Genotypic and Phenotypic Characterization of Novel Sequence Types of Carbapenem-Resistant Acinetobacter baumannii, With Heterogeneous Resistance Determinants and Targeted Variations in Efflux Operons

Acinetobacter baumannii has emerged as one of the dominant nosocomial human pathogens associated with high morbidity and mortality globally. Increased incidences of carbapenem-resistant A. baumannii (CRAB) have resulted in an enormous socioeconomic burden on health-care systems. Here, we report the genotypic and phenotypic characterization of novel ST1816 and ST128 variants in A. baumannii strains belonging to International clone II (GC2) with capsule types KL1:OCL8 and KL3:OCL1d from India. Sequence analysis revealed the presence of diverse virulome and resistome in these clinical strains, in addition to islands, prophages, and resistance genes. The oxacillinase blaOXA–23detected in the genomic island also highlighted the coexistence of blaOXA–66/blaOXA–98, blaADC73/blaADC–3, and blaTEM–1D in their mobile scaffolds, which is alarming. Together with these resistance-determining enzymes, multidrug efflux transporters also harbored substitutions, with increased expression in CRAB strains. The hotspot mutations in colistin resistance-conferring operons, PmrAB, LpxACD, and AdeRS, were additionally confirmed. Phenotype microarray analysis indicated that multidrug-resistant strains A. baumannii DR2 and A. baumannii AB067 preferred a range of antimicrobial compounds as their substrates relative to the other. To our knowledge, this is the first comprehensive report on the characterization of A. baumannii variants ST1816 and ST128, with different genetic makeup and genome organization. The occurrence of CRAB infections worldwide is a severe threat to available limited therapeutic options; hence, continued surveillance to monitor the emergence and dissemination of such novel ST variants in A. baumannii is imperative.

Genomic comparison and phenotypic profiling of small colony variants of Burkholderia pseudomallei

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific.

The Lrp/AsnC-Type Regulator PA2577 Controls the EamA-like Transporter Gene PA2576 in Pseudomonas aeruginosa

The regulatory network of gene expression in Pseudomonas aeruginosa, an opportunistic human pathogen, is very complex. In the PAO1 reference strain, about 10% of genes encode transcriptional regulators, many of which have undefined regulons and unknown functions. The aim of this study is the characterization of PA2577 protein, a representative of the Lrp/AsnC family of transcriptional regulators. This family encompasses proteins involved in the amino acid metabolism, regulation of transport processes or cell morphogenesis. The transcriptome profiling of P. aeruginosa cells with mild PA2577 overproduction revealed a decreased expression of the PA2576 gene oriented divergently to PA2577 and encoding an EamA-like transporter. A gene expression analysis showed a higher mRNA level of PA2576 in P. aeruginosa ΔPA2577, indicating that PA2577 acts as a repressor. Concomitantly, ChIP-seq and EMSA assays confirmed strong interactions of PA2577 with the PA2577/PA2576 intergenic region. Additionally, phenotype microarray analyses indicated an impaired metabolism of ΔPA2576 and ΔPA2577 mutants in the presence of polymyxin B, which suggests disturbances of membrane functions in these mutants. We show that PA2576 interacts with two proteins, PA5006 and PA3694, with a predicted role in lipopolysaccharide (LPS) and membrane biogenesis. Overall, our results indicate that PA2577 acts as a repressor of the PA2576 gene coding for the EamA-like transporter and may play a role in the modulation of the cellular response to stress conditions, including antimicrobial peptides, e.g., polymyxin B.

McpT, a broad range carboxylate chemoreceptor in Sinorhizobium meliloti

Chemoreceptors enable the legume symbiont Sinorhizobium meliloti to detect and respond to specific chemicals released from their host plant alfalfa, which allows the establishment of a nitrogen-fixing symbiosis. The periplasmic region (PR) of transmembrane chemoreceptors act as the sensory input module for chemotaxis systems via binding of specific ligands, either directly or indirectly. S. meliloti has six transmembrane and two cytosolic chemoreceptors. However, only the function of three of the transmembrane receptors have been characterized so far, with McpU, McpV, and McpX serving as general amino acid, short-chain carboxylate, and quaternary ammonium compound sensors, respectively. In the present study, we analyzed the S. meliloti chemoreceptor McpT. High-throughput differential scanning fluorimetry assays, using Biolog Phenotype Microarray TM plates, identified fifteen potential ligands for McpT PR , the majority classified as mono-, di-, and tri-carboxylates. S. meliloti exhibited positive chemotaxis toward seven selected carboxylates, namely, α-ketobutyrate, citrate, glyoxylate, malate, malonate, oxalate, and succinate. These carboxylates were detected in seed exudates of the alfalfa host. Deletion of mcpT resulted in a significant decrease of chemotaxis to all carboxylates except for citrate. Isothermal titration calorimetry revealed that McpT PR bound preferentially to the monocarboxylate glyoxylate, and with lower affinity to the dicarboxylates malate, malonate and oxalate. However, no direct binding was detected for the remaining three carboxylates that elicited an McpT-dependent chemotaxis response. Taken together, these results demonstrate that McpT is a broad range carboxylate chemoreceptor that mediates chemotactic response via direct ligand binding and an indirect mechanism that yet needs to be identified. IMPORTANCE Nitrate pollution is one of the most widespread and challenging environmental problems, mainly caused by the agricultural over-application of nitrogen fertilizers. Biological nitrogen fixation by the endosymbiont Sinorhizobium meliloti enhances the growth of its host Medicago sativa (alfalfa), which also efficiently supplies the soil with nitrogen. Establishment of the S. meliloti - alfalfa symbiosis relies on the early exchange and recognition of chemical signals. The present study contributes to the disclosure of this complex molecular dialogue by investigating the underlying mechanisms of carboxylate sensing in S. meliloti . Understanding individual steps that govern S. meliloti -alfalfa molecular cross-talk helps in the development of efficient, commercial bacterial inoculants that promote the growth of this most cultivated forage legume in the world and improves soil fertility.

Insight into phenotypic and genotypic differences between vaginal Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12 to unravel nutritional and stress factors influencing their metabolic activity

The vaginal microbiota, normally characterized by lactobacilli presence, is crucial for vaginal health. Members belonging to L. crispatus and L. gasseri species exert crucial protective functions against pathogens, although a total comprehension of factors that influence their dominance in healthy women is still lacking. Here we investigated the complete genome sequence and comprehensive phenotypic profile of L. crispatus strain BC5 and L. gasseri strain BC12, two vaginal strains featured by anti-bacterial and anti-viral activities. Phenotype microarray (PM) results revealed an improved capacity of BC5 to utilize different carbon sources as compared to BC12, although some specific carbon sources that can be associated to the human diet were only metabolized by BC12, i.e. uridine, amygdalin, tagatose. Additionally, the two strains were mostly distinct in the capacity to utilize the nitrogen sources under analysis. On the other hand, BC12 showed tolerance/resistance towards twice the number of stressors (i.e. antibiotics, toxic metals etc.) with respect to BC5. The divergent phenotypes observed in PM were supported by the identification in either BC5 or BC12 of specific genetic determinants that were found to be part of the core genome of each species. The PM results in combination with comparative genome data provide insights into the possible environmental factors and genetic traits supporting the predominance of either L. crispatus BC5 or L. gasseri BC12 in the vaginal niche, giving also indications for metabolic predictions at the species level.

Characterization of Cold-Tolerant Saccharomyces cerevisiae Cheongdo Using Phenotype Microarray

The cold-tolerant yeast Saccharomyces cerevisiae is industrially useful for lager fermentation, high-quality wine, and frozen dough production. S. cerevisiae Cheongdo is a recent isolate from frozen peach samples which has a good fermentation performance at low temperatures and desirable flavor profiles. Here, phenotype microarray was used to investigate industrial potentials of S. cerevisiae Cheongdo using 192 carbon sources. Compared to commercial wine yeast S. cerevisiae EC1118, Cheongdo showed significantly different growth rates on 34 substrates. The principal component analysis of the results highlighted that the better growth of Cheongdo on galactose than on EC1118 was the most significant difference between the two strains. The intact GAL4 gene and the galactose fermentation performance at a low temperatures suggested that S. cerevisiae Cheongdo is a promising host for industrial fermentation rich in galactose, such as lactose and agarose.

Probiotic and Metabolic Characterization of Vaginal Lactobacilli for a Potential Use in Functional Foods

The main aim of this work was to verify the metabolic and functional aptitude of 15 vaginal strains belonging to Lactobacillus crispatus, Lactobacillus gasseri, and Limosilactobacillus vaginalis (previously Lactobacillus vaginalis), already characterized for their technological and antimicrobial properties. In order to evaluate the metabolic profile of these vaginal strains, a phenotype microarray analysis was performed on them. Functional parameters such as hydrophobicity, auto-aggregation, deconjugation of bile salts, adhesion to an intestinal cell line (Caco-2), and a simulated digestion process were evaluated for these strains. A good number of these strains showed hydrophobicity values higher than 70%. Regarding the auto-aggregation assay, the most promising strains were L. crispatus BC9 and BC1, L. gasseri BC10 and BC14, and L. vaginalis BC17. Moreover, L. crispatus BC4, BC6, BC7, and BC8 were characterized by strong bile salts hydrolase activity (BHS). In addition, L. crispatus BC8 and L. vaginalis BC17 were characterized by a medium ability to adhere to Caco-2 cells. Data related to digestion process showed a strong ability of vaginal lactobacilli to withstand this stress. In conclusion, the data collected show the metabolic versatility and several exploitable functional properties of the investigated vaginal lactobacilli.

Genome-scale model reconstruction of the methylotrophic yeast Ogataea polymorpha

Background Ogataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. While previously mainly used for protein synthesis, it also holds promise for producing platform chemicals. O. polymorpha has the distinct advantage of using methanol as a substrate, which could be potentially derived from carbon capture and utilization streams. Full development of the organism into a production strain and estimation of the metabolic capabilities require additional strain design, guided by metabolic modeling with a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha. Results To overcome this limitation, we used a published reconstruction of the closely related yeast Komagataella phaffii as a reference and corrected reactions based on KEGG and MGOB annotation. Additionally, we conducted phenotype microarray experiments to test the suitability of 190 substrates as carbon sources. Over three-quarter of the substrate use was correctly reproduced by the model and 27 new substrates were added, that were not present in the K. phaffii reference model. Conclusion The developed genome-scale metabolic model of O. polymorpha will support the engineering of synthetic metabolic capabilities and enable the optimization of production processes, thereby supporting a sustainable future methanol economy.

Development of a Genome-Scale Metabolic Model and Phenome Analysis of the Probiotic Escherichia coli Strain Nissle 1917

Escherichia coli Nissle 1917 (EcN) is an intestinal probiotic that is effective for the treatment of intestinal disorders, such as inflammatory bowel disease and ulcerative colitis. EcN is a representative Gram-negative probiotic in biomedical research and is an intensively studied probiotic. However, to date, its genome-wide metabolic network model has not been developed. Here, we developed a comprehensive and highly curated EcN metabolic model, referred to as iDK1463, based on genome comparison and phenome analysis. The model was improved and validated by comparing the simulation results with experimental results from phenotype microarray tests. iDK1463 comprises 1463 genes, 1313 unique metabolites, and 2984 metabolic reactions. Phenome data of EcN were compared with those of Escherichia coli intestinal commensal K-12 MG1655. iDK1463 was simulated to identify the genetic determinants responsible for the observed phenotypic differences between EcN and K-12. Further, the model was simulated for gene essentiality analysis and utilization of nutrient sources under anaerobic growth conditions. These analyses provided insights into the metabolic mechanisms by which EcN colonizes and persists in the gut. iDK1463 will contribute to the system-level understanding of the functional capacity of gut microbes and their interactions with microbiota and human hosts, as well as the development of live microbial therapeutics.