APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1992, p. 2420-2425
Vol. 58, No. 8
0099-2240/92/082420-06$02.00/0 Copyright C) 1992, American Society for Microbiology
Relationship between Legionella pneumophila and Acanthamoeba polyphaga: Physiological Status and Susceptibility to Chemical Inactivation JOHN BARKER,1 MICHAEL R. W. BROWN,2* PHILLIP J. COLLIER,2 IAN FARRELL,1 AND PETER GILBERT3 Regional Public Health Laboratory, East Binningham Hospital, Birmingham B9 5ST, 1 Department of Pharmaceutical Sciences, Pharnaceutical Sciences Institute, Aston University, Aston Triangle, Birningham B4 7ET,2 and Department of Pharnacy, University of Manchester,
Manchester M13 9PL,3 United Kingdom Received 31 January 1992/Accepted 20 May 1992
Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1x MIC) gave 99.99%o reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.
Legionella pneumophila is ubiquitous in aquatic environ-
chlorine (25). It is likely that, as with Pseudomonas aeruginosa infections in humans (3), engulfed L. pneumophila cells are subject to an amoeba-imposed iron restriction. Such conditions might alter the phenotypic response of the legionellae and affect the susceptibility of the released organism. By utilizing a chemically defined culture medium for L. pneumophila (31), we evaluated the separate contributions of iron deprivation and intra-amoebal growth to the susceptibility of this organism to the thiol-interactive isothiazolone biocides (8) and to polyhexamethylene biguanide (PHMB), which causes injury to the cell envelope (6, 17).
ments and may serve as a source of human infection when
found in association with air conditioning machinery, cooling towers, and water systems in large buildings. Rigorous regimes of temperature control and chlorination of hot and cold water systems (7, 11) and chemical treatment of cooling towers (24, 27) are generally employed to combat the presence of microorganisms but have failed to eradicate legionellae from such plants. Contributory to the recalcitrance of legionellae in water systems is its growth within an adherent biofilm comprising numerous other bacterial species, protozoa, and ciliates (25). Together, these form a complex balanced ecosystem in which the legionellae are able to express several physiological states: as planktonic cells, as free-living components of the biofilm ecosystem, and in association with amoebae, which may become parasitized by this organism (32). In the biomedical field it has been clearly demonstrated that slow growth rates within biofilms and particular nutrient insufficiences cause the expression of distinct phenotypes that are often resistant to chemical (10) and antibiotic (2, 5, 16) agents. Similar instances of expressed phenotype might well contribute to the properties of legionellae in situ and also to their apparent recalcitrance to chemical inactivation. Whereas the majority of studies considering the susceptibility of L. pneumophila to disinfectants have utilized cells grown in complex, nutrient-rich media (15, 18, 35), there is recent evidence (22, 30) to suggest that intra-amoebal growth significantly enhances the resistance of this organism to
*
MATERLALS AND METHODS
Biocides. PHMB (Vantocil) and benzisothiazolone (BIT; Proxel) were obtained from ICI, Organics Division, Blackley, Manchester, United Kingdom. 5-Chloro-N-methylisothiazolone (CMIT), the active ingredient of Kathon (Rohm and Haas, Rochester, N.Y.), was synthesized and its purity was confirmed as previously described (26). Organisms and culture maintenance. Acanthamoeba polyphaga was obtained from T. Rowbotham, Leeds Public Health Laboratory, Leeds, U.K. A human isolate of L. pneumophila serogroup 1, subtype Knoxville (19), was used throughout. Continual passage was avoided by storage on polystyrene beads at -70°C (1). Organisms were recovered on buffered charcoal-yeast extract agar (BCYE) (14) and then incubated at 35°C for 4 days in a moist atmosphere. In vitro preparation of L. pneumophila suspensions. Fourday BCYE cultures of L. pneumophila were scraped from the agar into 0.25x Ringer's solution (RS; Oxoid) (pH 7.0), pelleted by centrifugation (2,080 x g, 15 min, room temper-
Corresponding author. 2420
VOL.
58,1992
PHYSIOLOGY AND SUSCEPTIBILITY OF L. PNEUMOPHILA
sterile double-distilled water as described above before being used. Chelex treatment of media also reduced iron to growth-limiting levels (13, 20). Suspensions for susceptibility testing were prepared from ABCD broth and iron-depleted cultures after 4 days of incubation. Cells were harvested by centrifugation (2,080 x g, 15 min, room temperature), washed, and finally resuspended in RS. Culture of A. polyphaga. A. polyphaga was grown axenically at 35°C in PYG broth (32) as monolayers in 75-cm2 tissue culture flasks containing shallow levels of liquid. After 3 days of incubation, the amoebae were harvested by centrifugation (400 x g, 6 min, room temperature), washed twice, and resuspended in sterile amoebal saline (32) to give cell densities of ca. 105 cells per ml as assessed with a hemocytometer. Intra-amoebal culture of L. pneumophila. For biocide susceptibility studies, L. pneumophila cells were grown in amoebae in two stages. Suspensions of A. polyphaga (105 trophozoites per ml) were inoculated with water-washed ABCD broth cultures of L. pneumophila (102 bacteria per ml) and incubated for 10 days at 35°C. Viable counts were performed on BCYE agar, and the mixed suspension was centrifuged (400 x g, 6 min, room temperature) to remove amoebal cells. Legionellae were harvested from the supernatants by further centrifugation (2,080 x g, 15 min, room temperature). The resultant pellets were washed twice and resuspended in amoebal saline. The second-stage culture within amoebae was as described above, but the initial inoculum levels were increased to 105 cells per ml for the legionellae. Cultures were monitored by phase-contrast microscopy, which indicated the presence of infective, highly motile intra- and extra-amoebal legionellae after 3 days of incubation. At this point the bacterial cells were harvested as described above and resuspended in RS. Biocide susceptibility. The MICs of BIT, PHMB, and CMIT were determined by serial (1/2) dilution of the agents in ABCD broth and inoculation with L. pneumophila (106 cells per ml). Tubes were assessed for visible signs of growth at 60 h, and the MIC was determined as the lowest concentration of biocide that prevented growth. The MICs were 1, 4, and 32 ,ug/ml for PHMB, BIT, and CMIT, respectively. After 60 h of incubation at 35°C, aliquots (0.1 ml) were removed and spread on the surfaces of BCYE agar plates. Minimum concentrations that effected 99.9% reduction of viability within these constraints were taken as the MBCs,
9
8
E
7
5
4
0
1
2
3
4
2421
5
Time (Days) FIG. 1. Viable counts of L. pneumophila in amoebal saline (A), with metabolic products of lysed A. polyphaga (A), in coculture with A. polyphaga trophozoites (O) and total cell count of A. polyphaga (M) in coculture with L. pneumophila. Bars represent the standard errors of the means associated with three replicate experiments.
ature), and resuspended in sterile, double-distilled water to anA660 of 1.0. Aliquots (750 ,ul) were inoculated into 25 ml of defined synthetic broth (ABCD broth [31]) and incubated at 35°C in a shaking incubator (100 oscillations per min). Iron-depleted cultures were also prepared in ABCD broth, except that iron (Fe2SO4) and heme were omitted and the inoculation was with washed suspensions prepared from ABCD broth cultures (4 days) rather than from BCYE agar cultures. The inocula were harvested by centrifugation (2,080 x g, 15 min, room temperature) and resuspended in
TABLE 1. Growth-inhibitory activity and cytopathic effects, over various time periods, of BIT, CMIT, and PHMB against A. polyphaga grown in PYG broth at 35°C
Cytopathic effect' (cell morphology)
Growth-inhibitory concn'
Biocide
BIT CMIT PHMB
(jig/ml) after exposure for: 3 days
5 days
5.0 10 0.3
5.0 10 0.6
after exposure for:
Concn used for treatment
(pg/ml) 8 16 2
20 hC
100% rounded 100% rounded 95% rounded
24 h
(after
recoveryd)
95% trophozoites 100% rounded 70% rounded
30% trophozoites
Control a
50% rounded 50% trophozoites
Minimum concentration of biocide inhibiting the formation of cell monolayers in PYG broth at 350C.
b As observed by phase-contrast inverted microscopy. c The response of confluent monolayers to biocide treatment at 35°C; the control was amoebal saline only. dRecovery was determined by removing the biocide and washing and resuspending the cells in PYG broth.
95% trophozoites
APPL. ENVIRON. MICROBIOL.
BARKER ET AL.
2422
1000
100
100 10
10 .-I
S
=
I..
1
C,, 0 -j
0 -j
.1 .1
.01
.01 0
1
2
3
4
5
6
7
Time (Hours) FIG. 2. Survival of stationary-phase ABCD broth cultures of L. pneumophila (-, A, *) and amoeba-grown L. pneumophila (El, A, O) after exposure to various concentrations of PHMB (0.5 ,ug/ml [O, U], 1.0 ,ug/ml [A, A], 2.0 p,g/ml [O, *]) at 35'C in RS. Controls (x) for both treatments were coincident. Bars represent the standard error of the mean associated with each data point.
which were 1, 16, and 64 ,ug/ml for PHMB, BIT, and CMIT, respectively. Time survival data were determined for variously grown L. pneumophila suspensions by exposure of cells (10 cells per ml) to various concentrations of the biocides, selected on the basis of their MICs and MBCs. Aliquots (0.1 ml) of suspensions were taken at the commencement of the exposure and at 2, 4, 6, and 24 h. Serial dilutions were made initially in sodium thioglycollate (0.01 M) to inactivate the isothiazolone biocides (9) and then in sterile RS. The dilutions were spread onto the surface of predried BCYE agar plates. Colony counts were made after 9 days of incubation at 35°C, and the results were expressed as the percent survival. The data were subjected to analysis of variance and Student t tests were applied to 4- and 6-h survival data by utilizing an SPSS statistics package and an IBM 386 PC computer.
Antiamoebal activity was determined by preparing serial (1/2) dilutions of the biocides in PYG broth and distributing into flat-bottomed microtiter trays. These were inoculated with washed suspensions of A. polyphaga to give ca. 103 amoebae per ml. The suspensions were incubated at 35°C and observed through an inverted microscope after 1, 3, and 5 days. The MIC was taken as the minimum concentration of biocide that inhibited the formation of a confluent layer of trophozoites on the bases of the chambers after 5 days. In addition, the cytopathic effects of the biocides on established monolayers of trophozoites were determined by observing
0
1
2
3
4
5
6
7
Time (Hours) FIG. 3. Survival of stationary-phase ABCD broth cultures of L. pneumophila (-, A, *) and amoeba-grown L. pneumophila (l, A, O) after exposure to various concentrations of BIT (16 ,ug/ml [K>, *], 32 ,ug/ml [O, *], 64 ,ug/ml [A and A]) at 35'C in RS. Controls (x) for both treatments were coincident. Bars represent the standard error of the mean associated with each data point.
the ability of the organism to recover after treatment and transfer to fresh medium. RESULTS
Figure 1 shows the release of L. pneumophila from A. polyphaga during the second stage of culture. Initial inoculum levels were set at ca. 5 x 105 cells per ml for both bacteria and trophozoites. After 3 days of incubation, the number of L. pneumophila cells had increased to 108 cells per ml and the number of A. polyphaga had decreased by 90%; the majority of the surviving amoebae were encysted. The legionellae did not multiply in amoebal saline without A. polyphaga trophozoites or in amoebal saline containing metabolic products released from viable amoebal trophozoites by ultrasonic disintegration. King et al. (23) also found that L. pneumophila would not grow extracellularly when separated by a 0.2-,um-pore-size membrane filter in media containing viable Hartmanella verniformis trophozoites. L. pneumophila cells obtained after 3 days of growth in A. polyphaga were highly motile and generally smaller (ca. 1 pum) than their broth-grown counterparts (ca. 2 ,um). After continued incubation for an additional 24 h, the legionellae were nonmotile and the amoebae were predominantly encysted. This two-stage culture, with L. pneumophila being harvested at 3 days in the second stage, minimized the numbers of legionellae that had not been passaged through amoebae in subsequent tests. The antiamoebal properties of the biocides BIT, CMIT,
PHYSIOLOGY AND SUSCEPTIBILITY OF L. PNEUMOPHILA
VOL. 58, 1992
2423
c 100
a 100
10
10
L-
1
cn
-
CO)
cm
cm
0 -J
1
.1
.1
.01 0
2
1
3
5
4
6
7
0
Time (Hours)
2
3
4
5
6
7
Time (Hours) FIG. 4. Survival of L. pneumophila after growth at 35°C in ABCD broth (El) or iron-depleted ABCD broth (-) or passage through amoebae (A) after exposure to BIT (16 ,ug/ml) (a), PHMB (1.0 ,ug/ml) (b), or CMIT (32 ,ug/ml) (c) with controls (x). Bars represent the standard errors of the means for three replicate
b 1000
experiments.
100
aI
1
10
1 -1
.1
.01
0
1
2
3
4
5
6
7
Time (Hours)
and PHMB are summarized in Table 1. The MICs on days 3 and 5 were identical for the isothiazolone biocides, the MIC for PHMB was increased from 0.3 ,ug/ml to 0.6 ,ug/ml on continued incubation. However, the activities for PHMB were significantly greater than those for the isothiazolones. Direct exposure of the amoebae to these agents caused them
to lose their characteristic trophozoite appearance. Rounding of the cells but failure to encyst may be taken as an indication of reduced metabolic activity of the protozoa and is also seen on starvation. The ability of the rounded amoebae to revert to the trophozoite form after treatment with BIT and PHMB, but not after treatment with CMIT, indicates that irreversible damage to the amoebal cell is caused by CMIT. When cells were exposed to biocides for 20 h and tested for their ability to recover on transfer to fresh medium, a different pattern of susceptibilities emerged. The majority of cells recovered from BIT treatment, few (if any) cells recovered from CMIT treatment, and ca. 30% of cells recovered from PHMB exposure. However, such data should be viewed within the context of the individual biocide use concentrations. In this respect, the concentrations of CMIT employed in these experiments are higher than those generally used in water treatment (ca. 10 ,ug/ml) (27) and the concentrations of PHMB are 10 to 20% of those employed in
its major application of swimming pool sanitation (ca. 10 p,g/ml) (38). The BIT concentrations used in these procedures were much lower than their use concentrations of around 150 ppm; sometimes BIT is used as part of a complex with lauryldimethyl benzylammonium chloride (34). The relative susceptibilities of ABCD-broth-grown and amoeba-grown L. pneumophila to BIT and PHMB are shown in Fig. 2 and 3. The activities of both biocides were greatly reduced in amoeba-grown cells. Student t tests (paired with respect to amoeba- and ABCD-grown cells) showed the amoebal cells to be significantly less susceptible
2424
BARKER ET AL.
(for PHMB, t = 5.44 and 0 = 6; for BIT, t = 7.06 and 4) = 6). Bactericidal activities reflected the MIC data in that PHMB was more active at concentrations well below the recommended use concentrations for swimming pool applications (ca. 10 ,ug/ml). Concentrations of PHMB equivalent to 4 times the MIC gave 4 log 10 cycles of killing within 6 h and no detectable survivors at 24 h for the broth-grown cultures. This bactericidal activity was reduced to 90% killing at 6 h and 99.9% killing at 24 h against the amoebagrown organisms. Concentrations of BIT equivalent to 4 times its MIC resulted in reductions in viability of only 3 log 10 cycles within 6 h for broth-grown cells and by ca. 40% for amoeba-grown cells. After 24 h of contact, there were no detectable survivors from the broth-grown inoculum, whereas 30 to 50% of the amoeba-grown cells survived. In
Fig. 4 the relative effects of iron deprivation, intra-amoebal growth, and nutrient-rich conditions on susceptibility of L. pneumophila to BIT (Fig. 4a), PHMB (Fig. 4b), and CMIT (Fig. 4c) are compared. BIT was relatively ineffective against both iron-depleted and amoeba-grown cells but not against ABCD broth-grown (P = 0.95) cells. Whereas the activity of PHMB was once again reduced by intra-amoebal growth, iron depletion of the culture increased the susceptibility of L. pneumophila beyond that of the ABCD brothgrown organisms. However, the difference between the susceptibility of ABCD broth-grown cells and that of irondepleted cells was not significant (P = 0.95). A similar pattern of susceptibilities was also indicated for CMIT; iron-deprived cells were the most susceptible. DISCUSSION In the aquatic environment, L. pneumophila infects and multiplies within a wide range of amoebal hosts (32, 33). Therefore, the environmental stresses imposed upon such cells and their physiological responses and phenotypes are unlikely to be represented by typical broth cultures (4, 16). In common with infections of humans (3), intra-amoebal growth is likely to subject the bacterial cells to iron insufficiency, causing them to express iron-deprived phenotypes. These phenotypes were found to differ significantly in their susceptibility to chemical inactivation. CMIT (the active ingredient of Kathon CG, a biocide recommended for water treatment) was profoundly bactericidal to iron-restricted and ABCD-broth grown legionellae, but it was relatively ineffective against amoeba-grown cultures even at concentrations far in excess of those recommended for use (15, 27). This is in spite of conducting the tests in a thiol-free environment to reduce the potentially neutralizing effects of the medium (9). These data suggest either that iron restriction does not apply within the amoeba or that some other factor confers resistance to CMIT on amoeba-grown cells. Such resistance of amoeba-grown cultures of L. pneumophila to chlorine has also been reported (22, 30) and has been suggested to account for the recalcitrance of this organism to conventional water treatment. Curiously, PHMB, an agent routinely used in swimming pool sanitization, was effective not only against the amoeba-grown legionellae but also against the amoebae. Such activity was demonstrable at PHMB concentrations of 10% of the recommended-use level (10 p,g/ml) (38). These results are similar to those observed by Kilvington (21). The toxicity profiles of the isothiazolone biocides greatly limit the applications to which they can be deployed and also the concentrations to which humans may be exposed (28, 37). This is not the case for PHMB, which is relatively nontoxic and has recently found application as a
APPL. ENVIRON. MICROBIOL.
preservative of ophthalmic lens solutions (12). The recalcitrance of L. pneumophila grown intra-amoebally might relate to the increased levels of poly-p-hydroxybutyric acid inclusions (32, 36) that are associated with this mode of growth. Such lipophilic moeties might act as a sink for the relatively hydrophobic isothiazolone biocides and reduce their interaction with cytosolic thiol groups (8). Such inclusions are unlikely to affect the action of PHMB, which is hydrophilic and acts solely at the level of the cell membrane (6, 17). In a similar fashion, changes in the L. pneumophila cell wall, which is characteristically rich in branched-chain fatty acids (29), would affect isothiazolone activity to a far greater extent than it would affect PHMB activity. In conclusion, these studies clearly indicate the profound effect of intra-amoebal growth on the physiological status and antimicrobial susceptibility of L. pneumophila and suggest that such phenomena might account for the recalcitrance of the organism in situ. Particularly, these data show PHMB to be a potential biocide for water treatment with useful activities against legionellae in all physiological states and against the host amoebae. ACKNOWLEDGMENTS This study was funded in part by a grant awarded to J.B. from the Public Health Laboratory Service, England and Wales. We thank T. Rowbotham for helpful discussions on this topic. REFERENCES 1. Barker, J., and D. H. Till. 1986. Survival of Legionella pneumophila. Med. Lab. Sci. 43:388-389. 2. Brown, M. R. W., D. G. Allison, and P. Gilbert. 1988. Resistance of bacterial biofilms to antibiotics: a growth rate related effect? Antimicrob. Chemother. 22:777-783. 3. Brown, M. R. W., H. Anwar, and P. A. Lambert. 1984. Evidence that mucoid Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron-restricted conditions. FEMS Microbiol. Lett. 21:113-117. 4. Brown, M. R. W., P. J. Collier, and P. Gilbert. 1990. Influence of growth rate on susceptibility to antimicrobial agents: modification of the cell envelope and batch and continuous culture studies. Antimicrob. Agents Chemother. 34:1623-1628. 5. Brown, M. R. W., and P. Williams. 1985. Influence of substrate limitation and growth phase on sensitivity to antimicrobial agents. J. Antimicrob. Chemother. 15(Suppl. A):7-14. 6. Broxton, P., P. M. Woodcock, and P. Gilbert. 1984. Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli. J. Appl. Bacteriol. 57:115-124. 7. Chartered Institution of Building Service Engineers: 1987. Technical memorandum (TM 13). Minimising the risk from Legionaires disease. Chartered Institution of Building Service Engineers, London. 8. Collier, P. J., P. Austin, and P. Gilbert. 1990. Uptake and distribution of some isothiazolone biocides into Escherichia coli ATCC8739 and Schizosaccharomyces pombe NCYC1354. Int. J. Pharm. 66:201-206. 9. Collier, P. J., A. J. Ramsey, P. Austin, and P. Gilbert. 1990. Growth inhibiting and biocidal activity of some isothiazolone biocides. J. Appl. Bacteriol. 69:569-577. 10. Costerton, J. W., T. J. Marrie, and K. J. Cheng. 1985. Phenomena of bacterial adhesion, p. 3-43. In D. C. Savage and M. Fletcher (ed.), Bacterial adhesion. Plenum Publishing Corp., New York. 11. Department of Health and Social Security and the Welsh Office. 1988. The control of legionellae in health care premises: a code of practice. Her Majesty's Stationery Office, London. 12. Dexter, D., M. B. Ficke, and B. T. Decicco. 1989. Effectiveness of polyhexamethylene biguanide against bacteria of product, clinical and environmental origins, abstr. A122. Abstr. 89th Annu. Meet. Am. Soc. Microbiol. 1989. American Society for Microbiology, Washington, D.C.
VOL. 58, 1992
PHYSIOLOGY AND SUSCEPTIBILITY OF L. PNEUMOPHILA
13. Domingue, P. A. G., B. Mottle, D. W. Morck, M. R. W. Brown, and J. W. Costerton. 1990. A simplified and rapid method for the removal of iron and other cations from complex media. J. Microbiol. Methods 12:13-22. 14. Edelstein, P. H. 1981. Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J. Clin. Microbiol. 14:298-303. 15. Elsmore, R. 1986. Biocidal control of legionellae. Isr. J. Med. Sci. 22:647-654. 16. Gilbert, P., P. J. Collier, and M. R. W. Brown. 1990. Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob. Agents Chemother. 34:1865-1868. 17. Gilbert, P., D. Pemberton, and D. E. Wilkinson. 1990. Barrier properties of the Gram-negative cell envelope towards high molecular weight polyhexamethylene biguanides. J. Appl. Bacteriol. 69:585-592. 18. Hollis, C. G., and D. L. Smalley. 1980. Resistance of Legionella pneumophila to microbiocides. Dev. Ind. Microbiol. 21:265271. 19. Joly, J. R., R. M. McKinney, J. O'H. Tobin, W. F. Bibb, I. D. Watkins, and D. R. Ramsey. 1986. Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies. J. Clin. Microbiol. 23:768-771. 20. Kadurugamuwa, J. L., H. Anwar, M. R. W. Brown, G. H. Shand, and K. H. Ward. 1987. Media for study of growth kinetics and envelope properties of iron-deprived bacteria. J. Clin. Microbiol. 25:849-855. 21. Kilvington, S. 1990. Activity of water biocide chemicals and contact lens disinfectants on pathogenic free-living amoebae. Int. Biodeterior. 26:127-138. 22. Kilvington, S., and J. Price. 1990. Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. J. Appl. Bacteriol. 68:519-525. 23. King, C. H., B. S. Fields, E. B. Shotts, Jr., and E. H. White. 1991. Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells. Infect. Immun. 59:758-763. 24. Kurtz, J. B., C. L. R. Bartlett, U. A. Newton, R. A. White, and N. L. Jones. 1982. Legionella in cooling water systems. Report of a survey of cooling towers in London and a pilot trial of selected biocides. J. Hyg. Camb. 88:369-381. 25. Lee, J. V., and A. A. West. 1991. Survival and growth of Legionella species in the environment. J. Appl. Bacteriol. (Symp. Suppl.) 70:121s-129s.
2425
26. Lewis, S. N., and G. A. Miller. June 1974. U.S. patent 3,835,150. 27. McCoy, W. F., J. W. Wireman, and E. S. Lashen. 1986. Efficacy of methylchloroisothiazolone biocide against Legionella pneumophila in cooling tower water. Chim. Oggi. 4:79-83. 28. Monte, W. C., S. H. Ashoor, and B. J. Lewis. 1983. Mutagenicity of two non-formaldehyde-forming antimicrobial agents. Food Chem. Toxicol. 21:695-697. 29. Moss, C. W., R. E. Weaver, S. B. Dees, and W. B. Cherry. 1977. Cellular fatty acid composition of isolates from Legionaires' disease. J. Clin. Microbiol. 6:140-143. 30. Navratil, J. S., R. H. Palmer, S. States, J. M. Kuchta, R. M. Wadowsky, and R. B. Yee. 1990. Increased chlorine resistance of Legionella pneumophila released after growth in amoeba Hartmanella vermiformis, abstr. Q82. Abstr. 90th Annu. Meet. Am. Soc. Microbiol. 1990. American Society for Microbiology, Washington, D.C. 31. Pine, L., P. S. Hoffman, G. B. Malcolm, R. F. Benson, and M. J. Franzus. 1986. Role of keto acids and reduced oxygen-scavenging enzymes in the growth of Legionella species. J. Clin. Microbiol. 23:33-42. 32. Rowbotham, T. J. 1983. Isolation of Legionella pneumophila from clinical specimens via amoebae, and the interaction of those and other isolates with amoebae. J. Clin. Pathol. 36:978986. 33. Rowbotham, T. J. 1986. Current views on the relationship between amoeba, legionellae and man. Isr. J. Med. Sci. 22:678689. 34. Singer, M. 1976. Laboratory procedures for assessing the potential of antimicrobial agents as industrial biocides. Process Biochem. 11:30-35. 35. Skaliy, P., T. A. Thompson, J. G. W. Gorman, G. K. Morris, H. V. McEachem, and D. C. Mackel. 1980. Laboratory studies of disinfectants against Legionella pneumophila. Appl. Environ. Microbiol. 40:697-700. 36. Vandenesh, F., M. Surgot, N. Bornstein, J. C. Paucod, D. Marmet, P. Isoard, and J. Fleurette. 1990. Relationship between free amoeba and Legionella: studies in vitro and in vivo. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 272:265-275. 37. Weaver, J. E., C. W. Cardin, and H. I. Maibach. 1985. Doseresponse assessments of Kathon biocide. Contact Dermatitis 12:141-145. 38. Yang, W., and G. S. Banker. 1981. Biguanide-induced staining in oral hygeine. Drug Dev. Ind. Pharm. 7:113-133.
Vol. 58, No. 8
0099-2240/92/082420-06$02.00/0 Copyright C) 1992, American Society for Microbiology
Relationship between Legionella pneumophila and Acanthamoeba polyphaga: Physiological Status and Susceptibility to Chemical Inactivation JOHN BARKER,1 MICHAEL R. W. BROWN,2* PHILLIP J. COLLIER,2 IAN FARRELL,1 AND PETER GILBERT3 Regional Public Health Laboratory, East Binningham Hospital, Birmingham B9 5ST, 1 Department of Pharmaceutical Sciences, Pharnaceutical Sciences Institute, Aston University, Aston Triangle, Birningham B4 7ET,2 and Department of Pharnacy, University of Manchester,
Manchester M13 9PL,3 United Kingdom Received 31 January 1992/Accepted 20 May 1992
Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1x MIC) gave 99.99%o reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.
Legionella pneumophila is ubiquitous in aquatic environ-
chlorine (25). It is likely that, as with Pseudomonas aeruginosa infections in humans (3), engulfed L. pneumophila cells are subject to an amoeba-imposed iron restriction. Such conditions might alter the phenotypic response of the legionellae and affect the susceptibility of the released organism. By utilizing a chemically defined culture medium for L. pneumophila (31), we evaluated the separate contributions of iron deprivation and intra-amoebal growth to the susceptibility of this organism to the thiol-interactive isothiazolone biocides (8) and to polyhexamethylene biguanide (PHMB), which causes injury to the cell envelope (6, 17).
ments and may serve as a source of human infection when
found in association with air conditioning machinery, cooling towers, and water systems in large buildings. Rigorous regimes of temperature control and chlorination of hot and cold water systems (7, 11) and chemical treatment of cooling towers (24, 27) are generally employed to combat the presence of microorganisms but have failed to eradicate legionellae from such plants. Contributory to the recalcitrance of legionellae in water systems is its growth within an adherent biofilm comprising numerous other bacterial species, protozoa, and ciliates (25). Together, these form a complex balanced ecosystem in which the legionellae are able to express several physiological states: as planktonic cells, as free-living components of the biofilm ecosystem, and in association with amoebae, which may become parasitized by this organism (32). In the biomedical field it has been clearly demonstrated that slow growth rates within biofilms and particular nutrient insufficiences cause the expression of distinct phenotypes that are often resistant to chemical (10) and antibiotic (2, 5, 16) agents. Similar instances of expressed phenotype might well contribute to the properties of legionellae in situ and also to their apparent recalcitrance to chemical inactivation. Whereas the majority of studies considering the susceptibility of L. pneumophila to disinfectants have utilized cells grown in complex, nutrient-rich media (15, 18, 35), there is recent evidence (22, 30) to suggest that intra-amoebal growth significantly enhances the resistance of this organism to
*
MATERLALS AND METHODS
Biocides. PHMB (Vantocil) and benzisothiazolone (BIT; Proxel) were obtained from ICI, Organics Division, Blackley, Manchester, United Kingdom. 5-Chloro-N-methylisothiazolone (CMIT), the active ingredient of Kathon (Rohm and Haas, Rochester, N.Y.), was synthesized and its purity was confirmed as previously described (26). Organisms and culture maintenance. Acanthamoeba polyphaga was obtained from T. Rowbotham, Leeds Public Health Laboratory, Leeds, U.K. A human isolate of L. pneumophila serogroup 1, subtype Knoxville (19), was used throughout. Continual passage was avoided by storage on polystyrene beads at -70°C (1). Organisms were recovered on buffered charcoal-yeast extract agar (BCYE) (14) and then incubated at 35°C for 4 days in a moist atmosphere. In vitro preparation of L. pneumophila suspensions. Fourday BCYE cultures of L. pneumophila were scraped from the agar into 0.25x Ringer's solution (RS; Oxoid) (pH 7.0), pelleted by centrifugation (2,080 x g, 15 min, room temper-
Corresponding author. 2420
VOL.
58,1992
PHYSIOLOGY AND SUSCEPTIBILITY OF L. PNEUMOPHILA
sterile double-distilled water as described above before being used. Chelex treatment of media also reduced iron to growth-limiting levels (13, 20). Suspensions for susceptibility testing were prepared from ABCD broth and iron-depleted cultures after 4 days of incubation. Cells were harvested by centrifugation (2,080 x g, 15 min, room temperature), washed, and finally resuspended in RS. Culture of A. polyphaga. A. polyphaga was grown axenically at 35°C in PYG broth (32) as monolayers in 75-cm2 tissue culture flasks containing shallow levels of liquid. After 3 days of incubation, the amoebae were harvested by centrifugation (400 x g, 6 min, room temperature), washed twice, and resuspended in sterile amoebal saline (32) to give cell densities of ca. 105 cells per ml as assessed with a hemocytometer. Intra-amoebal culture of L. pneumophila. For biocide susceptibility studies, L. pneumophila cells were grown in amoebae in two stages. Suspensions of A. polyphaga (105 trophozoites per ml) were inoculated with water-washed ABCD broth cultures of L. pneumophila (102 bacteria per ml) and incubated for 10 days at 35°C. Viable counts were performed on BCYE agar, and the mixed suspension was centrifuged (400 x g, 6 min, room temperature) to remove amoebal cells. Legionellae were harvested from the supernatants by further centrifugation (2,080 x g, 15 min, room temperature). The resultant pellets were washed twice and resuspended in amoebal saline. The second-stage culture within amoebae was as described above, but the initial inoculum levels were increased to 105 cells per ml for the legionellae. Cultures were monitored by phase-contrast microscopy, which indicated the presence of infective, highly motile intra- and extra-amoebal legionellae after 3 days of incubation. At this point the bacterial cells were harvested as described above and resuspended in RS. Biocide susceptibility. The MICs of BIT, PHMB, and CMIT were determined by serial (1/2) dilution of the agents in ABCD broth and inoculation with L. pneumophila (106 cells per ml). Tubes were assessed for visible signs of growth at 60 h, and the MIC was determined as the lowest concentration of biocide that prevented growth. The MICs were 1, 4, and 32 ,ug/ml for PHMB, BIT, and CMIT, respectively. After 60 h of incubation at 35°C, aliquots (0.1 ml) were removed and spread on the surfaces of BCYE agar plates. Minimum concentrations that effected 99.9% reduction of viability within these constraints were taken as the MBCs,
9
8
E
7
5
4
0
1
2
3
4
2421
5
Time (Days) FIG. 1. Viable counts of L. pneumophila in amoebal saline (A), with metabolic products of lysed A. polyphaga (A), in coculture with A. polyphaga trophozoites (O) and total cell count of A. polyphaga (M) in coculture with L. pneumophila. Bars represent the standard errors of the means associated with three replicate experiments.
ature), and resuspended in sterile, double-distilled water to anA660 of 1.0. Aliquots (750 ,ul) were inoculated into 25 ml of defined synthetic broth (ABCD broth [31]) and incubated at 35°C in a shaking incubator (100 oscillations per min). Iron-depleted cultures were also prepared in ABCD broth, except that iron (Fe2SO4) and heme were omitted and the inoculation was with washed suspensions prepared from ABCD broth cultures (4 days) rather than from BCYE agar cultures. The inocula were harvested by centrifugation (2,080 x g, 15 min, room temperature) and resuspended in
TABLE 1. Growth-inhibitory activity and cytopathic effects, over various time periods, of BIT, CMIT, and PHMB against A. polyphaga grown in PYG broth at 35°C
Cytopathic effect' (cell morphology)
Growth-inhibitory concn'
Biocide
BIT CMIT PHMB
(jig/ml) after exposure for: 3 days
5 days
5.0 10 0.3
5.0 10 0.6
after exposure for:
Concn used for treatment
(pg/ml) 8 16 2
20 hC
100% rounded 100% rounded 95% rounded
24 h
(after
recoveryd)
95% trophozoites 100% rounded 70% rounded
30% trophozoites
Control a
50% rounded 50% trophozoites
Minimum concentration of biocide inhibiting the formation of cell monolayers in PYG broth at 350C.
b As observed by phase-contrast inverted microscopy. c The response of confluent monolayers to biocide treatment at 35°C; the control was amoebal saline only. dRecovery was determined by removing the biocide and washing and resuspending the cells in PYG broth.
95% trophozoites
APPL. ENVIRON. MICROBIOL.
BARKER ET AL.
2422
1000
100
100 10
10 .-I
S
=
I..
1
C,, 0 -j
0 -j
.1 .1
.01
.01 0
1
2
3
4
5
6
7
Time (Hours) FIG. 2. Survival of stationary-phase ABCD broth cultures of L. pneumophila (-, A, *) and amoeba-grown L. pneumophila (El, A, O) after exposure to various concentrations of PHMB (0.5 ,ug/ml [O, U], 1.0 ,ug/ml [A, A], 2.0 p,g/ml [O, *]) at 35'C in RS. Controls (x) for both treatments were coincident. Bars represent the standard error of the mean associated with each data point.
which were 1, 16, and 64 ,ug/ml for PHMB, BIT, and CMIT, respectively. Time survival data were determined for variously grown L. pneumophila suspensions by exposure of cells (10 cells per ml) to various concentrations of the biocides, selected on the basis of their MICs and MBCs. Aliquots (0.1 ml) of suspensions were taken at the commencement of the exposure and at 2, 4, 6, and 24 h. Serial dilutions were made initially in sodium thioglycollate (0.01 M) to inactivate the isothiazolone biocides (9) and then in sterile RS. The dilutions were spread onto the surface of predried BCYE agar plates. Colony counts were made after 9 days of incubation at 35°C, and the results were expressed as the percent survival. The data were subjected to analysis of variance and Student t tests were applied to 4- and 6-h survival data by utilizing an SPSS statistics package and an IBM 386 PC computer.
Antiamoebal activity was determined by preparing serial (1/2) dilutions of the biocides in PYG broth and distributing into flat-bottomed microtiter trays. These were inoculated with washed suspensions of A. polyphaga to give ca. 103 amoebae per ml. The suspensions were incubated at 35°C and observed through an inverted microscope after 1, 3, and 5 days. The MIC was taken as the minimum concentration of biocide that inhibited the formation of a confluent layer of trophozoites on the bases of the chambers after 5 days. In addition, the cytopathic effects of the biocides on established monolayers of trophozoites were determined by observing
0
1
2
3
4
5
6
7
Time (Hours) FIG. 3. Survival of stationary-phase ABCD broth cultures of L. pneumophila (-, A, *) and amoeba-grown L. pneumophila (l, A, O) after exposure to various concentrations of BIT (16 ,ug/ml [K>, *], 32 ,ug/ml [O, *], 64 ,ug/ml [A and A]) at 35'C in RS. Controls (x) for both treatments were coincident. Bars represent the standard error of the mean associated with each data point.
the ability of the organism to recover after treatment and transfer to fresh medium. RESULTS
Figure 1 shows the release of L. pneumophila from A. polyphaga during the second stage of culture. Initial inoculum levels were set at ca. 5 x 105 cells per ml for both bacteria and trophozoites. After 3 days of incubation, the number of L. pneumophila cells had increased to 108 cells per ml and the number of A. polyphaga had decreased by 90%; the majority of the surviving amoebae were encysted. The legionellae did not multiply in amoebal saline without A. polyphaga trophozoites or in amoebal saline containing metabolic products released from viable amoebal trophozoites by ultrasonic disintegration. King et al. (23) also found that L. pneumophila would not grow extracellularly when separated by a 0.2-,um-pore-size membrane filter in media containing viable Hartmanella verniformis trophozoites. L. pneumophila cells obtained after 3 days of growth in A. polyphaga were highly motile and generally smaller (ca. 1 pum) than their broth-grown counterparts (ca. 2 ,um). After continued incubation for an additional 24 h, the legionellae were nonmotile and the amoebae were predominantly encysted. This two-stage culture, with L. pneumophila being harvested at 3 days in the second stage, minimized the numbers of legionellae that had not been passaged through amoebae in subsequent tests. The antiamoebal properties of the biocides BIT, CMIT,
PHYSIOLOGY AND SUSCEPTIBILITY OF L. PNEUMOPHILA
VOL. 58, 1992
2423
c 100
a 100
10
10
L-
1
cn
-
CO)
cm
cm
0 -J
1
.1
.1
.01 0
2
1
3
5
4
6
7
0
Time (Hours)
2
3
4
5
6
7
Time (Hours) FIG. 4. Survival of L. pneumophila after growth at 35°C in ABCD broth (El) or iron-depleted ABCD broth (-) or passage through amoebae (A) after exposure to BIT (16 ,ug/ml) (a), PHMB (1.0 ,ug/ml) (b), or CMIT (32 ,ug/ml) (c) with controls (x). Bars represent the standard errors of the means for three replicate
b 1000
experiments.
100
aI
1
10
1 -1
.1
.01
0
1
2
3
4
5
6
7
Time (Hours)
and PHMB are summarized in Table 1. The MICs on days 3 and 5 were identical for the isothiazolone biocides, the MIC for PHMB was increased from 0.3 ,ug/ml to 0.6 ,ug/ml on continued incubation. However, the activities for PHMB were significantly greater than those for the isothiazolones. Direct exposure of the amoebae to these agents caused them
to lose their characteristic trophozoite appearance. Rounding of the cells but failure to encyst may be taken as an indication of reduced metabolic activity of the protozoa and is also seen on starvation. The ability of the rounded amoebae to revert to the trophozoite form after treatment with BIT and PHMB, but not after treatment with CMIT, indicates that irreversible damage to the amoebal cell is caused by CMIT. When cells were exposed to biocides for 20 h and tested for their ability to recover on transfer to fresh medium, a different pattern of susceptibilities emerged. The majority of cells recovered from BIT treatment, few (if any) cells recovered from CMIT treatment, and ca. 30% of cells recovered from PHMB exposure. However, such data should be viewed within the context of the individual biocide use concentrations. In this respect, the concentrations of CMIT employed in these experiments are higher than those generally used in water treatment (ca. 10 ,ug/ml) (27) and the concentrations of PHMB are 10 to 20% of those employed in
its major application of swimming pool sanitation (ca. 10 p,g/ml) (38). The BIT concentrations used in these procedures were much lower than their use concentrations of around 150 ppm; sometimes BIT is used as part of a complex with lauryldimethyl benzylammonium chloride (34). The relative susceptibilities of ABCD-broth-grown and amoeba-grown L. pneumophila to BIT and PHMB are shown in Fig. 2 and 3. The activities of both biocides were greatly reduced in amoeba-grown cells. Student t tests (paired with respect to amoeba- and ABCD-grown cells) showed the amoebal cells to be significantly less susceptible
2424
BARKER ET AL.
(for PHMB, t = 5.44 and 0 = 6; for BIT, t = 7.06 and 4) = 6). Bactericidal activities reflected the MIC data in that PHMB was more active at concentrations well below the recommended use concentrations for swimming pool applications (ca. 10 ,ug/ml). Concentrations of PHMB equivalent to 4 times the MIC gave 4 log 10 cycles of killing within 6 h and no detectable survivors at 24 h for the broth-grown cultures. This bactericidal activity was reduced to 90% killing at 6 h and 99.9% killing at 24 h against the amoebagrown organisms. Concentrations of BIT equivalent to 4 times its MIC resulted in reductions in viability of only 3 log 10 cycles within 6 h for broth-grown cells and by ca. 40% for amoeba-grown cells. After 24 h of contact, there were no detectable survivors from the broth-grown inoculum, whereas 30 to 50% of the amoeba-grown cells survived. In
Fig. 4 the relative effects of iron deprivation, intra-amoebal growth, and nutrient-rich conditions on susceptibility of L. pneumophila to BIT (Fig. 4a), PHMB (Fig. 4b), and CMIT (Fig. 4c) are compared. BIT was relatively ineffective against both iron-depleted and amoeba-grown cells but not against ABCD broth-grown (P = 0.95) cells. Whereas the activity of PHMB was once again reduced by intra-amoebal growth, iron depletion of the culture increased the susceptibility of L. pneumophila beyond that of the ABCD brothgrown organisms. However, the difference between the susceptibility of ABCD broth-grown cells and that of irondepleted cells was not significant (P = 0.95). A similar pattern of susceptibilities was also indicated for CMIT; iron-deprived cells were the most susceptible. DISCUSSION In the aquatic environment, L. pneumophila infects and multiplies within a wide range of amoebal hosts (32, 33). Therefore, the environmental stresses imposed upon such cells and their physiological responses and phenotypes are unlikely to be represented by typical broth cultures (4, 16). In common with infections of humans (3), intra-amoebal growth is likely to subject the bacterial cells to iron insufficiency, causing them to express iron-deprived phenotypes. These phenotypes were found to differ significantly in their susceptibility to chemical inactivation. CMIT (the active ingredient of Kathon CG, a biocide recommended for water treatment) was profoundly bactericidal to iron-restricted and ABCD-broth grown legionellae, but it was relatively ineffective against amoeba-grown cultures even at concentrations far in excess of those recommended for use (15, 27). This is in spite of conducting the tests in a thiol-free environment to reduce the potentially neutralizing effects of the medium (9). These data suggest either that iron restriction does not apply within the amoeba or that some other factor confers resistance to CMIT on amoeba-grown cells. Such resistance of amoeba-grown cultures of L. pneumophila to chlorine has also been reported (22, 30) and has been suggested to account for the recalcitrance of this organism to conventional water treatment. Curiously, PHMB, an agent routinely used in swimming pool sanitization, was effective not only against the amoeba-grown legionellae but also against the amoebae. Such activity was demonstrable at PHMB concentrations of 10% of the recommended-use level (10 p,g/ml) (38). These results are similar to those observed by Kilvington (21). The toxicity profiles of the isothiazolone biocides greatly limit the applications to which they can be deployed and also the concentrations to which humans may be exposed (28, 37). This is not the case for PHMB, which is relatively nontoxic and has recently found application as a
APPL. ENVIRON. MICROBIOL.
preservative of ophthalmic lens solutions (12). The recalcitrance of L. pneumophila grown intra-amoebally might relate to the increased levels of poly-p-hydroxybutyric acid inclusions (32, 36) that are associated with this mode of growth. Such lipophilic moeties might act as a sink for the relatively hydrophobic isothiazolone biocides and reduce their interaction with cytosolic thiol groups (8). Such inclusions are unlikely to affect the action of PHMB, which is hydrophilic and acts solely at the level of the cell membrane (6, 17). In a similar fashion, changes in the L. pneumophila cell wall, which is characteristically rich in branched-chain fatty acids (29), would affect isothiazolone activity to a far greater extent than it would affect PHMB activity. In conclusion, these studies clearly indicate the profound effect of intra-amoebal growth on the physiological status and antimicrobial susceptibility of L. pneumophila and suggest that such phenomena might account for the recalcitrance of the organism in situ. Particularly, these data show PHMB to be a potential biocide for water treatment with useful activities against legionellae in all physiological states and against the host amoebae. ACKNOWLEDGMENTS This study was funded in part by a grant awarded to J.B. from the Public Health Laboratory Service, England and Wales. We thank T. Rowbotham for helpful discussions on this topic. REFERENCES 1. Barker, J., and D. H. Till. 1986. Survival of Legionella pneumophila. Med. Lab. Sci. 43:388-389. 2. Brown, M. R. W., D. G. Allison, and P. Gilbert. 1988. Resistance of bacterial biofilms to antibiotics: a growth rate related effect? Antimicrob. Chemother. 22:777-783. 3. Brown, M. R. W., H. Anwar, and P. A. Lambert. 1984. Evidence that mucoid Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron-restricted conditions. FEMS Microbiol. Lett. 21:113-117. 4. Brown, M. R. W., P. J. Collier, and P. Gilbert. 1990. Influence of growth rate on susceptibility to antimicrobial agents: modification of the cell envelope and batch and continuous culture studies. Antimicrob. Agents Chemother. 34:1623-1628. 5. Brown, M. R. W., and P. Williams. 1985. Influence of substrate limitation and growth phase on sensitivity to antimicrobial agents. J. Antimicrob. Chemother. 15(Suppl. A):7-14. 6. Broxton, P., P. M. Woodcock, and P. Gilbert. 1984. Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli. J. Appl. Bacteriol. 57:115-124. 7. Chartered Institution of Building Service Engineers: 1987. Technical memorandum (TM 13). Minimising the risk from Legionaires disease. Chartered Institution of Building Service Engineers, London. 8. Collier, P. J., P. Austin, and P. Gilbert. 1990. Uptake and distribution of some isothiazolone biocides into Escherichia coli ATCC8739 and Schizosaccharomyces pombe NCYC1354. Int. J. Pharm. 66:201-206. 9. Collier, P. J., A. J. Ramsey, P. Austin, and P. Gilbert. 1990. Growth inhibiting and biocidal activity of some isothiazolone biocides. J. Appl. Bacteriol. 69:569-577. 10. Costerton, J. W., T. J. Marrie, and K. J. Cheng. 1985. Phenomena of bacterial adhesion, p. 3-43. In D. C. Savage and M. Fletcher (ed.), Bacterial adhesion. Plenum Publishing Corp., New York. 11. Department of Health and Social Security and the Welsh Office. 1988. The control of legionellae in health care premises: a code of practice. Her Majesty's Stationery Office, London. 12. Dexter, D., M. B. Ficke, and B. T. Decicco. 1989. Effectiveness of polyhexamethylene biguanide against bacteria of product, clinical and environmental origins, abstr. A122. Abstr. 89th Annu. Meet. Am. Soc. Microbiol. 1989. American Society for Microbiology, Washington, D.C.
VOL. 58, 1992
PHYSIOLOGY AND SUSCEPTIBILITY OF L. PNEUMOPHILA
13. Domingue, P. A. G., B. Mottle, D. W. Morck, M. R. W. Brown, and J. W. Costerton. 1990. A simplified and rapid method for the removal of iron and other cations from complex media. J. Microbiol. Methods 12:13-22. 14. Edelstein, P. H. 1981. Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J. Clin. Microbiol. 14:298-303. 15. Elsmore, R. 1986. Biocidal control of legionellae. Isr. J. Med. Sci. 22:647-654. 16. Gilbert, P., P. J. Collier, and M. R. W. Brown. 1990. Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob. Agents Chemother. 34:1865-1868. 17. Gilbert, P., D. Pemberton, and D. E. Wilkinson. 1990. Barrier properties of the Gram-negative cell envelope towards high molecular weight polyhexamethylene biguanides. J. Appl. Bacteriol. 69:585-592. 18. Hollis, C. G., and D. L. Smalley. 1980. Resistance of Legionella pneumophila to microbiocides. Dev. Ind. Microbiol. 21:265271. 19. Joly, J. R., R. M. McKinney, J. O'H. Tobin, W. F. Bibb, I. D. Watkins, and D. R. Ramsey. 1986. Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies. J. Clin. Microbiol. 23:768-771. 20. Kadurugamuwa, J. L., H. Anwar, M. R. W. Brown, G. H. Shand, and K. H. Ward. 1987. Media for study of growth kinetics and envelope properties of iron-deprived bacteria. J. Clin. Microbiol. 25:849-855. 21. Kilvington, S. 1990. Activity of water biocide chemicals and contact lens disinfectants on pathogenic free-living amoebae. Int. Biodeterior. 26:127-138. 22. Kilvington, S., and J. Price. 1990. Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. J. Appl. Bacteriol. 68:519-525. 23. King, C. H., B. S. Fields, E. B. Shotts, Jr., and E. H. White. 1991. Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells. Infect. Immun. 59:758-763. 24. Kurtz, J. B., C. L. R. Bartlett, U. A. Newton, R. A. White, and N. L. Jones. 1982. Legionella in cooling water systems. Report of a survey of cooling towers in London and a pilot trial of selected biocides. J. Hyg. Camb. 88:369-381. 25. Lee, J. V., and A. A. West. 1991. Survival and growth of Legionella species in the environment. J. Appl. Bacteriol. (Symp. Suppl.) 70:121s-129s.
2425
26. Lewis, S. N., and G. A. Miller. June 1974. U.S. patent 3,835,150. 27. McCoy, W. F., J. W. Wireman, and E. S. Lashen. 1986. Efficacy of methylchloroisothiazolone biocide against Legionella pneumophila in cooling tower water. Chim. Oggi. 4:79-83. 28. Monte, W. C., S. H. Ashoor, and B. J. Lewis. 1983. Mutagenicity of two non-formaldehyde-forming antimicrobial agents. Food Chem. Toxicol. 21:695-697. 29. Moss, C. W., R. E. Weaver, S. B. Dees, and W. B. Cherry. 1977. Cellular fatty acid composition of isolates from Legionaires' disease. J. Clin. Microbiol. 6:140-143. 30. Navratil, J. S., R. H. Palmer, S. States, J. M. Kuchta, R. M. Wadowsky, and R. B. Yee. 1990. Increased chlorine resistance of Legionella pneumophila released after growth in amoeba Hartmanella vermiformis, abstr. Q82. Abstr. 90th Annu. Meet. Am. Soc. Microbiol. 1990. American Society for Microbiology, Washington, D.C. 31. Pine, L., P. S. Hoffman, G. B. Malcolm, R. F. Benson, and M. J. Franzus. 1986. Role of keto acids and reduced oxygen-scavenging enzymes in the growth of Legionella species. J. Clin. Microbiol. 23:33-42. 32. Rowbotham, T. J. 1983. Isolation of Legionella pneumophila from clinical specimens via amoebae, and the interaction of those and other isolates with amoebae. J. Clin. Pathol. 36:978986. 33. Rowbotham, T. J. 1986. Current views on the relationship between amoeba, legionellae and man. Isr. J. Med. Sci. 22:678689. 34. Singer, M. 1976. Laboratory procedures for assessing the potential of antimicrobial agents as industrial biocides. Process Biochem. 11:30-35. 35. Skaliy, P., T. A. Thompson, J. G. W. Gorman, G. K. Morris, H. V. McEachem, and D. C. Mackel. 1980. Laboratory studies of disinfectants against Legionella pneumophila. Appl. Environ. Microbiol. 40:697-700. 36. Vandenesh, F., M. Surgot, N. Bornstein, J. C. Paucod, D. Marmet, P. Isoard, and J. Fleurette. 1990. Relationship between free amoeba and Legionella: studies in vitro and in vivo. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 272:265-275. 37. Weaver, J. E., C. W. Cardin, and H. I. Maibach. 1985. Doseresponse assessments of Kathon biocide. Contact Dermatitis 12:141-145. 38. Yang, W., and G. S. Banker. 1981. Biguanide-induced staining in oral hygeine. Drug Dev. Ind. Pharm. 7:113-133.
