ABSTRACT Acinetobacter baumannii is one of the most frequent Gram-negative opportunistic pathogens associated with hospital-acquired infection worldwide. We briefly describe A. baumannii isolates that were recovered from surrounding ICU bed surfaces, exhibiting multidrug resistance phenotype and belonging to some widely spread clonal complexes of clinical A. baumannii isolates.

Abstract The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates.

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

In vitro activity of doripenem and comparator antimicrobial agents was evaluated against Gram-negative bacilli recently isolated from Brazilian private hospitals that were enrolled in the INVITA-A-DORI Brazilian Study. A total of 805 unique Gram-negative bacilli were collected from patients hospitalized at 18 medical centers between May/08 and March/09. Each hospital was asked to submit 50 single Gram-negative bacilli isolated from blood, lower respiratory tract or intraabdominal secretions. Bacterial identification was confirmed and antimicrobial susceptibility testing was performed using Clinical Laboratory Standards Institute (CLSI) microdilution method at a central laboratory. CLSI M100-S21 (2011) or US-FDA package insert criteria (tigecycline) was used for interpretation of the antimicrobial susceptibility results. Doripenem was as active as meropenem and more active than imipenem against E. coli and K. pneumoniae isolates. A total of 50.0% of Enterobacter spp. isolates were resistant to ceftazidime but 85.7% of them were inhibited at doripenem MICs < 1 µg/mL. Polymyxin B was the only agent to show potent activity against Acinetobacter spp. (MIC50/90, < 0.5/1 µg/mL) and P. aeruginosa (MIC50/90, 1/2 µg/mL). Although high rates of imipenem (53.1%) and meropenem (44.5%) resistance were detected among P. aeruginosa, doripenem showed MIC50 of 16 µg/mL against imipenem-resistant P. aeruginosa and inhibited a greater number of imipenem-resistant P. aeruginosa (10.5%) at MIC values of < 4 µg/mL than did meropenem (0.0%). In this study, doripenem showed similar in vitro activity to that of meropenem and retained some activity against imipenem-resistant P. aeruginosa isolated from Brazilian medical centers.

Ceftobiprole is a broad-spectrum cephalosporin with potent activity against staphylococci, including those resistant to oxacillin, as well as against most Gram-negative bacilli including Pseudomonas aeruginosa. In this study, the in vitro activity of ceftobiprole and comparator agents was tested against bacterial isolates recently collected from Brazilian private hospitals. A total of 336 unique bacterial isolates were collected from hospitalized patients between February 2008 and August 2009. Each hospital was asked to submit 100 single bacterial isolates responsible for causing blood, lower respiratory tract or skin and soft tissue infections. Bacterial identification was confirmed and antimicrobial susceptibility testing was performed using CLSI microdilution method at a central laboratory. The CLSI M100-S21 (2011) was used for interpretation of the antimicrobial susceptibility results. Among the 336 pathogens collected, 255 (75.9%) were Gram-negative bacilli and 81 (24.1%) were Gram-positive cocci. Although ceftobiprole MIC50 values for oxacillin resistant strains were two-fold higher than for methicillin susceptible S. aureus, ceftobiprole inhibited 100% of tested S. aureus at MICs < 4 µg/mL. Polymyxin B was the only agent to show potent activity against Acinetobacter spp. (MIC50/90, 0.5/1 µg/mL), and P. aeruginosa (MIC50/90, 1/2 µg/mL). Resistance to broad-spectrum cephalosporins varied from 55.3-68.5% and 14.3-28.5% among E. coli and Klebsiella spp. isolates, respectively; with ceftobiprole MIC50 > 6 µg/mL for both species. Our results showed that ceftobiprole has potent activity against staphylococci and E. faecalis, which was superior to that of vancomycin. Our data also indicates that ceftobiprole demonstrated potency comparable to that of cefepime and ceftazidime against key Gram-negative species.

Pseudomonas sp. é um bacilo gram-negativo ubíquo de vida livre e freqüente em ambientes hospitalares. Bactérias produtoras de metalo-betalactamases (MBLs) são em grande parte resistentes aos betalactâmicos de largo espectro, incluindo cefalosporinas e carbapenens. Este trabalho objetivou detectar cepas de Pseudomonas spp. resistentes ao imipenem e à ceftazidima, assim como identificar aquelas produtoras de MBLs. Foram estudadas (entre junho de 2002 e junho de 2003) 311 cepas isoladas de diversas amostras clínicas no Hospital Geral de Fortaleza (HGF), bem como foram realizados testes de identificação e sensibilidade pelo sistema de automação MicroScan®/WalkAway, sendo as cepas multirresistentes confirmadas através do método de difusão em disco. A triagem para detecção de amostras produtoras de MBLs foi realizada pelo método de dupla difusão, utilizando discos com mercaptoacetato de sódio. Entre essas amostras, 24 (7,71%) demonstraram produção de MBLs e padrão de multirresistência entre as cepas estudadas. Os antimicrobianos para os quais as cepas apresentaram maior sensibilidade foram a piperacilina/tazobactam com 255 (82%) de sensibilidade, seguido da piperacilina isoladamente, com 229 (73,63%); imipenem com 195 (62,70%); ticarcilina/ácido clavulânico com 193 (62,05%); e ceftazidima com 138 (44,37%). A detecção dessas amostras configura um problema emergente, com importantes implicações na terapêutica antimicrobiana.

Pseudomonas sp. is a ubiquitous gram-negative bacilli, of free and frequent life in hospital environment. Metallo-betalactamases (MBLs) productive bacteria are largely resistant to betalactamics of wide spectrum, including cephalosporin and carbapenem. The objective of this work was to detect Pseudomonas spp. strains resistant to imipenem and ceftazidime, as well as to identify the MBLs producer ones. It was studied 311 isolated strains from several clinical samples at Fortaleza General Hospital (FGH), from June 2002 to June 2003. Identification and sensibility tests were done by the MicroScan®/WalkAway automation system. The multiresisting strains were confirmed by the diffusion method in disk. The triage to detect MBLs productive samples was accomplished by the double diffusion method, using disks containing sodium mercaptoacetat. Among these samples, 24 (7.71%) indicated production of MBLs and multiresistance standard in the midst of the studied strains. The antimicrobials to which the strains presented larger sensibility were piperacillin/tazobactam, with 255 (82%) of sensibility, followed by isolated piperacillin with 229 (73.63%); imipenem with 195 (62.70%); ticarcillin/clavulanic acid with 193 (62.05%); and ceftazidime with 138 (44.37%). The detection of these samples configures an emerging problem, with important implications in the antimicrobial therapeutic.

Bactérias produtoras de metalo-beta-lactamases (MBLs) são em grande parte resistentes aos betalactâmicos de largo espectro, incluindo oximino-aminotiazol cefalosporinas e também aos carbapenens. O objetivo deste trabalho foi detectar cepas de Pseudomonas aeruginosa resistentes ao imipenem e à ceftazidima, assim como identificar aquelas produtoras de MBLs. Foram estudadas 198 linhagens não-repetitivas isoladas de diversas amostras clínicas, hospitalares e comunitárias, identificadas bioquimicamente por técnicas de rotina. A triagem para a detecção de amostras produtoras de MBLs foi realizada pelo método de dupla difusão proposto por Arakawa et al. (2000) e modificado por Nakajima et al. (2001), utilizando discos contendo mercaptoacetato de sódio. Foi detectado um percentual de resistência de 19,7% (39/198) ao imipenem e de 15,2% (30/198) à ceftazidima, com 10,1% (20/198) de resistência cruzada aos dois antimicrobianos. Entre estas amostras, 2% (4/198) demonstraram produção de MBLs e padrão de multirresistência. A detecção destas amostras configura um problema emergente, com importantes implicações na terapêutica antimicrobiana, necessitando, portanto, de maior investigação através de metodologia molecular, para melhor caracterizar a extensão do problema.

Producers of metallo-beta-lactamases (MBLs) have been shown to confer resistance to carbapenems. The present study was undertaken to evaluate the presence of resistance to imipenem and oxymino cephalosporins, as well as MBLs production among clinical isolates of Pseudomonas aeruginosa in João Pessoa-PB, Brazil. A total of 198 non-repetitive strains of P. aeruginosa identified by routine techniques were studied. These strains were screened for MBL production, using the double disk diffusion test previously described by Arakawa et al. (2000), as modified by Nakajima et al. (2001), using disks containing sodium mercaptoacetate as inhibitor of MBL. Strains were considered to be MBL producer when they demonstrated a typical expansion in the inhibitory zone by the presence of thiol compound. A total of four strains (2%) showed positive results among the isolates tested. Our findings indicate that the appearance of metallo-beta-lactamase in Pseudomonas aeruginosa strains is an emerging problem, with important therapeutic implications, and this fact needs further investigation including the use of molecular methodologies to fully characterize the extent of the problem.