Adherence of Virulent and Avirulent Legionella to Hydrocarbons
The hydrophobic nature of the outermost surface of microbial
cells has been implicated in their interaction with phagocytes and attachment
to host cells. The interaction between seven isolates of Legionella
pneumophila, of differing virulence and n-hexadecane and n-octane
was investigated. Virulent strains had a higher affinity to hydrocarbons
than avirulent strains. The hydrophobicity of strains appeared to be related
to LD50 hyperbolically and an empirical expression relating
the two variables was derived. This report extends the use of microbial
adherence to hydrocarbons (MATH) as a possible tool for distinguishing
between pathogenic and non-pathogenic Legionella strains. In the
context of this study, the terms hydrophobic Legionella and hydrophilic
Legionella are used to indicate the affinity of the organism to
Legionella pneumophila, the causative agent of Legionnaires`
disease, is a facultative intracellular parasite that is capable of multiplying
in human phagocytes and protozoa (Horwitz, 1988; Halablab et al.,
1990a). The first step in phagocytic ingestion is adhesion of the invading
particle to the phagocyte surface. Once attachment has taken place, a
cohesive force prevents their separation despite their circulation in
the blood stream. The physicochemical mechanism of this adhesion may be
either a Van der Waals-type attraction (van Oss et al., 1983),
a hydrophobic attraction and/or (more rarely) an electrostatic attraction
(Nagura et al., 1977), or a receptor mediated bond. Albertsson
(1971) introduced the use of immiscible aqueous dextran and polyethylene
glycol solution in partition separation of microorganisms. Similar techniques
were used (Stendahl et al., 1973) to demonstrate that the more
hydrophobic (rough forms) of salmonellae are phagocytized more readily
than smooth forms. Later, an interesting and novel approach for measuring
cell surface hydrophobicity based on microbial adhesion to hydrocarbons
(MATH) (Rosenberg et al., 1980, 1981, 1982; Rosenberg and Rosenberg,
1981) was introduced. The inherent simplicity of their technique, which
involves mixing washed suspension of cells with hydrocarbons and observing
their adhesion, has made it a popular assay for testing surface hydrophobicity
of microorganisms (Wojnicz and Jankowski, 2007; Szabelska et al.,
The majority of legionellae isolated from the environment are non-pathogenic
but estimating their virulence in terms of their LD50, with
guinea pigs for example, is laborious and expensive. In this study, we
report a novel application of the MATH assay to virulence estimation which
is relatively inexpensive, rapid and may be a convenient way of assessing
the pathogenicity of virulent and avirulent isolates of Legionella.
MATERIALS AND METHODS
A total of seven strains (Table 1) of Legionella
pneumophila serogroup 1 were used in this study. The Corby Av strain
was isolated in our laboratory following two passages of the Corby strain
(LD50 1x102.2 as estimated by aerosol infection
of guinea pigs (Jepras et al., 1985) on Mueller-Hinton agar (Difco)
supplemented with 0.025% ferric citrate and 0.025% cysteine (Sigma). This
has been reported to act as a selective medium for growing avirulent L.
pneumophila (Catrenich and Johnson, 1988). The virulence of the latter
derivative was estimated to be 4.9x104, using a previously
established technique (Halablab et al., 1990b).
Multiple sub-culture of strains was avoided by maintaining original cultures
at -70°C on glass beads and inoculating onto buffered charcoal yeast
extract agar supplemented with α-ketoglutarate (Edelstein, 1981)
(α-BCYE) for 72 h at 37°C only once for each experiment. Cells
were then harvested from the plates, washed three times in saline (1.25%
NaCl) and adjusted to an optical density (OD400 nm) of 0.8
units (final volume 3 mL).
Math: For the MATH assay, a modification of the method of Rosenberg
and colleagues developed in 1980 was used. To the washed cells (3 mL)
in acid-cleaned test tubes, 0.2 mL of either n-hexadecane or n-octane
was added. The mixture was then mixed by vortexing for timed periods.
After phase separation, the optical density (400 nm) of the aqueous phase
was determined. Results were recorded as the percentage change in turbidity
which was assumed to reflect the number of cells that partitioned into
the aqueous phase.
Light microscopy: After vortexing the cells with hydrocarbons,
a drop of n-octane-associated organisms was taken onto a glass slide and
was examined under an Olympus BH2 microscope fitted with a 100X phase
||Characteristics of strains used in this study
|‡Additional details in text
RESULTS AND DISCUSSION
Figure 1a shows the effect of vortexing time on virulent
isolate (1400), an attenuated strain of the same isolate (1400 Av) and
an avirulent strain (1397) and on surface hydrophilicity in n-hexadecane
(a) and n-octane (b). In both hydrocarbons the hydrophilicity of the virulent
strain decreased with increased vortexing time while the avirulent strain
remained relatively constant. However, the avirulent isolate was at all
times more hydrophilic than the virulent strain. The Corby Av variant
showed intermediate attachment to the hydrocarbons used. Cell-coated hydrocarbon
droplets were stable at room temperature for several days. This was confirmed
by microscopic examination of the samples (Fig. 2) which
related a significant number of virulent cells associated with hydrocarbon
droplets. This resulted in a greater decrease in the optical density of
the aqueous phase than when avirulent microorganisms were used.
Longer mixing times than that indicated in Fig. 1b resulted
in fluctuating turbidity readings. This is probably due to coalescence
of the hydrocarbon droplets and desorption of the bacteria into the bulk
aqueous phase. More legionellae appeared to adhere to octane than to n-hexadecane,
an observation which is in agreement with previous reports (Rosenberg
et al., 1982, 1983).
It is noteworthy that attachment of microorganisms to hydrocarbons was
closely related to adhesion to other substrata of interest (Rosenberg
and Doyle, 1990). Several microorganisms with pathogenic properties have
been shown to adhere to different hydrocarbons (Rosenberg et al.,
1980; Magnusson and Johansson, 1977). Other studies have proposed (Ofek
et al., 1983) that Streptococcus pyogenes cells are
hydrophobic during adhesion and colonization and subsequently elaborate
a hydrophilic capsule which protects the organism against phagocytosis.
L. pneumophila has not been reported to colonize humans
(Bridge and Edelstein, 1983). A high percentage of clinical isolates,
from different sources including infected catheters and tracheal and bladder
devices, have shown affinity to hydrocarbons (Boujaafar et al.,
1990). It is, therefore, likely that virulent legionellae, which have
a tendency to adhere, might behave in a similar manner and hence be more
hydrophobic. Surface hydrophobicity of microbial cells influences their
uptake by phagocytes; bacteria that are more hydrophobic than the phagocytes
are readily phagocytozed; bacteria that are more hydrophilic than the
phagocytic cells resist phagocytosis. Avirulent L. pneumophila
have been shown to resist phagocytosis (Horwitz, 1987). Legionellae multiply
in eukaryotic cells.
||Surface hydrophobicity test, using n-hexadecane (0.2
mL), of virulent (Corby) (•), Corby Av (Δ) and avirulent
strain Philadelphia-1 (1397) (Ο)
||Surface hydrophobicity test, using n-octane (0.2 mL),
of virulent (Corby) (•), Corby Av (Δ) and avirulent strain
Philadelphia-1 (1397) (Ο)
Phagocyte-phagocytes-hydrophobicity interaction might be necessary for
such organisms to attach and gain entry into mammalian cells and subsequently,
to initiate infection. If avirulent isolates fail to attach then they
will not be ingested and will ultimately be removed from the body.
Figure 3 shows the relationship between hydrophilicity
after 30 sec vortexing and LD50 as estimated by aerosol infection
of guinea pigs. For the more virulent strains (lower LD50s),
there is a rapid decrease in hydrophilicity but at higher LD50
values relatively little change occurs. The dependence of % hydrophilicity
(H) on LD50 (L) can be described in terms of a simple, empirical
saturation function of the form:
||Adhesion of avirulent (a) and virulent (b) Legionella
cells to droplets of octane. Fewer avirulent cells, which were mostly
filamentous, associated with the hydrocarbon. Magnification was X10000
(a) or X2250 (b)
||Adherence of six strains of L. pneumophila to
n-octane as a function of LD50. Cells were vortexed with
the hydrocarbon for 30 sec
where, Hm and k are constants. The theoretical value of Hm,
the maximum % hydrophilicity, is 100. Using the Marquardt algorithm (Marquardt,
1963) available in the computer package Regression (Blackwell Scientific
Software, Oxford, 1989) we computed a value for Hm of 100.85/7%
and an LD50 for k of 1.7/0.7x104.
Rearrangement of Eq. 1 gives:
This equation has the potential of forming the basis of predicting the
virulence of Legionella strains in terms of their LD50
values from hydrophilicity data. For such purposes it has the particular
advantage that it is more discriminatory for virulent organisms and consequently
might be of significant practical value.
Present results indicate the possibility that a surface component(s)
of L. pneumophila mediates attachment to hydrocarbons.
Although the MATH assay does not determine specific surface properties
of microorganisms, the results were in general agreement with the so-called
hydrophobicity tests reviewed by Rosenberg and Doyle (1990). In addition,
organisms which do adhere in the MATH assay often tend to adhere to solid
surfaces (Busscher et al., 1990). The lipopolysaccharide of virulent
and avirulent L. pneumophila of the same serogroup have
very similar SDS-PAGE pattern (Horwitz, 1987; Conlan et al., 1988).
However, a better understanding of its configuration with regard to hydrophobicity
is still to be determined.
Until the surface component(s), or other factors, which mediate the MATH
assay are known, the data presented in this report offer a simple and
rapid system for differentiating between virulent and avirulent legionellae
Albertsson, P.A., 1971.
Partition of Cells, Particles and Macromolecules. 1st Edn., Wiley (Interscience), New York
Boujaafar, N., J. Freney, P.J.M. Bouvet and Jeddi, 1990.
Cell surface hydrophobicity of 88 clinical strains of Acinetobacter baumannii
. Res. Microbiol., 141: 477-482.PubMed | Direct Link |
Bridge, J.A. and P.H. Edelstein, 1983.
Oropharyngeal colonization with Legionella pneumophila
. J. Clin. Microbiol., 18: 1108-1112.Direct Link |
Busscher, H.J., J. Sjollema and H.C. van der Mei, 1990.
Relative Importance of Surface Free Energy as a Measure of Hydrophobicity in Bacterial Adhesion to Solid Surfaces. In: Microbial Cell Surface Hydrophobicity, Doyle, R.J. and M. Rosenberg (Eds.). ASM., Washington, DC
Catrenich, C.E. and W. Johnson, 1988.
Virulence conversion of Legionella pneumophila
: A one-way phenomenon. Infect. Immunol., 56: 3121-3125.Direct Link |
Conlan, J.W., A. Williams and L.A.E. Ashworth, 1988.
In vivo production of a tissue-destructive protease by Legionella pneumophila
in the lungs of experimentally infected guinea pigs. J. Gen. Microbiol., 134: 143-149.PubMed |
Edelstein, P.H., 1981.
Improved semi selective medium for isolation of Legionella pneumophila
from contaminated clinical and environmental specimens. J. Clin. Microbiol., 14: 298-303.PubMed | Direct Link |
Halablab, M.A., M. Bazin and L. Richards, 1990.
Estimation of Legionella pneumophila
virulence by nitro-blue tetrazolium reduction. Lancet, 335: 240-240.CrossRef | Direct Link |
Halablab, M.A., L. Richards and M. Bazin, 1990.
Phagocytosis of Legionella pneumophila
. J. Med. Microbiol., 33: 75-83.
Horwitz, M.A., 1987.
Characterization of avirulent mutant Legionella pneumophila
that survive but do not multiply within human monocytes. J. Exp. Med., 166: 1310-1328.PubMed | Direct Link |
Horwitz, M.A., 1988.
Phagocytosis of Intracellular Biology of Legionella pneumophila
. In: Bacteria-Host Cell Interactions. Horwitz, M., (Ed.). Alen R. Liss, Inc., New York, pp: 283-302.
Jepras, R.I., R.B. Fitzgeorge and A. Baskerville, 1985.
A comparison of virulence of two strains of Legionella pneumophila
based on experimental aerosol infection of guinea-pigs. J. Hyg., 95: 29-38.PubMed | Direct Link |
Magnusson, K.E. and G. Johansson, 1977.
Probing the surface of Salmonella typhimurium
and Salmonella minnesota SR and R bacteria by aqueous biphasic partitioning in systems containing hydrophobic and charged polymers. FEMS. Microbiol. Lett., 2: 225-228.CrossRef | Direct Link |
Marquardt, W.D., 1963.
An algorithm for the least-squares estimation of nonlinear parameters. SIAM J. Applied Math., 11: 431-441.CrossRef | Direct Link |
Nagura, H., J. Asai and K. Kojima, 1977.
Studies on mechanism of phagocytosis I. Effect of electric surface-charge on phagocytic activity of macrophages for fixed red cells. Cell Struct. Function, 2: 11-28.
Ofek, I., E. Whitnack and E.H. Beachey, 1983.
Hydrophobic interactions of group A streptococci with hexadecane droplets. J. Bacteriol., 154: 139-145.PubMed | Direct Link |
Rosenberg, M., D. Gutnick and E. Rosenberg, 1980.
Adherence of bacteria to hydrocarbons: A simple method for measuring cell-surface hydrophobicity. FEMS Microbiol. Lett., 9: 29-33.CrossRef | Direct Link |
Rosenberg, M. and E. Rosenberg, 1981.
Role of adherence in growth of Acinetobacter calcoaceticus
RAG-1 on hexadecane. J. Bacteriol., 148: 51-57.PubMed | Direct Link |
Rosenberg, M., A. Perry, E.A. Bayer, D. Gutnick, E. Rosenberg and I. Ofek, 1981.
Adherence of Acinetobacter calcoaceticus
RAG-1 to human epithelial cells and to hexadecane. Infect. Immunol., 33: 29-33.PubMed | Direct Link |
Rosenberg, M., S. Rottem and E. Rosenberg, 1982.
Cell surface hydrophobicity of smooth and rough Proteus mirabilis
strains as determined by adherence to hydrocarbons. FEMS. Microbiol. Lett., 13: 167-169.CrossRef | Direct Link |
Rosenberg, M., H. Judes and E. Weiss, 1983.
Cell surface hydrophobicity of dental plaque microorganisms in situ
. Infect. Immunol., 42: 831-834.Direct Link |
Rosenberg, M. and R.J. Doyle, 1990.
Microbial Cell Surface Hydrophobicity: History, Measurement and Significance. In: Microbial Cell Surface Hydrophobicity, Doyle, R.J. and M. Rosenberg (Eds.). ASM Publications, Washington, DC
Stendahl, O., C. Tagesson and L. Edebo, 1973.
Partition of Salmonella typhimurium
in a two-polymer aqueous phase system in relation to liability to phagocytosis. Infect. Immunol., 8: 36-41.PubMed | Direct Link |
Szabelska, M., E. Gospodarek and E. Ciok-Pater, 2006.
Influence of incubation conditions on cell surface hydrophobicity of Candida
species fungi. Medycyna Doswiadczalna i Mikrobiologia, 58: 253-260.Direct Link |
Van Oss, C.J., D.R. Absolom and A.W. Newmann, 1983.
Interaction of phagocytes with other blood cells and with pathogenic and nonpathogenic microbes. Ann. N. Y. Acad. Sci., 416: 332-350.CrossRef | Direct Link |
Wojnicz, D. and S. Jankowski, 2007.
Effects of subinhibitory concentrations of amikacin and ciprofloxacin on the hydrophobicity and adherence to epithelial cells of uropathogenic Escherichia coli
strains. Int. J. Antimicrob. Agents, 29: 700-704.Direct Link |