Epidemiological and clinical characterization of community, healthcare-associated and nosocomial colonization and infection due to carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in Spain.

BACKGROUND: Community-acquired (CA) and healthcare-associated (HCA) infections caused by carbapenemase-producing Enterobacterales (CPE) are not well characterized. The objective was to provide detailed information about the clinical and molecular epidemiological features of nosocomial, HCA and CA infections caused by carbapenemase-producing Klebsiella pneumoniae (CP-Kp) and Escherichia coli (CP-Ec). METHODS: A prospective cohort study was performed in 59 Spanish hospitals from February to March 2019, including the first 10 consecutive patients from whom CP-Kp or CP-Ec were isolated. Patients were stratified according to acquisition type. A multivariate analysis was performed to identify the impact of acquisition type in 30-day mortality. RESULTS: Overall, 386 patients were included (363 [94%] with CP-Kp and 23 [6%] CP-Ec); in 296 patients (76.3%), the CPE was causing an infection. Acquisition was CA in 31 (8.0%) patients, HCA in 183 (47.4%) and nosocomial in 172 (48.3%). Among patients with a HCA acquisition, 100 (54.6%) had been previously admitted to hospital and 71 (38.8%) were nursing home residents. Urinary tract infections accounted for 19/23 (82.6%), 89/130 (68.5%) and 42/143 (29.4%) of CA, HCA and nosocomial infections, respectively. Overall, 68 infections (23%) were bacteremia (8.7%, 17.7% and 30.1% of CA, HCA and nosocomial, respectively). Mortality in infections was 28% (13%, 14.6% and 42.7% of CA, HCA and nosocomial, respectively). Nosocomial bloodstream infections were associated with increased odds for mortality (adjusted OR, 4.00; 95%CI 1.21-13.19). CONCLUSIONS: HCA and CA infections caused by CPE are frequent and clinically significant. This information may be useful for a better understanding of the epidemiology of CPE.

Deciphering mechanisms affecting cefepime-taniborbactam in vitro activity in carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas spp. isolates recovered during a surveillance study in Spain.

PURPOSE: To characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates. METHODS: CPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing. RESULTS: A reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers. CONCLUSIONS: The reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

Corrigendum: CARB-ES-19 multicenter study of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli from all Spanish provinces reveals interregional spread of high-risk clones such as ST307/OXA-48 and ST512/KPC-3.

[This corrects the article DOI: 10.3389/fmicb.2022.918362.].

The CARBA-MAP study: national mapping of carbapenemases in Spain (2014-2018).

Introduction: Infections caused by carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa, including isolates producing acquired carbapenemases, constitute a prevalent health problem worldwide. The primary objective of this study was to determine the distribution of the different carbapenemases among carbapenemase-producing Enterobacterales (CPE, specifically Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae complex, and Klebsiella aerogenes) and carbapenemase-producing P. aeruginosa (CPPA) in Spain from January 2014 to December 2018. Methods: A national, retrospective, cross-sectional multicenter study was performed. The study included the first isolate per patient and year obtained from clinical samples and obtained for diagnosis of infection in hospitalized patients. A structured questionnaire was completed by the participating centers using the REDCap platform, and results were analyzed using IBM SPSS Statistics 29.0.0. Results: A total of 2,704 carbapenemase-producing microorganisms were included, for which the type of carbapenemase was determined in 2692 cases: 2280 CPE (84.7%) and 412 CPPA (15.3%), most often using molecular methods and immunochromatographic assays. Globally, the most frequent types of carbapenemase in Enterobacterales and P. aeruginosa were OXA-48-like, alone or in combination with other enzymes (1,523 cases, 66.8%) and VIM (365 cases, 88.6%), respectively. Among Enterobacterales, carbapenemase-producing K. pneumoniae was reported in 1821 cases (79.9%), followed by E. cloacae complex in 334 cases (14.6%). In Enterobacterales, KPC is mainly present in the South and South-East regions of Spain and OXA-48-like in the rest of the country. Regarding P. aeruginosa, VIM is widely distributed all over the country. Globally, an increasing percentage of OXA-48-like enzymes was observed from 2014 to 2017. KPC enzymes were more frequent in 2017-2018 compared to 2014-2016. Discussion: Data from this study help to understand the situation and evolution of the main species of CPE and CPPA in Spain, with practical implications for control and optimal treatment of infections caused by these multi-drug resistant organisms.

Cefiderocol resistance genomics in sequential chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients.

OBJECTIVE: To evaluate the activity of cefiderocol against sequential P. aeruginosa isolates from chronically-infected cystic fibrosis patients as well as to investigate the potential mechanisms involved in resistance through whole genome sequencing. METHODS: Three sequential P. aeruginosa isolates from each of 50 chronically-colonized cystic fibrosis patients were studied. MICs for novel and classical antipseudomonal agents were determined by broth microdilution and whole genome sequences (n = 150) were obtained to investigate the presence of mutations within a set of chromosomal genes involved in P. aeruginosa antibiotic resistance (n = 40) and iron uptake (n = 120). RESULTS: Cefiderocol showed the lowest MIC50/90 values and its susceptibility rate was comparable to other novel antipseudomonal agents. Clinical resistance was documented in 9 isolates from 6 patients. Resistance genes associated with a statistically significant increase in cefiderocol MICs included ampC, pmrAB, galU, fusA1 and those coding the penicillin-binding proteins PBP2 and PBP3. Likewise, mutations within several genes participating in different iron-uptake systems were found to be significantly associated with resistance, including genes participating in the pyochelin and pyoverdin biosynthesis and several tonB-dependent receptors. Mutator and small colony variants isolates were also associated with increased cefiderocol MICs. DISCUSSION: Cefiderocol resistance is modulated by a complex mutational resistome, potentially conferring cross-resistance to novel beta-lactam beta-lactamase combinations, as well as an extended list of mutated iron-uptake genes. Monitoring the acquisition of mutations in all these genes will be helpful to guide treatments and mitigate the emergence and spread of resistance to this novel antibiotic.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC ß-Lactamases in Pseudomonas aeruginosa.

In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3.

Objectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain. Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were bla OXA-48 (263/377), bla KPC-3 (62/377), bla VIM-1 (28/377), and bla NDM-1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3.

Underlying Polar and Nonpolar Modification MOF-Based Factors that Influence Permanent Porosity in Porous Liquids.

It is increasingly apparent that porous liquids (PLs) have unique use cases due to the combination of ready liquid handling and their inherently high adsorption capacity. Among the PL types, those with permanent porosity are the most promising. Although Type II and III PLs have economic synthetic methods and can be made from a huge variety of metal-organic frameworks (MOFs) and solvents, these nanocomposites still need to be stable to be useful. This work aims to systematically explore the possibilities of creating PLs using different MOF modification methods. This delivered underpinning insights into the molecular-level influence between solvent and MOF on the overall nanocomposite stability. Zirconium-based metal-organic frameworks were combined with two different solvents of varying chemistry to deliver CO2 sorption capacities as high as 2.9 mmol g-1 at 10 bar. The results of the study could have far-reaching ramifications for future investigations in the PL field.

Practical considerations in the design and use of porous liquids.

The possibility of creating well-controlled empty space within liquids is conceptually intriguing, and from an application perspective, full of potential. Since the concept of porous liquids (PLs) arose several years ago, research efforts in this field have intensified. This review highlights the design, synthesis, and applicability of PLs through a thorough examination of the current state-of-the-art. Following a detailed examination of the fundamentals of PLs, we examine the different synthetic approaches proposed to date, discuss the nature of PLs, and their pathway from the laboratory to practical application. Finally, possible challenges and opportunities are outlined.

Impact of Peptidoglycan Recycling Blockade and Expression of Horizontally Acquired ß-Lactamases on Pseudomonas aeruginosa Virulence.

In the current scenario of antibiotic resistance magnification, new weapons against top nosocomial pathogens like Pseudomonas aeruginosa are urgently needed. The interplay between ß-lactam resistance and virulence is considered a promising source of targets to be attacked by antivirulence therapies, and in this regard, we previously showed that a peptidoglycan recycling blockade dramatically attenuated the pathogenic power of P. aeruginosa strains hyperproducing the chromosomal ß-lactamase AmpC. Here, we sought to ascertain whether this observation could be applicable to other ß-lactamases. To do so, P. aeruginosa wild-type or peptidoglycan recycling-defective strains (ΔampG and ΔnagZ) harboring different cloned ß-lactamases (transferable GES, VIM, and OXA types) were used to assess their virulence in Galleria mellonella larvae by determining 50% lethal doses (LD50s). A mild yet significant LD50 increase was observed after peptidoglycan recycling disruption per se, whereas the expression of class A and B enzymes did not impact virulence. While the production of the narrow-spectrum class D OXA-2 entailed a slight attenuation, its extended-spectrum derivatives OXA-226 (W159R [bearing a change of W to R at position 159]), OXA-161 (N148D), and principally, OXA-539 (D149 duplication) were associated with outstanding virulence impairments, especially in recycling-defective backgrounds (with some LD50s being >1,000-fold that of the wild type). Although their exact molecular bases remain to be deciphered, these results suggest that mutations affecting the catalytic center and, therefore, the hydrolytic spectrum of OXA-2-derived enzymes also drastically impact the pathogenic power of P. aeruginosa. This work provides new and relevant knowledge to the complex topic of the interplay between the production of ß-lactamases and virulence that could be useful to build future therapeutic strategies against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is one of the leading nosocomial pathogens whose growing resistance makes the development of therapeutic options extremely urgent. The resistance-virulence interplay has classically aroused researchers' interest as a source of therapeutic targets. In this regard, we describe a wide array of virulence attenuations associated with different transferable ß-lactamases, among which the production of OXA-2-derived extended-spectrum ß-lactamases stood out as a dramatic handicap for pathogenesis, likely as a side effect of mutations causing the expansion of their hydrolytic spectrums. Moreover, our results confirm the validity of disturbing peptidoglycan recycling as a weapon to attenuate P. aeruginosa virulence in class C and D ß-lactamase production backgrounds. In the current scenario of dissemination of horizontally acquired ß-lactamases, this work brings out new data on the complex interplay between the production of specific enzymes and virulence attenuation that, if complemented with the characterization of the underlying mechanisms, will likely be exploitable to develop future virulence-targeting antipseudomonal strategies.

Biomimetic metal-organic frameworks as protective scaffolds for live-virus encapsulation and vaccine stabilization.

The invaluable health, economic and social impacts of vaccination are hard to exaggerate. The ability to stabilize vaccines is urgently required for their equitable distribution without the dependence on the 'cold-chain' logistics. Herein, for the first time we report biomimetic-mineralization of live-viral vaccines using metal-organic frameworks (MOFs) to enhance their storage stability from days to months. Applying ZIF-8 and aluminium fumarate (Alfum), the Newcastle Disease Virus (NDV) V4 strain and Influenza A WSN strain were encapsulated with remarkable retention of their viral titre. The ZIF-8@NDV, ZIF-8@WSN and Alfum@WSN composites were validated for live-virus recovery using a tissue culture infectious dose (TCID50) assay. With the objective of long-term stabilization, we developed a novel, trehalose (T) and skim milk (SM) stabilized, freeze-dried MOF@Vaccine composite, ZIF-8@NDV+T/SM. The thermal stability of this composite was investigated and compared with the control NDV and non-encapsulated, freeze-dried NDV+T/SM composite at 4 °C, RT, and 37 °C over a period of 12 weeks. We demonstrate the fragility of the control NDV vaccine which lost all viability at RT and 37°C by 12 and 4 weeks, respectively. Comparing the freeze-dried counterparts, the MOF encapsulated ZIF-8@NDV+T/SM demonstrated significant enhancement in stability of the NDV+T/SM composite especially at RT and 37 °C upto 12 weeks. STATEMENT OF SIGNIFICANCE: Vaccination is undoubtedly one of the most effective medical interventions, saving millions of lives each year. However, the requirement of 'cold-chain' logistics is a major impediment to widespread immunization. Live viral vaccines (LVVs) are widely used vaccine types with proven efficacy and low cost. Nonetheless, their complex composition increases their susceptability to thermal stress. Several LVV thermostabilization approaches have been investigated, including their complex engineering and the facile addition of stabilizers. Still, the lack of a universal approach urgently requires finding a stabilization technique especially when additives alone may not be sufficient. Herein, we demonstrate MOF biomimetic-mineralization technology to encapsulate LVVs developing an optimised composite which significantly preserves vaccines without refrigeration for extended periods of time.

In Vitro Activity of Cefepime-Taniborbactam against Carbapenemase-Producing Enterobacterales and Pseudomonas aeruginosa Isolates Recovered in Spain.

Novel ß-lactam-ß-lactamase inhibitor combinations currently approved for clinical use are poorly active against metallo-ß-lactamase (MBL)-producing strains. We evaluated the in vitro activity of cefepime-taniborbactam (FTB [formerly cefepime-VNRX-5133]) and comparator agents against carbapenemase-producing Enterobacterales (n = 247) and carbapenem-resistant Pseudomonas species (n = 170) clinical isolates prospectively collected from different clinical origins in patients admitted to 8 Spanish hospitals. FTB was the most active agent in both Enterobacterales (97.6% MICFTB, ≤8/4 mg/L) and Pseudomonas (67.1% MICFTB, ≤8/4 mg/L) populations. The MICFTB was >8 mg/L in 6/247 (2.4%) Enterobacterales isolates (3 KPC-producing Klebsiella pneumoniae isolates, 1 VIM-producing Enterobacter cloacae isolate, 1 IMP-producing E. cloacae isolate, and 1 NDM-producing Escherichia coli isolate) and in 56/170 (32.9%) Pseudomonas isolates, 19 of them carbapenemase producers (15 producers of VIM, 2 of GES, 1 of GES+VIM, and 1 of GES+KPC). Against the Enterobacterales isolates with meropenem MICs of >2 mg/L (138/247), FTB was the most active agent against both serine-ß-lactamases (107/138) and MBL producers (31/138) (97.2 and 93.5% MICFTB, ≤8/4 mg/L, respectively), whereas the activity of comparators was reduced, particularly against the MBL producers (ceftazidime-avibactam, 94.4 and 12.9%, meropenem-vaborbactam, 85.0 and 64.5%, imipenem-relebactam, 76.6 and 9.7%, ceftolozane-tazobactam, 1.9 and 0%, and piperacillin-tazobactam, 0 and 0%, respectively). Among the meropenem-resistant Pseudomonas isolates (163/170; MIC, >2 mg/L), the activities of FTB against serine-ß-lactamase (35/163) and MBL (43/163) producers were 88.6 and 65.1%, respectively, whereas the susceptibilities of comparators were as follows: ceftazidime-avibactam, 88.5 and 16.0%, meropenem-vaborbactam, 8.5 and 7.0%, imipenem-relebactam, 2.9 and 2.3%, ceftolozane-tazobactam, 0 and 2.3%, and piperacillin-tazobactam, 0 and 0%, respectively. Microbiological results suggest FTB as a potential therapeutic option in patients infected with carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas isolates, including MBL producers.

Hetero-metallic metal-organic frameworks for room-temperature NO2 sensing.

Metal-organic frameworks (MOFs) with exceptional features such as high structural diversity and surface area as well as controlled pore size has been considered a promising candidate for developing room temperature highly-sensitive gas sensors. In comparison, the hetero-metallic MOFs with redox-active open-metal sites and mixed metal nodes may create peculiar surface properties and synergetic effects for enhanced gas sensing performances. In this work, the Fe atoms in the Fe3 (Porous coordination network) PCN-250 MOFs are partially replaced by transition metal Co, Mn, and Zn through a facile hydrothermal approach, leading to the formation of hetero-metallic MOFs (Fe2IIIMII, M = Co, Mn, and Zn). While the PCN-250 framework is maintained, the morphological and electronic band structural properties are manipulated upon the partial metal replacement of Fe. More importantly, the room temperature NO2 sensing performances are significantly varied, in which Fe2Mn PCN-250 demonstrates the largest response magnitude for ppb-level NO2 gas compared to those of pure Fe3 PCN-250 and other hetero-metallic MOF structures mainly attributed to the highest binding energy of NO2 gas. This work demonstrates the strong potential of hetero-metallic MOFs with carefully engineered substituted metal clusters for power-saving and high-performance gas sensing applications.

Emergence of Resistance to Novel Cephalosporin-ß-Lactamase Inhibitor Combinations through the Modification of the Pseudomonas aeruginosa MexCD-OprJ Efflux Pump.

A ceftolozane-tazobactam- and ceftazime-avibactam-resistant Pseudomonas aeruginosa isolate was recovered after treatment (including azithromycin, meropenem, and ceftolozane-tazobactam) from a patient that had developed ventilator-associated pneumonia after COVID-19 infection. Whole-genome sequencing revealed that the strain, belonging to ST274, had acquired a nonsense mutation leading to truncated carbapenem porin OprD (W277X), a 7-bp deletion (nt213Δ7) in NfxB (negative regulator of the efflux pump MexCD-OprJ), and two missense mutations (Q178R and S133G) located within the first large periplasmic loop of MexD. Through the construction of mexD mutants and complementation assays with wild-type nfxB, it was evidenced that resistance to the novel cephalosporin-ß-lactamase inhibitor combinations was caused by the modification of MexD substrate specificity.

In Vivo Evolution of GES ß-Lactamases Driven by Ceftazidime/Avibactam Treatment of Pseudomonas aeruginosa Infections.

The mechanisms underlying an in vivo switch in the resistance phenotype of P. aeruginosa after ceftazidime-avibactam treatment was investigated. The initial isolate (a blood culture) was resistant to meropenem but remained susceptible to antipseudomonal cephalosporins and combinations with ß-lactamase inhibitors. One week after ceftazidime-avibactam therapy, a subsequent isolate (a rectal swab) recovered from the same patient showed the opposite phenotype. Whole-genome sequence analysis revealed a single SNP difference between both (ST235) isolates, leading to a P162S change in blaGES-5, creating blaGES-15. Thus, blaGES-1, blaGES-5, and blaGES-15 were cloned and expressed in the wild-type strain PAO1. Susceptibility profiles confirmed the P162S substitution reverted the carbapenemase phenotype determined by the G170S change of GES-5 back into the ESBL phenotype of GES-1.

Validation of MALDI-TOF for the early detection of the ST175 high-risk clone of Pseudomonas aeruginosa in clinical isolates belonging to a Spanish nationwide multicenter study.

INTRODUCTION: Pseudomonas aeruginosa causes severe infections, particularly in healthcare settings and immunocompromised patients in whom MDR and XDR isolates are more prevalent. The aim of this study is to validate a method based on MALDI-TOF spectra analysis for early detection of the ST175 high-risk clone (HRC). METHODS: The MALDI-TOF spectra of the first 10 P. aeruginosa clinical isolates from each of the 51 participating Spanish hospitals were analyzed (n=506). Resistance profiles were determined by broth microdilution, and clonal epidemiology was assessed by PFGE analysis and multilocus sequence typing (MLST) in a previous study. RESULTS: Among all the isolates, 14.2% were XDR and 26.9% were non-susceptible to meropenem, while rates of resistance to ceftolozane/tazobactam (3.6%) and colistin (5.7%) were low. Up to 41.7% of all XDR isolates belonged to the ST175 clone, and most of them were only susceptible to ceftolozane/tazobactam and colistin. However, most of the resistance to ceftolozane/tazobactam among isolates belonging to this HRC was observed in carbapenemase-producing isolates. A model based on the presence of two MALDI-TOF biomarker peaks at m/z 6911 and 7359 yielded a negative predictive value (NPV) and a positive predictive value (PPV) of 99.8% and 91.9%, respectively, and sensitivity and specificity values of 97.1% and 99.4%, respectively. CONCLUSIONS: MALDI-TOF spectra analysis using a model based on the presence of two biomarker peaks proved to maintain high sensitivity and specificity for early detection of the ST175 HRC in a large collection of isolates from all Spanish regions. These data support the use of this model in a clinical setting; however, the consequences of detection of the ST175 HRC, such as choice of empirical antibiotic therapy, must be consistent with local epidemiology and the prevalence of certain resistance patterns of this HRC, such as carbapenemase production, in a given geographical area.

Validation of MALDI-TOF for the early detection of the ST175 high-risk clone of Pseudomonas aeruginosa in clinical isolates belonging to a Spanish nationwide multicenter study / Validación del MALDI-TOF para la detección precoz del clon de alto riesgo ST175 de Pseudomonas aeruginosa en aislados clínicos pertenecientes a un estudio multicéntrico nacional

Introduction: Pseudomonas aeruginosa causes severe infections, particularly in healthcare settings and immunocompromised patients in whom MDR and XDR isolates are more prevalent. The aim of this study is to validate a method based on MALDI-TOF spectra analysis for early detection of the ST175 high-risk clone (HRC). Methods: The MALDI-TOF spectra of the first 10 P. aeruginosa clinical isolates from each of the 51 participating Spanish hospitals were analyzed (n=506). Resistance profiles were determined by broth microdilution, and clonal epidemiology was assessed by PFGE analysis and multilocus sequence typing (MLST) in a previous study. Results: Among all the isolates, 14.2% were XDR and 26.9% were non-susceptible to meropenem, while rates of resistance to ceftolozane/tazobactam (3.6%) and colistin (5.7%) were low. Up to 41.7% of all XDR isolates belonged to the ST175 clone, and most of them were only susceptible to ceftolozane/tazobactam and colistin. However, most of the resistance to ceftolozane/tazobactam among isolates belonging to this HRC was observed in carbapenemase-producing isolates. A model based on the presence of two MALDI-TOF biomarker peaks at m/z 6911 and 7359 yielded a negative predictive value (NPV) and a positive predictive value (PPV) of 99.8% and 91.9%, respectively, and sensitivity and specificity values of 97.1% and 99.4%, respectively. Conclusions: MALDI-TOF spectra analysis using a model based on the presence of two biomarker peaks proved to maintain high sensitivity and specificity for early detection of the ST175 HRC in a large collection of isolates from all Spanish regions. These data support the use of this model in a clinical setting; however, the consequences of detection of the ST175 HRC, such as choice of empirical antibiotic therapy, must be consistent with local epidemiology and the prevalence of certain resistance patterns of this HRC, such as carbapenemase production, in a given geographical area.(AU)

Introducción: P. aeruginosa causa infecciones graves, particularmente asociadas a cuidados sanitarios y en pacientes inmunodeprimidos, donde los aislamientos MDR o XDR son más frecuentes. El objetivo de este estudio es validar el método basado en el análisis de espectros MALDI-TOF para la detección precoz del clon de alto riesgo ST175. Métodos: Se analizaron los espectros de MALDI-TOF de los primeros 10 aislados clínicos de P. aeruginosa pertenecientes a cada uno de los 51 hospitales españoles participantes (n=506). En un trabajo previo se determinaron los perfiles de resistencia mediante microdilución en caldo y se estableció su relación clonal mediante electroforesis en campo pulsante (PFGE) y multilocus sequence typing (MLST). Resultados: Del total de los aislamientos el 14,2% fueron XDR y el 26,9% resultaron ser no sensibles a meropenem, mientras que la resistencia al ceftolozano-tazobactam (3,6%) y la colistina (5,7%) fue baja. Hasta el 41,7% de todos los aislamientos XDR pertenecieron al clon ST175 y la mayoría de ellos solo resultaron ser sensibles a ceftolozano-tazobactam y a colistina. No obstante, la mayor parte de la resistencia a ceftolozano-tazobactam observada entre los aislados pertenecientes a este clon de alto riesgo se debió a la producción de carbapenemasas. El modelo basado en la presencia de dos picos de biomarcadores MALDI-TOF en m/z 6911 y 7359 obtuvo un valor predictivo negativo y positivo (VPN/VPP) del 99,8/91,9% y valores de sensibilidad y especificidad del 97,1/99,4%, respectivamente. Conclusiones: El análisis de los espectros de MALDI-TOF utilizando el modelo basado en la presencia de dos picos de biomarcadores ha demostrado poseer una alta sensibilidad y especificidad para la detección precoz del clon de alto riesgo ST175 en una gran colección de aislados clínicos representando todo el territorio español.(AU)

Plasmonic metal-organic framework nanocomposites enabled by degenerately doped molybdenum oxides.

Metal-organic frameworks (MOFs) nanocomposites are under the limelight due to their unique electronic, optical, and surface properties for various applications. Plasmonic MOFs enabled by noble metal nanostructures are an emerging class of MOF nanocomposites with efficient solar light-harvesting capability. However, major concerns such as poor photostability, sophisticated synthesis processes, and high fabrication cost are raised. Here, we develop a novel plasmonic MOF nanocomposite consisting of the ultra-thin degenerately doped molybdenum oxide core and the flexible iron MOF (FeMOF) shell through a hydrothermal growth, featuring low cost, facile synthesis, and non-toxicity. More importantly, the incorporation of plasmonic oxides in the highly porous MOF structure enhances the visible light absorbability, demonstrating improved photobleaching performances of various azo and non-azo dyes compared to that of pure FeMOF without the incorporation of oxidative agents. Furthermore, the nanocomposite exhibits enhanced sensitivity and selectivity towards NO2 gas at room temperature, attributed to the electron-rich surface of plasmonic oxides. This work possibly broadens the exploration of plasmonic MOF nanocomposites for practical and efficient solar energy harvesting, environmental remediation, and environmental monitoring applications.

Multicenter Performance Evaluation of MALDI-TOF MS for Rapid Detection of Carbapenemase Activity in Enterobacterales: The Future of Networking Data Analysis With Online Software.

In this study, we evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories during a multicenter networking validation study. The study was divided into three different stages: "software design," "intercenter evaluation," and "clinical validation." First, a standardized procedure with an online software for data analysis was designed. Carbapenem resistance was detected by measuring imipenem hydrolysis and the results were automatically interpreted using the Clover MS data analysis software (Clover BioSoft, Spain). Second, a series of 74 genotypically characterized Enterobacterales (46 carbapenemase-producers and 28 non carbapenemase-producers) were analyzed in 8 international centers to ensure the reproducibility of the method. Finally, the methodology was evaluated independently in all centers during a 2-month period and results were compared with the reference standard for carbapenemase detection used in each center. The overall agreement rate relative to the reference method for carbapenemase resistance detection in clinical samples was 92.5%. The sensitivity was 93.9% and the specificity, 100%. Results were obtained within 60 min and accuracy ranged from 83.3 to 100% among the different centers. Further, our results demonstrate that MALDI-TOF MS is an outstanding tool for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories. The use of a simple in-house procedure with online software allows routine screening of carbapenemases in diagnostics, thereby facilitating early and appropriate antimicrobial therapy.

Whole-genome sequence-guided PCR for the rapid identification of the Pseudomonas aeruginosa ST175 high-risk clone directly from clinical samples.

OBJECTIVES: Pseudomonas aeruginosa frequently show MDR/XDR profiles, which are associated with worldwide-disseminated high-risk clones (HRCs). We developed a PCR assay for the detection in clinical samples of ST175, an HRC that is widespread in European countries. METHODS: The whole-genome sequence was obtained for one ST175 isolate using a PacBio RSII sequencer. Reads from multiple isolates belonging to ST175 and the PAO1 reference strain were mapped against the ST175 genome to identify potentially specific regions. Once curated, using the BLAST database to search for the presence of those regions in any other organism, we designed a specific PCR for the detection of ST175. RESULTS: Assembly of the ST175 PacBio-sequenced genome resulted in three contigs with a total length of 7 087 985 bases, encoding 6566 coding sequences. Specific regions for ST175 genomes were detected and a PCR targeting a 318 bp fragment located within a 3177 bp ORF coding for a putative reverse transcriptase was designed. The PCR test was first evaluated in silico against 229 XDR P. aeruginosa genomes (73 ST175) from two multicentre studies, yielding 100% sensitivity and specificity. Then, the PCR was evaluated in vitro in 25 isolates (12 ST175) and in 120 clinical samples (30 urine samples, 30 blood cultures, 30 sputum samples and 30 rectal swabs) of which 10% contained ST175, yielding again 100% sensitivity and specificity. CONCLUSIONS: The PCR assay developed, showing high sensitivity and specificity for the detection of the ST175 HRC directly from clinical samples, could become a useful tool for guiding infection control and treatment strategies in areas with a high prevalence of this clone.