LAS CUOTAS

3.4.1. Susceptibilities to tetracyclines of varying hydrophobicities

One of the ways of assessing the result of an experimental variation or mutational event relies on the relative susceptibility of the altered bacteria and their controls to a wide variety of unrelated antibiotics, detergents and dyes (Coleman and Leive, 1979; Nikaido, 1976). The model proposed by Minikin (1982) for the physical organisation of the mycobacterial cell wall with lipids assembled in two leaflets of different fluidity, is reminiscent of the outer membrane of the Gram negative bacterial For this reason, the permeability of the cell wall of the mycobacterial strains to hydrophobic solutes was experimentally determined using a method described for measurement of outer membrane permeability of E. colt (Leive et a l, 1984). This assay measures changes in hydrophobic permeability based on the susceptibility of the microorganism to tetracyclines of various hydrophobicities and, therefore, should prove useful in distinguishing mutants with alterations of the cell wall.

To compare the degree of hydrophobic permeability of ms629 relative to ms627 and

M. smegmatis mc^l55, the susceptibility to tetracycline and minocycline was

determined. These tetracyclines are equally effective against gram-positive organisms (Blackwood and English, 1977) and apparently share the same mechanism of action (Levy, 1981). It is important to note that minocycline has a higher degree of hydrophobicity than tetracycline.

^ The Gram negative outer membrane forms an asymmetric lipid bilayer with an outer leaflet com posed exclu sively o f lipopolysaccharide (LPS) and an inner leaflet containing phospholipids.

The three mycobacterial strains were grown to stationary phase and then diluted to 0.01 ODggQ. A 1/100 dilution of this suspension was used to inoculate 4 ml of liquid Sautons containing antibiotics in the following concentrations: 4 , 2 , 1 , 0.5, 0.25, 0.125, 0.06 and 0.03 )Lig/ml (the appropriate concentration range of had been previously determined). The cultures were incubated shaking at 37°C for four days and growth values were recorded spectrophotometrically at 580 nm after 48, 72 and 96 hours. Two separate determinations were done. Table 3.1 shows the minimal inhibitory concentrations (MIC) obtained.

Table 3.1. Susceptibility of M. smegmatis mc^l55, ms627 and ms629 to tetracyclines as a function of hydrophobocity Antibiotic M. smegmatis mc^l55 ms 627 ms 629 Tetracycline 48h 72h 96h 48h 72h 96h 48h 72h 96h 0.25 0.5 1 0.125 0.5 1 0.25 0.5 1 Minocycline 0.06 0.25 0.5 0.03 0.125 0.25 0.06 0.25 0.5

MICs are expressed as micrograms of antibiotic per milliliter. MIC was defined as the lowest concentration of antimicrobial agent at which the organism showed no visible growth.

The MICs of tetracycline and minocycline for M. smegmatis, ms627 and ms629 were very similar indicating that these strains are equally susceptible to each of the antibiotics. Differences in susceptibility to drugs sharing the same mode o f action but with diverse hydrophobicities presumably reflect differences in cell wall permeability. From the results obtained, there is no evidence that the lipid composition of the cell wall of ms629 has been altered.

All three strains showed greater susceptibility to minocycline than tetracycline. These results are in accordance with previous observations that the most active drugs against mycobacteria are hydrophobic compounds of low polarity due to their solubility into the fats of the cell wall (David et a l, 1987). Antibacterial agents of the lipophilic classes are thought to utilise the lipid bilayer pathway to cross the cell wall of mycobacteria rather than the porin pathway which is extremely inefficient in

these microorganisms (Trias et a l, 1992; Trias and Benz, 1994). As a consequence, within each class of antibiotic, the more lipophilic derivative proves to be more active against mycobacteria.

3.4.2. Hydrophobicity measurements

The aim of the present series of experiments was to determine possible changes in the overall lipid composition of the cell wall of the strains under study, on the basis of their physicochemical cell-surface properties. A number of methods for studying hydrophobic interactions of cells has been reported in the literature although no single technique adequately describes cell-surface hydrophobicity since the experimental conditions employed and various non-hydrophobic effects interfere with the end result. It is likely, however, that the various methods used to measure hydrophobicity detect quantitative and qualitative differences of the lipophilic residues present on bacterial surfaces.

The outer cell surface of M. smegmatis mc^l55, ms627 and ms629 was studied by microbial adhesion to hydrocarbon (MATH), a simple and rapid quantitative assay based on the affinity of cells to n-hexadecane. This method was originally developed by Rosenberg et a l (1980) and later modified by Ofek et a l (1983). The modification introduced permits the recording of turbidity of the bacterial suspensions in the same tube during the assay, and also to follow the kinetics of the mycobacteria-hexadecane interaction. Briefly, the strains were grown to stationary phase in plain Sautons and in the presence of 0.9 and 1.8 U/ml of avidin. The cells were harvested, washed, resuspended in PBS, and adjusted to an optical density of about 0.5 at 580 nm. Then, 50 |xl of n-hexadecane was added, the suspensions were vortexed and left standing to allow separation of layers, at which time the optical density of the aqueous phase was again measured. This was repeated another three times until a total volume of 200 |il of n-hexadecane was reached. The cumulative percentage of bacteria that adhered to n-hexadecane was estimated and the results obtained are illustrated on Figure 3.4.

As a common feature, most of the cell population adhered to the organic phase in the first two stages of the assay, the removal rate reaching the peak at 1 0 0 |il o f n-

0.7 0.6 0.5 0.4 0.3 0.2 mc2 155 ms627 ms629 n-H exadecane (ml) 120 100 b 60 < 40 mc2 155 ms627 ms629 20 n-H exadecane (ml) B 0.7 0.6 0.5 0.4 0.3 0.2 mc2 155 m s627 m s629 n-H exadecane (ml) 120 100 40 m c2 155 m s627 m s629 20 n-H exadecane (ml) 0.7 0.6 0.5 F 0.4 0 0.3 0 0.2 0.1 0 mc2 155 0 0.05 0.1 0.15 0.2 n-Hexadecane (ml) 8 c 2 0) sz < 120 100 80 60 40 mc2 155 ms627 ms629 20 0 0 0.05 0.1 0.15 0.2 n-H exadecane (ml)

Figure 3.4. Adherence to n-hexadecane of M. smegmatis mc^l55, ms627 and ms629.

S trains w er e g ro w n u n til stationary p h ase in liq u id S au ton s (A ) and in S a u to n s co n ta in in g 0 .9 U /m l (B) and 1.8 U /m l (C ) o f avid in . C e lls w er e h arvested , w a sh e d and su sp e n d e d in P B S . T h e bacterial su sp e n sio n w a s adjusted to O D5 8 0 o f ab out 0 .5 (Ao) and 4 x 5 0 |xl o f n-

h e x a d e c a n e w a s ad ded and m ix e d u n ifo rm ly . A fter p h ase separation , the a b so rb a n c e o f the lo w e r aq u eo u s p h a se w a s recorded in the sa m e tub e (At). T h e p ercen ta g e o f b a cteria that ad hered to n -h e x a d e c a n e (A o-A ,/A o x 100) w a s p lotted again st th e n -h e x a d e c a n e v o lu m e .

affinity towards the organic phase regardless of the plasmid content and culture conditions. The MATH profile of the strains grown in the presence of 0.9 and 1.8 U/ml of avidin indicates that the chemical composition of the cell surface does not seem to have been affected by the unavailability of extracellular biotin. These results suggest that the potential biotin depletion, which was expected to cause a reduction in the lipid synthesis, failed to produce changes in the phenotypic appearance of the cell surface of the strains under study.

One last remark on these experiments concerns the easy detachment of adhering mycobacterial cells from the organic phase upon gentle movements of the test tube. On the contrary, Rosenberg et al. (1980) while studying the adherence of

Acinetobacter calcoaceticus to hydrocarbons, described this upper layer as a cell-

stabilised emulsion that did not break even after several days. Although unexpected, this indicates that other factors than hydrophobicity interfere with MATH for mycobacteria. It has been demonstrated that adhesion-based methods to assess microbial cell-surface hydrophobicity actually measure an interplay o f the hydrophobic and electrostatic cell-surface properties (Busscher et a l, 1995; Van der Mei et a l, 1995), since hydrocarbon droplets in most buffers appear negatively charged. Hydrophilic microorganisms do not adhere to the hydrocarbon phase regardless of the absence or presence of electrostatic repulsion (Van der Mei et a l, 1993), while hydrophobic cells, such as mycobacterial cells, only adhere to the hydrocarbon phase when electrostatic repulsion is moderate or absent (Geertsema- D oom bushe/fl/., 1993).