a Laboratoire de Bactériologie, UMR 5558, Faculté de Médecine Lyon-Sud, BP 12, 69921 Oullins; b Laboratoire de Bactériologie, EA 1655, Faculté Médecine Laennec, 69372 Lyon Cedex 08, France
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Abstract |
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Introduction |
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During the last decade, efforts were directed towards increasing the understanding of the epidemiology and evolution of MRSA strains, using molecular approaches. In this study, we moved to the analysis of physiological characteristics (defined as metabolic functions related to biochemical activities) as opposed to genetic analysis in order to compare the new gentamicin-susceptible French MRSA clones with the previous French gentamicin-resistant MRSA clones. Fitness of bacteria is defined as the ability to adjust metabolism to suit environmental conditions, in order to survive and grow, and is a major physiological determinant. The measure of the maximal growth rate in batch culture offers a good model for evaluating fitness and the deduced generation time has been considered as the optimal marker to compare fitness of strains.58 This study compared the fitness of 67 French MRSA strains belonging to the new and old phenotypes and 12 methicillin-susceptible S. aureus (MSSA) isolates used as controls.
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Materials and methods |
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Sixty-seven MRSA isolates were collected from 24 hospitals in France between 1996 and 1998. Each participating hospital sent one to three MRSA isolates exhibiting susceptibility to gentamicin, resistance to tobramycin and heterogeneous resistance to methicillin (GS-MRSA) or resistance to gentamicin and tobramycin and homogeneous resistance to methicillin (GR-MRSA). Thirty-eight GS-MRSA and 29 GR-MRSA isolates were collected. Twelve unrelated MSSA strains isolated in Lyon-Sud Hospital (Lyon, France) and Louis Pradel Hospital (Lyon, France) were included as control strains. All isolates were stored in liquid nitrogen in brain heart infusion (BHI) broth (bioMérieux, Marcy-l'Etoile, France) containing 10% glycerol. All the strains were identified as S. aureus by their ability to coagulate citrated rabbit plasma (bioMérieux) and produce clumping factor (Staphyslide Test, bioMérieux).
Antimicrobial susceptibility testing
The antimicrobial susceptibilities were tested by the disc diffusion method on MuellerHinton (MH) agar (bioMérieux) according to NCCLS recommendations9 with the following antibiotics: erythromycin, gentamicin, kanamycin, lincomycin, pefloxacin and tobramycin. Methicillin resistance was tested using MH agar containing oxacillin 6 mg/L according to NCCLS guidelines.9 Detection of the mecA gene by PCR was performed as described previously.10 Heterogeneous resistance to methicillin was differentiated from homogeneous resistance by incubation of the plates with 5 µg oxacillin discs at 30 and 37°C. Strains showing a clear zone of inhibition around the oxacillin disc at 37°C and growing in the near vicinity of the disc at 30°C, were considered to have heterogeneous phenotypic expression of resistance to methicillin. Strains showing no detectable inhibition of growth around the disc at 30 and 37°C were considered to have homogeneous phenotypic expression of resistance to methicillin.11
Pulsed-field gel electrophoresis
SmaI macrorestriction patterns were obtained using a contour-clamped homogeneous electric field system with a CHEF DR-II (Bio-Rad, Ivry-sur-Seine, France) apparatus as described previously.2,12 Comparisons of resolved macrorestriction patterns were made using the recommendations of Tenover et al.13 If no difference was observed between banding patterns, strains were considered identical. Strains differing in up to three fragments were considered clonally related, and were described as subtypes of a given clonal type. Strains differing by four or more fragments were considered separate types. Letters were used to denote major genotypes, and each variant subtype was indicated by a numerical suffix.
Estimation of growth rate parameters
An MS2 Research System (Abbot Laboratories, Dallas, TX, USA) was used to monitor growth continuously. This automated system can process 16 x 11 growth-chamber cartridges. Each bacterial strain was subcultured twice on Columbia sheep blood agar (bioMérieux) at 35°C for 24 h in order to obtain cells in stationary phase. Bacteria were suspended in T2 buffer14 [70 mM NaCl, 30 mM K2SO4, 10 mM KH2PO4, 20 mM Na2HPO42H2O, 1 mM MgSO4 7H2O, 1 mM CaCl22H2O, 0.001% gelatin (w/v)] using a nephelometer (ATB 1550, bioMérieux) to adjust the density to that of a no. 1 McFarland standard. Fifty microlitres of this suspension was added to 5 mL BHI broth to achieve an initial bacterial concentration of 105 cfu/mL. Other growth media [MH broth, trypticase soy (TS) broth and LuriaBertani (LB) broth] were tested and shown to give similar results. Each growth chamber was filled with 1 mL of the above bacterial suspension. The cartridges were inserted into the system and maintained for 24 h at 37°C with constant agitation. Optical density (OD) was measured at 5 min intervals, and evolution of OD was calculated from the resulting data.15 The instantaneous growth rates, estimated every 5 min on the basis of the 12 previous OD measurements (1 h period), were analysed with the non-linear regression model X = X0e µt, where t is time (h), X is the biomass at t, X0 is the biomass at t = 0 and µ is the growth rate (h1). The outcome of instantaneous growth rate was plotted against time to estimate the maximal growth rate of each strain. The generation time () was calculated according to the formula
= ln 2/µ.
For each growth curve, a non-linear regression of MS2 transmission measurements using the Mathematica software (Wolfram Research Inc., Champaign, IL, USA) allowed the estimation of maximal growth rate of each strain. The deduced generation time is an average of two different regressions of duplicate cultures. For each PFGE group of strains, mean and standard error of mean (S.E.M.) of generation time were calculated. Statistical analysis was obtained using the ANOVA procedure of the Statistical Analysis System (SAS Institute Inc., Cary, NC, USA).16 Means were compared using the Bonferroni comparison test.
Competitive cultures
Competitive cultures with GS-MRSA-A1 and GR-MRSA strains were performed by two different methods allowing end point analysis of growth or measurement of growth kinetics.
(i) End point analysis.
Three isolates belonging to each of the GS-MRSA-A1 and GR-MRSA groups were selected randomly. Each isolate was tested against each of the others for competitive growth in mixed culture (nine pairs). Bacteria were cultured on Columbia sheep blood agar at 35°C for 24 h, suspended in T2 buffer14 to obtain a no. 1 McFarland standard density, and 30 µL of each suspension were inoculated into 3 mL of BHI broth. Wells were then incubated at 37 ± 0.1°C with gentle agitation using a magnetic stirrer in Uvikon 922 with thermostatted chamber (Kontron Instruments Ltd, Watford, UK). The number of viable cells was monitored at t = 0 and at stationary phase (OD620 = 1.5, t 8 h) by dilution plating in quintuplicate on to Columbia agar (bioMérieux) with or without gentamicin (32 mg/mL). The colony count was carried out after 18 h incubation at 37°C. This experimental design allowed a comparison of the ratio GS-MRSA-A1/GR-MRSA immediately after inoculation and at the end of growth.
(ii) Study of growth kinetics.
Two representative isolates of GR-MRSA and GS-MRSA-A1 groups were selected randomly to study the kinetics of competitive growth. The isolates were grown on Columbia sheep blood agar at 35°C for 24 h and inoculated at a density of 105 cfu/L into a 250 mL Erlenmeyer flask containing 200 mL of BHI broth. Spinal needles (Becton Dickinson, Meylan, France) in the caps of the flasks allowed inoculation of the medium and sampling under sterile conditions during growth. The flasks were incubated at 37 ± 0.1°C in thermostat-controlled waterbaths and the medium was aerated using a magnetic stirrer. At each sampling time (every 30 min), the number of viable cells was determined in duplicate by plating two 0.1 mL portions of appropriate dilutions of the sample on to Columbia agar with or without gentamicin (32 mg/L). The ratio GS-MRSA-A1/GR-MRSA during growth was thus evaluated. This experiment was performed in triplicate.
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Results |
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MRSA isolates were grouped according to their PFGE types and antibiotic resistance patterns. PFGE profiles of the 29 GR-MRSA isolates were distributed into six different pulsotypes, of which pulsotype A1 was the most frequent (7/29 isolates, 24%). The majority of the GRMRSA isolates were resistant to erythromycin, lincomycin and pefloxacin (25/29, 86%) (Table I). PFGE profiles of the 38 GS-MRSA were mostly clustered into two major PFGE types, type A1 (20 isolates) and type B (14 isolates). Type A1 isolates were usually resistant to erythromycin, lincomycin and pefloxacin (18/20, 90%), in contrast to type B isolates which were most frequently resistant to pefloxacin but susceptible to erythromycin and lincomycin (12/14, 86%) (Table I
).
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The generation times of the GR-MRSA, GS-MRSA and MSSA isolates in BHI broth were compared (Table I, Figure 1
). The generation times of the GR-MRSA and MSSA isolates presented a homogeneous distribution whereas a bimodal distribution was observed for GS-MRSA isolates (Figure 1
). This bimodal distribution corresponded to the two subpopulations of GS-MRSA individualized by their PFGE patterns (A1 and B). GS-MRSA isolates with pulsotype A1 (GS-MRSA-A1) displayed the shortest generation times [
= 23.7 ± 0.1 min (mean ± 2 S.E.M.)] while GS-MRSA isolates with pulsotype B (GS-MRSA-B) had the longest,
= 32.5 ± 1.0 min). The difference between the generation times of GS-MRSA-A1 and GS-MRSA-B was highly significant (P < 0.0001). The generation times of MSSA isolates (
= 22.9 ± 0.1 min) and GS-MRSA-A1 were short and were not different (P > 0.5). Similarly the generation times of GR-MRSA isolates (
= 30.3 ± 0.4 min) and GS-MRSA-B isolates were long and were not different (P > 0.5) (Table I
).
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Competitive culture
(i) End point analysis.
The outcome of population equilibrium during competitive experiments conducted with six randomly selected isolates belonging to the GR-MRSA and GS-MRSA-A1 groups is presented in Table II. While GS-MRSA-A1 and GR-MRSA isolates were inoculated initially in almost equal numbers (µratio GS-MRSA-A1/GR-MRSA = 1.7,
= 0.7), the GS-MRSA-A1 isolates represented 8897% of the population (µratio GS-MRSA-A1/GR-MRSA = 18.9;
= 11.4), after a 8 h incubation period (Table II
). These data demonstrated that GS-MRSA-A1 strains can outgrow GR-MRSA strains in mixed culture. The ratio increase resulted from the fitness advantage of GS-MRSA-A1 strains over GR-MRSA strains.
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Discussion |
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Several studies have demonstrated that genetic modifications conferring antibiotic resistance induce a decrease in growth rate by interfering with normal physiological processes.17,20,21 The genetic basis of the fitness modifications can involve plasmid transfer,6,20,22,23 insertion sequences,24 transposons22 or punctuation mutations,5,25 all of which could be involved in the case of MRSA. Since the aac6'-aph2" gene is frequently carried by Tn4001,2 it is likely that certain GS-MRSA strains could have emerged from GR-MRSA strains by transposon excision or deletion, a phenomenon that was reproduced in vitro by long-term storage at room temperature of GR-MRSA strains.2 The fact that both GR- and GS-MRSA were of closely related PFGE types,2 would suggest a recent common ancestor for certain GS- and GR-MRSA isolates, and supports this hypothesis. Our results could reflect the capacity of some MRSA to undergo genetic adaptations to increase their ability to grow and to be able to re-invade their environment after having lost a potentially useless antibiotic resistance determinant.
Of course, in vitro results must be carefully interpreted. Some authors have demonstrated that fitness in vivo (in mice) and in vitro (in laboratory medium) imposes different constraints especially on the translation machinery.26 Nevertheless, in vitro data offer a model to study and compare bacterial fitness. Modifications of bacterial fitness are likely in clinical practice without clinicians and microbiologists being aware of them. Nevertheless, such fitness advantages are probably as important in the emergence of new clones as selection of clones with particular resistance profiles or virulence factors. They merit scientific interest because they may have significance for hospital epidemiology and infections. In any case, fitness benefit should now be considered as one of the factors involved in the epidemiology of MRSA clones.
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Acknowledgments |
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Notes |
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References |
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Received 12 July 2000; returned 4 September 2000; revised 19 October 2000; accepted 14 November 2000