1 Antibiotic Research Unit, Department of Medical Sciences, Clinical Bacteriology, Box 552, Uppsala University, S-751 22 Uppsala; 2 Swedish Institute for Infectious Disease Control, Solna, Sweden
Received 7 February 2003; returned 6 April 2003; revised 9 May 2003; accepted 18 May 2003
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Abstract |
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Objective: We have developed a human competition model to investigate the relative fitness of antibiotic-resistant and -susceptible bacteria.
Materials and methods: A non-epidemic Staphylococcus epidermidis strain was isolated from skin, and a rifampicin-resistant (RifR) clone was selected. The RifR marker was used to distinguish the inoculated strains from the resident population of coagulase-negative staphylococci. The RifR strains were further selected for resistance to ciprofloxacin (CipR) and fusidic acid (FusR). A 1:1 mix of susceptible and resistant bacteria was applied on the forearms of 12 volunteers. Competition was monitored by sampling bacteria from skin and determining their relative numbers.
Results: Resistance to ciprofloxacin due to parC mutations did not decrease the growth rate in vitro, and the CipR/CipS ratio was close to 1 during day 1 and 3 in the in vivo competition experiments. In contrast, fusidic acid resistance due to fusA mutations resulted in a decrease in the growth rate in vitro and a considerable loss of fitness in the competition. The FusR/FusS ratio diminished from 1.3 to 0.023 in 3 days.
Conclusions: These data show that human volunteers can be used as a simple and relevant model to study the biological cost of resistance.
Keywords: antibiotic resistance, biological fitness cost, human in vivo model
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Introduction |
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Coagulase-negative staphylococci (CoNS) belong to the microflora of human skin and mucous membranes, and are easily dispersed by skin scales. CoNS are considered opportunistic pathogens and, in immunocompetent adults, are mostly associated with endocarditis, osteomyelitis, surgical site and foreign body infections.9 CoNS, particularly Staphylococcus epidermidis, are one of the major causes of nosocomial infections, especially nosocomial bacteraemia. CoNS express several virulence factors including those involved in the ability to adhere to and accumulate as a biofilm on a variety of surfaces, including prosthetic devices and transcutaneous catheters.10 Prophylaxis and treatment have also become more difficult since CoNS have developed resistance to several antibiotics over the past years.11,12
In this study, antibiotic-susceptible and -resistant strains were allowed to compete on the forearms of healthy volunteers where the indigenous flora is present. A genetically marked clone of S. epidermidis was used to distinguish between the inoculated strains and the resident CoNS strains. Competitions were carried out between a susceptible parent strain and otherwise isogenic resistant strains that were resistant to ciprofloxacin or fusidic acid.
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Materials and methods |
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A coagulase-negative staphylococcus was isolated from the skin of one of the authors (IG). It was verified by negative Dnase-test (Acumedia) and identified as S. epidermidis by API-staph (bioMérieux SA, France). In order to distinguish between the inoculated strains in the competition experiments and the resident CoNS, rifampicin resistance was used as a marker. A rifampicin resistant (RifR) clone was selected by plating 108 bacterial cells from a fresh broth culture on Luria-Bertani (LB) plates supplemented with 0.8 mg/L rifampicin (Rifadin, Aventis Pharma). One fast-growing colony was chosen for further resistance selection to ciprofloxacin (Bayer AG, Wuppertal, Germany) and fusidic acid (Leo Pharma, Denmark). Thirty 1 mL LB broth cultures were inoculated with the RifR strain and inoculated for 6 h at 35°C in 5% CO2 to a cell density of approximately 1 x 108 cfu/mL. The cells were concentrated by a short centrifugation and the pellets were resuspended in 0.2 mL PBS pH 7.2. Ten cultures each were plated on LB agar containing ciprofloxacin 1.25 mg/L and fusidic acid 5 and 25 mg/L. The plates were incubated at 35°C in 5% CO2 for 2 days. The bacteria isolated from each antibiotic selection were stored at 70°C.
Generation time
To be able to select four resistant strains with different biological fitness, an initial screen for generation times was carried out using BioscreenC (Lab Systems, Finland). This is an incubator and turbidimetric reader where growth curves are monitored continuously. Ten FusR strains and nine CipR strains isolated as described above were inoculated at a density of 1 x 105 cfu/mL into the wells with 0.4 mL LB broth. The growth was followed by optical density at 540 nm for 8 h at 35°C. Based on the generation times from this initial screening, four strains were chosen for the in vivo competition experiments. A more precise generation time was determined by viable count on the four chosen strains, the parental strain and the RifR strain. The cultures were grown for 7 h in LB broth, and samples were withdrawn every hour for viable counts. The experiments were done in triplicate.
MIC determination
Minimal inhibitory concentrations for rifampicin, ciprofloxacin and fusidic acid were determined by Etest (Biodisk AB, Solna, Sweden) on Iso-sensitest agar (Oxoid Ltd., Basingstoke, Hampshire, UK) according to the manufacturers instructions.
Identification of mutations in the rpoB, grlA and fusA genes
The resistance mutations in the in vitro selected resistant mutants were identified by PCR amplification and sequencing of expected target genes. Primers used are shown in Table 1. The primers for the ß-subunit of rpoB were constructed from Staphylococcus warneri (GenBank accession number AF 325895) and fusA primers were constructed from Staphylococcus aureus (GenBank accession number AJ 237696). Primers for grlA have previously been described.13 DNA was prepared by DNeasy tissue kit (Qiagen). Five microlitres of sample was added to 45 µL PCR reaction mixture, consisting of PCR buffer (Amersham Bioscience), 0.2 mmol/L deoxynucleotide triphosphate, 1 U/50 µL Taq-polymerase (Amersham Bioscience), and 2 pmol/µL of each primer. Amplification was carried out in a thermal cycler (GeneAmp PCR system 9700, Applied Biosystems) with an initial denaturing step of 94°C for 5 min followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s. An additional extension step of 72°C for 7 min completed the PCR. For the primers topot3 and topot7, the annealing temperature was 50°C instead of 55°C, and the fusA reaction was carried out with 40 cycles. Negative controls were included and the reactions were evaluated in 1% agarose gel electrophoresis.
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Ethics
The study was approved by the Ethics Committee at the Faculty of Medicine, Uppsala University.
Competition experiments on skin
The five bacterial strains were grown overnight in LB broth and the number of bacteria was verified by viable counts. The RifR strain was mixed 1:1 with each mutant strain (RifR, CipR or RifR, FusR) and centrifuged briefly. The pellets were resuspended in 0.050 mL PBS and applied on the forearms of volunteers and air-dried. The study included 12 volunteers (24 arms) and each of the four different bacterial combinations was applied on six arms. The volunteers were instructed to engage in their normal daily activities, including washing, and the application site was not covered. A skin sampling method previously described was used to recover the bacteria.14 Preliminary experiments were done to determine whether the normal skin flora contained rifampicin-resistant bacteria that would give false results, but no rifampicin-resistant strains were isolated. Sampling from the competition experiment was done on days 1, 3 and 10. The samples were taken with a pad manufactured from 85% viscose and a 15% mixture of polyester and polyamide fibres (Kg Henry Hulthen, GmbH and Company, Hamburg, Germany). The pads measured 5 x 5 cm and were attached to a holder by Velcro thus making it possible to take the sample without touching the pad. The pads were moistened with PBS supplemented with 2% Tween 80 and 0.3% lecithin before autoclaving. Each pad was rubbed back and forth 10 times over an area of 5 x 10 cm at the site of bacterial application. The pad was processed in a Stomacher Lab-Blender 400 (Seward Laboratories, London, UK) for 1 min in a sterile plastic bag with 20 mL phosphate buffer pH 7.4 (PBS with 2% Tween 80 and 0.3% lecithin) to recover the bacteria. The extraction was cultured in two dilutions, 5 mL and 0.5 mL, on blood agar plates containing rifampicin 50 mg/L. Plates were dried and incubated overnight at 35°C in 5% CO2. Colonies were counted and replicated over to selective plates containing ciprofloxacin 1.25 mg/L or fusidic acid 25 mg/L. The replicated plates were incubated as described above and counted. The competition ratios were calculated from the number of resistant/susceptible bacteria. Samples were also cultured on blood agar at a volume of 0.5 mL to obtain the total number of recovered bacteria.
Isolation of mutants with increased fitness from a fusidic acid resistant strain
To examine whether fitness compensation would occur in the environment of the skin, strain fusR40 with resistance to fusidic acid and a considerable fitness loss was inoculated on 24 forearms of the same volunteers. The experiment was done 4 months after the competition experiments to avoid any interference from the previously inoculated bacteria. Control experiments showed that no RifR strains could be found 4 months after completion of the initial competition experiments. An overnight culture was prepared in 25 mL LB broth. The number of bacteria was determined by viable counts. The culture was aliquotted into 24 tubes of 1 mL and centrifuged briefly to concentrate the bacteria. The pellets (5 x 109 bacteria) were resuspended in 30 µL PBS and applied on the forearms of 12 volunteers. Sampling was carried out as described above on days 1, 10 and 30. The pad was processed for 1 min in a sterile plastic bag with 12 mL PBS with 2% Tween 80 and 0.3% lecithin, 10 mL were transferred to a centrifuge tube and centrifuged at 1400g for 10 min. The pellets were plated on blood agar containing rifampicin 50 mg/L. On day 1, the pellets were diluted 1:10 in PBS and 0.5 mL was plated. Plates were dried and incubated overnight at 35°C in 5% CO2. Colonies were counted and putative larger colonies were further investigated for MICs to fusidic acid and cultured on ordinary blood agar plates together with strain fusR40 for visual inspection of colony size. Previous experiments have shown that growth rate differences of >10% can easily be detected by visual inspection of colony size.7,8,15
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Results |
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The MIC values and the generation times for the different mutants are presented in Table 2. The ciprofloxacin-resistant strains (46 and 51) had similar generation times, 38 to 39 min, and were similar to the parent RifR strain. Both the fusidic acid-resistant strains, with MICs of 96 and >256 mg/L, showed a considerable decrease in fitness, with generation times of 49 and 62 min, respectively.
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A point mutation in rpoB was identified at amino acid 526 with a substitution from CAC (His) to TAC (Tyr). This corresponds to a mutation in cluster I in E. coli as previously described.16 In the CipR strains, the sequence analysis of the grlA gene (corresponding to parC in E. coli) revealed identical mutations in the two strains at codon 80, TCT to TAT which results in an exchange of Ser to Tyr.13 The gyrA gene was also sequenced in these two strains and no mutations were found. The sequence of the fusA gene in the fusidic acid-resistant strains 40 and 45 showed point mutations TTC (Phe) to TTA (Leu) and CAA (Gln) to CTA (Leu), respectively. This corresponds to the previously described amino acid substitutions, F88L and Q115L, in the fusA gene in S. aureus.8 The genotypes are presented in Table 2.
Competition experiments
The method we used for skin sampling is established for studies of colonization and spread of staphylococci in hospital infections. Our results showed a mean yield of 205 cfu per cm2 of skin, range 17694, which is in accordance with other studies.17,18 There was a large individual variation in adherence of the S. epidermidis between the volunteers. The bacterial counts on most of the volunteers on day 10 were too low to allow (<100 bacteria/5 mL) a determination of a competition ratio. Because of this, the competition ratios on day 10 were excluded, except for mutant cipR51.
The competition indexes for the two ciprofloxacin-resistant strains were similar and there was no indication of loss of fitness compared to the susceptible strain (Figure 1a). The CipR/CipS ratio for strain cipR46 was 1.0 on day 0 (at inoculation), 0.68 (0.491.29) on day 1 and 0.90 (0.413.1) on day 3. The figures represent the geometrical mean of six competitions with the range in parenthesis. The CipR/CipS ratio for strain cipR51 was 1.0 on day 0, 0.69 (0.351.15) on day 1, 1.06 (0.671.60) on day 3 and 1.62 (0.535.2) on day 10. In contrast, fusidic acid resistance conferred a clear fitness loss (Figure 1b). The fusR40 strain, with a generation time in vitro of 62 min, diminished rapidly on skin. Thus, the initial competition ratio of 1.3 on day 0 decreased to 0.16 (0.080.24) and 0.025 (0.0060.07) on day 1 and 3, respectively. The corresponding numbers for fusR45, with a generation time of 49 min, was 1.2, 0.72 (0.442.33) and 0.33 (0.260.4) on day 0, 1 and 3, respectively.
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For many types of resistance caused by chromosomal mutations, the fitness cost can be compensated by second-site mutations that restore fitness without loss of resistance.15,19,20 To examine if the cost of fusidic acid resistance could be compensated in a natural environment, we inoculated the low fitness fusR40 mutant on several volunteers. From an overnight culture, 4.6 x 109 bacteria were applied on each of 24 arms. On day 10, rifampicin-resistant bacteria were recovered from 16/24 arms with a total number of 1849 colonies. Ten putative larger colonies were further examined. Five had the same generation time as the original fusR40 strain on blood agar, and five strains had the same generation time as the RifR parent strain and were fully susceptible to fusidic acid. On day 30, 82 RifR colonies were isolated from only two arms. We examined 70 of these colonies and all were susceptible to fusidic acid and showed the same generation time as the RifR parent strain.
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Discussion |
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Any theoretical prediction of the rate and extent of reversibility will depend on a correct experimental assessment of the costs of resistance but unfortunately there are very few studies of the costs in vivo and the presumed correlation between in vitro and in vivo measurements. It is reasonable to assume that if a cost is seen in vitro under favourable conditions there will also be a cost associated with the resistance in vivo where conditions typically are more stressful. However, if no cost is seen in vitro, there might still exist in vivo conditions where a cost is incurred. Here, we describe a human in vivo competition model that is useful for obtaining relevant numbers for the costs of resistance. The bacteria competed in their normal human habitat and were exposed to natural environmental factors. It should also be noted that we used a non-epidemic S. epidermidis strain isolated from one of the authors. Clonal spread of fusidic acid-resistant S. aureus has been reported26 but in this study we have not considered any genetic changes associated with epidemic strains. It is satisfying that the fitness measured in vitro as generation time was correlated with the fitness measured on human skin. Thus, the ciprofloxacin-resistant mutants, with mutations in the parC gene, showed no fitness loss either in vitro or in vivo. Conversely, the fusidic acid-resistant mutants, with mutations in the fusA gene, showed fitness losses both in vitro and in vivo and the extent of fitness loss in vitro and in vivo was correlated. The fitness effects of these particular fusA mutations have also been examined in S. typhimurium and S. aureus.8,15 Similar to the results presented here for S. epidermidis, fitness of the S. typhimurium and S. aureus fusA mutants was decreased in both culture medium and experimental animals. Thus, for fusidic acid resistance, the costs were similar in three bacterial species using three different assay systems. However, these data should not be used as conclusive evidence that fusidic acid-resistant bacteria do not survive on human skin.
There was a large variation between the volunteers in how well S. epidermidis persisted on their arms. In general, there was a very rapid loss of bacteria from the forearms of the volunteers. Thus, 108 bacteria were inoculated on skin. After 1 day, there were only 102104 bacteria remaining on the arms and after 10 days generally less than 100 bacteria per sample was detected (range 01000 bacteria). This suggests that very little net growth occurred on the arms and that this assay mainly measures adhesion and/or survival rather than growth rate. In spite of this, the generation times in culture medium and adhesion/survival on skin appeared as correlated variables for these mutants. We suspected that the different resistance mechanisms could affect surface proteins and adherence of the bacteria to skin. However, hydrophobicity tests27 did not show any differences between the strains (not shown). Finally, we were able to isolate clones with increased fitness after 10 and 30 days of incubation on the arms. All of these fast-growing clones had reverted to wild-type in the fusA gene and become fully susceptible to fusidic acid. Similar results were previously obtained with fusidic acid-resistant Salmonella typhimurium grown in mice.15 These results indicate that fusidic acid-resistant bacteria, at least when they are growing in vivo, regain their fitness via reversion of the original resistance mutation rather than by second-site compensatory mutations.
In conclusion, this work shows the use of human volunteers to obtain relevant measurements of biological fitness, and other bacteria could also be studied in a similar manner. For example, -streptococci, E. coli, enterococci and several intestinal anaerobes would be amenable to competition studies in humans.28 However, it is necessary to genetically mark the investigated strains to be able to distinguish them from the resident microflora when the sampling is carried out. In this study, rifampicin resistance was used as a marker and it could also be used for a number of other bacterial species.
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Acknowledgements |
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Footnotes |
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References |
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