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Antimicrobial Agents and Chemotherapy, January 2008, p. 337-339, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00939-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Fitness Cost and Impaired Survival in Penicillin-Resistant Streptococcus gordonii Isolates Selected in the Laboratory{triangledown} ,{dagger}

Marisa Haenni and Philippe Moreillon*

Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

Received 20 July 2007/ Returned for modification 15 August 2007/ Accepted 30 October 2007


   ABSTRACT
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 ABSTRACT
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Cycling of Streptococcus gordonii (115 times) with penicillin resulted in a MIC increase of more than 100-fold, from 0.008 to 2 µg/ml. The 2-µg/ml MIC maximum was already reached after 36 passages but resulted in impaired fitness. Although the MIC did not increase further, fitness was partially recovered during the 79 additional cycles.


   TEXT
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In Streptococcus spp., β-lactam resistance is related mainly to alterations in penicillin-binding protein (PBP) genes (6, 7, 10). Those mutations lower the affinity of the penicillin-binding enzymes for penicillin and permit bacterial growth in the presence of the antibiotic. However, while essential for survival under conditions selective for penicillin resistance, PBP mutations may affect bacterial fitness and constitute a biological disadvantage in the absence of the drug, as shown for other antibiotics (4, 14; for a review, see reference 1). Such counterselection may result in the disappearance of resistant bacteria after treatment arrest due to overgrowth by other microorganisms or susceptible revertants. Here, we assessed the fitness cost of penicillin resistance in laboratory-generated resistant S. gordonii strains by determining alterations in their growth rates and viability compared to those of the susceptible parent strain.

Penicillin-resistant mutants were obtained in a follow-up study by cycling liquid cultures of S. gordonii in increasing concentrations of penicillin (9). The evolution toward resistance was monitored over 115 cycles (Fig. 1). It was characterized by an initial stepwise increase from an MIC of 0.008 µg/ml to an MIC of 2 µg/ml during the first 36 cycles (mutant PR1_2) (Fig. 1), followed by MIC stabilization despite 79 additional cycles (mutant PR1_2evolved) (Fig. 1). Both mutants presented two mutations in PBP 2X (G545S and Q548E). Moreover, PR1_2evolved harbored two supplementary mutations in PBP 2B (T450A and V596F), without any associated MIC evolution (8).


Figure 1
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  		FIG. 1. Evolution of penicillin resistance in a culture of susceptible S. gordonii exposed to penicillin. Liquid cultures were inoculated into series of tubes containing increasing concentrations of the drug, as for MIC determination. Bacteria from the last tube showing visible growth were reinoculated into a new series of tubes, and the increase in the MIC was monitored. Numbers on the graph represent steps at which mutants were purified and stored for further characterization. Adapted from reference 9.

 
Since resistance development may be associated with fitness cost, we determined the stability, generation times (tgen), and survival rates of PR1_2 and PR1_2evolved compared to those of the parent strain. The MICs for the two mutants remained stable throughout 100 consecutive passages in drug-free medium. The tgen were calculated during the mid-logarithmic phase of growth. tgen were 48.4 min (95% confidence interval, 45.16 to 51.64 min) for the parent strain, 60.6 min (54.74 to 66.46 min) for mutant PR1_2, and 45.0 min (37.2 to 50.8 min) for PR1_2evolved, resulting in relative fitness scores (tgen for the wild type/tgen for the mutant [3]) of 1, 0.8, and 1.06, respectively (P, <0.01 for PR1_2 versus the parent strain and PR1_2evolved by Tukey's multiple-comparison test). Fitness impairment of PR1_2 could also be visualized on plates, since it formed colonies smaller than the parent and PR1_2evolved strains (see the supplemental material). Four additional strains (PR2 to PR5) were characterized (9). Two of them presented the same pattern of fitness loss and recovery.

We next determined whether the resistant mutants might also be impaired in their ability to survive during prolonged stationary phase. When the strains were grown individually, 70 h of stationary phase resulted in a viability loss of 2 log10 CFU/ml for the parent strain, >4 log10 CFU/ml for PR1_2, and 2 log10 CFU/ml for PR1_2evolved (Fig. 2). These findings indicate that the resistant PR1_2 was made more fragile than the wild type, not only in growth fitness, but also in long-term survival. Note that viability loss was not correlated with a drop in the optical density at 620 nm—which would be proof of culture lysis—suggesting that nonlytic mechanisms of cell death were involved (12).


Figure 2
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  		FIG. 2. Long-term survival of the parent S. gordonii strain and the resistant mutants PR1_2 and PR1_2evolved in individual cultures. Squares represent the susceptible wild type, triangles represent the mutant PR1_2, and diamonds represent the mutant PR1_2evolved. Bacteria were inoculated into brain heart infusion medium, and samples were taken at various times during the stationary phase and plated onto plain agar. The generation times in the logarithmic phase of growth are specified in the text. Experiments were performed strictly in parallel and repeated on ≥5 individual occasions. All yielded the same profile, with a relative variation of ≤15%.

 
In the natural milieu, susceptible and resistant bacteria may occupy similar niches and grow in complex communities. Therefore, we further tested the growth and survival fitness of PR1_2evolved when the strain was grown in coculture with its susceptible parent. Equal amounts (106 CFU/ml) of the parent and mutant strains were coinoculated into prewarmed antibiotic-free medium and incubated at 37°C. To differentiate between susceptible and resistant strains, aliquots taken for viable-cell counts were plated in parallel onto antibiotic-free plates and onto plates containing 0.5 µg of penicillin G/ml. Numerous controls (including parallel plating on selective and nonselective medium or restreaking from nonselective plates onto selective ones) showed that the method reliably differentiated between the two isolates. The total number of CFU on nonselective agar and the number of penicillin-resistant CFU on penicillin-containing plates were determined. The number of CFU of the susceptible parent strain was calculated by subtracting the number of resistant CFU from the total number of CFU. PR1_2evolved grew at the same rate as the parent strain up to the stationary phase (data not shown). However, upon 15 h in the stationary phase, it started to lose viability at a much greater rate than the parent strain (Fig. 3). Indeed, after 70 h of coculture, it had accumulated a ≥1,000-fold deficit in viable-cell counts compared to those of the parent strain. The experiment was repeated eight times and yielded similar results.


Figure 3
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  		FIG. 3. Competition experiments with the parent S. gordonii strain and the resistant mutant PR1_2evolved. Squares represent the susceptible wild type, and diamonds represent the mutant PR1_2evolved. Equal amounts of parent and mutant cultures (ca. 106 CFU/ml) were inoculated into antibiotic-free brain heart infusion medium. Samples were taken at various times during the stationary phase and plated either onto plain agar to determine the total number of CFU or onto selective agar containing 0.5 µg of penicillin/ml in order to detect penicillin-resistant mutants. Experiments were performed strictly in parallel and repeated on ≥5 individual occasions. All yielded the same profile, with a relative variation of ≤15%.

 
Explorative experiments with exhausted medium (16) were attempted to determine whether the factor responsible for the parent strain-induced eradication of the mutant might be associated with nutrient exhaustion or toxin production by the parent strain. However, PR1_2evolved was not killed more rapidly than the parent strain under these conditions. Moreover, backcross DNA transformation, using PR1_2evolved as the donor and competent PR1_2 as the recipient, could not restore fitness in PR1_2. As a control, the levels of competency of the parent and mutant strains were monitored and found to be similar. This finding indicated that, under the experimental conditions used (9, 13), the transformation frequency of the fitness determinants was lower than that of a single marker (e.g., if a single marker transforms at a frequency of 10–3, two individual markers transform at a frequency of 10–6), and thus, the determinants involved more than one mutation.

Altered fitness is often regarded as the price of resistance. Nevertheless, several examples indicate that fitness may recover during prolonged exposure to the selecting drug (2, 3, 11). This seesaw effect also occurs during penicillin resistance development in S. gordonii. However, despite growth rate recovery, the evolved PR1_2evolved strain still suffered a deficit in survival fitness when cultivated with the parent, an aspect that was not examined in previous studies.

As in other studies (1, 15), we could not unravel the fitness mutations by using simple genetic tools. Likewise, we could not explain the survival deficit of PR1_2evolved in cocultures by simple medium purification techniques. Fitness mutations are important because they may reveal new targets to eradicate resistant bacteria. They will most appropriately be identified by total genome sequencing plus comparative genomics. Similarly, it is critical to understand the phenomenon of bacterial eradication in cocultures, because interactions in complex communities are the everyday life of most microbes in their natural milieu. The results of the present study are reminiscent of a reverse example by Ender et al. (5), who observed that faster-growing resistant mutants of Staphylococcus aureus could be selected in the presence of competitors. Thus, while the seminal observation of fitness cost and recovery is experimentally simple, it relies on complex interactions involving both multiple mutations and environmental determinants.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Fundamental Microbiology, Quartier UNIL-Sorge, Biophore Building, CH-1015 Lausanne, Switzerland. Phone: 41.21.692.56.01/00. Fax: 41.21.692.56.05. E-mail: philippe.moreillon{at}unil.ch Back

{triangledown} Published ahead of print on 12 November 2007. Back

{dagger} Supplemental material for this article may be found at http://aac.asm.org/. Back


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Antimicrobial Agents and Chemotherapy, January 2008, p. 337-339, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00939-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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