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1 Institute for Experimental Treatment of Cystic Fibrosis, Scientific Institute H. S. Raffaele, Milano, Italy
2 Servicio de Microbiologìa Hospital Son Dureta, Palma de Mallorca, Spain
3 Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy
4 Klinische Forschergruppe, OE 6710, Medizinische Hochschule Hannover, Hannover, Germany
5 Ospedale Maggiore Policlinico, CF Clinic, Milano, Italy
6 Institute of Medical Microbiology and Hygiene, Universitätsklinikum Tübingen, Tübingen, Germany
Correspondence
Alessandra Bragonzi
bragonzi.alessandra{at}hsr.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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mutS were subjected to competition in vitro and in the agar-beads mouse model of chronic airway infection. When tested in competition assay in vitro, the wild-type outcompeted four clinical hypermutable strains and the PAO1mutS strain. In vivo, all of the hypermutable strains were less efficient at establishing lung infection than their wild-type clones. These results suggest that P. aeruginosa hypermutation is associated with a biological cost, reducing the potential for colonization of new environments and therefore strain transmissibility.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ratjen & Döring, 2003) contribute to the selection of multiple P. aeruginosa phenotypes by genetic variation (Luzar et al., 1985; Deretic et al., 1995; Barth & Pitt, 1996; Oliver et al., 2000). Two mechanisms are known to accelerate mutation and/or recombination rates in bacterial populations: stress-inducible wild-type genes, usually part of the SOS regulon, and genes whose functional loss increases the rate of genetic variability (hypermutable or mutator strains). The bacterial SOS response is switched on only under strong selective pressure, and as soon as growth conditions are restored, either by a genetic adaptation or by a favourable environmental change, the mutator and hyper-recombination activities are repressed by the LexA repressor (Friedberg et al., 1995; Radman et al., 2000). Hypermutable strains are those with an increased random spontaneous mutation rate, due to defect(s) in genes involved in the DNA-repair or error-avoidance systems (Miller, 1996). Of these, the methyl-directed mismatch-repair (MMR) system (MutS, MutL, MutH or UvrD) has been found to be the most frequently affected in naturally hypermutable bacterial populations (LeClerc et al., 1996; Oliver et al., 2002). P. aeruginosa (Oliver et al., 2000; Ciofu et al., 2005), Staphylococcus aureus (Prunier et al., 2003), Haemophilus influenzae (Roman et al., 2004; Watson et al., 2004) and, recently, Burkholderia cepacia complex (Burns, 2005) hypermutable phenotypes have been observed repeatedly in a high proportion of isolates from patients with CF, in contrast to what is observed in acute processes (Gutiérrez et al., 2004; Oliver et al., 2000), suggesting that this mechanism may play a crucial role in the pathogenesis of chronic lung infection. The acquisition of a stable mutator phenotype may confer a selective advantage for bacteria, particularly in stressful and/or fluctuating environments, because this may increase the probability of generating adaptive variants (Chao & Cox, 1983; Taddei et al., 1997). However, as demonstrated extensively in Escherichia coli, mutator bacteria accumulate numerous mutations in their genomes that are neutral or adaptive in a given environment, but are often deleterious in and affect the ability of the mutator bacteria to adapt to other environments, suggesting that transmissibility may be impaired (Funchain et al., 2000; Giraud et al., 2001). Furthermore, hypermutable strains are favoured by selection only when the advantage of beneficial mutations is greater than the cost of being a mutator, due to the overproduction of lethal and deleterious mutations (Taddei et al., 1997).
The in vivo consequences of hypermutation in terms of virulence have been investigated in a few micro-organisms in addition to E. coli. The inactivation of the MMR system in the intracellular pathogen Listeria monocytogenes was found to determine attenuation of virulence in a mouse model of infection (Merino et al., 2002). On the other hand, whilst mutS inactivation in the facultative intracellular pathogen Salmonella typhimurium had no observable effect on bacterial virulence (Campoy et al., 2000), mutS recD double mutants showed slower disease progression compared with the isogenic wild-type strain (Zahrt et al., 1999) when administered intraperitoneally in BALB/c mice. In addition, the mutator S. typhimurium LT2 strain showed enhanced adaptability, measured as significantly increased fitness after several passages in a mouse model of infection compared with wild-type (Nilsson et al., 2004). Nevertheless, the consequent niche specialization was associated with an important biological cost, determined by the loss of fitness in secondary environments, as seen by reduced bacterial metabolic capacity (Nilsson et al., 2004).
Whilst it is clear that hypermutation may be beneficial for P. aeruginosa populations, favouring the selection of particular adaptive mutations, as shown for antimicrobial resistance (Oliver et al., 2004; Macia et al., 2005), it remains unknown whether the mutator phenotype produced by the MMR deficiency determines, either directly or through the increased accumulation of mutations, a biological cost or benefit for the colonization of secondary environments and therefore for transmissibility in P. aeruginosa. To tackle this problem, we performed in vitro and in vivo competition experiments by using pairs of wild-type/hypermutable, clonally related P. aeruginosa strains isolated from patients with CF and the isogenic laboratory strains PAO1/PAOmutS. We present evidence that increased mutagenesis in P. aeruginosa is associated with an important biological cost, reducing the potential for colonization of new environments and therefore strain transmissibility.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Additional data of P. aeruginosa strains were published previously (Bragonzi et al., 2006). The clinical P. aeruginosa strains PAO1 (Stover et al., 2000) and PAO1mutS (Oliver et al., 2004) and complemented strains used in this study are listed in Table 1.
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Strain genotyping was performed by PFGE and analysis of single-nucleotide polymorphisms (SNPs) in ten loci as described previously (Bragonzi et al., 2006).
Mutation-frequency measurement.
A single P. aeruginosa colony was grown in 5 ml TSB overnight at 37 °C. Thereafter, aliquots from serial dilutions were plated on TSB–agar plates, 100–200 µl undiluted inoculum was plated in the presence of rifampicin (300 µg ml–1) and colonies were counted after 36 h at 37 °C. The mutation frequencies on rifampicin were determined relative to the total count of viable organisms plated according to previously established criteria (Oliver et al., 2000).
Complementation with the MMR genes.
Plasmid pUCPMS (Oliver et al., 2004) harbouring the wild-type mutS gene from the PAO1 strain was electroporated into the PAO1, PAO1mutS, BST44, BT1, NN84 and SG2 strains. Transformants for pUCPMS were selected on TSB–agar plates containing gentamicin (PAO1mutS+ and PAO1mutS mutS+, 50 µg ml–1; BST44mutS+ and NN84mutS+, 100 µg ml–1; BT1mutS+, 1000 µg ml–1; SG2mutS+, 250 µg ml–1). Plasmids pJMML and pJMMU, harbouring the mutL and uvrD genes, respectively (Oliver et al., 2002), were transferred by conjugation from E. coli XL1Blue to the RP74, NN83 and MF2 strains. Transconjugants for pJMML (RP74mutL+ and NN83mutL+) and pJMMU (MF2uvrD+) were selected on PIA plates containing kanamycin at 4000 µg ml–1. As control, the empty plasmid pUCP24 (Oliver et al., 2004) was electroporated into the PAO1 strain and selected on PIA plates containing gentamicin (50 µg ml–1) (PAO1Ø). Plasmid DNA extraction of transformed colonies confirmed the presence of plasmids, and mutation frequencies were measured.
Sequencing of the MMR genes.
Genomic DNA of P. aeruginosa strains was extracted by using a Qiagen DNA isolation kit. The mutS, mutL and uvrD genes of the strains were screened for variations from the PAO1 sequence (Stover et al., 2000) by sequencing of PCR-amplified genomic DNA. The following primers (MWG-Biotech) for mutS, mutL and uvrD were used: mutS-1F, 5'-GTTCCGAACGGACCCGACA-3', and mutS-1R, 5'-GCGATCGAAGTCCCATGGC-3'; mutS-2F, 5'-CCGAGCTGCTGATTCCAGAC-3', and mutS-2R, 5'-CAGGTCCATCAGGAATTGCCC-3'; mutS-3F, 5'-ACCTGCAGAACGCCATGA-3', and mutS-3R, 5'-GCGTGCAGCCGGAACGAAGC-3'; mutS-4F, 5'-TCCGAACATGGGCGGTAAATC-3', and mutS-4R, 5'-TCTAGTTCTCTCCTCAGGCGG-3'; mutL-1F, 5'-ACAGCCTGTCCAGCGACAA-3', and mutL-1R, 5'-GCGCCCGTTCACATAGAAG-3'; mutL-2F, 5'-CACAACGGCAAGACCATCTT-3', and mutL-2R, 5'-ATGTAGATGCCCTTGAGCTGC-3'; mutL-3F, 5'-TGGCGCCTACAAGGCCTACT-3', and mutL-3R, 5'-CCATGAGAAAGATGGCGGGA-3'; uvrD-1F, 5'-AGAGCATTTTTCCGCAGCCA-3', and uvrD-1R, 5'-AATCCGCAGCCAGGCGTATT-3'; uvrD-2F, 5'-TATCCTGGTGGACGAGTTCCA-3', and uvrD-2R, 5'-TCTTCCTCGCTGTTCTCGAA-3'; uvrD-3F, 5'-ATCTTTCGGCGAAGGTCATGG-3', and uvrD-3R, 5'-AGGATGCCAGCATGTCGTCCT-3'.
Growth rates.
P. aeruginosa strains were grown overnight in TSB and each culture was standardized to an OD600 of 0.05. Bacterial growth was monitored at 37 °C for 24 h. Samples were taken every 1 h and the OD600 was determined. The readings were plotted on semi-log paper and the growth rate was determined from the slope of the straight line drawn through the points during exponential growth. The doubling time of each lineage tested was compared with those of clonal wild-type, hypermutable and complemented P. aeruginosa strains.
Antibiotic-susceptibility testing.
MICs were determined for amikacin, ceftazidime, levofloxacin, meropenem, colistin, ciprofloxacin, piperacillin/tazobactam, ticarcillin/clavulanic acid and tobramycin. A 0.5 McFarland standard of P. aeruginosa strains was plated on Müller–Hinton agar plates (Difco) and MICs were determined by using Etest strips (AB Biodisk) after 36 h incubation at 37 °C following standard procedures. The Clinical Laboratory Standards Institute document M100-S15 (CLSI, 2005) was used as a qualitative report of susceptible or resistant phenotype. In addition, for P. aeruginosa strains used in competition experiments, the antibiotic-resistance profile was defined exactly by the broth microdilution method and on TSB–agar plates containing different antibiotic dilutions, as detailed below.
In vitro competition experiments.
Competition experiments between wild-type and hypermutable clonal P. aeruginosa strains were performed in vitro. Strains were grown overnight in TSB, each culture was diluted to a starting OD600 of 0.025 and cultured for an additional 24 h into fresh medium in the presence of competitor. The starting ratio of hypermutable : wild-type was 1 : 1 and the ratio of the mixed inoculum after 24 h was determined on selective TSB–agar plates, taking advantage of different antibiotic-resistance patterns identified within the pairs. Therefore, susceptible strains BST2, BT2, RP73, MF1 and NN83mutL+ and resistant strains BST44 and BST44muS+ (amikacin, 10 µg ml–1), BT1 and BT1muS+ (amikacin, 30 µg ml–1), RP74 and RP74mutL+ (ciprofloxacin, 1 µg ml–1), MF2 and MF2uvrD+ (ticarcillin/clavulanic acid, 10 µg ml–1), NN2 (amikacin, 3 µg ml–1) and NN83 (amikacin, 30 µg ml–1) were tested in competition. For PAO1mutS, resistance to kanamycin (250 µg ml–1), for PAO1Ø and PAO1mutS+, resistance to gentamicin (50 µg ml–1), and for PAO1mutSmutS+, double resistance to kanamycin (250 µg ml–1) and gentamicin (50 µg ml–1) were used.
Mouse model.
The agar-beads P. aeruginosa mouse model was used (Cash et al., 1979). A starting amount of 5x109 hypermutable/wild-type bacteria, mixed at a 1 : 1 ratio, was used for inclusion in the agar beads prepared according to a method described previously (Bragonzi et al., 2005). C57Bl/6 male mice (20–22 g; Charles River Laboratories) were infected with 2x106 c.f.u. P. aeruginosa as described previously (Bragonzi et al., 2005). Fourteen days after infection, murine lungs were excised, homogenized and plated onto TSB–agar plates in the presence or absence of antibiotics.
Statistical analysis.
Data were analysed for statistical significance by using Student's t-test. A P value of <0.05 was considered significant. The SAS statistical package was used for statistical analysis.
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RESULTS |
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. The collection was screened for hypermutability by evaluating the mutation rate to rifampicin (Oliver et al., 2000) and seven hypermutable P. aeruginosa strains (18 %) were identified. Non-hypermutable clinical P. aeruginosa strains had a range of mutation frequencies between 1.6x10–9 and 8.7x10–8, whereas those of the hypermutable strains were between 1.2x10–6 and 3.7x10–5. As controls, mutation frequencies of 1.5x10–8 and 1.6x10–6 were measured for the wild-type laboratory strain PAO1 and the PAO1mutS isogenic variant, respectively. The hypermutable strains possessed widely different levels of mutability, ranging between 1682- and 111-fold higher than their wild-type clonal strains (BST44/BST2, 1682-fold; RP74/RP73, 880-fold; NN83/NN2, 853-fold; BT1/BT2, 131-fold; MF2/MF1, 111-fold; and PAO1/PAO1mutS, 107-fold).
By using PFGE and SNPs in ten loci, the seven hypermutable P. aeruginosa strains were assigned to five different genotypes (Table 1), of which strains SG2, NN2 and NN83 belong to the abundant clone C and strains BT1, BT2 and NN84 to clone PA14 (Dinesh et al., 2003; Bragonzi et al., 2006; Lee et al., 2006). Six of 10 patients (60 %) with CF carried at least one hypermutable strain isolated at different stages of infection. Two hypermutable strains (BT1 and MF2: 0 years of colonization) were collected at the onset of chronic colonization and five later, i.e. after colonization (RP74, NN83, NN84, BST44 and SG2: 2.1–17.5 years of colonization) (Bragonzi et al., 2006).
Characterization of clinical P. aeruginosa hypermutable strains
The genetic basis for hypermutability was investigated by complementation of the clinical hypermutable P. aeruginosa strains with mutS, mutL and uvrD. Three of these strains (BT1, BST44 and NN84) complemented the mutS defect, reducing their mutation frequency to that of a wild-type strain when harbouring plasmid pUCPMS (BT1mutS+, BST44mutS+, NN84mutS+); two strains (RP74 and NN83) complemented the mutL defect when transformed with pJMML (RP74mutL+ and NN83mutL+), and one strain, MF2, complemented the uvrD defect when transformed with pJMMU (MF2uvrD+) (Table 1). Strain SG2 partially reduced its mutation frequency only when complemented with plasmid pUCPMS (SG2mutS+).
Sequence analysis of mutS revealed that the BST44 and NN84 strains carried a G-to-A transition at nt 543 and a C-to-T transition at 2299, whilst the BT1 strain carried an A insertion at nt 1271, resulting in a premature stop codon. Strain SG2 carried a G-to-T transition at nt 559 and an A-to-C transition at nt 859, resulting in two amino acid changes (Table 1). When the mutS sequence of the hypermutable strains was compared with those of the wild-type, clonally related strains BST2, BT2 and NN2, none of these stop mutations or amino acid changes were found, suggesting that both were implicated directly in the mutator phenotype. In the case of strains SG2 and NN84, their mutS sequences were compared with those of clonally related strains NN2 and BT2 (Bragonzi et al., 2006), isolated from different patients with CF (data not shown). In mutL, strain RP74 carried a deletion of a G at position 1369, resulting in a frameshift from aa 457, whilst strain NN83 carried one G-to-T transition at nt 1117, resulting in a stop codon (Table 1). These mutations were not found in the wild-type clonally related strains RP73 or NN2. However, the C-to-T transition at nt 1139 of strain RP74 was also detected in the clonally related strain RP73. In uvrD, strain MF2 carried a GT-to-AC transition at nt 1985 and an A-to-G transition at nt 1997, resulting in two amino acid changes that were also detected in strain MF1 (Table 1). Whether other mutations in the regulatory regions of uvrD were implicated in the mutator phenotype, which is complementable by uvrD, needs to be explored further.
Competition between wild-type and hypermutable P. aeruginosa strains in vitro
Competition experiments between clonal pairs of wild-type and hypermutable strains were carried out to investigate whether mutations in the MMR genes are associated with a general benefit or cost for the bacterial population. Therefore, we prepared co-cultures, each containing a mixture of the wild-type and hypermutable strain at a starting ratio of 1 : 1, and the final ratio was determined after 24 h growth. Each strain from the co-cultures was distinguished on different indicator plates, taking advantage of resistance patterns that were originally present in each lineage or carried by plasmids. The wild-type outcompeted the hypermutable strain(s) in the laboratory strain PAO1mutS/PAO1 and in four of the clinical clonal pairs tested (BT1/BT2, BST44/BST2, NN83/NN2 and RP74/RP73), indicating that the hypermutable strains are at a general disadvantage when growing in the same environment (Table 2; Fig. 2). In contrast, the MF2/MF1 pair showed an advantage for the hypermutable strain MF2 when grown in the presence of its wild-type clonal strain, MF1.
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mutS, NN83 and RP74 with a wild-type copy of the mutS and mutL genes reduced the cost of the hypermutable strains significantly (t-test: P=0.03, PAO1mutS/PAO1 versus PAO1mutSmutS+/PAO1; P=0.02, NN83/NN2 versus NN83mutL+/NN2; P=0.03, RP74/RP73 versus RP74mutL+/RP73), suggesting a direct effect of MMR inactivation on fitness reduction (Table 2; Fig. 2). The cost of the other hypermutable P. aeruginosa strains (BST44 and BT1) was not reduced significantly by complementation, indicating that these strains may have accumulated fitness-compromising mutations in genes other than those of the MMR system (P=0.17, BT1/BT2 versus BT1mutS+/BT2; P=0.18, BST44/BST2 versus BST44mutS+/BST2). Similarly, the advantage acquired by strain MF2 is also maintained after complementation with the uvrD gene (P=0.25, MF2/MF1 versus MF2uvrD+/MF1). To verify further that the plasmids themselves did not confer a selective advantage, we performed competition experiments between PAO1mutSmutS+/PAO1mutS+ and PAO1mutSmutS+/PAO1Ø, showing no statistically significant differences when compared with PAO1mutSmutS+/PAO1 (P=0.11 and 0.70, respectively) (Fig. 2).
Additionally, to find out whether the results of the competitions were affected by the strains' growth rates, fitness was also measured in single cultures. The doubling times of each lineage, including wild-type, hypermutable and complemented clonal strains, were compared with each other (Table 2). Most of the clinical hypermutable P. aeruginosa strains (BT1, BST44, NN83 and MF2) showed significantly reduced growth rates when compared with their respective clonal wild-type strains, indicating that the reduced in vitro fitness of natural mutator strains BT1, BST44 and NN83 was due at least partially to growth defects. As determined by competition experiments, defective growth was not modified in the complemented strains BT1mutS+ and BST44mutS+, again arguing in favour of the accumulation of fitness-compromising mutations in genes other than those of the MMR system for these strains. Nevertheless, as was also observed in competition experiments, a certain direct effect of MMR deficiency on growth rate was observed for some natural lineages: complementation with the wild-type MMR genes recovered the growth rate of NN83mutL+ and MF2uvrD+ strains significantly, conferring growth similar to that of clonally related strains NN2 and MF1. Furthermore, the growth rates of the lineage RP (RP73, RP74 and RP74mutL+) and the reference strains PAO1, PAO1mutS and PAO1mutSmutS+ were essentially identical, supporting the conclusion that biological cost was not linked exclusively to defective growth (Table 2).
Virulence of hypermutable P. aeruginosa strains in a murine model of chronic infection
To test whether increased mutation rates contribute to the organisms' transmissibility and colonization of secondary environments, competition experiments between wild-type and mutator bacteria were performed in the agar-beads murine model of chronic airway infection, which provides bacterial growth conditions similar to those of CF mucus (Bragonzi et al., 2005; Worlitzsch et al., 2002). C57Bl/6 mice were inoculated with 2x106 c.f.u. wild-type P. aeruginosa and clonal pairs of hypermutable strains were mixed at a 1 : 1 ratio in the agar beads. When the population sizes of hypermutable and wild-type clinical P. aeruginosa strains were measured after 2 weeks infection, the wild-type population outcompeted the mutator strains (Table 3; Fig. 3). As shown in Fig. 3, the competition index (CI) ranged between 8.12x10–5 and 0.17 (BT1/BT2, 8.12x10–5; RP74/RP73, 3.60x10–4; NN83/NN2, 0.05; MF2/MF1, 0.05; BST44/BST2, 0.17), indicating that, in general, the hypermutable strains were disadvantaged when colonizing the murine lung. The hypermutable/wild-type bacterial populations recovered after 2 weeks infection were significantly different for the BT1/BT2 (P=0.03), BST44/BST2 (P=0.04) and RP73/RP74 (P=0.003) pairs of strains when compared with the starting inocula. In addition, whilst the wild-type P. aeruginosa strains were capable of establishing chronic lung infection in all animals, the number of mice infected with the hypermutable strains was reduced (Table 3).
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mutS/PAO1, with a CI of 2.10x10–3 at 14 days after challenge. Significant reduction of PAO1mutS/PAO1 was observed when compared with the initial inoculum (P=0.019). The cost of the hypermutable PAO1mutS strain was also seen at earlier time points after infection (CI at day 1, 0.055; at day 3, 0.134). Overall, the results indicated that the hypermutable strains are at a general disadvantage and less efficient at colonizing murine airways when compared with wild-type under conditions of infection similar to those provided by CF mucus. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ciofu et al., 2005) and also, recently, with other underlying chronic respiratory diseases (Macia et al., 2005), in contrast to what is documented for acute infections (Gutiérrez et al., 2004), sustains the hypothesis that hypermutation may play a crucial role in the intense adaptation process required for long-term persistence in the lung environment. In vitro and in vivo experiments have shown that mutator populations are indeed selected during bacterial adaptations to stressful environments because they generate adaptive mutations at a higher rate than the regular population, therefore playing a role in bacterial evolution (Sniegowski et al., 1997; Taddei et al., 1997; Giraud et al., 2001).
Our results confirm previous findings showing that a high percentage of patients with CF are infected with hypermutable strains. The percentage of CF patients (60 %) carrying at least one hypermutable strain was higher than that first reported (36.7 %) by Oliver et al. (2000), but closer to the results of a more recent investigation (54.4 %) by Ciofu et al. (2005). Recent reports also showed similar percentages (57 %) in patients with non-CF chronic respiratory infections (Macia et al., 2005). Genotyping of P. aeruginosa by PFGE and multilocus SNP analysis showed that the seven hypermutable isolates belonged to five different genotypes, suggesting that there is no association between hypermutability and background genotype. Our clinical and epidemiological data documented that three P. aeruginosa hypermutable strains (BST44, NN83 and RP74) had developed in the CF lung habitat. At least two strains (NN84 and SG2) had probably been acquired from another patient. The hypermutable strain NN84 was the patient's first strain to belong to the major P. aeruginosa clone represented by the sequenced strain PA14 (Lee et al., 2006). The patient had been harbouring P. aeruginosa clone C strains (Römling et al., 1997) chronically for more than 17 years and the first PA14-related strain, NN84, was isolated at a time when the patient had prolonged contact with another patient who was chronically carrying a clone PA14 strain of identical genotype. The hypermutable clone C strain SG2 was isolated from a CF patient 25 months after the onset of colonization. This patient belonged to a family with three CF siblings, where the oldest sister had become chronically infected with clone C strains several years before and frequent cross-infection between the siblings had been documented by meticulous strain typing (Tümmler et al., 1991). As an exceptional case, we document for the first time the isolation of one P. aeruginosa mutator strain (MF2) at the onset of chronic colonization. As prior bacteriological analysis, taken at regular 8–12 week intervals, had been negative for P. aeruginosa during the previous 3 years and the patient had had no contact with other CF patients or centres of CF care, nosocomial acquisition of the hypermutable strain from a CF-related source is extremely unlikely. Hence, mutator and non-mutator phenotypes co-evolved within the patient's airways in a short period of 10 weeks or less. Clonal diversification during adaptation to the airways of CF patients has also been observed for other phenotypes, such as mucoidy (Deretic et al., 1995) and small-colony variants (Häussler et al., 1999), which are typical for P. aeruginosa infections in CF.
Gene-sequence analysis revealed high heterogeneity of mutations in the MMR genes responsible for the hypermutable phenotype in our isolates, with a high frequency of mutation in mutS followed by mutL, and rare mutations in uvrD. Although these frequencies are in agreement with previous findings, none of these stop mutations and only two amino acid substitutions have been described previously (Oliver et al., 2002; Hogardt et al., 2006), arguing against the presence of hot spots in the MMR genes, e.g. as those documented in mucA responsible for the development of the mucoid phenotype (Anthony et al., 2002; Bragonzi et al., 2006).
To find out whether the mutator phenotype produced by the MMR deficiency determines a biological cost or benefit for the colonization of secondary environments and therefore for transmissibility in P. aeruginosa, five pairs of clinical wild-type/hypermutable, clonally related strains and the isogenic laboratory strains PAO1/PAOmutS were tested in competition assays in vitro and in the agar-beads mouse model of chronic airway infection. PAOmutS was outcompeted by the wild-type strain both in vitro and in vivo, suggesting that MMR deficiency determines an important direct fitness cost for P. aeruginosa. As expected, when the wild-type mutS gene was restored in the in vitro competition experiments, the biological cost was reduced considerably, demonstrating the direct effect of the MMR deficiency on phenotype. These results are in agreement with those obtained by the screening of a collection of 7968 P. aeruginosa mutants in a rat model of chronic respiratory infection, where a transposition event into PA3620, corresponding to mutS, was shown to be attenuated in virulence (Potvin et al., 2003). Four of the five naturally hypermutable strains were also outcompeted by their respective wild-type in vitro, but in only two of them was the fitness cost reduced significantly by complementation with the wild-type gene. This finding suggests that the accumulation of mutations in secondary loci determines an important further biological cost in naturally hypermutable strains. Furthermore, as found in the in vivo competition experiments, all five naturally hypermutable strains showed reduced potential for establishing a chronic respiratory infection in the mouse model. These results show that although hypermutation may be beneficial for the fixation of adaptive mutations during long-term persistence in the lung environment of patients with CF, it does not at all determine a benefit for the subsequent colonization of a similar environment (the mouse lung), but rather the opposite. Taken together, these findings suggest that the accumulation of mutations in naturally hypermutable strains reduces their potential for transmissibility considerably. Similar results were obtained by Giraud et al. (2001) in a mouse model of intestinal colonization by MMR-deficient E. coli strains. In that work, hypermutation was found to be advantageous, as it increased bacterial adaptation to the gut environment, but after long-term colonization, the transmissibility of the hypermutable strains was reduced considerably.
Although our results show clearly that increased mutagenesis of P. aeruginosa is associated with an important biological cost, i.e. reducing the potential for the colonization of new environments when no selective pressure is applied, the picture could be completely different in the presence of strong selective pressure, such as antibiotic treatment. Indeed, previous studies on naturally hypermutable P. aeruginosa strains have shown a clear link between hypermutation and the development of antibiotic resistance (Oliver et al., 2000; Ciofu et al., 2005; Macia et al., 2005). Furthermore, a recent report showed that when mice infected with PAOmutS were treated with antibiotics, resistant mutants able to maintain a chronic infection were selected, whereas the PAO1 wild-type strain was killed rapidly (Macia et al., 2006). Whether the advantage demonstrated by the P. aeruginosa hypermutable laboratory strain PAOmutS under antibiotic treatment also applies to natural strains needs to be investigated further.
In summary, our results show that although hypermutable P. aeruginosa strains are indeed very prevalent in chronic infections, which is probably linked to their co-selection with adaptive mutations such as those conferring antibiotic resistance, the increased mutagenesis is associated with an important biological cost, reducing potential for the colonization of new environments and therefore strain transmissibility. These findings are certainly a step forward in our understanding of the epidemiology of chronic infections by hypermutable micro-organisms.
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ACKNOWLEDGEMENTS |
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Edited by: W. Bitter
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Received 18 October 2006;
revised 10 January 2007;
accepted 11 January 2007.
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