Langmuir 1995,11, 2991-2995
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Bacteria Flocculation and Death by Cationic Vesicles S. M. Sicchierolli,t E. M. Mamizuka,$and A. M. Carmona-Ribeiro*lt Departamento de Bioquimica, Instituto de Quimica, Universidade de Sdo Paulo, CP 26077, Sdo Paulo, Sdo Paulo, Brazil, and Departamento de Ancilises Clinicas e Toxicolbgicas, Faculdade de Cibncias Farmacbuticas, Universidade de S d o Paulo, CP 66083, Sdo Paulo, Sdo Paulo, Brazil Received December 16, 1994. In Final Form: May 19, 1995@ Dioctadecyldimethylammonium bromide (DODAB)vesicles kill Escherichia coli in the micromolar range of DODAB concentrations. At 1.2 x lo6 bacteridml, the minimum bactericidal concentration is smaller than 0.5 pM DODAB for an interaction time of 24 h. DODAB effects on the cells are described using microelectrophoresis, viable counts and turbidimetry over a range of bacterial number densities, and DODAB concentrations. Electrophoretic mobility for the cells as a function of DODAB concentration establishes the DODAB amount required to attain charge neutralization on the cell surface and maximal flocculation rate and extent. From los bacteridml, there is rapid bacteria flocculation induced by DODAB. However, cell death is not related to aggregation taking place also for nonaggregated cells at much smaller bacteria number densities. The results may be of importance for improving water quality in contaminated reservoirs.
Introduction Quaternary ammonium compounds have been widely usedl since their introduction as germicides in 1915.2 Dioctadecyldimethylammonium bromide (DODAB) or chloride (DODAC) are quaternary ammonium compounds that form stable and closed bilayers (vesicles) in aqueous s ~ l u t i o n . ~These - ~ vesicles can be seen as highly charged polycations. Polyelectrolytes in general have been used to flocculate bacteria at very low ionic strength or in water.'J' The practical utility of introducing a flocculant and bactericidal polyelectrolyte effective at low ionic strength is evident in the case of water deposits contaminated with enteropathogenic bacteria. Recently, physicochemical aspects of the interaction between cationic DODAC or DODAB vesicles a n d Escherichia coli were d e ~ c r i b e d .Adsorption ~ isotherms of high affinity (nonreversible adsorption)were obtained for DODAB o r DODAC adsorption fromvesicles onto E . At limiting adsorption, calculations for adhesion of a palisade of vesicles on bacteria yielded figures consistent
* Author to whom correspondence may be addressed at Departamento de Bioquimica,Instituto de Quimica, Universidade de Sa0 Paulo, CP 26077, Si40 Paulo, SP, Brazil. Fax: 55 11 815-5579. Electronic mail address:
[email protected]. Departamento de Bioquimica. Departamento de halises Clinicas e Toxicologicas. Abstract published in Advance ACSAbstracts, July 15,1995. (1)Merianos, J. J. Quaternaryammonium compounds. In Disinfection, Sterilization and Preservation, 4th ed.; Block, S. S., Ed.; Lea & Farbiger, Philadelphia & London, 1991;pp 225-255. (2)Jacobs, W. A.; Heidelberg, M. The quaternarysalts ofhexamethylenetetramine. I . The problem of the chemotherapy of experimental @
bacterial infections. J. Exp. Med. 1915,23,563-568. (3)Carmona-Ribeiro, A. M. Synthetic amphiphile vesicles. Chem. SOC.Rev. 1992,21(3),209-214. (4)Carmona-Ribeiro, A. M.; Chaimovich, H. Preparation and characterization of large dioctadecyldimethylammonium chloride liposomes and comparisonwith small sonicated vesicles. Biochim. Biophys. Acta 1983,733, 172-179. 15) Carmona-Ribeiro,A. M.;Yoshida, L. S.; Chaimovich,H. Salt effects on the stability of dioctadecyldimethylammonium chloride and sodium dihexadecylphosphate vesicles. J.Phys. Chem. 1986,89,2928-2933. (6)Carmona-Ribeiro, A. M. Interactions between charged spheric vesicles. J. Phys. Chem. 1993,97, 11843-11846. (7)Jones, G.D. InPolyelectrolytes; Frich, K. C., Klempner, D., Patsis, A. V., Ed.; Technomic: Westerport, CT, 1976. (8)Eriksson, B.;Haerdin, A.-M. InFlocculation in biotechnologyand separation systems;Attia, Y. A,, Ed.; Elsevier Science Publishers B. V.: Amsterdam, 1987. (9)Tbpias, G.N.;Sicchierolli, S. M.; Mamizuka, E. M.; CarmonaRibeiro, A. M. Langmuir 1994,10 (lo),3461-3465.
with the experimental value obtained from the adsorption isotherms. Also, bacteria aggregation induced by these cationic vesicles w a s observed turbidimetrically a n d microsc~pically.~ Here w e characterize DODAB vesicles as bactericides, quantify their action as flocculants, a n d observe that there is n o relationship between both effects. O u r strategy is to determine viable counts, turbidity kinetics, and electrophoretic mobility for bacteria in mixtures of bacteria and vesicles over a wide range of bacteria number densities and DODAB concentrations. Over a 24-h interaction time with DODAB vesicles, there was cell death over the entire range of DODAB M) a n d bacteria concentrations tested (lo4- lo8cells/ mL) whereas aggregation is observed only from lo8 cells/
mL. Material and Methods Organism and Culture Conditions. Escherichia coli serogroup 0lll:H- is an enteropathogenic, nonmotile strain chosen as our model biocolloid because of these two characteristics. After isolation from human feces in MacConkey agar, it was grown overnight at 37 "C in a tube containing 15 mL of trypticase soy broth (Difco Laboratories, Detroit, MI). Thereafter, the culture was transferred to 150 mL of the same nutritive broth and incubated on a shaker (150 rpm) at 37 "C for 5 h. The turbid suspension thus obtained was centrifuged at 8000 rpm for 15 min and the pellet was resuspended in phosphate-buffered saline, pH 7.2 (PBS). This last procedure was repeated twice before finally resuspending the pellet in PBS containing 2% formaldehyde for fixation and preservation of the cells. Before interaction of these dead cells with DODAB vesicles a final wash in 0.264 M D-glucose eliminated the extracellular formaldehyde from solution. Fixation and preservation of the bacteria by 2% formaldehyde did not alter significantly the DODAB effect on the surface charge of the cells (see Results). Therefore, fured cells were used for determination of DODAB effects on electrophoretic mobilities and cell aggregation. For the colony forming units (cfu) counting the final pellet resuspension was in a aqueous solution of 0.264M D-glucose, instead of resuspension in PBS containing 2% formaldehyde. Bacteria number densities were obtained from the MacFarland scale.I0 Chemicals. Dioctadecyldimethylammonium bromide (DODAB)was obtained from Fluka ChemieAG(Switzer1and)and used (10) Paik, G. Reagents, Stains and Miscellaneous Test Procedures. In Manual of clinical microbiology, 3rd ed.; Lennette, E. H., Balows, A., Hausler, W. J., Jr., Truant, J. P., Eds.; American Society for Microbiology: Washington, DC, 1980;p 1004.
0743-746319512411-2991$09.00/00 1995 American Chemical Society
Sicchierolli et al.
2992 Langmuir, Vol. 11, No. 8, 1995 without further purification. All other reagents were analytical grade and were used without further purification. Water was Milli-Q quality. Vesicle Preparation. Small unilamellar DODAB vesicles (SV) with 86 nm mean z-average diameterll were prepared by sonication with tip in a 0.264 M D-glucose solution.4 sv were centrifuged at 104gfor 1h at 15 "C to precipitate multilamellar liposomes and titanium particles ejected from the titanium probe during sonication. The supernatant containing the unilamellar vesicles was used within 1 h of the preparation. DODAB concentration was determined by microtitration.12 Interactions between Vesicles and Bacteria. Due to the low stability of DODAB vesicles in the presence of ~ a l t ,before ~-~ mixing bacteria and vesicles, bacteria were centrifuged at 5000 rpm for 15 min and the pellet was resuspended in 0.264 M D-glUCOSe. Thereafter, any desired dilution ofvesicles or bacteria was done using a 0.264 M D-glucose solution so that cells and vesicleswere kept in a perfectly isotonicenvironment throughout and the vesicles presented high colloidal stability. Interaction between vesicles and bacteria was induced by adding the vesicles to the bacteria. Bacteria Microelectrophoresis. Equal volumes of the bacteria suspensions (2.1 x 105 to 2.1 x lo9 bacteridml) and vesicles (10-6-10-3 M DODAB) were mixed and allowed to interact at 25 "C for 1 h before measuring electrophoretic mobilities (EM)of the bacteria. Mobilitieswere measured using a Rank Brothers microelectrophoresis apparatus with a flat cell at 25 "C. The sample to be measured was placed into the electrophoresis cell, electrodes were connected, and a voltage of 60 Vwas applied across the cell. Velocities of individual bacteria over a given tracking distance were recorded, as was direction of bacteria movement. Average velocities were calculated from data on at least 20 individual bacteria. EM was calculated according to the equation EM = cm(uN)(l/t), where u is the distance over which the particle is tracked (micrometers), cm is the interelectrodes distance (7.27 cm), Vis the voltage applied (&60 V), and t is the average time in seconds required to track one particle a given distance u . Flocculation Experiments. Turbidity at 400 nm was continuously recorded as a function of time after vesicle addition to the bacteria using a Hitachi U-2000 spectrophotometer. At lo9 bacteridml, kinetics were obtained against a blank of bacteria in 0.264 M D-glucose. At lo8bacteridml, kinetics were obtained in the single beam mode (no blank was used). The time lag between mixing and recording was usually smaller than 10 s. The extent of flocculation (AA)was taken as the difference between the initial turbidity and turbidity at 3 min after mixing. The initial turbidity was taken as the turbidity of the bacteria suspension in absence of vesicles. The initial flocculation rate (uo) was taken as the first derivative of the turbidity vs time curve at t = 10 s. CFU Counting. In the survival experiments, equal volumes of the bacterial suspensions (2.4 x lo4 to 2.4 x lo8bacteridml) and vesicles (10-7-10-3 M DODAB) were mixed and allowed to interact for 2 and 24 h before spreading 0.1 mL of the mixtures on agar plates which were then incubated for 24 h at 37 "C. cfu counts were made using a colony counter. Viability curves were expressed as cfu as a function of the logarithm of the DODAB molarity. Minimum bactericidal concentrations (MBC) were estimated from the minimum DODAB concentration yielding less than 20 cfu per plate in the viability curves.
Results and Discussion The electrophoretic mobility of the bacterial cell increased as a function of the DODAB concentration (Figure 1). There is a DODAB concentration, CO, where the electrophoretic mobility of t h e cell is zero. COincreases as a function of the bacteria number density (Figure 1). This is expected for an interaction driven by the electrostatic attraction between the negatively charged bacterium (11)Carmona-Ribeiro, A. M.; Midmore, B. R. Synthetic bilayer adsorption onto polystyrene microspheres. Langmuir 1992,8(3),801806. (12)Schales, 0.; Schales, S. S. A simple and accurate method for the determination ofchloride in biological fluids. J.Biol. Chem. 1941,140, 879-883.
9-
a?C
6-
:: 5 -
d
4-
I'
3 -,'
2I
I
and the oppositely charged vesicle. As the number density of cells ( n ) increases so increases the total number of negatively charged sites that have to be neutralized by the DODAB vesicles to yield zero as electrophoretic mobility. Indeed there was a linear dependence between log n a n d log CO(Figure2). The DODAB amount required for charge neutralization on the cell surface is the same both for dead, preserved cells and for live, viable ones
(Figure 2). The survival of E. coli was investigated by incubating DODAB SV and bacterial suspensions for 2 a n d 24 h (Figure3). There was cell death in the micromolar range of DODAB concentrations over the entire range ofbacteria concentrations tested, a n d there was an effect of time on the cfu % titers. They are lower at 24 h than at 2 h contact time. This would be consistent with blockade by the vesicles of the transport of essential nutrients that takes place through t h e external bacterial wall. The vesicles indeed remain adhered as such to the external cell ~ u r f a c e . ~ A n obstruction of pores by the vesicles at the porins level would possibly lead to an effect of time on the cfu titers. The porins blockade hypothesis is presently under investigation in our laboratory. The minimum bactericidal concentration (MBC) obtained from t h e viability curves in Figure 3 is shown over a range of bacteria number densities. For larger contact
Bacteria Flocculation and Death by Cationic Vesicles
Langmuir, Vol. 11, No. 8, 1995 2993 0.551
0.35
C
1
, too
0
, 2 01
T I M E (s)
Figure 4. Turbidity at 400 nm as a function of time, in s, after mixing E.coli and DODAB vesicles in 0.264 M D-glucose at 25 "C. Final DODAB concentration is 5 x (a), 5 x (b), and 5 x M (c). Final bacteria number densityin the cuvette is 2.1 x lo8 bacteridml.
0
to an increase of the particle mass that overcomes the effect of the decrease in particle number density and leads to an increase in turbidityg at very early stages ofbacteria flocculation (Figure 4). Assuming neutralization of the cell surface charge by DODAB, flocculation is purely diffision-controlled and the time needed to reduce bacteria number density by 50% (Tu21 can be calculated from the initial bacteria number density ( n )in su~pension'~
-8 -7 -6 -5 -4 - 3 log c
Figure 3. cfu counting (%) as a function of DODAB concentration (C) in a logarithmic scale. Bacteria were killed from 1.2 x lo4 (A), 1.2 x lo5 (B), 1.2 x lo6 (C),1.2 x lo7 (D),and 1.2 x lo8 cells per mL (E). Mixtures of bacterialvesicles were left interacting for 2 (0)and 24 h (0). Table 1. Minimal Bactericidal Concentration over a Range of Bacteria Number Densities
bacteria concn (per mL) 1.2 x 105 1.2 x 106 1.2 107
minimum bactericidal concn" 2h >5.0 10-7 >2.5 x 5.0 x
24 h > 5 10-7 > 5 10-7 25 10-7
a Concentration is expressed as molarity and 5 equivalent to 0.3 ppm and to 0.3 pg/mL.
x
M is
times, MBC can be lowered by orders of magnitude. For the sake of comparison with other quaternary ammonium compounds, MBC may be converted to pg/mL and ppm. A MBC of 0.3 ppm (5 x M DODAB) at 1.2 x lo6 bacteridml (Table 1)compares well with MBC values previously reported for other quaternary ammonium compounds.l The DODAB vesicles seem to remain adsorbed at the bacterium surface in contrast to other surfactants. In fact, a palisade model for adhesion of DODAB vesicles to E. coli is consistent with figures obtained at limiting a d s ~ r p t i o n .Thus, ~ binding to and disruption of the cytoplasmic membrane would not occur. The most commonly accepted mechanism for the bactericidal action of quaternary ammonium compounds would not explain the DODAB effect on E . coli. At 2.1 x lo8bacteridml, turbidity at 400 nm increases as a function of time aRer mixing bacteria and vesicles (Figure 4). This increase in turbidity is consistent with bacteria aggregation induced by DODAB.9 Turbidity measurements are more nearly a measure ofparticle mass than of particle numbers.13 Bacterial aggregation leads
T,
= 2 x 101l/n
s
Therefore, Tm for flocculation of lo4bacteridml neutralized by DODAB would be approximately 233 days! Indeed, rapid flocculation taking place in a few minutes was only obtained at and above lo* bacteridml (Figure 4). At 2 x lo8 bacteridml the expected Tu2 calculated from the equation above is 16 min, in good agreement with the time scale used to monitor the turbidity increase. log uo increases linearly with log C until a maximal initial flocculation rate (UO is attained at a critical coagulation concentration, CCC (parts A and C of Figure 5 ) . For C > CCC the initial flocculation rate remains constant and maximal (parts A and C of Figure 5). This behavior is typical for colloids in the presence of electrolytes over a much higher concentration range and was predicted from the DLVO theory for colloidal ~tabi1ity.l~ From Figure 5, bacteria suspensions in the presence of DODAB vesicles behave as colloids. Most interestingly the CCC is attained when the electrophoretic mobility of the bacteria is zero, i.e., at CO(Figure 5A,C). Thus, charge neutralization triggers bacteria flocculation at maximum rate. The degree of flocculation estimated from A4 displays a bell-shaped dependence as a function of DODAB concentration (Figure 5B,D). It goes through a maximum at the same DODAB concentration where the electrophoretic mobility of the cells is zero. A similar result was obtained for flocculation of large negative polystyrene (13)Koch, A. L. Some calculations on the turbidity of mitochondria and bacteria. Biochim. Biophys. Acta 1961,51, 423-441. (14)Overbeek, J.Th.G. Recent developments in the understanding of colloid stability. J. CoZZoid Interface Sci. 1977,58, 408-422. (15)Reerink, H.; Overbeek, J. Th. G . The rate of coagulation as a measure of the stability of silver iodide sols. Discuss.Faraday SOC. 1954,18, 74-84.
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2994 Langmuir, Vol. 11, No. 8, 1995
-7 -6 -5 - 4 - 3 -2
0
-7 -6 -5 -L -3
log
0
c
Figure 5. Initial flocculation rate (UO)and flocculation extent (AA)for E . coli aggregation as a function of DODAB concentration, C. Bacteria number densities are 2.1 x lo8(Aand B)and 2.1 x lo9bacteridml (Cand D).Arrows indicate the DODAB concentration where electrophoretic mobility for bacteria is zero, Co. For bacteria at 2.1 x lo9 bacteridml, turbidity kinetics upon DODAB addition were obtained against a blank containing 2.1 x lo9 bacteridml in 0.264 M D-glucose.
particles (with 2 pm diameter) by small, positive ones (with 0.2 pm diameter) at low ionic strength (adsorption of high aEnity).16 As the number of small particles adsorbed increases, so the electrostatic repulsion between the large particles decreases, and their mutual van der Waals attraction becomes more dominant, until, at the point of zero charge, there is no repulsion and the large particles are strongly flocculated. At this point the sediment volume reaches a maximum. Adsorption of further small particles results in the large particles having a net positive charge and a corresponding decrease in the degree offlocculation. This is accompaniedby a reduction in sediment volume. Analogously to the heteroflocculation process described for these latexes,16 bacteria are aggregated by small DODAB vesicles followingvery similar patterns. The degree of flocculation goes through a maximum at CO(Figure 5B,D)and bacteria aggregates at Co were larger than those obtained at C > Coor at C
