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Background: The optimal antibiotic regimen for Pseudomonas aeruginosa bacteremia is controversial. Although β-lactam monotherapy is common, data to guide the choice between antibiotics are scarce. We aimed to compare ceftazidime, carbapenems, and piperacillin-tazobactam as definitive monotherapy., Methods: A multinational retrospective study (9 countries, 25 centers) including 767 hospitalized patients with P. aeruginosa bacteremia treated with β-lactam monotherapy during 2009-2015. The primary outcome was 30-day all-cause mortality. Univariate and multivariate, including propensity-adjusted, analyses were conducted introducing monotherapy type as an independent variable., Results: Thirty-day mortality was 37/213 (17.4%), 42/210 (20%), and 55/344 (16%) in the ceftazidime, carbapenem, and piperacillin-tazobactam groups, respectively. Type of monotherapy was not significantly associated with mortality in either univariate, multivariate, or propensity-adjusted analyses (odds ratio [OR], 1.14; 95% confidence interval [CI], 0.52-2.46, for ceftazidime; OR, 1.3; 95% CI, 0.67-2.51, for piperacillin-tazobactam, with carbapenems as reference in propensity adjusted multivariate analysis; 542 patients). No significant difference between antibiotics was demonstrated for clinical failure, microbiological failure, or adverse events. Isolation of P. aeruginosa with new resistance to antipseudomonal drugs was significantly more frequent with carbapenems (36/206 [17.5%]) versus ceftazidime (25/201 [12.4%]) and piperacillin-tazobactam (28/332 [8.4%] (P = .007)., Conclusions: No significant difference in mortality, clinical, and microbiological outcomes or adverse events was demonstrated between ceftazidime, carbapenems, and piperacillin-tazobactam as definitive treatment of P. aeruginosa bacteremia. Higher rates of resistant P. aeruginosa after patients were treated with carbapenems, along with the general preference for carbapenem-sparing regimens, suggests using ceftazidime or piperacillin-tazobactam for treating susceptible infection., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

This study aimed to evaluate risk factors for 30-day mortality among hospitalised patients with Pseudomonas aeruginosa bacteraemia, a highly fatal condition. A retrospective study was conducted between 1 January 2009 and 31 October 2015 in 25 centres (9 countries) including 2396 patients. Univariable and multivariable analyses of risk factors were conducted for the entire cohort and for patients surviving ≥48 h. A propensity score for predictors of appropriate empirical therapy was introduced into the analysis. Of the 2396 patients, 636 (26.5%) died within 30 days. Significant predictors (odds ratio and 95% confidence interval) of mortality in the multivariable analysis included patient-related factors: age (1.02, 1.01-1.03); female sex (1.34, 1.03-1.77); bedridden functional capacity (1.99, 1.24-3.21); recent hospitalisation (1.43, 1.07-1.92); concomitant corticosteroids (1.33, 1.02-1.73); and Charlson comorbidity index (1.05, 1.01-1.93). Infection-related factors were multidrug-resistant Pseudomonas (1.52, 1.15-2.1), non-urinary source (2.44, 1.54-3.85) and Sequential Organ Failure Assessment (SOFA) score (1.27, 1.18-1.36). Inappropriate empirical therapy was not associated with increased mortality (0.81, 0.49-1.33). Among 2135 patients surviving ≥48 h, hospital-acquired infection (1.59, 1.21-2.09), baseline endotracheal tube (1.63, 1.13-2.36) and ICU admission (1.53, 1.02-2.28) were additional risk factors. Risk factors for mortality among patients with P. aeruginosa were mostly irreversible. Early appropriate empirical therapy was not associated with reduced mortality. Further research should be conducted to explore subgroups that may not benefit from broad-spectrum antipseudomonal empirical therapy. Efforts should focus on prevention of infection, mainly hospital-acquired infection and multidrug-resistant pseudomonal infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

BackgroundAntibiotic resistance, either intrinsic or acquired, is a major obstacle for treating bacterial infections.AimOur objective was to compare the country-specific species distribution of the four Gram-negative species Escherichia coli , Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter species and the proportions of selected acquired resistance traits within these species.MethodWe used data reported for 2016 to the European Antimicrobial Resistance Surveillance Network (EARS-Net) by 30 countries in the European Union and European Economic Area.ResultsThe country-specific species distribution varied considerably. While E. coli accounted for 31.9% to 81.0% (median: 69.0%) of all reported isolates, the two most common intrinsically resistant species P. aeruginosa and Acinetobacter spp. combined (PSEACI) accounted for 5.5% to 39.2% of isolates (median: 10.1%). Similarly, large national differences were noted for the percentages of acquired non-susceptibility to third-generation cephalosporins, carbapenems and fluoroquinolones. There was a strong positive rank correlation between the country-specific percentages of PSEACI and the percentages of non-susceptibility to the above antibiotics in all four species (rho > 0.75 for 10 of the 11 pairs of variables tested).ConclusionCountries with the highest proportion of P. aeruginosa and Acinetobacter spp. were also those where the rates of acquired non-susceptibility in all four studied species were highest. The differences are probably related to national differences in antibiotic consumption and infection prevention and control routines.

Objectives: We evaluated the RAPIDEC(®) CARBA NP assay (bioMérieux SA, Marcy-l'Étoile, France), a colorimetric test for rapid detection of carbapenemases, at two sites: Karolinska University Laboratory and PHE's national reference laboratory., Methods: A total of 138 bacterial isolates previously characterized as positive for class A, B and/or D carbapenemase genes and 138 supposed non-carbapenemase producers were tested with RAPIDEC(®) CARBA NP according to the manufacturer's protocol. Two carbapenemase-producing isolates carried both NDM and OXA-48-like genes. Molecular detection of the expected carbapenemase gene(s) was used as the gold standard, and was performed by conventional and real-time PCR in-house assays., Results: The RAPIDEC(®) CARBA NP assay detected 135 of 138 carbapenemase producers; one OXA-48-producing Klebsiella pneumoniae and two Acinetobacter baumannii producing OXA-23 or OXA-24 were not detected. Among 'negative' controls, 135 of 138 isolates were negative by RAPIDEC(®) CARBA NP. The exceptions were one Klebsiella oxytoca, which was later found to produce GES-5 carbapenemase, one Pseudomonas aeruginosa with OprD loss and increased efflux, and one Enterobacter cloacae with impermeability. When numbers were adjusted for the GES-5 producer, the overall sensitivity of the RAPIDEC(®) CARBA NP test was 97.8% and its specificity was 98.5%., Conclusions: The assay took less than 2.5 h to carry out, was user-friendly and had a high overall performance, making it an attractive option for clinical laboratories., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: The increase in carbapenemase producing Enterobacteriaceae and Pseudomonas aeruginosa is a significant threat to modern medicine. A rapid detection of carbapenemase production in Klebsiella pneumoniae and Pseudomonas aeruginosa is of importance for the institution of correct antibiotic treatment and infection control measures., Results: Standardised inoculums of K. pneumoniae or P. aeruginosa were incubated at 37 °C with ertapenem in 15 and 120 min followed by centrifugation. The supernatant was applied on a steel target plate, covered with HCCA matrix and analysed using a Microflex(TM) (Bruker Daltonics) in the mass range of 4-600 Da. The assay detected and separated KPC from other carbapenemases in K. pneumoniae after only 15 min incubation. In P. aeruginosa, however, only 8/14 isolates of VIM-producing P. aeruginosa were detected. None of the tested carbapenemase negative isolates displayed a pattern of hydrolysis of ertapenem., Conclusions: This assay allows for a very rapid detection and verification of KPC (45 min including the preparation steps) and MBL production (150 min) in K. pneumoniae and can be performed using standard matrix. However, the study revealed the need for optimization of the substrate/species combination in assays for the detection of carbapenemases in P. aeruginosa using MALDI-TOF.

Several molecular typing schemes have been proposed to differentiate among isolates and clonal groups, and hence establish epidemiological or phylogenetic links. It has been widely accepted that multi-locus sequence typing (MLST) is the gold standard for phylogenetic typing/long-term epidemiological surveillance, but other recently described methods may be easier to carry out, especially in settings with limited access to DNA sequencing. Comparing the performance of such techniques to MLST is therefore of relevance. A study was therefore carried out with a collection of P. aeruginosa strains (n = 133) typed by four typing schemes: MLST, multiple-locus variable number tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE) and the commercial DiversiLab microbial typing system (DL). The aim of this study was to compare the results of each typing method with MLST. The Simpson's indices of diversity were 0.989, 0.980, 0.961 and 0.906 respectively for PFGE, MLVA, DL and MLST. The congruence between techniques was measured by the adjusted Wallace index (W): this coefficient indicates the probability that a pair of isolates which is assigned to the same type by one typing method is also typed as identical by the other. In this context, the congruence between techniques was recorded as follow: MLVA-type to predict MLST-type (93%), PFGE to MLST (92%), DL to MLST (64.2%), PFGE to MLVA (63.5%) and PFGE to DL (61.7%). Conversely, for all above combinations, prediction was very poor. The congruence was increased at the clonal complex (CC) level. MLST is regarded the gold standard for phylogenetic classification of bacteria, but is rather laborious to carry out in many settings. Our data suggest that MLVA can predict the MLST-type with high accuracy, and even higher when studying the clonal complex level. Of the studied three techniques MLVA was therefore the best surrogate method to predict MLST.

Herein, we describe the phenotypic and genotypic characterization of a multiresistant clone of Pseudomonas aeruginosa disseminating in a burn unit in Orumieh, Iran. A total of 58 isolates of P. aeruginosa were collected during August 2007 and June 2008. Minimum inhibitory concentrations (MICs) of P. aeruginosa isolates were determined against 11 antimicrobial agents by E test. Serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were used for studying the clonal relationship among the isolates. Antibiotic susceptibility testing revealed that most of the isolates were multidrug resistant and colistin was the antibiotic with the highest activity. Pseudomonas aeruginosa isolates fell into nine different serotypes, and O10 and O11 were the most common. PFGE analyses showed 12 different genotypes and 68.1% of isolates showed more than 80% similarity, indicating possible clonal relatedness. These isolates were found to belong to the same sequence type, ST773. This sequence type has earlier been reported from China, and a double locus variant of this ST has been found earlier in France in a PER-1 extended-spectrum β-lactamase-producing P. aeruginosa., (© 2012 The Authors APMIS © 2012 APMIS.)

Several studies in recent years have provided evidence that Pseudomonas aeruginosa has a non-clonal population structure punctuated by highly successful epidemic clones or clonal complexes. The role of recombination in the diversification of P. aeruginosa clones has been suggested, but not yet demonstrated using multi-locus sequence typing (MLST). Isolates of P. aeruginosa from five Mediterranean countries (n = 141) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping and PCR targeting the virulence genes exoS and exoU. The occurrence of multi-resistance (≥ 3 antipseudomonal drugs) was analyzed with disk diffusion according to EUCAST. MLST was performed on a subset of strains (n = 110) most of them had a distinct PFGE variant. MLST data were analyzed with Bionumerics 6.0, using minimal spanning tree (MST) as well as eBURST. Measurement of clonality was assessed by the standardized index of association (I(A) (S)). Evidence of recombination was estimated by ClonalFrame as well as SplitsTree4.0. The MST analysis connected 70 sequence types, among which ST235 was by far the most common. ST235 was very frequently associated with the O11 serotype, and frequently displayed multi-resistance and the virulence genotype exoS⁻/exoU⁺. ClonalFrame linked several groups previously identified by eBURST and MST, and provided insight to the evolutionary events occurring in the population; the recombination/mutation ratio was found to be 8.4. A Neighbor-Net analysis based on the concatenated sequences revealed a complex network, providing evidence of frequent recombination. The index of association when all the strains were considered indicated a freely recombining population. P. aeruginosa isolates from the Mediterranean countries display an epidemic population structure, particularly dominated by ST235-O11, which has earlier also been coupled to the spread of ß-lactamases in many countries.

Scandinavia is considered a region with a low prevalence of antimicrobial resistance. However, the number of multidrug-resistant (MDR) Gram-negative bacteria is increasing, including metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa. In this study MBL-producing P. aeruginosa isolates identified in Norway (n = 4) and Sweden (n = 9) from 1999 to 2007 were characterized. Two international clonal complexes (CC), CC111 (n = 8) and CC235 (n = 2), previously associated with MBL-producing isolates, were dominant. CC111 isolates (ST111/229; serotype O12; bla(VIM-2)) included clonally related isolates identified in Skåne County, Sweden (n = 6), and two isolates associated with importation from Greece and Denmark. In all CC111 isolates, bla(VIM-2) was located in integron In59.2 or In59 variants. The two CC235 isolates (ST235/ST230; serotype O11; bla(VIM-4)) were imported from Greece and Cyprus, were possibly clonally related, and carried bla(VIM-4) in two different integron structures. Three isolates imported from Ghana (ST233; serotype O6; bla(VIM-2)), Tunisia (ST654; serotype O11; bla(VIM-2)), and Thailand (ST260; serotype O6; bla(IMP-14)) were clonally unrelated. ST233 was part of a new CC (CC233) that included other MBL-producing isolates, while ST654 could also be part of a new CC associated with MBL producers. In the isolates imported from Ghana and Tunisia, bla(VIM-2) was part of unusual integron structures lacking the 3' conserved segment and associated with transposons. The bla(VIM) gene was found to be located on the chromosome in all isolates. Known risk factors for acquisition of MBL were reported for all patients except one. The findings suggest that both import of successful international clones and local clonal expansion contribute to the emergence of MBL-producing P. aeruginosa in Scandinavia.

Objectives: To evaluate four phenotypic tests for the detection of metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa in a low MBL prevalence setting., Methods: Sixty clinical isolates of P. aeruginosa resistant to imipenem and/or meropenem and seven MBL-positive control strains were examined by: (i) MBL Etest; (ii) combined imipenem discs supplemented with EDTA (IPM-EDTA); (iii) beta-lactam discs on dipicolinic acid plates (DF-DIPI); and (iv) the Cica-beta test. Spectrophotometric analysis of crude cell extracts for imipenem hydrolysis along with consensus PCRs for bla(VIM) and bla(IMP) was used as reference methods., Results: Two clinical isolates (3%) were MBL-positive. The MBL Etest and IPM-EDTA test scored positive for all MBL-positive isolates, but showed specificities of 86% and 91%, and positive predictive values (PPVs) of only 20% and 29%, respectively. Adding resistance to ceftazidime (MIC >8 mg/L) as a criterion for MBL testing would reduce the number of isolates to be screened by 50% and increase the PPVs of the MBL Etest and IMP-EDTA test to 29% and 40%, respectively. The Cica-beta test correctly identified all MBL-negative isolates, but misidentified one MBL-positive clinical isolate as an extended-spectrum beta-lactamase (ESBL)-producer and one as inconclusive (producing multiple beta-lactamases). No reliable breakpoints could be defined for the DF-DIPI test due to overlapping inhibition zone diameters for MBL-positive and -negative isolates., Conclusions: None of the phenotypic tests were optimal due to low sensitivity or specificity, resulting in low PPVs. Including ceftazidime resistance to the MBL-screening criteria would significantly improve the performance of the MBL Etest and IPM-EDTA disc test.

Carbapenem-resistant clinical isolates of Pseudomonas aeruginosa from Sweden and Norway (n=27) were characterized regarding transcription of genes encoding OprD, efflux pumps MexAB-OprM and MexCD-OprJ, as well as penicillin-binding proteins (PBP) 2 and 3. Quantification of mRNA was performed with real-time RT-PCR. Levels of mRNA in clinical isolates were compared to ATCC 27853 and average transcription levels of four carbapenem-susceptible clinical isolates of P. aeruginosa. Sequencing of oprD, pbp 2, and pbp 3 was performed in selected isolates. An efflux inhibition assay was performed with Phe-Arg-beta-naphtylamide. Most carbapenem-resistant isolates had decreased levels of oprD mRNA. Among six isolates with normal amount of oprD mRNA, half of them had significant OprD sequence alterations. Increased transcription of mexB was observed in 16 of 23 meropenem-resistant isolates, and one isolate showed mexD hyperproduction. Decreased amount of pbp 2 and pbp 3 was found in two and three isolates, respectively. Sequencing of pbp 2 and 3 revealed no amino acid changes potentially leading to conformational changes. In conclusion, OprD changes were the predominant mechanism of high-level carbapenem resistance, and increased transcription of mexB was often present in meropenem-resistant isolates. Reduced transcription of pbp 2 and 3 may contribute to the carbapenem resistance.

Pseudomonas aeruginosa is 1 of the bacteria most adaptive to anti-bacterial treatment. Previous studies have shown nosocomial spread and transmission of clonal strains of P. aeruginosa in European hospitals. In this study we investigated antibiotic susceptibility and clonality in 101 P. aeruginosa isolates from 88 patients admitted to 8 Swedish ICUs during 2002. We also compared phenotypes and genotypes of P. aeruginosa and carried out cluster analysis to determine if phenotypic data can be used for surveillance of clonal spread. All isolates were collected on clinical indication as part of the NPRS II study in Sweden and were subjected to AFLP analysis for genotyping. 68 isolates with unique genotypes were found. Phenotyping was performed using MIC values for 5 anti-pseudomonal agents. Almost 6% of the isolates were multi-drug resistant (MDR), and this figure rose to almost 8% when intermediate isolates were also included. We found probable clonal spread in 9 cases, but none of them was found to be an MDR strain. Phenotypical cluster analysis produced 40 clusters. Comparing partitions did not demonstrate any significant concordance between the typing methods. The conclusion of our study is that cross-transmission and clonal spread of MDR P. aeruginosa does not present a clinical problem in Swedish ICUs, but probable cross-transmission of non-MDR clones indicate a need for improved hygiene routines bedside. The phenotype clusters were not concordant with genotype clusters, and genotyping is still recommended for epidemiological tracking.

Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-beta-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE>RAPD>MLST>fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying blaVIM-1-like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different blaVIM-containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.

Imipenem resistance in Pseudomonas aeruginosa is considered to be associated with loss of the porin OprD combined with activity of chromosomal beta-lactamase (AmpC), while overexpression of multidrug efflux pumps is considered to confer meropenem resistance. Carbapenem resistance can also result from production of metallo-beta-lactamases. Transcription of oprD and efflux pump genes mexB, mexY and mexF was analysed in 23 clinical isolates of P. aeruginosa by quantitative RT-PCR. oprD was sequenced in all, and mexR, regulator of efflux pump MexAB-OprM, in selected isolates. Four isolates that were imipenem susceptible had significant reduction of oprD mRNA and presence of oprD mutations causing frameshift or translational stop. In strains only resistant to imipenem no significant difference in transcription of oprD was observed between low-level and high-level resistant isolates. The differences could not be explained by either pattern of oprD mutations. Increased transcription of mexB generally correlated well with meropenem resistance. One high-level meropenem-resistant isolate showed no significant change in mexB mRNA, but sequencing confirmed presence of a nalB mutation. Furthermore, one meropenem-susceptible isolate showed significant increase in mexB transcription, but no mexR mutations. In summary, our findings indicate that the resistance patterns observed cannot be fully explained by the currently described carbapenem resistance mechanisms.