Laura Zamorano

Hospital Universitari Son Espases, Palma, Balearic Islands, Spain

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Publications (42)158.17 Total impact

  • [Show abstract] [Hide abstract] ABSTRACT: Carbapenemase-producing Pseudomonas aeruginosa 14 is increasing world-wide linked to 15 wide-spread international high-risk clones (such as ST235, ST111 or ST175), thus representing 16 a major epidemiological and therapeutical challenge (1, 2).…
    No preview · Article · Mar 2016 · Antimicrobial Agents and Chemotherapy
  • [Show abstract] [Hide abstract] ABSTRACT: Pseudomonas aeruginosa , a major cause of nosocomial and chronic infections, is considered a paradigm of antimicrobial resistance development. However, the evolutionary trajectories of antimicrobial resistance and the impact of mutator phenotypes remain mostly unexplored. Therefore, whole genome sequencing (WGS) was performed in lineages of wild-type and mutator (Δ mutS ) strains exposed to growing concentrations of relevant antipseudomonal agents. WGS provided a privileged perspective of the dramatic effect of mutator phenotypes on the accumulation of random mutations, most of which were transitions as expected. Moreover, a frameshift mutagenic signature, consistent with error-prone DNA polymerase activity consequence of SOS system induction, was also seen. This effect was evidenced for all antibiotics tested, but it was higher for fluoroquinoles than for cephalosporins or carbapenems. Analysis of genotype vs phenotype confirmed expected resistance evolution trajectories but also revealed new pathways. Classical mechanisms included multiple mutations leading to AmpC overexpression (ceftazidime), QRDR mutations (ciprofloxacin), oprD inactivation (meropenem) or efflux pump overexpression (ciprofloxacin and meropenem). Groundbreaking findings included gain of function mutations leading to the structural modification of AmpC (ceftazidime), a novel DNA gyrase (GyrA) modification (ciprofloxacin) or the alteration of the β-lactam binding site of PBP3 (meropenem). A further striking finding was seen in the evolution of meropenem resistance, selecting for specific extremely large (>250 Kb) genomic deletions providing a growth advantage in the presence of the antibiotic. Finally, fitness and virulence varied within and across evolved antibiotic resistant populations, but mutator lineages showed a lower biological cost for some antibiotics.
    No preview · Article · Jan 2016 · Antimicrobial Agents and Chemotherapy
  • [Show abstract] [Hide abstract] ABSTRACT: Pseudomonas aeruginosa multidrug resistance, and particularly the production of carbapenemases linked to international high-risk clones, is of growing concern. While high levels of carbapenem resistance (>60 %) have been reported in Lithuania, so far, there is no information on the underlying mechanisms. Thus, the aim of this work was to determine the molecular epidemiology and prevalence of acquired carbapenemases among 73 carbapenem-resistant P. aeruginosa isolates recovered in a hospital from Kaunas, Lithuania in 2011–2012. The presence of acquired carbapenemases was evaluated through phenotypic (modified Hodge test, cloxacillin inhibition test, double-disc synergy test) and genetic methods [polymerase chain reaction (PCR) and sequencing]. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Acquired β-lactamases were detected in 19 (26 %) of the isolates, whereas resistance was exclusively chromosomal (OprD inactivation ± AmpC hyperproduction) in the remaining 54 (74 %) isolates. The acquired β-lactamases detected included 16 VIM-2, one PER-1 and two GES enzymes. PFGE revealed that 15 of the 16 VIM-2 isolates belonged to a single clone, identified as the international high-risk clone ST235 by MLST. bla VIM-2 was preceded by aacA7 in a class I integron, similar to epidemic ST235 isolates described in nearby countries. Additionally, sequencing of bla GES revealed the presence of the carbapenem-hydrolysing enzyme GES-5 in one of the isolates and a novel GES variant, designated GES-27, in the other. GES-27 differed from GES-5 by a single amino acid substitution, proline 167, that was replaced by glutamine. Increasing emergence and dissemination of concerning resistance mechanisms and international clones warrants global surveillance and control strategies.
    No preview · Article · Dec 2015 · European Journal of Clinical Microbiology
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    [Show abstract] [Hide abstract] ABSTRACT: Background: Most available information on carbapenemase-producing Enterobacteriaceae (CPE) is usually associated with specific types of infection or patient or with descriptions of outbreaks. The aim of this study was to comprehensively analyse the clinical epidemiology, clinical features and outcomes of colonisation and infections due to CPE in Spain. Methods: A multicentre prospective cohort study was carried out in 34 Spanish hospitals from February to May 2013. All new patients testing positive for CPE in clinical samples were included. Logistic regression was used to identify predictors of mortality. Results: Overall, 245 cases were included. The most frequent organism was Klebsiella pneumoniae (74%) and the carbapenemases belonged to the OXA-48 (74%), metallo-β-lactamase (MBL) (24%) and KPC (2%) groups. Acquisition was nosocomial in 145 cases (60%) and health care-associated (HCA) in 91 (37%); 42% of the latter were nursing home residents, in whom OXA-48-producing K. pneumoniae ST405 predominated. MBLs and OXA-48 predominated in ICU and medical patients, respectively. Overall, 67% of patients had infections. The most frequent infections identified in this study were urinary tract (43%) and skin structure (21%) infections, and 10% of infections were bacteraemic. Crude mortality was 20%. Inappropriate antibiotic therapy was independently associated with an increased risk of death (OR=3.30; 95% CI: 1.34-8.11). Conclusions: We found some differences in the epidemiology of CPE depending on the type of carbapenemase produced. Although a low proportion of CPE infections were bacteraemic, active antibiotic therapy was a protective factor for reducing mortality.
    Full-text · Article · Nov 2015 · The Journal of infection
  • [Show abstract] [Hide abstract] ABSTRACT: Objectives: The main objective of this study was to investigate the relationship among the in vivo acquisition of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates, the underlying molecular mechanisms and previous exposure to antipseudomonal agents. Methods: PFGE was used to study the molecular relatedness of the strains. The MICs of ceftazidime, cefepime, piperacillin/tazobactam, imipenem, meropenem, ciprofloxacin and amikacin were determined. Outer membrane protein profiles were assessed to study OprD expression. RT-PCR was performed to analyse ampC, mexB, mexD, mexF and mexY expression. The presence of mutations was analysed through DNA sequencing. Results: We collected 17 clonally related paired isolates [including first positive samples (A) and those with MICs increased ≥4-fold (B)]. Most B isolates with increased MICs of imipenem, meropenem and ceftazidime became resistant to these drugs. The most prevalent resistance mechanisms detected were OprD loss (65%), mexB overexpression (53%), ampC derepression (29%), quinolone target gene mutations (24%) and increased mexY expression (24%). Five (29%) B isolates developed multidrug resistance. Meropenem was the most frequently (71%) received treatment, explaining the high prevalence of oprD mutations and likely mexB overexpression. Previous exposure to ceftazidime showed a higher impact on selection of increased MICs than previous exposure to piperacillin/tazobactam. Conclusions: Stepwise acquisition of resistance has a critical impact on the resistance phenotypes of P. aeruginosa, leading to a complex scenario for finding effective antimicrobial regimens. In the clinical setting, meropenem seems to be the most frequent driver of multidrug resistance development, while piperacillin/tazobactam, in contrast to ceftazidime, seems to be the β-lactam least associated with the selection of resistance mechanisms.
    No preview · Article · Aug 2015 · Journal of Antimicrobial Chemotherapy
  • No preview · Article · Jul 2015 · Journal of Antimicrobial Chemotherapy
  • [Show abstract] [Hide abstract] ABSTRACT: We examined the genetic context of 74 acquired ampC genes and 17 carbapenemase genes from 85 out of 640 Enterobacteriaceae isolates collected in 2009. Using S1-PFGE and Southern hybridization, 37 out of 74 blaAmpC genes were located on large plasmids of different sizes belonging to six Inc groups. We used sequencing and PCR mapping to investigate the regions flanking the acquired ampC genes. The blaCMY-2like genes were associated with ISEcp1, the surrounding blaDHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons, and blaACC-1 genes were associated with IS26 elements inserted into ISEcp1. All the carbapenemase genes (blaVIM-1, two blaIMP-22 and blaIMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination of ampC genes among Enterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons are described in this study (In846 to In848). Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    No preview · Article · Jun 2015 · Antimicrobial Agents and Chemotherapy
  • [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to determine the impact of the carbapenemase-producing Enterobacteriaceae (CPE) in Spain in 2013 by describing their prevalence, dissemination and geographic distribution of CPE clones, their population structure and antibiotic susceptibility. From February 2013 to May 2013, 83 hospitals (about 40,000 hospital beds) prospectively collected non-duplicate Enterobacteriaceae using the screening cut-off recommended by EUCAST. Carbapenemase characterisation was performed by phenotypic methods and confirmed by PCR and sequencing. MLST types were determined for Klebsiella pneumoniae and Escherichia coli. A total of 702 Enterobacteriaceae isolates met the inclusion criteria; 379 (54%) were CPE. OXA-48 (71.5%) and VIM-1 (25.3%) were the most frequent carbapenemases, and K. pneumoniae (74.4%), Enterobacter cloacae (10.3%), and E. coli (8.4%) were the species most affected. Susceptibility to colistin, amikacin and meropenem was 95.5%, 81.3%, and 74.7%, respectively. The most prevalent STs were ST11 and ST405 in K. pneumoniae, and ST131 in E. coli. Forty-five (54.1%) of the hospitals had at least one CPE case. In K. pneumoniae, ST11/OXA-48, ST15/OXA-48, ST405/OXA-48, and ST11/VIM-1 were detected in two or more Spanish provinces. ST11 carried four carbapenemases (VIM-1, OXA-48, KPC-2, and OXA-245), but ST405 carried OXA-48 only. A wide interregional spread of CPE in Spain was observed mainly due to a few successful clones of OXA-48-producing K. pneumoniae (e.g. ST11 and ST405). Dissemination of OXA-48-producing E. coli is a new finding of public health concern. According to in vitro susceptibilities, most of the CPE (94.5%) had three or more options of antibiotic treatment. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    No preview · Article · Mar 2015 · Antimicrobial Agents and Chemotherapy
  • [Show abstract] [Hide abstract] ABSTRACT: Background: The type III secretion system (TTSS) is a major virulence determinant of Pseudomonas aeruginosa. The objective of this study was to determine whether the TTSS genotype is a useful prognostic marker of P. aeruginosa bacteremia mortality. We also studied the potential association between TTSS genotypes and multidrug-resistant (MDR) profiles, and how this interaction impacts the outcome of bloodstream infections. Methods: We performed a post hoc analysis of a published prospective multicenter cohort of P. aeruginosa bloodstream infections. The impact in mortality of TTSS genotypes (exoS, exoT, exoU, and exoY genes) and resistance profiles was investigated. Cox regression analysis was used to control for confounding variables. Results: Among 590 patients, the 30-day mortality rate was 30% (175 patients), and 53% of them died in the first 5 days (early mortality). The unadjusted probabilities of survival until 5 days was 31.4% (95% confidence interval [CI], 17.4%-49.4%) for the patients with exoU-positive isolates and 53.2% (95% CI, 44.6%-61.5%) for exoU-negative isolates (log rank P = .005). After adjustment for confounders, exoU genotype (adjusted hazard ratio [aHR], 1.90 [95% CI, 1.15-3.14]; P = .01) showed association with early mortality. In contrast, late (30-day) mortality was not influenced by TTSS genotype but was independently associated with MDR profiles (aHR,1.40 [95% CI, 1.01-1.94]; P = .04). Moreover, the exoU genotype (21% of all isolates) was significantly less frequent (13%) among MDR strains (particularly among extensively drug-resistant isolates, 5%), but was positively linked to moderately resistant (1-2 antipseudomonals) phenotypes (34%). Conclusions: Our results indicate that the exoU genotype, which is associated with specific susceptibility profiles, is a relevant independent marker of early mortality in P. aeruginosa bacteremia.
    No preview · Article · Nov 2014 · Clinical Infectious Diseases
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    [Show abstract] [Hide abstract] ABSTRACT: During a Spanish surveillance study, two natural variants of DHA β-lactamases, DHA-6 and DHA-7, were found, with the replacements Ala226Thr and Phe322Ser, respectively, with respect to DHA-1. The DHA-6 and DHA-7 enzymes were isolated from Escherichia coli and Enterobacter cloacae clinical isolates, respectively. The aim of this study was to genetically, microbiologically, and biochemically characterize the DHA-6 and DHA-7 β-lactamases. The blaDHA-6 and blaDHA-7 genes were located in the I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The genetic contexts of blaDHA-6 and blaDHA-7 were similar to that already described for the blaDHA-1 gene and included the qnrB4 and aadA genes. The MICs for cephalothin, aztreonam, cefotaxime, and ceftazidime were 8- to 32-fold lower for DHA-6 than for DHA-1 or DHA-7 expressed in the same isogenic E. coli TG1 strain. Interestingly, the MIC for cefoxitin was higher in the DHA-6-expressing transformant than in DHA-1 or DHA-7. Biochemical studies with pure β-lactamases revealed slightly lower catalytic efficiencies of DHA-6 against cephalothin, ceftazidime, and cefotaxime than those of DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed that the DHA-6 protein displayed significantly higher stability than the DHA-1 and DHA-7 enzymes. The proximity of Thr226 to the N terminus in the tertiary protein structure in DHA-6 may promote this stabilization and, consequently, may induce a slight reduction in the dynamic of this enzyme that primarily affects the hydrolysis of some of the bulkiest antibiotics.
    Full-text · Article · Aug 2014 · Antimicrobial Agents and Chemotherapy
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    [Show abstract] [Hide abstract] ABSTRACT: Pseudomonas aeruginosa is a ubiquitous versatile environmental microorganism with a remarkable ability to grow under diverse environmental conditions. Moreover, P. aeruginosa is responsible for life threatening infections in immunocompromised and cystic fibrosis patients, where the extraordinary capacity of this pathogen to develop antimicrobial resistance dramatically limits our therapeutic arsenal. Its large genome carries an outstanding number of genes belonging to regulatory systems, including multiple two component sensor-regulator systems modulating the response to the different environmental stimuli. Here we show that one of such systems, designated CreBC (carbon-source responsive) or BlrAB (beta -lactam resistance), might be of particular relevance. We first identified the stimuli triggering the activation of the CreBC system, specifically responding to PBP4 inhibition by certain β-lactam antibiotics. Second, through the analysis of a large comprehensive collection of mutants we demonstrate an intricate interconnection between the CreBC system, the peptidoglycan recycling pathway and the expression of the concerning chromosomal β-lactamase AmpC. Third, we show that the CreBC system, and particularly its effector inner membrane protein CreD, plays a major role in bacterial fitness and biofilm development, especially in the presence of subinhibitory concentrations of β-lactams. Finally, global transcriptomics reveals broad regulatory functions of CreBC in basic physiological aspects, particularly anaerobic respiration, both in the presence and absence of antibiotics. Therefore, the CreBC system is envisaged as a potentially interesting target for improving the efficacy of β-lactams against P. aeruginosa infections.
    Full-text · Article · Jun 2014 · Antimicrobial Agents and Chemotherapy
  • [Show abstract] [Hide abstract] ABSTRACT: We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, mutS-) Pseudomonas aeruginosa. The strains were incubated for 24h with 0.5-64x MIC concentrations of each antibiotic in triplicate experiments. Tubes from the highest antibiotic concentration showing growth were re-inoculated in fresh medium containing concentrations up to 64x MIC for 7 consecutive days. Susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic and strain. Ceftolozane-tazobactam resistant mutants were further characterized by whole genome analysis through RNA-seq. High-level resistance development was fastest for ceftazidime followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed crossresistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower and high-level resistance was only observed for the mutator strain. Ceftolozane-tazobactam moderately resistant (MICs 4-8 μg/ml) PAO1 derivatives showed only 2-4 mutations, that determined global pleiotropic effects associated with a severe fitness cost. High-level resistant (MICs 32-128 μg/ml) PAOMS derivatives showed 45-53 mutations. Major changes in global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1-4 mutations in conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, when compared with wild-type ampC. Therefore, development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.
    No preview · Article · Mar 2014 · Antimicrobial Agents and Chemotherapy
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    [Show abstract] [Hide abstract] ABSTRACT: A limited number of Pseudomonas aeruginosa genotypes (mainly ST-111, ST-175, and ST-235), known as high-risk clones, are responsible for epidemics of nosocomial infections by multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains worldwide. We explored the potential biological parameters that may explain the success of these clones. A total of 20 isolates from each of 4 resistance groups (XDR, MDR, ModR [resistant to 1 or 2 classes], and MultiS [susceptible to all antipseudomonals]), recovered from a multicenter study of P. aeruginosa bloodstream infections performed in 10 Spanish hospitals, were analyzed. A further set of 20 XDR isolates belonging to epidemic high-risk clones (ST-175 [n = 6], ST-111 [n = 7], and ST-235 [n = 7]) recovered from different geographical locations was also studied. When unknown, genotypes were documented through multilocus sequence typing. The biological parameters evaluated included twitching, swimming, and swarming motility, biofilm formation, production of pyoverdine and pyocyanin, spontaneous mutant frequencies, and the in vitro competition index (CI) obtained with a flow cytometry assay. All 20 (100%) XDR, 8 (40%) MDR, and 1 (5%) ModR bloodstream isolate from the multicenter study belonged to high-risk clones. No significant differences were observed between clonally diverse ModR and MultiS isolates for any of the parameters. In contrast, MDR/XDR high-risk clones showed significantly increased biofilm formation and mutant frequencies but significantly reduced motility (twitching, swimming, and swarming), production of pyoverdine and pyocyanin, and fitness. The defined biological markers of high-risk clones, which resemble those resulting from adaptation to chronic infections, could be useful for the design of specific treatment and infection control strategies.
    Full-text · Article · Nov 2013 · Antimicrobial Agents and Chemotherapy
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    [Show abstract] [Hide abstract] ABSTRACT: A novel class C β-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant.
    Full-text · Article · Jul 2013 · Antimicrobial Agents and Chemotherapy
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    [Show abstract] [Hide abstract] ABSTRACT: A panel of 29 multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from seven hospitals as part of a country-wide surveillance of antimicrobial resistance in Bulgarian hospitals was studied. Molecular typing through multiple-locus variable number tandem-repeat analysis (MLVA6) yielded 23 different profiles. Phenotypic and genotypic tests for the detection of acquired carbapenemases yielded negative results in all cases. In contrast, 76% of the isolates produced other acquired β-lactamases, including extended-spectrum β-lactamases (ESBLs). Namely, 6 of the isolates (21%) produced a VEB-1 ESBL; 14 (48%) produced an OXA-10-type enzyme (7 OXA-10 and 7 OXA-10 ESBL variants, including 2 OXA-17 [A218G], 2 OXA-74 [C197T, A218G], and 3 OXA-142 [A218G, G470A]); 8 (28%) an OXA-2-type enzyme (all OXA-2); and 1 (3%) a PSE-1 carbenicillinase. Further analysis through multilocus sequence typing (MLST) revealed that the six VEB-1-producing strains, recovered from four hospitals, belonged to ST111 or ST244 international high-risk clones. Additionally, nearly all of the isolates (97%) lacked OprD production, explaining carbapenem resistance. Overexpression of AmpC was documented in 5 (17%) of the isolates, including most of the MDR isolates not producing any acquired β-lactamase. Particularly noteworthy was the very high prevalence of MexXY-OprM overexpression, documented in 72% of the isolates, whereas the prevalence of MexAB-OprM overexpression was lower (21%). In summary, while the production of metallo-β-lactamases is uncommon among P. aeruginosa isolates from Bulgarian hospitals, MDR profiles frequently result from the production of ESBLs combined with the lack of production of the carbapenem porin OprD and the overexpression of the MexXY-OprM efflux pump.
    Full-text · Article · Apr 2013 · Microbial drug resistance (Larchmont, N.Y.)
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    Dataset: Table S1
    No preview · Dataset · Feb 2013
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    Dataset: Figure S1
    [Show abstract] [Hide abstract] ABSTRACT: Prevalence of plasmid-mediated AmpC β-lactamases and carbapenemases in different Spanish Autonomous Communities
    No preview · Dataset · Feb 2013
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    Full-text · Article · Oct 2012 · European Journal of Clinical Microbiology
  • [Show abstract] [Hide abstract] ABSTRACT: The purpose of this investigation was to determine the prevalence of plasmid-mediated AmpC (pAmpC) and carbapenemases in Enterobacteriaceae collected from 35 hospitals in Spain and to establish their epidemiological relationships. We conducted a prospective multi-centre study on pAmpC- or carbapenemase-producing Enterobacteriaceae isolates from clinical samples collected from February to July 2009. The strains suspected to carry pAmpC were resistant or showed intermediate susceptibility to co-amoxiclav and second- or third-generation cephalosporins. Strains suspected to carry a carbapenemase were selected because they showed a minimum inhibitory concentration (MIC) to imipenem >1 mg/L. Polymerase chain reaction (PCR) and a sequencing strategy were used to characterise the enzymes. The clonal relationships between isolates was analysed by pulsed field gel electrophoresis (PFGE). Among 100,132 Enterobacteriaceae isolates collected, 1,654 were compatible with the production of pAmpC or carbapenemases. We found a prevalence of 0.64 % of pAmpC (n = 635) and 0.04 % of carbapenemases (n = 43). The most prevalent pAmpC enzymes were CMY-type (78.3 %), DHA-type (19.5 %), ACC-type (1.6 %) and FOX-type (0.6 %). The CMY-type was the most frequent in Escherichia coli and Proteus mirabilis species, whereas the DHA-type was mainly found in Klebsiella spp. The enzymes involved in carbapenem resistance were VIM-1, IMP-22 and the new IMP-28. Nine new bla genes were described: bla CMY-54, bla CMY-55, bla CMY-56, bla CMY-57, bla CMY-96, bla DHA-6, bla DHA-7, bla FOX-8 and bla IMP-28. The prevalence of pAmpC or carbapenemases found is not negligible. The CMY-types were the predominant pAmpC, whereas the VIM or IMP enzymes were the predominant carbapenemases. Furthermore, we observed a great genetic diversity among pAmpC-producing strains and a close clonal relationship between carbapenemase-producing strains.
    No preview · Article · Sep 2012 · European Journal of Clinical Microbiology
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    [Show abstract] [Hide abstract] ABSTRACT: We investigated the mechanisms leading to Pseudomonas aeruginosa pan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence of oprD, ampD, and dacB mutations was explored by PCR followed by sequencing and the expression of ampC and efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC50s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressed ampC and one or several efflux pumps, particularly mexB and/or mexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n = 1) or diminished expression (n = 1) of PBP1a and a lack of PBP4 expression (n = 4), which correlated with AmpC overexpression driven by dacB mutation. Analysis of the essential PBP IC50s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC50s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in the in vivo emergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.
    Full-text · Article · Jun 2012 · Antimicrobial Agents and Chemotherapy