TY - JOUR
AU - Fraise, A., P.
AB - Abstract Glutaraldehyde-resistant Mycobacterium chelonae have been isolated from endoscope washer disinfectors and endoscope rinse water. The mechanism of glutaraldehyde resistance is not well understood. Two spontaneous, glutaraldehyde-resistant mutants of the sensitive type strain, NCTC 946, were investigated. The colony morphology of the two mutants differed from that of the the type strain: colonies of the former were dry and waxy whereas those of the latter were smooth and shiny. Increased resistance to glutaraldehyde of the mutants was matched by small increases in the MICs of rifampicin and ethambutol but not isoniazid. Both mutants showed increased surface hydrophobicity. No changes were identified in the extractable fatty acids or the mycolic acid components of the cell wall but a reduction in each of the resistant strains in the arabinogalactan/arabinomannan portion of the cell wall was detected. Introduction In 1903 Friedman first isolated Mycobacterium chelonae, an atypical, environmentally associated mycobacterium. It is capable of multiplying in both natural and treated waters, 1,2 and has been reported as a problematic organism in the hospital, being associated with haemodialysis fluids and some disinfectant solutions. 1 It has also been isolated from decontaminated (i.e. cleaned and disinfected) flexible endoscopes and endoscope washer disinfectors with increasing frequency. 3,4,5 The intrinsic resistance of M. chelonae to a wide range of commonly used antibiotics and disinfectants is believed to be due to the presence of a waxy envelope which gives the cell surface its hydrophobic nature. 6 Resistant strains of M. chelonae have been isolated from endoscope washer disinfectors after decontamination with 2% alkaline glutaraldehyde solution. These organisms are resistant to 2% alkaline glutaraldehyde, 7 i.e. 60 min exposure to the disinfectant causes a ≤1 log 10 reduction in mycobacteria. Since 2% alkaline glutaraldehyde is the most widely used disinfectant for flexible fibre-optic endoscopes, this resistance is of great concern. M. chelonae can cause opportunistic infections 8 and immunologically compromised patients could become infected with resistant strains if these strains are not eradicated during cleaning and disinfection procedures. The mechanism of glutaraldehyde resistance in M. chelonae has not yet been elucidated. It is likely that the cell wall of M. chelonae can reduce the organism's affinity for glutaraldehyde or decrease the permeability to glutaraldehyde. Alteration in cell wall structure may affect antimicrobial susceptibility. For example, ethambutol acts on arabinogalactan, so changes in this component of the cell wall might affect the MIC of ethambutol. Similarly, isoniazid acts on mycolic acids, so mycolic acid content might influence the MIC of isoniazid. This paper describes our efforts to study the chemical nature of the cell wall and its association with resistance to glutaraldehyde and antibiotics. Materials and methods Strains and selection of glutaraldehyde-resistant mutants The strains investigated were M. chelonae NCTC 946 (the type strain), MCNC3 and MCNC4 (glutaraldehyde-resistant mutants derived from NCTC 946). MCNC3 was isolated by growing the type strain, NCTC 946, over many subcultures, on Middlebrook 7H11 agar (Becton Dickinson, Cockeysville, MD, USA) containing 5-175 mg/L of acidic glutaraldehyde (BDH Laboratory Supplies, Poole, UK) in 5 mg/L increments. The plates were inoculated with 20 µ L spots containing 10 9 cfu/mL of M. chelonae test suspension, prepared in sterile distilled water. Plates were then sealed and incubated for 5 days at 30°C. MCNC4 was derived from MCNC3 by a similar process, on Middlebrook 7H11 agar (Becton Dickinson) containing 60- 175 mg/L of acidic glutaraldehyde (BDH Laboratory Supplies) in 5 mg/L increments. The resistant phenotypes of MCNC3 and MCNC4 were stable when subcultured on 7H11 medium containing no glutaraldehyde; no sensitive revertants were detected. MIC determination The MICs of rifampicin, ethambutol and isoniazid were determined using the agar dilution method, 10 using 7H11 agar plates (containing no OADC supplement). The plates were then inoculated with 12 spots of M. chelonae test suspension (20 µL) at a concentration of approximately 10 5 cfu/mL, and incubated for 5 days at 30°C. The MIC was taken as the lowest concentration at which no growth was visible. Susceptibility to aldehydes Growth was harvested from the surface of a 7H11 agar plate and suspended in a bottle containing glass beads and 10 mL of sterile water. The suspension of cells was shaken vigorously for 5 min and then allowed to settle for 30 min. The supernatant was removed to a sterile container and allowed to settle for a further 2 h. The resulting supernatant was again removed to a sterile container and sonicated in a sonicating water bath (Ultra Wave Ltd., Cardiff, UK) operating at 40-50 Hz for 5 min before use. 5 Challenges with aldehydes, as described below, were performed by the method of Griffiths et al.9 A 100 µL volume of test suspension was added to 900 µL of aldehyde for a set period of time. A 10 µL aliquot of the mix was then removed after 1, 4, 10, 20 and 60 min and immediately neutralized in 990 µL of Ringer's/Tween solution (0.5% (v/v) Tween 80 in 1/4 strength Ringer's solution). Aliquots were then serially diluted in 1/4 strength Ringer’ s solution with a maximum dilution of 10−3. One hundred microlitres of the undiluted mixture and subsequent dilutions were plated on 7H11 agar (no OADC added) and cfu enumerated. Control experiments were performed in parallel in which the aldehyde was replaced by water. Aldehydes used to compare the susceptibility of these strains were: 1% and 2% alkaline glutaraldehyde (BDH Laboratory Supplies); 10% succine-dialdehyde and formaldehyde mix (Gigasept; Sanofi Winthrop Medicare, Guildford, UK); 10% ethanedial (Glyoxal; Sigma Chemical Co., Poole, UK). Hydrophobicity measurements The hydrophobicity of each strain was determined using the bacterial adherence to hydrocarbons (BATH) method. 11 A 1.2 mL volume of M. chelonae test suspension (approximately 109 cfu/mL), prepared in PUM buffer, pH 7.1 (22.2 g K 2HPO4·3H2O, 7.26 g KH2PO 4, 1.8 g urea, 0.2 g MgSO4·7H2O in 1 L of distilled water), was mixed with a range of volumes of n-octane or n-hexadecane. The suspensions were incubated at 30°C for 10 min then agitated by vortex mixing and left to stand to allow the hydrocarbon phase to rise. The lower aqueous layer was then removed and the absorbance measured at 400 nm using a Perkin Elmer Lambda 2 UV/visible spectrophotometer. Absorbance was then expressed as a percentage of the absorbance of the test suspension without hydrocarbon. The decrease in absorbance of the aqueous phase was taken as a measure of hydrophobicity. Extractable fatty acid analysis Fatty acids were extracted from the cell walls of M. chelonae strains by placing a loopful of organisms (containing approximately 109 cfu/mL) from a single agar plate into a screw-capped glass tube. Fatty acids were then converted to methyl esters using the method of White et al.12 The resulting extracts were resuspended in 100 µL of hexane and injected on to a Hewlett Packard HP-1 capillary column on a Unicam 610 series gas chromatograph operating with 1:100 sample splitting and flame ionizing detector. The injector temperature was set at 200°C and the detector temperature at 280°C; the temperature programme was set at 4°C/min from 150°C to 225°C. A sample of standard fatty acid methyl esters (bacterial acid methyl esters CP mix; Sigma-Aldrich, Poole, UK) was also run on the column for comparison of the components. Mycolic acid analysis This method was adapted from that of Butler et al.13 and Butler & Kilburn. 14 Confluent colonies containing a total of approximately 10 9 cfu were removed from a single 7H11 agar plate and placed into a screw-capped test tube containing 2 mL of 50% ethanol containing 25% (w/v) potassium hydroxide. Cells were saponified in the sealed tubes for 2 h at 100°C before adding 1.5 mL of HCl (50% (v/v) in water) and 2 mL of chloroform. After mixing, the solutions were allowed to settle and the lower layer was transferred to a clean tube and air dried. One hundred microlitres of potassium bicarbonate (2 g/L (w/v) in water) was then added to the tubes and the samples air dried again before the addition of 1 mL chloroform containing 30 µL p- bromophenacyl-8 reagent (Pierce Chemical Co., Rockford, IL, USA). Samples were heated at 85°C for 20 min and then allowed to cool to room temperature before the addition of 0.5 mL HCl (50% (v/v) in water) and 0.5 mL methanol. The solutions were mixed well, using a vortex mixer for 20 s, allowed to separate and the lower layer transferred to a clean tube and air dried. Samples were resuspended in 100 µL of chloroform before injection on to a C 18 ODS-Hypersil column on a Hewlett Packard series 1100 high-performance liquid chromotography (HPLC) system operating with 1:100 sample splitting and UV detection at 254 nm. A dichloromethane- methanol solvent system was used, starting at 10% dichloromethane/90% methanol to equilibrate the column. The sample was then injected and the gradient changed linearly over 1 min to 25% dichloromethane/75% methanol. This was further changed, linearly, over a 20 min period to 70% dichloromethane/30% methanol. The flow rate was set at 0.6 mL/ min and the detection at 254 nm. The mycolic acid bromophenacyl esters were separated using acetonitrile/ dichloromethane. Mass spectral detection of the liquid samples eluted from the column were analysed using a Hewlett Packard HP 59987A API-electrospray LC/MS integrated system. Arabinogalactan/arabinomannan analysis Cell wall arabinogalactan/arabinomannan content was estimated by the method of Takayama & Kilburn 15 using 20 inoculated plates of 7H11 agar for each M. chelonae strain. Plates were incubated at 30°C for 5 days before use. The arabinogalactan/arabinomannan extracts dissolved in water were purified by passage through a Sep-pack C18 column (Waters Ltd., Watford, UK). The samples were rinsed in 2 mL of 10% (v/v) acetonitrile (in water) and eluted from the column with 2 mL of 40% (v/v) acetonitrile (in water). Samples were then dried under air and resuspended in 100 µL of chloroform before being injected on to a Supelco SP 2380 column on a Unicam 610 series GC operating with 1:100 sample splitting and flame ionizing detector. Authentic standards (Sigma-Aldrich) were also run on the column for identification of the monosaccharides. Results Morphology of strains Figure 1 shows the colonial appearance of the strains on 7H11 agar plates. The mutants were markedly drier and waxier than the parent strain, which appeared smooth and shiny, suggesting an alteration in surface composition. Susceptibility to aldehydes The bactericidal activity of a range of aldehydes, measured as the time taken to obtain a 5 log reduction, is shown in Table I. Both mutants were more resistant to alkaline glutaraldehyde and glyoxal than the type strain, but all three were equally resistant to Gigasept. MCNC4 was more resistant than MCNC3 to 1% alkaline glutaraldehyde only. MIC determination The rifampicin, isoniazid and ethambutol MICs are shown in Table II. Reduction in glutaraldehyde susceptibility was associated with reduction in rifampicin and ethambutol susceptibility. The isoniazid MIC of strain NCTC 946 was maintained in its mutant derivatives. Hydrophobicity measurements Surface hydrophobicity measurements are shown in Figure 2. The type strain was less hydrophobic than the two mutant strains. The results for the two mutant strains depended upon the hydrocarbon used with octane showing a greater difference between the two mutants. MCNC3 appeared to be slightly more hydrophobic than MCNC4. Extractable fatty acid analysis GLC traces of the extractable fatty acids are shown in Figure 3. The trace shows that there was no significant quantitative or qualitative difference in the extractable fatty acids for all three strains. Mycolic acid analysis HPLC traces of the mycolic acid analysis for NCTC 946 and one of the mutants, MCNC3, are shown in Figure 4. The profiles were essentially the same, with major peaks at masses of 926, 940, 1106 and 1135, indicating that there was no difference in mycolic acid composition between these strains (MCNC4 was not examined as the method was time consuming and the results from MCNC3 were sufficient to draw appropriate conclusions). Arabinogalactan/arabinomannan analysis The amounts of monosaccharide derived by hydrolysis of the arabinogalactan fraction (precipitated by 65-85% ethanol) and hydrolysis of the arabinomannan fraction (precipitated by 55-65% ethanol), from 1 g wet weight of cells of the three M. chelonae strains is shown in Table III. In the arabinogalactan fraction both mutants contained lower amounts of all monosaccharides, as compared with the type strain. This decrease was more noticeable as the level of reduced susceptibility to glutaraldehyde increased. In the arabinomannan fraction, both mutants had less arabinose, mannose and galactose and more glucose than did the type strain. MCNC4 had slightly more arabinose and galactose than MCNC3, but slightly less glucose. No mannose was detected in either mutant. Discussion The changes in colonial morphology, susceptibility to unrelated antibiotics and hydrophobicity found in the mutants of NCTC 946 suggest a change in cell wall structure or composition. Increase in hydrophobicity could be due to overproduction of mycolic acids or shorter-chain fatty acids. However, analysis of mycolic acids indicated that there were no differences between MCNC3 and the type strain, in either the proportion or amounts of these cell wall components. These results indicate that mycolic acids and extractable fatty acids are unlikely to play a role in glutaraldehyde resistance in these strains. It is thought that isoniazid acts by inhibiting the biosynthesis of mycolic acids by preventing synthesis of very long chain fatty acid precursors. 16,17 Since no differences between the type and mutant strains were detected in the MIC of isoniazid, these findings support the view that, although isoniazid MICs are very high, altered mycolic acids are not the reason for increased glutaraldehyde resistance. The transfer of arabinogalactan into the cell wall of Mycobacterium smegmatis is inhibited by ethambutol. 15 As a slight increase in the resistance to ethambutol was observed in the two mutant strains, the arabinogalactan and arabinomannan components of the cell wall were analysed by GLC. Changes in the monosaccharide contents of the cell wall arabinogalactan and arabinomannan were observed. Decreases in the monosaccharides of arabinogalactan were associated with an increase in resistance to glutaraldehyde. This suggests that the arabinogalactan and arabinomannan portions of the cell wall could be involved in the mechanism of increased resistance to glutaraldehyde. Work is currently being carried out on the cell wall proteins to determine if this portion of the cell has been altered and is linked with an increased level of resistance to glutaraldehyde. Glutaraldehyde has widespread chemical reactivity in its unpolymerized state, as it contains two aldehyde groups which may react singly or together, 18 combining with −NH2, − COOH and −SH groups as well as many cell components containing these groups, i.e. cell wall polysaccharides, proteins and lipids. 19 Glutaraldehyde is known to react with the ε-amino group of lysine, forming internal protein cross-linking. This cross-linking could result in the impaired function of membrane transport proteins and porins. 19 It is likely that the lipid-rich layers of the cell wall act as a permeability barrier to many solutes, such as glutaraldehyde and ethambutol, preventing entry into the cell. Changes in the monosaccharides of the arabinogalactan and arabinomannan fractions may have further reduced the permeability of the cell wall, hindering the penetration of glutaraldehyde and ethambutol, thus delaying its action upon its target site. This theory would correlate with our results as both mutant strains were more hydrophobic than the type strain and both had reduced susceptibility to glutaraldehyde and ethambutol. Also, since ethambutol has been shown to inhibit the formation of arabinogalactan, 15 a reduced permeability of the cell wall would delay the penetration of it, and its action upon the arabinogalactan precursors. Table I. Susceptibility to aldehydes of M. chelonae strains . Time (in minutes) taken to obtain a ≥5 log . . reduction in viable count . Aldehyde used . NCTC 946 . MCNC3 . MCNC4 . 1% Alkaline glutaraldehyde 4 10 60 2% Alkaline glutaraldehyde 1 4 4 10% Gigasept 10 10 10 10% Glyoxal 20 >60 >60 . Time (in minutes) taken to obtain a ≥5 log . . reduction in viable count . Aldehyde used . NCTC 946 . MCNC3 . MCNC4 . 1% Alkaline glutaraldehyde 4 10 60 2% Alkaline glutaraldehyde 1 4 4 10% Gigasept 10 10 10 10% Glyoxal 20 >60 >60 Open in new tab Table I. Susceptibility to aldehydes of M. chelonae strains . Time (in minutes) taken to obtain a ≥5 log . . reduction in viable count . Aldehyde used . NCTC 946 . MCNC3 . MCNC4 . 1% Alkaline glutaraldehyde 4 10 60 2% Alkaline glutaraldehyde 1 4 4 10% Gigasept 10 10 10 10% Glyoxal 20 >60 >60 . Time (in minutes) taken to obtain a ≥5 log . . reduction in viable count . Aldehyde used . NCTC 946 . MCNC3 . MCNC4 . 1% Alkaline glutaraldehyde 4 10 60 2% Alkaline glutaraldehyde 1 4 4 10% Gigasept 10 10 10 10% Glyoxal 20 >60 >60 Open in new tab Table II. Antibiotic susceptibility of M. chelonae strains . MIC (mg/L) . Antibiotic . NCTC 946 . MCNC3 . MCNC4 . Rifampicin 50 75 100 Isoniazid >300 >300 >300 Ethambutol 10 15 20 . MIC (mg/L) . Antibiotic . NCTC 946 . MCNC3 . MCNC4 . Rifampicin 50 75 100 Isoniazid >300 >300 >300 Ethambutol 10 15 20 Open in new tab Table II. Antibiotic susceptibility of M. chelonae strains . MIC (mg/L) . Antibiotic . NCTC 946 . MCNC3 . MCNC4 . Rifampicin 50 75 100 Isoniazid >300 >300 >300 Ethambutol 10 15 20 . MIC (mg/L) . Antibiotic . NCTC 946 . MCNC3 . MCNC4 . Rifampicin 50 75 100 Isoniazid >300 >300 >300 Ethambutol 10 15 20 Open in new tab Table III. Arabinogalactan and arabinomannan analysis of M. chelonae . Amount of monosaccharide (µmol)b . Cell wall polysaccharidesa . NCTC 946 . MCNC3 . MCNC4 . ND, not detected. aArabinogalactan was precipitated in 65– 85% ethanol, and arabinomannan in 55– 65% ethanol. bThe amount of monosaccharide from the arabinogalactan and the arabinomannan precipitates, in 1 g wet weight. Arabinogalactan components  arabinose 0.15 0.06 0.0022  mannose 0.05 0.02 ND  galactose 0.0069 0.0025 0.0038  glucose 0.0024 ND ND Arabinomannan components  arabinose 0.031 ND 0.0075  mannose 0.081 ND ND  galactose 0.014 0.002 0.0053  glucose 0.0027 0.0085 0.0078 . Amount of monosaccharide (µmol)b . Cell wall polysaccharidesa . NCTC 946 . MCNC3 . MCNC4 . ND, not detected. aArabinogalactan was precipitated in 65– 85% ethanol, and arabinomannan in 55– 65% ethanol. bThe amount of monosaccharide from the arabinogalactan and the arabinomannan precipitates, in 1 g wet weight. Arabinogalactan components  arabinose 0.15 0.06 0.0022  mannose 0.05 0.02 ND  galactose 0.0069 0.0025 0.0038  glucose 0.0024 ND ND Arabinomannan components  arabinose 0.031 ND 0.0075  mannose 0.081 ND ND  galactose 0.014 0.002 0.0053  glucose 0.0027 0.0085 0.0078 Open in new tab Table III. Arabinogalactan and arabinomannan analysis of M. chelonae . Amount of monosaccharide (µmol)b . Cell wall polysaccharidesa . NCTC 946 . MCNC3 . MCNC4 . ND, not detected. aArabinogalactan was precipitated in 65– 85% ethanol, and arabinomannan in 55– 65% ethanol. bThe amount of monosaccharide from the arabinogalactan and the arabinomannan precipitates, in 1 g wet weight. Arabinogalactan components  arabinose 0.15 0.06 0.0022  mannose 0.05 0.02 ND  galactose 0.0069 0.0025 0.0038  glucose 0.0024 ND ND Arabinomannan components  arabinose 0.031 ND 0.0075  mannose 0.081 ND ND  galactose 0.014 0.002 0.0053  glucose 0.0027 0.0085 0.0078 . Amount of monosaccharide (µmol)b . Cell wall polysaccharidesa . NCTC 946 . MCNC3 . MCNC4 . ND, not detected. aArabinogalactan was precipitated in 65– 85% ethanol, and arabinomannan in 55– 65% ethanol. bThe amount of monosaccharide from the arabinogalactan and the arabinomannan precipitates, in 1 g wet weight. Arabinogalactan components  arabinose 0.15 0.06 0.0022  mannose 0.05 0.02 ND  galactose 0.0069 0.0025 0.0038  glucose 0.0024 ND ND Arabinomannan components  arabinose 0.031 ND 0.0075  mannose 0.081 ND ND  galactose 0.014 0.002 0.0053  glucose 0.0027 0.0085 0.0078 Open in new tab Figure 1. Open in new tabDownload slide The type strain (NCTC 946; centre) and two mutant strains (MCNC3 (left) and MCNC4 (right)) of M. chelonae on 7H11 agar after incubation at 30°C for 5 days. Figure 1. Open in new tabDownload slide The type strain (NCTC 946; centre) and two mutant strains (MCNC3 (left) and MCNC4 (right)) of M. chelonae on 7H11 agar after incubation at 30°C for 5 days. Figure 2. Open in new tabDownload slide Hydrophobicity measurements of M. Chelonae strains (NCTC 946 ( ), MCNC3 ( ) and MCNC4 ( )), measured by the BATH method. The absorbance of the exposed suspension is expressed as a percentage of the suspension unexposed to (a) n-hexadecane or (b) n-octane Figure 2. Open in new tabDownload slide Hydrophobicity measurements of M. Chelonae strains (NCTC 946 ( ), MCNC3 ( ) and MCNC4 ( )), measured by the BATH method. The absorbance of the exposed suspension is expressed as a percentage of the suspension unexposed to (a) n-hexadecane or (b) n-octane Figure 3. Open in new tabDownload slide GLC traces of the extractable fatty acids from three M. chelonae strains, NCTC 946, MCNC3 and MCNC4. The time scale is shown in minutes. Figure 3. Open in new tabDownload slide GLC traces of the extractable fatty acids from three M. chelonae strains, NCTC 946, MCNC3 and MCNC4. The time scale is shown in minutes. Figure 4. Open in new tabDownload slide HPLC trace of the mycolic acid bromophenacyl esters of (a) M. chelonae NCTC 946 and (b) M. chelonae MCNC3. Peaks are measured in milli-absorbance units (mAU) at 254 nm. The time scale is in minutes. Figure 4. Open in new tabDownload slide HPLC trace of the mycolic acid bromophenacyl esters of (a) M. chelonae NCTC 946 and (b) M. chelonae MCNC3. Peaks are measured in milli-absorbance units (mAU) at 254 nm. The time scale is in minutes. * Corresponding author. Part of this work was presented at the Third Federation of Infection Societies (FIS) meeting in November 1996. References 1. Carson, L. A., Petersen, N. J., Favero, M. S. & Aguero, S. M. ( 1978 ). Growth characteristics of atypical mycobacteria in water and their comparative resistance to disinfectants. Applied and Environmental Microbiology 36 , 839 –46. 2. Goslee, S. & Wolinsky, E. ( 1976 ). Water as a source of potentially pathogenic mycobacteria. 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E. & Mdluli, K. ( 1996 ). Drug sensitivity and environmental adaptation of mycobacterial cell wall components. Trends in Microbiology 4 , 275 –81. 18. Hugo, W. B. & Russell, A. D. (1992). Types of antimicrobial agents. In Principles and Practice of Disinfection, Preservation and Sterilisation, 2nd edn (Russell, A. D., Hugo, W. B. & Ayliffe, G. A. J., Eds), pp. 7–88. Blackwell Scientific, London. 19. Hugo, W. B. (1992). Disinfection mechanisms. In Principles and Practice of Disinfection, Preservation and Sterilisation, 2nd edn (Russell, A. D., Hugo, W. B. & Ayliffe, G. A. J., Eds), pp. 187–210. Blackwell Scientific, London. The British Society for Antimicrobial Chemotherapy
TI - Reduced glutaraldehyde susceptibility in Mycobacterium chelonae associated with altered cell wall polysaccharides
JF - Journal of Antimicrobial Chemotherapy
DO - 10.1093/jac/43.6.759
DA - 1999-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/reduced-glutaraldehyde-susceptibility-in-mycobacterium-chelonae-3XuT6Qr0G8
SP - 759
VL - 43
IS - 6
DP - DeepDyve
ER -