Characterization of carbapenem-resistant Pseudomonas aeruginosa clinical isolates, carrying multiple genes coding for this antibiotic resistance

Background Carbapenemase genes are one of the most frequent mechanisms reported in carbapenem-resistant P. aeruginosa; however, description of P. aeruginosa co-harbouring two or more carbapenemases is unusual. Methods In this study we evaluated the presence of carbapenemase genes and the clonality of P. aeruginosa isolates obtained from a hospital over a 12-year period. A total of 127 isolates of carbapenem-resistant P. aeruginosa recovered from 109 patients feces (four samples), rectal swab (three samples), nasal swab (one sample) and anal abscess (one sample), were evaluated. Minimum inhibitory concentrations of the following antibiotics imipenem, meropenem and polymyxin E were determined by broth microdilution. The molecular profile of isolates was evaluated by pulsed field gel electrophoresis (PFGE). PCR for the following carbapenemase genes blaIMP;blaSPM;blaVIM;blaSIM;blaNDM;blaKPC;blaGES and nucleotide sequencing to confirm the enzyme gene types were performed and compared with the database available on the Internet (BLAST-http://www.ncbi.nlm.nhi.gov/blast/). Results All isolates were carbapenem-resistant, their MIC50 and MIC90 were respectively 64 μg/mL and 256 μg/mL to imipenem and 32 μg/mL and 256 μg/mL to meropenem, all isolates except one (MIC = 8 mg/L) were susceptible to polymyxin E. The most frequent carbapenemase genes identified were blaSPM identified in 41 isolates (32%), followed by 10 with blakpc and 5 with blaVIM (3.9%). All belonged to the class SPM-1 and VIM-2. In 2011, one isolate harbouring three carbapenemase genes (SPM-1, VIM-2 and KPC-2) that belonged to a new clone was identified in a hematopoietic stem cell transplanted patient. Then, 19 carbapenem-resistant P. aeruginosa were identified in an outbreak that occurred in the bone marrow transplant unit, all positive for SPM-1 gene, and 9 (47.3%) harbored both SPM-1 and KPC. Conclusion Our findings showed that PCR for KPC gene should be performed to evaluate carbapenem resistance in P. aeruginosa and that this agent can harbor more than one carbapenemase gene. Attention should be focused on the possible rapid spread of KPC in P. aeruginosa isolates and for the fact that P. aeruginosa may become a reservoir of this transmissible resistance mechanism.


Background
Carbapenem-resistant P. aeruginosa has become an important problem all over the world challenging the current diagnostic approaches. Carbapenemase genes are one of the most frequent mechanisms reported in carbapenemresistant P. aeruginosa [1][2][3][4][5]. It is important to identify carbapenemase genes transmitted on mobile genetic elements which can lead to the spread of resistance of P. aeruginosa to carbapenem, which are the main drugs used to treat infections caused by this agent. In Brazil, the most common carbapenemase is the metallo-betalactamase, SPM, however, recently P. aeruginosa harboring KPC was identified [2].
In this study, we evaluated the presence of carbapenemase genes and the clonality of carbapenem-resistant P. aeruginosa isolates obtained from a teaching hospital over a 12-year period.

Study setting
The study was conducted in The Central Institute of Hospital das Clinicas (ICHC -FMUSP), Brazil, a teaching hospital with 1,000 beds, ten intensive care units totalizing 110 beds and a bone marrow transplant ward with 20 beds.

Isolates
A total of 129 P. aeruginosa carbapenem-resistant clinical isolates identified over a 12-year period, from 1998 to 2012, recovered from 109 patients, hospitalized in the Clinical and Surgical nursery, Intensive Care, Burned, Haematology and Bone Marrow units at Hospital das Clinicas-FMUSP were evaluated.

Susceptible profile
Minimum inhibitory concentrations (MICs) of imipenem, meropenem and polymixin E were determined by broth microdilution according with Clinical Laboratory Standards Institute (CLSI 2012).

Molecular typing
Bacterial isolates were grown on blood agar overnight at 37°C. Gel blocks were made by using equal volumes of 2% low-melting-point agar (BioRad, USA) and a bacterial suspension of 9 × 10 8 cells. Genomic DNA was digested with 10U of XBAI (Fermentas, USA), [6] (Sekiguchi ref). DNAs were separated by pulsed-field gel electrophoresis (PFGE) using a CHEF-DR III system (Bio-Rad, USA). Running conditions were 21 h at 14°C, with and initial switching time of 1 s and final time of 30 s, at 6 V/cm. PFGE patterns were interpreted according to Tenover et al. 1997 [7].

Carbapenemases genes
PCR for the following carbapenemase genes bla IMP; bla SPM; bla VIM; bla SIM; bla NDM; bla KPC; bla GES was done as described previously [8][9][10][11]. The following reference strains were used as control in this study: P. aeruginosa that produced IMP-1, VIM-2, SIM-1, SPM-1 [10], KPC ATCC and E. coli NDM. Nucleotide sequencing to confirm the enzyme gene types was performed by MegaBACE 1000. The sequences were analyzed using the software Sequence Analyzer with the Base Caller Cimarron 3.12. The genetic sequence was compared with the database available on the Internet (BLAST-http://www.ncbi.nlm.nhi.gov/blast/). The KPC sequence was also comparing with KPC lahey databases (http://www.lahey.org/Studies/other.asp#table1).

Discussion
It is the first report of P. aeruginosa co-harbouring bla KPC and bla SPM genes and the first report in Brazil of P. aeruginosa carrying KPC-2, VIM-2 and SPM-1. SPM-1 was the most frequent carbapenemase identified in our hospital, followed by KPC-2. KPC in P. aeruginosa is rare and occurs mainly in the American continent [1][2][3][4][5]12]. Recently, P. aeruginosa harbouring KPC was described in Argentina [13] and Iran [14], showing the potential rapid dissemination of this mechanism of resistance to the world.  Even though P. aeruginosa harbouring KPC was identified in 2010 in Brazil [2], no other report was published since then.
Only 50 of 129 P. aeruginosa carbapenem-resistant harboured a carbapenemase evaluated in this study. Thus, the carbapenem resistance could be related to other mechanism of resistance such as outer-membrane protein alteration, efflux system overexpression or new carbapenemase not yet identified [15][16][17].
Pseudomonas aeruginosa co-harbouring carbapenemase is uncommon; there are few reports in the literature [1;3]. Pseudomonas aeruginosa isolate co-harbouring KPC and a metallo-β-lactamase (IMP-8) was recently reported in Puerto Rico [3] and isolates co-harbouring KPC and VIM gene were identified in Colombia [1]. We described a new clone of P. aeruginosa co-harbouring SPM-1 and KPC-2 that caused an outbreak in a Bone Marrow transplantation unit, and an isolate co-harbouring three carbapenemase (SPM-1; KPC-2 and VIM-2) that belonged to a different clone then previous described in two outbreaks that occurred in this unit and were controlled with reinforcement of hand hygiene and contact precautions, one due to P. aeruginosa harbouring SPM-1 and other harbouring VIM-2 [18].
This P. aeruginosa isolate harbouring KPC-2 was identified for the first time in our hospital in 2011 in a hematopoietic stem cell transplanted patient. It harboured three carbapanemase genes (SPM-1, VIM-2 and KPC-2), and belonged to a new clone (J A ) not identified before in the hospital. This isolate showed a resistant profile to both imipenem and meropenem with MIC of 64 μg/mL and 32 μg /mL, respectively, but was susceptible to polymyxin and colistin with a MIC of 2 μg /mL for both drugs.
In 2012 during an outbreak that occurred in the Bone Marrow Transplant unit, 19 carbapemem-resistant P. aeruginosa were identified (clone K, L, L1, L2, L3 and M), all positive for SPM-1 gene, and 10 (47.3%) harbored both SPM-1 and KPC-2. The majority of the KPC, six isolates of ten, belong to the clone of the outbreak (K B ). Our data showed different clones circulating in our hospital and a new one predominate clone harboring KPC. A recent study also described dissemination of a new clone of P. aeruginosa harbouring KPC in a hospital in Argentina after a K. pneumonia-KPC positive outbreak [13].
Other interesting findings of our study are that GES-5 was restricted to the Burned Intensive Care Unit and was identified only in 2001. VIM-1 was first identified in the Emergency Room and then in the Bone Marrow Transplant Unit, and SPM is spread in different units in the hospital. These results were similar with previous Brazilian reports that showed that SPM-1 is endemic in several hospitals in the country [19,20].
The complete sequence of two KPC-harbouring plasmids, Plasmid pCOL-1 (31 529 bp), IncP-6 replicon group and Plasmid pPA-2 (7995 bp) from P. aeruginosa showed that they differing in size and in incompatibility group, and harbouring different genetic structures containing the blaKPC-2 genes [21]. These findings suggest that the carbapenemase resistance dissemination due to KPC in P. aeruginosa will be similar to that seen in Enterobacteriaceae. Thus, it is very important to understand the epidemiology of these multiresistant isolates, in order to achieve early implementation of adequate control measures to contain and reduce their dissemination in the hospital setting. Pseudomonas aeruginosa can acquired this transmissible resistance mechanism, going unnoticed and be a source of spread of KPC to other genus and species of bacteria. Besides carbapenem-resistance in P. aeruginosa can be due to two or more carbapenemase genes, including KPC-gene.
In conclusion, our findings showed that SPM-1 is the most frequent carbapenemase identified in our hospital, followed by KPC-2. Thus, PCR for KPC gene should be performed to evaluate carbapenem resistance in P. aeruginosa and this agent can harbor more than one carbapenemase gene. Attention should be focused on the possible rapid spread of KPC in P. aeruginosa isolates and for the fact that P. aeruginosa may become a reservoir of this transmissible resistance mechanism.