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Objectives: Based on pulsed-field gel electrophoresis (PFGE) profile, whole-genome sequencing (WGS) of eight carbapenem-resistant Pseudomonas aeruginosa isolates from a bone marrow transplant unit in São Paulo, Brazil, was performed to investigate the presence of resistance and virulence genes as well as to determine the sequence type (ST) by multilocus sequence typing (MLST)., Methods: The initial phenotypic susceptibility pattern of the isolates was determined by VITEK ® 2. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method for amikacin, meropenem and colistin. WGS was performed using an Illumina MiSeq system. A Galleria mellonella infection model was used to evaluate the virulence of the strains., Results: WGS demonstrated that mutations in genes encoding outer membrane proteins and efflux pumps in an isolate harbouring bla VIM-36 (ST308) differed from those in isolates harbouring bla SPM (ST277). The mexT gene harboured a mutation resulting in a frameshift in all isolates; in addition, the oprD gene of the bla VIM-36 -carrying isolate had an insertion leading to a frameshift. Virulence genes did not differ between ST277 and ST308 strains. Moreover, only two isolates harbouring bla SPM showed virulence in the G. mellonella model, killing 100% of larvae after 18-24h., Conclusions: P. aeruginosa carrying bla VIM-36 belonging to ST308 was identified for the first time in our hospital. Although the virulence gene profiles were similar in isolates carrying bla SPM and the isolate carrying bla VIM-36 , only two isolates harbouring bla SPM showed virulence in the G. mellonella model., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Purpose: Carbapenem resistance in P. aeruginosa is increasing worldwide. In Brazil, SPM-1 is the main P. aeruginosa carbapenemase identified. Little is known about the virulence factor in SPM-1 clones.Methodolgy. We describe a carbapenem-resistant P. aeruginosa bloodstream infection (CRPa-BSI) outbreak in a bone marrow transplant Unit (BMT). Twenty-nine CRPa-BSI cases were compared to 58 controls. Microbiological characteristics of isolates, such as sensitivity, carbapenemase gene PCR for P. aeruginosa, and PFGE are described, as well as the whole-genome sequence (WGS) of three strains.Results/Key findings. The cultures from environmental and healthcare workers were negative. Some isolates harboured KPC and SPM. The WGS showed that the 03 strains belonged to ST277, presented the same mutations in outer membrane protein, efflux pump, and virulence genes such as those involved in adhesion, biofilm, quorum-sensing and the type III secretion system, but differ regarding the carbapenemase profile. A predominant clone-producing SPM harbouring Tn 4371 was identified and showed cross-transmission; no common source was found. Overall mortality rate among cases was 79 %. The first multivariate analysis model showed that neutropenia (P=0.018), GVHD prophylaxis (P=0.016) and prior use of carbapenems (P=0.0089) were associated with CRPa-BSI. However, when MASCC>21 points and platelets were added in the final multivariate analysis, only prior use of carbapenems remained as an independent risk factor for CRPa-BSI (P=0.043)., Conclusions: The predominant clone belonging to ST277 showed high mortality. Carbapenem use was the only risk factor associated with CRPa-BSI. This finding is a wake-up call for the need to improve management in BMT units.

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: The use of combined antibiotic therapy has become an option for infections caused by multidrug-resistant (MDR) bacteria. The time-kill (TK) assay is considered the gold standard method for the evaluation of in vitro synergy, but it is a time-consuming and expensive method. The purpose of this study was to evaluate two methods for testing in vitro antimicrobial combinations: the disk diffusion method through disk approximation (DA) and the agar gradient diffusion method via the MIC:MIC ratio. The TK assay was included as the gold standard. MDR Gram-negative clinical isolates (n = 62; 28 Pseudomonas aeruginosa, 20 Acinetobacter baumannii, and 14 Serratia marcescens) were submitted to TK, DA, and MIC:MIC ratio synergy methods. Results: Overall, the agreement between the DA and TK assays ranged from 20 to 93%. The isolates of A. baumannii showed variable results of synergism according to TK, and the calculated agreement was statistically significant in this species against fosfomycin with meropenem including colistin-resistant isolates. The MIC:MIC ratiometric agreed from 35 to 71% with TK assays. The kappa test showed good agreement for the combination of colistin with amikacin (K = 0.58; P = 0.04) among the colistin-resistant A. baumannii isolates. Conclusions: The DA and MIC:MIC ratiometric methods are easier to perform and might be a more viable tool for clinical microbiology laboratories. [ABSTRACT FROM AUTHOR]