxylamine (NH₂OH), and sodium nitrite (NaNO₂) were measured by tube dilution in M7H9 broth containing 0.5% (vol/vol) glycerol and 10% (vol/vol) OAA . A twofold dilution series of each compound was prepared in 5-ml volumes contained in optically matched, 16- by 125-mm screw-cap tubes, inoculated with 0.5 ml of each strain, and incubated at 37°C in a slanted position on a rotator (6 rpm) . Turbidity (absorbance at 580 nm) of each culture was measured daily, and values were compared to those for the antimicrobial-free control . The concentration of the antimicrobial agent reducing turbidity by 50% after 7 days of incubation was defined as the MIC .

There were significant differences in the susceptibilities to chlorine (expressed at CT_99.9), Tween 80, para-nitrobenzoate, hydroxylamine, and sodium nitrite of the strains representing the three species (Table 1) . M . scrofulaceum was significantly (t test, P < 0.002) more susceptible to chlorine than either M . avium or M . intracellulare (Table 1) . The responses of individual strains within each species were similar, if not identical . The relatively chlorine-sensitive M . scrofulaceum strains were more sensitive to all four compounds than the more chlorine-resistant strains of M . avium and M . intracellulare (Table 1) . Statistical analysis also illuminated a correlation (r² = 0.82) between chlorine susceptibility and 9-day culture turbidity for the five strains of M . avium and three strains of M . scrofulaceum .

 
There was a striking difference in the chlorine susceptibilities of cells collected during early log, log, and stationary phases (Table 2) . The M . avium, M . intracellulare, and M . scrofulaceum strains displayed two exponential growth phases, early log (0.5 generation/day) and log (1.0 generation/day), under the growth conditions used here, as has been reported by others (24, 29) . The CT_99.9 values for cells in the early log, log, and stationary phases were significantly different for all three strains (t test with Welch correction, P < 0.05), with the exception of the values for log- and stationary-phase cells of M . avium strain A5 . The magnitude of the differences means that even cells collected from cultures within a rather narrow time frame (e.g., one generation) may differ markedly in their susceptibilities to chlorine . This undoubtedly contributes to the variance in chlorine susceptibility values . At all three growth stages, the susceptibility of the M . scrofulaceum strain was significantly greater (t test with Welch correction, P < 0.001) than that of either M . avium or M . intracellulare (Table 2) .

 
Because cells in different growth phases grow at different rates, the influence of growth rate on the chlorine susceptibility of M . avium strain A5 was examined . To alter the rate of growth, cells were grown under the same conditions in the M7H9 medium and M7H9 containing glycerol (0.1 and 0.5%) or sodium acetate (0.1, 0.2, and 0.3%) . In all instances, cells were harvested during log phase immediately after the end of the early log phase . The chlorine susceptibility of M . avium strain A5 was mildly influenced by growth in the different media (Table 3) . Some of the differences were statistically significant . However, cell metabolism and composition were likely altered by growth in the different media . Therefore, it is not possible to ascribe the differences in chlorine susceptibility only to differences in growth rate .

 
Colony type strongly influenced susceptibility to chlorine (Table 4) . M . avium strain 2812 (P) was more susceptible to chlorine than its isogenic variant 2812 (U) . The pigmented variant has the opaque colony morphology, whereas the unpigmented variant has the transparent colony morphology (32) . M . intracellulare strain Va14 (O) was more susceptible to chlorine than Va14 (T) . Fewer than 1% of the cells of the cultures and cell suspensions used for these experiments were of the opposite colony type . Growth stage also strongly influenced the chlorine susceptibilities of both colonial variants of the two strains (Table 4) .

 
Because of reports of M . avium infections associated with exposure in M . avium-containing hot tubs and spas (9, 16), the effects of growth and chlorine exposure on the chlorine susceptibility of M . avium strain A5 was measured . Both growth and exposure temperature affected the chlorine susceptibility of M . avium strain A5 (Table 5) . Cells grown at 30°C were significantly more susceptible to chlorine (by a factor of 20 [t test with Welch correction, P = <0.0001]) when they were exposed at 40°C than when they were exposed at 30°C . Cells grown at 40°C were also more sensitive, but to a reduced extent, if they were exposed at 40°C (t test with Welch correction, P = 0.023) . If cells were exposed to chlorine at 30°C, cells grown at 40°C were significantly more chlorine sensitive (by a factor of 8 [t test with Welch correction, P = <0.0001]) than cells grown at 30°C . Although the M . avium A5 strain grew more rapidly at 40°C than at 30°C, the length of exposure to chlorine was a small proportion of the generation time and would not have contributed to the differences in chlorine susceptibility . As was the case for all experiments, the concentration of chlorine was monitored at every time point . Interestingly, chlorine concentrations fell, especially for cells grown and exposed at 40°C (Table 5) .

 
Mycobacteria in natural waters and drinking water distribution systems are often exposed to low oxygen levels, and M . avium numbers are higher in waters of low oxygen content (11, 18) . Therefore, the effect of growth at a low oxygen level (i.e., 6% oxygen) on chlorine susceptibility was measured (Table 6) . CT_99.9 values for M . avium strain A5 and M . intracellulare TMC 1406^T grown in 6% oxygen were significantly lower than for air-grown cells (t test with Welch correction, P = <0.001) . Growth in 6% oxygen did not significantly reduce the chlorine susceptibility of M . scrofulaceum strain TMC 1323^T (Table 6) . The differences were all the more significant because the growth rate of cells in 6% oxygen is approximately half that of cells growing in aerated media (data not shown) .

 
In summary, the data have identified a number of factors strongly influencing the susceptibilities of MAIS strains to chlorine, among which there were significant differences (Table 1) . The fact that the CT_99.9 values for the individual strains within each species were similar (Table 1) suggests that the levels of chlorine resistance of the three species were characteristic . M . scrofulaceum is approximately threefold more sensitive to chlorine than either M . avium or M . intracellulare, whose chlorine susceptibilities are similar (Table 1) . The greater susceptibility of the M . scrofulaceum strains to para-nitrobenzoate, hydroxylamine, and sodium nitrite suggests that susceptibility to hydrophilic compounds is characteristic of that species . Evidence of the hydroxylamine sensitivity of M . scrofulaceum compared to those of M . avium and M . intracellulare confirmed previously published work (5) . The correlation between culture turbidity at 9 days for M . avium and M . scrofulaceum and chlorine susceptibility led to measurements of the influence of growth stage and rate upon chlorine susceptibility . Because the relatively chlorine-susceptible M . scrofulaceum strains were more sensitive to Tween 80, the effect of growth in Tween 80 on chlorine susceptibility was measured to determine whether permeability was a determinant of chlorine sensitivity . Although Tween 80-grown cells of M . avium strain A5 were more susceptible to chlorine (data not shown), it was impossible to ascribe those differences to permeability alone because of the multiple changes in detergent-grown cells (30) .

The extent of the differences in the chlorine susceptibilities of cells collected during early log, log, and stationary phases were striking (Table 2) . Those differences explain, in part, the high variance in measures of the chlorine susceptibility of mycobacteria . The gradient was very steep, and small differences in culture age resulted in very high differences in chlorine susceptibility (Table 2) . However, the variance in the data reported here suggests that even laboratory-grown mycobacterial populations are heterogeneous in their response to chlorine and, perhaps, other antimicrobial agents . It is unlikely that aggregation was a major contributor to the variation . Suspensions lacking large aggregates were used, and suspensions were dried and spread to disrupt any small aggregates, as was described earlier (35) . Growth stage-dependent differences in antimicrobial susceptibility are not restricted to chlorine; they have been shown to affect antibiotic susceptibility (23, 39) and may involve susceptibility to hydrophilic antimicrobial agents and other disinfectants . In those reports, rapidly growing cells were more susceptible to antibiotics than cells in nongrowing cultures (23, 39) . Growth phase-associated changes in the protein profiles of Mycobacterium smegmatis have previously been reported (25) . Membrane lipid composition may also change as cultures age . It is also possible that the differences in chlorine susceptibility are due to differences in growth rate, because the cells in different phases grow at different rates .

One contributor to the differences in the chlorine susceptibility of MAIS cells in early log, log, and stationary phases may be growth rate . Faster-growing cells of M . avium strain A5 were more susceptible to chlorine than were cells growing more slowly (Table 3) . The greater chlorine resistance of M . avium grown in water (35) is likely due, in part, to a reduced growth rate . Cells of Legionella pneumophila grown in water were also more resistant to chlorine than cells grown in medium (19), and rapidly growing E . coli cells were more sensitive to chlorine dioxide than slowly growing cells (2) . The same relationship between growth rate and chlorine susceptibility was found whether glycerol or acetate was added . However, it is likely that changes in cell metabolism or cell wall composition brought on by the different concentrations of carbon sources also contributed to the differences in chlorine susceptibility .

As expected, colony type strongly influenced susceptibility to chlorine (Table 4) . This finding extends observations that cells in opaque colony variants are more sensitive to a variety of antimicrobial agents than their isogenic transparent variants (22, 38) . Unpigmented variants of pigmented M . avium strains are more hydrophobic and grow more slowly than their isogenic pigmented parents (32) . Either factor, or both, may be responsible for the differences in chlorine susceptibility . Based on these results, we expect drinking water treatment with chlorine to select for transparent (unpigmented) variants of M . avium and M . intracellulare . Unfortunately, the transparent variants are more virulent (31) .

More cells of M . avium strain A5 grown at 30°C were killed if they were exposed to chlorine at 40°C than if they were exposed at 30°C (Table 5) . Those data suggest that membrane fluidity influences susceptibility to chlorine . Clearly, cells grown at 30°C and exposed at 40°C would have been more permeable . The lipid compositions of mycobacterial membranes are different in cells grown at low as opposed to high temperatures to provide cells with a fluid membrane (17, 33, 34) . Exposure of mycobacterial cells grown at 30 to 40°C will result in a more fluid and permeable membrane (20) . The increased fluidity might be responsible for the increased susceptibility to chlorine . The magnitude of the increase in killing suggests that simultaneous exposure of M . avium and other mycobacterial cells to chlorine at elevated temperatures might result in increased eradication of M . avium from public water supplies . Another tactic would be to chlorinate cells that have been exposed to reduced oxygen levels . Residual disinfectant levels may kill MAIS cells in portions of drinking water distribution systems where oxygen concentrations are low more effectively than they kill cells in aerated portions .

Marjorie Beggs of the McClellan Veterans Hospital, Little Rock, Arkansas, generously supplied M . avium strain A5 . I acknowledge the skilled technical assistance of Myra Williams and the suggestion of Bradley J . Eldred of Analytical Services, Inc . (Williston, Vt.), to examine the effect of temperature on chlorine susceptibility .

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