Heteroresistance to colistin can be defined as the presence of resistant subpopulations in an isolate that is susceptible to this antibiotic. Colistin resistance in Gram-negative bacteria is more frequently related to chromosomal mutations and insertions. This work aimed to study heteroresistance in nine clinical isolates of Klebsiella pneumoniae producing OXA-48 and to describe genomic changes in mutants with acquired resistance in vitro. Antimicrobial susceptibility was determined by broth microdilution (BMD) and heteroresistance by population analysis profiling (PAP). The proteins related to colistin resistance were analyzed for the presence of mutations. Additionally, PCR of the mgrB gene was performed to identify the presence of insertions. In the nine parental isolates, the PAP method showed colistin heteroresistance of colonies growing on plates with concentrations of up to 64 mg/L, corresponding to stable mutant subpopulations. The MICs of some mutants from the PAP plate containing 4×MIC of colistin had absolute values of ≤2 mg/L that were higher than the parental MICs and were defined as persistent variants. PCR of the mgrB gene identified an insertion sequence that inactivated the gene in 21 mutants. Other substitutions in the investigated mutants were found in PhoP, PhoQ, PmrB, PmrC, CrrA and CrrB proteins. Colistin heteroresistance in K. pneumoniae isolates was attributed mainly to insertions in the mgrB gene and point mutations in colistin resistance proteins. The results of this study will improve understanding regarding the mechanisms of colistin resistance in mutants of K. pneumoniae producing OXA-48.

Heteroresistance to colistin can be defined as the presence of resistant subpopulations in an isolate that is susceptible to colistin. The aim of this work was to study heteroresistance in 10 wild-type Klebsiella pneumoniae isolates of clinical origin, to determine the importance of persisters variants and stable mutants in heteroresistance, and to describe genomic changes observed in mutants obtained in population analysis profiling (PAP) assays. Mutations in genes related to colistin resistance (including pmr A, pmr B, pmr C, pmr D, pho P, pho Q, crr A, ccr B, mgr B, ram A, rom A, lax A, lpx C, lpx D, acr A, and acr B) were investigated by whole-genome sequencing with an Illumina platform. Additionally, PCR and Sanger sequencing of the mgr B gene of the selected mutants were also performed. PAP showed that the investigated K. pneumoniae isolates present heteroresistance to colistin, which is related to both the growth of persisters variants and the selection of resistant mutants. A total of 31-point mutations were identified in proteins PhoP, PhoQ, PmrB, and CrrB of which novel mutations were reported in PhoP (E22K, L12Q, and M175K), PhoQ (V24G and L105Q), PmrB (G207D), and CrrB (G183V). MgrB changes included amino acid substitutions (D31N and L19R), deletions (∆C28, ∆C39, ∆Q22, and ∆W6) or the presence of insertion sequences belonging to the IS1 (ISKpn14) and IS5 (ISKpn74) families that inactivated the gene. The results of this study will help to understand the mechanisms involved in colistin heteroresistance among wild-type K. pneumoniae isolates. IMPORTANCE: Colistin is one of the last remaining therapeutic options for dealing with Enterobacteriaceae. Unfortunately, heteroresistance to colistin is also rapidly increasing. We described the prevalence of colistin heteroresistance in a variety of wild-type strains of Klebsiella pneumoniae and the evolution of these strains with colistin heteroresistance to a resistant phenotype after colistin exposure and withdrawal. Resistant mutants were characterized at the molecular level, and numerous mutations in genes related to lipopolysaccharide formation were observed. In colistin-treated patients, the evolution of K. pneumoniae heteroresistance to resistance phenotype could lead to higher rates of therapeutic failure.

Sparkling wine is a distinctive wine. Saccharomyces cerevisiae flor yeasts is innovative and ideal for the sparkling wine industry due to the yeasts’ resistance to high ethanol concentrations, surface adhesion properties that ease wine clarification, and the ability to provide a characteristic volatilome and odorant profile. The objective of this work is to study the proteins in a flor yeast and a conventional yeast that are responsible for the production of the volatile compounds released during sparkling wine elaboration. The proteins were identified using the OFFGEL fractionator and LTQ Orbitrap. We identified 50 and 43 proteins in the flor yeast and the conventional yeast, respectively. Proteomic profiles did not show remarkable differences between strains except for Adh1p, Fba1p, Tdh1p, Tdh2p, Tdh3p, and Pgk1p, which showed higher concentrations in the flor yeast versus the conventional yeast. The higher concentration of these proteins could explain the fuller body in less alcoholic wines obtained when using flor yeasts. The data presented here can be thought of as a proteomic map for either flor or conventional yeasts which can be useful to understand how these strains metabolize the sugars and release pleasant volatiles under sparkling wine elaboration conditions.

Acetic acid bacteria (AAB) and other members of the complex microbiotas, whose activity is essential for vinegar production, display biodiversity and richness that is difficult to study in depth due to their highly selective culture conditions. In recent years, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has emerged as a powerful tool for rapidly identifying thousands of proteins present in microbial communities, offering broader precision and coverage. In this work, a novel method based on LC–MS/MS was established and developed from previous studies. This methodology was tested in three studies, enabling the characterization of three submerged acetification profiles using innovative raw materials (synthetic alcohol medium, fine wine, and craft beer) while working in a semicontinuous mode. The biodiversity of existing microorganisms was clarified, and both the predominant taxa (Komagataeibacter, Acetobacter, Gluconacetobacter, and Gluconobacter) and others never detected in these media (Asaia and Bombella, among others) were identified. The key functions and adaptive metabolic strategies were determined using comparative studies, mainly those related to cellular material biosynthesis, energy-associated pathways, and cellular detoxification processes. This study provides the groundwork for a highly reliable and reproducible method for the characterization of microbial profiles in the vinegar industry.