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Antimicrobial Agents and Chemotherapy, April 2009, p. 1552-1560, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01264-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Azithromycin in Pseudomonas aeruginosa Biofilms: Bactericidal Activity and Selection of nfxB Mutants

Xavier Mulet, María D. Maciá, Ana Mena, Carlos Juan, José L. Pérez, and Antonio Oliver^*

Servicio de Microbiología and Unidad de Investigación, Hospital Son Dureta, Instituto Universitario de Investigación en Ciencias de la Salud, Palma de Mallorca, Spain

Received 22 September 2008/ Accepted 24 January 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Azithromycin (AZM) has shown promising results in the treatment of Pseudomonas aeruginosa chronic lung infections such as those occurring in cystic fibrosis (CF) patients. We evaluated the effect of hypermutation and alginate hyperproduction on the bactericidal activity and resistance development to AZM in P. aeruginosa biofilms. Strains PAO1, its mucA mutant (PAOMA), and their respective mutS-deficient hypermutable derivatives (PAOMS and PAOMSA) were used. Biofilms were incubated with several AZM concentrations for 1, 2, 4, or 7 days, and the numbers of viable cells were determined. During the first 2 days, AZM showed bactericidal activity for all the strains, but in extended AZM incubation for strain PAOMS and especially strain PAOMSA, a marked increased in the number of viable cells was observed, particularly at 4 µg/ml. Biofilms formed by the lineages recovered from the 7-day experiments showed enhanced AZM resistance. Furthermore, most of the independent lineages studied, including those obtained from biofilms treated with AZM concentrations as low as 0.5 µg/ml, showed MexCD-OprJ hyperexpression and mutations in nfxB. The role of nfxB mutation in AZM resistance was further confirmed through the characterization of nfxB and mexD knockout mutants. Results from this work show that, although AZM exhibits bactericidal activity against P. aeruginosa biofilms, resistant mutants are readily selected and that, furthermore, they frequently show cross-resistance to other unrelated antipseudomonal agents such as ciprofloxacin or cefepime but hypersusceptibility to others such as imipenem or tobramycin. Therefore, these results should help guide the selection of appropriate antipseudomonal therapies in CF patients under AZM maintenance treatment.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The establishment of Pseudomonas aeruginosa chronic respiratory infection is mediated by a complex adaptive process that includes physiological changes, mainly represented by the transition from a planktonic to a biofilm mode of growth (3) and by the selection of an important number of adaptive mutations required for long-term persistence (42). The biofilm mode of growth is one of the most important factors in the persistence of chronic lung infections due to its increased resistance to the host defense mechanisms, including mechanical clearance and clearance mediated by complement, antibodies, or phagocytes, and to its inherent resistance to antibiotics (14).

A common feature of P. aeruginosa chronic lung infections, including those occurring in patients suffering from cystic fibrosis (CF), bronchiectasias, or chronic obstructive pulmonary disease, is the very high prevalence (30 to 60% of patients) of hypermutable (or mutator) strains deficient in the DNA mismatch repair system in contrast to what is observed in acute infections (<1%) (2, 8, 23, 31, 32). The presence of hypermutable strains has been found to be linked to the high antibiotic resistance rates of P. aeruginosa strains isolated from patients with chronic lung infections (23, 32). It has also been shown by in vitro and in vivo experiments that hypermutation dramatically favors resistance development during antibiotic exposure (24, 33, 37). A recent work has also shown that hypermutation favors the establishment of chronic colonization in the CF mouse model, likely by facilitating the acquisition of adaptive mutations (27). One of the most frequent and relevant adaptive mutations in P. aeruginosa strains causing chronic lung infections is the inactivation of mucA that leads to alginate hyperproduction (7). Mucoid variants are found in 80 to 90% of chronically infected patients and are associated with a poorer pulmonary outcome. Alginate hyperproduction is also known to reduce bacterial clearance (35) and to inhibit phagocytosis (34), complement activation (36), antibiotic penetration (9), and neutralization of oxygen radicals (40).

Therefore, once chronic infection by P. aeruginosa is established, its eradication using conventional antibiotic treatments is extremely difficult, if not impossible.

In the last years, azithromycin (AZM), a macrolide antibiotic, has shown promising results in the treatment of chronic infections by P. aeruginosa. Although macrolides are not active against P. aeruginosa in standard in vitro susceptibility tests, these antibiotics show bactericidal activity on stationary phase bacteria (16). Furthermore, AZM is known to inhibit biofilm growth and the production of alginate and other virulence factors, likely due to its interaction with the quorum-sensing (QS) system (15, 44, 45). In fact, AZM is widely used currently as maintenance therapy in P. aeruginosa chronic respiratory infections since several studies have demonstrated its clinical benefit (4, 46).

Nevertheless, it has not yet been evaluated whether AZM treatment creates a pressure for the selection of resistant mutants or whether this potential effect might be favored by the hypermutable and/or mucoid phenotypes. Therefore, in this work, we evaluated the individual and combined effect of hypermutation and alginate hyperproduction on the bactericidal activity and development of resistance to AZM in P. aeruginosa biofilms in vitro.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1. PAO1 single or double knockout mutants in mucA, mutS, nfxB, and mexD were constructed following the procedure previously described by Quénée et al. (38) for gene deletion and antibiotic marker recycling in P. aeruginosa. Upstream and downstream PCR products (Table 2) were digested with either BamHI or EcoRI and HindIII and cloned by two-way ligation into pEX100Tlink with the HindIII site deleted and opened by EcoRI and BamHI. Resulting plasmids were transformed into Escherichia coli XL1-Blue, and transformants were selected on MacConkey agar plates containing 50 µg/ml ampicillin. The cloned PCR products were sequenced to verify the absence of mutations generated during PCR amplification. The lox-flanked gentamicin resistance cassette (aacC1) obtained by HindIII restriction of plasmid pUCGmlox was cloned into the single site for this enzyme formed by ligation of the two flanking fragments, and resulting plasmids were transformed into E. coli XL1-Blue. Transformants were selected in 30 µg/ml ampicillin and 5 µg/ml gentamicin on Luria Bertani (LB) agar plates. These plasmids were then transformed into E. coli helper strain S17-1. Knockout mutants were generated by conjugation followed by selection of double recombinants using LB agar plates supplemented with 5% sucrose, 1 µg/ml cefotaxime, and 30 µg/ml gentamicin. Double recombinants were checked first by screening for carbenicillin (200 µg/ml) susceptibility and afterwards by PCR amplification and sequencing. For the recycling of the gentamicin resistance cassettes, plasmid pCM157 was electroporated into the different mutants as previously described (41). Transformants were selected in LB agar plates supplemented with 250 µg/ml tetracycline. One transformant for each mutant was grown overnight in LB broth with 250 µg/ml tetracycline in order to allow the expression of the cre recombinase. Plasmid pCM157 was then cured from the strains by successive passages on LB broth. Selected colonies were then screened for tetracycline (250 µg/ml) and gentamicin (30 µg/ml) susceptibility and checked by PCR amplification and DNA sequencing to ascertain that the corresponding genes were properly disrupted. The double mutants were then constructed from the single mutants following the same procedure. The appropriate phenotypes of the constructed mutants were ascertained by the mucoid phenotype (mucA), the approximately 2-log increase in the rifampin resistance mutation rates (mutS) (33), or the expression of mexD (nfxB and mexD).

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TABLE 1. Strains and plasmids used in this work

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TABLE 2. Primers used in this work

Antimicrobial susceptibility studies on planktonically growing cells. Conventional MICs of ceftazidime, cefepime, aztreonam, imipenem, ciprofloxacin, tobramycin, and colistin were determined in Müller-Hinton agar (MHA) plates using Etest strips (AB Biodisk, Sweden) following the manufacturer's recommendations. AZM MICs were determined by microdilution (range of AZM concentrations from 0.5 to 1,024 µg/ml) in Müller-Hinton broth (MHB) using a final bacterial inoculum of 5 x 10⁵ log-phase CFU/ml. The effect of long-term exposure of planktonically growing cells on subinhibitory concentrations of AZM was also investigated. For this purpose, strain PAO1 or PAOMS (a mutS mutant of PAO1) was inoculated into six 10-ml MHB tubes containing 4 µg/ml AZM. Cultures were incubated for 7 days at 37°C and 180 rpm with daily dilution (1/100) into a fresh tube. After the 7 days, serial dilutions of the cultures were seeded in MHA plates. Two colonies from each of the six independent replicate experiments per strain were stored frozen at –80°C for further studies.

Antimicrobial susceptibility studies on biofilms. Biofilms were formed following a modified protocol of that previously described by Moskowitz et al. (28). Briefly, biofilms were grown by incubating bacteria (statically for 24 h at 37°C) on pegs that protrude from the lids (Nunc, Denmark) and extend into the microtiter plates containing approximately 2 x 10⁸ cells/ml of strains PAO1, PAOMA (a mucA mutant of PAO1), PAOMS, or PAOMSA (a mucA-mutS double mutant of PAO1) in MHB. Then, biofilms on the pegged lids were incubated in MHB with 0, 0.5, 1, 4, 16, 64, or 256 µg/ml of AZM. At this stage, four independent experiments were initiated in which biofilms were incubated in the presence of each of the AZM concentrations for 1, 2, 4, or 7 days, with daily rinsing and antibiotic renewal. Afterwards, biofilms were rinsed and transferred to MHB by centrifugation (for 20 min at 1,000 rpm and 4°C). Serial 1/10 dilutions were then plated in MHA to determine the number of viable cells. The established lower limit of detection was 2 CFU. Six replicate experiments were performed for each strain, antibiotic concentration, and incubation period. Two colonies per experiment, strain, and AZM concentration from the 7-day biofilm assays were stored frozen at –80°C after passage into MHA for further studies. Because of the obtained results (see Results section), AZM biofilm experiments were also conducted afterwards using the nfxB and mexD knockout of PAO1 or PAOMS. The activity of ciprofloxacin on biofilms formed by these mutants was also assessed using the procedure described above (24-h incubation experiments) and concentrations of 0.125, 0.25, 0.5, 1, and 2 µg/ml.

Investigation and characterization of AZM-resistant mutants. To determine if resistance to AZM activity on biofilm growth was developed during exposure, stored lineages were retested in the biofilm AZM treatment assay (24-h incubation experiments), and the survival percentages were compared with those of the parent strains. Six replicate experiments were performed for each of the stored lineages. Likewise, to evaluate whether cross-resistance to conventional antipseudomonal agents was developed during AZM exposure, MICs of ciprofloxacin, ceftazidime, cefepime, aztreonam, imipenem, tobramycin, and colistin were determined by Etest in the stored lineages.

Characterization of mutants hyperexpressing the MexCD-OprJ efflux pump. The level of expression of mexD was determined by real-time PCR following a modified protocol of that previously described by Oh et al. (30). Briefly, total RNA from cultures grown to logarithmic phase was obtained with an RNeasy minikit (Qiagen, Hilden, Germany) and was adjusted to a final concentration of 50 ng/µl. Fifty nanograms of purified RNA was then used for one-step reverse transcription and real-time PCR amplification using a QuantiTect SYBR green reverse transcription-PCR kit (Qiagen, Hilden, Germany) in a SmartCycler II (Cepheid, Sunnyvale, CA) instrument. Previously described conditions and the primer pair MxD-U and MxD-L and the pair RpsL-1 and RpsL-2 were used for the amplification of mexD and rpsL (used as a reference to calculate the relative amount of mRNA of the efflux pump protein), respectively (30). In all cases, the mean values of mRNA expression obtained in two experiments were considered. Additionally, nfxB (encoding the regulator of MexCD-OprJ expression) was PCR amplified using a PTC-200 Peltier Thermal Cycler with AmpliTaq Gold polymerase and 10x PCR Buffer II (Applied Biosystems, Branchburg, NJ). Primers NfxB-1 and NfxB-2 (Table 2) were used for PCR amplification under the following conditions: 12 min at 94°C; 35 cycles of 45 s at 94°C, 45 s at 62°C, and 1 min at 72°C; and a final extension step of 10 min at 72°C. PCR products were sequenced on both strands. A BigDye Terminator kit (PE-Applied Biosystems) was used to perform sequencing reactions that were analyzed with an ABI Prism 3100 DNA sequencer (PE-Applied Biosystems). Finally, the nfxB sequences were compared with those of PAO1 using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST).

Statistical analysis. Survival percentages after AZM treatment of mutants and their respective parent strains were compared using a Mann-Whitney U test. A P value of <0.05 was considered statistically significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of mutS and/or mucA inactivation on AZM activity against planktonic cells and biofilms. As expected, P. aeruginosa planktonically grown cells were found to be highly resistant to AZM. Furthermore, the MIC in the microdilution susceptibility assay was 128 µg/ml for the four strains (PAO1, PAOMA, PAOMS, and PAOMSA), showing that neither mucA nor mutS had major effects on AZM susceptibility.

The dynamics over time (1 to 7 days) of bacterial populations in biofilms treated with different AZM concentrations for the four strains are shown in Fig. 1. The median initial (time 0) bacterial load in biofilms was similar for the four strains and ranged from 5.2 x 10⁵ to 4.0 x 10⁶ CFU (Fig. 1). In the 24-h (day 1) biofilm assays, AZM exhibited concentration-dependent bactericidal activity, and concentrations as low as 0.5 µg/ml produced a significant reduction in the bacterial load (Fig. 1). For PAO1, the reduction in the bacterial load at day 1 ranged from 2 logs with 0.5 µg/ml AZM to 4 logs with 256 µg/ml AZM (Fig. 1). Results were similar for PAOMS, but AZM activity at day 1 tended to be lower for the mucA strains, particularly for the mucA-mutS double mutant (PAOMSA); the reduction in the bacterial load for PAOMSA ranged from approximately twofold with 0.5 µg/ml AZM to 2 logs with 256 µg/ml AZM (Fig. 1). The reduction in the bacterial load observed at day 1 for all strains and AZM concentrations was further increased at day 2 in all cases. Again, the overall reductions in the bacterial loads were lowest for PAOMSA. After extended AZM incubation (for 4 and 7 days), the bacterial load was stabilized (similar to day 2 to day 7) for the lower concentrations (0.5 and 1 µg/ml) in the four strains (Fig. 1). Nevertheless, the reduction in the bacterial load continued up to day 7 for strains PAO1 and PAOMA for concentrations 4 µg/ml. On the other hand, extended AZM incubation (for 4 and 7 days) for strain PAOMS and, especially, PAOMSA produced a marked increase in the number of viable cells for 4 µg/ml AZM; at day 7, the number of bacteria was approximately 50-fold higher for PAOMS than for PAO1 and approximately 5,000-fold higher for PAOMSA than PAOMA (Fig. 1). Furthermore, for PAOMSA, extended incubation with 16 µg/ml AZM also produced a slight increase in the number of viable cells (Fig. 1).

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FIG. 1. Dynamics over time (0, 1, 2, 4, and 7 days) of bacterial populations in biofilms treated with different concentrations of AZM (0, 0.5, 1, 4, 16, 64, or 256 µg/ml) for strains PAO1, PAOMS, PAOMA, and PAOMSA. The total CFU counts are the median values for six different experiments.

Mutants showing resistance to AZM activity on biofilms are selected during treatment. The above results suggested that AZM-resistant populations were being selected in biofilms of the mutS strains during exposure to 4 µg/ml AZM. Therefore, 12 stored lineages per strain (PAOMS and PAOMSA) from the 7-day, 4 µg/ml AZM biofilm experiments were investigated. To determine whether the large increase in the numbers of viable cells was due to the selection of a stable AZM resistance mechanism, the lineages were retested in the biofilm treatment assay (day 1) with 4 µg/ml AZM. As shown in Fig. 2, AZM bactericidal activity was indeed significantly lower in the majority of these lineages; the survival percentages (viable cells in treated versus nontreated biofilms) were 1 to 2 log higher than those of their respective parent strains. Differences in survival percentages compared with the respective parent strains were statistically significant (P < 0.05) in 12 of 12 lineages of strain PAOMS and in 9 of 12 lineages of PAOMSA (Fig. 2).

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FIG. 2. Survival percentages (viable cells in treated versus nontreated biofilms) after 24 h of AZM 4 µg/ml (AZM4) treatment for 12 PAOMS (A) and 12 PAOMSA (B) lineages recovered from the 7-day, 4 µg/ml AZM biofilm assays (black bars) and their respective parent strains (white bars). Median values (bars) and ranges (error bars) for 6 (mutants) or 24 (control strains) different experiments are shown. In the strain designations, numerals I to VI stand for the number of the experiment, and A or B represents the two colonies stored from each experiment, strain, and AZM concentration. *, P < 0.05 for survival percentages compared with values for the respective parent strains.

nfxB mutants showing cross-resistance to ciprofloxacin and cefepime are selected during treatment of biofilms with AZM. To explore whether cross-resistance to conventional antipseudomonal agents was developed during AZM exposure, MICs of ciprofloxacin, ceftazidime, cefepime, aztreonam, tobramycin, and colistin were initially determined in the 12 above-described lineages of PAOMS and PAOMSA. Indeed, the antibiotic susceptibility profile showed that 10 of 12 and 6 of 12 lineages of PAOMS and PAOMSA, respectively, had increased MICs to ciprofloxacin and cefepime. Considering these results, we decided to evaluate whether cross-resistance was also being selected in the nonhypermutable strains (PAO1 and PAOMA) and at the other AZM concentrations. Thus, we determined the MICs of ciprofloxacin in the 12 stored lineages of the four strains, recovered from 7-day biofilms treated with AZM at concentrations of 0, 0.5, 1, or 4 µg/ml. The ciprofloxacin resistance screening was considered positive when the documented MIC for this antibiotic was more than one twofold dilution higher than for the wild-type strains (MIC > 0.25 µg/ml). As shown in Table 3, for strains PAO1 and PAOMA a small number (one or two) of the tested lineages met this criterion, indicating, nevertheless, that resistant mutants were indeed selected in nonhypermutable strains even at concentrations as low as 0.5 µg/ml. In strains PAOMS and PAOMSA, this proportion was much larger, and most of the lineages recovered from the 7-day AZM-treated biofilms showed ciprofloxacin resistance, even those treated with 0.5 µg/ml (Table 3). In contrast, all the lineages recovered from nontreated biofilms of the four strains showed wild-type ciprofloxacin MICs.

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TABLE 3. Results for the ciprofloxacin resistance screening in isolates recovered from the 7-day biofilms incubated in the presence of several concentrations of AZM

To explore the susceptibility profile of the detected ciprofloxacin-resistant lineages, the MICs of ceftazidime, cefepime, aztreonam, imipenem, tobramycin, and colistin were determined. As shown in Table 4, all the mutants showed increased resistance to ciprofloxacin (MICs ranging from 0.5 to 4 µg/ml) and cefepime (MICs ranging from 6 to 16 µg/ml). The MICs of ceftazidime and colistin did not significantly differ from those of the parent strains, but most of the lineages showed increased imipenem and tobramycin susceptibility. Overall, the susceptibility results suggested that the mechanism involved might be MexCD-OprJ hyperexpression, and therefore the level of mexD mRNA was evaluated in all the lineages. Indeed, as shown in Table 4, all the lineages showed MexCD-OprJ hyperexpression, with mexD levels ranging from 30 to 1,300-fold higher than PAO1 basal expression. In order to investigate the mechanisms leading to MexCD-OprJ hyperexpression, nfxB was sequenced in these lineages, revealing in all cases the presence of mutations in this regulatory gene (Table 4). Eighteen different types of mutations in nfxB were detected in the studied collection. The most frequently found were missense mutations, which were detected in 19 of the 25 studied lineages. Frequently (10 of 19), the missense mutations involved a Leu-to-Pro change occurring in several different residues of NfxB (Table 4). On the other hand, directly inactivating mutations (nonsense mutations, frameshifts, insertions, or deletions) occurred in only five of the lineages. Finally, one of the lineages contained a T562C mutation that eliminates the stop codon, leading to the origination of a longer predicted protein of 235 amino acids (the regular NfxB contains 187 amino acids).

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TABLE 4. Antimicrobial resistance patterns, levels of mexD expression, and mutations in nfxB for the AZM-resistant mutants recovered from the 7-day biofilm experiments

It should be noted at this stage, however, that a 100% correlation between development of AZM resistance (Fig. 1) and the acquisition of nfxB mutations (Table 4) was not observed. As described above and shown in Fig. 1, most of the lineages tested (21 of the 24) from 4 µg/ml AZM 7-day biofilms showed AZM resistance, and all of the 16 nfxB mutants (Table 4) were among the 21 resistant lineages. Nevertheless, it was of interest that 5 of the 21 resistant lineages had no nfxB mutations (or NfxB phenotype). These results indicate that even though nfxB mutants are clearly positively selected during AZM treatment, there are yet unidentified mechanisms that can also drive AZM resistance development in biofilms.

Characterization of nfxB and mexD knockout mutants. In order to further understand the role of the efflux pump MexCD-OprJ in AZM resistance, we constructed and characterized the nfxB and mexD knockout mutants, leading to the constitutive upregulation and the inactivation of the efflux pump, respectively. As could be expected, the inactivation of nfxB dramatically increased mexD expression and produced the characteristic NfxB phenotype with increased MICs of ciprofloxacin and cefepime and decreased MICs of tobramycin (Table 4). On the other hand, as also could be expected, the inactivation of mexD did not significantly modify the MICs of quinolones, β-lactams, or aminoglycosides (Table 4). Moreover, the inactivation of nfxB increased further (to 512 µg/ml) the already high AZM MICs of wild-type strains (128 µg/ml), whereas mexD inactivation had no effect. In accordance with the results of this work, the nfxB knockout mutant of PAO1 showed significantly enhanced AZM resistance in biofilm growth (1- to 2-log increased survival rates) at all concentrations ranging from 0.5 to 4 µg/ml (Fig. 3A). As shown in Fig. 3B, the effect of nfxB inactivation on resistance was even more pronounced when ciprofloxacin was tested. On the other hand, the inactivation of mexD did not increase susceptibility to AZM or ciprofloxacin (Fig. 3), showing that MeCD-OprJ does not play a significant role unless it is overexpressed through nfxB mutation.

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FIG. 3. Survival percentages after a 24-h incubation of PAO1, PAONB, or PAOMxD biofilms in the presence of several concentrations of AZM or ciprofloxacin.

Selection of mutants showing cross-resistance to ciprofloxacin during AZM treatment is exclusively mediated by MexCD-OprJ overexpression. We have shown above that long-term exposure to AZM of P. aeruginosa biofilms frequently leads, particularly in hypermutable strains, to the selection of mutants showing cross-resistance to ciprofloxacin and that in all cases this resistance is mediated by MexCD-OprJ overexpression due to nfxB mutation. In order to determine whether AZM exposure may select other resistance mechanisms (for instance MexAB-OprM overexpression) showing cross-resistance to ciprofloxacin, we performed the same 7-day AZM treatment experiments, now with the mexD mutants of PAO1 (PAOMxD) and PAOMS (PAOMSMxD). Again, 12 lineages (from six independent experiments) per AZM concentration and strain were screened for ciprofloxacin resistance through the determination of the MICs by Etest. As shown in Table 3, not a single lineage (even for the mutS strain) of the mexD mutants showed resistance to ciprofloxacin. These results obviously suggest that the selection of mutants showing cross-resistance to ciprofloxacin during AZM treatment is exclusively mediated by MexCD-OprJ overexpression.

Selection of nfxB mutants during AZM treatment of planktonically growing cells. To evaluate whether long-term exposure to subinhibitory concentrations of AZM may select nfxB mutants also in planktonically growing cells, six independent replicates of the PAOMS strain were incubated in MHB tubes containing 4 µg/ml of AZM for 7 days at 37°C and 180 rpm with daily dilution (1/100) into a fresh tube. After the 7 days, serial dilutions of the cultures were seeded in MHA plates, and two colonies from each of the six independent replicate experiments were screened for ciprofloxacin susceptibility and MexCD-OprJ overexpression. Two of the 12 PAOMS lineages tested were indeed nfxB mutants, showing that selection may occur also in planktonic growth although apparently to a lower extent than in biofilm growth in which most of the PAOMS lineages (10 of 12 for all concentrations tested) were nfxB mutants.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The failure to achieve the eradication of P. aeruginosa by classical antimicrobial therapies once chronic respiratory infection is established has generated the need for alternative treatment strategies. The high prevalence of mutator strains in this context (23, 32), due to their extraordinary capacity for antibiotic resistance development (33), increases the severity of the problem. Several studies have demonstrated the clinical benefit of AZM treatment in patients with CF or diffuse panbronchiolitis, based on increased lung function and body weight (4, 46). However, the exact mechanism that mediates the AZM anti-Pseudomonas activity remains uncertain. Several studies have shown the effect of this antibiotic on QS-dependent virulence factor production (13, 29, 44, 45), biofilm formation (5, 15), and cell killing in stationary phase (13, 16). It has also been suggested that AZM directly disrupts the outer membrane of P. aeruginosa, probably by displacement of divalent cations from their binding sites on the lipopolysaccharide on the outer membrane, contributing to its bactericidal activity (16). In another recent work it was found that AZM killing requires interaction with the ribosome (20). The authors from this work found a consensus between their results and those from Imamura et al. (16) by proposing a two-step process in which AZM first permeabilizes the outer membrane and then causes cell death by inhibiting protein synthesis and/or ribosome assembly (20).

Data concerning antibiotic susceptibility assays in biofilm are scarce. A work by Moskowittz et al. (28) showed that the biofilm inhibitory concentrations were much higher than the corresponding conventionally determined MICs for all the antibiotics tested (β-lactams, tetracycline, quinolones, and aminoglycosides) except for AZM, which appeared much more active against biofilm-grown P. aeruginosa. Nevertheless, there were no studies that had evaluated AZM bactericidal activity in biofilms of typical chronic P. aeruginosa phenotypes, like hypermutable or mucoid, or determined whether treatment with this antibiotic created pressure for resistance development. In this work, we provide information about the individual or combined effect of hypermutation and alginate hyperproduction on the bactericidal activity and the development of resistance to AZM in P. aeruginosa biofilms in vitro.

The results of this work show that AZM exhibits bactericidal activity on P. aeruginosa biofilms in vitro, as demonstrated by the marked reduction in the numbers of viable cells even at concentrations as low as 0.5 µg/ml. Overall, alginate hyperproduction did not significantly modify AZM activity although the reduction in the bacterial loads tended to be lower for the mucA strains in 1-day experiments, suggesting that alginate may protect P. aeruginosa biofilms from AZM to some extent. Nevertheless, the most significant differences in the dynamics over time of bacterial populations in AZM-treated biofilms were observed between nonhypermutable and hypermutable strains. Indeed, a marked selection of AZM-resistant mutants was demonstrated for PAOMS and especially for PAOMSA. Remarkably, in most cases AZM resistance was found to be mediated by the selection of mutants that hyperproduce the multidrug efflux pump MexCD-OprJ. Given the high prevalence of P. aeruginosa hypermutable strains in chronic respiratory infections, the notable amplification of AZM-resistant mutants that are also resistant to ciprofloxacin and cefepime raises an alert about the impact of AZM maintenance treatment in these patients. Furthermore, although to a much lower extent, MexCD-OprJ-hyperproducing mutants were also selected in the nonhypermutable strains. It has also been shown previously that hyperexpression of MexCD-OprJ is a frequent fluoroquinolone resistance mechanism among P. aeruginosa strains isolated from CF patients while it is uncommon in other settings (10, 12, 17, 18, 19, 26). A positive in vivo selection of mutants that hyperproduce MexCD-OprJ has also been documented in a mouse model of hypermutable P. aeruginosa chronic infection treated with ciprofloxacin (24). Further studies should be performed to determine whether there is a specific link between MexCD-OprJ hyperexpression and chronic respiratory infections, which could be eventually related to the effects of this efflux system in QS regulation and biofilm formation (6, 21, 22). Although the impact of the AZM maintenance therapy on the prevalence of resistance-mediated MexCD-OprJ hyperexpression in P. aeruginosa isolates from CF patients remains to be investigated, some practical information might be extracted from the findings of this work. Since AZM treatment selects MexCD-OprJ-hyperproducing mutants, the use of ciprofloxacin or cefepime in patients under AZM maintenance therapy should probably be avoided in favor of antipseudomonal agents such as ceftazidime, carbapenems, or aminoglycosides that are not affected by this efflux pump.

In all cases, MexCD-OprJ hyperexpression was found to be driven by mutations in its negative regulator, nfxB. We detected up to 18 types of different mutations in this gene in the 25 mutants studied. As observed for the few P. aeruginosa clinical strains that have been investigated so far (10, 11, 18, 19), the most frequently found mutations were missense mutations, detected in 19 of the 25 studied lineages. Frequently (10 of 19), the missense mutations involved a Leu-to-Pro change, occurring in several different residues of NfxB. On the other hand, directly inactivating mutations (nonsense mutations, frameshifts, insertions, or deletions) occurred only in five of the lineages. Finally, one of the lineages contained a T562C mutation that eliminates the stop codon, leading to the origination of a longer predicted protein of 235 amino acids. Interestingly, we have found (unpublished results) this same mutation in four independent MexCD-OprJ-hyperproducing mutants that were recovered in a previous study by our group from mice treated with ciprofloxacin (24).

In partial agreement with our findings, in a recent work by Gillis et al. (6) MexCD-OprJ overexpression was found to be specifically selected during biofilm growth in the presence of AZM. Nevertheless, in contrast to our results, when they isolated AZM-exposed lineages, Gillis et al. failed to detect nfxB mutations and the characteristic NfxB phenotype with cross-resistance to ciprofloxacin and cefepime. These findings led them to the conclusion that AZM induces a stable MexCD-OprJ overexpression phenotype specific to biofilm growth and not dependent on nfxB mutations. Our results clearly offer a different perspective for MexCD-OprJ overexpression in AZM-treated biofilms. We show that after exposure to AZM, nfxB mutants are selected, accounting for 10 to 20% of the lineages of PAO1 populations (close to 100% for populations of mutS strains). This proportion of nfxB mutants in biofilm populations should certainly be enough to account for the MexCD-OprJ overexpression phenotype documented by Gillis et al. and also explains why they did not find nfxB mutants; still, most (80 to 90%) PAO1 lineages are not nfxB mutants. These data, together with the documented lack of increased susceptibility of mexD mutants, strongly suggest that MexCD-OprJ plays a role in AZM resistance, but it is driven by the selection of nfxB mutants and not by a biofilm-specific induction of MexCD-OprJ.

In summary, we show that although AZM exhibits bactericidal activity against P. aeruginosa biofilms, resistant mutants are readily selected, particularly for hypermutable strains. The main AZM resistance mechanism is found to be the hyperexpression of MexCD-OprJ due to mutations in its negative regulator gene, nfxB. This mechanism confers resistance to relevant unrelated antipseudomonal agents such as ciprofloxacin or cefepime and may confer hypersusceptiblity to others such as aminoglycosides. Therefore, these results may certainly help in guiding the selection of appropriate antipseudomonal therapies in CF patients under AZM maintenance treatment.

 
This work was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III through the Spanish Network for the Research in Infectious Diseases (REIPI C03/14 and RD06/0008) and by the Ministerio de Educación y Ciencia of Spain (SAF2006-08154). M.D.M. is recipient of a PostMIR fellowship from the Instituto de Salud Carlos III.

 
* Corresponding author. Mailing address: Servicio de Microbiología and Unidad de Investigación, Hospital Son Dureta, C. Andrea Doria no. 55, 07014 Palma de Mallorca, Spain. Phone: 34 971 175 148. Fax: 34 971 175 185. E-mail: antonio.oliver{at}ssib.es

Published ahead of print on 2 February 2009.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, April 2009, p. 1552-1560, Vol. 53, No. 4
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