Fitness and competitive growth advantage of new gentamicin-susceptible MRSA clones spreading in French hospitals

Frédéric Laurenta,*, Hervé Lelièvreb, Marie Cornua, François Vandeneschb, Gérard Carreta, Jerome Etienneb and Jean-Pierre Flandroisa

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since 1991, new epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains characterized by the unexpected reappearance of heterogeneous phenotypic expression of resistance to methicillin and by susceptibility to gentamicin and various other antibiotics (GS-MRSA) have been reported in France. GS-MRSA strains have progressively replaced MRSA clones expressing homogeneous resistance to methicillin and resistance to gentamicin (GR-MRSA). In this study, we investigated the physiological characteristics of these new clones. In particular, we evaluated and compared the maximal growth rate and the deduced generation times (related to fitness of strains) of the major French epidemic MRSA clones. The population studied consisted of 79 isolates including (i) GR-MRSA that comprised six different types on the basis of PFGE; (ii) GS-MRSA the majority of which clustered into two PFGE types, A1 (usually resistant to erythromycin) and B (usually susceptible to erythromycin); (iii) methicillin-susceptible S. aureus (MSSA). GS-MRSA-A1 and MSSA strains were shown to have a significant fitness benefit (about 20%) with shorter generation times ({theta} = 23.7 ± 0.1 and 22.9 ± 0.05 min, respectively) than GR-MRSA and GS-MRSA-B strains ({theta} = 30.3 ± 0.2 and 32.5 ± 0.5 min, respectively). These data suggest that a link exists between genetic patterns, resistance profiles and physiological properties. In vitro competitive experiments indicated that GS-MRSA- A1 strains were able to rapidly outgrow GR-MRSA strains. The growth advantage observed should be taken into account in understanding the spread of some new clones of MRSA.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemic methicillin-resistant Staphylococcus aureus (MRSA) that predominated in Western European countries until 1990 were resistant to multiple antibiotics and expressed a homogeneous resistance towards methicillin.1 In 1992, a new phenotype of MRSA arose in France, characterized by heterogeneous expression of resistance to methicillin and susceptibility to various antibiotics including gentamicin, tetracycline, minocycline, lincomycin, pristinamycin, co-trimoxazole, rifampicin and fusidic acid.13 During the last 7 years, the incidence of isolation of strains of this new phenotype has increased steadily throughout France, often replacing the endogenous classical MRSA, whilst the overall prevalence of methicillin resistance among S. aureus isolates remained stable, or varied only slightly.2 A similar outcome has been reported recently in other European countries.4

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

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 Mueller–Hinton (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 Na2HPO4•2H2O, 1 mM MgSO4 7H2O, 1 mM CaCl2•2H2O, 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 Luria–Bertani (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 = X0•e 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 (h–1). The outcome of instantaneous growth rate was plotted against time to estimate the maximal growth rate of each strain. The generation time ({theta}) was calculated according to the formula {theta} = 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 {approx} 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PFGE patterns versus antibiotic resistance profiles

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 GR–MRSA isolates were resistant to erythromycin, lincomycin and pefloxacin (25/29, 86%) (Table IGo). 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 IGo).


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Table I. Resistance profiles and generation times in BHI broth of the S. aureus strains studied
 
Generation times versus PFGE patterns

The generation times of the GR-MRSA, GS-MRSA and MSSA isolates in BHI broth were compared (Table IGo, Figure 1Go). 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 1Go). 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 [{theta} = 23.7 ± 0.1 min (mean ± 2 S.E.M.)] while GS-MRSA isolates with pulsotype B (GS-MRSA-B) had the longest, {theta} = 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 ({theta} = 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 ({theta} = 30.3 ± 0.4 min) and GS-MRSA-B isolates were long and were not different (P > 0.5) (Table IGo).



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Figure 1. Histogram of the generation times of strains grown in BHI broth. GR-MRSA, methicillin-resistant S. aureus with homogeneous methicillin resistance, {blacksquare}; GS-MRSA-A1, methicillin-resistant S. aureus with heterogeneous methicillin resistance and pulsotype A1, GS-MRSA-B, methicillin-resistant S. aureus with heterogeneous methicillin resistance and pulsotype B, MSSA, methicillin-susceptible S. aureus, .

 
The same experiments were performed in MH, LB and TS broth with three randomly selected isolates of each of the GR-MRSA, GS-MRSA-A1, GS-MRSA-B and MSSA groups. GS-MRSA-A1 and GS-MRSA-B isolates showed non-significantly different generation times in MH broth; however, analysis of variance considering the results obtained with the four media, revealed that each group can be individualized on the basis of its generation times (P < 0.001).

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 IIGo. While GS-MRSA-A1 and GR-MRSA isolates were inoculated initially in almost equal numbers (µratio GS-MRSA-A1/GR-MRSA = 1.7, {sigma} = 0.7), the GS-MRSA-A1 isolates represented 88–97% of the population (µratio GS-MRSA-A1/GR-MRSA = 18.9; {sigma} = 11.4), after a 8 h incubation period (Table IIGo). 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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Table II. Ratio of gentamicin-susceptible and -resistant MRSA in competitive culture in BHI broth
 
(ii) Growth kinetics.
Growth kinetics of the competitive cultures performed with two randomly selected GR-MRSA and GS-MRSA-A1 isolates inoculated initially in about equal numbers revealed that the GS-MRSA-A1 isolate exhibited faster and more sustained growth (Figure 2Go). In the stationary phase, the GS-MRSA-A1 strain comprised 96% of the total population. Each isolate had identical growth curves in mixed and pure cultures (data not shown), establishing that the growth rate of each was not affected by the presence of the other. These results were in agreement with those deduced from the individual growth rate parameters of each strain in pure culture. The fitness benefit of the GS-MRSA-A1 strain should therefore be considered as an intrinsic character of the strain.



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Figure 2. Outcome of the number of GS-MRSA-A1 and GR-MRSA cfu/mL in mixed culture. {triangleup}, GR-MRSA, methicillin-resistant S. aureus with homogeneous methicillin resistance; {circ}, GS-MRSA-A1, methicillin-resistant S. aureus with heterogeneous methicillin resistance and pulsotype A1.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The differences in generation times observed in the present study provide a rational explanation for the increase and the current predominance of GS-MRSA strains in French hospitals, by indicating their potential to outgrow other MRSA strains and gradually replace them. In France,the new GS-MRSA clones represented 3% of the MRSA strains in 1992 and 58% in 1998 (and more than 80% in our institution in 2000; F. Laurent, personal data), irrespective of the overall incidence of MRSA.13 Moreover, one would predict that the GS-MRSA-B strains, which are also erythromycin susceptible and do not present a fitness benefit over the GR-MRSA, should no longer persist in hospitals. This hypothesis is in agreement with statistical data concerning erythromycin susceptibility among GS-MRSA strains in our institution: the low incidence of erythromycin-susceptible GS-MRSA (20% of 1210 GS-MRSA isolates in 1998) probably reflects the low prevalence of the GS-MRSA-B clone for the benefit of the GS-MRSA-A1 clone (mostly resistant to erythromycin and lincomycin). According to Aubry-Damon1 the recent emergence and rapid spread of GS-MRSA in France resulted from a long-term decrease in the consumption of gentamicin over a period of 5 years or more. Several elements indicated that such an event cannot be considered alone. Although a marked decrease in the consumption of gentamicin was reported in the Henri Mondor Hospital (located near Paris) between 1983 and 1987,1 data documenting changes in the consumption of aminoglycosides have not been reported in all French hospitals. Furthermore, changes in aminoglycoside consumption alone cannot explain the increase in susceptibility to other antibiotics (e.g. lincomycin, rifampicin, minocycline and fusidic acid) and the reappearance of heterogeneous resistance to methicillin observed with the new MRSA phenotypes.2 Finally, the shift of antibiotic selective pressure cannot explain the replacement of a resistant subpopulation by a susceptible one.17 Only positive selective advantage(s) of GS-MRSA strains over GR-MRSA strains can be involved in the current MRSA subpopulation dynamics. In the Darwinian sense of the term, these selective advantages allow the expression of differences in fitness in a given environment, resulting in the differential proliferation of some organisms. Fitness of bacteria, by allowing individuals with greater growth abilities to predominate, can be considered as one of the determinants of the spread of bacteria. Our data revealed that GS-MRSA-A1 have a significant fitness benefit (about 20%) over GR-MRSA and GS-MRSA-B strains. In vitro competition experiments using mixed cultures confirmed that the GS-MRSA-A1 strains rapidly outgrew the GR-MRSA strains (Figure 2Go). Such a model was recently illustrated by Wang et al.,18 who demonstrated that among Helicobacter pylori clinical isolates, an A-to-G point mutation within 23S rRNA conferring resistance to clarithromycin also conferred a competitive growth advantage over other kinds of mutants. They concluded that their results could explain the number of A-to-G mutants among clarithromycin-resistant clinical isolates. In the same way, Billington et al.19 showed that the variation in frequency of mutations to rifampicin resistance in Mycobacterium tuberculosis is a function of their Darwinian fitness. Our results suggest that a close relationship exists between the antibiotic resistance profiles, the PFGE patterns and the growth abilities among S. aureus clinical isolates. This is the first description of a linkage between molecular epidemiology and physiological properties among MRSA.

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.


    Acknowledgments
 
The following colleagues actively participated in isolate and data collection for our study: H. Chardon (Aix en Provence), S. Blanc (Annecy), V. Blanc (Antibes), G. Couetdic (Besançon), P. Maroye (Bordeaux), C. Jurand (Boulogne), C. Grasmick (Cahors), L. Bret (Clermont-Ferrand), A. Pechinot (Dijon), K. Amal (Kremlin-Bicètre), E. L. Dehecq (Lille), M. C. Ploy (Limoges), M. Morillon (Marseilles), C. Lion (Nancy), A. Gouby (Nîmes), V. Jarlier (Paris), P. Y. Donnio (Rennes), A. VernetGarnier (Reims), A. Carricajo-Druetta (Saint Etienne), F. Saheb (Suresnes), Y. Piemont (Strasbourg), H. Monteil (Strasbourg), J. Lemozy (Toulouse), P. Lanotte (Tours), B. Pangon (Versailles), E. Deliere (Villers-le Bel). We gratefully acknowledge Dr S. Tigaud from Croix-Rousse Hospital (Lyon, France) for his statistical data concerning antimicrobial susceptibility of MRSA strains in our institution. We are grateful to C. Courtier, G. Fardel, C. Gardon and C. Pichat for technical assistance.


    Notes
 
* Corresponding author. Tel: +33-4-78-86-31-67; Fax: +33-4-78-86-31-49; E-mail: laurent{at}lyon-sud.univ-lyon1.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 12 July 2000; returned 4 September 2000; revised 19 October 2000; accepted 14 November 2000