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Minimal inhibitory concentration methodology in aquaculture: the temperature effect
Aquaculture 196 Ž2001. 311–318 www.elsevier.nlrlocateraqua-online
Review article
Minimal inhibitory concentration methodology in aquaculture: the temperature effect Christian Michel a,) , Guillaume Blanc b a
Unite´ de Virologie et Immunologie Moleculaires, Centre de Recherche de Jouy-en-Josas, INRA, 78352 ´ Jouy-en-Josas cedex, France b Laboratoire d’Aquaculture et de Pathologie Aquacole, ENVN, BP 40707, 44307 Nantes cedex 3, France Received 22 May 2000; accepted 30 August 2000
Abstract Although antimicrobial drugs testing and minimal inhibitory concentration ŽMIC. assessment have become of common use for guiding bacterial disease treatments, it soon appeared that temperature conditions under which tests were performed could result in strong variations and cause misinterpretations. This is specially inconvenient with fish pathogenic bacteria, many of which require low temperature incubation and do not fit easily with the standardized procedures generally accepted in medical laboratories. Temperature may affect both antimicrobial drugs and bacterial physiology through a variety of mechanisms such as stability and diffusion kinetics of the drug species, growth rate, membrane properties, enzymatic activity and genetic regulations of the bacterial cells. Interference and combination of so many effects make any prediction unrealistic. In MIC studies, however, dilution methods appear much less susceptible than diffusion methods to temperature-induced fluctuations. Provided interpretation is done carefully they should always be preferred to perform routinely antimicrobial tests. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Aquatic bacteria; Antimicrobial drugs; Antibiogramme; MIC; Diffusion methods; Dilution methods; Temperature effects
1. Historical background : the diffusion method Although theoretical studies on the efficacy of antimicrobial compounds had to focus on accurate estimation of effective doses against different varieties of microorganisms, it was the development of microbial resistances and the subsequent difficulties encountered in practice that urged diagnostic laboratories to perform systematically antimicro)
Corresponding author. E-mail addresses: [email protected] ŽC. Michel., [email protected] ŽG. Blanc..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 4 4 - 0
312
C. Michel, G. Blanc r Aquaculture 196 (2001) 311–318
bial susceptibility testing. Minimum inhibitory concentration ŽMIC. early appeared as a convenient concept to provide quantitative and meaningful indications. However, the large number of examinations performed in routine medical practice should lead to adopt preferentially those of the relevant methods which were better adapted to rapid and easy screening procedures. This, and the fact that medical interest was limited at first to human or homeothermic animal species, may explain why the diffusion technique was almost exclusively used for a long time. Indeed, guidelines and standardized procedures defined by international committees ŽBauer et al., 1966; Ericsson and Sherris, 1971. were readily available for bacteria of medical significance. However, during the 1970s, an increasing interest for the diseases of poikilotherms, and even vegetal pathogens, led to consider the application of antimicrobial testing to bacteria normally found in the environment, from which optimal temperature could display a large range of variability. Agar diffusion method provides only an indirect estimation of MICs relying upon a linear relationship between the concentration of the antimicrobial product and the diameter of an area of visible inhibition it produces on bacterial growth. The mathematical theory of the antibiogramme had been first established by Cooper and Woodman Ž1946., who proposed the following formula : x log m 0 y log m t s 4 Dt where m 0 and m t are the concentrations of the drug at the source and at a distance x from the source at the reading time, t, the reading time and D the diffusion coefficient. Passive diffusion itself, formulated by the Fick’s law when a flux of chemical species occurs through a concentration gradient, may be ultimately attributed to the thermal energy of the product molecules. This let us expect a critical role for temperature, although many other factors, for instance, the composition and thickness of the culture medium, the bacterial inoculum size, the dose and the diffusion coefficient of the product itself, also exert strong effects. In disk diffusion antibiogramme method performed in standard conditions at a temperature of 378C ŽBauer et al., 1966; Ericsson and Sherris, 1971., regression lines had been established without too much difficulty to relate the MIC values of different molecules to the inhibition zone diameters measured after 24 h incubation. So, it was first expected that similar procedures could apply to bacteria growing at different temperatures, even though delayed reading and resulting hazy limits of inhibition areas could not be completely avoided with slow growing species. In fact, it soon appeared that in addition to these difficulties, the situation could vary greatly according to the molecules ŽHahnel and Gould, 1982. and the organisms tested, and that no simple extrapolation could be figured out ŽMichel and Bassalert, 1982..
2. The dilution methods In practice, establishing regression curves for every couple bacteriumrantibiotic did not seem realistic in non-specialized laboratories. Conversely, with the advantage of
C. Michel, G. Blanc r Aquaculture 196 (2001) 311–318
313
permitting direct measurement of the MICs, dilution methods performed in liquid or preferentially in solid media had always been considered the more reliable methods, and their results were not then considered to depend closely on temperature. Certainly, serial dilution of active products could represent a repetitive and cumbersome work, but the development of microsystems, which was very active at that time, helped to overcome the challenge. Many papers at the turn of the 1970s had reported on the possibility of using micromethods for antimicrobial susceptibility testing of particular groups of bacteria, and some were specially developed for fish-associated bacteria ŽColorni, 1985; Michel et al., 1984.. In the following decade, with the development of active research on methodological aspects and needs for standardization of antibiotic testing, many results, and many new questions too, were put forwards. Dilution methods did not confirm as independent from temperature as it had been supposed, and it was realised that temperature could take greater importance and act through more complex modalities than it had been formerly admitted. It so appeared that temperature influence on the MICs of the antimicrobial products could not be explained merely by the physical characteristics of some experimental system, but that many factors, including physical and chemical properties of the drugs as well as biological behaviour of the bacteria could be put in competition, with a rather unpredictable issue.
3. Different aspects of the temperature effects 3.1. Stability and conserÕation of the chemicals According to the manufacturers, all antimicrobial drugs must be stored in appropriate conditions, generally at low temperatures, and so it is to be feared that these complex molecules may be subject to some degree of decaying. The risk of denaturation is higher when the products have been diluted, and some of them, for instance some beta-lactames and chlortetracycline, may prove so unstable that their use should not be encouraged in fish farms where storing conditions are rarely convenient and result in quick inactivation. A similar phenomenon may be expected when unstable products are used in testing systems, inasmuch as incubation temperatures are generally chosen as high as possible. It is noticeable that objective studies on the effects of high temperature on drugs conservation and activity have been chiefly carried out with thermophilic bacteria of industrial interest ŽPeteranderl et al., 1990., or to address the possibility of autoclaving ready-for-use media ŽTraub and Leonhard, 1995.. The results could differ slightly according to the target micoorganism, but most of the drugs proved fairly stable although generally more active when exposed to the lowest temperatures. 3.2. Bacterial metabolism and physiology In most of the studies conducted with medically important bacteria, the cultures are supposed to be grown in optimal conditions. This is no more the case for fastidious
314
C. Michel, G. Blanc r Aquaculture 196 (2001) 311–318
organisms ŽReisinger et al., 1996. or slow-growing bacteria isolated from aquatic animals. In fish microbiology, the required temperatures being lower than 378C the stability of the chemicals should be favoured, but conversely the slower metabolism of the bacteria often obliges to delay the time of reading, so increasing the risk of drug inactivation. Furthermore, it seems that temperature is not the only parameter to determine the degradation rate of certain drugs. Ray and Newton Ž1991. monitored the stability of tetracycline and chlortetracycline grown in trypticase soy broth by high performance liquid chromatography. Chlortetracycline appeared to be actively degraded in less than 5 days in TBS, while both tetracyclines proved far more stable in water. It was inferred that in this case, the cause for inactivation of the drugs was to be found in the medium composition, rather than in temperature effects. Optimal temperatures follow the same rules in bacteria as in other living beings and they may vary in a range of values limited by critical thresholds, with the only difference that bacterial growth provides an easy way of observing the effect of any variation. Reaching upper limits generally results in sudden interruption of metabolic reactions, whereas lower values are more likely to allow progressive slowering of metabolic activity before total inhibition occurs. Although most of the bacterial strains found in fish and aquatic habitat are specially adapted to low temperatures, the optimal conditions they require are far more variable than in the case of higher animals pathogens. Such a diversity does not make standardization of experimental conditions very easy. Moreover, detailed studies have shown that growth rate was not the only process to respond to temperature fluctuations. In the case of antimicrobial substances, which must penetrate in the cells before being distributed in the different cell compartments and reaching their metabolic target, it came out that each of these sequential steps may be affected differently. Considering the complexity of the situations, the available documentation about these aspects still appears very partial. However, some examples may serve to illustrate the many difficulties MIC assessment may be faced to. Antibiotic penetration into bacterial cell must occur through cellular membranes. In certain cases, as exemplified by erythromycin action on Staphylococcus aureus, selective intake does not seem to occur, and it has been demonstrated that instead, passive diffusion represented the normal way of penetration ŽDette et al., 1987.. Logically, low temperature tends to slow down the diffusion according to strict chemical equilibrium laws. However, it was also observed, in enterobacteria at first ŽDunnick and O’Leary, 1970., that the chemical composition of the membrane, namely the phospholipid rate, was likely to change according to the temperature and correlatively influence the antimicrobial susceptibility of the bacteria. Unsaturated fatty acids appeared to increase and cyclopropanic acid to decrease in proportion when bacteria were grown at low temperature. A similar effect, resulting in enhancement of drug efficacy at higher temperatures, was suspected in several studies involving mainly Pseudomonas species ŽMackowiak et al., 1982; Papapetropoulou et al., 1994; Wheat et al., 1985.. In other cases, active membrane mechanisms could be involved. It is known that complex physiological mechanisms involving ionic pumps and structural proteins called porins are responsible for the selective transport of foreign molecules, and it is sensible to suspect such mechanisms in the case of antimicrobial molecules. Although tempera-
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ture would basically exert the same effect as in the previous case, optimal functioning of porins could require slightly different thermic conditions, a situation that possible interference with other parameters such as the pH could contribute to complicate still more. In Aeromonas salmonicida, outer-membrane proteins modifications have been associated with the expression of low-level multiple resistance involving, namely, quinolones and tetracyclines ŽBarnes et al., 1990a; Wood et al., 1986.. The A-layer of the virulent forms of A. salmonicida is suspected to play a part in the higher susceptibility of the A q strains to chloramphenicol and streptonigrin ŽGarduno ˜ et al., 1994.. Little is known for the moment about such points, apart from indirect evidences, but it is clear that modifications in the membrane and cell–wall structure resulting from an increase in temperature could be of consequence in the susceptibility of many bacteria to chemotherapy ŽMackowiak et al., 1982.. Inside the bacterial cell, the fate of the molecule depends upon its ability to reach target structures and accumulate at sufficient concentration to interfere with the cell functions. Once again, the cell metabolism will command the kinetics of this critical step. However, two kinds of mechanisms may also result in partial neutralisation or degradation of the antibiotic. On the one hand, passive bounding of the product to intracellular proteins may occur, sometimes in combination with a lowered affinity for the target structures, and it seems that low temperatures are likely to favour these mechanisms, probably due to the extension of time required to reach effective concentrations. Decrease in drug activity at low temperature was reported in several cases, namely, with the 4-quinolones studied by Barnes et al. Ž1990b. whose observations were confirmed and tentatively commented by Martinsen et al. Ž1992.. On the other hand, degrading enzymes produced by the bacterium are likely to modify and neutralize the product. It has been noted by some authors that enzymatic activity could be temperature dependent, as in the case of a cephalosporinase activity observed in Yersinia kristensenii ŽBejar et al., 1986.. In the aforementioned paper, Martinsen et al. Ž1992. noticed that, compared to the quinolones, oxytetracycline showed opposite properties in response to temperature variations, and called for a possible thermodependence of the ribosomal activity. 3.3. Microbial genetics Another modality of temperature effects could concern the genetic regulations of the cell. Thermodependent expression of certain characteristics has often been described in enterobacteria, and we have the personal experience ŽMichel et al., 1984. of a Serratia strain which expressed strong multiple resistance at 228C, whereas it proved constantly susceptible at 378C, using both diffusion or dilution methods. Expression of b-lactamase genes located on the chromosome in Aeromonas spp. has been shown to differ according to the temperature ŽWalsh et al., 1997.. All species were more likely to produce depressed mutants at 308C than at 37 8C. At last, the frequency of spontaneous mutations supporting antimicrobial resistance should not be disregarded. This has been considered in several studies, and some of them tried to objectivize possible relations between the mutation rate and temperature. Attempts at selecting mutant quinolone-resistant strains at 258C, 308C or 378C produced mutation rates varying greatly according to the bacterial species but clearly related to the temperature ŽParte and Smith, 1996..
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4. Practical consequences It is concluded from this brief review that temperature may affect the results of MIC testing through multiple mechanisms which may act synergistically, or antagonistically. To add to the complexity of this background, it is important to consider other parameters, namely, salinity, composition of the assay medium, inoculum size, lag time and growth rate, which may themselves condition the drug efficacy at different degrees according to the temperature. It has been pointed out that the agar diffusion method was specially responsive to any variation of these parameters, among which temperature is probably the most difficult to control. Certainly, some species of fish bacteria are likely to grow at 378C and, for routine purpose, could be tested under usual standard conditions. However, it is not sure that such properties correspond to optimal conditions in all cases. Adaptation of the pathogenic species to their host generally fulfil specific requirements, and it might appear hazardous to get quantitative data and draw practical implications from bacterial cultures experiencing stress or physiological distress ŽGilbert et al., 1990; Guerin-Faublee ´ ´ et al., 1995.. Diffusion methods should then be given up when aiming to assess antibiotic MIC values for bacteria other than those known to be associated to homeotherms, unless regression curves have been clearly established. In fact, it is possible that such an endeavour would meet with new limitations, because while higher vertebrates pathogens may display some degree of homogeneity among the isolates of a recognized species, the same would not be true for all environmental species, quite more plastic, having adapted to diverse habitats, or remaining insufficiently defined in taxonomical guidelines. Assessing regression lines for every strain would not be totally unrealistic but would require long-term developments! Suggestions have been made to modify the agar diffusion method and replace the classical MIC estimation by other paramers in E-test ŽBrown and Brown, 1991. or Inhibitory Concentration in Diffusion test ŽICD. ŽDelignette-Muller and Flandrois, 1994.. However, application of these methods to fish pathogenic bacteria did not produce consistent results with all products ŽGuerin-Faublee ´ ´ et al., 1996.. With regard to the objective consequences of incubation temperature on the results of dilution methods, observations and conclusions may differ significantly according to the studied bacteria and to the workers. In several cases, it seems that the numerous causes of variation may counteract each other and result in slightly different but roughly comparable results ŽManzella et al., 1980., and in no way the amplitude of the fluctuations may be compared to the variability observed in diffusion tests. It is clear that temperature directly affects the lag time and growth rate of the culture, and that when addressing dynamic endpoint studies the influence of temperature must be taken into consideration. In single endpoint studies, constraints are not so critical, and the common opinion is that, due to compensatory mechanisms, MIC determination assessed according to this way should be endowed with some reliability. For instance, Smith et al. Ž1987. concluded that delaying the reading time could minimize the deviations resulting at first from different incubation temperatures. This may be right at least when the molecules are quite stable in solution and do not combine to medium components.
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Eventually, it is rather comforting to perceive that whatever the microbial organism to be studied and the temperature required for its development, dilution methods may offer convenient possibilities for MIC assessment. Indeed, it seems logical to accept quantitative values obtained at temperatures next to those which prevail in the daily environment of the fish and their pathogens. As in many in vitro biological systems, it will just be necessary to remember that data collected under the form of MICs only represent a convenient quantitative representation of the bacterial susceptibility to drugs, which in no way is intended to entirely tally with what occurs in animal organism. References Barnes, A.C., Lewin, C.S., Hastings, T.S., Amyes, S.G.B., 1990a. Cross resistance between oxytetracycline and oxolinic acid in Aeromonas salmonicida associated with alterations in outer membrane proteins. FEMS Microbiol. Lett. 72, 337–340. Barnes, A.C., Lewin, C.S., Hastings, T.S., Amyes, S.G.B., 1990b. In vitro activities of 4-quinolones against the fish pathogen Aeromonas salmonicida. Antimicrob. Agents Chemother. 34, 1819–1820. Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45, 493–496. Bejar, V., Calvo, C., Ramos Corvenzana, A., 1986. In vitro susceptibility of Yersinia kristensenii strains to b-lactam antibiotics. Ann. Inst. PasteurrMicrobiol. 137A, 169–177. Brown, D.J., Brown, L., 1991. Evaluation of the E-test, a novel method of quantifying antimicrobial activity. J. Antimicrob. Chemother. 27, 183–190. Colorni, A., 1985. A practical method for determining the susceptibility of aquatic Vibrio and Aeromonas spp. to antimicrobial agents. Aquaculture 46, 263–266. Cooper, K.E., Woodman, D., 1946. The diffusion of antiseptics through agar gels, with special reference to the agar cup assay method of estimating the activity of penicillin. J. Pathol. Bacteriol. 58, 75–84. Delignette-Muller, M.L., Flandrois, J.P., 1994. An accurate diffusion method for determining bacterial sensitivity to antibiotics. J. Antimicrob. Chemother. 33, 73–81. Dette, G.A., Knothe, H., Kaula, S., 1987. Modifying effects of pH and temperature on Ž14 C.erythromycin uptake into Staphylococcus aureus —relation to antimicrobial activity. Zentralbl. Bakteriol. Hyg., A 265, 393–403. Dunnick, J.D., O’Leary, W.M., 1970. Correlation of bacterial lipid composition with antibiotic resistance. J. Bacteriol. 101, 892–900. Ericsson, H.M., Sherris, J.C., 1971. Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol. Microbiol. Scand., B, Suppl. No. 217, 1–90. Garduno, ˜ R.A., Phipps, B.M., Kay, W.W., 1994. Physiological consequences of the S-layer of Aeromonas salmonicida in relation to growth, temperature, and outer membrane permeation. Can. J. Chemother. 40, 622–629. Gilbert, P., Collier, P.J., Brown, M.R.W., 1990. Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob. Agents Chemother. 34, 1865–1868. Guerin-Faublee, ´ ´ V., Rosso, L., Vigneulle, M., Flandrois, J.-P., 1995. The effect of incubation temperature and sodium chloride concentration on the growth kinetics of Vibrio anguillarum and Vibrio anguillarum-related organisms. J. Appl. Bacteriol. 78, 621–629. Guerin-Faublee, ´ ´ V., Delignette-Muller, M.L., Vigneulle, M., Flandrois, J.-P., 1996. Application of a modified disc diffusion technique to antimicrobial susceptibility testing of Vibrio anguillarum and Aeromonas salmonicida clinical isolates. Vet. Microbiol. 51, 137–149. Hahnel, G.B., Gould, R.W., 1982. Effects of temperature on biochemical reactions and drug resistance of virulent and avirulent Aeromonas salmonicida. J. Fish Dis. 5, 329–337. Mackowiak, P.A., Marling-Cason, M., Cohen, R.L., 1982. Effects of temperature on antimicrobial susceptibility of bacteria. J. Infect. Dis. 145, 550–553.
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Manzella, J.P., Roberts, N.J.J., Robertson, R.G., Hoder, G.B., 1980. Temperature elevation, bacterial growth and antibiotic activity. J. Antimicrob. Chemother. 6, 795–798. Martinsen, B., Oppegaard, H., Wichstrøm, R., Myhr, E., 1992. Temperature-dependent in vitro antimicrobial activity of four 4-quinolones and oxytetracycline against bacteria pathogenic to fish. Antimicrob. Agents Chemother. 36, 1738–1743. Michel, C., Bassalert, J.-F., 1982. Influence de la temperature sur les resultats de l’antibiogramme pratique´ par ´ ´ la methode de diffusion en ichtyopathologie. Ann. Rech. Vet. 13, 245–250. ´ Michel, C., Suprin, L., De Kinkelin, P., 1984. A microdilution method for drug sensitivity testing of fish associated bacteria at 228C. J. Appl. Bacteriol. 57, 15–21. Papapetropoulou, M., Rodopoulou, G., Giannoulaki, E., Stergiopoulos, P., 1994. Effect of temperature on antimicrobial susceptibilities of Pseudomonas species isolated from drinking water. J. Chemother. 6, 404–407. Parte, A.C., Smith, J.T., 1996. Influence of temperature on mutational resistance to 4-quinolones. Arzneim.Forsch. 46 ŽI., 429–432. Peteranderl, R., Shotts, E.B.J., Wiegel, J., 1990. Stability of antibiotics under growth conditions for thermophilic anaerobes. Appl. Environ. Microbiol. 56, 1981–1983. Ray, A., Newton, V., 1991. Use of high-performance liquid chromatography to monitor stability of tetracycline and chlortetracycline in susceptibility determinations. Antimicrob. Agents Chemother. 35, 1264– 1266. Reisinger, E., Wendelin, I., Gasser, R., Halwachs, G., Wilders-Truschnig, M., Krejs, G., 1996. Antibiotics and increased temperature against Borrelia burgdorferi in vitro. Scand. J. Infect. Dis. 28, 155–157. Smith, J.A., Henry, D.A., Bourgault, A.-M., Bryan, L., Harding, G.J., Hoban, D.J., Horsman, G.B., Marrie, T., Turgeon, P., 1987. Comparison of agar disk diffusion, microdilution broth, and agar dilution for testing antimicrobial susceptibility of coagulase-negative staphylococcci. J. Clin. Microbiol. 25, 1741–1746. Traub, W.H., Leonhard, B., 1995. Heat stability of the antimicrobial activity of sixty-two antibacterial agents. J. Antimicrob. Chemother. 35, 149–154. Walsh, T.R., Stunt, R.A., Nabi, J.A., MacGowan, A.P., Bennett, P.M., 1997. Distribution and expression of b-lactamase genes among Aeromonas spp. J. Antimicrob. Chemother. 40, 171–178. Wheat, P.F., Winstanley, T.G., Spencer, R.C., 1985. Effect of temperature on antimicrobial susceptibility of Pseudomonas maltophilia. J. Clin. Pathol. 38, 1055–1058. Wood, S.C., McCashion, R.N., Lynch, W.H., 1986. Multiple low-level antibiotic resistance in Aeromonas salmonicida. Antimicrob. Agents Chemother. 29, 992–996.
Review article
Minimal inhibitory concentration methodology in aquaculture: the temperature effect Christian Michel a,) , Guillaume Blanc b a
Unite´ de Virologie et Immunologie Moleculaires, Centre de Recherche de Jouy-en-Josas, INRA, 78352 ´ Jouy-en-Josas cedex, France b Laboratoire d’Aquaculture et de Pathologie Aquacole, ENVN, BP 40707, 44307 Nantes cedex 3, France Received 22 May 2000; accepted 30 August 2000
Abstract Although antimicrobial drugs testing and minimal inhibitory concentration ŽMIC. assessment have become of common use for guiding bacterial disease treatments, it soon appeared that temperature conditions under which tests were performed could result in strong variations and cause misinterpretations. This is specially inconvenient with fish pathogenic bacteria, many of which require low temperature incubation and do not fit easily with the standardized procedures generally accepted in medical laboratories. Temperature may affect both antimicrobial drugs and bacterial physiology through a variety of mechanisms such as stability and diffusion kinetics of the drug species, growth rate, membrane properties, enzymatic activity and genetic regulations of the bacterial cells. Interference and combination of so many effects make any prediction unrealistic. In MIC studies, however, dilution methods appear much less susceptible than diffusion methods to temperature-induced fluctuations. Provided interpretation is done carefully they should always be preferred to perform routinely antimicrobial tests. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Aquatic bacteria; Antimicrobial drugs; Antibiogramme; MIC; Diffusion methods; Dilution methods; Temperature effects
1. Historical background : the diffusion method Although theoretical studies on the efficacy of antimicrobial compounds had to focus on accurate estimation of effective doses against different varieties of microorganisms, it was the development of microbial resistances and the subsequent difficulties encountered in practice that urged diagnostic laboratories to perform systematically antimicro)
Corresponding author. E-mail addresses: [email protected] ŽC. Michel., [email protected] ŽG. Blanc..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 4 4 - 0
312
C. Michel, G. Blanc r Aquaculture 196 (2001) 311–318
bial susceptibility testing. Minimum inhibitory concentration ŽMIC. early appeared as a convenient concept to provide quantitative and meaningful indications. However, the large number of examinations performed in routine medical practice should lead to adopt preferentially those of the relevant methods which were better adapted to rapid and easy screening procedures. This, and the fact that medical interest was limited at first to human or homeothermic animal species, may explain why the diffusion technique was almost exclusively used for a long time. Indeed, guidelines and standardized procedures defined by international committees ŽBauer et al., 1966; Ericsson and Sherris, 1971. were readily available for bacteria of medical significance. However, during the 1970s, an increasing interest for the diseases of poikilotherms, and even vegetal pathogens, led to consider the application of antimicrobial testing to bacteria normally found in the environment, from which optimal temperature could display a large range of variability. Agar diffusion method provides only an indirect estimation of MICs relying upon a linear relationship between the concentration of the antimicrobial product and the diameter of an area of visible inhibition it produces on bacterial growth. The mathematical theory of the antibiogramme had been first established by Cooper and Woodman Ž1946., who proposed the following formula : x log m 0 y log m t s 4 Dt where m 0 and m t are the concentrations of the drug at the source and at a distance x from the source at the reading time, t, the reading time and D the diffusion coefficient. Passive diffusion itself, formulated by the Fick’s law when a flux of chemical species occurs through a concentration gradient, may be ultimately attributed to the thermal energy of the product molecules. This let us expect a critical role for temperature, although many other factors, for instance, the composition and thickness of the culture medium, the bacterial inoculum size, the dose and the diffusion coefficient of the product itself, also exert strong effects. In disk diffusion antibiogramme method performed in standard conditions at a temperature of 378C ŽBauer et al., 1966; Ericsson and Sherris, 1971., regression lines had been established without too much difficulty to relate the MIC values of different molecules to the inhibition zone diameters measured after 24 h incubation. So, it was first expected that similar procedures could apply to bacteria growing at different temperatures, even though delayed reading and resulting hazy limits of inhibition areas could not be completely avoided with slow growing species. In fact, it soon appeared that in addition to these difficulties, the situation could vary greatly according to the molecules ŽHahnel and Gould, 1982. and the organisms tested, and that no simple extrapolation could be figured out ŽMichel and Bassalert, 1982..
2. The dilution methods In practice, establishing regression curves for every couple bacteriumrantibiotic did not seem realistic in non-specialized laboratories. Conversely, with the advantage of
C. Michel, G. Blanc r Aquaculture 196 (2001) 311–318
313
permitting direct measurement of the MICs, dilution methods performed in liquid or preferentially in solid media had always been considered the more reliable methods, and their results were not then considered to depend closely on temperature. Certainly, serial dilution of active products could represent a repetitive and cumbersome work, but the development of microsystems, which was very active at that time, helped to overcome the challenge. Many papers at the turn of the 1970s had reported on the possibility of using micromethods for antimicrobial susceptibility testing of particular groups of bacteria, and some were specially developed for fish-associated bacteria ŽColorni, 1985; Michel et al., 1984.. In the following decade, with the development of active research on methodological aspects and needs for standardization of antibiotic testing, many results, and many new questions too, were put forwards. Dilution methods did not confirm as independent from temperature as it had been supposed, and it was realised that temperature could take greater importance and act through more complex modalities than it had been formerly admitted. It so appeared that temperature influence on the MICs of the antimicrobial products could not be explained merely by the physical characteristics of some experimental system, but that many factors, including physical and chemical properties of the drugs as well as biological behaviour of the bacteria could be put in competition, with a rather unpredictable issue.
3. Different aspects of the temperature effects 3.1. Stability and conserÕation of the chemicals According to the manufacturers, all antimicrobial drugs must be stored in appropriate conditions, generally at low temperatures, and so it is to be feared that these complex molecules may be subject to some degree of decaying. The risk of denaturation is higher when the products have been diluted, and some of them, for instance some beta-lactames and chlortetracycline, may prove so unstable that their use should not be encouraged in fish farms where storing conditions are rarely convenient and result in quick inactivation. A similar phenomenon may be expected when unstable products are used in testing systems, inasmuch as incubation temperatures are generally chosen as high as possible. It is noticeable that objective studies on the effects of high temperature on drugs conservation and activity have been chiefly carried out with thermophilic bacteria of industrial interest ŽPeteranderl et al., 1990., or to address the possibility of autoclaving ready-for-use media ŽTraub and Leonhard, 1995.. The results could differ slightly according to the target micoorganism, but most of the drugs proved fairly stable although generally more active when exposed to the lowest temperatures. 3.2. Bacterial metabolism and physiology In most of the studies conducted with medically important bacteria, the cultures are supposed to be grown in optimal conditions. This is no more the case for fastidious
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organisms ŽReisinger et al., 1996. or slow-growing bacteria isolated from aquatic animals. In fish microbiology, the required temperatures being lower than 378C the stability of the chemicals should be favoured, but conversely the slower metabolism of the bacteria often obliges to delay the time of reading, so increasing the risk of drug inactivation. Furthermore, it seems that temperature is not the only parameter to determine the degradation rate of certain drugs. Ray and Newton Ž1991. monitored the stability of tetracycline and chlortetracycline grown in trypticase soy broth by high performance liquid chromatography. Chlortetracycline appeared to be actively degraded in less than 5 days in TBS, while both tetracyclines proved far more stable in water. It was inferred that in this case, the cause for inactivation of the drugs was to be found in the medium composition, rather than in temperature effects. Optimal temperatures follow the same rules in bacteria as in other living beings and they may vary in a range of values limited by critical thresholds, with the only difference that bacterial growth provides an easy way of observing the effect of any variation. Reaching upper limits generally results in sudden interruption of metabolic reactions, whereas lower values are more likely to allow progressive slowering of metabolic activity before total inhibition occurs. Although most of the bacterial strains found in fish and aquatic habitat are specially adapted to low temperatures, the optimal conditions they require are far more variable than in the case of higher animals pathogens. Such a diversity does not make standardization of experimental conditions very easy. Moreover, detailed studies have shown that growth rate was not the only process to respond to temperature fluctuations. In the case of antimicrobial substances, which must penetrate in the cells before being distributed in the different cell compartments and reaching their metabolic target, it came out that each of these sequential steps may be affected differently. Considering the complexity of the situations, the available documentation about these aspects still appears very partial. However, some examples may serve to illustrate the many difficulties MIC assessment may be faced to. Antibiotic penetration into bacterial cell must occur through cellular membranes. In certain cases, as exemplified by erythromycin action on Staphylococcus aureus, selective intake does not seem to occur, and it has been demonstrated that instead, passive diffusion represented the normal way of penetration ŽDette et al., 1987.. Logically, low temperature tends to slow down the diffusion according to strict chemical equilibrium laws. However, it was also observed, in enterobacteria at first ŽDunnick and O’Leary, 1970., that the chemical composition of the membrane, namely the phospholipid rate, was likely to change according to the temperature and correlatively influence the antimicrobial susceptibility of the bacteria. Unsaturated fatty acids appeared to increase and cyclopropanic acid to decrease in proportion when bacteria were grown at low temperature. A similar effect, resulting in enhancement of drug efficacy at higher temperatures, was suspected in several studies involving mainly Pseudomonas species ŽMackowiak et al., 1982; Papapetropoulou et al., 1994; Wheat et al., 1985.. In other cases, active membrane mechanisms could be involved. It is known that complex physiological mechanisms involving ionic pumps and structural proteins called porins are responsible for the selective transport of foreign molecules, and it is sensible to suspect such mechanisms in the case of antimicrobial molecules. Although tempera-
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ture would basically exert the same effect as in the previous case, optimal functioning of porins could require slightly different thermic conditions, a situation that possible interference with other parameters such as the pH could contribute to complicate still more. In Aeromonas salmonicida, outer-membrane proteins modifications have been associated with the expression of low-level multiple resistance involving, namely, quinolones and tetracyclines ŽBarnes et al., 1990a; Wood et al., 1986.. The A-layer of the virulent forms of A. salmonicida is suspected to play a part in the higher susceptibility of the A q strains to chloramphenicol and streptonigrin ŽGarduno ˜ et al., 1994.. Little is known for the moment about such points, apart from indirect evidences, but it is clear that modifications in the membrane and cell–wall structure resulting from an increase in temperature could be of consequence in the susceptibility of many bacteria to chemotherapy ŽMackowiak et al., 1982.. Inside the bacterial cell, the fate of the molecule depends upon its ability to reach target structures and accumulate at sufficient concentration to interfere with the cell functions. Once again, the cell metabolism will command the kinetics of this critical step. However, two kinds of mechanisms may also result in partial neutralisation or degradation of the antibiotic. On the one hand, passive bounding of the product to intracellular proteins may occur, sometimes in combination with a lowered affinity for the target structures, and it seems that low temperatures are likely to favour these mechanisms, probably due to the extension of time required to reach effective concentrations. Decrease in drug activity at low temperature was reported in several cases, namely, with the 4-quinolones studied by Barnes et al. Ž1990b. whose observations were confirmed and tentatively commented by Martinsen et al. Ž1992.. On the other hand, degrading enzymes produced by the bacterium are likely to modify and neutralize the product. It has been noted by some authors that enzymatic activity could be temperature dependent, as in the case of a cephalosporinase activity observed in Yersinia kristensenii ŽBejar et al., 1986.. In the aforementioned paper, Martinsen et al. Ž1992. noticed that, compared to the quinolones, oxytetracycline showed opposite properties in response to temperature variations, and called for a possible thermodependence of the ribosomal activity. 3.3. Microbial genetics Another modality of temperature effects could concern the genetic regulations of the cell. Thermodependent expression of certain characteristics has often been described in enterobacteria, and we have the personal experience ŽMichel et al., 1984. of a Serratia strain which expressed strong multiple resistance at 228C, whereas it proved constantly susceptible at 378C, using both diffusion or dilution methods. Expression of b-lactamase genes located on the chromosome in Aeromonas spp. has been shown to differ according to the temperature ŽWalsh et al., 1997.. All species were more likely to produce depressed mutants at 308C than at 37 8C. At last, the frequency of spontaneous mutations supporting antimicrobial resistance should not be disregarded. This has been considered in several studies, and some of them tried to objectivize possible relations between the mutation rate and temperature. Attempts at selecting mutant quinolone-resistant strains at 258C, 308C or 378C produced mutation rates varying greatly according to the bacterial species but clearly related to the temperature ŽParte and Smith, 1996..
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4. Practical consequences It is concluded from this brief review that temperature may affect the results of MIC testing through multiple mechanisms which may act synergistically, or antagonistically. To add to the complexity of this background, it is important to consider other parameters, namely, salinity, composition of the assay medium, inoculum size, lag time and growth rate, which may themselves condition the drug efficacy at different degrees according to the temperature. It has been pointed out that the agar diffusion method was specially responsive to any variation of these parameters, among which temperature is probably the most difficult to control. Certainly, some species of fish bacteria are likely to grow at 378C and, for routine purpose, could be tested under usual standard conditions. However, it is not sure that such properties correspond to optimal conditions in all cases. Adaptation of the pathogenic species to their host generally fulfil specific requirements, and it might appear hazardous to get quantitative data and draw practical implications from bacterial cultures experiencing stress or physiological distress ŽGilbert et al., 1990; Guerin-Faublee ´ ´ et al., 1995.. Diffusion methods should then be given up when aiming to assess antibiotic MIC values for bacteria other than those known to be associated to homeotherms, unless regression curves have been clearly established. In fact, it is possible that such an endeavour would meet with new limitations, because while higher vertebrates pathogens may display some degree of homogeneity among the isolates of a recognized species, the same would not be true for all environmental species, quite more plastic, having adapted to diverse habitats, or remaining insufficiently defined in taxonomical guidelines. Assessing regression lines for every strain would not be totally unrealistic but would require long-term developments! Suggestions have been made to modify the agar diffusion method and replace the classical MIC estimation by other paramers in E-test ŽBrown and Brown, 1991. or Inhibitory Concentration in Diffusion test ŽICD. ŽDelignette-Muller and Flandrois, 1994.. However, application of these methods to fish pathogenic bacteria did not produce consistent results with all products ŽGuerin-Faublee ´ ´ et al., 1996.. With regard to the objective consequences of incubation temperature on the results of dilution methods, observations and conclusions may differ significantly according to the studied bacteria and to the workers. In several cases, it seems that the numerous causes of variation may counteract each other and result in slightly different but roughly comparable results ŽManzella et al., 1980., and in no way the amplitude of the fluctuations may be compared to the variability observed in diffusion tests. It is clear that temperature directly affects the lag time and growth rate of the culture, and that when addressing dynamic endpoint studies the influence of temperature must be taken into consideration. In single endpoint studies, constraints are not so critical, and the common opinion is that, due to compensatory mechanisms, MIC determination assessed according to this way should be endowed with some reliability. For instance, Smith et al. Ž1987. concluded that delaying the reading time could minimize the deviations resulting at first from different incubation temperatures. This may be right at least when the molecules are quite stable in solution and do not combine to medium components.
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Eventually, it is rather comforting to perceive that whatever the microbial organism to be studied and the temperature required for its development, dilution methods may offer convenient possibilities for MIC assessment. Indeed, it seems logical to accept quantitative values obtained at temperatures next to those which prevail in the daily environment of the fish and their pathogens. As in many in vitro biological systems, it will just be necessary to remember that data collected under the form of MICs only represent a convenient quantitative representation of the bacterial susceptibility to drugs, which in no way is intended to entirely tally with what occurs in animal organism. References Barnes, A.C., Lewin, C.S., Hastings, T.S., Amyes, S.G.B., 1990a. Cross resistance between oxytetracycline and oxolinic acid in Aeromonas salmonicida associated with alterations in outer membrane proteins. FEMS Microbiol. Lett. 72, 337–340. Barnes, A.C., Lewin, C.S., Hastings, T.S., Amyes, S.G.B., 1990b. In vitro activities of 4-quinolones against the fish pathogen Aeromonas salmonicida. Antimicrob. Agents Chemother. 34, 1819–1820. Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45, 493–496. Bejar, V., Calvo, C., Ramos Corvenzana, A., 1986. In vitro susceptibility of Yersinia kristensenii strains to b-lactam antibiotics. Ann. Inst. PasteurrMicrobiol. 137A, 169–177. Brown, D.J., Brown, L., 1991. Evaluation of the E-test, a novel method of quantifying antimicrobial activity. J. Antimicrob. Chemother. 27, 183–190. Colorni, A., 1985. A practical method for determining the susceptibility of aquatic Vibrio and Aeromonas spp. to antimicrobial agents. Aquaculture 46, 263–266. Cooper, K.E., Woodman, D., 1946. The diffusion of antiseptics through agar gels, with special reference to the agar cup assay method of estimating the activity of penicillin. J. Pathol. Bacteriol. 58, 75–84. Delignette-Muller, M.L., Flandrois, J.P., 1994. An accurate diffusion method for determining bacterial sensitivity to antibiotics. J. Antimicrob. Chemother. 33, 73–81. Dette, G.A., Knothe, H., Kaula, S., 1987. Modifying effects of pH and temperature on Ž14 C.erythromycin uptake into Staphylococcus aureus —relation to antimicrobial activity. Zentralbl. Bakteriol. Hyg., A 265, 393–403. Dunnick, J.D., O’Leary, W.M., 1970. Correlation of bacterial lipid composition with antibiotic resistance. J. Bacteriol. 101, 892–900. Ericsson, H.M., Sherris, J.C., 1971. Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol. Microbiol. Scand., B, Suppl. No. 217, 1–90. Garduno, ˜ R.A., Phipps, B.M., Kay, W.W., 1994. Physiological consequences of the S-layer of Aeromonas salmonicida in relation to growth, temperature, and outer membrane permeation. Can. J. Chemother. 40, 622–629. Gilbert, P., Collier, P.J., Brown, M.R.W., 1990. Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob. Agents Chemother. 34, 1865–1868. Guerin-Faublee, ´ ´ V., Rosso, L., Vigneulle, M., Flandrois, J.-P., 1995. The effect of incubation temperature and sodium chloride concentration on the growth kinetics of Vibrio anguillarum and Vibrio anguillarum-related organisms. J. Appl. Bacteriol. 78, 621–629. Guerin-Faublee, ´ ´ V., Delignette-Muller, M.L., Vigneulle, M., Flandrois, J.-P., 1996. Application of a modified disc diffusion technique to antimicrobial susceptibility testing of Vibrio anguillarum and Aeromonas salmonicida clinical isolates. Vet. Microbiol. 51, 137–149. Hahnel, G.B., Gould, R.W., 1982. Effects of temperature on biochemical reactions and drug resistance of virulent and avirulent Aeromonas salmonicida. J. Fish Dis. 5, 329–337. Mackowiak, P.A., Marling-Cason, M., Cohen, R.L., 1982. Effects of temperature on antimicrobial susceptibility of bacteria. J. Infect. Dis. 145, 550–553.
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Manzella, J.P., Roberts, N.J.J., Robertson, R.G., Hoder, G.B., 1980. Temperature elevation, bacterial growth and antibiotic activity. J. Antimicrob. Chemother. 6, 795–798. Martinsen, B., Oppegaard, H., Wichstrøm, R., Myhr, E., 1992. Temperature-dependent in vitro antimicrobial activity of four 4-quinolones and oxytetracycline against bacteria pathogenic to fish. Antimicrob. Agents Chemother. 36, 1738–1743. Michel, C., Bassalert, J.-F., 1982. Influence de la temperature sur les resultats de l’antibiogramme pratique´ par ´ ´ la methode de diffusion en ichtyopathologie. Ann. Rech. Vet. 13, 245–250. ´ Michel, C., Suprin, L., De Kinkelin, P., 1984. A microdilution method for drug sensitivity testing of fish associated bacteria at 228C. J. Appl. Bacteriol. 57, 15–21. Papapetropoulou, M., Rodopoulou, G., Giannoulaki, E., Stergiopoulos, P., 1994. Effect of temperature on antimicrobial susceptibilities of Pseudomonas species isolated from drinking water. J. Chemother. 6, 404–407. Parte, A.C., Smith, J.T., 1996. Influence of temperature on mutational resistance to 4-quinolones. Arzneim.Forsch. 46 ŽI., 429–432. Peteranderl, R., Shotts, E.B.J., Wiegel, J., 1990. Stability of antibiotics under growth conditions for thermophilic anaerobes. Appl. Environ. Microbiol. 56, 1981–1983. Ray, A., Newton, V., 1991. Use of high-performance liquid chromatography to monitor stability of tetracycline and chlortetracycline in susceptibility determinations. Antimicrob. Agents Chemother. 35, 1264– 1266. Reisinger, E., Wendelin, I., Gasser, R., Halwachs, G., Wilders-Truschnig, M., Krejs, G., 1996. Antibiotics and increased temperature against Borrelia burgdorferi in vitro. Scand. J. Infect. Dis. 28, 155–157. Smith, J.A., Henry, D.A., Bourgault, A.-M., Bryan, L., Harding, G.J., Hoban, D.J., Horsman, G.B., Marrie, T., Turgeon, P., 1987. Comparison of agar disk diffusion, microdilution broth, and agar dilution for testing antimicrobial susceptibility of coagulase-negative staphylococcci. J. Clin. Microbiol. 25, 1741–1746. Traub, W.H., Leonhard, B., 1995. Heat stability of the antimicrobial activity of sixty-two antibacterial agents. J. Antimicrob. Chemother. 35, 149–154. Walsh, T.R., Stunt, R.A., Nabi, J.A., MacGowan, A.P., Bennett, P.M., 1997. Distribution and expression of b-lactamase genes among Aeromonas spp. J. Antimicrob. Chemother. 40, 171–178. Wheat, P.F., Winstanley, T.G., Spencer, R.C., 1985. Effect of temperature on antimicrobial susceptibility of Pseudomonas maltophilia. J. Clin. Pathol. 38, 1055–1058. Wood, S.C., McCashion, R.N., Lynch, W.H., 1986. Multiple low-level antibiotic resistance in Aeromonas salmonicida. Antimicrob. Agents Chemother. 29, 992–996.