a Division of Infectious Diseases, University Hospitals of Geneva, CH-1211 Geneva 14; b Institute of Medical Microbiology, University of Zürich, Switzerland
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Abstract |
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Introduction |
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A number of in vitro studies have documented the stepwise development of resistance to either vancomycin or teicoplanin in S. aureus,6,7,9,1925 but in vivo conditions promoting emergence of glycopeptide resistance have not been described experimentally. The molecular mechanisms of glycopeptide resistance in S. aureus have not yet been elucidated, yet they are clearly different from those found in enterococcal strains.5,12 In vivo emergence of glycopeptide resistance in S. aureus was first reported during teicoplanin therapy of either severely infected patients22,26,27 or rabbits with experimental endocarditis.22,28 Such in vivo teicoplanin-resistant isolates frequently exhibit a greater increase in teicoplanin compared with vancomycin MICs. A similar observation was made after in vitro selection of resistant organisms to teicoplanin compared with vancomycin.22,23,27 Despite these glycopeptide-specific differences, teicoplanin- and vancomycin-resistant mutants of S. aureus resulting from in vivo or in vitro exposure have some biochemical and morphological changes in common, in particular significant cell wall thickening, increased penicillin-binding protein 2 (PBP2) production and an increased binding capacity for glycopeptides by peptidoglycan.6,7,9,19,2325,27,29
We reported previously that a methicillin-resistant strain of S. aureus recovered from subcutaneous implant exudates in a rat model of chronic foreign body infection included subpopulations that would grow on agar containing 10 times the teicoplanin MIC.30 The subpopulations growing on teicoplanin-supplemented agar represented >105 of the total number of organisms cultivated from tissue cage fluids, after 3 weeks of infection, in contrast to the same strain grown in vitro, which yielded a <107 teicoplanin-resistant colony. Emergence of subpopulations of S. aureus growing on teicoplanin-supplemented agar during experimental foreign body infection occurred in the absence of any prior antibiotic exposure and was not promoted further by high-dose teicoplanin therapy. Furthermore, conventional MIC testing of colonies removed from teicoplanin-supplemented agar failed to demonstrate any significant increase in teicoplanin MICs following subcultures in antibiotic-free liquid medium.
This report describes further characteristics of the emerging in vivo glycopeptide-resistant subpopulations. Conditions affecting the stability of the resistance phenotype of these subpopulations are described. Finally, a selection of subclones derived from the in vivo-selected teicoplanin-resistant subpopulations and expressing stable high levels of glycopeptide resistance is also reported.
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Materials and methods |
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The MRSA strain MRGR3,31,32 used for in vitro and animal studies, and expressing heterogeneous resistance to methicillin, was isolated in 1979 from a patient with catheter-related sepsis and selected for its virulence properties in the rat model of chronic S. aureus tissue cage infection.31 Strain MRGR3 is also resistant to penicillin, gentamicin, chloramphenicol, erythromycin, tetracycline and polymyxin, but not to fluoroquinolones. The average MIC and MBC of teicoplanin (Lepetit Research Center, Varese, Italy) for strain MRGR3 grown in cation-adjusted MuellerHinton broth (MHB; Difco Laboratories, Detroit, MI, USA) were reported previously as 1 and 2 mg/L, respectively, as determined by a macrodilution method.30 Identical values were found with vancomycin (Laboratory Lilly, Giessen, Germany).31 Standard overnight cultures in MHB of strain MRGR3 showed the absence of any glycopeptide-resistant subpopulation growing on MuellerHinton agar (MHA) containing 2 mg/L of teicoplanin or vancomycin at a limit of detection of 107.
Detection of bacterial subpopulations growing on glycopeptide-supplemented agar
Experiments involving rats were approved by the Ethics Committee of the Faculty of Medicine of the University of Geneva and by the Veterinary Office of the State of Geneva. The in vivo emergence of subpopulations of S. aureus growing on either teicoplanin- or vancomycin-supplemented agar was studied in a rat model of S. aureus chronic tissue cage infections, composed of four tissue cages subcutaneously implanted in Wistar rats as described previously.31 At 3 weeks post-implantation, tissue cage fluid was aspirated and checked for sterility, then tissue cages were inoculated with 0.1 mL of saline containing 0.22 x 106 cfu of strain MRGR3 as described previously.3133 Three weeks later, all tissue cages were punctured and quantitative cultures of 10-fold serially diluted tissue cage fluids performed on either glycopeptide-free MHA or MHA containing 10 mg/L of either teicoplanin or vancomycin. To optimize the yield of viable bacteria, tissue cage fluids were briefly (60 W, 1 min) sonicated (model 2200; Brandson Ultrasonics, Branburry, CT, USA) to disrupt the biofilm and phagocytic cells before the serial dilutions and plating. Plates were incubated for 2448 h at 37°C. The detection limit was one colony equivalent to 2 log10 cfu/mL of tissue cage fluid.
A very similar procedure was used to record the population analysis profiles of tissue cage fluid bacteria. In this case, quantitative cultures of tissue cage fluid organisms were performed on MHA containing either 0, 2, 4 or 8 mg/L of teicoplanin as described above.
Determination of MICs
MICs of teicoplanin and vancomycin for tissue cage bacterial colonies grown on glycopeptide-supplemented MHA were determined by the broth microdilution method in cation-adjusted MHB according to the standards of the National Committee for Clinical Laboratory Standards (NCCLS).34 Suspensions of one to several bacterial colonies removed from either teicoplanin- or vancomycin-supplemented agar, or of strain MRGR3 grown on glycopeptide-free agar as a control, were prepared in phosphate-buffered saline (PBS) and adjusted to a turbidity equal to McFarland 0.5 (c. 108 cfu/mL). Thereafter, 100 µL portions of 100-fold diluted bacterial suspensions, containing an average inoculum of 105 cfu as checked by routine plating, were added to 100 µL portions containing increasing concentrations (0.516 mg/L) of either teicoplanin or vancomycin in microtitre plates. MICs were read after 48 h of incubation at 37°C.
Population analysis
Suspensions of one to several bacterial colonies removed from teicoplanin-supplemented agar, or of strain MRGR3 grown on glycopeptide-free MHA, were prepared in PBS and adjusted to a turbidity equal to McFarland 0.5. One hundred microlitre portions of either 10- or 104-fold diluted bacterial suspensions were spread on MHA plates containing teicoplanin in doubling concentrations ranging from 1 to 16 mg/L or glycopeptide-free MHA, and enumerated after 48 h of incubation at 37°C.
Survival kinetic studies
Suspensions of one to a few bacterial colonies removed from teicoplanin-supplemented agar, or of strain MRGR3 grown on glycopeptide-free MHA, were prepared in PBS and adjusted to a turbidity equal to McFarland 0.5. Thereafter, 50 µL portions of each bacterial suspension were added to glass tubes containing 10 mL of MHB containing 8 mg/L of teicoplanin in a shaking waterbath at 37°C. The total number of viable organisms was determined by subculturing 50 µL of 10-fold serially diluted portions on MHA after 0, 2, 4, 6 and 24 h of incubation. For each time point, 50 µL of 10-fold serially diluted portions were spread in parallel on agar plates containing 2, 4 or 8 mg/L of teicoplanin for population analysis of surviving bacteria. Colonies were enumerated after 48 h of incubation at 35°C. The detection limit was 2 log10 cfu/mL.
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Results |
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Among 24 tissue cages that developed a significant MRSA infection at 3 weeks, none of the 10 cages with the lowest numbers of plated bacteria (<4 x 104 cfu), yielded any colony growing on MHA containing 10-fold the MIC of teicoplanin or vancomycin for strain MRGR3. All the other 14 cages in which numbers of plated bacteria exceeded 105 cfu yielded colonies on teicoplanin- or vancomycinsupplemented agar with average frequencies of 4 x 105 or 2 x 105, respectively. For eight cages with intermediate bacterial titres, the number of bacteria growing on either teicoplanin-supplemented (Figure 1a) or vancomycinsupplemented (Figure 1b
) MHA was directly correlated with the number of plated bacteria (range: 4 x 104 to 5 x 105 cfu), with average frequencies of 1.5 x 104 and 1.3 x 104 on teicoplanin- (r = 0.84, P < 0.01) and vancomycin-containing (r = 0.86, P < 0.01) MHA, respectively. Paradoxically, much lower average frequencies of bacteria growing on either teicoplanin-supplemented (8.1 x 106) or vancomycin-supplemented (3.2 x 106) MHA were recorded in six cages with the highest titres of strain MRGR3 (not shown). Thus, the average frequency of tissue cage bacteria growing on glycopeptide-supplemented MHA was not artificially increased by spreading a larger number of organisms on agar plates.
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Stability of the glycopeptide resistance phenotypes of tissue cage bacteria
Direct loop transfers of colonies from 12 different cages cultured on teicoplanin-supplemented MHA, then subcultured on to equivalent antibiotic-containing media, led to positive subcultures in all cases. Identical results were obtained with colonies grown on vancomycin-supplemented MHA. In contrast, when the stability of glycopeptide resistance was tested on subcultures of the glycopeptide-selected colonies that were first briefly suspended in saline and then plated at a concentration of c. 106 cfu, none of these subcultures was positive on either teicoplanin- or vancomycin-containing MHA. This indicated that the glycopeptide resistance phenotypes were unstable and strongly influenced by the methodology used for subcultures.
To analyse in a different way the stability of the resistance phenotypes of the 12 tissue cage bacterial subpopulations grown on glycopeptide-supplemented agar, these colonies were briefly suspended in saline and their teicoplanin or vancomycin MICs evaluated by the broth microdilution method. Compared with the parent strain MRGR3 grown on glycopeptide-free agar, the teicoplanin MIC of which was 0.5 mg/L, all subclones of teicoplanin-selected tissue cage bacteria showed four- to 16-fold increases in teicoplanin MICs, namely 2 mg/L for four subclones, 4 mg/L for five subclones and 8 mg/L for three subclones. In contrast, only three out of 12 vancomycin-selected subclones showed a minor two-fold increase in vancomycin MICs (2 mg/L), whereas nine other subclones showed MICs identical to that of the parent strain MRGR3 (1 mg/L).
These data indicated that expression of the resistance phenotype by tissue cage bacterial subpopulations selected on glycopeptide-containing agar was more frequent and more stable with teicoplanin than vancomycin.
Further characteristics of teicoplanin-selected subpopulations
Four subclones of tissue cage bacterial subpopulations grown on glycopeptide-supplemented MHA that showed the highest increase in teicoplanin MICs (48 mg/L) were tested further by population analysis profiles. Figure 2 demonstrates that all four subclones survived much better than the parental strain MRGR3 in teicoplanin concentrations ranging from 2 to 8 mg/L. Despite almost identical population analysis profiles, subclones 14-4 and 15-4 exhibited a markedly different colonial morphology. Subclone 15-4 was the only one to exhibit a uniform small colony variant morphology and was not studied further, in contrast to subclones 14-4, 16-3 and 17-2, which showed heterogeneous colonies ranging from small to essentially normal size.
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Selected colonies from subclones 14-4, 16-3 and 17-2 that were removed from teicoplanin-supplemented agar were briefly suspended in saline and evaluated for survival or killing kinetics in the presence of 8 mg/L teicoplanin in MHB. For subclones 14-4 and 17-2, which grew on agar containing 8 mg/L of teicoplanin, a complete growth arrest but no significant killing was observed for at least 6 h in the liquid medium having an equivalent glycopeptide concentration, followed by significant growth from 6 to 24 h (Figure 3a). In contrast, subclone 16-3, which was initially selected on agar containing 4 mg/L of teicoplanin, remained growth-arrested for 24 h.
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Selection of stable teicoplanin-resistant subclones
The stability of subclones 14-4 and 17-2 was tested further for a period of 3 months by weekly passages on to three different media, namely MHA supplemented with 8 mg/L teicoplanin or 2 mg/L vancomycin, or glycopeptide-free MHA. This latter medium was used to assay the stability of the resistance phenotype during repeated passages. For each passage, the saline-suspended subclones, plated at an average inoculum of 106 cfu on either glycopeptidesupplemented or plain MHA, yielded consistently positive subcultures on either growth medium. After 1, 2 and 3 months of weekly subculture on either glycopeptide-containing or plain MHA, teicoplanin and vancomycin MICs of passaged subclones were assayed by the macrodilution method and found to be consistently equivalent on glycopeptide-containing and glycopeptide-free MHA. The average teicoplanin and vancomycin MICs of the stable subclones 14-4 and 17-2 were 16 and 4 mg/L, respectively.
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Discussion |
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In view of the intensive use of vancomycin and teicoplanin for several years, the rarity with which GISA strains are isolated is surprising. Two opposing explanations might be considered. First, detection of heterogeneous resistance to glycopeptides may be viewed as essentially an in vitro phenomenon, in vivo expression of which is poorly documented and clinical relevance uncertain (see discussions in references 11 and 18), with the exception of sporadic cases of clinical isolates highly resistant to teicoplanin.22,26,27 Conversely, it has been suggested that some in vivo conditions might promote glycopeptide resistance and compromise the outcome of antimicrobial therapy. In vivo expression of glycopeptide resistance might be too unstable to be detected by laboratory antimicrobial assays, because of the reversion of glycopeptide-resistant into glycopeptide-susceptible organisms after repeated in vitro passages in glycopeptide-free growth media.15,16,18 The results recorded in the tissue cage model of S. aureus infection seem to support the latter hypothesis. First, we found in infected fluids of the subcutaneous implants the emergence of subpopulations characterized by growth on agar containing 10-fold the MICs of either teicoplanin or vancomycin for the original MRSA strain MRGR3, which under in vitro growth conditions was devoid of any glycopeptide-resistant subpopulation. In vivo emergence of subpopulations growing on teicoplanin-containing agar was repeatedly observed in several experiments performed between 1993 and 1999, and the frequency of these subpopulations was consistently >105 on MHA containing eight- to 10-fold the MIC of teicoplanin for strain MRGR3. Further characterization of the subpopulations growing on glycopeptide-containing agar was hampered by the unstable phenotypes of subclones, which reverted to a teicoplanin-susceptible state, as defined by MIC testing, after passage in antibiotic-free media, as reported previously.30
To improve the characterization of the resistance phenotypes of the tissue cage bacterial subpopulations grown on glycopeptide-supplemented MHA, we tried to avoid or minimize passages in antibiotic-free growth media that were suspected of promoting reversion of glycopeptide-resistance expression. MIC testing and population analysis profiles were performed on colonies removed directly from glycopeptide-containing agar, and suspended in saline. This procedure yielded MICs of teicoplanin that increased consistently, whereas those of vancomycin were much less affected. These data fit well with the more frequent emergence of teicoplanin- versus vancomycin-resistant isolates occurring in vivo or in in vitro conditions used to select glycopeptide-resistant organisms. We can speculate that this ex vivo phenomenon of increased teicoplanin resistance may result from some in vivo stimulation of cell wall production, with the resulting thickened cell wall affording protection.
The striking difference between plating and survival of the resistant subclones in liquid versus solid phase may reflect either differential expression of cell wall-associated genes in planktonic versus sessile bacteria, or be due to the dilution of an inducer of teicoplanin resistance in liquid medium, which would not occur on agar surfaces in the colonial mode of growth. More than 90% of cells in suspension were unable to grow, but still survived quite well for a prolonged period of time in the presence of teicoplanin, and were thus defined as glycopeptide-tolerant rather than glycopeptide-resistant. These data may indicate an alternative expression of tolerance versus resistance in liquid versus solid growth medium by subpopulations of S. aureus. Two recent reports indicate that glycopeptide tolerance is a frequent phenomenon in S. aureus, particularly amongst MRSA isolates, which might compromise glycopeptide therapy for serious staphylococcal infection.35,36 In our tissue cage model of S. aureus infection, we also found that in vivo-grown organisms exhibited a broad-spectrum tolerance to different antibiotics in several therapeutic studies.30,31,33,37,38 This in vivo-induced tolerance, which is either not expressed or rapidly disappears under in vitro conditions, was therefore referred to as phenotypic tolerance.39 In therapeutic studies, the highest phenotypic tolerance expressed by strain MRGR3 infecting tissue cages was against teicoplanin.30 Thus, expression of phenotypic tolerance and emergence of in vivo glycopeptide-resistant subpopulations may explain to some extent the poor therapeutic activity of teicoplanin recorded previously in tissue cages chronically infected with S. aureus.30
In conclusion, we have provided evidence that emergence of resistance to glycopeptides can occur in vivo in a well-defined experimental model of S. aureus infection. These data suggest that some in vivo conditions may exert a selective pressure on S. aureus, thus leading to emergence of subpopulations exhibiting reduced glycopeptide susceptibility and allowing their growth in the presence of otherwise inhibitory concentrations of glycopeptide. The conditions leading eventually to emergence of stable teicoplanin- or vancomycin-resistant subpopulations are still unknown and deserve further investigation. We hope that this experimental infection model will help to identify some of the up- and down-regulated genes either induced or constitutively expressed in vivo by glycopeptideresistant S. aureus, as reported recently under in vitro conditions.29
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Acknowledgments |
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Notes |
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References |
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Received 6 June 2000; returned 24 August 2000; revised 4 October 2000; accepted 6 November 2000