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. 2009 Sep 4;191(22):6968–6974. doi: 10.1128/JB.00748-09

The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in Pseudomonas aeruginosa ▿

Alfredo Castañeda-García 1, Alexandro Rodríguez-Rojas 1, Javier R Guelfo 1, Jesús Blázquez 1,*
PMCID: PMC2772468  PMID: 19734311

Abstract

Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10−6. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10−11). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.

Pseudomonas aeruginosa is an opportunistic, life-threatening bacterial pathogen that especially affects critically ill patients in intensive care units or those suffering from chronic respiratory diseases such as cystic fibrosis (19, 40). Its 6.3-Mb genome supports its enormous metabolic versatility and, consequently, its adaptability to almost any challenging environment. One of the consequences of this versatility is the rapid adaptation to stressful environmental conditions, including starvation, desiccation, and antibiotic treatments (14, 40). Mutants resistant to one or several antibiotics will evolve during sufficiently prolonged treatments, this being a process facilitated by the presence of hypermutable alleles (31, 32). After years of treating cystic fibrosis patients with antibiotics, P. aeruginosa became unavoidably resistant to many or all of them (5). Multidrug-resistant strains of P. aeruginosa are an important problem for the treatment of nosocomial outbreaks and cystic fibrosis patients (27, 37). Currently, the treatment of multidrug-resistant P. aeruginosa requires the combination of various antimicrobial agents. Fosfomycin (Fos) has been reported to be effective in combination with other antipseudomonal agents (6, 29, 42, 44). The proportion of Fos-resistant (Fos-R) strains in clinical isolates of P. aeruginosa currently is not well known, and even the mechanisms that support Fos resistance in P. aeruginosa are not clear. Thus, the knowledge of the molecular bases involved in the development of spontaneous Fos resistance in P. aeruginosa is of particular interest.

Fos is a unique broad-spectrum bactericidal antibiotic that is chemically unrelated to any other known antimicrobial agent used to treat urinary tract and gastrointestinal infections in humans (9, 35). It binds UDP-GlcNAc enol-pyruvyltransferase (MurA), acting as a phosphoenolpyruvate analogue and avoiding the formation of UDP-N-acetylglucosamine-3-O-enolpyruvate from UDP-N-acetylglucosamine and phosphoenolpyruvate (12, 33). Fos is taken up actively into bacterial cells via transport systems. In Escherichia coli, Fos is imported through two nutrient transport systems, the glycerol-3-phosphate (glycerol-3-P) transporter (GlpT) and glucose-6-phosphate (glucose-6-P) transporter (UhpT), to achieve its target and inhibits the initial step in cell wall synthesis (12, 17). The expression of these transport systems is induced by their substrates (glycerol-3-P and glucose-6P) and requires the presence of the cyclic AMP receptor protein (cAMP-CRP) complex (23, 30). Additionally, the high-level expression of UhpT requires the regulatory genes uhpA, uhpB, and uhpC (12, 30). Therefore, Fos-R strains isolated in E. coli contain mutations that prevent Fos transport using GlpT or UhpT (23, 30). Plasmid-encoded resistance also has been described previously (4, 41).

In this paper, we describe the screening and analysis of Fos-R clones in a P. aeruginosa PA14 ordered insertional library (18). In addition, we studied the mutations responsible for the spontaneous resistance to Fos in P. aeruginosa PA14, the effect of these mutations on the in vitro growth rate, and the uniqueness of the mutational target.

MATERIALS AND METHODS

Bacteria and media.

Table 1 shows the E. coli and P. aeruginosa strains used in this work. The P. aeruginosa mutant library PA14 glpT::MAR2xT7 and its wild-type strain PA14 were kindly provided by N. T. Liberati (18). All P. aeruginosa strains were cultured at 37°C in Luria-Bertani (LB) or M9 medium (38) containing gentamicin (15 μg/ml) or tetracycline (final concentration of 150 μg/ml for plasmid selection) when appropriate. E. coli K-12 mutant strains were obtained from the NARA institute (3) and were cultured routinely at 37°C in LB supplemented with (40 μg/ml) when appropriate.

TABLE 1.

Fos MICs for mutants of E. coli K-12 BW25113 and P. aeruginosa PA14 in LB alone or with 0.05% glucose-6-phosphatea (G6P)

Strain Fos MIC (μg/ml) for E. coli BW25113 with:
 Strain Fos MIC (μg/ml) for P. aeruginosa PA14 with:
 Identityb (%)
LB G6P LB G6P
Wild type 2 2 Wild type 8 8
glpT 32 8 glpT 1,024 1,024 74.76
uhpT 8 8 hexP1(PA14_49610) 8 8 23.04
hexP2(PA14_44190) 8 8 24.01
hexP3(PA14_38560) 8 8 26.49
uhpA 8 8
uhpB 8 8
uhpC 4 4
cyaA 16 8 cyaA 8 8 36.94
ptsI 4 4 ptsI 8 8 39.72
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a

E. coli strains are deletion mutants with a kanamycin resistance cassette. P. aeruginosa are insertional mutants harboring an MAR2xT7 gentamicin resistance transposon.

b

Identity indicates a comparison between E. coli and P. aeruginosa protein sequences encoded by homologous genes.

Screening of the P. aeruginosa PA14 insertional library.

The PA14 MAR2xT7 nonredundant insertional library (18) was screened for Fos resistance. The library contains 5,459 mutants, each in a well of 96-well plates. The library was replicated in 96-well plates containing LB plus gentamicin (15 μg/ml) and incubated overnight at 37°C. Aliquots of 5 μl from each well were spotted onto LB plates containing either 128 μg/ml Fos or no antibiotic. Plates were incubated for 24 h at 37°C and immediately submitted to screening for the resistance phenotype. Mutants showing resistance or producing a much higher number of colonies than the wild-type strain on Fos-containing plates were isolated from the frozen 96-well plates and submitted to a second round of verification. To retest candidate strains, three independent colonies from each mutant were grown in LB broth, and appropriate dilutions were plated onto LB and LB-Fos to verify resistance.

Estimation of spontaneous Fos-R mutant frequencies.

For spontaneous mutation frequency measurements of PA14, mutS and mutT mutant derivatives, and E. coli BW25113, approximately 102 cells from overnight cultures were inoculated into five tubes, each containing 1 ml of LB, and the tubes were incubated at 37°C with strong shaking for 24 h. Aliquots from successive dilutions were plated onto LB plates with Fos (final concentration, 128 μg/ml) or without any antibiotic to estimate viability. The numbers of colonies growing after 24 h of incubation were determined, and the mutation frequency was estimated as the number of Fos-R colonies divided by the number of viable cells.

Generation and analysis of spontaneous Fos-R mutants.

To test the linkage between glycerol-3-P use and Fos resistance, 20 flasks containing M9-glucose were inoculated with about 103 wild-type PA14 cells. Flasks were incubated overnight with shaking at 37°C. About 109 cells from each culture were plated on plates of M9 agar containing glycerol-3-P plus Fos (128 μg/ml) and on M9 agar plates containing glycerol, instead of glycerol-3-P, plus Fos. Appropriate dilutions from these flasks also were plated on M9 agar-glycerol-3-P and M9 agar-glycerol plates without Fos to estimate the number of viable cells.

For the isolation and characterization of Fos-R mutants, about 103 cells of PA14 were inoculated in 10 25-ml flasks containing 5 ml of LB and were incubated overnight at 37°C with shaking. One hundred microliters of each of the 10 independent overnight cultures was plated on petri dishes containing LB agar supplemented with Fos (128 μg/ml) and incubated at 37°C for 24 h. From each plate, a single colony was isolated and purified on LB agar containing Fos at the same concentration. Chromosomal DNA was isolated from each of them and the wild-type strain. The corresponding glpT genes were PCR amplified and sequenced using the oligonucleotides glpT-P14-F1 (5′-AGCGGAGCTCGCGATGTTC-3′) and glpT-P14-R1 (5′-TCAGCCGGCTTGCTGCGG-3′) as forward and reverse primers, respectively.

Construction of plasmid pBBR-glpT and complementation of Fos-R mutants.

A DNA fragment containing the glpT gene from the wild-type PA14 genomic DNA was generated by PCR using the oligonucleotides glpT-P14-F1 and glpT-P14-R1. The wild-type glpT gene was cloned directly into the vector pBBR1MCS-3 (15). For this purpose, the plasmid was digested with SmaI and converted in a T-vector as previously described (36). The resulting plasmid was named pBBR-glpT. Finally, pBBR-glpT was introduced by electroporation into the wild-type PA14, the glpT insertional mutant (PA14 glpT::MAR2xT7), and all of the isolated Fos-R mutants. Transformants were selected on LB agar supplemented with 150 μg/ml tetracycline.

Antibiotic susceptibility testing.

MICs were determined by the broth microdilution method as recommended by the CLSI (7), except that LB was used instead of Mueller-Hinton medium. To stimulate hexose phosphate transporter expression, 0.05 mM glucose-6-phosphate was added, as an inducer, to the LB broth when necessary.

Growth curves.

Growth curves of wild-type PA14 and glpT mutant strains were determined in rich medium (LB broth) and in minimal medium (M9). Strains were grown overnight at 37°C with shaking in LB or M9-glucose, respectively. These cultures were diluted 100-fold in the same medium and incubated for 3 h until they reached exponential-phase growth. Approximately 104 cells of each strain then were inoculated in quadruplicate into the wells of a 96-well microplate containing 100 μl of LB or M9 broth, using 0.1% glucose, 0.5% glucose-6-phosphate, 0.1% glycerol, or 0.5% glycerol-3-phospate as the sole carbon source. Cultures were incubated at 37°C with shaking in an Infinite M200 multiwell fluorimeter (Tecan, Switzerland). The absorbance at 595 nm was monitored every 15 min. The growth rates were calculated with values from the exponential part of each one of the growth curves (the regression coefficient was >0.95 in all cases).

Biofilms.

An abiotic solid-surface biofilm formation assay was performed in 96-well polystyrene microtiter plates after 20 h of incubation at 37°C, as described previously (36). After crystal violet staining, the absorbance was measured at 595 nm. Forty independent replicas were carried out for each strain.

RESULTS

Screening of the PA14 insertional library of mutants for Fos resistance.

The 5,459 mutants of the P. aeruginosa PA14 MAR2xT7 insertional library (18) were screened to identify genes whose inactivation conferred resistance to Fos (128 μg/ml), as described in Materials and Methods. Putative resistance mutants were retested, and those able to grow on Fos-containing LB agar plates were submitted to further analysis. Several mutants showed an increased number of colonies compared to the that of wild-type strain PA14 due to a hypermutator phenotype (32). Only two additional mutants, 30348 and 39942 (mutant identification numbers are from the PA14 collection) (18), grew clearly on this concentration of Fos. Both mutants carried insertions that interrupted the same gene, glpT (PA14_69130), and we thus conclude that glpT inactivation plays an essential role in resistance to the antibiotic Fos in P. aeruginosa.

In P. aeruginosa, the glpT gene encodes a putative glycerol-3-P permease protein that belongs to the organophosphate:phosphate antiporter (OPA) family of the major facilitator superfamily (MFS). Hydrophobicity sequence analysis revealed that the GlpT protein of P. aeruginosa contained the 12 highly conserved regions typical of all glycerol-3-P permeases, and they were arranged as putative transmembrane α-helices (named TM1 to TM12 in this study). Proteins within the OPA family identified in different bacterial species conserve significant sequence homology. In this sense, the P. aeruginosa PA14 GlpT protein shares the same sequence with the P. aeruginosa PAO1 ortholog, and it shows a high degree of homology to GlpT of E. coli K-12 (75% identity) and B. subtilis (62% identity). In E. coli, GlpT acts as a secondary active transporter for glycerol-3-P uptake that exchanges a phosphate ion for a glycerol-3-P molecule (8, 11). This transporter also provides an entrance mechanism for Fos inside cells owing to this antibiotic mimicking glycerol-3-P. Thus, the impaired function of GlpT produces Fos resistance in E. coli (12).

Effect of inactivation in P. aeruginosa of known mutational targets that confer Fos resistance in E. coli.

In E. coli, Fos can enter cells via the GlpT or UhpT transporter; thus, defects blocking either one of these transport systems decrease Fos susceptibility (11). UhpT is involved in the transport of glucose-6-P into the cell (2), and the expression of this protein is regulated by uhpA, uhpB, and uhpC (10, 26). The expression of both UhpT and GlpT is positively regulated by cAMP; thus, mutations in cyaA and ptsI that lower the cAMP levels will produce decreased Fos sensitivity in this species (1, 43). Therefore, apart from those in glpT, mutations in the genes uhpA, uhpB, uhpC, uhpT, cyaA, or ptsI confer Fos resistance in E. coli (26).

To study the effect of the inactivation of these genes on Fos resistance in P. aeruginosa, we first performed a BLAST search to identify their homologues in the PA14 genomic sequence database (http://www.pseudomonas.com). We detected proteins that maintain a high degree of identity to E. coli CyaA and PtsI (Table 2), but we failed to identify clear Uhp homologues encoded by the PA14 genome sequence, indicating that P. aeruginosa probably lacks a specific glucose-6-P transport system. When we compared the E. coli UhpT sequence to the PA14 genome database, the most similar protein was P. aeruginosa GlpT. Nevertheless, three different predicted MFS proteins (named here as HexP1, HexP2, and HexP3, for putative hexose phosphate transport) retain a certain homology with the E. coli UhpT protein (Table 2) (E value, ≤1.0 × 10−5), reaching the highest levels of similarity among all of the transporters encoded in the P. aeruginosa PA14 genome (23 to 26% identity).

TABLE 2.

Genotypic and phenotypic characteristics of Fos-R mutants and their positions in the glpT gene in 10 independent Fos-R mutantsa

Strain Fos MIC (μg/ml) for strain harboring plasmid:
 Resistance mutation
pBBR1MCS-3 pBBR-glpT
PA14 8 8
glpT::MAR2xT7 1,024 8 Insertional inactivation
FosR-7 1,024 8 A59 deletion, 1 bp (frameshift); stop TGA318
FosR-9 1,024 8 C219ATCGC225 deletion, 6 bp; Δ (Ile74-Ala75); TM2
FosR-1 1,024 8 A220TCGCC226 deletion, 6 bp; Δ (Ile74-Ala75); TM2
FosR-2 1,024 8 T365CATGTT371 deletion, 7 bp (frameshift); stop TGA507
FosR-3 1,024 8 G to A; Gly137(GG410C)→ Asp (GAC); TM4
FosR-5 1,024 8 G596 deletion, 1 bp (frameshift); stop TAG600
FosR-10 512 8 C975GG insertion, 3 bp; +Gly326; TM9
FosR-4 1,024 8 A to C; Thr336 (A1006CC)→ Pro (CCC); TM9
FosR-8 1,024 8 C to G; Tyr362 (TAC1086)→ stop TAG; TM10
FosR-6 512 8 G to A; Met 366(ATG1098)→ Ile (ATA); TM10
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a

The levels of resistance when harboring the empty vector pBBR1MCS-3 or the complementing plasmid pBBR-glpT also are shown. The mutants are ordered in the table according to the position of their mutations in the glpT gene.

The level of Fos resistance conferred by the inactivation of these genes in P. aeruginosa PA14 was studied and compared to that conferred by their inactivation in E. coli K-12 BW25113 (Table 1). P. aeruginosa and E. coli mutants were obtained from the available libraries (PA14 mutant collections from Harvard Medical School and the NARA institute, respectively) (3, 18). Interestingly, the inactivation of glpT produced a dramatic increase in Fos resistance in P. aeruginosa with respect to the wild type (MICs of 1,024 and 8 μg/ml, respectively), whereas the inactivation of this gene produced only a moderate increase in Fos resistance in E. coli (MICs of 32 and 2 μg/ml, respectively). The inactivation of uhpT conferred a moderate Fos resistance in E. coli (MIC of 8 μg/ml). However, the inactivation of any of the three genes that encoded MFS proteins related to UhpT in P. aeruginosa did not produce any effect on Fos resistance. Also, the inactivation of cyaA and ptsI decreased susceptibility in E. coli but had no effect in P. aeruginosa (Table 2).

As stated before, UhpT expression is induced by its substrate, glucose-6-P. Thus, it is expected that, in the presence of glucose-6-P, resistance to Fos conferred by glpT mutations will be suppressed or attenuated due to an increase in Fos transport via UhpT permease. Table 2 shows that the level of Fos resistance conferred by the deletion of glpT decreased in E. coli when glucose-6-P was present. However, glucose-6-P had no effect on resistance in P. aeruginosa. These results strongly suggest that Fos enters P. aeruginosa cells using solely the GlpT permease, which also is essential for glycerol-3-P uptake, whereas in E. coli Fos can use both UhpT and GlpT permeases.

Fos-R spontaneous mutants do not grow in minimal medium with glycerol-3-P as the sole carbon source.

To test if mutations in genes other than glpT can confer Fos resistance in P. aeruginosa, 20 flasks containing M9-glucose medium were inoculated with about 103 wild-type PA14 cells and incubated overnight at 37°C with shaking. About 109 cells from each culture were plated on M9 agar plates containing Fos (128 μg/ml) and glycerol-3-P as the sole carbon source and on M9 agar plates containing Fos plus glycerol instead of glycerol-3-P. Appropriate dilutions from these flasks also were plated on these media without Fos to estimate the number of viable cells. While about 1,000 Fos-R colonies grew on glycerol-Fos plates (frequency of Fos-R mutants, 1 × 10−6), none grew on glycerol-3-P plus Fos (frequency, ≤5 × 10−11). This result demonstrates that all mutations conferring Fos resistance affected the capacity to use glycerol-3-P as the sole carbon source. Therefore, it seems that Fos is imported into P. aeruginosa cells exclusively via the glycerol-3-P transport system. Thus, the spontaneous acquisition of Fos resistance requires the inactivation of GlpT permease, leading to a glycerol-3-phosphate-defective growth phenotype in all mutant isolates.

Analysis of spontaneous PA14 Fos-R mutants.

The inability to select Fos-R mutants in minimal medium with glycerol-3-P as the sole carbon source prompted us to analyze the glpT sequence carried by spontaneous PA14 Fos-R mutants to know whether the glpT gene is the only target for Fos resistance in P. aeruginosa.

Ten independent PA14 Fos-R mutants were obtained in LB-Fos as described in Materials and Methods. Chromosomal DNA was extracted from each resistant mutant, and their glpT sequences were PCR amplified and sequenced. The analysis of their respective sequences showed that all of them contained mutations in the glpT gene sequence and putatively were able to impair GlpT function. This strongly suggests that glpT inactivation is essential for acquiring Fos resistance in P. aeruginosa. Table 2 shows the type of mutations, their positions in the glpT gene, and the level of resistance conferred.

A detailed analysis of the type of mutations identified in these isolates reveals that five mutants contained deletions in the glpT gene, four mutants contained point mutations, and one harbored an insertion. Two mutants, 1 and 9, contained 6-bp deletions in the 6-bp repeated sequence CATCGCCATCGC, yet each displaced 1 bp (the deletion of ATCGCC and CATCGC in mutants 1 and 9, respectively). These deletions lead, in both cases, to the loss of amino acids Ile-74 and Ala-75 in TM2, conferring a highly Fos-R phenotype and a MIC of 1,024 μg/ml (Table 2). Three other mutants also carried deletions (1 bp in mutants 5 and 7 and 7 bp in mutant 2) producing frameshifts that led to premature stop codons. Truncated proteins putatively are nonfunctional and reach a high level of Fos resistance (MIC of 1,024 μg/ml in all three cases) (Table 2). Four mutants contained point mutations, two transitions (mutants 3 and 6), and two transversions (mutants 4 and 8). Mutant 3 contained the mutation G to A, leading to a Gly-137-Asp change in the middle of the TM4 region. The appearance of a negatively charged amino acid may drastically distort the conformation of this transmembrane helix. Mutant 4 contained an A-to-C mutation originating the change Thr-336-Pro inside TM9, probably producing a great structural distortion in this region. Mutant 8 carried a C-to-G mutation, creating a stop codon at position 362, which is located in the middle of TM10. This produces the loss of the last two α-helices of the protein. These three mutants also are highly resistant to Fos, which has a MIC of 1,024 μg/ml for all three. Mutant 6 contained a G-to-A mutation, originating the conservative change Met-366-Ile, which, in principle, maintains the hydrophobic nature in the TM10 region. This change, however, produces high Fos resistance (MIC of 512 μg/ml), although slightly less than that of the previous mutants. This suggests that methionine in this position of TM10 is important for the function of the protein. Finally, mutant 10 contained an in-frame insertion of the triplet CGG after another CGG codon, originating the insertion of a Gly at position 326 (inside TM9). The level of Fos resistance is similar to that of mutant 6 (MIC of 512 μg/ml). Mutations carried by the last two isolates demonstrate that even small changes in the GlpT amino acid sequence can produce considerable increases in Fos resistance levels in P. aeruginosa.

Finally, none of the Fos-R mutants, including both insertional glpT::MAR2xT7 and spontaneous mutants, was able to grow in glycerol-3-P as the sole carbon source, even after prolonged incubation for 4 days in either solid or liquid medium (data not shown).

Only glpT mutations are responsible for the Fos-R phenotype and the inability to grow in glycerol-3-P as the sole carbon source.

To further prove that only the mutations found in the glpT gene are responsible for the Fos-R phenotype, we introduced the plasmid pBBR-glpT, containing the wild-type glpT gene, into the 10 spontaneous Fos-R mutants and also into both the insertion mutant glpT::MAR2xT7 and the wild-type PA14. In all cases, the wild-type level of Fos susceptibility was recovered upon complementation with the wild-type glpT gene, with no effect on the MIC of Fos for the wild-type strain (Table 2). The ability to grow in glycerol-3-P was recovered after the introduction of the wild-type glpT gene in these isolates (data not shown).

On the other hand, glucose-6-P did not have any effect on the level of the Fos resistance of the spontaneous Fos-R isolates (Table 2), suggesting again the lack of a glucose-6-P-specific transport in P. aeruginosa.

Effect of Fos-R mutations on growth and biofilm formation.

To know the possible effect of Fos-R mutations on the growth rate of P. aeruginosa, we compared the growth curves of the Fos-R mutants, including that of the insertion mutant glpT::MAR2xT7, to that of the wild-type PA14 strain. Fos-R mutations seem to have no apparent effect on growth rates in LB (Table 3) and in M9 minimal medium with glucose or glycerol as the sole carbon sources compared to those for the wild type (data not shown), although more accurate experiments should be performed. In addition, neither wild-type nor mutant derivatives were able to grow in minimal medium with glucose-6-P as the sole carbon source (data not shown). These results suggest that, unlike E. coli Fos-R mutants (26), mutations conferring Fos resistance in P. aeruginosa do not have an effect on fitness in vitro. On the other hand, we studied the effect of Fos-R mutations on the ability of P. aeruginosa to form biofilm on an abiotic surface. Our results demonstrate that, under our conditions, the Fos-R mutants produce biofilm at a level similar to that of the wild type (Table 3).

TABLE 3.

Effect of Fos-R mutations on growth rates and biofilm formationa

Strain Relative growth rate (%) SD Relative biofilm formation (%) SD
Wild type 100 4.84 100 7.13
glpT::MAR2xT7 106.79 6.47 95.34 9.21
Fos-R7 99.92 8.45 103.79 7.23
Fos-R9 97.20 4.78 95.37 11.34
Fos-R1 105.93 4.80 94.78 9.00
Fos-R2 106.47 5.05 97.84 12.20
Fos-R3 106.31 5.05 91.84 9.12
Fos-R5 100.40 3.18 94.24 6.10
Fos-R10 96.77 7.43 101.79 2.05
Fos-R4 104.10 6.53 98.32 13.40
Fos-R8 98.24 5.46 116.13 12.20
Fos-R6 100.17 5.55 92.85 8.70
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a

All values are relative to that for the wild-type strain (100%). In all cases, the Student's t test indicates no differences between wild-type and mutant strains (P > 0.05). The mutants are ordered in the table according to the position of their mutations in the glpT gene. SD, standard deviations.

Frequency of mutants conferring resistance to Fos in hypermutator strains.

The frequency of Fos-R mutants was calculated for wild-type and hypermutator P. aeruginosa strains with deficiency in mutS and mutT. The mutS allele is more frequently found in isolates from cystic fibrosis patients (20, 31). A P. aeruginosa hypermutator strain with deficiency in mutT recently was isolated in a cystic fibrosis patient (22). Table 4 shows that the frequency of P. aeruginosa Fos-R mutants was considerably increased in the mutS strain (close to two orders of magnitude). The frequency also was increased, although moderately (20-fold), in the mutT strain. Thus, the acquisition of Fos resistance is a very easy task for P. aeruginosa hypermutator strains, suggesting that even very small populations, such as those that probably colonize the lungs of cystic fibrosis patients at the beginning of the natural history of P. aeruginosa infections, contain Fos-R mutants. On the other hand, the mutant frequency for Fos-R (128 μg/ml) was higher in the P. aeruginosa wild-type strain (1.4 × 10−6) (Table 4) than in E. coli BW25113 (≤2.6 × 10−10) (not shown). Thus, it seems very difficult to reach a high level of Fos resistance (128 μg/ml) by single spontaneous mutations in E. coli, as it has two different Fos import systems.

TABLE 4.

Frequency of mutants resistant to Fos (128 μg/ml) of the wild-type strain PA14 and its hypermutable variants mutS and mutT

Strain Mutant frequency Increase (fold)
PA14 1.4 × 10−6 1
mutS::MAR2xT7 1.2 × 10−4 86
mutT::MAR2xT7 2.8 × 10−5 20
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DISCUSSION

Fos is active against both gram-positive and gram-negative bacteria and is structurally unrelated to any other antibiotic, minimizing the possibility of cross-resistance with them (35). Interestingly, despite its long-term use in E. coli urinary tract infections, the frequency of Fos resistance in uropathogenic E. coli strains remains very low (13). Resistance mutations have been demonstrated to confer a fitness cost that prevents resistant strains from being established in the bladder, suggesting that this cost is responsible for the low frequency of resistance (26).

In cystic fibrosis patients, after continuous exposure to antibiotics, P. aeruginosa develops resistance to many or all of them (5). Prolonged therapy courses also lead to the selection of hypermutator strains that, indirectly, favor the acquisition of resistance (31, 32). Therefore, the treatment of multidrug-resistant P. aeruginosa infections requires the combination of various antimicrobial agents (7, 29, 42, 44). Most antibiotic resistance mechanisms in cystic fibrosis isolates of P. aeruginosa are based on mutation (31); therefore, it is expected that Fos resistance follows the same principle. In this sense, the molecular bases of Fos resistance in this species has been extrapolated from E. coli, suggesting that Fos could be transported into P. aeruginosa through the same permeases, i.e., via glucose-6-P and glycerol-3-P transport systems (10, 24, 28, 39). Thus, a systematic study to unveil the molecular bases involved in the development of Fos resistance in P. aeruginosa was of particular interest.

In E. coli, spontaneous Fos resistance is caused by mutations in genes involved in glycerol-3-P transport (glpT) or glucose-6-P transport (uhpA, uhpB, uhpC, and uhpT) (23). However, P. aeruginosa does not appear to contain the UhpT system. Four different predicted proteins, including GlpT, share weak homology with the E. coli UhpT protein in P. aeruginosa. However, the inactivation of the genes encoding these additional MFS proteins did not produce any effect on Fos resistance. In addition, no uhpA, uhpB, or uhpC homologues were found in the PA14 genome sequence. Finally, although cyaA and ptsI mutants are resistant to Fos in E. coli (26), the inactivation of cyaA and ptsI homologues did not confer Fos resistance in P. aeruginosa. These results suggest that multiple components whose disruption leads to the acquisition of Fos resistance in E. coli are not conserved in P. aeruginosa.

We have demonstrated that only insertions and spontaneous mutations affecting glpT produce resistance to Fos in P. aeruginosa PA14. In addition, all Fos-R mutants are unable to use glycerol-3-P as the sole carbon source. Spontaneous Fos-R mutants appeared at a frequency of 10−6 on both rich and minimal medium with glycerol as the sole carbon source. However, no mutants were isolated on minimal medium with glycerol-3-P as the sole carbon source (frequency, ≤5 × 10−11), which is consistent with a deficient transport of glycerol-3-P.

The analysis of the glpT sequence of 10 independent P. aeruginosa Fos-R mutants confirmed that all of them contained mutations in glpT. The identity of these mutations provides additional insight into the structure-function relationship of the GlpT permease (producing Fos-R mutants with different levels of resistance). For instance, it has become clear that even subtle changes in the GlpT amino acid sequence produce a considerable effect on the GlpT activity (impairing both growth in glycerol-3-P and Fos transport) in P. aeruginosa. In this sense, all changes in the amino acid sequence caused by point mutations always are located in any of the 12 TM domains of the GlpT permease, distorting the conformation of these α-helices. In E. coli GlpT, these 12 conserved TM domains are arranged in two compact bundles of six α-helices around a central pore, and the maintenance of the structure of these regions is essential for substrate translocation (11). Additionally, proteins within the OPA transporter family retain a high degree of sequence homology, with a considerable number of amino acids whose change impairs the transporter function (16).

On the other hand, generally it has been assumed that P. aeruginosa has a specific glucose-6-P transport system. Furthermore, it has been stated that glucose-6-P should be added to the growth medium for Fos sensitivity in in vitro testing to allow Fos efficacy (i.e., to induce the expression of glucose-6-P transport) (see, for instance, references 24 and 28) and references therein). However, four lines of evidence strongly suggest that, in contrast with E. coli, P. aeruginosa lacks a specific glucose-6-P transport system: (i) the absence of the detectable growth of wild-type PA14 and its mutant derivatives when cultured in minimal medium with glucose-6-P as the sole carbon source, (ii) the absence of uhp transport genes in the P. aeruginosa genome, (iii) the lack of an effect of the inactivation of the three putative hexose phosphate transporters, with low homology to the E. coli UhpT transporter, on Fos resistance, and (iv) the absence of an effect of glucose-6-P on the Fos MIC for the P. aeruginosa glpT Fos-R mutants.

Finally, we studied the effect of these mutations on the in vitro growth rate and the ability to form biofilms. Although all of the Fos-R mutants studied in this work showed a clear fitness cost when grown on glycerol-3-P as the sole carbon source, no apparent cost was observed when it was grown in rich medium or in minimal medium with glucose or glycerol, unlike the E. coli Fos-R mutants. Because of its metabolic versatility, P. aeruginosa can use a wider variety of carbon compounds than E. coli to obtain energy. Its ability to use a large number of compounds could allow P. aeruginosa to overcome the lack of glycerol-3-P supply, leading to the absence of a fitness cost. In contrast to P. aeruginosa, E. coli uses primarily sugars as energy and carbon sources through glycolysis. Thus, E. coli Fos-R mutants unable to provide glucose-6-P and/or glycerol-3-P could suffer an energy shortage, which might explain their reduced growth ability. However, competition experiments between Fos-R mutants and the wild-type strain should be performed to fully disprove a cost for these mutations in P. aeruginosa. In addition, these mutations did not affect the ability to form biofilm.

In summary, our results strongly suggest that glpT inactivation is the only mutational mechanism leading to Fos resistance in P. aeruginosa. Both the apparent lack of a fitness cost and the high frequency of the spontaneous Fos-R mutants suggest that Fos-R variants will be obtained easily in vivo upon the Fos treatment of P. aeruginosa infections. Furthermore, considering the frequency of Fos-R mutants in the mutS hypermutable strain (10−4) found in this study and the reported frequency of mutants resistant to other antipseudomonal antibiotics (21, 25, 34), a dangerous frequency of spontaneous mutants with simultaneous resistance to the combination of Fos with other antibiotics can be expected even with combined treatments. Obviously, exploring both the frequency of P. aeruginosa Fos-R mutants in vivo and the ability of these mutants to colonize and survive in the lungs is essential.

Acknowledgments

We thank N. T. Liberati and F. Ausubel for the P. aeruginosa strain PA14 and the PA14::MAR2xT7 library of insertions.

This work was supported by grant PI070215 and the Spanish Network for Research in Infectious Diseases (REIPI RD06/0008), both from the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III.

Footnotes

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Published ahead of print on 4 September 2009.

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