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Efficacy of Common Disinfectants against Mycobacterium

marinum

Article  in  Journal of Aquatic Animal Health · September 2005

DOI: 10.1577/H04-051.1

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Journal of Aquatic Animal Health 17:284–288, 2005
q Copyright by the American Fisheries Society 2005
[Communication]
DOI: 10.1577/H04-051.1

Efficacy of Common Disinfectants against

Mycobacterium marinum
MARY E. MAINOUS AND STEPHEN A. SMITH*
Aquatic Medicine Laboratory, Department of Biomedical Sciences and Pathobiology,
Virginia–Maryland Regional College of Veterinary Medicine,
Virginia Polytechnic Institute and State University,
Duck Pond Drive, Blacksburg, Virginia 24061-0442, USA

Abstract.—Mycobacteriosis is an important bacterial and Cohen 1962; Black et al. 1971; Dunlap et al.
disease of freshwater, brackish-water, and marine fishes. 1972; Wilson 1976; Ashburner 1977; Holmes et
In addition to affecting many species of wild and cul-
al. 1999). Infections of humans generally result in
tured fish, the aquatic mycobacterial species present a
potentially important zoonotic risk to humans. Reduc- localized, cutaneous granulomas that are com-
tion or elimination of the causative pathogen from an monly called ‘‘fish handler’s disease’’ or ‘‘swim-
aquarium or aquaculture facility is therefore paramount. ming pool granulomas.’’
This study examined a variety of commercially available It is generally thought that transmission of my-
disinfectants for their efficacy in reducing or eliminating cobacteriosis between fish occurs via ingestion of
Mycobacterium marinum. In this study, ethyl alcohol infected fish tissues or through contaminated food
(50% and 70%), benzyl-4-chlorophenol/phenylphenol
(1%), and sodium chlorite (mixed as 1:5:1 or 1:18:1 or debris in the water (Smith 1997). However, wa-
[base : water : activator]) were the most effective disin- ter may become contaminated through defecation
fectants evaluated; each reduced or eliminated the num- of infected material or by the death and decom-
ber of detectable M. marinum within 1 min of contact position of infected fish. As a result, the water
time. Sodium hypochlorite (50,000 mg/L) was moder- itself may act as a potential source of infection to
ately effective but required a minimum contact time of other fish or humans, and an outbreak of myco-
10 min to reduce bacterial counts. Ethyl alcohol (30%),
N-alkyl dimethyl benzyl ammonium chloride (1:256;
bacteriosis in a home aquarium or aquaculture fa-
two formulations), and potassium peroxymonosulfate– cility can be a serious health hazard to both. Un-
sodium chloride (1%) did not substantially reduce bac- fortunately, little is known about the efficacies of
terial counts even after 60 min of contact time. common disinfectants on aquatic mycobacterial
species.
Disinfection is the process that eliminates many
Fish mycobacteriosis, commonly known as ‘‘pi-
or all pathogenic microorganisms (with the ex-
scine tuberculosis,’’ is an important, progressively
ception of bacterial spores) from inanimate objects
fatal bacterial disease of wild and cultured fish
(APIC 1996). High-level disinfection can be ex-
(Dulin 1979; Smith 1997). The most frequently
pected to destroy all microorganisms, while inter-
isolated species of Mycobacterium from fish are M.
mediate-level disinfection inactivates M. tuber-
marinum, M. fortuitum, and M. piscium (Ashburner
1977; Shieszko 1978; Hedrick et al. 1987; Hum- culosis, vegetative bacteria, most viruses, and most
phrey et al. 1987; Bragg et al. 1990; Shamsudin fungi but does not necessarily kill bacterial spores.
et al. 1990; Landsell et al. 1993). Over 160 species Low-level disinfection can kill most bacteria,
of ornamental, tropical, bait, and food fish have some viruses, and some fungi but cannot be relied
been reported as being susceptible to infection upon to kill resistant microorganisms, such as My-
with these bacterial pathogens. In addition, the cobacterium species or bacterial spores. In this
prevalence of infection may reach as high as 100% study, an effective disinfectant is defined as one
in fish populations that are subjected to intensive that results in a 3-log reduction in bacterial growth
culture practices (Smith 1997). Besides causing within 10 min of contact time (Best et al. 1990).
morbidity and mortality in fish, these organisms There are many methods of disinfection avail-
have a zoonotic potential for humans, especially able for aquaculture, including chemical treatment,
in individuals who are immunosuppressed (Swift ultraviolet irradiation, and ozonation. The most
commonly recommended chemicals for mycobac-
terial disinfection (phenolics, glutaraldehyde) are
* Corresponding author: [email protected]. toxic to animals and are not readily available out-
Received October 20, 2004; accepted April 3, 2005 side a laboratory or hospital setting. There are only
Published online August 18, 2005 a few chemical disinfectants approved by the U.S.

284
 COMMUNICATION 285

TABLE 1.—Disinfectants and concentrations used in a study of disinfectant efficacy against Mycobacterium marinum.
All disinfectants were mixed in sterile distilled water immediately prior to use except for Clidox-S (sodium chlorite),
which was mixed at least 15 min before use as per the manufacturer’s instructions.

Disinfectant Concentration
Ultra-Clorox 50,000, 200, 100, and 50 mg/L
Roccal-D Plus 1:256 (manufacturer’s recommendation)
Lysol concentrate 1% (manufacturer’s recommendation)
Ethyl alcohol 70, 50, and 30%
Clidox-S 1:18:1, 1:5:1 (manufacturer’s recommendation)
Micronex 1:256 (manufacturer’s recommendation)
Virkon-S 1% (manufacturer’s recommendation)

Environmental Protection Agency for use with vania) at 258C for 10 d before harvesting. Bacteria
food fish aquaculture; of these disinfectants, none were harvested by centrifugation at 800 3 gravity
is specifically labeled for mycobacteria. Therefore, (g) for 10 min and were washed twice with 10 mL
seven readily available disinfectants were exam- of DW; the final pellet was resuspended in 5 mL
ined for their efficacy against a common species of DW. One to two milliliters of this stock solution
of aquatic mycobacteria, M. marinum. The goal were then added to 5 mL of DW to give a final
was to determine which disinfectants could be working solution of 1–4 3 105 bacteria with an
used to disinfect the water, aquatic surfaces, and optical density of approximately 0.100 at 660 nm.
equipment of an aquarium or aquaculture facility. Experimental exposures were performed at
room temperature (218C) with a modification of a
Methods previously described disinfection protocol (Best et
The disinfectants and their working concentra- al. 1988, 1990). We used sterile 2.0-mL micro-
tions used in this investigation are listed in Table centrifuge tubes (Fisher Scientific Co.) for expo-
1. These included sodium hypochlorite (Ultra-Clo- sures. Each disinfectant was tested in duplicate
rox; The Clorox Co., Oakland, California), benzyl- with two replicate trials. Briefly, we used a vol-
4-chlorophenol2phenylphenol (Lysol; Reckitt- umetric pipette to transfer 100 mL of the working
Colman, Inc., Wayne, New Jersey), ethyl alcohol solution of M. marinum to sterile microcentrifuge
(AAPER Alcohol and Chemical Co., Shelbyville, tubes and then added 900 mL of either sterile DW
Kentucky), sodium chlorite (Clidox-S; Pharmacal (control) or the working concentration of the dis-
Research Laboratories, Naugatuck, Connecticut), infectant. After 1 min, 0.1-mL aliquots were taken
two formulations of N-alkyl dimethyl benzyl am- from each microcentrifuge tube, added to 9.9 mL
monium chloride (Roccal-D Plus by Pharmacia, of DW, and mixed well by inversion to maximize
Kalamazoo, Michigan; Micronex by ZEP, Atlanta, exposure to the disinfectant. A sterile spreader
Georgia), and potassium peroxymonosulfa- (Fisher Scientific Co.) was used to plate 0.1 mL
te2sodium chloride (Virkon-S; Pharmacal Re- of the 100-fold dilution onto Middlebrook 7H11
search Laboratories). All working concentrations agar (Fisher Scientific Co.); plates were incubated
were made with sterile distilled water (DW) im- at 258C for a minimum of 12 d, and the resulting
mediately prior to use, except sodium chlorite, colonies were counted and recorded. This process
which was prepared at least 15 min before use as was repeated at 5, 10, 20, 30, and 60 min after the
per the manufacturer’s recommendation. Sterile addition of each disinfectant.
DW was used as a diluent because municipal water
has been described as a habitat of free-living my- Results
cobacteria and is considered to be an important The efficacies of the various disinfectants
potential source of mycobacterial infections against M. marinum are shown in Tables 2 and 3.
(Schulze-Robbecke and Fischeder 1989). Sodium hypochlorite (Clorox) was considered to
Stock cultures of M. marinum (American Type be moderately effective against M. marinum when
Culture Collection 927) were maintained over the mixed according to manufacturer’s instructions
long term on Lowenstein-Jensen medium (Remel, (50,000 mg/L); at least 10 min of contact time were
Lenexa, Kansas). Cultures used for the disinfec- required to reduce bacterial counts, while 20 min
tion assays were subcultured in Middlebrook 7H9 were necessary to eliminate bacterial growth.
broth (Fisher Scientific Co., Pittsburgh, Pennsyl- When sodium hypochlorite was mixed at lower
286 MAINOUS AND SMITH

TABLE 2.—Bacterial plate counts of Mycobacterium marinum following exposure to various doses of Ultra-Clorox
(sodium hypochlorite) and ethanol. All samples were run in duplicate with replicate trials; the numbers of colony-
forming units/mL indicate the average bacterial counts of replicates. There was no loss of bacterial growth in distilled-
water controls. All disinfectants were mixed in sterile distilled water immediately prior to use. Abbreviations are as
follows: TNTC 5 too numerous to count; NG 5 no growth.

Ultra-Clorox (mg/L) Ethanol

Time
(min) 50,000 200 100 50 70% 50% 30%
1 TNTC TNTC TNTC TNTC NG NG TNTC
5 2 3 103 TNTC TNTC TNTC NG NG TNTC
10 6 3 102 TNTC TNTC TNTC NG NG TNTC
20 NG TNTC TNTC 2.9 3 103 NG NG TNTC
30 NG 5 3 102 1.5 3 102 4 3 102 NG NG TNTC
60 NG NG NG NG NG NG 1.15 3 103

concentrations (50, 100, and 200 mg/L), at least 256, Micronex) was equally ineffective against M.
20–30 min were required to reduce bacterial marinum, and did not affect bacterial growth with-
counts and 60 min were required to achieve com- in 30 min of contact time. Potassium peroxymo-
plete disinfection of M. marinum. Benzyl-4-chlo- nosulfate2sodium chloride (1%, Virkon-S) was
rophenol2phenylphenol (Lysol), when mixed at also not effective against M. marinum under these
the manufacturer’s recommended concentration experimental conditions.
(1%), was extremely effective as a disinfection
agent against M. marinum, killing all bacteria with- Discussion
in 1 min of contact time. Ethyl alcohol at a con- Mycobacterial species are generally considered
centration of either 50% or 70% was effective to be more resistant to chemical disinfection than
within 1 min of contact time, but a 30% concen- other microorganisms and are second only to bac-
tration was ineffective against M. marinum. So- terial spores in terms of resistance (Russell et al.
dium chlorite (Clidox-S) as a high-level disinfec- 1986; Best et al. 1990; APIC 1996). This is be-
tant (mixed as 1 part base : 5 parts water : 1 part lieved to be partly attributable to their unusually
activator) or an intermediate disinfectant (mixed high cell wall lipid content and the resultant hy-
as 1:18:1) was very effective against M. marinum, drophobicity (Best et al. 1990). Since mycobac-
killing all bacterial organisms within 1 min of con- terial species pose a significant potential health
tact time. threat to fish, aquaculturists, and home aquarists,
N-alkyl dimethyl benzyl ammonium chloride (1: an effective method of disinfection is highly de-
256, Roccal-D Plus) was not effective against M. sirable.
marinum, even when the contact time was as long There are many factors that can affect the ef-
as 60 min. The other commercial formulation of ficacy of liquid disinfectants. These include tem-
n-alkyl dimethyl benzyl ammonium chloride (1: perature, time of contact, pH, concentration and

TABLE 3.—Bacterial plate counts of Mycobacterium marinum after exposure to Lysol (benzyl-4-chlorophenol–phen-
ylphenol), Clidox-S (sodium chlorite), Micronex (N-alkyl dimethyl benzyl ammonium chloride), Roccal-D Plus (same
active ingredients as Micronex), and Virkon-S (potassium peroxymonosulfate–sodium chloride). All samples were run
in duplicate with replicate trials; the numbers of colony forming units/mL indicate the average bacterial counts of
replicates. There was no loss of bacterial growth in distilled water controls. All disinfectants were mixed in sterile
distilled water immediately prior to use except Clidox-S, which was mixed 15 min before use as per the manufacturer’s
instructions. Abbreviations are as follows: TNTC 5 too numerous to count; NG 5 no growth.

Clidox-S
Time Lysol Micronex Roccal-D Plus Virkon-S
(min) (1%) (1:5:1) (1:18:1) (1:256) (1:256) (1%)
1 NG NG NG TNTC TNTC TNTC
5 NG NG NG TNTC TNTC TNTC
10 NG NG NG TNTC 4.6 3 103 TNTC
20 NG NG NG TNTC 1.13 3 103 TNTC
30 NG NG NG 7 3 103 3.3 3 102 TNTC
60 NG NG NG 1.95 3 103 1.0 3 102 TNTC
 COMMUNICATION 287

presence of suspended solids, and organic and in- and Epidemiology, Inc.). 1996. Guideline for se-
organic constituents. Quaternary ammoniums, lection and use of disinfectants. (Reprinted from the
American Journal of Infection Control 24:313–
such as Roccal-D Plus and Micronex, are rapidly
342.) APIC, Washington, D.C.
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effective against organisms such as Pseudomonas, film formation and biocide susceptibility testing of
Proteus, and other gram-negative bacteria. Chlo- Mycobacterium fortuitum and Mycobacterium mar-
rine compounds require a contact time of 10–30 inum. Current Microbiology 46:28–32.
min to be effective and are highly corrosive and Best, M., S. A. Sattar, V. S. Springthorpe, and M. E.
toxic. Phenolic compounds, such as Lysol, are ir- Kennedy. 1988. Comparative mycobactericidal ef-
ficacy of chemical disinfectants in suspension and
ritating to the skin and eyes, relatively toxic, and carrier tests. Applied Environmental Microbiology
corrosive. Alcohols at concentrations of 70–95% 54:2856–2858.
are very effective against vegetative bacteria and Best, M., S. A. Sattar, V. S. Springthorpe, and M. E.
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eyes, and toxic (USEPA 1998: F2-19–22). Ulti- against Mycobacterium tuberculosis. Journal of
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cobacterium marinum infections acquired from trop-
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sodium chlorite (Clidox-S) were the most effective genotypically defined strain in an immunocompro-
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Copeland. 1987. The disease status of Australian
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Acknowledgments
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The authors would like to acknowledge S. Schulze-Robbecke, R., and R. Fischeder. 1989. My-
Brown and L. Link for technical assistance and G. cobacteria in biofilms. International Journal of
Flick for critical review of the manuscript. Hygiene and Environmental Medicine 188:385–
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Shamsudin, M., K. Tajima, T. Kimura, M. Shariff, and
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