Identification and characterization of CIM-1, a carbapenemase that adds to the family of resistance factors against last resort antibiotics.

The increasing rate of carbapenem-resistant bacteria within healthcare environments is an issue of great concern that needs urgent attention. This resistance is driven by metallo-ß-lactamases (MBLs), which can catalyse the hydrolysis of almost all clinically available ß-lactams and are resistant to all the clinically utilized ß-lactamase inhibitors. In this study, an uncharacterized MBL is identified in a multidrug resistant isolate of the opportunistic pathogen, Chryseobacterium indologenes. Sequence analysis predicts this MBL (CIM-1) to be a lipoprotein with an atypical lipobox. Characterization of CIM-1 reveals it to be a high-affinity carbapenemase with a broad spectrum of activity that includes all cephalosporins and carbapenems. Results also shown that CIM-1 is potentially a membrane-associated MBL with an uncharacterized lipobox. Using prediction tools, we also identify more potentially lipidated MBLs with non-canonical lipoboxes highlighting the necessity of further investigation of lipidated MBLs.

Exploiting the aggregation propensity of beta-lactamases to design inhibitors that induce enzyme misfolding.

There is an arms race between beta-lactam antibiotics development and co-evolving beta-lactamases, which provide resistance by breaking down beta-lactam rings. We have observed that certain beta-lactamases tend to aggregate, which persists throughout their evolution under the selective pressure of antibiotics on their active sites. Interestingly, we find that existing beta-lactamase active site inhibitors can act as molecular chaperones, promoting the proper folding of these resistance factors. Therefore, we have created Pept-Ins, synthetic peptides designed to exploit the structural weaknesses of beta-lactamases by causing them to misfold into intracellular inclusion bodies. This approach restores sensitivity to a wide range of beta-lactam antibiotics in resistant clinical isolates, including those with Extended Spectrum variants that pose significant challenges in medical practice. Our findings suggest that targeted aggregation of resistance factors could offer a strategy for identifying molecules that aid in addressing the global antibiotic resistance crisis.

The chloroplast protein HCF164 is predicted to be associated with Coffea SH9 resistance factor against Hemileia vastatrix.

To explore the connection between chloroplast and coffee resistance factors, designated as SH1 to SH9, whole genomic DNA of 42 coffee genotypes was sequenced, and entire chloroplast genomes were de novo assembled. The chloroplast phylogenetic haplotype network clustered individuals per species instead of SH factors. However, for the first time, it allowed the molecular validation of Coffea arabica as the maternal parent of the spontaneous hybrid "Híbrido de Timor". Individual reads were also aligned on the C. arabica reference genome to relate SH factors with chloroplast metabolism, and an in-silico analysis of selected nuclear-encoded chloroplast proteins (132 proteins) was performed. The nuclear-encoded thioredoxin-like membrane protein HCF164 enabled the discrimination of individuals with and without the SH9 factor, due to specific DNA variants linked to chromosome 7c (from C. canephora-derived sub-genome). The absence of both the thioredoxin domain and redox-active disulphide center in the HCF164 protein, observed in SH9 individuals, raises the possibility of potential implications on redox regulation. For the first time, the identification of specific DNA variants of chloroplast proteins allows discriminating individuals according to the SH profile. This study introduces an unexplored strategy for identifying protein/genes associated with SH factors and candidate targets of H. vastatrix effectors, thereby creating new perspectives for coffee breeding programs.

Mismatch Repair system protein deficiency as a resistance factor for locally advanced rectal adenocarcinoma patients receiving neoadjuvant chemo-radiotherapy.

BACKGROUND: Available data on Mismatch Repair system (MMR) deficiency are conflicting and derived from small studies. Our study aimed to evaluate the therapeutic implications of MMR status in patients with locally advanced rectal cancer (LARC). METHODS: We retrospectively collected data from 318 patients affected by LARC treated in Italy at the Medical Oncology Units of the University Hospital of Cagliari, Istituto Nazionale dei Tumori Milan, and AOU Ospedali Riuniti Ancona. All patients underwent neoadjuvant chemoradiotherapy. The primary objective was major TRG while secondary objectives were pathological complete response, disease-free survival (DFS) and overall survival (OS). RESULTS: One hundred sixty patients (148 pMMR and 12 dMMR) were included in the exploratory cohort and 158 (146 pMMR and 12 dMMR) were included in the validation cohort. A major TRG has been shown in 42.6% and 43.1% patients with pMMR in exploratory and validation cohort, respectively; while no major TRG have been shown in dMMR patients in both cohorts. Exploratory and validation cohorts showed a statistically significant higher mDFS in pMMR patients compared to dMMR: NR vs. 14 months and NR vs. 17 months, respectively. CONCLUSION: Our results indicated an association between dMMR and poor response to preoperative chemoradiotherapy and they represent a hypothesis-generating data for new neoadjuvant strategies.

Genome sequence of Vibrio anguillarum isolates carrying a novel class A ß-lactamase VAN-1: do migratory fish transport novel resistance factors?

OBJECTIVES: The aim of the study was to understand the genetic basis of resistance of five ß-lactam resistant Vibrio anguillarum isolates obtained from the gut content of Atlantic mackerel (Scomber scomberus), using whole genome sequencing and to characterize a novel ß-lactamase (VAN-1) from these isolates. METHOD: Antibiotic sensitivity pattern was determined using Sensititre™ plates and whole genome sequencing was carried out using Illumina MiSeq-based sequencing. The blaVAN-1 gene was synthesized and expressed in Escherichia coli Top10 cells. RESULTS: Five isolates obtained (out of 73) from the gut content of Atlantic mackerel were identified as Vibrio anguillarum. Whole genome assemblies ranged from 3.894 to 3.906 million bases in length with an average of 50 contigs. A novel ß-lactamase blaVAN-1, sharing 77.7% nucleotide identity with a known mobile ß-lactamase from Vibrio species was detected. The blaVAN-1 gene in these isolates is flanked by a truncated IS5 family transposase on one end and a hypothetical protein and outer membrane protein followed by another IS5 family transposase on the other end, suggesting its potential for mobility. The blaVAN-1 gene was absent in V. anguillarum type strain (ATCC 14181) and V. anguillarum isolates from bivalves and sea water in Norway. VAN-1 conferred ampicillin resistance when expressed in E. coli, thus confirming the functionality of this gene. CONCLUSIONS: Our study highlights the importance of the marine environment as a reservoir of new antibiotic resistance genes. Our results suggest that migratory fish may transport novel antibiotic resistance determinants over long distances.

Efecto del extracto hexánico de Tagetes lucida sobre la inhibición del crecimiento de enterobacterias Shigella flexneri y Salmonella typhi / Effect of the hexanic extract of Tagetes lucida on the inhibition of the growth of enterobacteria Shigella flexneri and Salmonella typhi

La resistencia a los antibióticos aumenta la búsqueda de nuevas estrategias para combatir las enfermedades que causan, y el uso de plantas medicinales representa una estrategia altamente efectiva y valiosa, como el uso de Tagetes lucida con diferentes bacterias gram positivas y gram negativas.Objetivo: Evaluar la actividad biológica que tiene el extracto hexanico de la planta Tagetes lucida a diferentes concentraciones sobre la inhibición del crecimiento en placa y tubo de dos enterobacterias, Shigella flexneri y Salmonella typhiMétodos: En el siguiente trabajo, se evaluó un extracto de hexano de Tagetes lucida sobre la inhibición del crecimiento de dos enterobacterias, Shigella flexneri y Salmonella typhi utilizando diferentes concentraciones de vehículo para evaluar si afectaba el crecimiento bacteriano y también diferentes concentraciones de extracto para evaluar la actividad.Resultados: Realizados los estudios por triplicado se logró concretar que a partir de 75µl/µg de extracto se logra una inhibición casi total del crecimiento de ambas bacterias, tanto en método de placa, como en método de tubo. Y a partir de 100 µl/µg se logra una inhibición total.Conclusiones: Los resultados favorables obtenidos con 75 µl/µg, permiten confirmar que los extractos de plantas medicinales son una estrategia importante para combatir infecciones bacterianas multi-resistentes. Por otro lado permite dar paso a un estudio para evaluar los metabolitos más activos del extracto, así como, el mecanismo de acción sobre la inhibición del crecimiento de las bacterias en estudio. (AU)

Antibiotic resistance increases the search for new strategies to combat the diseases they cause, and the use of medicinal plants represents a highly effective and valuable strategy, such as the use of Tagetes lucida with different gram positive and gram negative bacteria.Objective: To evaluate the biological activity of the hexane extract of the Tagetes lucida plant at different concentrations on the inhibition of growth in plaque and tube of two enterobacteriaceae, Shigella flexneri and Salmonella typhiMethods: In the following work, a hexane extract from Tagetes lucida was evaluated on the growth inhibition of two enterobacteriaceae, Shigella flexneri and Salmonella typhi using different concentrations of vehicle to evaluate if it affected bacterial growth and also different concentrations of extract to evaluate activity.Results: Once the studies were carried out in triplicate, it was possible to specify that from 75µl/µg of extract, almost total inhibition of the growth of both bacteria was achieved, both in the plate method and in the tube method. And from 100 µl/µg total inhibition is achieved.Conclusions: The favorable results obtained with 75 µl/ µg, confirm that medicinal plant extracts are an important strategy to combat multi-drug resistant bacterial infections. On the other hand, it allows a study to be carried out to evaluate the most active metabolites of the extract, as well as the mechanism of action on the inhibition of the growth of the bacteria under study. (AU)

Genes involucrados con resistencia antimicrobiana en hospitales del Ecuador / Genes involved with antimicrobial resistance in Ecuadorian hospitals

INTRODUCCIÓN. La resistencia a los antimicrobianos es un problema de salud pública actual asociado con alta mortalidad, hospitalización prolongada, alternativas terapéuticas reducidas, mayores costos económicos y la posibilidad de brotes hospitalarios. OBJETIVO. Describir los principales genes involucrados con resistencia antimicrobiana en hospitales del Ecuador. MATERIALES Y MÉTODOS. Se realizó una descripción retrospectiva no experimental, de artículos indexados relacionados con resistencia antimicrobiana en hospitales del Ecuador, con evidencia desde el año 2009 al 2022. La revisión de bibliografías se llevó a cabo en bases de datos como Pubmed, Science Direct y Google Scholar. RESULTADOS. De un grupo original de 77 artículos, se seleccionaron 33 documentos. En Ecuador, varios estudios han descrito los mecanismos moleculares involucrados en la resistencia bacteriana. Sin embargo, en bacterias menos comunes, falta investigación sobre los genes asociados. CONCLUSIONES. Las principales bacterias multirresistentes descritas en Ecuador son Klebsiella pneumoniae, Escherichia coli y Acinetobacter baumanni, las cuales presentan genes involucrados en la producción de carbapenemasas (blaKPC, blaNDM, blaOXA-48). Estas bacterias presentan altos niveles de resistencia a los antibióticos y son objeto de vigilancia epidemiológica por parte del sistema nacional de salud. A nivel local, otras bacterias presentan mecanismos de resistencia a los carbapenémicos (Pseudomonas aeruginosa, Enterobacter sp., Serratia marcescens, Citrobacter sp.), pero no existen descripciones detalladas del genotipo, sus características microbiológicas o la clínica del paciente. El conocimiento de las tasas de resistencia a los antimicrobianos en los diferentes hospitales, la implementación de un plan de administración de antibióticos, el uso correcto de los equipos de protección personal, el aislamiento de las personas con infecciones multirresistentes, así como el trabajo colaborativo entre las diferentes áreas del hospital, son esenciales para reducir la propagación de estos patógenos.

INTRODUCTION. Antimicrobial resistance is a current public health problem associated with high mortality, prolonged hospitalization, reduced therapeutic alternatives, increased economic costs, and the potential for hospital outbreaks. OBJECTIVE. To describe the main genes involved with antimicrobial resistance in hospitals in Ecuador. MATERIALS AND METHODS. A retrospective non-experimental description of indexed articles related to antimicrobial resistance in hospitals in Ecuador was carried out, with evidence from 2009 to 2022. The review of bibliographies was carried out in databases such as Pubmed, Science Direct and Google Scholar. RESULTS. From an original group of 77 articles, 33 papers were selected. In Ecuador, several studies have described the molecular mechanisms involved in bacterial resistance. However, in less common bacteria, research on the associated genes is lacking. CONCLUSIONS. The main multidrug-resistant bacteria described in Ecuador are Klebsiella pneumoniae, Escherichia coli and Acinetobacter baumanni, which present genes involved in the production of carbapenemases (blaKPC, blaNDM, blaOXA-48). These bacteria present high levels of antibiotic resistance and are subject to epidemiological surveillance by the national health system. Locally, other bacteria present mechanisms of resistance to carbapenemics (Pseudomonas aeruginosa, Enterobacter sp., Serratia marcescens, Citrobacter sp.), but there are no detailed descriptions of the genotype, their microbiological characteristics or the patient's clinic. Knowledge of antimicrobial resistance rates in different hospitals, the implementation of an antibiotic stewardship plan, the correct use of personal protective equipment, the isolation of individuals with multidrug-resistant infections, as well as collaborative work between different areas of the hospital, are essential to reduce the spread of these pathogens.

Genomic Amplification of UBQLN4 Is a Prognostic and Treatment Resistance Factor.

Ubiquilin-4 (UBQLN4) is a proteasomal shuttle factor that directly binds to ubiquitylated proteins and delivers its cargo to the 26S proteasome for degradation. We previously showed that upregulated UBQLN4 determines the DNA damage response (DDR) through the degradation of MRE11A. However, the regulatory mechanism at DNA level, transcriptionally and post-transcriptional levels that control UBQLN4 mRNA levels remains unknown. In this study, we screened 32 solid tumor types and validated our findings by immunohistochemistry analysis. UBQLN4 is upregulated at both mRNA and protein levels and the most significant values were observed in liver, breast, ovarian, lung, and esophageal cancers. Patients with high UBQLN4 mRNA levels had significantly poor prognoses in 20 of 32 cancer types. DNA amplification was identified as the main mechanism promoting UBQLN4 upregulation in multiple cancers, even in the early phases of tumor development. Using CRISPR screen datasets, UBQLN4 was identified as a common essential gene for tumor cell viability in 81.1% (860/1,060) of the solid tumor derived cell lines. Ovarian cancer cell lines with high UBQLN4 mRNA levels were platinum-based chemotherapy resistant, while they were more sensitive to poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Our findings highlight the utilities of UBQLN4 as a significant pan-cancer theranostic factor and a precision oncology biomarker for DDR-related drug resistance.

Anticoagulant rodenticide blood-clotting dose-responses and resistance factors for Tyrosine139Cysteine (Y139C) heterozygous- and homozygous-resistant house mice (Mus musculus).

BACKGROUND: The house mouse (Mus musculus) is a globally distributed rodent pest species against which anticoagulant rodenticides are widely used for the protection of human and animal health and the conservation of threatened wildlife. Anticoagulant-resistant house mice have been known for more than half a century. A house mouse strain was developed in the laboratory that was homozygous resistant for the single nucleotide polymorphism (SNP) Tyrosine139Cysteine (Y139C) and, subsequently, heterozygous resistant animals were produced from this strain by crossing with the homozygous susceptible strain. RESULTS: Using blood clotting response tests, resistance factors at the ED50 level in the homozygous resistant strain for the first-generation anticoagulants warfarin, chlorophacinone, diphacinone and coumatetralyl were in the range 31.5 to 628.0 for males (M) and 21.6 to 628.0 for females (F), thus indicating that Y139C house mice are substantially resistant to all these substances. Resistance factors at the ED50 level for the homozygous strain generated against the second-generation compounds were: brodifacoum (M, 1.7; F, 1.9), bromadiolone (M, 16.6; F, 21.0), difenacoum (M, 1.2; F, 2.7), difethialone (M, 1.5; F, 1.5), and flocoumafen (M, 0.9; F, 1.2). Equivalent values for the heterozygous strain were: brodifacoum (M, 1.6; F, 1.4), bromadiolone (M, 5.6; F, 6.5), difenacoum (M, 1.0; F, 1.3), difethialone (M, 1.1; F, 1.1), flocoumafen (M, 0.9; F, 1.1). CONCLUSION: Y139C SNP homozygous resistant mice are more resistant to anticoagulants than heterozygous resistant animals. All first-generation anticoagulants are highly resisted and, among the second-generation compounds, Y139C mice are resistant to bromadiolone and sometimes to difenacoum. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Regionalization and Shaping Factors for Microbiomes and Core Resistomes in Atmospheric Particulate Matters.

Antimicrobial resistance (AMR) seriously threatens public health by reducing antibiotic effectiveness in curing bacterial infections. Atmospheric particulate matter (APM) is a common environmental hazard that affects human health by causing various diseases and disseminating bacterial pathogenesis, of which pathogenic bacteria and AMR are essential parts. The properties of APM microbiomes and resistomes, along with their shaping factors and mutual relationships, need further examination. To address this, we analyzed APMs collected from 13 cities within four clusters (North and South China, Inner Mongolia, and Tibet). Significant regionalization was found for both the microbiomes (P < 0.001) and core resistomes (P < 0.001) for APMs, with statistical analyses showing significant differences in different regions. Principal coordinate analysis (PCoA) and accompanying ANOSIM analyses showed that microbiomes and core resistomes followed the same regional subclustering hierarchy patterns. This finding, together with response analysis of APM microbiomes and core resistomes to environmental parameters that showed similar response patterns, as well as Procrustes analysis (M2 = 0.963, P < 0.05) between APM microbiomes and core resistomes, strongly suggested that APM microbiomes and core resistomes are correlated. Co-occurrence network analysis further revealed key taxa and antimicrobial resistance determinants in the interactions between APM microbiomes and core resistomes. Thus, it was concluded that APM microbiome and resistome compositions were highly regional, that environmental pollutants and APM levels impacted APM microbiomes and resistomes, and that microbiomes and resistomes in APMs are significantly correlated (P < 0.05). IMPORTANCE Bacteria associated with atmospheric particulate matter (APMs) can transmit over long distances. A large portion of these bacteria can potentially threaten human health. The antimicrobial resistance (AMR) of pathogenic bacteria carried by APMs prevents curing from infections. Therefore, both the pathogenic bacteria in APMs and their AMR are receiving more attention. The literature suggests a knowledge gap that exists for bacterial AMR and bacterial pathogenesis in APMs, including their distribution patterns, mutual relationships, and factors influencing their compositions. This work aimed to bridge this knowledge gap by studying APM samples collected from 13 cities. The results demonstrated that both bacteria and antibiotic resistance determinants were highly regional and that their composition patterns were significantly correlated, and influenced by the same group of environmental factors. This study thus determined the relationship between the two important aspects of bacterial pathogenesis in APMs and represents significant progress in understanding bacterial pathogenesis in APMs.

Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp.

Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.

T Cell Defects: New Insights Into the Primary Resistance Factor to CD19/CD22 Cocktail CAR T-Cell Immunotherapy in Diffuse Large B-Cell Lymphoma.

Despite impressive progress, a significant portion of patients still experience primary or secondary resistance to chimeric antigen receptor (CAR) T-cell immunotherapy for relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). The mechanism of primary resistance involves T-cell extrinsic and intrinsic dysfunction. In the present study, a total of 135 patients of DLBCL treated with murine CD19/CD22 cocktail CAR T-therapy were assessed retrospectively. Based on four criteria (maximal expansion of the transgene/CAR-positive T-cell levels post-infusion [Cmax], initial persistence of the transgene by the CAR transgene level at +3 months [Tlast], CD19+ B-cell levels [B-cell recovery], and the initial response to CAR T-cell therapy), 48 patients were included in the research and divided into two groups (a T-normal group [n=22] and a T-defect [n=26] group). According to univariate and multivariate regression analyses, higher lactate dehydrogenase (LDH) levels before leukapheresis (hazard ratio (HR) = 1.922; p = 0.045) and lower cytokine release syndrome (CRS) grade after CAR T-cell infusion (HR = 0.150; p = 0.026) were independent risk factors of T-cell dysfunction. Moreover, using whole-exon sequencing, we found that germline variants in 47 genes were significantly enriched in the T-defect group compared to the T-normal group (96% vs. 41%; p<0.0001), these genes consisted of CAR structure genes (n=3), T-cell signal 1 to signal 3 genes (n=13), T cell immune regulation- and checkpoint-related genes (n=9), cytokine- and chemokine-related genes (n=13), and T-cell metabolism-related genes (n=9). Heterozygous germline UNC13D mutations had the highest intergroup differences (26.9% vs. 0%; p=0.008). Compound heterozygous CX3CR1I249/M280 variants, referred to as pathogenic and risk factors according to the ClinVar database, were enriched in the T-defect group (3 of 26). In summary, the clinical characteristics and T-cell immunodeficiency genetic features may help explain the underlying mechanism of treatment primary resistance and provide novel insights into CAR T-cell immunotherapy.

Isolation of a Colistin-Susceptible MDR Pantoea calida Harboring the mcr-9 Gene Suggests the Silent Spread of the Resistance Factor.

Pantoea spp. are bacteria that are often detected in the environment and as symbionts of arthropods. They sporadically cause infections in humans and recently extended spectrum beta-lactamases (ESBL)- and carbapenemase-producing strains have started to emerge. In this study, we report the isolation and the complete genome sequence of a strain of Pantoea calida encoding the colistin-resistance gene mcr-9. The strain was isolated from a preterm newborn in a neonatal pathology ward. On clinical examination, his vital signs were normal and blood culture was negative. Rectal swab screening for ESBL-producing Enterobacterales allowed to isolate the bacterium, and a complete genome was obtained using both short and long read sequencing. The mcr-9 gene was found to be encoded on a IncHI2 superplasmid, which confers resistance to six classes of antibiotics, including beta lactams (ESBL). Despite the presence of mcr-9, the isolate retains susceptibility to colistin, which could be explained by the absence of compatible regulatory genes (qseBC) from the genome. The presence of the resistance gene is undetectable with the routine clinical procedures, that is, phenotypic tests. This suggests that a silent spread might be ongoing in the ward. To our knowledge, this is the first description of an MDR P. calida and of a Pantoea spp. encoding any mobile colistin resistance gene.

Clinically healthy household dogs and cats as carriers of multidrug-resistant Salmonella enterica with variable R plasmids.

Introduction. Antimicrobial resistance (AMR) is a One Health issue concerning humans, animals and the environment and a unified One Health approach is required to contain this problematic issue. Dogs and cats are popular pet animals and are known to carry many bacterial pathogens that are of public health importance, including Salmonella. However, data on AMR in companion animals is limited.Gap statement. Scant AMR data from bacteria originating from companion animals limits an accurate assessment of the impacts of pet-animal-related AMR on public health.Purpose. This study aimed to phenotypically and genetically investigate AMR in Salmonella isolated from pet dogs and cats in Thailand.Methodology. Salmonella enterica were isolated from pet dogs (n=159) and cats (n=19) in Thailand between 2016 and 2019. All isolates were serotyped. Phenotypic and genotypic antimicrobial resistance was examined. PCR-based replicon typing, replicon sequence typing and plasmid multilocus sequence typing were conducted to characterize plasmids.Results. Seventy-seven serovars were identified, with serovars Weltevreden (9.6%) and Stockholm (9.0%) the most common. Most of the isolates (34.3%) were multidrug-resistant. The serovar Stockholm was an ESBL-producer and carried the ß-lactamase genes bla TEM-1 and bla CTX-M-55. The plasmid-mediated quinolone resistance (PMQR) gene, qnrS, was also detected (10.1%). Class 1 integrons carrying the dfrA12-aadA2 cassette array were most frequent (45.9%). Five plasmid replicon types as IncA/C (0.6%), N (1.1%), IncFIIA (28.7%), IncHI1 (2.2%), and IncI1 (3.4%) were identified. Based on the pMLST typing scheme (n=9), plasmids were assigned into five different STs including IncA/C-ST6 (n=1), IncH1-ST16 (n=4), IncI1-ST3 (n=1), IncI1-ST60 (n=1) and IncI1-ST136 (n=1). The ST 16 of IncHI1 plasmid was a novel plasmid ST. Subtyping F-type plasmids using the RST scheme (n=9) revealed four different combinations of replicons including S1:A-:B- (n=4), S1:A-:B22 (n=2), S3:A-:B- (n=1) and S-:A-:B47 (n=1).Conclusions. Our findings highlight the role of clinically healthy household dogs and cats as carriers of AMR Salmonella strains with different R plasmid. The implementation of AMR phenotypes instigation and genotypic monitoring and surveillance programmes in companion animals are imperative as integral components of the One Health framework.

Prevalence of insertion sequence elements in plasmids relating to mgrB gene disruption causing colistin resistance in Klebsiella pneumoniae.

Colistin is a last resort antibiotic for the treatment of carbapenemase producing Klebsiella pneumoniae. The disruption of the mgrB gene by insertion sequences (ISs) is a mechanism mediating colistin resistance. Plasmids encode mobilizable IS elements which integrate into the mgrB gene in K. pneumoniae causing gene inactivation and colistin resistance. The species prevalence of mgrB-gene disrupting insertion elements ISL3 (ISKpn25), IS5 (ISKpn26), ISKpn14, and IS903B present on plasmids were assessed. IS containing plasmids were also scanned for antimicrobial resistance genes, including carbapenem resistant genes. Plasmids encoding ISs are abundant in K. pneumoniae. IS903B was found in 28 unique Inc groups, while ISKpn25 was largely carried by IncFIB(pQil) plasmids. ISKpn26 and ISKpn14 were most often found associated with IncFII(pHN7A8) plasmids. Of the 34 unique countries which contained any of the IS elements, ISKpn25 was identified from 26. ISKpn26, ISKpn14, and IS903B ISs were identified from 89.3%, 44.9%, and 23.9% plasmid samples from China. Plasmids carrying ISKpn25, ISKpn14, and ISKpn26 IS have a 4.6-, 6.0-, and 6.6-fold higher carbapenemase gene count, respectively, relative to IS903B-carrying plasmids. IS903B bearing plasmids have a 20-, 5-, and 5-fold higher environmental source isolation count relative to ISKpn25, ISKpn14, and ISKpn26 bearing plasmids. ISKpn25 present on IncFIB(pQil) sourced from clinical settings is established across multiple countries, while ISKpn26, ISKpn14, and IS903B appear most often in China. Carbapenemase presence in tandem with IS elements may help promote an extensively drug resistant profile in K. pneumoniae limiting already narrow treatment options.

Sensitizing Staphylococcus aureus to antibacterial agents by decoding and blocking the lipid flippase MprF.

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens.

The inverse paradigm and the ancestral cell of IDH-wildtype glioblastoma

Rethinking IDH-wildtype glioblastoma through its unique features can help researchers find innovative and effective treatments. It is currently emerging that, after decades of therapeutic impasse, some traditional concepts regarding IDH-wildtype glioblastoma need to be supplemented and updated to overcome therapeutic resistance. Indeed, multiple clinical aspects and recent indirect and direct experimental data are providing evidence that the supratentorial brain parenchyma becomes entirely and quiescently micro-infiltrated long before primary tumor bulk growth. Furthermore, they are indicating that the known micro-infiltration that occurs during the IDH-wildtype glioblastoma growth and evolution is not at the origin of distant relapses. It follows that the ubiquitous supratentorial brain parenchyma micro-infiltration as a source for the development of widespread distant recurrences is actually due to the silent stage that precedes tumor growth rather than to the latter. All this implies that, in addition to the heterogeneity of the primary bulk, there is a second crucial cause of therapeutic resistance that has never hitherto been identified and challenged. In this regard, the ancestral founder cancer stem cell (CSC) appears as the key cell that can link the two causes of resistance.

Serum resistance factors in avian pathogenic Escherichia coli mediated by ETT2 gene cluster.

Serum resistance is a poorly understood but common trait of some systemic disease pathogenic strains of bacteria. In this study, we analysed the role Escherichia coli type III secretion system 2 (ETT2) of avian pathogenic Escherichia coli (APEC) in serum resistance by bacteria survival number in serum culture, mRNA Seq and Tandem Mass Tag™ (TMT™) detection, lipopolysaccharide (LPS) extraction, and biofilm formation detection. We found that the ETT2 gene cluster deletion strain (ΔETT2) is more resistant to the killing effect of serum than wild-type APEC40. The analysis of ΔETT2 compared to APEC40 in the transcriptomics and proteomics data showed that ETT2 has a negative effect in the ATP-binding cassette (ABC) translator system and quorum sensing system and a positive effect in purine metabolism. ETT2 may affect the LPS, biofilm, flagella, and fimbriae which may affect the serum resistance. These results could lead to effective strategies for managing the infection by APEC. The mRNA Seq data of this study are available in the Sequence Read Archive of the National Center for Biotechnology Information under the BioProject PRJNA757182, and proteomic raw data have been deposited under the accession number IPX0003420000 at iProX.

Use of corneal hysteresis and corneal resistance factor in target intraocular pressure estimation in patients with early primary open-angle glaucoma.

PURPOSE: To introduce a new method for estimation of the target intraocular pressure (TIOP) in naïve eyes with early primary open-angle glaucoma (POAG) using corneal hysteresis (CH) and corneal resistance factor (CRF). METHODS: A prospective quasi-experimental study was conducted on naïve 90 eyes of 45 patients who were newly diagnosed with early primary open-angle glaucoma (POAG). They were compared to 72 eyes of 36 normal subjects. The TIOP was determined for each eye. The IOP Goldmann (IOPg), IOP corneal-compensated (IOPcc), CH and CRF were estimated by ocular response analyzer (ORA, Reichert) device. Measurements were taken for each patient prior to treatment and after 1, 3, 6, 9 and 12 months of receiving medications; either monotherapy or combination therapy. RESULTS: For all patients, there was a significant negative correlation (p < 0.05) between IOP, either IOPg or IOPcc, and CH, while a significant positive relationship (p < 0.05) existed between IOP and CRF. For patients with early POAG, the CH was significantly increased (p ≤ 0.001), while CRF was significantly decreased (p ≤ 0.001) when TIOP was achieved. At IOP levels higher than TIOP, CH value was lower than CRF with a significant negative correlation between them in contrast to controls. This correlation was reversed on reaching TIOP and CH values became higher than CRF similar to controls. CONCLUSION: CH, CRF and IOP measured by ORA can be used for TIOP estimation. This provides us with a guide for assessing the effectiveness of medications introduced to patients with POAG.

Patrones de resistencia en agentes bacterianos involucrados en otitis caninas en Medellín, Colombia, durante 2019: análisis retrospectivo / Patterns of resistance in bacterial agents involved in canine otitis at Medellín, Colombia, during 2019: retrospective analysis

RESUMEN Dentro de los agentes patógenos en los procesos otíticos bacterianos, se destacan microorganismos como Staphylococcus pseudintermedius, Pseudomona auriginosa, Proteus mirabi-lis, Escherichia coli, Corynebacterium spp., Enterococcus spp. y Streptococcus spp., para los cuales se ha descrito resistencia frente a los antibióticos empleados para combatirlos. En Colombia son pocos los reportes acerca de la resistencia antibiótica de microorganismos causantes de otitis. Por ello, el objetivo de esta investigación fue determinar los agentes bacterianos más frecuentemente aislados en infecciones otíticas de caninos remitidas a un laboratorio veterinario de Medellín durante el 2019 y su resistencia a antibióticos. Para llevarlo a cabo, se realizó un estudio descriptivo transversal retrospectivo. Se analizaron los resultados de los antibiogramas realizados a partir de cultivos bacterianos en muestras óticas remitidas a un laboratorio de referencia de la ciudad de Medellín. Además, se efectuó un análisis de frecuencias para la muestra total. Se encontró que los principales microorganismos bacterianos aislados fueron Staphylococcus pseudintermedius, Pseudomona auriginosa, Proteus mirabili y Staphylococcus aureus. La gentamicina fue el medicamento que mayor porcentaje de resistencia presentó y la cefalexina el que menos resistencia presentó. Se pudo concluir que el Staphylococcus pseudintermedius está presente en más del 60% de los casos de otitis bacteriana. Adicionalmente, se observó una variación de la resistencia presentada por los microorganismos en el tiempo. Estos presentaron mayor resistencia ante los antibióticos aminoglucósidos.

ABSTRACT Among the pathogens in bacterial otic processes, microorganisms such as Staphylococcus pseudintermedius, Pseudomona auriginosa, Proteus mirabilis, Escherichia coli, Corynebac-terium spp., Enterococcus spp., and Streptococcus spp. stand out, for which resistance to antibiotics has been described employed to combat them. In Colombia there are few reports about the antibiotic resistance of microorganisms that cause otitis. For that reason, the purpose of this study was to determine the bacterial agents most frequently isolated from canine ear infections and their resistance to antibiotics from samples of ear secretions sent to a veterinary laboratory in Medellín during 2019. In order to do that, an cross-sectional, retrospective descriptive study was done. The results of the antibiograms performed from bacterial cultures obtained from ear samples sent to a reference laboratory in the city of Medellín were analyzed. A frequency analysis was carried out for the total sample. It was found that the main isolated bacterial microorganisms were Staphylococcus pseudintermedius, Pseudomona auriginosa, Proteus mirabili and Staphylococcus aureus. Gentamicin was the drug with the highest percentage of resistance and cephalexin the one with the least resistance. It was possible to conclude that Staphylococcus pseudintermedius is linked in more than 60% of cases of bacterial otitis and the resistance presented by microorganisms varies over time. The group of aminoglycosides antibiotics was the one which microorganisms are manifesting more percentage of resistance.