Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions.

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress.

Impact of multidrug resistance on the virulence and fitness of Pseudomonas aeruginosa: a microbiological and clinical perspective.

Pseudomonas aeruginosa is one of the most common nosocomial pathogens and part of the top emergent species associated with antimicrobial resistance that has become one of the greatest threat to public health in the twenty-first century. This bacterium is provided with a wide set of virulence factors that contribute to pathogenesis in acute and chronic infections. This review aims to summarize the impact of multidrug resistance on the virulence and fitness of P. aeruginosa. Although it is generally assumed that acquisition of resistant determinants is associated with a fitness cost, several studies support that resistance mutations may not be associated with a decrease in virulence and/or that certain compensatory mutations may allow multidrug resistance strains to recover their initial fitness. We discuss the interplay between resistance profiles and virulence from a microbiological perspective but also the clinical consequences in outcomes and the economic impact.

A case-control study of the clinical and economic impact of infections caused by Carbapenemase-producing Enterobacterales (CPE).

PURPOSE: The aim was to analyse the clinical and economic impact of carbapenemase-producing Enterobacterales (CPE) infections. METHODS: Case-control study. Adult patients with CPE infections were considered cases, while those with non-CPE infections were controls. Matching criteria were age (± 5 years), sex, source of infection and microorganism (ratio 1:2). Primary outcome was 30-day mortality. Secondary outcomes were 90-day mortality, clinical failure, hospitalisation costs and resource consumption. RESULTS: 246 patients (82 cases and 164 controls) were included. Klebsiella pneumoniae OXA-48 was the most common microorganism causing CPE infections. CPE cases had more prior comorbidities (p = 0.007), septic shock (p = 0.003), and were more likely to receive inappropriate empirical and definitive antibiotic treatment (both p < 0.001). Multivariate analysis identified septic shock and inappropriate empirical treatment as independent predictors for 7-day and end-of-treatment clinical failure, whereas Charlson Index and septic shock were associated with 30- and 90-day mortality. CPE infection was independently associated with early clinical failure (OR 2.18, 95% CI, 1.03-4.59), but not with end-of-treatment clinical failure or 30- or 90-day mortality. In terms of resource consumption, hospitalisation costs for CPE were double those of the non-CPE group. CPE cases had longer hospital stay (p < 0.001), required more long-term care facilities (p < 0.001) and outpatient parenteral antibiotic therapy (p = 0.007). CONCLUSIONS: The CPE group was associated with worse clinical outcomes, but this was mainly due to a higher comorbidity burden, more severe illness, and more frequent inappropriate antibiotic treatment rather than resistance patterns as such. However, the CPE group consumed more healthcare resources and incurred higher costs.

Studying Macromolecular Interactions of Cellular Machines by the Combined Use of Analytical Ultracentrifugation, Light Scattering, and Fluorescence Spectroscopy Methods.

Cellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and their modulation in response to specific or unspecific factors. We also illustrate, with several examples taken from studies on bacterial processes, the advantages of the combined use of subsets of these techniques as orthogonal analytical methods to analyze protein oligomerization and polymerization, interactions with ligands, hetero-associations involving membrane proteins, and protein-nucleic acid complexes.

Significant increase of CTX-M-15-ST131 and emergence of CTX-M-27-ST131 Escherichia coli high-risk clones causing healthcare-associated bacteraemia of urinary origin in Spain (ITUBRAS-2 project).

OBJECTIVES: To assess the microbiological characteristics of Escherichia coli causing healthcare-associated bacteraemia of urinary origin (HCA-BUO) in Spain (ITUBRAS-2 project), with particular focus on ESBL producers and isolates belonging to ST131 high-risk clone (HiRC). Clinical characteristics and outcomes associated with ST131 infection were investigated. METHODS: A total of 222 E. coli blood isolates were prospectively collected from patients with HCA-BUO from 12 tertiary-care hospitals in Spain (2017-19). Antimicrobial susceptibility and ESBL/carbapenemase production were determined. ST131 subtyping was performed. A subset of 115 isolates were selected for WGS to determine population structure, resistome and virulome. Clinical charts were reviewed. RESULTS: ESBL-producing E. coli prevalence was 30.6% (68/222). ST131 represented 29.7% (66/222) of E. coli isolates and accounted for the majority of ESBL producers (46/68, 67.6%). The C2/H30-Rx subclone accounted for most ST131 isolates (44/66) and was associated with CTX-M-15 (37/44) and OXA-1 enzymes (27/44). Cluster C1-M27 was identified in 4/10 isolates belonging to subclade C1/H30-R1 and associated with CTX-M-27. Additionally, ST131 isolates showed a high content of other acquired resistance genes, and clade C/ST131 isolates carried characteristic QRDR mutations. They were categorized as uropathogenic E. coli and had higher aggregate virulence scores. ST131 infection was associated with more complex patients, prior use of cephalosporins and inadequate empirical treatment but was not associated with worse clinical outcomes. CONCLUSIONS: ST131 HiRC is the main driver of ESBL-producing E. coli causing HCA-BUO in Spain, mainly associated with the expansion of subclade CTX-M-15-C2/H30-Rx and the emergence of CTX-M-27-C1/H30-R1 (Cluster C1-M27).

Lipid Surfaces and Glutamate Anions Enhance Formation of Dynamic Biomolecular Condensates Containing Bacterial Cell Division Protein FtsZ and Its DNA-Bound Regulator SlmA.

Dynamic biomolecular condensates formed by liquid-liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the formation of FtsZ-containing condensates when compared to potassium chloride in crowded solutions. These condensates accumulated on supported lipid bilayers and eventually fused, resulting in a time-dependent increase in the droplet size. Moreover, the accumulated condensates were dynamic, capturing protein from the external phase. FtsZ partitioned into the condensates at the lipid surface only in its guanosine diphosphate (GDP) form, regardless of whether it came from FtsZ polymer disassembly upon guanosine triphosphate (GTP) exhaustion. These results provide insights into the behavior of these GTP-responsive condensates in minimal membrane systems, which suggest how these membraneless assemblies may tune critical bacterial division events during the cell cycle.

Comparison of Hospitalized Coronavirus Disease 2019 and Influenza Patients Requiring Supplemental Oxygen in a Cohort Study: Clinical Impact and Resource Consumption.

BACKGROUND: To compare clinical characteristics, outcomes, and resource consumption of patients with coronavirus disease 2019 (COVID-19) and seasonal influenza requiring supplemental oxygen. METHODS: Retrospective cohort study conducted at a tertiary-care hospital. Patients admitted because of seasonal influenza between 2017 and 2019, or with COVID-19 between March and May 2020 requiring supplemental oxygen were compared. Primary outcome: 30-day mortality. Secondary outcomes: 90-day mortality and hospitalization costs. Attempted sample size to detect an 11% difference in mortality was 187 patients per group. RESULTS: COVID-19 cases were younger (median years of age, 67; interquartile range [IQR] 54-78 vs 76 [IQR 64-83]; P < .001) and more frequently overweight, whereas influenza cases had more hypertension, immunosuppression, and chronic heart, respiratory, and renal disease. Compared with influenza, COVID-19 cases had more pneumonia (98% vs 60%, <.001), higher Modified Early Warning Score (MEWS) and CURB-65 (confusion, blood urea nitrogen, respiratory rate, systolic blood pressure, and age >65 years) scores and were more likely to show worse progression on the World Health Organization ordinal scale (33% vs 4%; P < .001). The 30-day mortality rate was higher for COVID-19 than for influenza: 15% vs 5% (P = .001). The median age of nonsurviving cases was 81 (IQR 74-88) and 77.5 (IQR 65-84) (P = .385), respectively. COVID-19 was independently associated with 30-day (hazard ratio [HR], 4.6; 95% confidence interval [CI], 2-10.4) and 90-day (HR, 5.2; 95% CI, 2.4-11.4) mortality. Sensitivity and subgroup analyses, including a subgroup considering only patients with pneumonia, did not show different trends. Regarding resource consumption, COVID-19 patients had longer hospital stays and higher critical care, pharmacy, and complementary test costs. CONCLUSIONS: Although influenza patients were older and had more comorbidities, COVID-19 cases requiring supplemental oxygen on admission had worse clinical and economic outcomes.

Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA.

Macromolecular condensation resulting from biologically regulated liquid-liquid phase separation is emerging as a mechanism to organize intracellular space in eukaryotes, with broad implications for cell physiology and pathology. Despite their small size, bacterial cells are also organized by proteins such as FtsZ, a tubulin homolog that assembles into a ring structure precisely at the cell midpoint and is required for cytokinesis. Here, we demonstrate that FtsZ can form crowding-induced condensates, reminiscent of those observed for eukaryotic proteins. Formation of these FtsZ-rich droplets occurs when FtsZ is bound to SlmA, a spatial regulator of FtsZ that antagonizes polymerization, while also binding to specific sites on chromosomal DNA. The resulting condensates are dynamic, allowing FtsZ to undergo GTP-driven assembly to form protein fibers. They are sensitive to compartmentalization and to the presence of a membrane boundary in cell mimetic systems. This is a novel example of a bacterial nucleoprotein complex exhibiting condensation into liquid droplets, suggesting that phase separation may also play a functional role in the spatiotemporal organization of essential bacterial processes.

Epidemiology and Treatment of Multidrug-Resistant and Extensively Drug-Resistant Pseudomonas aeruginosa Infections.

In recent years, the worldwide spread of the so-called high-risk clones of multidrug-resistant or extensively drug-resistant (MDR/XDR) Pseudomonas aeruginosa has become a public health threat. This article reviews their mechanisms of resistance, epidemiology, and clinical impact and current and upcoming therapeutic options. In vitro and in vivo treatment studies and pharmacokinetic and pharmacodynamic (PK/PD) models are discussed. Polymyxins are reviewed as an important therapeutic option, outlining dosage, pharmacokinetics and pharmacodynamics, and their clinical efficacy against MDR/XDR P. aeruginosa infections. Their narrow therapeutic window and potential for combination therapy are also discussed. Other "old" antimicrobials, such as certain ß-lactams, aminoglycosides, and fosfomycin, are reviewed here. New antipseudomonals, as well as those in the pipeline, are also reviewed. Ceftolozane-tazobactam has clinical activity against a significant percentage of MDR/XDR P. aeruginosa strains, and its microbiological and clinical data, as well as recommendations for improving its use against these bacteria, are described, as are those for ceftazidime-avibactam, which has better activity against MDR/XDR P. aeruginosa, especially strains with certain specific mechanisms of resistance. A section is devoted to reviewing upcoming active drugs such as imipenem-relebactam, cefepime-zidebactam, cefiderocol, and murepavadin. Finally, other therapeutic strategies, such as use of vaccines, antibodies, bacteriocins, anti-quorum sensing, and bacteriophages, are described as future options.

Zinc Differentially Modulates the Assembly of Soluble and Polymerized Vimentin.

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress. We previously showed that vimentin interacts with zinc, which affects its assembly and redox sensing. Here, we used vimentin wt and C328S, an oxidation-resistant mutant showing improved NaCl-induced polymerization, to assess the impact of zinc on soluble and polymerized vimentin by light scattering and electron microscopy. Zinc acts as a switch, reversibly inducing the formation of vimentin oligomeric species. High zinc concentrations elicit optically-detectable vimentin structures with a characteristic morphology depending on the support. These effects also occur in vimentin C328S, but are not mimicked by magnesium. Treatment of vimentin with micromolar ZnCl2 induces fibril-like particles that do not assemble into filaments, but form aggregates upon subsequent addition of NaCl. In contrast, when added to NaCl-polymerized vimentin, zinc increases the diameter or induces lateral association of vimentin wt filaments. Remarkably, these effects are absent or attenuated in vimentin C328S filaments. Therefore, the zinc-vimentin interaction depends on the chemical environment and on the assembly state of the protein, leading to atypical polymerization of soluble vimentin, likely through electrostatic interactions, or to broadening and lateral association of preformed filaments through mechanisms requiring the cysteine residue. Thus, the impact of zinc on vimentin assembly and redox regulation is envisaged.

Impact of De-escalation on Prognosis of Patients With Bacteremia due to Enterobacteriaceae: A Post Hoc Analysis From a Multicenter Prospective Cohort.

BACKGROUND: More data are needed about the safety of antibiotic de-escalation in specific clinical situations as a strategy to reduce exposure to broad-spectrum antibiotics. The aims of this study were to investigate predictors of de-escalation and its impact on the outcome of patients with bloodstream infection due to Enterobacteriaceae (BSI-E). METHODS: A post hoc analysis was performed on a prospective, multicenter cohort of patients with BSI-E initially treated with ertapenem or antipseudomonal ß-lactams. Logistic regression was used to analyze factors associated with early de-escalation (EDE) and Cox regression for the impact of EDE and late de-escalation (LDE) on 30-day all-cause mortality. A propensity score (PS) for EDE vs no de-escalation (NDE) was calculated. Failure at end of treatment and length of hospital stay were also analyzed. RESULTS: Overall, 516 patients were included. EDE was performed in 241 patients (46%), LDE in 95 (18%), and NDE in 180 (35%). Variables independently associated with a lower probability of EDE were multidrug-resistant isolates (odds ratio [OR], 0.50 [95% confidence interval {CI}, .30-.83]) and nosocomial infection empirically treated with imipenem or meropenem (OR, 0.35 [95% CI, .14-.87]). After controlling for confounders, EDE was not associated with increased risk of mortality; hazard ratios (HR) (95% CIs) were as follows: general model, 0.58 (.25-1.31); model with PS, 0.69 (.29-1.65); and PS-based matched pairs, 0.98 (.76-1.26). LDE was not associated with mortality. De-escalation was not associated with clinical failure or length of hospital stay. CONCLUSIONS: De-escalation in patients with monomicrobial bacteremia due to Enterobacteriaceae was not associated with a detrimental impact on clinical outcome.

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition.

Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/ß domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.

Gbp2 interacts with THO/TREX through a novel type of RRM domain.

Metazoan SR and SR-like proteins are important regulatory factors in RNA splicing, export, translation and RNA decay. We determined the NMR structures and nucleic acid interaction modes of Gbp2 and Hrb1, two paralogous budding yeast proteins with similarities to mammalian SR proteins. Gbp2 RRM1 and RRM2 recognise preferentially RNAs containing the core motif GGUG. Sequence selectivity resides in a non-canonical interface in RRM2 that is highly related to the SRSF1 pseudoRRM. The atypical Gbp2/Hrb1 C-terminal RRM domains (RRM3) do not interact with RNA/DNA, likely because of their novel N-terminal extensions that block the canonical RNA binding interface. Instead, we discovered that RRM3 is crucial for interaction with the THO/TREX complex and identified key residues essential for this interaction. Moreover, Gbp2 interacts genetically with Tho2 as the double deletion shows a synthetic phenotype and preventing Gbp2 interaction with the THO/TREX complex partly supresses gene expression defect associated with inactivation of the latter complex. These findings provide structural and functional insights into the contribution of SR-like proteins in the post-transcriptional control of gene expression.

Genomics and Susceptibility Profiles of Extensively Drug-Resistant Pseudomonas aeruginosa Isolates from Spain.

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% (n = 101) of the XDR isolates, distantly followed by ST244 (n = 16), ST253 (n = 12), ST235 (n = 8), and ST111 (n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital (n = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the ß-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in ß-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

MIC of amoxicillin/clavulanate according to CLSI and EUCAST: discrepancies and clinical impact in patients with bloodstream infections due to Enterobacteriaceae.

Objectives: To compare results of amoxicillin/clavulanate susceptibility testing using CLSI and EUCAST methodologies and to evaluate their impact on outcome in patients with bacteraemia caused by Enterobacteriaceae. Patients and methods: A prospective observational cohort study was conducted in 13 Spanish hospitals. Patients with bacteraemia due to Enterobacteriaceae who received empirical intravenous amoxicillin/clavulanate treatment for at least 48 h were included. MICs were determined following CLSI and EUCAST recommendations. Outcome variables were: failure at the end of treatment with amoxicillin/clavulanate (FEAMC); failure at day 21; and 30 day mortality. Classification and regression tree (CART) analysis and logistic regression were performed. Results: Overall, 264 episodes were included; the urinary tract was the most common source (64.7%) and Escherichia coli the most frequent pathogen (76.5%). Fifty-two isolates (19.7%) showed resistance according to CLSI and 141 (53.4%) according to EUCAST. The kappa index for the concordance between the results of both committees was only 0.24. EUCAST-derived, but not CLSI-derived, MICs were associated with failure when considered as continuous variables. CART analysis suggested a 'resistance' breakpoint of > 8/4 mg/L for CLSI-derived MICs; it predicted FEAMC in adjusted analysis (OR = 1.96; 95% CI: 0.98-3.90). Isolates with EUCAST-derived MICs >16/2 mg/L independently predicted FEAMC (OR = 2.10; 95% CI: 1.05-4.21) and failure at day 21 (OR= 3.01; 95% CI: 0.93-9.67). MICs >32/2 mg/L were only predictive of failure among patients with bacteraemia from urinary or biliary tract sources. Conclusions: CLSI and EUCAST methodologies showed low agreement for determining the MIC of amoxicillin/clavulanate. EUCAST-derived MICs seemed more predictive of failure than CLSI-derived ones. EUCAST-derived MICs >16/2 mg/L were independently associated with therapeutic failure.

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits.

The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5'-[(α,ß)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection.

Impact of the MIC of piperacillin/tazobactam on the outcome for patients with bacteraemia due to Enterobacteriaceae: the Bacteraemia-MIC project.

OBJECTIVE: Our objective was to evaluate the impact of low versus borderline MIC of piperacillin/tazobactam on the clinical outcomes of patients with bacteraemia caused by Enterobacteriaceae who were treated with that antimicrobial. PATIENTS AND METHODS: A prospective observational multicentre cohort study was conducted in 13 Spanish university hospitals. Patients >17 years old with bacteraemia due to Enterobacteriaceae who received empirical piperacillin/tazobactam treatment for at least 48 h were included. Outcome variables were clinical response at day 21, clinical response at end of treatment with piperacillin/tazobactam and all-cause 30 day mortality. Univariate and multivariate logistic regression analyses were performed. RESULTS: Overall, 275 patients were included in the analysis; 248 (90.2%) in the low MIC group (≤ 4 mg/L) and 27 (9.8%) in the borderline MIC group (8-16 mg/L). The biliary tract was the most common source of infection (48.4%) and Escherichia coli was the most frequent pathogen (63.3%). Crude 30 day mortality rates were 10.5% and 11.1% for the low MIC group and the borderline MIC group, respectively (relative risk = 1.06, 95% CI = 0.34-3.27, P = 1). Multivariate analysis of failure at day 21 and at end of treatment with piperacillin/tazobactam and 30 day mortality showed no trend towards increased clinical failure or mortality with borderline MICs (OR = 0.96, 95% CI = 0.18-4.88, P = 0.96; OR = 0.47, 95% CI = 0.10-2.26, P = 0.35; OR = 1.48, 95% CI = 0.33-6.68, P = 0.6). CONCLUSIONS: We did not find that higher piperacillin/tazobactam MIC within the susceptible or intermediate susceptibility range had a significant influence on the outcome for patients with bacteraemia due to Enterobacteriaceae.

Influence of virulence genotype and resistance profile in the mortality of Pseudomonas aeruginosa bloodstream infections.

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.

Prospective observational study of prior rectal colonization status as a predictor for subsequent development of Pseudomonas aeruginosa clinical infections.

The potential role of Pseudomonas aeruginosa (PA) intestinal colonization in the subsequent development of infections has not been thoroughly investigated. The aims of this study were to assess the role of PA intestinal colonization as a predictor of subsequent infections and to investigate the risk factors associated with the development of PA infection in patients in the intensive care unit (ICU). For this purpose, a prospective study was conducted that included (i) active surveillance of PA rectal colonization at ICU admission and weekly until ICU discharge, (ii) detection of PA clinical infections, and (iii) genotypic analysis by pulsed-field gel electrophoresis (PFGE). A total of 414 patients were included, of whom 179 (43%) were colonized with PA. Among the 77 patients who developed PA infection, 69 (90%) had prior PA colonization, and 60 (87%) of these showed genotyping concordance between rectal and clinical isolates. The probability of PA infection 14 days after ICU admission was 26% for carriers versus 5% for noncarriers (P < 0.001). Cox regression analysis identified prior PA rectal colonization as the main predictor of PA infection (hazard ratio [HR], 15.23; 95% confidence interval [CI], 6.9 to 33.7; P < 0.001). Prior use of nonantipseudomonal penicillins was also identified as an independent variable associated with PA infection (HR, 2.15; 95% CI, 1.3 to 3.55; P < 0.003). Our study demonstrated that prior PA rectal colonization is a key factor for developing PA infection.

Control by potassium of the size distribution of Escherichia coli FtsZ polymers is independent of GTPase activity.

The influence of potassium content (at neutral pH and millimolar Mg(2+)) on the size distribution of FtsZ polymers formed in the presence of constantly replenished GTP under steady-state conditions was studied by a combination of biophysical methods. The size of the GTP-FtsZ polymers decreased with lower potassium concentration, in contrast with the increase in the mass of the GDP-FtsZ oligomers, whereas no effect was observed on FtsZ GTPase activity and critical concentration of polymerization. Remarkably, the concerted formation of a narrow size distribution of GTP-FtsZ polymers previously observed at high salt concentration was maintained in all KCl concentrations tested. Polymers induced with guanosine 5'-(α,ß-methylene)triphosphate, a slowly hydrolyzable analog of GTP, became larger and polydisperse as the potassium concentration was decreased. Our results suggest that the potassium dependence of the GTP-FtsZ polymer size may be related to changes in the subunit turnover rate that are independent of the GTP hydrolysis rate. The formation of a narrow size distribution of FtsZ polymers under very different solution conditions indicates that it is an inherent feature of FtsZ, not observed in other filament-forming proteins, with potential implications in the structural organization of the functional Z-ring.