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In a prospective study (2009–2011) in healthcare institutions from the Canary Islands (Spain), 6 out of 298 carbapenem non-susceptible Pseudomonas aeruginosa isolates produced a metallo-β-lactamase: four IMP-15, two VIM-2 (including one IMP-15-positive isolate) and one VIM-1. Multilocus sequence typing identified the single VIM-1-producing isolate as clone ST111 and two IMP-15-producing isolates as ST606, but, strikingly, bacterial re-identification revealed that the other three isolates (producing IMP-15 and/or VIM-2) were actually Pseudomonas putida. Further retrospective analysis revealed a very high prevalence (close to 50%) of carbapenem resistance in this environmental species. Hence, we report the simultaneous emergence in hospitals on the Canary Islands of P. putida and P. aeruginosa strains producing IMP-15, a metallo-β-lactamase not previously detected in Europe, and suggest an underestimated role of P. putida as a nosocomial reservoir of worrying transferable resistance determinants.
Pseudomonas aeruginosa is one of the most relevant nosocomial pathogens, particularly in the Intensive Care Unit setting , as well as the first cause of chronic respiratory infections in patients with underlying diseases such as cystic fibrosis . One of the most striking features of this pathogen is its outstanding capacity for the development of antimicrobial resistance, through the selection of chromosomal mutations and the acquisition of horizontally transferred resistance determinants in genetic elements such as integrons located in transposons and/or plasmids. Particularly noteworthy among these determinants are class B carbapenemases (also called metallo-β-lactamases: MBLs), hydrolysing all β-lactams with the exception of monobactams [3-5]. In this scenario, the prevalence of multidrug-resistant and extensively drug-resistant P. aeruginosa strains is globally increasing, significantly compromising our anti-pseudomonal arsenal . Moreover, recent reports have provided evidence of the existence of multidrug-resistant/extensively drug resistant clones disseminated in several hospitals worldwide and for that reason denominated high-risk clones . Among them, ST235, ST111 and ST175 are probably more widespread, linked to multiple transferable and mutational resistance mechanisms . Furthermore, although some environmental Pseudomonas species such as Pseudomonas putida may not have the clinical relevance of P. aeruginosa, their potential role as reservoirs of transferable β-lactamases has recently been suggested [9, 10]. In this work, we report the simultaneous emergence of P. putida and P. aeruginosa strains harbouring an MBL not previously detected in Europe (IMP-15), and highlight the potentially underestimated role of P. putida as a nosocomial reservoir of worrying transferable resistance determinants.
A prospective study, from 2009 to 2011, was carried out to determine the prevalence of carbapenem non-susceptible P. aeruginosa in healthcare institutions of Gran Canaria, Canary Islands (Spain), including Hospital Universitario de Gran Canaria Dr Negrín, Hospital Universitario Materno Infantil and the primary-care centres. The P. aeruginosa isolates showing an intermediate or resistant clinical category (following CLSI breakpoints) to at least one of the tested carbapenems (imipenem and meropenem) were included . The GN card of Vitek2 (bioMérieux, Marcy l'Étoile, France) was used for initial identification and susceptibility testing. From a total of 298 non-susceptible isolates (14.7% of all the P. aeruginosa isolates recovered in the study period), six yielded a positive result with the MBL-Etest (bioMérieux). To confirm the presence of MBL determinants and determine the specific genes involved, PCR followed by sequencing of the complete coding regions was performed using previously described primers and protocols [12, 13]. Sequencing results revealed the presence of blaIMP-15 in four isolates, blaVIM-2 in two (including one of the blaIMP-15-positive isolates) and blaVIM-1 in one. Strikingly, the re-identification using the Api-20NE strips (bioMérieux) and 16S rDNA sequencing showed that three of the six isolates (producing blaVIM-2 and/or blaIMP-15) were actually P. putida.
Clonal relatedness was evaluated by pulsed-field gel electrophoresis (PFGE) following described protocols . Additionally, P. aeruginosa isolates were further analysed through multilocus sequence typing (MLST), using described procedures and available databases (http://pubmlst.org/paeruginosa/). The PFGE, MLST and resistance profiles (imipenem, meropenem, ceftazidime, cefepime, aztreonam, piperacillin-tazobactam, amikacin, gentamicin, tobramycin, colistin and ciprofloxacin MICs determined by Etest) documented for the six isolates are shown in Table 1. The single VIM-1-producing P. aeruginosa isolate was found to belong to the internationally spread high-risk clone ST111, previously linked to multiple different integron-borne acquired β-lactamases, from narrow to extended spectrum and carbapenemases . Although VIM-2 production has been recently linked to ST111 in several Spanish hospitals , this is the first association of this clone with VIM-1 in our territory. On the other hand, the two P. aeruginosa isolates producing blaIMP-15 showed an identical PFGE pattern, identified as ST606 clone through MLST (Table 1). ST606 has been reported in a few P. aeruginosa isolates , but has never been related to an acquired β-lactamase, and it is not yet considered one of the high-risk clones . Interestingly, one of the two isolates was recovered from a sputum sample of a patient with cystic fibrosis, accounting for the first documentation of colonization by MBL-producing P. aeruginosa among Spanish cystic fibrosis patients (Table 1). The two IMP-15-producing P. putida isolates also belonged to a single clone, showing an identical PFGE pattern (despite one of the isolates additionally produced VIM-2), completely different to that of the remaining VIM-2-producing strain (Table 1). Additionally, the three P. putida isolates were found to be aztreonam resistant. As shown in Table 1, MICs performed in Müller–Hinton plates containing the efflux pump inhibitor Phe-Arg β-naphthylamide dihydrochloride (final concentration, 20 mg/L)  suggested the involvement of efflux in the resistance phenotype.
Table 1. Characteristics of the metallo-β-lactamase (MBL) -producing strains studied
|Species||Isolate||PFGE clone||MLST clone||Date of isolation (mm/dd/yy)||Clinical sample (colonization/infection)||Ward||MBL||MIC (mg/L)|
|PP||364223||PP-A||–||02/09/09||Urine (C)||Internal medicine||VIM-2/IMP-15||>32||>32||>256||>256||64||6||>256||6||0.5||12||0.25||>32|
|447490||PP-A||–||09/14/10||Urine (I)||Domiciliary admission||IMP-15||>32||>32||>256||>256||32||4||>256||3||0.5||6||0.38||>32|
|PA||394851||PA-A||ST606||09/07/09||Sputum (I) (CF)||Pneumology||IMP-15||>32||>32||64||>256||2||ND||>256||4||0.5||1||0.094||2|
|496312||PA-B||ST111||08/25/11||Tracheal aspirate (C)||ICU||VIM-1||>32||>32||>256||>256||1||ND||>256||12||64||>256||2||>32|
The re-identification of three of the six isolates as P. putida prompted us to retrospectively review the carbapenem-resistance rates in this species during the study period, yielding quite alarming results: up to 22 of 48 isolates (45.8%) were found to be carbapenem resistant. Moreover, carbapenem resistance in P. putida significantly increased during the study period reaching 71.4% (10 of 13) in 2011. Unfortunately, a screening for MBL production in the 22 isolates could not be performed retrospectively because the isolates were no longer available, but the resistance patterns of many of them suggest a high prevalence of MDR determinants that could be transferred to P. aeruginosa, which is consistent with previous evidence .
This is the first time that blaIMP-15 has been detected in Europe, as well as, to our knowledge, the first report of the simultaneous presence of VIM and IMP enzymes in P. putida isolates. IMP-15, showing a 90% identity with IMP-1, was originally detected in a P. aeruginosa strain from Thailand (GenBank accession no. AY553333), and later in a strain recovered from a surgical wound of a patient admitted to a US hospital after surgery in a Mexican centre . Other reports have documented an endemic presence in Mexico of P. aeruginosa strains harbouring blaIMP-15 in several integron structures related to In95 [19-21]. To analyse whether blaIMP-15 of the strains from the Canary Islands were located in the same genetic element, PCR and sequencing of the complete integron was performed following described protocols . However, a different integron structure (designated In589, GenBank accession no. KC310496), which contained blaIMP-15 and blaOXA-4, was detected, arguing against a direct importation of the Mexican P. aeruginosa strains. Finally, regarding the potential plasmid/chromosomal location of blaIMP-15 in the studied strains, all attempts to transfer the plasmid DNA (obtained using the Ultraclean Plasmid Prep Kit; MO BIO Laboratories Inc., Carlsbad, CA, USA) to the PAO1 reference strain through electroporation/conjugation yielded negative results. Furthermore, the southern blot hybridization following described protocols  and using the North2South Complete Biotin Random Primer labelling and detection kit (Thermo Scientific, Rockford, IL, USA), over the I-CeuI/S1 nuclease-digested genomes, suggested the chromosomal location of blaIMP-15 in all the strains, given that the blaIMP-15 probe hybridized with bands that also hybridized with the rRNA gene probe (data not shown).
In summary, we report the simultaneous emergence in hospitals from the Canary Islands of P. putida and P. aeruginosa strains producing IMP-15, an MBL not previously detected in Europe. Moreover, our results suggest a relevant role of P. putida as a nosocomial reservoir of worrying transferable resistance determinants, which is probably underestimated because of the lack of active surveillance in environmental Pseudomonas species and their misidentification as P. aeruginosa.