Journal of Antimicrobial Chemotherapy (1992) 29, 121-127
Maintenance requirements of Escherichia coli ATCC 25922 in the presence of sub-inhibitory concentrations of various antibiotics
'Laboratoire de Biomttrie, CNRS URA 243, Universiti Claude Bernard, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbarme; bLaboratoire de bactiriologie, CNRS URA 243, Faculti de Midecine Lyon sud, 69288 Lyon Cedex 02, France Escherichia coli ATCC 25922 was grown in minimal medium M63, with glucose 0-1 gL as limiting energy substrate, in the presence of sub-inhibitory concentrations of netilmicdn, habekacin, tobramycin, dibekacin, amikacin, kanamycin, amoxycillin, ampidllin, cephalothin, cefoxitin and nalidixic acid. Maintenance requirements were determined with a simple relationship derived from batch growth curves. The apparent relationship between maintenance requirements and antibiotic concentration is an exponential increase, log m(c) = log ma+k-c, where m is maintenance, c antibiotics concentration, m0 maintenance without antibiotics, and k a constant Values for k were found to be in the range 0-5-2-0 mg~'.L.
Introduction
In presence of an antibiotic, a microbial population can grow in accordance with three mutually exclusive patterns: (i) growth observed is the same or enhanced as compared with the control without antibiotic; (ii) no growth is observed; (iii) inhibited growth, intermediary between these two extremes, is observed. Patterns (i) and (ii) are easily summarized: the antibiotic concentration is respectively less and greater than the MIC. In pattern (iii) growth curves may be so complex they cannot be rehably summarized by such a simple yes/no answer. The utilization of the maximum specific growth rate is not easy because the exponential growth model does not always hold (Comby et al., 1988, 1989). However, maintenance requirements of the population c"an be determined when growth is limited by a single energy substrate. This offers a simple way to determine antibiotic activity in a research setting. Maintenance requirements may be defined (Pirt, 1965) as the amount of energy substrate consumed per unit biomass per unit time to maintain a microbial population. In his pioneering work, Monod (1941) indicated that the energy of maintenance of Escherichia coli during growth in normal culture conditions is virtually zero. This result has been confirmed as maintenance requirements have been found to be negligible (about 7/ig glucose/mg biomass/h) by various authors (McGrew & Mallette, 1962; Mallette, 1963; Wallace & Holms, 1986). When maintenance requirements are negligible, all the substrate consumed is devoted to growth so that, in a batch experiment, a constant portion of substrate is consumed per unit of biomass formed. Final biomass is thus proportional to the initial substrate 121 0305-7453/92/020121 +07 $02.00/0
© 1992 The Britiih Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
J. R. Lobrv*, G. Carrot* and J. P. Flandrois*
122
J. R. Lobry et aL
concentration, i.e.:
•r
where m(c) is maintenance requirements in presence of a concentration c of antibiotics, to the beginning of the experiment, and tm the time at which the biomass x{c) reaches its final value in presence of antibiotics. In this expression, the integral term represents the area under the growth curve from to to tm. Combining the two previous equations gives the expression:
*(c)-dt ^
Jlo
This expression describes the maintenance requirements in the presence of a subinhibitory concentration of antibiotics. All the terms can be easily obtained from batch growth data, but this implies the time-sampling interval must be small enough for a good estimation of the integral term. Methods The reference strain E. coli ATCC 25922 was stored at -196°C and subcultured on Sheep Blood Columbia agar (bioMerieux, Charbpnnieres les Bains, France) before each experiment. The inocula (3x10* cfu/ml) were standardized by nephelometry (ATB 1550, bioMerieux). Growth, as indicated by increases in the optical density (670 nm) of the culture was measured with an MS-2 system (Abbott Laboratories, Dallas Texas, USA), incubated at 36°C using 1 ml of culture (pH = 7-2). The synthetic medium M63 (Herzenberg, 1959) was supplemented with glucose (0-1 g/L) as the sole carbon and energy source and also with thiamine (40/ig/L). For ATCC 25922, in these experimental conditions, the linearity of response has been confirmed up to an absorbance reading of 0-8 UA (units of absorbance), always
Maintenance requirements of Escherichia coli ATCC 25922 in the presence of sub-inhibitory concentrations of various antibiotics
'Laboratoire de Biomttrie, CNRS URA 243, Universiti Claude Bernard, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbarme; bLaboratoire de bactiriologie, CNRS URA 243, Faculti de Midecine Lyon sud, 69288 Lyon Cedex 02, France Escherichia coli ATCC 25922 was grown in minimal medium M63, with glucose 0-1 gL as limiting energy substrate, in the presence of sub-inhibitory concentrations of netilmicdn, habekacin, tobramycin, dibekacin, amikacin, kanamycin, amoxycillin, ampidllin, cephalothin, cefoxitin and nalidixic acid. Maintenance requirements were determined with a simple relationship derived from batch growth curves. The apparent relationship between maintenance requirements and antibiotic concentration is an exponential increase, log m(c) = log ma+k-c, where m is maintenance, c antibiotics concentration, m0 maintenance without antibiotics, and k a constant Values for k were found to be in the range 0-5-2-0 mg~'.L.
Introduction
In presence of an antibiotic, a microbial population can grow in accordance with three mutually exclusive patterns: (i) growth observed is the same or enhanced as compared with the control without antibiotic; (ii) no growth is observed; (iii) inhibited growth, intermediary between these two extremes, is observed. Patterns (i) and (ii) are easily summarized: the antibiotic concentration is respectively less and greater than the MIC. In pattern (iii) growth curves may be so complex they cannot be rehably summarized by such a simple yes/no answer. The utilization of the maximum specific growth rate is not easy because the exponential growth model does not always hold (Comby et al., 1988, 1989). However, maintenance requirements of the population c"an be determined when growth is limited by a single energy substrate. This offers a simple way to determine antibiotic activity in a research setting. Maintenance requirements may be defined (Pirt, 1965) as the amount of energy substrate consumed per unit biomass per unit time to maintain a microbial population. In his pioneering work, Monod (1941) indicated that the energy of maintenance of Escherichia coli during growth in normal culture conditions is virtually zero. This result has been confirmed as maintenance requirements have been found to be negligible (about 7/ig glucose/mg biomass/h) by various authors (McGrew & Mallette, 1962; Mallette, 1963; Wallace & Holms, 1986). When maintenance requirements are negligible, all the substrate consumed is devoted to growth so that, in a batch experiment, a constant portion of substrate is consumed per unit of biomass formed. Final biomass is thus proportional to the initial substrate 121 0305-7453/92/020121 +07 $02.00/0
© 1992 The Britiih Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
J. R. Lobrv*, G. Carrot* and J. P. Flandrois*
122
J. R. Lobry et aL
concentration, i.e.:
•r
where m(c) is maintenance requirements in presence of a concentration c of antibiotics, to the beginning of the experiment, and tm the time at which the biomass x{c) reaches its final value in presence of antibiotics. In this expression, the integral term represents the area under the growth curve from to to tm. Combining the two previous equations gives the expression:
*(c)-dt ^
Jlo
This expression describes the maintenance requirements in the presence of a subinhibitory concentration of antibiotics. All the terms can be easily obtained from batch growth data, but this implies the time-sampling interval must be small enough for a good estimation of the integral term. Methods The reference strain E. coli ATCC 25922 was stored at -196°C and subcultured on Sheep Blood Columbia agar (bioMerieux, Charbpnnieres les Bains, France) before each experiment. The inocula (3x10* cfu/ml) were standardized by nephelometry (ATB 1550, bioMerieux). Growth, as indicated by increases in the optical density (670 nm) of the culture was measured with an MS-2 system (Abbott Laboratories, Dallas Texas, USA), incubated at 36°C using 1 ml of culture (pH = 7-2). The synthetic medium M63 (Herzenberg, 1959) was supplemented with glucose (0-1 g/L) as the sole carbon and energy source and also with thiamine (40/ig/L). For ATCC 25922, in these experimental conditions, the linearity of response has been confirmed up to an absorbance reading of 0-8 UA (units of absorbance), always
