Inactivation of Mycobacterium tuberculosis and

Mycobacterium bovis by 14 Hospital Disinfectants

WILLIAM A. RUTALA, Ph.D., M.P.H., EUGENE C. COLE, D.P.H., NANCY S. WANNAMAKER, MS.,
DAVID J. WEBER, M.‘D., M.P.H., ch&i WV, North caroka

Epidemics of mycobacteria due to contami- complete inactivation of M. tuberculosis (O+

nation of medical devices continue to occnr. P) and good inactivation of M. bovis (l-3 +
For this reason, we assessed the ability of P). Two disinfectants, hydrogen peroxide
disinfectants, generally used in hospitals for and ethyl alcohol, provided differing results
disinfecting noncritical and semicritical pa- against M. tuberculosis and M. bovis. These
tient care items, to inactivate mycobacteria. studies have important implications for dis-
A modified Association of Official Analytical infecting semicritical patient care items.
Chemists’ (AOAC) Tuberculocidal Activity
Test, using Middlebrook 7H9 broth as the
primary subculture
tion by dilution,
medium and neutraliza-
was used to assess the abil-
ity of 14 hospital disinfectants to inactivate
D uring the past decade, multiple epidemics and
pseudoepidemics have been reported due to
Mycobacterium tuberculosis and nontuberculous
about lo6 Mycobacterium tuberculosis and mycobacteria [ 11. Indirect contact transmission
about lo6 Mycobacterium bovis at 20°C using involving bronchoscopes and airborne transmis-
lo- or 20-minute exposure. All products sion have been responsible for M. tuberculosis
were tested for each organism using 10 pen- epidemics [l-4]. Epidemics and pseudoepidemics
icylinders (P) and were prepared at the of nontuberculous mycobacteria have resulted from
manufacturers’ recommended use-dilution. contamination followed by inadequate disinfection
Chlorine dioxide, 0.80% hydrogen peroxide of medical instruments [1,5]. The source of contam-
plus 0.06% peroxyacetic acid, and an io- ination has been either patients or environmental
dophor achieved complete inactivation (O+ reservoirs, especially water supplies. This is not
P) of both M. tuberculosis and M. bouis. One surprising, since nontuberculous mycobacteria may
quaternary ammonium compound with a be isolated from tap water [1,51.
tuberculocidal label claim, a quaternary The absence of preregistration and postregistra-
ammonium compound without a tubercu- tion testing for antimicrobial activity of disinfec-
locidal label claim, chlorine (approximately tants by the Environmental Protection Agency
100 ppm) and 0.13% glutaraldehyde/0.44% (EPA) and the presence of epidemics secondary to
phenol/O.O(l% phenate were not effective ineffective disinfectants or disinfection procedures
(lo+ P) against both M. tuberculosis and M. have emphasized the need for independent evalua-
bovis. Another quaternary ammonium com- tion of the antimicrobial activity of disinfectants
pound with a tuberculocidal label claim was [2--51. Mycobacteria were selected as the test organ-
tested against only M. bouis and found inef- ism for several reasons. First, mycobacteria are
fective (lo+ P). Glutaraldehydes (2% alka- generally more resistant to chemical disinfection
line and 2% acid), a phenolic and chlorine than all other microorganisms with the exception
(approximately 1,000 ppm) demonstrated of bacterial endospores. Second, nontuberculous
mycobacteria are ubiquitous in the environment,
especially water supplies, and therefore likely to
contaminate semicritical and noncritical patient
care items. Finally, we can expect increasing num-
From the Division of Infectious Diseases, Department of Medicine,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, bers of hospitalized patients to be infected with
and Department of Hospital Epidemiology, University of North Carolina mycobacteria, since 5-10% of patients infected
Hospitals, Chapel Hill, North Carolina. with the human immunodeficiency virus type 1
Requests for reprints should be addressed to William A. Rutala, Ph.D.,
M.P.H., Division of Infectious Diseases, 547 Clinical Sciences Bldg, CB #
(HIV-11 will develop infection with M. tuberculosis
7030, University of North Carolina School of Medicine, Chapel Hill, North and as many as 50% will develop infection with
Carolina 27599-7030. nontuberculous mycobacteria [61.
Current address of Dr. Eugene C. Cole is Research Triangle Institute,
Research Triangle Park, North Carolina 27709.2194.
For the aforementioned reasons, this study was
initiated to compare the mycobactericidal activity

September 16, 1991 The American Journal of Medicine Volume 91 (suppl 3B) 30-267s
CONFERENCE ON NOSOCOMIAL INFECTIONS / RUTALA
of 14 hospital disinfectants. The disinfectants tested
are commonly used in hospitals for disinfecting
noncritical (such as floors, walls, and furniture
that touch intact skin) or semicritical (such as
endoscopes, respiratory therapy equipment, which
touch mucous membranes) patient care items.
METHODS AND MATERIALS
Comparative Evaluation of 13 Disinfectants
A modified Association of Official Analytical
Chemists’ (AOAC) Tuberculocidal Activity Test
[7], using Middlebrook 7H9 broth as the primary
subculture medium and neutralization by dilution,
was used to assess the antimicrobial activity of the
disinfectants against 2.4 x lo6 M. tuberculosis and
3.3 x lo5 Mycobacterium bovis at 20°C and 20
minutes’ exposure. In brief, porcelain penicylin-
ders (carriers) were inoculated by soaking in a
24-day old culture of test mycobacteria. After
removal and drying, the inoculated penicylinders
were placed in the disinfection solution and ex-
posed for 10 or 20 minutes. Each penicylinder was
removed and placed in a 20-ml tube of 7H9 broth,
removed, and transferred to a second 20-ml tube of
7H9 broth. The tubes were capped and incubated
at 37°C for 60 days and then examined for growth.
M. tuberculosis was used because it is a recog-
nized human pathogen that has been associated
with infections caused by ineffective disinfectants
or disinfection procedures [2-41, and M. bouis was
selected because it is the organism required by the
AOAC for tuberculocidal activity testing. M. bouis
BCG (ATCC 35743, Tice strain) was obtained from
the American Type Culture Collection (Rockville,
Maryland) and the M. tuberculosis was a clinical
isolate obtained from the University of North
Carolina Hospitals’ Mycobacteriology Laboratory.
This isolate was sensitive to the first-line antituber-
culous drugs: isoniazid, rifampin, ethambutol, and
streptomycin. Detailed information on organisms,
media, growth conditions, neutralization, and the
tuberculodical activity test has previously been
reported [71.
All products were tested on the same day for
each organism using 10 inoculated porcelain peni-
cylinders prepared from the same standardized cell
suspension. The number of organisms on inocu-
lated penicylinders did not vary significantly ( I 0.6
log of the mean) during a test day. All disinfectants
were purchased for this study and were used
within their specified use-life. Sterile distilled wa-
ter, USP (Travenol Laboratories, Deerfield, Illi-
nois) was used for disinfectant dilution and all
disinfectants were diluted according to the manu-
facturers’ instructions. The chemical class of disin-
38-268s September 16, 1991 The American Journal
ET AL
fectant, use-dilution, and active ingredients before
dilution were: glutaraldehyde, undiluted, and 2%
alkaline glutaraldehyde for disinfectant A, glutaral-
dehyde, undiluted, and 2% acid glutaraldehyde for
disinfectant B; hydrogen peroxide, undiluted, and
6% hydrogen peroxide, 0.85% phosphoric acid for
disinfectant C; hydrogen peroxide plus peroxyace-
tic acid, undiluted, and 0.80% hydrogen peroxide,
0.06% peroxyacetic acid for disinfectant D; glutaral-
dehyde-phenol-phenate, 1: 16 and 2.00% glutaralde-
hyde, 7.05% phenol, 1.20% sodium phenate for
disinfectant E; phenolic, 1:lOO and 5.7% o-phe-
nylphenol, 1.8% p-tertiary amylphenol, 10.8% so-
dium xylene sulfonate, 6.3% triethanolamine dode-
cylbenzene sulfonate, 3.0% trisodium ethylene
diamine tetracetate for disinfectant F; quaternary
ammonium, 1:256 and 5.36% alkyl (Cl4 50%, Cl2
40%, Cl6 10%) dimethyl benzyl ammonium chlo-
ride, 4.02% octyl decyl dimethyl ammonium chlo-
ride, 2.71% didecyl dimethyl ammonium chloride,
2.01% dioctyl dimethyl ammonium chloride for
disinfectant G; quaternary ammonium, 1:256 and
3.750% octyl decyl dimethyl ammonium chloride,
1.875% dioctyl dimethyl ammonium chloride,
1.875% didecyl dimethyl ammonium chloride,
5.00% alkyl (Cl4 50%, Cl2 40%, Cl6 10%) benzyl
dimethyl ammonium chloride, 3.420% tetrasodium
ethylenediaminetetracetate for disinfectant H; chlo-
rine, 1:500 and 5.25% sodium hypochlorite or
household bleach for disinfectant I; chlorine, 1:50
and 5.25% sodium hypochlorite or household bleach
for disinfectant J; chlorine dioxide, 1 base:10 wa-
ter:l activator and 2.73% sodium chlorite (base),
15.1% organic acid (activator) for disinfectant K;
iodophor, 1:213 and 9.10% polyethoxy polypropoxy
polyethoxy ethanol-iodine complex, 8.74% no-
nylphenoxypolyethyleneoxyethanol-iodine com-
plex (provides 1.6% minimum titratable iodine) for
disinfectant L; alcohol, undiluted and 70% ethyl
alcohol for disinfectant M.
Tuberculocidal Activity of Two Quaternary
Ammonium Compounds
Two quaternary ammonium compounds with
tuberculocidal label claims were evaluated by the
modified AOAC method using M. bovis at lo- and
20-minute exposure time. The two disinfectants
were tested on the same day using 10 inoculated
porcelain penicylinders prepared from the same
standardized cell suspension. The use-dilution and
active ingredients before dilution for disinfectant
G have already been listed, and the same informa-
tion for disinfectant N is: 1:128 and 12% N, N-bis
(2-[omega-hydroxypoly (oxyethylene)] ethyl] alkyl-
amine, 8% alkyl (50% C14, 40% C12, 10% C16)
dimethyl benzyl ammonium chloride.
of Medicine Volume 91 (suppl 3B)
 CONFERENCE ON NOSOCOMIAL INFECTIONS / RUTALA ET AL

RESULTS TABLE I
Our data demonstrate that chlorine dioxide (K),
Tuberculocidal Activity of 13 Hospital DisinfectantsUsing a Modified
0.80% hydrogen peroxide/0.06% peroxyacetic acid AOACMethod
(D), and an iodophor (L) demonstrated excellent
mycobactericidal activity, whereas two quaternary Numberof Positive
Penicylinders/NumberReplicates*
ammonium compounds (G, H), chlorine (1500
Disinfectant,concentration M. bovis M. tuberculosis
household bleach, approximately 100 ppm chlo-
rine) (I) and 0.13% glutaraldehyde/0.44% phenol/ A: Glutaraldehyde, 2% alkaline7 l/10 Oil0
B: Glutaraldehyde, 2% acid7
0.08% phenate (E) were not effective against either C: Hydrogen peroxide, 6%t i;iz %i
D: Hydrogen peroxide, 0.80%- 0/10 o/10
M. tuberculosis or M. bovis (Table I). Glutaralde- peroxyacetic acid, O.O6%t
hydes (2% alkaline [Al and 2% acid [BI), a phenolic E: Glutaraldehvde. 0.13%- lo/lo lo/lo
(F) and chlorine (150 household bleach, approxi- phenol, d.4&-phenate, O.O8%/,t
F: Phenolic, UDi l/10 o/10
mately 1,000 ppm chlorine) (J) demonstrated com- G: Quaternary ammonium, UDt IO/10
H: Quaternary ammonium, UD :iiti lo/lo
plete inactivation of M. tuberculosis and good I : Chlorine, - 100 ppm lO/lO lO/lO
inactivation of M. bovis. Two disinfectants pro- 1 : Chlorine, - l,,OOO ppm l/10
vided differing results against M. tuberculosis and K: Chlorine dioxide, UDt i;ii
L : lodophor, UD :;i;
M. bovis. Hydrogen peroxide (C) demonstrated M: Ethyl alcohol, 70% lO/lO 8:
I
excellent activity against M. bovis but poor activity UD = manufacturers’ recommended use-dilution(see text).
against M. tuberculosis. In contrast, 70% ethyl *Temperature = 20°C; exposure time, = 20 minutes; mean number of colony forming units per
penicylinderwas 3.3 x 10 fork!. bows and 2.4 x 106forM. tuberculosis.
alcohol (M) provided excellent activity against M. tlndicates tuberculocidal label claim, but the tuberculocidal label claim of disinfectant A is 45
minutes at 25°C.
tuberculosis but poor activity against M. bovis.
Another experiment using two quaternary am-
monium compounds (G, N) with tuberculocidal TABLE II
label claims was performed and demonstrated that Tuberculocidal Activity of Two Quaternary Ammonium Compoundswith
these products were not effective against M. bovis Tuberculocidal Label Claims Using a Modified AOACTest at 20°C
using the modified AOAC method with a lo- or
Numberof tpenicylindersl
20-minute exposure time (Table II). ExposureTime NumberReplicates,
Disinfectant,concentration (minutes) M. bovis
COMMENTS G: Quaternary ammonium, UD
:t :s!i
Since 1966, the AOAC tuberculocidal activity N: Quaternary ammonium, UD lo/lo
test has been used to assess the tuberculocidal :i lo/lo
activity of chemical disinfectants. This qualitative UD = manufacturers’ recommended use-dilution (see text).
carrier method has been criticized because it pro-
duces results that are neither accurate nor repro- have been published in the scientific literature
ducible [8,9]. A modified AOAC test, which substi- [12]. The majority of these 14 commonly used
tuted Middlebrook 7H9 broth for modified hospital disinfectants had not been independently
Proskauer-Beck as the primary subculture me- tested against the human pathogen M. tuberculo-
dium and used neutralization by dilution, has been sis or M. bouis until a recent study by Best and
used and found to have dramatic effects on test coworkers U31. Nonetheless, the good to excellent
results. For example, in a number of trials the tuberculocidal activity exhibited by the iodophor,
standard AOAC test provided no positive penicylin- chlorine dioxide, chlorine (approximately 1,000
ders per 10 replicates and the modified AOAC test ppm), phenolic, two 2% glutaraldehydes and the
provided 10 positive penicylinders per 10 replicates poor activity of the three quaternary ammonium
171. The EPA pass criterion for a tuberculocidal compounds, chlorine (approximately 100 ppm) and
label claim is 0 positive penicylinders/ 10 replicates 0.13% glutaraldehyde/0.44% phenol/0.08% phe-
tested. The explanation for this improved sensitiv- nate were largely predictable based on the antimi-
ity is that Middlebrook 7H9 broth yields signifi- crobial activity of the chemical class of disinfec-
cantly improved recovery of mycobacteria com- tants tested, the concentration of the disinfectants
pared with the recommended modified Proskauer- tested [12], and/or a few published studies involv-
Beck broth [7,10,111. Obviously, the use of a ing other mycobacterial species or strains [9,14,X5].
recovery medium (modified Proskauer-Beck) that Interestingly, there was no measurable difference
can neither efficiently support the growth of healthy in the tuberculocidal activity of two quaternary
mycobacteria nor resuscitate sublethally damaged ammonium compounds with tuberculocidal label
cells can provide misleading results. claims when compared with a quaternary ammo-
Unfortunately, only a few independent investiga- nium compound without a tuberculocidal label
tions of the antimicrobial activity of disinfectants claim. All three quaternary ammonium com-

September 16, 1991 The American Journal of Medicine Volume 91 (suppl3B) 38-2698
 CONFERENCE ON NOSOCOMIAL INFECTIONS / RUTALA ET AL

pounds demonstrated no tuberculocidal activity by ingly tested their own product, and three of the
the modified AOAC test. It is unclear why the 6% four failed their product against one or more of the
hydrogen peroxide and 70% ethyl alcohol provided test organisms. Although the same study showed
differing results against M. tuberculosis and M. extreme variability of test results among laborato-
bouis; further investigations are underway. Since ries testing identical products 1161 these results
mycobacteria are generally more resistant to chem- showed that EPA registration does not guarantee
ical disinfection than other microorganisms, these that products meet microbiocidal label claims. A
data may be used to predict the antimicrobial recent report from the General Accounting Office
activity of these disinfectants against nonspore- also concluded that the EPA does not know whether
forming microorganisms. Thus, if a disinfectant disinfectants kill the microorganisms claimed on
has excellent tuberculocidal activity, it is likely to the product labels 1171.
inactivate lipophilic and hydrophilic viruses, yeasts, The increased use of invasive procedures involv-
fungi, and bacteria. ing semicritical patient care items on infected and
Our data are of particular concern because sev- highly susceptible patients increases the risk of
eral disinfectants with tuberculocidal label claims infection. Disinfection has had a significant role in
(e.g., 0.13% glutaraldehyde/0.44% phenol/0.08% reducing these risks, but total reliance on manufac-
phenate [El, a quaternary ammonium compound turers’ microbiocidal data is not prudent. Until
[GI), when tested by a modified AOAC method, there is preregistration testing that is supple-
failed to inactivate a clinical strain of M. tuberculo- mented by random enforcement (postregistration)
sis (Table I). These same disinfectants, as well as testing of disinfectants using standardized tests,
another quaternary ammonium compound (N), did users should not depend entirely on the manufac-
not inactivate the M. bovis strain that is recom- turers’ label claims but should augment those
mended for use in the standard AOAC test; how- claims with articles in the scientific literature
ever, this strain was found to exceed marginally and/or disinfection guidelines 112,181.
the AOAC phenol resistance requirements [7].
These failures occurred despite the fact that we ACKNOWLEDGMENT
used a longer exposure time (20 minutes) than This work was supported in part by the U.S. Environmental Protection Agency,

required by the AOAC tuberculocidal activity test Office of Pesticides Program, under cooperative agreement CR813006030.

(10 minutes). These results are in agreement with

Best et al 1131, who were unable to demonstrate
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September 16, 1991 The American Journal of Medicine Volume 91 (suppl3B) 30-271s