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Antimicrobial Agents and Chemotherapy, June 2004, p . 2043-2048, Vol . 48, No . 6

Biochemical Characterization of the Naturally Occurring Oxacillinase OXA-50 of Pseudomonas aeruginosa

Delphine Girlich, Thierry Naas, and Patrice Nordmann*

Service de Bactériologie-Virologie, Université Paris XI, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France

Received 1 August 2003/ Returned for modification 9 November 2003/ Accepted 12 February 2004


   ABSTRACT

 
The blaOXA-50 gene (formerly known as the PA5514 gene) is an oxacillinase gene identified in silico in the genome of Pseudomonas aeruginosa PAO1 . By using a mutant strain of P . aeruginosa PAO1 that had an inactivated blaAmpC cephalosporinase gene, the blaOXA-50 gene was shown to be expressed constitutively in P . aeruginosa . This ß-lactamase gene was cloned onto a multicopy plasmid and expressed in P . aeruginosa and Escherichia coli . It conferred decreased susceptibility to ampicillin and ticarcillin and, interestingly, to moxalactam and meropenem in P . aeruginosa but not in E . coli . Overexpression and purification enabled us to determine the molecular mass (25 kDa), the pI value (8.6), and the hydrolysis spectrum of the OXA-50 ß-lactamase . It is a narrow-spectrum oxacillinase that uncommonly hydrolyzes imipenem, although at a low level . Very similar oxacillinase genes were identified in all P . aeruginosa isolates from various geographical origins tested . The weak variability of the nucleotide sequence of this gene (0 to 2%) corresponded to that found for the naturally occurring blaAmpC cephalosporinase gene of P . aeruginosa . The study indicated that P . aeruginosa harbors two naturally encoded ß-lactamase genes, one of which encodes an inducible cephalosporinase and the other of which encodes a constitutively expressed oxacillinase .


   INTRODUCTION

 
Pseudomonas aeruginosa is an opportunistic pathogen that mainly causes severe pneumonia in immunocompromised hosts and may colonize the lungs of patients with cystic fibrosis (31) . A mutant strain of P . aeruginosa PAO1 that had an inactivated cephalosporinase gene was constructed in a previous study (20) . The resulting ß-lactam resistance profile was oversusceptibility to all ß-lactam antibiotics, demonstrating the main role of cephalosporinase in naturally occurring resistance in that species . However, in silico analysis of the P . aeruginosa PAO1 genome (32) identified a putative ß-lactamase gene, named PA5514, whose sequence was consistent with those of several oxacillinase genes . Thus, the purpose of the present work was to clone and overexpress the gene, characterize its biochemical properties, and analyze its diversity among P . aeruginosa isolates .


   MATERIALS AND METHODS

 
Bacterial strains and plasmids. P . aeruginosa PAO1 was used as the template (32) for PCR amplification and cloning experiments (29) . P . aeruginosa KG2505 (20), an AmpC-deficient PAO1 mutant (ampC::{Omega}Sm mexA::res{Omega}Sm), was used as the host in the cloning experiments with the multicopy shuttle vector pBBR1MCS.3 (13) . A series of clinical P . aeruginosa isolates was analyzed, i.e., P . aeruginosa COL-1 (23) and P . aeruginosa ED-1 (24), which were obtained from the Hospital Bicêtre, Paris, France; P . aeruginosa GW-1 (26), which was from South Africa; P . aeruginosa Ka.209 (27), which was from Spain; P . aeruginosa strain 1, which was from Thailand (8); and P . aeruginosa IND, which was from India (this study) . Escherichia coli reference strains DH10B and BL21(DE3), as well as the TOPO plasmid (Invitrogen, Life Technologies, Cergy-Pontoise, France), were used for the cloning experiments; and pET9a (Stratagene, Amsterdam, The Netherlands) was used as an expression vector .

Susceptibility testing. The antimicrobial agents and their sources have been referenced elsewhere (22) . Antibiotic-containing disks were used to detect antibiotic susceptibility with Mueller-Hinton agar plates and by a disk diffusion assay (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) . The results were interpreted according to the guidelines of the French Society for Microbiology (http://www.sfm.asso.fr/nouv/general.php?pa=2) . The MICs of selected ß-lactams were determined by an agar dilution technique, as described elsewhere (22) . MIC results were interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards (18) .

PCR amplification and cloning experiments. Whole-cell DNAs of P . aeruginosa PAO1 and clinical isolates were extracted as described previously (22, 29) and were used as templates for PCR amplification . PCR products of 869 bp, including the complete sequence of the blaOXA-50 gene (previously named PA5514), were generated with primers S (5'-AATCCGGCGCTCATCCATC-3') and AS (5'-GGTCGGCGACTGAGGCGG-3'), the sequences of which are located at each end of the blaOXA-50 gene of the P . aeruginosa PAO1 genome (32) . The diversity of the oxacillinase genes in P . aeruginosa was studied by sequencing the PCR products obtained with whole-cell DNA of unrelated P . aeruginosa strains, as described previously (8) .

The PCR amplicon of the entire blaOXA-50 gene of P . aeruginosa PAO1 was then cloned into the pPCRBluntII-TOPO plasmid, as recommended by the manufacturer (Invitrogen, Life Technologies), and expressed in E . coli DH10B . The cloned insert was then removed by restriction with SpeI and XbaI (Amersham Pharmacia Biotech, Orsay, France) and subcloned into the SpeI- and XbaI-digested shuttle vector pBBR1MCS.3 (13), which replicates in P . aeruginosa and E . coli . The recombinant plasmid, named pBB-1, was introduced into P . aeruginosa strains PAO1 and KG2505, as described previously (13) .

The following primers were used to clone the blaOXA-50 gene into the expression vector pET9a: primer O50-ATG, which contained the initiation codon of the blaOXA-50 gene; ATG, along with an NdeI restriction site, which is underlined (5'-AAAAACATATGCGCCCTCTCCTCTTCAGT-3'); and primer O50-TGA, which contained the termination codon of the blaOXA-50 gene, TGA, along with a BamHI restriction site, which is underlined (5'-AAAAGGATCCATCAGGGCAGTATCCCGAGAGC-3') . Whole-cell DNA of P . aeruginosa PAO1 was used as the template . After PCR amplification, a 788-bp NdeI-BamHI-restricted fragment was ligated into the NdeI-BamHI-restricted pET9a expression vector and electroporated into E . coli BL21(DE3) . E . coli BL21(DE3) containing recombinant plasmid pET-1 was selected on kanamycin-containing agar plates (30 µg/ml) .

DNA sequencing and protein analysis. Both strands of the PCR products corresponding to the blaOXA-50 genes of unrelated P . aeruginosa isolates and all the insert region of the recombinant plasmids were sequenced with an automated sequencer (ABI 3100; Applied Biosystems, Les Ulis, France) . The nucleotide and protein sequences were analyzed with software available over the Internet at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) .

IEF analysis. ß-Lactamases were submitted to isoelectric focusing (IEF), as described previously (22), with culture extracts of strains P . aeruginosa PAO1, P . aeruginosa KG2505, and E . coli BL21(DE3) with and without recombinant plasmids (i.e., pBB-1 for the P . aeruginosa strains or pET-1 for the E . coli strain) and with the purified ß-lactamase OXA-50 .

ß-Lactamase purification, relative molecular mass determination, and N-terminal sequencing. Induction of an exponentially growing culture of E . coli BL21(DE3)(pET-1) with 0.4 mM isopropyl ß-D-thiogalactopyranoside (IPTG) was performed at 37°C for 5 h in Trypticase soy (TS) broth . Four liters of this culture was pelleted and resuspended in 30 ml of 20 mM Tris H2SO4 buffer (pH 9.0) . The protein extracts obtained were purified as described previously (24) . Briefly, the extracts were subjected to several purification steps, including ion-exchange chromatography on a Q-Sepharose column equilibrated with a 20 mM Tris-H2SO4 buffer (pH 9.0), followed by chromatography on an S-Sepharose column equilibrated with 25 mM malonate buffer (pH 5.6) . Elution of the ß-lactamase was performed with a linear K2SO4 gradient (0 to 500 mM) . Peaks of ß-lactamase activities were dialyzed overnight against 50 mM phosphate buffer (pH 7.0) . The protein content was measured by the Bio-Rad DC protein assay, and the specific activities of the crude extract and the purified ß-lactamase from E . coli BL21(DE3)(pET-1) were compared . The protein purification rate and the relative molecular mass of OXA-50 ß-lactamase were estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis (22) . The proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane (Problott; Applied Biosystems) by passive absorption, as described by Messer et al . (16), with some modifications . After the destaining step, the bands of interest were excised and dried in a Speed-Vac dryer for 30 min, and then the gel pieces were reswollen in 136 µl, which corresponded to the initial volume of the excised band (2% SDS in 0.2 M Tris-HCl [pH 8.5]), for 60 min . After the gel pieces were swollen, a fivefold volume of high-pressure liquid chromatography-grade water was added, and then a piece of prewetted (methanol) PVDF membrane (4 by 4 mm; Problott; Applied Biosystems) was added to the solution . The protein was transferred to the membrane by gentle shaking for 2 days at room temperature (23°C) . Subsequently, the membrane was washed five times with 1 ml of 10% methanol with vortexing . N-terminal Edman sequencing was performed on an Applied Biosystems Procise 494HT sequencer with the reagents and by the methods recommended by the manufacturer .

Kinetic studies. Purified ß-lactamase was used for kinetic measurements at 30°C in 100 mM Tris-H2SO4-300 mM K2SO4 (pH 7.0) (21, 24) . The kcat and Km values were determined by analyzing ß-lactam hydrolysis under initial-rate conditions with a UV spectrophotometer, as described previously (24) . The 50% inhibitory concentrations (IC50s) of clavulanic acid, tazobactam, sulbactam, and NaCl were determined (24) . Various concentrations of these inhibitors were preincubated with purified enzyme for 3 min at 30°C to determine the concentrations that decreased the rate of hydrolysis of 100 µmol of nitrocefin by 50% . The effect of carbon dioxide on the modulation of the enzymatic properties of OXA-50 was investigated by adding NaHCO3 to the reaction buffer at a final concentration of 10 mM . Specific activities and kcat and Km values were determined for most substrates in the presence and in the absence of bicarbonate (9, 15, 21) . The specific activities of the protein extracts and purified ß-lactamase from culture of E . coli BL21(DE3)(pET-1) were determined with 100 µM nitrocefin as the substrate . One unit of activity was defined as the amount of enzyme that hydrolyzes 1 µmol of nitrocefin per min (24) . The inducibility of the ß-lactamase content from the P . aeruginosa KG2505 culture was tested in TS broth at 37°C by the induction protocol described previously (19) with cefoxitin and imipenem as ß-lactam inducers at various concentrations (0.1, 0.5, and 1 µg/ml) .

Nucleotide sequence accession numbers. The nucleotide sequences of the OXA-50 variants reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and assigned accession numbers AY306130 to AY306135 .


   RESULTS AND DISCUSSION

 
Antibiotic susceptibility and ß-lactamase activity. P . aeruginosa KG2505 (ampC::{Omega}Sm mexA::res{Omega}Sm [20]), an AmpC-deficient derivative of wild-type strain P . aeruginosa PAO1, was hypersusceptible to all ß-lactams compared to the susceptibilities of any of the wild-type P . aeruginosa strains (Table 1), underlining an important role of the cephalosporinase in the natural resistance of P . aeruginosa to ß-lactams . The multicopy plasmid pBB-1, which contains the blaOXA-50 gene from P . aeruginosa PAO1, provided a slight additional level of resistance to amoxicillin, ticarcillin, and, interestingly, meropenem in P . aeruginosa KG2505 . Specific ß-lactamase activities of 0.2 and 20 mU/mg of protein were determined for P . aeruginosa KG2505 and P . aeruginosa KG2505(pBB-1), respectively, with 100 µM nitrocefin as the substrate . This result suggests that the blaOXA-50 gene is expressed in P . aeruginosa KG2505 and that it induces only minor phenotypic changes when expressed from a multicopy plasmid in P . aeruginosa KG2505 . The introduction of plasmid pBB-1 into parental strain P . aeruginosa PAO1, which had wild-type levels of AmpC and MexA-OprM expression, provided additional ß-lactam resistance, i.e., resistance to moxalactam and meropenem (Table 1) .


TABLE 1 . MICs of ß-lactams for P . aeruginosa PAO1 and its isogenic mutant, P . aeruginosa KG2505 (AmpC–, MexA–, OmpF–), with and without recombinant plasmid pBB-1

 
Plasmid pBB-1 was electroporated into E . coli DH10B . Antibiotic susceptibility testing revealed that E . coli DH10B(pBB-1) was susceptible to all the ß-lactams tested, even though ß-lactamase production could be demonstrated by nitrocefin hydrolysis (data not shown) . The blaOXA-50 gene of P . aeruginosa PAO1 was subsequently cloned into expression vector pET9a and expressed in E . coli BL21(DE3) . Similarly to E . coli DH10B (pBB-1), E . coli BL21(DE3)(pET-1) was fully susceptible to all the ß-lactams tested whether the bacterium was grown in the presence or in the absence of IPTG (data not shown) . Culture extracts of E . coli BL21(DE3)(pET-1) contained strong ß-lactamase activity, as assessed by nitrocefin hydrolysis activity (see Table 4) .


TABLE 4 . Purification steps of ß-lactamase OXA-50 produced by E . coli BL21(DE3) harboring recombinant plasmid pET-1

 
IEF analysis of P . aeruginosa strain KG2505 culture extracts revealed a single ß-lactamase band with a pI value of 8.6 focusing at the same pI value as the chromosomally encoded cephalosporinase of parental strain P . aeruginosa PAO1 (data not shown) . The identical pI values of the cephalosporinase and oxacillinase of P . aeruginosa might explain why this oxacillinase was unidentified for decades . IEF of IPTG-induced culture extracts of E . coli BL21(DE3) harboring recombinant plasmid pET-1 had ß-lactamase activity with a pI value of 8.6, which corresponds to that of the cloned oxacillinase OXA-50; the chromosome-encoded cephalosporinase AmpC from E . coli had a pI value of 9.2 .

Induction studies with cefoxitin or imipenem as the ß-lactam inducers at various concentrations failed to demonstrate any induction of ß-lactamase expression in P . aeruginosa KG2505 (data not shown) .

Detailed structural analysis of the blaOXA-50 gene and its surrounding DNA sequences. The G+C content of the blaOXA-50 gene open reading frame was 65%, which lies within the expected range of the G+C contents of P . aeruginosa genes, suggesting that the blaOXA-50 gene is naturally occurring in that species . The deduced amino acid sequence of the ß-lactamase OXA-50 contained a motif found in serine ß-lactamases (11), with a serine-threonine-tyrosine-lysine (S-T-Y-K) motif replacing the most commonly found serine-threonine-phenylalanine-lysine (S-T-F-K) motif at positions 70 to 73 (class D ß-lactamase [DBL] numbering) (4) (Fig . 1) . The five structural elements characteristic of class D ß-lactamases were found: S-X-V at DBL positions 119 to 120, W-X-X-X-X-L-X-I-X at DBL positions 164 to 172, Q-X-X-X-L at DBL positions 176 to 190, K-T-G at DBL positions 216 to 219, and Y-G-N at DBL positions 144 to 146 (4) . The amino acid sequence of OXA-50 was compared with those of known oxacillinases (Fig . 1; Table 2) . A K-T-G motif (DBL positions 216 to 219) was found in OXA-50, as in the carbapenem-hydrolyzing ß-lactamases OXA-23 and OXA-27, whereas it was replaced by a K-S-G motif in the second group of carbapenem-hydrolyzing oxacillinases, made up of OXA-24, OXA-25, OXA-26, and OXA-40 . The fifth structural element of oxacillinases, Y-G-N, was not replaced by an F-G-N motif in OXA-50, as in the most of the carbapenem-hydrolyzing oxacillinases (10) . As illustrated in Fig . 1, the oxacillinase genes exhibit many different structural features, and little is known about the relevance of these differences for explaining the variabilities of the hydrolysis spectra (7) . Indeed, OXA-50 is the only oxacillinase containing an S-T-Y-K motif that replaces the classical S-T-F-K motif of oxacillinases . Further investigations may demonstrate whether this structural feature plays a role in the biochemistry of OXA-50 . OXA-50 was weakly related to other oxacillinases; the highest identity was with the carbapenem-hydrolyzing ß-lactamases OXA-23 (44%) (6) and OXA-27 (43%) (1) from Acinetobacter baumannii, OXA-SHE (41%) from Shewanella algae, and ß-lactamase all2480 (40%) (12) from a Nostoc cyanobacterial sp . A phylogenetic tree constructed with the known oxacillinases and based on amino acid sequence identity (2) showed that OXA-50 (formerly PA5514) could not be included in any of the five defined groups (30) . However, OXA-50 might share a common ancestor with the group of enzymes that includes OXA-10 and OXA-5, referred to as "group I" by Sanschagrin et al . (30) .


FIG . 1 . Comparison of the OXA-50 sequence (in boldface) with the sequences of other class D ß-lactamases representative of the principal lineages of this group of ß-lactamases . The previously proposed class D ß-lactamase consensus sequence and numbering (DBL) (4) is indicated below the alignment (uppercase letters are the currently recognized invariant residues, and lowercase letters are the others) . Boxed amino acid sequences are those conserved for oxacillinases . The vertical arrow indicates the cleavage site for the leader peptide of OXA-50 . The enzymes included in the alignment are described in detail in Table 2.

 

TABLE 2 . Sources of oxacillinases for protein sequence comparison

 
The nucleotide sequences of the flanking regions of the blaOXA-50 gene did not show any inverted or repeated sequences indicating the presence of a transposable element . In addition, the blaOXA-50 gene was not inserted into a classical integron, as indicated by the absence of 59-base elements linked to the ß-lactamase gene . No open reading frame that shared significant sequence identity with known ß-lactamase regulator genes was found immediately upstream of the blaOXA-50 gene .

A blaOXA-50-like gene was identified in all the P . aeruginosa isolates tested (Table 3) . Comparison of the nucleotide sequences of these genes from unrelated P . aeruginosa isolates in different geographical locations showed 2 to 7 nucleotide changes compared to the nucleotide sequence of the blaOXA-50 gene (32) . Most of these nucleotide changes remained silent, and only a few led to amino acid changes (Table 3) . The deduced amino acid sequences of these oxacillinases showed 98 to 100% identity (Table 3) . The low nucleotide substitution rate of the blaOXA-50-like genes corresponded to that found for ampC-like ß-lactamase genes in that species (99.6% identity) (5, 14, 31) .


TABLE 3 . Comparison of amino acid sequences of the chromosome-encoded oxacillinases of several P . aeruginosa isolates

 
Biochemical properties of OXA-50. After purification, the specific activity of the ß-lactamase OXA-50 against nitrocefin was 75.5 U/mg of protein, and its purification factor was 47-fold (Table 4) . On SDS-PAGE, the purified protein appeared as single band of ca . 25 kDa and was estimated to be >95% pure (Fig . 2) . N-terminal amino acid sequencing of the mature protein revealed that the cleavage site for the leader peptide is between amino acid positions 18 and 19 (A-SEWND) . OXA-50 had a narrow-spectrum hydrolysis profile that included ampicillin, benzylpenicillin, cephaloridine, cephalothin, nitrocefin, piperacillin, and, surprisingly, imipenem (Table 5) . OXA-50 did not significantly hydrolyze moxalactam or meropenem, although parental strain P . aeruginosa PAO1 harboring the multicopy plasmid pBB-1 had decreased susceptibilities to moxalactam and meropenem (Table 1) .


FIG . 2 . SDS-PAGE analysis of purified enzymes from E . coli BL21(pET-1) (lane 1) and the culture extract (lane 2) . A total of 25 µg of each sample was submitted to electrophoresis . Lane M, molecular mass markers, with sizes indicated on the left.

 

TABLE 5 . Kinetic parameters for purified ß-lactamase OXA-50a

 
Hydrolysis of oxacillin or cloxacillin by OXA-50 was not detected, as is the case for ß-lactamase OXA-24 (3) . The kcat/Km values were low for all ß-lactams except benzylpenicillin and nitrocefin; this was mostly due to the low affinity (high Km) of this enzyme for these substrates . The best affinity was obtained for imipenem (Km, 19 µM) . The level of imipenem hydrolysis was very low . Compared to carbapenem-hydrolyzing oxacillinases, the catalytic activity (kcat) for imipenem was twofold lower for OXA-50 than for OXA-40 and the affinity of OXA-50 for imipenem was similar to those of OXA-27 (1) and OXA-24 (3) . Surprisingly, hydrolysis of meropenem was very weak, although the MICs were significantly increased for P . aeruginosa PAO1(pBB1) and P . aeruginosa KG2505(pBB1) (Table 1) . Hydrolysis of meropenem was detected at a very low level, as is the case for OXA-25, OXA-26, and OXA-27; and the weak affinity of OXA-50 for this substrate did not allow determination of a kcat value . OXA-50 was found to have similar weak affinities for other substrates, such as ampicillin, ticarcillin, piperacillin, cephaloridine, cefsulodin, moxalactam, and oxacillin (Table 5) . Biphasic kinetics were seen for ampicillin, cefsulodin, and piperacillin . For these substrates, kcat and Km were determined for the steady-state part of the kinetics (the second part of the curve) . As described for OXA-10 (9, 15, 21), CO2 may strongly influence the kinetics of oxacillinases . In the case of OXA-50, NaHCO3 transformed the hydrolysis to a linear curve only for cefsulodin, whereas biphasic turnover kinetics were still observed for ampicillin and piperacillin . Kinetic parameters were also determined for cefsulodin in the presence of 10 mM NaHCO3 . Since the Km value was still very high for these substrates, the kcat/Km value could not be determined more precisely . The specific activities of all ß-lactams with and without sodium bicarbonate were compared . Addition of NaHCO3 did not significantly modify the catalytic efficiency of OXA-50 except for catalytic efficiencies for imipenem and meropenem, for which the specific activities were increased by 4.5- and 2.5-fold, respectively (data not shown) . As for cefsulodin, kcat and Km values for imipenem were both increased after bicarbonate addition, resulting in a kcat/Km value that remained almost unchanged (data not shown) .

Studies of activity inhibition, as measured by determination of IC50s, showed that OXA-50 is weakly inhibited by clavulanic acid (500 µM), tazobactam (350 µM), and sulbactam (>2 mM), as is found for most of the oxacillinases (17) . OXA-50 was inhibited by NaCl (IC50, 50 mM), as is observed for most oxacillinases (17) .

This study indicates that P . aeruginosa harbors two chromosomally and naturally encoded ß-lactamase genes; the first one encodes an inducible cephalosporinase (class C) (14), whereas the second one encodes a constitutively expressed oxacillinase (class D) . Both ß-lactamase genes display minor strain-to-strain variations (5, 31), suggesting their possible use in combination for PCR-based P . aeruginosa identification . The oxacillinase may not contribute significantly to the overall ß-lactam susceptibility pattern of P . aeruginosa except for that to moxalactam . Furthermore, as opposed to most oxacillinase genes, the blaOXA-50-like genes were not part of class 1 integrons, and unlike the chromosomally located and naturally occurring oxacillinase genes from Aeromonas spp . (17, 28) and Ralstonia pickettii (19), blaOXA-50 gene expression was expressed constitutively . Finally, this work underlines the fact that an important reservoir of oxacillinase genes is likely gram-negative environmental species such as R . pickettii (19), Aeromonas spp . (28), Shewanella spp . (25), and now P . aeruginosa .

 


   ACKNOWLEDGMENTS

 
We thank N . Gotoh for providing P . aeruginosa strain KG2505 . We are indebted to Laurent Poirel, who was at the origin of this study .

This work was funded by a grant (UPRES-EA3539) from the Ministère de l'Education Nationale et de la Recherche, Université Paris XI, Paris, France, and by the European Community (6th PCRD, LSHM-CT-2003-503-335) .


   FOOTNOTES

 
* Corresponding author . Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France . Phone: 33-1-45-21-36-32 . Fax: 33-1-45-21-63-40 . E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr .


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