JAC Advance Access originally published online on October 8, 2002
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Journal of Antimicrobial Chemotherapy (2002) 50, 657-664
© 2002 The British Society for Antimicrobial Chemotherapy
Fitness of in vitro selected Pseudomonas aeruginosa nalB and nfxB multidrug resistant mutants
1 Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid; 2 Museo Nacional de Ciencias Naturales (CSIC), Madrid; 3 Servicio de Microbiología, Hospital Ramón y Cajal, Madrid, Spain
Received 14 May 2002; accepted 18 July 2002
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Abstract |
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Overproduction of multidrug resistance (MDR) efflux pumps is involved in the resistance to a wide range of compounds in bacteria. These determinants extrude antibiotics, but also bacterial metabolites like quorum-sensing signals. Non-regulated extrusion of bacterial metabolites might produce a metabolic burden, so that MDR-overproducing mutants could have a reduced fitness when compared with their parental strains. To test such a possibility, we have compared the behaviour of two MDR Pseudomonas aeruginosa in vitro selected mutants (nalB and nfxB) with their isogenic parental strain with respect to some properties with potential relevance for the survival in the environment and the virulence of this bacterial species. Overproduction of the MDR determinants MexABOprM (nalB mutant) and MexCDOprJ (nfxB mutant) decreased the survival in water, the production of phenazines and proteases, and the virulence (using a Caenorhabditis elegans model system) of the P. aeruginosa mutants. In contrast, the capability of forming biofilms was not impaired. The simple models tested in the present work can enable the analysis of the fitness of large numbers of antibiotic-resistant bacteria by using more realistic approaches than the in vitro competition assays currently used.
Keywords: multidrug resistance, efflux pump, Pseudomonas aeruginosa, bacterial fitness, cost of resistance
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Introduction |
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Pseudomonas aeruginosa is an increasingly relevant opportunistic pathogen,1 with a prominent role in the morbidity and mortality of cystic fibrosis patients.2 One of the most cumbersome features of P. aeruginosa resides in its characteristic low susceptibility to antibiotics.1 Intrinsic antibiotic resistance in P. aeruginosa is mainly due to the presence of several multidrug resistance (MDR) efflux pumps encoded in its genome.3,4 These pumps are present and actively working in all analysed strains of P. aeruginosa irrespective of whether or not the isolate originates from an environmental or clinical source.5 The expression of MDR pumps is strictly down-regulated, at least under laboratory growing conditions, in P. aeruginosa.6,7 However, mutants overexpressing MDR determinants are easily selected in the laboratory under antibiotic selective pressure,8,9 and these types of mutant are frequently encountered in clinical post-therapy bacterial isolates.10–12 This indicates that de-repression of MDR determinant expression has an important role in mutationally acquired antibiotic resistance during therapy.13 Among the various MDR determinants characterized in P. aeruginosa so far, epidemiological analysis has demonstrated that mutants with an increased expression of mexABOprM14 (nalB mutants) or mexCDOprJ15 (nfxB mutants) are the most frequently encountered among clinical isolates.11,12
Changes in the antibiotic susceptibility patterns of bacterial species are frequently due to the displacement of the susceptible populations by a few well adapted, resistant clones. In the case of P. aeruginosa, however, MDR-overproducing mutants are isolated ‘after’ therapy.10–12 Since these mutants are not present before antibiotic treatment, it seems that, at least for the moment, they have not displaced the wild-type strains in the environment. One plausible explanation for this potential lack of competitiveness could be that MDR overproduction reduces bacterial fitness. It is generally accepted that expression of antibiotic resistance determinants may have a physiological cost for bacteria (see Andersson & Levin16 and references therein), and in some cases it has been established that antibiotic-resistant bacteria can be counter-selected in the field.17
MDR systems differ from other antibiotic resistance determinants in that they are quite non-specific, as they are capable of extruding an ample range of antibiotics and non-antibiotic compounds, including some, like quorum-sensing signals,18,19 that are extremely relevant for bacterial physiological adaptation to changing environments.20,21 The presence of several different MDR determinants in the chromosome of all bacterial species so far analysed22 suggests that these transporters have not arisen recently in pathogens in response to antimicrobial chemotherapy, and indicates that their primary function should be something other than antibiotic resistance.22,23 Although the precise functional role of MDR determinants remains obscure in most cases, the ample range of substrates, together with their potential role in bacterial adaptation for growing under stress conditions and their strict down-regulation, supports the concept that overexpression of these determinants should have a detrimental effect on bacterial fitness.
In the present work, we analyse the behaviour of two in vitro selected nalB and nfxB mutants in comparison with their isogenic strain P. aeruginosa ML5087 with respect to some properties potentially relevant for their survival in the environment and their virulence. We have determined the capability of forming biofilms and the maintenance of these P. aeruginosa strains in environments such as water or dry surfaces, which can be important reservoirs for the dissemination of P. aeruginosa. We have also studied their virulence in an in vivo model system such as the killing of Caenorhabditis elegans,24 as well as the production of virulence determinants like proteases and phenazines that have been shown to be relevant virulence factors. With the exception of biofilm formation, the mexABOprM- and mexCDOprJ-overproducing mutants were impaired for all analysed traits when compared with the wild-type strain.
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Materials and methods |
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Bacterial strains and growth conditions
The P. aeruginosa mutants nalB strain K1112 and nfxB strain K1111, as well as their parent strain ML5087,25 were obtained from Dr K. Poole. Strain ML5087 is an auxotroph derivative obtained from the reference P. aeruginosa strain PAO1.26 Strain K1111 is a spontaneous mutant obtained from ML5087 by single-step selection on plates containing ciprofloxacin as the selective agent. Strain K1112 is a spontaneous mutant obtained from ML5087 by single-step selection on plates containing ciprofloxacin and cefoperazone. The strains had previously been confirmed as nfxB and nalB by antimicrobial susceptibility testing and by western immunoblotting of cell envelopes with antibodies specific to OprJ, OprM and MexA.25 Escherichia coli strain OP50, used as a control for the killing assays, was from the laboratory collection. The media used for culturing bacterial strains were Luria–Bertani (LB), Pseudomonas medium ACC and potato dextrose agar (PDA).27 Bacteria were grown at 37°C and with agitation unless otherwise specified.
DNA manipulations
P. aeruginosa genomic DNAs were prepared using standard protocols.28 P. aeruginosa mexR and nfxB genes were amplified by PCR using Vent DNA Polymerase (New England Biolabs). Primers used to amplify the mexR gene were mexR1 (5'-GCGCCATGGCCCATATTCAG-3') and mexR2 (5'-GGCATTCGCCAGTAAGCGG-3'); and for nfxB, nfxB1 (5'-CGATCCTTCCTATTGCACG-3') and nfxB2 (5'- GCCAAGTG -CCAGTATCG-3').15 Reaction mixtures (50 µL) contained 0.2 mM each deoxynucleotide triphosphate, 0.5 µM each primer, 2 mM MgSO4, 1 x PCR buffer [10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris–HCl pH 8.8, 2 mM MgSO4, 0.1% Triton X-100 final concentration] (New England Biolabs), 100 ng of genomic DNA and 1 U of DNA polymerase. The mixtures were treated for 4 min at 94°C followed by 35 cycles of 60 s at 94°C, 45 s at 57°C and 45 s at 72°C for mexR, and 45 s at 94°C, 60 s at 56°C and 2 min at 72°C for nfxB. Finally, in both cases, the mixtures were treated for 10 min at 72°C before finishing the reaction. PCR products were examined on 1.0% (w/v) agarose gels and purified using the Prep-A-Gene DNA Purification Kit (Bio-Rad). Purified PCR products were sequenced by the Sequencing Service of the CNB using primers mexR1, mexR2, nfxB1 and nfxB2.
Survival in water
Overnight cultures of the P. aeruginosa strains were washed in PBS buffer twice and finally resuspended in water to reach a concentration of 108 cfu/mL. Aliquots (1 mL) of these bacterial suspensions were used to inoculate 1 L bottles containing 19 mL of autoclaved sterile tap water. Incubation took place at 22°C with gentle agitation. The survival rate in water was evaluated by removing 0.1 mL from each bottle at different times, and determining the cfu/mL by plating sequential dilutions of the bacterial suspension onto LB plates. The experiment was repeated four times in duplicate.
Maintenance on dry surfaces
Overnight cultures of the bacterial strains were pelleted by centrifugation, washed with water three times and concentrated 10-fold (final concentration of ~5 x 1010 cfu/mL). Aliquots (10 µL) of the concentrated bacterial suspensions were seeded onto P24 flat-bottom multiwell plates, and left to dry at room temperature. Independent wells were sampled at fixed times, by suspending the dried bacteria in 0.1 mL of a PBS solution containing Triton X-100 [0.25% (v/v)]. The number of bacteria present in each sample was estimated as described above. The experiments were performed in triplicate.
Biofilm formation
Biofilm formation was quantified as previously described.29 Overnight cultures of the bacterial strains were diluted by 1/100 in fresh LB broth (final concentration of ~5 x 107 cfu/mL), and 0.1 mL of the bacterial suspensions was poured into Falcon 3911 MicroTest III silicone flexible assay plates. The plates were incubated for 24 h at 37°C without agitation. Bacteria were briefly stained with Crystal Violet, and rinsed thoroughly and repeatedly with water. The biofilm-forming bacteria were detached with ethanol containing Triton X-100 (0.25%), and the absorbance was determined at 560 nm. Eight different wells were tested in each experiment.
Nematode-killing assays
C. elegans wild-type Bristol strain N2 (provided by the Caenorhabditis Genetics Center, University of Minnesota, Minneapolis, MN, USA) was used in the study. The strain was maintained under standard culture conditions at 20°C using E. coli OP50 as a food source. Once the C. elegans stocks were obtained, the bacterial-mediated killing of the nematode was studied as described with some modifications.24 Briefly, a fresh culture of the bacterial strain to be tested was spread onto a 55 mm diameter plate containing 5 mL of PDA. After spreading the bacterial culture, plates were incubated at 37°C for 24 h, and the plates were kept at room temperature for 8 h. Bacterial plates were then seeded with five adult hermaphrodite nematodes and incubated at 24 h. The number of worms was scored immediately after plating, 4 h later and every 24 h during the 8 days. Each independent assay consisted of five replicates.
Assay of pyocyanin and pyoverdin production
Production of these phenazines was tested mainly as described previously.30 Bacterial strains were grown at 37°C in Pseudomonas medium ACC broth27 for 40 h. At this time, bacteria were pelleted by centrifugation, and the amount of the blue pigment pyocianin was evaluated by measuring the absorbance of the supernatants at 690 nm. The amount of pyoverdin was measured by fluorescence by exciting the supernatants at 400 nm and measuring the emission at 460 nm. Each experiment was performed in triplicate.
Quantification of proteases
Bacteria were grown overnight in LB broth at 37°C, pelleted by centrifugation and the proteolytic activity was tested in the culture supernatants. Caseinase activity was tested using azocasein31 as the substrate. A 0.1 mL aliquot of culture supernatant was mixed with 1 mL of a suspension of azocasein (3 mg/mL) in 0.1 M Tris–HCl/0.5 mM CaCl2 (pH 7.4). The mixture was incubated at 37°C under agitation for 30 min. A 0.1 mL aliquot of trichloroacetic acid (50%) was then added to each reaction tube, and 30 min later the tubes were spun for 10 min in a microfuge at 15 700g. The absorbance of the supernatants at 400 nm indicates the caseinase activity. Each experiment was performed in triplicate. Elastolytic activity was determined using elastin–Congo Red as the substrate.32 A 0.1 mL aliquot of culture supernatant was mixed with 1 mL of a suspension of elastin–Congo Red (10 mg/mL) in 0.1 M Tris–HCl (pH 7.4). The mixture was incubated at 37°C under agitation for 6 h. Then, the tubes were spun for 10 min in a microfuge at 1500g. The absorbance of the supernatants at 495 nm indicates the elastolytic activity. Each experiment was performed in triplicate.
Statistical analysis
Mean and standard deviations were obtained using the Microsoft Excel program. Sets of data were normalized and statistically compared by analysis of variance (ANOVA). Comparison of the means was performed by Bonferroni’s multiple comparisons test. These analyses were performed using the PRISM statistical package. In all cases the level of significance was P < 0.05.
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Results |
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Molecular characterization of nalB and nfxB mutations
Overexpression of P. aeruginosa MDR determinants is usually due to mutations in the genes encoding the proteins that regulate the expression of the structural operons.6 PCR amplification and further sequencing of the genes coding the synthesis of the regulatory proteins of mexABOprM and mexCDOprJ (mexR and nfxB, respectively) have shown that the nalB mutation is associated with the change of A by C at nucleotide 247, which renders the amino acid change T130P in MexR. The nfxB mutation consists of a deletion from positions 136 to 146. This deletion produces a frameshift, so that the resulting protein contains just the first 45 amino acids of NfxB.
Effect of nalB and nfxB mutations on the survival of P. aeruginosa in water
P. aeruginosa are nosocomial pathogens that can be present in water reservoirs.33 Their survival in this environment is relevant to their potential transmissibility. Thus, we analysed the survival of nalB and nfxB mutants in sterile tap water at 22°C. As shown in Figure 1, the survival of the nalB mutant in water was impaired as compared with the wild-type strain P. aeruginosa ML5087. The effect was significant at all analysed times. In contrast, the differences between ML5087 and K1111 were only significant at days 5 and 13, which indicates that a minor effect, if any, was found in the case of the nfxB mutant.
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Effect of nalB and nfxB mutations on the survival of P. aeruginosa on a dry surface
Resistance to dryness is also an important factor for the dissemination of bacteria, because it allows them to be maintained on surfaces.34,35 We evaluated the effect of nalB and nfxB mutations on the survival of P. aeruginosa cells on a dry surface. The results are shown in Figure 2. A strong reduction in viability was observed for the three strains, the loss of viability being slightly higher for the nalB and nfxB mutants. The differences were significant at all analysed times.
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Effect of nalB and nfxB mutations on biofilm formation
P. aeruginosa grows forming biofilms when attached to surfaces, and the style of biofilm growth is relevant to the virulence outcome36,37 as well as to the phenotypic antibiotic resistance of this bacterial species.38 The capability of forming biofilms of the wild-type strain ML5087 and the MDR nalB and nfxB mutants was compared. As shown in Figure 3, MDR mutations do not reduce the capability of P. aeruginosa for forming biofilms. In fact, the amount of biofilm produced by the nalB mutant was significantly higher when compared with ML5087.
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Effect of nalB and nfxB mutations on the killing capability of P. aeruginosa against C. elegans
It has been shown that P. aeruginosa is capable of killing the nematode C. elegans24 by using, at least in part, the same virulence determinants previously determined to be relevant for nosocomial infections. Thus, we have analysed the killing capabilities of wild-type and MDR-overproducing P. aeruginosa mutants over C. elegans. Our results clearly show that both nalB and nfxB mutants are unable to kill C. elegans under conditions in which 80% of the population is killed by the wild-type strain P. aeruginosa ML5087 (Figure 4). Significant differences were observed. It is noteworthy that previous analyses of P. aeruginosa mutants unable to interact with C. elegans have demonstrated that MexA is required for the killing of the nematode.39 Our results indicate that not only the lack of MexA, but also overproduction of MexABOprM, strongly reduced the killing capability of P. aeruginosa in the C. elegans model. This indicates that the presence of this MDR determinant is required, and its expression needs to be finely tuned for triggering P. aeruginosa virulence, at least in this non-mammalian model system.
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MDR mutations and expression of quorum-sensing regulated virulence determinants
It has been described previously that in vitro obtained nalB and nfxC (overproducing MexEFOprN) mutants are severely affected in their quorum-sensing response.18,19,40 Herein, we have analysed whether this situation might also arise in the case of nfxB mutants. For this purpose, we have analysed the level of production of proteases and phenazines, the expression of which is regulated by quorum sensing, in the wild-type strain and in the MDR mutants. As shown in Table 1, both the nalB and nfxB mutants are impaired in their quorum-sensing response. In all cases, significant differences were observed. These data, together with the previously published effect of MexEFOprN overproduction on P. aeruginosa quorum sensing,19 indicate that overexpression of MDR determinants might have a general effect on quorum sensing in P. aeruginosa.
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Two major signalling branches are responsible for the quorum-sensing response in P. aeruginosa. One is the rhl quorum-sensing system, which triggers the expression of exoproteases and phenazines, the other is the las quorum-sensing system, which triggers the formation of biofilms as well as the expression of multiple other genes.21 The data presented in our work indicate that overexpression of MDR determinants strongly affects rhl-controlled gene expression in P. aeruginosa, whereas the las-controlled formation of biofilms is not reduced, but even increased. The effect of MDR mutations on biofilm production has not been analysed previously. Our data indicate that the effect of these mutations on quorum sensing is specific to the rhl quorum-sensing pathway.
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Discussion |
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We have shown that in vitro obtained P. aeruginosa MDR mutants (mainly the nalB mutant) are impaired in terms of virulence as well as survival in water and on dried surfaces. However, they produce more biofilm than their isogenic susceptible parental strain. The capability of forming biofilms is relevant for the colonization of catheters and for the persistence in chronic infections (such as cystic fibrosis). Thus, the in vitro P. aeruginosa MDR mutants analysed in the present work could be well adapted for these types of infection. In fact, it has been described recently that P. aeruginosa isolates from cystic fibrosis patients accumulate pyoverdine-negative mutations, yet maintain their capability for forming biofilms.41 Also, data from our laboratory (G. Cobas, J. Ayala & J. L. Martínez, unpublished results) and others42 indicate a correlation between MDR, reduced expression of pyoverdine and increased production of biofilms in clinical P. aeruginosa isolates.
As previously stated by others,43 in vitro studies on the fitness of antibiotic-resistant bacteria must be carefully interpreted. In this regard, the testing of well defined isogenic mutants such as those used in our work is required before analysing clinical non-isogenic isolates. It should be noted that the large majority of the studies published so far on the effect of antibiotic resistance on bacterial fitness are just based on in vitro competition tests between antibiotic-resistant and -susceptible bacteria, usually growing in rich media. Only a handful of papers have been published analysing the effect of antibiotic resistance on bacterial fitness using in vivo models.44,45 We think that inferring the in vivo fitness of bacteria using in vitro competition tests is unrealistic. This view is supported by the fact that the mutations which compensate for fitness defects are different in vitro than in vivo.46 Within this scope, we propose that the simple models tested in the present work can enable the analysis of the fitness of large numbers of antibiotic-resistant bacteria by using more realistic approaches than the in vitro competition assays currently used.
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Note added in proof |
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At the time of reviewing this article two other works analysing the effect of overexpression of MDR pumps have been published.47,48
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Acknowledgements |
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We thank Dr Keith Poole (Queen’s University, Kingston, Ontario, Canada) for the kind gift of P. aeruginosa strains. This research was aided in part by grants QLRT-2000-01339, BIO2001-1081, QLRT-2000-00873 and CAM 08.2/0026/1999. P.S. is a recipient of a fellowship from Ministerio de Educacion y Ciencia (MEC). J.F.L. is a recipient of a fellowship from Ministerio de Ciencia y Tecnologia (MCyT). B.R.-D. is a recipient of a fellowship from Comunidad Autonoma de Madrid (CAM).
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Footnotes |
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* Corresponding author. Tel: +34-91-5854571; Fax: +34-91-5854506; E-mail: jlmtnez{at}cnb.uam.es
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