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Previous Article | Next Article 
Applied and Environmental Microbiology, April 2000, p. 1702-1705, Vol. 66, No. 4
Fralin Biotechnology Center, Virginia
Polytechnic Institute and State University, Blacksburg, Virginia
24061-0346,1 and American Water
Works Service Co., Inc., Belleville Laboratory, Belleville, Illinois
622202
Received 20 August 1999/Accepted 13 January 2000
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.
Mycobacterium avium is an
environmental, opportunistic human pathogen (8, 25) that
infects between 25 and 50% of advanced-stage AIDS patients in the
United States (15). M. avium has been isolated from drinking water and municipal water systems (6, 9, 10, 12, 14,
23) and grows in water (11). M. avium
isolates recovered from municipal water systems and local natural water sources have the same DNA fingerprints as those recovered from AIDS
patients exposed to the water (24). One reason for the persistence of M. avium in drinking water could be
resistance to disinfection methods (e.g., chlorination). A number of
environmental, opportunistic mycobacteria, including M. avium, have been shown to be relatively resistant to chlorine or
chloramine at concentrations used in municipal water systems for
disinfection (3, 4, 7, 14, 19, 20, 21). Unfortunately, those
earlier studies were flawed because strains were not completely
identified, different colony types were used, cells were grown on
different media and to different stages, and aggregates were not
excluded from the cell suspensions. Most mycobacterial species,
including M. avium, form aggregates or clumps during growth
in media (16, 18), and the presence of aggregates can lead
to spurious disinfection resistance (22) and variable colony
counts due to irregular dispersal of aggregates. The objective of the
studies described here was to develop a method to produce M. avium cell suspensions lacking large aggregates and to compare the
susceptibility of medium- and water-grown M. avium
suspensions to chlorine, monochloramine, chlorine dioxide, and ozone.
The M. avium strains A5 (1), 1508, 1060, 5002, and 5502 (24) were identified by DNA probe (Gen-Probe, San
Diego, Calif.). M. avium strain A5 was isolated from an AIDS
patient and was received from Marjorie Beggs McClellan Veterans
Hospital, Little Rock, Ark. Because A5 was not isolated by Marjorie
Beggs, it was undoubtedly subject to numerous transfers before receipt
in the Virginia Tech laboratory. Its inclusion in this study is based
on the fact that it is one of the few strains of M. avium
that have been transformed. Thus, it can serve as a host for genes
involved in chlorine resistance. In contrast, strains 1060, 1508, 5002, and 5502 were isolated as part of a study in which the Virginia Tech
laboratory participated. Cultures were inoculated from the original
slants. All strains had a transparent colony morphology, and the
frequency of opaque colony variants was less than 1 in 1,000. The
M. avium strains were grown to mid-log phase in either
Middlebrook 7H9 broth (BBL Microbiology Systems, Cockeysville, Md.)
containing 10% (vol/vol) oleic acid-albumin enrichment and 0.5%
(vol/vol) glycerol or in autoclaved water from the East St. Louis
Municipal Water system (assimilable organic carbon range, 500 to 750 mg/liter). Escherichia coli strain C was grown in nutrient
broth (Difco Laboratories, Detroit, Mich.) to mid-log phase at 37°C.
Cultures were then centrifuged (8,000 × g for 15 min
at 20°C) and washed in an equal volume of chlorine-demand-free
phosphate buffer (CDFPB) (5.3 g of
KH2PO4/liter, 8.29 g of
K2HPO4/liter [pH 7.0] [13])
three times and finally suspended in an equal volume of CDFPB (i.e.,
washed cultures). To prepare reduced aggregate fractions (RAFs), the
washed cultures were centrifuged at 1,300 × g for 5 min at 20°C and the supernatant, containing single cells and small
aggregates, was collected. Single cells (i.e., 1 to 5 cells), small
aggregates (i.e., those containing 5 to 25 cells), and large aggregates
(i.e., those containing more than 25 cells) of three independent
cultures were counted in triplicate by phase-contrast microscopy and
reported as percent total cells. Only washed cultures contained cells
in large aggregates (Table 1). Aggregates
did not form during disinfection (data not shown).
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Chlorine, Chloramine, Chlorine Dioxide, and Ozone
Susceptibility of Mycobacterium avium
ABSTRACT
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TABLE 1.
Characteristics of M. avium RAFs
To measure disinfectant susceptibility, 200 ml of CDFPB contained in a 250-ml glass, stoppered Erlenmeyer flask with a Teflon-coated magnetic stir bar was inoculated with 6 × 105 CFU from a washed culture or RAF. Disinfection was performed at 23°C, under reduced light levels and with gentle stirring (60 rpm). Chlorine, monochloramine, and chlorine dioxide were prepared and added to cell suspensions, and their free concentrations were measured by using published methods (13). Ozone was generated by an M-1500 corona discharge ozonator (Clearwater Technology, Inc., San Luis Obispo, Calif.) fed with oxygen gas. Ozone-demand-free glassware and ozone-demand-free phosphate buffer were prepared, and ozone concentrations were measured by using published methods (13). In all disinfection experiments whose results are reported here, the concentration of disinfectant did not vary by more than 75% of the initial value. Surviving M. avium cells were enumerated as CFU following inactivation of disinfectants with 0.1% sodium thiosulfate (13) on Middlebrook 7H10 agar medium (BBL Microbiology Systems) containing 10% (vol/vol) oleic acid-albumin enrichment and 0.5% (vol/vol) glycerol. Surviving E. coli cells were enumerated on plate count agar (Difco Laboratories).
The data presented are the averages of a minimum of three replicates. Linear regressions based on the logarithm of the percent survival with time (in minutes) for each strain were computed and used to calculate the product of disinfectant concentration (in parts per million) and time (in minutes) to reach 3 log units of cell death (CT99.9%) for each strain.
Because chlorine concentrations fell from 1 to 0.15 ppm in 10 min in
M. avium suspensions containing more than 5 × 106 CFU/ml (5), disinfection was measured in
suspensions of between 104 and 105 CFU/ml.
There was no difference in disinfection kinetics over the range of
103 to 106 CFU/ml for M. avium
strain A5 (data not shown). M. avium cells in RAFs were
significantly more resistant to chlorine, monochloramine, chlorine
dioxide, and ozone than E. coli cells (Table
2). The values for E. coli
agreed with published data (17). Strains of M. avium had chlorine CT99.9% values ranging from 51 to
204 (Fig. 1). Susceptibility to chlorine
correlated with the growth rate in the Middlebrook 7H9 medium. Though
the cultures of each M. avium strain were harvested in
mid-log phase, the turbidity of the cultures differed because strains
grew at different rates. The most chlorine-susceptible strain (i.e.,
strain 5502) had the highest turbidity, whereas the most
chlorine-resistant strain (i.e., strain 1060) had the lowest turbidity.
When chlorine CT99.9% values were plotted against
turbidity of 9-day, log-phase cultures for each strain, a straight line
was obtained with an R value of 
0.902 by linear
regression. Others have shown that antecedent growth conditions
influence susceptibility to chlorine-based disinfectants (2,
17).
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The M. avium strains were also resistant to monochloramine,
chlorine dioxide, and ozone (Table 2). The M. avium strains
could be divided into monochloramine-resistant and -susceptible groups, though that separation was not carried through for chlorine dioxide or
ozone susceptibility (Table 2). The chlorine dioxide (Fig. 2) and ozone disinfection kinetics for
M. avium strain 5002 were biphasic, unlike those for the
other strains tested.
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The chlorine susceptibility of RAFs and washed cultures of M. avium strain 1060 were measured to determine whether the presence of large aggregates would increase the resistance to disinfection. This strain was chosen because washed cultures had a high proportion of cells in large aggregates (75% [Table 1]). The average chlorine CT99.9% value for the washed cultures was 207 (standard deviation, ±43), based on triplicate measurements of two independent cultures. Because that value was approximately equal to that for the RAF (Table 1) and the variation was the same, the presence of cells in aggregates of more than 25 cells does not appear to influence chlorine susceptibility for that M. avium strain.
The chlorine susceptibility of water-grown cells was also measured to assess the effect of the growth medium on susceptibility, since M. avium is normally an inhabitant of water (11, 24). Water-grown cells of all five M. avium strains were significantly more chlorine resistant than were cells grown in medium (Table 2). That effect may be related to growth rate, because the growth rate of M. avium in water is much slower than that in medium (11).
This study documents the heretofore suspected disinfectant resistance of M. avium. Most of the M. avium strains were highly resistant to chlorine, monochloramine, chlorine dioxide, and ozone (Table 2). The M. avium strains possessed very high CT99.9% values; for example, chlorine CT99.9% values for the M. avium strains were 580 to 2,300 times greater than those for E. coli (Table 2). Similarly, the CT99.9% values of chlorine dioxide and ozone for the M. avium strains were at least 100- and 50-fold greater (respectively) than those for the E. coli strain (Table 2). In agreement with other studies (4), chlorine dioxide was a better mycobacterial disinfectant than chlorine at equal concentrations (Table 1).
The M. avium strains could be divided into two groups based upon their susceptibility to monochloramine. One of the susceptible M. avium strains (strain 5502) was a water isolate that had the same pulsed-field gel electrophoresis (PFGE) pattern as did an epidemiologically linked AIDS patient isolate, strain 5002 (24). The relative resistance of the AIDS patient isolate M. avium strain 5002 compared to its PFGE-matched and epidemiologically matched water isolate, strain 5502, was due to a difference in growth rate. Though the strains were isolated during the same study at approximately the same time in 1992 and retain the same, shared PFGE pattern (24) and colonial morphology, the water isolate, strain 5502, grew faster than did the patient isolate, strain 5002.
We believe the values reported here accurately document the disinfectant resistance of M. avium strains and are not artifacts of experimental variation. All strains were of the same colony type and grown in the same medium to the same stage of growth. None of the RAF suspensions used for disinfection contained aggregates consisting of more than 25 cells (Table 1). Further, the presence of cells in large aggregates did not increase chlorine CT99.9% values for one strain. All colony counts were obtained by spreading suspensions on the surface of M7H10 agar medium plates that were used 3 days after preparation. The suspensions were spread to dryness to ensure the disruption of aggregates (18). Such manipulations on all CFU measurements ensured the reproducibility of results. In spite of those precautions, there was variation in the results (Table 2). The source of that variation has not been identified. However, the magnitudes of the average CT99.9% values were so high that the relative resistance of M. avium to disinfectants was evident and the differences between M. avium strains could be demonstrated.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the American Water Works Association Research Foundation.
Marjorie Beggs of the McClellan Veterans Hospital generously supplied M. avium strain A5. We acknowledge the expert technical assistance of Myra D. Williams.
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FOOTNOTES |
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* Corresponding author. Mailing address: Fralin Biotechnology Center, Virginia Tech, Blacksburg, VA 24061-0346. Phone: (540) 231-5931. Fax: (540) 231-7126. E-mail: jofiii{at}vt.edu.
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