Raquel Girardello

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

BackgroundMulti-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.ResultsAztreonam 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 β-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.ConclusionsEfflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary β-lactamases play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates.

Comparative analysis of the complete genome of KPC-2-producing Klebsiella pneumoniae Kp13 reveals remarkable genome plasticity and a wide repertoire of virulence and resistance mechanisms

BackgroundKlebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial and community-acquired infections. A wide repertoire of virulence and antimicrobial resistance genes is present in K. pneumoniae genomes, which can constitute extra challenges in the treatment of infections caused by some strains. K. pneumoniae Kp13 is a multidrug-resistant strain responsible for causing a large nosocomial outbreak in a teaching hospital located in Southern Brazil. Kp13 produces K. pneumoniae carbapenemase (KPC-2) but is unrelated to isolates belonging to ST 258 and ST 11, the main clusters associated with the worldwide dissemination of KPC-producing K. pneumoniae. In this report, we perform a genomic comparison between Kp13 and each of the following three K. pneumoniae genomes: MGH 78578, NTUH-K2044 and 342.ResultsWe have completely determined the genome of K. pneumoniae Kp13, which comprises one chromosome (5.3 Mbp) and six plasmids (0.43 Mbp). Several virulence and resistance determinants were identified in strain Kp13. Specifically, we detected genes coding for six beta-lactamases (SHV-12, OXA-9, TEM-1, CTX-M-2, SHV-110 and KPC-2), eight adhesin-related gene clusters, including regions coding for types 1 (fim) and 3 (mrk) fimbrial adhesins. The rmtG plasmidial 16S rRNA methyltransferase gene was also detected, as well as efflux pumps belonging to five different families. Mutations upstream the OmpK35 porin-encoding gene were evidenced, possibly affecting its expression. SNPs analysis relative to the compared strains revealed 141 mutations falling within CDSs related to drug resistance which could also influence the Kp13 lifestyle. Finally, the genetic apparatus for synthesis of the yersiniabactin siderophore was identified within a plasticity region. Chromosomal architectural analysis allowed for the detection of 13 regions of difference in Kp13 relative to the compared strains.ConclusionsOur results indicate that the plasticity occurring at many hierarchical levels (from whole genomic segments to individual nucleotide bases) may play a role on the lifestyle of K. pneumoniae Kp13 and underlie the importance of whole-genome sequencing to study bacterial pathogens. The general chromosomal structure was somewhat conserved among the compared bacteria, and recombination events with consequent gain/loss of genomic segments appears to be driving the evolution of these strains.

Cation Concentration Variability of Four Distinct Mueller-Hinton Agar Brands Influences Polymyxin B Susceptibility Results

ABSTRACT Polymyxins have been the only alternative therapeutic option for the treatment of serious infections caused by multidrug-resistant Acinetobacter baumannii or Pseudomonas aeruginosa isolates. For this reason, it is of crucial importance that susceptibility tests provide accurate results when testing these drug-pathogen combinations. In this study, the effect of cation concentration variability found on different commercial brands of Mueller-Hinton agar (MHA) for testing polymyxin B susceptibility was evaluated. The polymyxin B susceptibilities determined using Etest and disk diffusion were compared to those determined by the CLSI reference broth microdilution method. In general, the polymyxin B MIC values were higher when determined by Etest than when determined by broth microdilution against both A. baumannii and P. aeruginosa isolates. A high very major error rate (10%) was observed, as well as a trend toward lower MICs, compared to those determined by broth microdilution when the Merck MHA was tested by Etest. Poor essential agreement rates (10 to 70%) were observed for P. aeruginosa when all MHA brands were tested by Etest. Although an excellent categorical agreement rate (100%) was seen between the disk diffusion and broth microdilution methods for P. aeruginosa, larger zones of inhibition were shown obtained using the Merck MHA. The high cation concentration variability found for the MHA brands tested correlated to the low accuracy, and discrepancies in the polymyxin B MICs were determined by Etest method, particularly for P. aeruginosa isolates.

Low Prevalence of blaOXA-143 in Private Hospitals in Brazil

We read with great interest C. S. Antonio et al.s letter describing the high prevalence of Acinetobacter baumannii carrying bla OXA-143 in Brazilian hospitals ([1][1]). Recently, we carried out a similar study, and although the bla OXA-143 gene was identified, its frequency was lower than that

Diversity of mechanisms conferring resistance to β-lactams among OXA-23-producing Acinetobacter baumannii clones.

A total of 31 unrelated OXA-23-producing Acinetobacter baumannii strains isolated from 14 hospitals located in distinct Brazilian regions were evaluated in this study. These isolates were grouped into 12 different sequence types (STs), of which 7 had unique allelic sequences (ST188, ST189, ST190, ST191, ST192, ST228, and ST299). Most isolates belonged to the clonal complex CC79 followed by CC15 and CC1. Only polymyxin B and minocycline showed good activity against the OXA-23-producing A. baumannii clones. The ISAba1 upstream blaOXA-23, blaOXA-51-like, or ampC was found in 100%, 54.8%, and 77.4% of the isolates, respectively. High resistance rates to ceftazidime and cefotaxime were observed among those isolates possessing ISAba1 upstream ampC, in contrast to those isolates that did not carry this configuration. Moreover, a ≥2 Log2 decrease in the MICs of meropenem and ceftazidime was observed in the presence of phenyl-arginine-β-naphthylamide for 80.6% and 54.8% of isolates, respectively. Overexpression of the adeB was observed in 61.3% of isolates, particularly among those isolates belonging to the ST1 (CC1). It was also verified that ompW was down-regulated in all isolates belonging to the ST15 (CC15). On the other hand, carO and omp33-36 genes were overexpressed in 48.4% and 58.1% of the isolates, respectively. In this study, we show that overexpression of AdeABC system could significantly contribute for resistance to meropenem and ceftazidime among OXA-23-producing A. baumannii clones in Brazil, demonstrating the complexity involved in the β-lactam resistance in such isolates.

Clinical and microbiological characterization of KPCproducing Klebsiella pneumoniae infections in Brazil

In 2008 isolates of KPC-producing Klebsiella pneumoniae (KPC-KPN) were detected for the first time at Hospital Heliópolis, São Paulo, Brazil. The aim of this study was to characterize the clinical and microbiological outcomes of infections caused by KPC-KPN. A historical cohort of patients from whom KPC-KPN strains were isolated was performed. Isolates were identified as resistant to ertapenem by automated broth microdilution system and screened as carbapenemase producers by the modified Hodge test. The beta-lactamase resistance gene blaKPC was detected by PCR. The genetic relatedness of isolates was determined by PFGE. The study provides early clinical experience in treating KPC-KPN infections in a Brazilian tertiary center.

Diversity of polymyxin resistance mechanisms among Acinetobacter baumannii clinical isolates.

Polymyxins have become drugs of last resort for treatment of multi-drug resistant (MDR) Gram-negative infections. However, the mechanisms of resistance to this compound have not been completely elucidated. In this study, we evaluated the mechanisms of resistance to this antimicrobial in two A. baumannii clinical isolates, respectively, susceptible (A027) and resistant (A009) to polymyxin B before and after polymyxin B exposure (A027ind and A009ind). The pmrAB and lpxACD were sequenced and their transcriptional levels were analyzed by qRT-PCR. The bacterial cell morphology was evaluated by transmission electronic microscopy (TEM) and the membrane potential was measured using Zeta-potential analyzer. The virulence of strains was studied using a Caenorhabditis elegans model. Both clinical isolates exhibited an elevation of the polymyxin B MIC after exposure to this compound. On the other hand, A027ind showed decreased values of MIC for β-lactams, aminoglycosides, vancomycin, teicoplanin, oxacillin and erythromycin. A027ind harbored two mutations in pmrB and the ISAba125 disrupting the lpxA. In contrast, A009ind strain exhibited increase of pmrB transcriptional level, after polymyxin B exposure, despite the absence of mutations in the pmrAB genes. The TEM images revealed a thicker and more electron-dense peptidoglycan layer for A009 than that of A027. The exposure to polymyxin B induced a strong condensation and darkening of intracellular material, mainly in A009ind. In addition, the surface charge of A009 was significantly less negative than the one of A027. Using the C. elegans model, only A027ind strain showed a reduction on virulence. The diversity of polymyxin B resistance mechanisms among A. baumannii strains evaluated in this study confirms the complexity of these mechanisms, which may vary depending of the background of each strain.

Antimicrobial activity of doripenem against Gram-negative pathogens: results from INVITA-A-DORI Brazilian study

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.

Antimicrobial activity of ceftobiprole against Gram-negative and Gram-positive pathogens: results from INVITA-A-CEFTO Brazilian study

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.

The polymyxin B-induced transcriptomic response of a clinical, multidrug-resistant Klebsiella pneumoniae involves multiple regulatory elements and intracellular targets

BackgroundThe emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling.ResultsWe sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13pH), magnesium deprivation (Kp13Mg), high concentrations of calcium (Kp13Ca) and iron (Kp13Fe), and a control condition with PB (Kp13PolB). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic.ConclusionsWe obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets.