1 Department of Pediatrics, University Hospital Innsbruck, Innsbruck; 2 Institute of Hygiene, University of Innsbruck, Innsbruck; 3 Department of Transplant Surgery, D. Swarovski Research Laboratory, University Hospital Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria
Received 14 January 2002; returned 24 May 2002; revised 8 July 2002; accepted 15 July 2002
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Abstract |
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Methods: We developed high-resolution respirometry for continuous monitoring of bacterial growth and inhibition kinetics. One hundred and ten clinical isolates from 90 paediatric patients were tested, including 48 isolates obtained from 28 patients with eradication failure. Respirometric inhibition curves were monitored at 4 mg/L penicillin G over a short 30 min period, corresponding to the drugs serum half-life.
Results: None of the clinical isolates exhibited penicillin tolerance. Latency in the respirometric response of S. pyogenes to penicillin increased significantly with decreasing strain-specific respirometric growth rate. No difference in inhibition kinetics was found in vitro for isolates from patients with or without bacteriological treatment failure.
Conclusions: In streptococcal pharyngotonsillitis, tolerance is not a relevant concept to explain bacteriological treatment failure. Definitions of tolerance should be reconsidered in the framework of growth-dependent antibiotic susceptibility.
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Introduction |
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The role of penicillin tolerance as a cause for eradication failure remains controversial. Tolerance occurs when, under defined conditions, a test substance that is usually bactericidal for the bacteria tested shows a diminished or absent bactericidal effect without loss of inhibitory action.10 Rates of penicillin tolerance reported for S. pyogenes depend on the definition of tolerance as the ratio of MBC to MIC of 327,11 or
16.5,10 Amsterdam12 questioned this rationale, since in view of the lack of reproducibility of conventional MBC methods, the definition [of tolerance] should be treated with caution.
An unequivocally tolerant isolate of S. pyogenes cannot be obtained from the ATCC, National Reference Centre for Streptococci (Institute of Medical Microbiology, Aachen, Germany) or from authors who described tolerant S. pyogenes strains.13,14 Whereas some studies on S. pyogenes pharyngotonsillitis failed to isolate any penicillin-tolerant strain,1517 Brett18 claimed penicillin tolerance in 29% (129 of 434) of clinical isolates of S. pyogenes tested in 1 year. Nevertheless, the clinical significance of tolerance has never been established.3 Whereas one study observed that penicillin-tolerant strains were more frequently isolated from patients with initial penicillin treatment failure,7 another investigation reported a lower treatment failure rate in patients with penicillin-tolerant strains.3 In a prospective study on children with clinical symptoms of S. pyogenes-associated pharyngotonsillitis, we found an eradication failure rate of 20% (30 of 149 patients).1 In the present study, we present the penicillin-inhibition kinetics in these streptococcal isolates, to elucidate the clinical significance of penicillin tolerance of S. pyogenes. One of our major concerns was the ill-defined antimicrobial activity during the long standard incubation time of 24 h for determination of MBC/MIC ratios, considering the short serum half-life of penicillin of 40 min.19 Using a new respirometric in vitro method, therefore, we analysed inhibition curves with high time resolution over periods of 30 min.
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Materials and methods |
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One hundred and ten clinical isolates of S. pyogenes were tested after collection from 90 paediatric patients with streptococcal pharyngotonsillitis. This prospective study included 48 isolates from 28 patients with eradication failure. The patients received penicillin V at a dose of 100 000 U/kg/day divided into three daily doses and given orally over a period of 10 days.1 Isolates were stored in tubes with Columbia agar (Becton Dickinson, Cockeysville, MD, USA) with 5% defibrinated sheep blood at 4°C. A penicillin-susceptible S. pyogenes (ATCC 19615; MIC = 0.03 mg/L, MBC = 0.06 mg/L) and a penicillin-tolerant Listeria monocytogenes (ATCC 19611; MIC = 0.125 mg/L, MBC > 128 mg/L) were used as reference strains. Listeria monocytogenes exerts intrinsic penicillin tolerance.20 A reportedly penicillin-tolerant streptococcal strain (L36K; MIC = 0.0156 mg/L, MBC = 0.5 mg/L) was kindly provided by Claas Schálen.14 These MIC and MBC values were determined with a macro broth dilution test.21 For respirometry, single colonies were incubated in 5 mL of culture medium 12 h before initiation of measurements. Todd-Hewitt and Columbia broth (Becton Dickinson) were used for S. pyogenes and L. monocytogenes, respectively. The NCCLS-recommended test medium for susceptibility testing of these organisms is MuellerHinton broth supplemented with 35% lysed horse blood.22 Preliminary test results yielded identical MICs for our quality control strains grown in MuellerHinton both supplemented with lysed horse blood and the culture media chosen for respirometry.
Antimicrobial agents
Screening for tolerance to penicillin G (Biochemie, Kundl, Austria) was performed at a final concentration of 4 mg/L. Since there is no NCCLS-recommended penicillin-resistant breakpoint for haemolytic streptococci (only susceptible: penicillin MIC 0.12 mg/L), the penicillin-resistant breakpoint for viridans streptococci was chosen in our study.22 Reference strains were additionally tested with clindamycin (Pharmacia & Upjohn, Puurs, Belgium; 3.3 mg/L final concentration), a drug with in vitro bactericidal action against S. pyogenes23 and with the bacteriostatic drug sulfadiazine (Sigma, St Louis, MO, USA; 120 mg/L final concentration). Sulfadiazine was dissolved in water (70°C) with 2.5 mol/L sodium hydroxide and added to Todd-Hewitt broth yielding a stock solution of 480 mg/L.
High-resolution respirometry
High-resolution respirometry was performed at 37°C in a two-chamber Oxygraph (OROBOROS Instruments, Innsbruck, Austria).2426 The 2 mL glass chambers with titanium stoppers and PEEK-coated stirrers26 were sterilized before every experiment with 3% formaldehyde for 10 min. The chambers were washed three times with distilled water and filled with culture medium saturated with air at 37°C. After closing the chamber, the stirring rate was set at 280310 rpm, and oxygen concentration was recorded every 5 s over experimental periods of 1.52 h (up to 8 h in specific cases).
Fifty microlitres of bacterial suspension was injected through a titanium cannula into the chamber, yielding an initial cell density of 105 cfu/mL. Respiration per volume is the negative time derivative of oxygen concentration, which was displayed online using the DatLab software.24,26 The increase of respiration over time (time derivative of respiration per volume) is the respiratory growth rate. Respiration reflects the total aerobic activity of the culture in the batch system, yielding an integrative measure of cell density and oxidative metabolic activity per cell. Oxygen concentration did not decline to <20% air saturation, thus preventing oxygen limitation of aerobic metabolism.24 Within 1030 min of incubation, respiration increased to 47 ± 17 pmol/s/mL (range 1590 pmol/s/mL). At this time, t0, 50 µL of antibiotic stock solution was injected. Respiration was expressed as a percentage of the rate at t0 (Figure 1). The rate of respiration at t0 did not correlate with any index of the respiratory inhibition curves (P = 0.70.9).
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Cell count
Viable counts were performed by removing samples of 25 µL from the oxygraph chambers at 30 min intervals. To prevent antibiotic carryover, these samples were diluted 1:100 in Todd-Hewitt broth before logarithmic plating of 50 µL of these dilutions on Columbia agar plates (plus 5% defibrinated sheep blood) using a Whitley automatic spiral plater (Don Whitley Scientific, West Yorkshire, UK). Colony forming units were counted after incubation at 37°C for 24 h.
Data analysis
Continuous records of oxygen concentration and respiratory rate were analysed using DatLab (OROBOROS Instruments). Statistical analysis was carried out using Pearsons correlation coefficient for continuous data. P < 0.05 was considered statistically significant. All statistical analyses were performed using the software SPSS 10.0 (SPSS Inc., Chicago, IL, USA).
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Results |
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Figure 1(b) shows respiration of S. pyogenes ATCC 19615 in comparison with L. monocytogenes ATCC 19611, an organism with intrinsic penicillin tolerance. Growth curves without antibiotics were identical in these organisms. The addition of penicillin resulted in a comparatively long latent period of 45 min in L. monocytogenes, when the baseline growth rate at the time of the addition of the drug was 68% per hour. Respiration increased up to a maximum of 150% and remained constant (at 140% for 8 h; data not shown), indicative of bacteriostasis due to penicillin tolerance. The MIC/MBC ratios of S. pyogenes ATCC 19615 and the reportedly penicillin-tolerant streptococcal strain L36K were 0.03/0.06 and 0.0156/0.5 mg/L, respectively. Their respirometric inhibition curves were indistinguishable (Figure 1; L36K data not shown).
To simulate a bacteriostatic mode of action of penicillin in S. pyogenes ATCC 19615, the temperature was lowered from 37 to 20°C, resulting in growth arrest. Bacterial growth was monitored over 3 h at 37°C, to yield a high microbial density and high respiration per volume (Figure 2). After a sudden switch to 20°C, respiration was reduced by 90% to a constant level, representing a non-proliferative state with constant cell density. Neither respiration nor cell density declined after the addition of penicillin G to these dormant bacteria. The addition of formaldehyde (1% final concentration), however, killed the bacteria and respiration ceased immediately.
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A positive correlation was obtained between the rate of inhibition after latency (measured over the time period from maximal respiration until 30 min past exposure to penicillin) and growth rate before the addition of the drug (Figure 3c; correlation coefficients 0.59 and 0.44, for treatment success and failure). In all parameters, the 48 strains obtained from 28 patients with eradication failure did not separate from the 62 strains acquired from patients with successful penicillin therapy (Figure 3).
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Discussion |
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The definition of tolerance has been criticized as arbitrary and purely dependent on methodology.11,12 MIC is measured over 1824 h at various initially added drug concentrations. The half-life of penicillin G, however, is 40 min in vivo.19 Respirometry requires a test window of merely 30 min for measurement of the inhibition kinetics and is thus superior to conventional MIC determination, since a continuous record is obtained of respiratory growth and inflection to respiratory inhibition (Figure 1). In addition, the MIC/MBC ratio does not take into account the relationship between the absolute MBC and the drug concentration clinically achieved in tissues. At a realistic penicillin concentration of 4 mg/L, killing curves were indistinguishable for the reportedly penicillin-tolerant S. pyogenes L36K (MBC 0.5 mg/L) and fully susceptible isolates. This was confirmed by respiratory inhibition kinetics of S. pyogenes L36K at a penicillin concentration of 4 mg/L, which reflected a bactericidal mode of action as seen in fully susceptible S. pyogenes ATCC 19615 and the clinical isolates. Our results with the bacteriostatic drug sulfadiazine and the bactericidal drug clindamycin provide the basis for a distinction between respiratory responses to non-growth conditions and to cell death, under conditions of identical metabolic substrate availability. Only cell death led to an immediate decline in respiration, whereas bacteriostasis caused respiration to level off at a constant rate, indicative of non-growth conditions and futile energy turnover (Figure 1b). Similarly, growth was completely arrested at 20°C. Under these conditions, a significant and constant level of respiration was maintained, and only killing by formaldehyde finally reduced the oxygen consumption rate to zero (Figure 2).
Continuous respirometric monitoring before and after application of the drug yielded information on the strain-specific growth rate, the lag time between exposure to the antibiotic and response (latency), the initial increase in respiration over this latent period and the subsequent rate of inhibition (Figure 3). High-resolution respirometry24 provides a prerequisite for the quantitative analysis of continuous changes of oxygen consumption rates (non-linear slopes of oxygen concentration over time) and online graphical display of inhibition curves.26 The identity of the simple patterns of these complex inhibition curves in isolates from patients with and without bacteriological eradication failure provides a strong argument against the clinical relevance of penicillin tolerance as previously defined in the isolates of S. pyogenes tested. Respirometric inhibition kinetics allows sensitive and meaningful distinction between tolerance and bactericidal modes of action. Independent of their origin from patients with or without eradication failure, none of the clinical isolates of S. pyogenes tested in our study was tolerant of penicillin at 4 mg/L. In agreement with our findings, various previous reports have questioned the significance of tolerance in streptococcal treatment failure.8,14,27 We propose to resolve the long-standing controversy on the implications of penicillin tolerance in bacteriological treatment failure.5,14 The dispute over definitions of tolerance should be replaced by the recognition of a continuity of quantitative relationships derived from respiratory inhibition kinetics.
The two phases of antimicrobial action observed by respirometry, i.e. minimal reduction in respiratory growth rate during latency followed by a rapid decline in oxygen consumption, may correspond to a sequence of two antimicrobial mechanisms exerted by penicillin: (i) interference with peptidoglycan synthesis, yielding a cell wall that cannot withstand osmotic forces, and (ii) activation of autolysins, which accelerates lysis by destroying the cell wall.
It is well known that ß-lactam antibiotics exert their bactericidal effects during the logarithmic growth phase, whereas apparent tolerance is observed during stationary phases caused by density effects, nutrient starvation or low temperature.28,29 When S. pyogenes is brought into a stationary growth phase, expression of penicillin binding proteins is reduced and the antimicrobial action of penicillin declines concomitantly.23 The unique finding in our study is the correlation between parameters derived from inhibition kinetics and the strain-specific growth rate. The paradigm of growth dependence of penicillin susceptibility is thus extended to the phenotypic variability of growth rate in a group of clinical isolates.
Bacteriological treatment failure in streptococcal pharyngotonsillitis may have multiple causes, including lack of compliance, re-infection and penicillin tolerance of the infecting streptococcal strain. Lack of compliance and re-infection are ruled out as the main factors for bacteriological treatment failure in our patient population.1 Our present results support the notion that tolerance is not a relevant factor to explain this clinical challenge. This suggests a physiological perspective, e.g. intracellular protection in human host cells12,30,31 or special growth conditions in affected tissue, to explain current eradication failures in S. pyogenes pharyngotonsillitis. Inhibition kinetics by high-resolution respirometry presents a new, sensitive and practical surveillance technique, well suited to meet the increasing challenge of assessing the potential emergence of microbial tolerance or resistance to antibiotics.32
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Acknowledgements |
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Footnotes |
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Corresponding author. Tel: +43-512-504-4623; Fax: +43-512-504-4625; E-mail: erich.gnaiger{at}uibk.ac.at
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References |
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