The yeast Xanthophyllomyces dendrorhous is one of the most promising and economically attractive natural sources of astaxanthin. The biosynthesis of this valuable carotenoid is a complex process for which the regulatory mechanisms remain mostly unknown. Several studies have shown a strong correlation between the carbon source present in the medium and the amount of pigments synthesized. Carotenoid production is especially low when high glucose concentrations are used in the medium, while a significant increase is observed with non-fermentable carbon sources. However, the molecular basis of this phenomenon has not been established.

Purpose: Colistin resistance mechanisms involving mutations in chromosomal genes associated with LPS modification are not completely understood. Mutations in genes coding for the MgrB regulator frequently account for colistin resistance in Klebsiella pneumoniae, whereas mutations in genes coding for PhoPQ and PmrAB are frequent in E. coli. Our aim was to perform a genetic analysis of chromosomal mutations in colistin-resistant (MIC ≥ 4 μg/mL) clinical isolates of K. pneumoniae (n = 8) and E. coli (n = 7) of different STs. Methods: Isolates were obtained in a 3-year period in a university hospital in Santiago, Chile. Susceptibility to colistin, aminoglycosides, cephalosporins, carbapenems and ciprofloxacin was determined through broth microdilution. Whole genome sequencing was performed for all isolates and chromosomal gene sequences were compared with sequences of colistin-susceptible isolates of the same sequence types. Results: None of the isolates carried mcr genes. Most of the isolates were susceptible to all the antibiotics analyzed. E. coli isolates were ST69, ST127, ST59, ST131 and ST14, and K. pneumoniae isolates were ST454, ST45, ST6293, ST380 and ST25. All the isolates had mutations in chromosomal genes analyzed. K. pneumoniae had mutations mainly in mgrB gene, whereas E. coli had mutations in pmrA, pmrB and pmrE genes. Most of the amino acid changes in LPS-modifying enzymes of colistin-resistant isolates were found in colistin-susceptible isolates of the same and/or different ST. Eleven of them were found only in colistin-resistant isolates. Conclusion: Colistin resistance mechanisms depend on genetic background, and are due to chromosomal mutations, which implies a lower risk of transmission than plasmid-mediated genes. Colistin resistance is not associated with multidrug-resistance, nor to high-risk sequence types.

Ceftazidime/Avibactam (CAZ/AVI) is frequently used to treat KPC-producing Pseudomonas aeruginosa (KPC-PA) and Enterobacterales. CAZ/AVI resistance is driven by several mechanisms. In P. aeruginosa this mainly occurs through alteration of AmpC, porins, and/or efflux pump overexpression, whereas in Enterobacterales it frequently occurs through D179Y substitution in the active site of KPC enzyme. This aminoacid change abolishes AVI binding to the KPC active site, hence inhibition is impaired. However, this substitution also decreases KPC-mediated resistance to carbapenems (“see-saw” effect). The goal of this work was to characterize the in vivo acquisition of CAZ/AVI resistance through D179Y substitution in a KPC-PA isolated from a hospitalized patient after CAZ/AVI treatment. Two KPC-PA isolates were obtained. The first isolate, PA-1, was obtained before CAZ/AVI treatment and was susceptible to CAZ/AVI. The second isolate, PA-2, was obtained after CAZ/AVI treatment and exhibited high-level CAZ/AVI resistance. Characterization of isolates PA-1 and PA-2 was performed through short and long-read whole genome sequencing analysis. The hybrid assembly showed that PA-1 and PA-2A had a single plasmid of 54,030 bp, named pPA-1 and pPA-2 respectively. Each plasmid harbored two copies of the bla_KPC-containing Tn4401b transposon. However, while pPA-1 carried two copies of bla_KPC-2, pPA-2 had one copy of bla_KPC-2 and one copy of bla_KPC-33, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the “see-saw” effect. The bla_KPC-33 allele was detected only through hybrid assembly using a long-read-first approach. The present work describes a KPC-PA isolate harboring a plasmid-borne CAZ/AVI resistance mechanism based on two copies of bla_KPC-2-Tn4401b and D179Y mutation in one of them, that is not associated with loss of resistance to carbapenems. These findings highlight the usefulness of a fine-tuned combined analysis of short and long-read data to detect similar emerging resistance mechanisms.

Salmonella enterica is a leading cause of foodborne illnesses globally, with significant mortality rates, especially among vulnerable populations. Traditional serotyping methods for Salmonella are accurate but expensive, resource-intensive, and time-consuming, necessitating faster and more reliable alternatives. This study evaluates the IR Biotyper, a Fourier-transform infrared spectroscopy system, in differentiating Salmonella serovars. We assessed 458 isolates of nine Salmonella serovars (Infantis, Enteritidis, Typhimurium, I,4,[5],12:i:-, Montevideo, Agona, Thompson, Panama, and Abony) from diverse sources. The IR Biotyper was used to acquire spectra from these isolates. Machine learning algorithms, including support vector machines, were trained to classify the isolates. The accuracy of classifiers was validated using a validation set to determine sensitivity, specificity, positive predictive value, and negative predictive value. Initial classifiers showed high accuracy for Abony, Agona, Enteritidis, and Infantis serovars, with sensitivities close to 100%. However, classifiers for S . Typhimurium, S . Panama, and S . Montevideo exhibited lower performance. Implementing a hierarchical classification system enhanced the accuracy of serogroup O:4 serovars, demonstrating that this approach offers a robust framework for Salmonella serovar identification. The hierarchical system enables progressive refinement of classification, minimizing misclassifications by focusing on serogroup-specific features, making it adaptable to complex data sets and diverse serovars. The IR Biotyper demonstrates high potential for rapid and accurate Salmonella serovar identification. This study supports its implementation as a cost-effective, high-throughput tool for pathogen typing, enhancing real-time epidemiological surveillance, and guiding treatment strategies for salmonellosis. This method establishes a robust and scalable framework for advancing Salmonella serotyping practices across clinical, industrial, and public health domains by leveraging hierarchical classification. IMPORTANCE: Early and accurate identification of Salmonella serovars is extremely important for epidemiological surveillance, public health, and food safety. Traditional serotyping is very successful but is laborious and costly. In this study, we demonstrate the promise of Fourier-transform infrared spectroscopy together with machine learning as a means for Salmonella serotyping. Using hierarchical classification, we attain optimal serovar identification accuracy, particularly for challenging-to-type serogroups. Our findings recognize the IR Biotyper as a high-throughput, scalable pathogen typing solution that offers real-time data that can enable enhanced outbreak response and prevention of foodborne disease. The approach bridges the gap between traditional microbiological practice and sophisticated analytical technology, the path to more effective, cost-saving interventions in the clinical, industrial, and regulatory settings. Application of these technologies can significantly improve Salmonella surveillance-control and Public Health outcomes.

Introduction: Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum β-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible.Methods: Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla_PER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla_PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Results: Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla_PER. One isolate carried bla_PER but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla_PER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum β-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla_PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other β-lactamsin the in vitro evolved isolate. Discussion: PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with bla_PER-3 gene implicated in resistance to CZA and other β-lactams.