Comparison of Mycobacterial Susceptibilities to Six Chemical

Chapter 21

Comparison of Mycobacterial Susceptibilities to Six Chemical Disinfectants

Downloaded by NORTH CAROLINA STATE UNIV on August 6, 2012 | http://pubs.acs.org Publication Date: September 7, 2007 | doi: 10.1021/bk-2007-0967.ch021

Chaowu Zhang and Guoqing Wang West China School of Public Health, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China

Objective: To compare the mycobacterial susceptibilities among 10 mycobacteria to six disinfectants. [Methods] 10 mycobacterial species and 6 disinfectants were used in this study. We first set up a quantitative suspension mycobactericidal test in the light of Chinese technique standard for disinfection, and then we used the test to evaluate the susceptibilities of each mycobacterium to the tested disinfectants. [Results] The 10 mycobacteria are similarly susceptible to glutaraldehyde, peracetic acid, ethanol and cresol disinfectants, M . fortuitum and M . chelonei subsp. abscessus are more resistant to chlorine disinfectant than the other 8 tested mycobacteria. [Conclusion] M . fortuitum and M. chelonei subsp. abscessus were more resistant than or at least as resistant as the other tested mycobacteria to disinfectant.

© 2007 American Chemical Society

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

439

440


1. Introduction Determinations of the incidence of tuberculosis word-wide are notoriously difficult, but according to World Hearth Organization (WHO) estimates, onethird of the world's population has been infected, about 100 million individuals are infected annually (1), non-tuberculosis mycobacteria (NTM) infection has been increasing rapidly since 1980's (2-3). Disinfection is an effective way to control mycobacterial infection. Because of the high lipid content in their cell walls, mycobacteria are more resistant to disinfectants than other bacterial vegetatives, hence the disinfectants used to prevent and control mycobacterial infection should be selected carefully.

2. Materials and methods 2.1 Strains and materials 2.7.7 Mycobacterial strains In this study, 10 species of mycobacteria were used, namely M. tuberculosis H37Ra CMCC (B) 93020, M . bovis CMCC (B) 93006, M . nonchromogenicum CMCC (B) 93311, M . xenopi CMCC (B) 93316, M. phlei CMCC (B) 93318, M. smegmatis CMCC (B) 93203, M . diernhoferi CMCC (B) 93320, M . flavescens CMCC (B) 93322, M . fortuitum CMCC (B) 93323 and M . chelonae subsp. abscessus CMCC (B) 93326.A11 mycobacteria were provided by Chinese Medical Culture Collection of Bacteria [CMCC (B)]. Among the 10 tested mycobacteria, M . chelonae subsp. Abscessus, M . fortuitum, M. smegmatis, M. phlei, M. diernhoferi and M. flavescens are rapid growing mycobacteria which can grow to be seen on the media within 7 days, the other 4 are slowly growing mycobacteia.

2.7.2 Disinfectants The six disinfectants used in this research were ERIC (an iodophor based disinfectant, manufactured by Chengdu Yongan Pharmacia CO), GA-50 (a glutaraldehyde based disinfectant, manufactured by Huaxi Center of Health Care Science and Technology), TC-101 disinfection tablet (a chlorine based disinfectant, manufactured by Chinese PL A 7018 factory), Ethanol (manufactured by Neijiang Kanghong sanitary articles factory), Peracetic acid and Ethanol. Peracetic acid (PAA) was prepared from hydrogen peroxide and acetic acid in our own laboratory as described previously. Alcohol was prepared by diluting dehydrated ethanol with sterile distilled water.


441 Tested concentrations of each disinfectant were prepared by diluting with sterile distilled water.


2.1.3 Neutralizes In this study, 0.03mol/L phosphate buffer solution (PBS) containing 1% tween 80, 1% lecithin and 1% glycine was used as neutralizer to glutaraldehyde disinfectant, 0.03mol/L PBS containing 0.5% tween 80 and 1% sodium thiosulfate to iodine, chlorine and peracetic acid disinfectants and 0.03mol/L PBS containing 0.5% tween 80 and 1% lecithin to cresol and ethanol disinfectants. All the neutralizers, according to Chinese technique standard for disinfection, were testified effective to stop the lasting effect of their corresponding disinfectants and harmless to the tested mycobacteria.

2.2 Mycobactericidal Tests Tested mycobacteria were incubated on slants of Mycobacteria Dehydrate Media at 37°C for 4 days and 20-30 days respectively for rapid growing mycobacteria and slow growing ones (M. xenopi incubated at 42°C for 20 days). Add 3-5 ml dilution (0.03mol/L phosphate buffer solution, pH 7.2) on to the slants and scrape the culture gently with a sterile incubation loop into the dilution. Then the mixture of dilution and culture were transferred to a small sterile flask containing glass pieces, shake vigorously for 5-10 min. Dilute the suspension to 107~108cfu/ml with sterile dilution. Pipette 5 ml disinfectant with different concentration into a sterile tube, incubate 5 min within 20~22°C water bath, and then add 0.1 ml of the tested mycobacterial suspension (107~108cfu/ml) into each of the tubes, vortex for 5 sec. After being exposed for selected time, 0.5 ml of the mixture was transferred to 4.5 ml sterile neutralizer (testified to neutralize the lasting effect of the corresponding tested disinfectant). After 10 minutes' neutralization, 0.2 ml of the mixture was inoculated onto the surface of L-J media plate and then incubated for viable count. The recovery cultural conditions (time, temperature etc) for each of the tested mycobacteria are consistent with the conditions mentioned above. Mycobacterial control plates were designated with exactly performance same as the tested plates with the exception of substituting of disinfectant with sterile water. Killing rate (KR) was calculated by the following formula.

Nc


442 In the formula, Nc means viable count of mycobacterial control (cfu/ml), and Nt means viable count of each bactericidal test (cfu/ml).


3. Results Results in Table 1 showed the bactericidal results of ERIC against the 10 tested mycobacteria. Data in this table suggest that the 10 tested mycobacteria would be killed by more than 99% after exposing to 40mg/L available iodine for 1 min. With the exposing time being prolonged, the killing rates are increasing. No tested mycobacteria survive after exposing to 40mg/L available iodine for 20 minutes. The killing rates of iodine disinfectant against M. chelonei subsp. abscessus are almost always lower than the other 9 myocbacteria when exposing to the same iodine concentration for the same time, which indicate that M. chelonei subsp. abscessus is comparatively less susceptible to iodine disinfectant than the other 9 tested mycobacteria. Table 2 to Table 5 showed the bactericidal results of glutaraldehyde, cresol, peracetic acid and ethanol against tested mycobacteria respectively. For each of these 4 disinfectants, the killing rates of the same concentration and exposing time are very similar among the 10 tested mycobacteria, which indicate that these 10 mycobacteria are similarly susceptible to glutaraldehyde, cresol, peracetic acid and ethanol disinfectant. The bactericidal results of chlorine disinfectant are listed in Table 6. TC101 solution with 40 mg/L available chlorine can kill M. tuberculosis H37Ra, M. bovis, M . nonchromogenicum, M . xenopi, M . phlei, M . smegmatis, M . diernhoferi and M. flavescens by more than almost 100% after exposing for 20 min, while 80 mg/L available chlorine is needed to kill M . fortuitum and M . chelonae subsp. abscessus by the same percentage after exposing for the same 20 min, which indicates, in order to acquire the same killing rate when exposing for the same time, M. fortuitum and M. chelonae subsp. abscessus need double dosage of available chlorine as the other 8 tested mycobacteria.

4. Conclusion and Discussion It is well accepted that mycobacteria are more resistant to disinfectants than most other bacterial vegetatives, owing to their high lipid contents in cell walls and complex cell wall structures. In order to guarantee the disinfection effect against mycobacteria, the disinfectants and disinfection procedures must be testified by a mycobactericidal evaluation test. Most countries and organizations have issued their own authoritative mycobactericidal evaluation tests, however, these tests differ in test strains, operation procedures and evaluation standards.


In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007. 100

99.61

99.99

99.97

99.98

99.66

M. xenopi

M. bovis

100

99.98

100 96.63 95.20

100 100

98.87 95.75

100

100

99.99

99.98

99.65

96.41

100

99.99

99.93

M. nonchromogenicum

89.00

M. tuberculosis H37Ra

98.53

100

96.87

100

100

98.60

100

99.99

100

98.73

99.99

99.96

99.61

M. smegmatis

100

100

1 min

99.95

99.99

99.93

M. fortuitum

100

20 min

99.99

99.99

99.94

M. phlei

10 min

M. diernhoferi 99.97 M. chelonae subsp. absces99.39 sus M. flavescens 99.58

5 min

40 mg/L

99.28

99.42

99.33

99.88

99.50

98.28

99.80

99.72

99.84

99.88

5 min

99.90

99.92

99.87

99.99

99.93

99.66

99.98

99.95

99.98

99.99

10 min

20 mg/L

99.99

99.99

99.99

99.99

99.99

99.96

99.99

99.99

100

100

20 min

81.66

81.72

91.02

78.66

77.44

75.30

73.59

81.82

76.94

81.64

1 min

95.78

96.35

96.66

95.80

95.30

90.59

96.07

97.18

95.86

96.98

5 min

98.65

99.15

99.37

99.16

98.99

98.39

99.29

99.10

99.13

99.27

10 min

10 mg/L

Average killing rate (%) of available iodine concentration (mg/L) after different exposing time (min)

1 min

Test mycobacteria

Table 1. Bactericidal efficacy of ERIC against test mycobacteria


99.76

99.85

99.86

99.85

99.78

99.66

99.86

99.82

99.79

99.91

20 min


1 min 99.93 M. phlei M. fortuitum 99.93 99.99 M smegmatis M. diernhoferi 99.95 99.93 M. chelonae subsp. abscessus 99.98 M. flavescens M. nonchromogenicum 99.93 99.92 M. tuberculosis H37Ra 99.92 M. xenopi 99.96 M bovis

Test mycobacteria

Average killing rate (%) of glutaraldehyde concentration after different exposing time (min) 0.5% 0.25% 1% 1 min 5 min 10 min 20 min 5 min 10 min 20 min 1 min 5 min 10 min 99.46 99.99 99.96 100 85.44 98.00 99.99 100 100 99.59 99.92 99.99 99.99 99.31 100 99.11 99.99 100 82.96 97.51 100 99.85 99.99 100 100 100 100 89.86 99.89 99.96 98.82 100 99.92 99.99 100 100 83.29 99.35 99.99 95.25 99.24 99.94 100 99.99 99.99 83.10 99.44 99.99 100 97.30 99.99 99.71 100 100 100 99.95 99.99 88.82 99.88 99.26 100 97.24 99.99 100 99.31 99.91 99.99 100 84.53 99.22 99.34 99.99 100 99.92 100 99.99 99.99 90.76 98.il 99.68 99.34 99.99 99.92 100 99.99 100 99.99 98.11 90.76 99.68 100 99.38 100 99.99 100 99.93 99.99 83.26 96.71 99.43

Table 2. Bactericidal efficacy of GA-50 against test mycobacteria


20 min 99.94 99.92 99.97 99.92 99.93 99.98 99.92 99.94 99.94 99.93

4t 4^

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007. 99.98 99.98 99.97 99.93

99.99 100 100

99.59

97.69

99.77

99.80

99.98

99.98

99.99

M. smegmatis

M. diernhoferi

99.94 M. chelonae subsp. abscessus

98.69

M. fortuitum

M. flavescens

M. nonchromogenicum


M. xenopi

M. bovis

99.98

99.99

99.73

M phlei

100

100

100

100

99.99

99.99

100

100

100

100

99.99

1 min

100

100

100

100

100

100

100

100

100

100

20 min

98.53

98.38

95.62

87.11

69.87

80.16

86.81

66.00

81.22

86.00

1 min

99.82

99.81

99.37

99.52

95.77

99.36

99.01

95.80

99.52

99.14

99.97

99.98

99.92

99.94

99.41

99.94

99.94

99.50

99.93

99.94

7.5% 10 min 5 min

99.99

99.99

99.99

99.99

99.94

99.99

99.99

99.98

99.99

99.99

20 min

64.90

67.08

59.53

53.45

54.68

55.35

64.69

50.94

40.00

54.43

88.56

89.83

82.78

83.77

76.54

83.32

84.02

77.36

80.84

85.42

1 min 5 min

96.34

97.20

94.45

97.39

93.03

97.75

98.25

93.61

97.01

97.80

5% 10 min

Average killing rate (%) of cresol concentration (%)after different exposing time (min) 10% 5 min 10 min

Test mycobacteria

Table 3. Bactericidal efficacy of cresol against test mycobacteria


99.30

99.61

98.72

99.72

97.71

99.63

99.79

97.96

99.71

99.76

20 min


1 min 99.93 M. phlei 99.94 M. fortuitum 99.97 M. smegmatis M. diernhoferi 99.95 99.97 M. chelonae subsp. abscessus 99.97 M. flavescens M nonchromogenicum 99.95 M. tuberculosis H37Ra 99.95 99.99 M. xenopi 99.98 M. bovis

Test mycobacteria 200 mg/L 10 min 5 min 100 99.99 100 99.99 100 100 100 100 100 100 100 99.99 99.99 99.99 99.99 99.99 100 99.99 100 99.99 20 min 100 100 100 100 100 100 100 100 100 100

1 min 98.71 97.72 99.49 98.50 98.06 99.23 98.46 98.52 99.08 99.16 99.81 99.97 99.90* 99.75 99.95 99.86 99.93 99.94 99.97

99.98 99.99 99.99 99.98 99.99 99.99 99.99 99.99 100

100 mg/L 10 min 5 min 99.82 99.98 1 min 86.07 85.07 94.71 84.11 86.46 93.62 86.50 86.98 90.45 90.21

20 min 99.99 99.99 100 100 99.99 100 99.99 100 100 100

50 mg/L 5 min 10 min 98.84 99.75 98.17 99.74 99.50 99.91 98.46 99.70 98.28 99.77 99.37 99.91 99.82 98.79 99.33 99.92 99.43 99.93 99.94 99.56

Average killing rate (%) of PAA concentration (mg/L) after different exposing time (min)

Table 4. Bactericidal efficacy of peracetic acid against test mycobacteria


99.96 99.98 99.96 99.98 99.99 99.97 99.99 99.99 99.99

20 min 99.97


M. phlei M. fortuitum

1 min 99.99 99.97 M. smegmatis 100 M. diernhoferi 99.99 M. chelonae subsp. abscessus 99.99 M. flavescens 99.99 M. nonchromogenicum 99.99 M. tuberculosis H37Ra 99.99 99.99 M. xenopi 99.94 M. bovis

Test mycobacteria S min 100 100 100 100 100 100 100 100 100 100

60

Average killing rate (%) of ethanol concentration (%)after different exposing time (min) 45 30 10 min 20 min 1 min 5 min 10 min 20 min 1 min 5 min 10 min 100 100 99.38 100 99.93 99.99 66.56 83.60 93.85 100 100 99.32 79.81 99.93 99.99 100 54.74 90.48 100 100 99.24 86.94 99.99 100 100 96.80 65.07 100 99.96 100 99.23 99.99 100 82.91 92.60 61.59 100 99.99 100 99.75 99.99 100 59.25 80.85 90.92 99.14 100 99.94 100 99.99 100 64.30 84.55 96.01 100 100 99.22 99.94 99.99 99.99 59.44 81.47 93.47 100 99.99 100 99.75 99.99 100 63.16 86.25 96.35 100 100 98.77 99.81 99.98 100 60.19 80.58 92.87 100 100 98.71 79.14 99.83 99.99 56.44 99.98 93.21

Table 5. Bactericidal efficacy of ethanol against test mycobacteria


20 min 97.43 96.79 98.75 98.55 97.76 98.83 98.43 98.47 98.23 97.91

-4


-

*: means not test

M. bovis

M. xenopi


M. nonchromogenicum

M. flavescens

-

-

98.00 M. chelonae subsp. abscessus

M. diernhoferi

M. smegmatis

-

99.92

-

-

-

99.99

-

99.99

-

-

-

100 -

99.94 99.92

99.13 98.06

99.52

99.49 99.98

99.96

99.99

98.98

89.36

99.75

99.94

99.98

98.89

99.58

99.99

99.99

100

99.99

99.99

99.86

99.99

100

100

100

100

99.99

100

99.99

100

100

99.98

99.84

98.71

88.93

99.90

100

M. fortuitum

96.54

100

20 min

99.99

10 min

40 mg/L 99.93

10 min 98.27

5 min

M. phlei

20 min

95.67

94.96

98.03

91.16

97.43

69.85

90.98

97.75

99.49

99.45

99.75

98.95

99.95

99.94

99.98

99.84

99.93

98.88

89.34 99.52

99.84

99.95

98.87

99.88

10 min

98.94

99.63

88.20

99.22

90.31 67.33

5 min

1 min

20 mg/L

99.99

99.99

99.99

99.99

99.99

99.83

99.98

99.99

99.72

99.99

20 min

71.38

74.32

89.47

68.45

75.15

-

69.31

76.48

74.83

1 min

Average killing rate (%) of available chlorine concentration (mg/L) after different exposeing time (min) 5 min

1 min

80 mg/L I min

Test mycobacteria

Table 6. Bactericidal efficacy of TC-101 against test mycobacteria


96.94

95.96

98.55

90.33

94.25

90.67 -

95.63

91.15

99.58

99.53

99.77

99.13

99.23

-

99.07

99.56

99.00 -

10 min

10 mg/L 5 min

99.95

99.94

99.99

99.90

99.94

99.91 -

99.97

99.82

20 min

00


449 Apparently, different test conclusion will be given to a given disinfectant by different method, which will puzzle the users. It is of great significance to use a wide-accepted standard evaluation tests in order to get a wide-accepted conclusion. The most significant thing to standardize the mycobactericidal test is to choose a reference mycobacterial strain. Because of its important status in mycobacterial infection, M. tuberculosis was widely used as the test strain in most mycobactericidal tests. But its high pathogenicity and low growing speed bring great inconvenience to conducting the test. An ideal test mycobacterial strain should be rapid growing, comparatively resistant to disinfectants and safe to the conductors and environment. In this study, we compared the susceptibilities of M. tuberculosis and some rapid growing NTMs by using a suspension quantitative bactericidal test. The results showed that M. chelonei subsp. abscessus and M. fortuitum are as resistant to the tested disinfectants as the other 8 tested mycobacteria or even more resistant, which suggests that M. chelonei subsp. abscessus and M . fortuitum have the potential to be used as reference strain in mycobacteicidal test. This result is consistent with that from Corinne Le Dantec et al [4]. Our study results also showed that somefrequentlyused disinfectants such as iodine, chlorine, glutaraldehyde, ethanol, peracetic acid and cresol disinfectants can effectively kill mycobacteria under concentrations lower than their routinely used concentrations. These results we got in this study were different with those from previous literatures (5-7), which need much higher disinfectants' concentrations. The possible reason, we think, lies in the difference in test methods. In this study, the quantitative bactericidal tests were conducted in a suspension without organic matters and tap water, while in the previous reports organic matters and tap water were used. Since organic matters can react to some disinfectants and provide protection for bacteria, so it can reduce disinfectants' bactericidal efficacy. Tap water can reduce disinfectants' bactericidal efficacy as well. These make the difference in results between our study and previous reports reasonable. Ethanol was and still is a widely andfrequentlyused disinfectant. In this study, we found that 45% (v/v) of ethanol, much lower than the commonly used concentration, and still possess the mycobactericidal potential. And this concentration is not higher than that have bactericidal effect against S. aureus and E. coli. The cell wall permeability may account for the result. Mycobacterial cell walls have high lipid's content, so lipid soluble disinfectants like ethanol can permeate the cell walls more easily to function on the targets.

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J. M . Grange. Mycobacterium tuberculosis; leprosy; David, Greenwood; Richard, C.B. Slack; John, F. Peutherer. Medical Microbiology; The edition first printed in China; 1999, 200-209.


450 2.

3.


4.

5.

6.

7.

Ramaswamy, A.A; Biswas, J; Bhaskar, V; et al. Postoperative Mycobacterium chelonae endophthalmitis after extracapsular cataract extraction and posterior chamber intraocular lens implantation. Ophthalmology. 2000, 107(7), 1283-1286. Massimo, B.; Diego, P.; Robert, C.A. Mycobacterium chelonae interface infection after endokeratoplasty. Am. J. Ophthalmol. 2003, 135(3), 393395. Dantec, C.L.; Duguet, J-P.; Montiel, A.; Dumoutier, N.; Dubrou, S.; Vincent, V. Chlorine Disinfection of Atypical Mycobacteria Isolatedfroma Water Distribution System. Appl Environ Microbiol, 2002, 68(3), 10251032 Dauendorffer, J.N.; Laurain, C.; Weber, M.; Dailloux, M. Evaluation of the bactericidal efficiency of a 2% alkaline glutaraldehyde solution on Mycobacterium xenopi. J. Hosp. Infect. 2000, 46 (1), 73-76. Fraud, S.; Hann, A.C; Maillard, J.Y; et al. Effects of ortho-phthalaldehyde, glutaraldehyde and chlorhexidine diacetate on Mycobacterium chelonae and Mycobacterium abscessus strains with modified permeability. J Antimicrob. Chemother. 2003, 51 (3), 575-584. Griffiths, P.A; Babb, J.R; Fraise, A.P. Mycobactericidal activity of selected disinfectants using a quantitative suspension test. J Hosp Infect. 1999, 41(2), 111-121.


Comparison of Mycobacterial Susceptibilities to Six Chemical

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