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Research Article

Adherence of Virulent and Avirulent Legionella to Hydrocarbons

M.A. Halablab and A. Al-Dahlawi
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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 hydrocarbons.

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M.A. Halablab and A. Al-Dahlawi, 2008. Adherence of Virulent and Avirulent Legionella to Hydrocarbons. Journal of Medical Sciences, 8: 234-238.

DOI: 10.3923/jms.2008.234.238



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., 2006).

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.


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 contrast objective.

Table 1: Characteristics of strains used in this study
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Additional details in text


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.

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Fig. 1a: Surface hydrophobicity test, using n-hexadecane (0.2 mL), of virulent (Corby) (•), Corby Av (Δ) and avirulent strain Philadelphia-1 (1397) (Ο)

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Fig. 1b: 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:

Image for - Adherence of Virulent and Avirulent Legionella to Hydrocarbons
Fig. 2: 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)

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Fig. 3: Adherence of six strains of L. pneumophila to n-octane as a function of LD50. Cells were vortexed with the hydrocarbon for 30 sec

Image for - Adherence of Virulent and Avirulent Legionella to Hydrocarbons

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:

Image for - Adherence of Virulent and Avirulent Legionella to Hydrocarbons

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


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