Efflux pumps expression and its association with porin down-regulation and beta-lactamase production among Pseudomonas aeruginosa causing bloodstream infections in Brazil.

BACKGROUND: Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile. RESULTS: Aztreonam exhibited the highest in vitro activity against the P. aeruginosa isolates studied (64.4% susceptibility), whereas susceptibility rates of imipenem and meropenem were both 47.5%. The MexXY-OprM and MexAB-OprM efflux systems were overexpressed in 50.8% and 27.1% of isolates studied, respectively. Overexpression of the MexEF-OprN and MexCD-OprJ systems was not observed. AmpC beta-lactamase was overexpressed in 11.9% of P. aeruginosa isolates. In addition, decreased oprD expression was also observed in 69.5% of the whole collection, and in 87.1% of the imipenem non-susceptible P. aeruginosa clinical isolates. The MBL-encoding genes blaSPM-1 and blaIMP-1 were detected in 23.7% and 1.7% P. aeruginosa isolates, respectively. The blaGES-1 was detected in 5.1% of the isolates, while blaGES-5 and blaCTX-M-2 were observed in 1.7% of the isolates evaluated. In the present study, we have observed that efflux systems represent an adjuvant mechanism for antimicrobial resistance. CONCLUSIONS: Efflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary beta-lactamases play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates.

SPM-1-producing Pseudomonas aeruginosa ST277 clone recovered from microbiota of migratory birds.

The production of São Paulo metallo-ß-lactamase (SPM-1) is the most common carbapenem resistance mechanism detected among multidrug-resistant Pseudomonas aeruginosa clinical isolates in Brazil. Dissemination of SPM-1-producing P. aeruginosa has been restricted to the nosocomial settings, with sporadic reports of environmental isolates due to contamination by hospital sewage. Herein, we described the detection and molecular characterization of SPM-1-producing P. aeruginosa recovered from the microbiota of migratory birds in Brazil. Three hundred gram-negative bacilli were recovered from cloacal and choanal swabs of Dendrocygna viduata during a surveillance study for detection of carbapenem-resistant isolates. All isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. MICs were determined by agar dilution, except for polymyxin B. Antibiotic resistance genes were detected by polymerase chain reaction (PCR) followed by DNA sequencing. Transcriptional levels of oprD and efflux system encoding genes were also carried out by quantitative real-time PCR. Nine imipenem-resistant P. aeruginosa isolates were recovered with 7 of them carrying blaSPM-1. Additional resistance genes (rmtD-1, blaOXA-56,aacA4, and aac(6')-Ib-cr) were also detected in all 9 isolates. The SPM-1-producing isolates showed high MICs for all ß-lactams, fluoroquinolones, and aminoglycosides, being susceptible only to polymyxin B. Interestingly, all isolates showed the same PFGE pattern and belonged to ST277. Overexpression of MexXY-OprM and MexAB-OprM was observed in those isolates that did not harbor blaSPM-1. Our results suggest that migratory birds might have played a role in the dissemination of SPM-1-producing P. aeruginosa within the Brazilian territory.

Intraclonal Genome Stability of the Metallo-ß-lactamase SPM-1-producing Pseudomonas aeruginosa ST277, an Endemic Clone Disseminated in Brazilian Hospitals.

Carbapenems represent the mainstay therapy for the treatment of serious P. aeruginosa infections. However, the emergence of carbapenem resistance has jeopardized the clinical use of this important class of compounds. The production of SPM-1 metallo-ß-lactamase has been the most common mechanism of carbapenem resistance identified in P. aeruginosa isolated from Brazilian medical centers. Interestingly, a single SPM-1-producing P. aeruginosa clone belonging to the ST277 has been widely spread within the Brazilian territory. In the current study, we performed a next-generation sequencing of six SPM-1-producing P. aeruginosa ST277 isolates. The core genome contains 5899 coding genes relative to the reference strain P. aeruginosa PAO1. A total of 26 genomic islands were detected in these isolates. We identified remarkable elements inside these genomic islands, such as copies of the blaSPM-1 gene conferring resistance to carbapenems and a type I-C CRISPR-Cas system, which is involved in protection of the chromosome against foreign DNA. In addition, we identified single nucleotide polymorphisms causing amino acid changes in antimicrobial resistance and virulence-related genes. Together, these factors could contribute to the marked resistance and persistence of the SPM-1-producing P. aeruginosa ST277 clone. A comparison of the SPM-1-producing P. aeruginosa ST277 genomes showed that their core genome has a high level nucleotide similarity and synteny conservation. The variability observed was mainly due to acquisition of genomic islands carrying several antibiotic resistance genes.

Frequency of plasmid-mediated AmpC in Enterobacteriaceae isolated in a Brazilian Teaching Hospital.

In Brazil, the presence of plasmid-mediated AmpC (pAmpC)-producing isolates has been sporadically reported. We evaluated the frequency of pAmpC among 133 Enterobacteriaceae clinical isolates. The bla CMY-2-like gene was detected in a single Klebsiella pneumoniae isolate. In our study, the pAmpC frequency was very low as previously reported.

Emergence of Klebsiella pneumoniae-producing KPC-2 carbapenemase in Paraíba, Northeastern Brazil.

The emergence of KPC-2 producing K. pneumoniae in hospitalized patients at the intensive care unit (ICU) of a teaching hospital located in the city of João Pessoa, Paraíba, Brazil, is reported. Seven carbapenem-resistant K. pneumoniae recovered from different body sites of infection were analyzed. Most isolates showed a multidrug-resistance phenotype. Genotypic analysis demonstrated the presence of two genotypes, with the predominance of genotype A, which belongs to ST 437. These isolates also carry the encoding genes of five other beta-lactamases.

Beta-lactam resistance mechanisms in Pseudomonas aeruginosa strains causing bloodstream infections: comparative results between Brazilian and American isolates.

This study evaluated the presence of distinct mechanisms of beta-lactam resistance in 122 Pseudomonas aeruginosa isolates, causing bloodstream infections at Hospital São Paulo (HSP, Brazil; 82 isolates) and Virginia Commonwealth University Medical Center (VCU, United States; 40 isolates). By Clinical Laboratory Standards Institute agar dilution, Brazilian P. aeruginosa isolates showed higher resistance rates to most antimicrobials tested than those collected from the United States, except for ciprofloxacin. Carbapenem hydrolysis was detected in seven P. aeruginosa from HSP, in which bla(SPM-1) (n=5), bla(IMP-1) (n=1), and bla(IMP-16) (n=1) were detected by polymerase chain reaction (PCR) followed by DNA sequencing. The production of GES-5 was observed in 1.25% of HSP isolates. No extended-spectrum beta-lactamase-encoding genes were detected in the VCU isolates. Expression of efflux systems genes (mexB, mexD, mexF, and mexY) was evaluated by quantitative reverse transcriptase-PCR. In HSP isolates MexXY-OprM (41.4%) efflux system was more frequently overexpressed, in contrast to what was observed in the VCU isolates, where both MexXY-OprM (25.0%) and MexAB-OprM (25.0%) were equally overexpressed. The oprD downregulation was similar among isolates collected from the HSP (92.7%) and VCU (95.0%). On the other hand, ampC overexpression was observed only among HSP isolates (31.7%). The distinct antimicrobial susceptibility profile and mechanisms of beta-lactam resistance found among P. aeruginosa isolated from teaching hospitals located in Brazil and the United States exemplify the importance of local epidemiology in determining antimicrobial resistance rates.