Genetic variation within a stick insect species associated with community-level traits.

Phenotypic variation within species can affect the ecological dynamics of populations and communities. Characterizing the genetic variation underlying such effects can help parse the roles of genetic evolution and plasticity in 'eco-evolutionary dynamics' and inform how genetic variation may shape patterns of evolution. Here we employ genome-wide association (GWA) methods in Timema cristinae stick insects and their co-occurring arthropod communities to identify genetic variation associated with community-level traits. Previous studies have shown that maladaptation (i.e., imperfect crypsis) of T. cristinae can reduce the abundance and species richness of other arthropods due to an increase in bird predation. Whether genetic variation that is independent from crypsis has similar effects is unknown and was tested here using genome-wide genotyping-by-sequencing data of stick insects, arthropod community information, and GWA mapping with Bayesian sparse linear mixed models. We find associations between genetic variation in stick insects and arthropod community traits. However, these associations disappeared when host-plant traits are accounted for. We thus use path analysis to disentangle interrelationships among stick-insect genetic variation, host-plant traits and community traits. This revealed that host-plant size has large effects on arthropod communities, while genetic variation in stick insects has a smaller, but still significant effect. Our findings demonstrate that: (1) genetic variation in a species can be associated with community-level traits, but that (2) interrelationships among multiple factors may need to be analyzed to disentangle whether such associations represent causal relationships. This work helps to build a framework for genomic studies of eco-evolutionary dynamics.

Transferable AmpCs in Klebsiella pneumoniae: interplay with peptidoglycan recycling, mechanisms of hyperproduction, and virulence implications.

Chromosomal and transferable AmpC ß-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to ß-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most ß-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable ß-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC ß-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for ß-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.

The balance between antibiotic resistance and fitness/virulence in Pseudomonas aeruginosa: an update on basic knowledge and fundamental research.

The interplay between antibiotic resistance and bacterial fitness/virulence has attracted the interest of researchers for decades because of its therapeutic implications, since it is classically assumed that resistance usually entails certain biological costs. Reviews on this topic revise the published data from a general point of view, including studies based on clinical strains or in vitro-evolved mutants in which the resistance phenotype is seen as a final outcome, i.e., a combination of mechanisms. However, a review analyzing the resistance/fitness balance from the basic research perspective, compiling studies in which the different resistance pathways and respective biological costs are individually approached, was missing. Here we cover this gap, specifically focusing on Pseudomonas aeruginosa, a pathogen that stands out because of its extraordinary capacity for resistance development and for which a considerable number of recent and particular data on the interplay with fitness/virulence have been released. The revised information, split into horizontally-acquired vs. mutation-driven resistance, suggests a great complexity and even controversy in the resistance-fitness/virulence balance in the acute infection context, with results ranging from high costs linked to certain pathways to others that are seemingly cost-free or even cases of resistance mechanisms contributing to increased pathogenic capacities. The elusive mechanistic basis for some enigmatic data, knowledge gaps, and possibilities for therapeutic exploitation are discussed. The information gathered suggests that resistance-fitness/virulence interplay may be a source of potential antipseudomonal targets and thus, this review poses the elementary first step for the future development of these strategies harnessing certain resistance-associated biological burdens.

Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysis.

Background: Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year time lapse. Methods: A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings: A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum ß-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem-resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation: While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the prevalence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance. Funding: MSD and the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU.

A stabilizing eco-evolutionary feedback loop in the wild.

There is increasing evidence that evolutionary and ecological processes can operate on the same timescale1,2 (i.e., contemporary time). As such, evolution can be sufficiently rapid to affect ecological processes such as predation or competition. Thus, evolution can influence population, community, and ecosystem-level dynamics. Indeed, studies have now shown that evolutionary dynamics can alter community structure3,4,5,6 and ecosystem function.7,8,9,10 In turn, shifts in ecological dynamics driven by evolution might feed back to affect the evolutionary trajectory of individual species.11 This feedback loop, where evolutionary and ecological changes reciprocally affect one another, is a central tenet of eco-evolutionary dynamics.1,12 However, most work on such dynamics in natural populations has focused on one-way causal associations between ecology and evolution.13 Hence, direct empirical evidence for eco-evolutionary feedback is rare and limited to laboratory or mesocosm experiments.13,14,15,16 Here, we show in the wild that eco-evolutionary dynamics in a plant-feeding arthropod community involve a negative feedback loop. Specifically, adaptation in cryptic coloration in a stick-insect species mediates bird predation, with local maladaptation increasing predation. In turn, the abundance of arthropods is reduced by predation. Here, we experimentally manipulate arthropod abundance to show that these changes at the community level feed back to affect the stick-insect evolution. Specifically, low-arthropod abundance increases the strength of selection on crypsis, increasing local adaptation of stick insects in a negative feedback loop. Our results suggest that eco-evolutionary feedbacks are able to stabilize complex systems by preventing consistent directional change and therefore increasing resilience.

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.

Susceptibility profiles and resistance genomics of Pseudomonas aeruginosa isolates from European ICUs participating in the ASPIRE-ICU trial.

OBJECTIVES: To determine the susceptibility profiles and the resistome of Pseudomonas aeruginosa isolates from European ICUs during a prospective cohort study (ASPIRE-ICU). METHODS: 723 isolates from respiratory samples or perianal swabs of 402 patients from 29 sites in 11 countries were studied. MICs of 12 antibiotics were determined by broth microdilution. Horizontally acquired ß-lactamases were analysed through phenotypic and genetic assays. The first respiratory isolates from 105 patients providing such samples were analysed through WGS, including the analysis of the resistome and a previously defined genotypic resistance score. Spontaneous mutant frequencies and the genetic basis of hypermutation were assessed. RESULTS: All agents except colistin showed resistance rates above 20%, including ceftolozane/tazobactam and ceftazidime/avibactam. 24.9% of the isolates were XDR, with a wide intercountry variation (0%-62.5%). 13.2% of the isolates were classified as DTR (difficult-to-treat resistance). 21.4% of the isolates produced ESBLs (mostly PER-1) or carbapenemases (mostly NDM-1, VIM-1/2 and GES-5). WGS showed that these determinants were linked to high-risk clones (particularly ST235 and ST654). WGS revealed a wide repertoire of mutation-driven resistance mechanisms, with multiple lineage-specific mutations. The most frequently mutated genes were gyrA, parC, oprD, mexZ, nalD and parS, but only two of the isolates were hypermutable. Finally, a good accuracy of the genotypic score to predict susceptibility (91%-100%) and resistance (94%-100%) was documented. CONCLUSIONS: An overall high prevalence of resistance is documented European ICUs, but with a wide intercountry variability determined by the dissemination of XDR high-risk clones, arguing for the need to reinforce infection control measures.

Comparative analysis of in vitro dynamics and mechanisms of ceftolozane/tazobactam and imipenem/relebactam resistance development in Pseudomonas aeruginosa XDR high-risk clones.

OBJECTIVES: To analyse the dynamics and mechanisms of stepwise resistance development to ceftolozane/tazobactam and imipenem/relebactam in XDR Pseudomonas aeruginosa clinical strains. METHODS: XDR clinical isolates belonging to ST111 (main resistance mechanisms: oprD-, dacB-, CARB-2), ST175 (oprD-, ampR-G154R) and ST235 (oprD-, OXA-2) high-risk clones were incubated for 24 h in Müeller-Hinton Broth with 0.125-64 mg/L of ceftolozane + tazobactam 4 mg/L or imipenem + relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated into fresh medium containing concentrations up to 64 mg/L for 7 consecutive days. Two colonies per strain from each of the triplicate experiments were characterized by determining the susceptibility profiles, whole genome sequencing (WGS), and in vitro fitness through competitive growth assays. RESULTS: Resistance development occurred more slowly and reached a lower level for imipenem/relebactam than for ceftolozane/tazobactam in all tested XDR strains. Moreover, resistance development to imipenem/relebactam remained low even for ST175 isolates that had developed ceftolozane/tazobactam resistance during therapy. Lineages evolved in the presence of ceftolozane/tazobactam showed high-level resistance, imipenem/relebactam hypersusceptibility and low fitness cost, whereas lineages evolved in the presence of imipenem/relebactam showed moderate (borderline) resistance, no cross-resistance to ceftolozane/tazobactam and high fitness cost. WGS evidenced that ceftolozane/tazobactam resistance was mainly caused by mutations in the catalytic centres of intrinsic (AmpC) or acquired (OXA) ß-lactamases, whereas lineages evolved in imipenem/relebactam frequently showed structural mutations in MexB or in ParS, along with some strain-specific mutations. CONCLUSIONS: Imipenem/relebactam could be a useful alternative for the treatment of XDR P. aeruginosa infections, potentially reducing resistance development during therapy.

In vitro evolution of cefepime/zidebactam (WCK 5222) resistance in Pseudomonas aeruginosa: dynamics, mechanisms, fitness trade-off and impact on in vivo efficacy.

OBJECTIVES: To study the dynamics, mechanisms and fitness cost of resistance selection to cefepime, zidebactam and cefepime/zidebactam in Pseudomonas aeruginosa. METHODS: WT P. aeruginosa PAO1 and its ΔmutS derivative (PAOMS) were exposed to stepwise increasing concentrations of cefepime, zidebactam and cefepime/zidebactam. Selected mutants were characterized for change in susceptibility profiles, acquired mutations, fitness, virulence and in vivo susceptibility to cefepime/zidebactam. Mutations were identified through WGS. In vitro fitness was assessed by measuring growth in minimal medium and human serum-supplemented Mueller-Hinton broth. Virulence was determined in Caenorhabditis elegans and neutropenic mice lung infection models. In vivo susceptibility to a human-simulated regimen (HSR) of cefepime/zidebactam was studied in neutropenic mice lung infection. RESULTS: Resistance development was lower for the cefepime/zidebactam combination than for the individual components and high-level resistance was only achieved for PAOMS. Cefepime resistance development was associated with mutations leading to the hyperexpression of AmpC or MexXY-OprM, combined with PBP3 mutations and/or large chromosomal deletions involving galU. Zidebactam resistance was mainly associated with mutations in PBP2. On the other hand, resistance to cefepime/zidebactam required multiple mutations in genes encoding MexAB-OprM and its regulators, as well as PBP2 and PBP3. Cumulatively, these mutations inflicted significant fitness cost and cefepime/zidebactam-resistant mutants (MIC = 16-64 mg/L) remained susceptible in vivo to the HSR. CONCLUSIONS: Development of cefepime/zidebactam resistance in P. aeruginosa required multiple simultaneous mutations that were associated with a significant impairment of fitness and virulence.

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.

Metagenomics Analysis Reveals an Extraordinary Inner Bacterial Diversity in Anisakids (Nematoda: Anisakidae) L3 Larvae.

L3 larvae of anisakid nematodes are an important problem for the fisheries industry and pose a potential risk for human health by acting as infectious agents causing allergies and as potential vectors of pathogens and microrganisms. In spite of the close bacteria-nematode relationship very little is known of the anisakids microbiota. Fresh fish could be contaminated by bacteria vectored in the cuticle or in the intestine of anisakids when the L3 larvae migrate through the muscles. As a consequence, the bacterial inoculum will be spread, with potential effects on the quality of the fish, and possible clinical effects cannot be discarded. A total of 2,689,113 16S rRNA gene sequences from a total of 113 L3 individuals obtained from fish captured along the FAO 27 fishing area were studied. Bacteria were taxonomically characterized through 1803 representative operational taxonomic units (OTUs) sequences. Fourteen phyla, 31 classes, 52 orders, 129 families and 187 genera were unambiguously identified. We have found as part of microbiome an average of 123 OTUs per L3 individual. Diversity indices (Shannon and Simpson) indicate an extraordinary diversity of bacteria at an OTU level. There are clusters of anisakids individuals (samples) defined by the associated bacteria which, however, are not significantly related to fish hosts or anisakid taxa. This suggests that association or relationship among bacteria in anisakids, exists without the influence of fishes or nematodes. The lack of relationships with hosts of anisakids taxa has to be expressed by the association among bacterial OTUs or other taxonomical levels which range from OTUs to the phylum level. There are significant biological structural associations of microbiota in anisakid nematodes which manifest in clusters of bacteria ranging from phylum to genus level, which could also be an indicator of fish contamination or the geographic zone of fish capture. Actinobacteria, Aquificae, Firmicutes, and Proteobacteria are the phyla whose abundance value discriminate for defining such structures.

Caracterización epidemiológica de pacientes con miopatía inflamatoria en un hospital de cuarto nivel en Cali, Colombia / Epidemiological characterization of patients with inflammatory myopathy in a fourth level hospital in Cali, Colombia

Introducción: Las miopatías inflamatorias idiopáticas (MII) constituyen un grupo heterogéneo de enfermedades que comprometen la musculatura esquelética y se manifiestan por debilidad y signos inflamatorios en la biopsia muscular. El objetivo de este estudio es hacer una caracterización epidemiológica de una cohorte de pacientes con MII en una población del suroccidente colombiano. Metodología: De forma retrospectiva, se revisaron las historias clínicas de pacientes con diagnóstico de MII que fueron tratados en un hospital de cuarto nivel de complejidad en Cali, Colombia, entre el 2011 y el 2017. Se recolectaron variables demográficas, clínicas, serológicas y de tratamiento. Resultados: Se identificaron 72 pacientes con MII, mayoritariamente mujeres (n = 54, 75%). La media de edad al inicio de los síntomas fue de 37,11 ± 19,18 años. Las principales MII fueron dermatomiositis (DM) y polimiositis, las cuales se presentaron en 35 (48,6%) y 25 pacientes (34,7%), respectivamente. Veintiocho pacientes (38,8%) presentaban enfermedad autoinmune asociada, siendo el lupus eritematoso sistémico la más frecuente, al presentarse en7 (9,72%) pacientes. La biopsia de músculo se realizó en 25 pacientes (34,7%), mientras que28 (38,8%) tenían anticuerpos antinucleares positivos. La mediana de la creatinfosfoquinasa fue de 877,5 mg/dL (163,5-4.358,3). Sesenta y siete pacientes (93,1%) fueron tratados con glucocorticoides y 18 (25%) con rituximab (RTX) como monoterapia o combinado con otro fármaco inmunosupresor. Conclusiones: La DM es la condición clínica más frecuente, es común en mujeres y se presenta en la cuarta década de vida. Los tratamientos con los que más se obtuvo mejoría clínica fueron los glucocorticoides, seguidos del RTX en monoterapia o combinado con otros inmunosupresores.

Background: Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of diseases characterised by skeletal muscle involvement, manifested by weakness and inflammatory signs in the muscle biopsy. The objective of this article is to describe the clinical, laboratory, and treatment features of a cohort of patients with IIM in southwest Colombia. Methods: A retrospective review was conducted on the medical records of patients diagnosed with IIM treated at a fourth-level complexity hospital in Cali, Colombia, from 2011 to 2017. Demographic, clinical, serological, and treatment data were collected. Results: A total of 72 patients with IIM were identified, mostly women (n = 54,75%). The mean age at onset of symptoms was 37.11 ± 19.18 years. The main subtypes of IIM were dermatomyositis (DM) and polymyositis, occurring in 35 patients (48.6%) and 25 patients (34.7%), respectively. Twenty-eight patients (38.8%) had associated autoimmune disease, with syste mic lupus erythematosus being the most frequent in 7 (9.72%) patients. Muscle biopsy was performed in 25 patients (34.7%), while 28 (38.8%) had positive antinuclear antibodies. The median creatine phosphokinase was 877.5 mg/dL (163.5-4358.3). Sixty-seven patients (93.1%) were treated with glucocorticoids, and 18 (25%) patients were treated with rituximab (RTX) as monotherapy or combined with another immunosuppressant drug. Conclusions: DM is the most frequent subtype of IIM, being common in women and occurring in the fourth decade of life. The most used treatments were glucocorticoids, followed by RTX monotherapy, or combined with other immunosuppressants.

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)

Molecular Analysis of the Contribution of Alkaline Protease A and Elastase B to the Virulence of Pseudomonas aeruginosa Bloodstream Infections.

Pseudomonas aeruginosa is a major cause of nosocomial bloodstream infections. This microorganism secretes two major proteases, alkaline protease A (AprA) and elastase B (LasB). Despite several in vitro studies having demonstrated that both purified proteases cleave a number of components of the immune system, their contribution to P. aeruginosa bloodstream infections in vivo remains poorly investigated. In this study, we used a set of isogenic mutants deficient in AprA, LasB or both to demonstrate that these exoproteases are sufficient to cleave the complement component C3, either soluble or deposited on the bacteria. Nonetheless, exoprotease-deficient mutants were as virulent as the wild-type strain in a murine model of systemic infection, in Caenorhabditis elegans and in Galleria mellonella. Consistently, the effect of the exoproteases on the opsonization of P. aeruginosa by C3 became evident four hours after the initial interaction of the complement with the microorganism and was not crucial to survival in blood. These results indicate that exoproteases AprA and LasB, although conferring the capacity to cleave C3, are not essential for the virulence of P. aeruginosa bloodstream infections.

Rituximab for Inflammatory Myopathies in a Colombian Cohort.

INTRODUCTION/OBJECTIVES: Rituximab (RTX) is a treatment for refractory inflammatory myopathies, such as dermatomyositis (DM) and polymyositis (PM). This study describes the characteristics of patients receiving RTX for myositis in our institution to evaluate its efficacy. METHOD: We collected demographic data from all patients diagnosed with DM or PM who received RTX between 2011 and 2018. Clinical and serological variables (including creatine phosphokinase [CPK] levels) were analyzed. Remission of disease was defined as no evidence of disease activity (active myositis) for longer than a 6-month continuous period while undergoing myositis therapy or no medication. RESULTS: Eighteen patients who had received first-line immunosuppressants were included. Fifteen (83%) had DM, 2 (11%) had PM, 1 had juvenile dermatomyositis, and 14 (77%) were women. All patients received glucocorticoids. Three patients (16.6%) were treated with RTX as monotherapy, and 15 (83.3%) were treated with RTX combined with other immunosuppressants. On average, there were 2 RTX treatment cycles. Improved muscular weakness was found in 13 cases (72%), and improved serum CPK levels were found in 15 cases (83%). Twelve patients (66%) achieved remission. CONCLUSIONS: Most patients experienced an objective improvement, as reflected in their serum CPK values and degree of muscular weakness. This suggests that RTX could be helpful in treating refractory myositis.

A Genome-Based Model to Predict the Virulence of Pseudomonas aeruginosa Isolates.

Variation in the genome of Pseudomonas aeruginosa, an important pathogen, can have dramatic impacts on the bacterium's ability to cause disease. We therefore asked whether it was possible to predict the virulence of P. aeruginosa isolates based on their genomic content. We applied a machine learning approach to a genetically and phenotypically diverse collection of 115 clinical P. aeruginosa isolates using genomic information and corresponding virulence phenotypes in a mouse model of bacteremia. We defined the accessory genome of these isolates through the presence or absence of accessory genomic elements (AGEs), sequences present in some strains but not others. Machine learning models trained using AGEs were predictive of virulence, with a mean nested cross-validation accuracy of 75% using the random forest algorithm. However, individual AGEs did not have a large influence on the algorithm's performance, suggesting instead that virulence predictions are derived from a diffuse genomic signature. These results were validated with an independent test set of 25 P. aeruginosa isolates whose virulence was predicted with 72% accuracy. Machine learning models trained using core genome single-nucleotide variants and whole-genome k-mers also predicted virulence. Our findings are a proof of concept for the use of bacterial genomes to predict pathogenicity in P. aeruginosa and highlight the potential of this approach for predicting patient outcomes.IMPORTANCEPseudomonas aeruginosa is a clinically important Gram-negative opportunistic pathogen. P. aeruginosa shows a large degree of genomic heterogeneity both through variation in sequences found throughout the species (core genome) and through the presence or absence of sequences in different isolates (accessory genome). P. aeruginosa isolates also differ markedly in their ability to cause disease. In this study, we used machine learning to predict the virulence level of P. aeruginosa isolates in a mouse bacteremia model based on genomic content. We show that both the accessory and core genomes are predictive of virulence. This study provides a machine learning framework to investigate relationships between bacterial genomes and complex phenotypes such as virulence.

In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa.

OBJECTIVES: We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. METHODS: PAO1 or PAOMS strains were incubated for 24 h in Mueller-Hinton Broth with 0.125-64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. RESULTS: Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. CONCLUSIONS: Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

Activity of Imipenem-Relebactam against a Large Collection of Pseudomonas aeruginosa Clinical Isolates and Isogenic ß-Lactam-Resistant Mutants.

Imipenem and imipenem-relebactam MICs were determined for 1,445 Pseudomonas aeruginosa clinical isolates and a large panel of isogenic mutants showing the most relevant mutation-driven ß-lactam resistance mechanisms. Imipenem-relebactam showed the highest susceptibility rate (97.3%), followed by colistin and ceftolozane-tazobactam (both 94.6%). Imipenem-relebactam MICs remained ≤2 µg/ml in all 16 isogenic PAO1 mutants and in 8 pairs of extensively drug-resistant clinical strains that had developed resistance to ceftolozane-tazobactam and ceftazidime-avibactam due to mutations in OXA-10 or AmpC.

Pathogenic characteristics of Pseudomonas aeruginosa bacteraemia isolates in a high-endemicity setting for ST175 and ST235 high-risk clones.

Multidrug-resistant (MDR) Pseudomonas aeruginosa represents a major clinical concern. The interplay between antimicrobial resistance and virulence of P. aeruginosa was investigated in in vitro and in vivo studies. Thirty-eight well-characterized (21 MDR and 17 non-MDR) P. aeruginosa strains from patients with bacteraemia were analysed. Resistance phenotype, carbapenemase production, clonal relatedness, type III secretion system genotype, O-antigen serotype, cytotoxicity (ability to lyse cells) on A549 cells, and virulence (lethality in nematodes) in a Caenorhabditis elegans model were investigated. MDR strains showed lower cytotoxicity (35.4 ± 21.30% vs. 45.0 ± 18.78 %; P = 0.044) and virulence (66.7% vs. 100%; P = 0.011) than non-MDR strains. However, the pathogenicity of MDR high-risk clones varied broadly, with ST235 and ST175 clones being the most and least cytotoxic (51.8 ± 10.59% vs. 11.0 ± 1.25%; P < 0.0001) and virulent ([100% vs. 73.1; P = 0.075] and [0% vs. 93.9%; P < 0.0001], respectively). The pathogenicity of the ST235 clone was similar to that of non-MDR strains, and its ability to lyse cells and high virulence were related with the exoU-positive genotype. Furthermore, the O11 serotype was more frequent among the ST235 clone and exoU-positive genotype strains and was also essential for the pathogenicity of P. aeruginosa. Our data suggest that the pathogenicity of MDR high-risk clones is the result not only of the resistance phenotype but also of the virulence genotype. These findings have implications for the clinical management of patients and infection control programmes.