The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans.

IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner-based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli. We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms.

IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli. Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.

Dietary protein residue can result in microbial generation of various toxic metabolites in the gut, such as ammonia. A prebiotic is "a substrate that is selectively utilised by host microorganisms conferring a health benefit" (G. R. Gibson, R. Hutkins, M. E. Sanders, S. L. Prescott, et al., Nat Rev Gastroenterol Hepatol 14:491–502, 2017, https://doi.org/10.1038/nrgastro.2017.75). Prebiotics are carbohydrates that may have the potential to reverse the harmful effects of gut bacterial protein fermentation. Three-stage continuous colonic model systems were inoculated with fecal samples from omnivore and vegetarian volunteers. Casein (equivalent to 105 g protein consumption per day) was used within the systems as a protein source. Two different doses of inulin-type fructans (Synergy1) were later added (equivalent to 10 g per day in vivo and 15 g per day) to assess whether this influenced protein fermentation. Bacteria were enumerated by fluorescence in situ hybridization with flow cytometry. Metabolites from bacterial fermentation (short-chain fatty acid [SCFA], ammonia, phenol, indole, and p-cresol) were monitored to further analyze proteolysis and the prebiotic effect. A significantly higher number of bifidobacteria was observed with the addition of inulin together with reduction of Desulfovibrio spp. Furthermore, metabolites from protein fermentation, such as branched-chain fatty acids (BCFA) and ammonia, were significantly lowered with Synergy1. Production of p-cresol varied among donors, as we recognized four high producing models and two low producing models. Prebiotic addition reduced its production only in vegetarian high p-cresol producers.

IMPORTANCE Dietary protein levels are generally higher in Western populations than in the world average. We challenged three-stage continuous colonic model systems containing high protein levels and confirmed the production of potentially harmful metabolites from proteolysis, especially replicates of the transverse and distal colon. Fermentations of proteins with a prebiotic supplementation resulted in a change in the human gut microbiota and inhibited the production of some proteolytic metabolites. Moreover, we observed both bacterial and metabolic differences between fecal bacteria from omnivore donors and vegetarian donors. Proteins with prebiotic supplementation showed higher Bacteroides spp. and inhibited Clostridium cluster IX in omnivore models, while in vegetarian modes, Clostridium cluster IX was higher and Bacteroides spp. lower with high protein plus prebiotic supplementation. Synergy1 addition inhibited p-cresol production in vegetarian high p-cresol-producing models while the inhibitory effect was not seen in omnivore models.

Vibralactone, a hybrid compound derived from phenols and a prenyl group, is a strong pancreatic lipase inhibitor with a rare fused bicyclic β-lactone skeleton. Recently, a researcher reported a vibralactone derivative (compound C1) that caused inhibition of pancreatic lipase with a half-maximal inhibitory concentration of 14 nM determined by structure-based optimization, suggesting a potential candidate as a new antiobesity treatment. In the present study, we sought to identify the main gene encoding prenyltransferase in Stereum vibrans, which is responsible for the prenylation of phenol leading to vibralactone synthesis. Two RNA silencing transformants of the identified gene (vib-PT) were obtained through Agrobacterium tumefaciens-mediated transformation. Compared to wild-type strains, the transformants showed a decrease in vib-PT expression ranging from 11.0 to 56.0% at 5, 10, and 15 days in reverse transcription-quantitative PCR analysis, along with a reduction in primary vibralactone production of 37 to 64% at 15 and 21 days, respectively, as determined using ultra-high-performance liquid chromatography-mass spectrometry analysis. A soluble and enzymatically active fusion Vib-PT protein was obtained by expressing vib-PT in Escherichia coli, and the enzyme’s optimal reaction conditions and catalytic efficiency (K_m/k_cat) were determined. In vitro experiments established that Vib-PT catalyzed the C-prenylation at C-3 of 4-hydroxy-benzaldehyde and the O-prenylation at the 4-hydroxy of 4-hydroxy-benzenemethanol in the presence of dimethylallyl diphosphate. Moreover, Vib-PT shows promiscuity toward aromatic compounds and prenyl donors.

IMPORTANCE Vibralactone is a lead compound with a novel skeleton structure that shows strong inhibitory activity against pancreatic lipase. Vibralactone is not encoded by the genome directly but rather is synthesized from phenol, followed by prenylation and other enzyme reactions. Here, we used an RNA silencing approach to identify and characterize a prenyltransferase in a basidiomycete species that is responsible for the synthesis of vibralactone. The identified gene, vib-PT, was expressed in Escherichia coli to obtain a soluble and enzymatically active fusion Vib-PT protein. In vitro characterization of the enzyme demonstrated the catalytic mechanism of prenylation and broad substrate range for different aromatic acceptors and prenyl donors. These characteristics highlight the possibility of Vib-PT to generate prenylated derivatives of aromatics and other compounds as improved bioactive agents or potential prodrugs.

Gastrointestinal (GI) or gut microbiotas play essential roles in host development and physiology. These roles are influenced partly by the microbial community composition. During early developmental stages, the ecological processes underlying the assembly and successional changes in host GI community composition are influenced by numerous factors, including dispersal from the surrounding environment, age-dependent changes in the gut environment, and changes in dietary regimes. However, the relative importance of these factors to the gut microbiota is not well understood. We examined the effects of environmental (diet and water sources) and host early ontogenetic development on the diversity of and the compositional changes in the gut microbiota of a primitive teleost fish, the lake sturgeon (Acipenser fulvescens), based on massively parallel sequencing of the 16S rRNA gene. Fish larvae were raised in environments that differed in water source (stream versus filtered groundwater) and diet (supplemented versus nonsupplemented Artemia fish). We quantified the gut microbial community structure at three stages (prefeeding and 1 and 2 weeks after exogenous feeding began). The diversity declined and the community composition differed significantly among stages; however, only modest differences associated with dietary or water source treatments were documented. Many taxa present in the gut were over- or underrepresented relative to neutral expectations in each sampling period. The findings indicate dynamic relationships between the gut microbiota composition and host gastrointestinal physiology, with comparatively smaller influences being associated with the rearing environments. Neutral models of community assembly could not be rejected, but selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for sturgeon conservation and aquaculture production specifically and applications of microbe-based management in teleost fish generally.

IMPORTANCE We quantified the effects of environment (diet and water sources) and host early ontogenetic development on the diversity of and compositional changes in gut microbial communities based on massively parallel sequencing of the 16S rRNA genes from the GI tracts of larval lake sturgeon (Acipenser fulvescens). The gut microbial community diversity declined and the community composition differed significantly among ontogenetic stages; however, only modest differences associated with dietary or water source treatments were documented. Selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for lake sturgeon and early larval ecology and survival in their natural habitat and for conservation and aquaculture production specifically, as well as applications of microbe-based management in teleost fish generally.

A longstanding awareness in generating resistance to common antimicrobial therapies by Gram-negative bacteria has made them a major threat to global health. The application of antimicrobial peptides as a therapeutic agent would be a great opportunity to combat bacterial diseases. Here, we introduce a new antimicrobial peptide (~8.3 kDa) from probiotic strain Lactobacillus acidophilus ATCC 4356, designated acidocin 4356 (ACD). This multifunctional peptide exerts its anti-infective ability against Pseudomonas aeruginosa through an inhibitory action on virulence factors, bacterial killing, and biofilm degradation. Reliable performance over tough physiological conditions and low hemolytic activity confirmed a new hope for the therapeutic setting. Antibacterial kinetic studies using flow cytometry technique showed that the ACD activity is related to the change in permeability of the membrane. The results obtained from molecular dynamic (MD) simulation were perfectly suited to the experimental data of ACD behavior. The structure-function relationship of this natural compound, along with the results of transmission electron microscopy analysis and MD simulation, confirmed the ability of the ACD aimed at enhancing bacterial membrane perturbation. The peptide was effective in the treatment of P. aeruginosa infection in mouse model. The results support the therapeutic potential of ACD for the treatment of Pseudomonas infections.

IMPORTANCE Multidrug-resistant bacteria are a major threat to global health, and the Pseudomonas bacterium with the ability to form biofilms is considered one of the main causative agents of nosocomial infections. Traditional antibiotics have failed because of increased resistance. Thus, finding new biocompatible antibacterial drugs is essential. Antimicrobial peptides are produced by various organisms as a natural defense mechanism against pathogens, inspiring the possible design of the next generation of antibiotics. In this study, a new antimicrobial peptide was isolated from Lactobacillus acidophilus ATCC 4356, counteracting both biofilm and planktonic cells of Pseudomonas aeruginosa. A detailed investigation was then conducted concerning the functional mechanism of this peptide by using fluorescence techniques, electron microscopy, and in silico methods. The antibacterial and antibiofilm properties of this peptide may be important in the treatment of Pseudomonas infections.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C_16:0 and n-C_18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes. Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2. In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes.

IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

To better understand how associated microorganisms ("microbiota") influence organismal aging, we focused on the model organism Drosophila melanogaster. We conducted a metagenome-wide association (MGWA) as a screen to identify bacterial genes associated with variation in the D. melanogaster life span. The results of the MGWA predicted that bacterial cysteine and methionine metabolism genes influence fruit fly longevity. A mutant analysis, in which flies were inoculated with Escherichia coli strains bearing mutations in various methionine cycle genes, confirmed a role for some methionine cycle genes in extending or shortening fruit fly life span. Initially, we predicted these genes might influence longevity by mimicking or opposing methionine restriction, an established mechanism for life span extension in fruit flies. However, follow-up transcriptome sequencing (RNA-seq) and metabolomic experiments were generally inconsistent with this conclusion and instead implicated glucose and vitamin B₆ metabolism in these influences. We then tested if bacteria could influence life span through methionine restriction using a different set of bacterial strains. Flies reared with a bacterial strain that ectopically expressed bacterial transsulfuration genes and lowered the methionine content of the fly diet also extended female D. melanogaster life span. Taken together, the microbial influences shown here overlap with established host genetic mechanisms for aging and therefore suggest overlapping roles for host and microbial metabolism genes in organismal aging.

IMPORTANCE Associated microorganisms ("microbiota") are intimately connected to the behavior and physiology of their animal hosts, and defining the mechanisms of these interactions is an urgent imperative. This study focuses on how microorganisms influence the life span of a model host, the fruit fly Drosophila melanogaster. First, we performed a screen that suggested a strong influence of bacterial methionine metabolism on host life span. Follow-up analyses of gene expression and metabolite abundance identified stronger roles for vitamin B₆ and glucose than methionine metabolism among the tested mutants, possibly suggesting a more limited role for bacterial methionine metabolism genes in host life span effects. In a parallel set of experiments, we created a distinct bacterial strain that expressed life span-extending methionine metabolism genes and showed that this strain can extend fly life span. Therefore, this work identifies specific bacterial genes that influence host life span, including in ways that are consistent with the expectations of methionine restriction.

The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, lytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytB_E585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either lytB or producing the inactive LytB_E585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytB_E585A mutant formed biofilm more rapidly than lytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed.

IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

Deep-subsurface hot brines in northwest Poland, extracted through boreholes reaching 1.6 and 2.6 km below the ground surface, were microbiologically investigated using culture-independent and culture-dependent methods. The high-throughput sequencing of 16S rRNA gene amplicons showed a very low diversity of bacterial communities, which were dominated by phyla Proteobacteria and Firmicutes. Bacterial genera potentially involved in sulfur oxidation and nitrate reduction (Halothiobacillus and Methylobacterium) prevailed in both waters over the sulfate reducers ("Candidatus Desulforudis" and Desulfotomaculum). Only one archaeal taxon, affiliated with the order Thermoplasmatales, was detected in analyzed samples. Bacterial isolates obtained from these deep hot brines were closely related to Bacillus paralicheniformis based on the 16S rRNA sequence similarity. However, genomic and physiological analyses made for one of the isolates, Bacillus paralicheniformis strain TS6, revealed the existence of more diverse metabolic pathways than those of its moderate-temperature counterpart. These specific traits may be associated with the ecological adaptations to the extreme habitat, which suggest that some lineages of B. paralicheniformis are halothermophilic.

IMPORTANCE Deep-subsurface aquifers, buried thousands of meters down the Earth’s crust, belong to the most underexplored microbial habitats. Although a few studies revealed the existence of microbial life at the depths, the knowledge about the microbial life in the deep hydrosphere is still scarce due to the limited access to such environments. Studying the subsurface microbiome provides unique information on microbial diversity, community structure, and geomicrobiological processes occurring under extreme conditions of the deep subsurface. Our study shows that low-diversity microbial assemblages in subsurface hot brines were dominated by the bacteria involved in biogeochemical cycles of sulfur and nitrogen. Based on genomic and physiological analyses, we found that the Bacillus paralicheniformis isolate obtained from the brine under study differed from the mesophilic species in the presence of specific adaptations to harsh environmental conditions. We indicate that some lineages of B. paralicheniformis are halothermophilic, which was not previously reported.

Biological nitrogen fixation can be an important source of nitrogen in tropical forests that serve as a major CO₂ sink. Extensive deforestation of the Amazon is known to influence microbial communities and the biogeochemical cycles they mediate. However, it is unknown how diazotrophs (nitrogen-fixing microorganisms) respond to deforestation and subsequent ecosystem conversion to agriculture, as well as whether they can recover in secondary forests that are established after agriculture is abandoned. To address these knowledge gaps, we combined a spatially explicit sampling approach with high-throughput sequencing of nifH genes. The main objectives were to assess the functional distance decay relationship of the diazotrophic bacterial community in a tropical forest ecosystem and to quantify the roles of various factors that drive the observed changes in the diazotrophic community structure. We observed an increase in local diazotrophic diversity (α-diversity) with a decrease in community turnover (β-diversity), associated with a shift in diazotrophic community structure as a result of the forest-to-pasture conversion. Both diazotrophic community turnover and structure showed signs of recovery in secondary forests. Changes in the diazotrophic community were primarily driven by the change in land use rather than differences in geochemical characteristics or geographic distances. The diazotroph communities in secondary forests resembled those in primary forests, suggesting that at least partial recovery of diazotrophs is possible following agricultural abandonment.

IMPORTANCE The Amazon region is a major tropical forest region that is being deforested at an alarming rate to create space for cattle ranching and agriculture. Diazotrophs (nitrogen-fixing microorganisms) play an important role in supplying soil N for plant growth in tropical forests. It is unknown how diazotrophs respond to deforestation and whether they can recover in secondary forests that establish after agriculture is abandoned. Using high-throughput sequencing of nifH genes, we characterized the response of diazotrophs’ β-diversity and identified major drivers of changes in diazotrophs from forest-to-pasture and pasture-to-secondary-forest conversions. Studying the impact of land use change on diazotrophs is important for a better understanding of the impact of deforestation on tropical forest ecosystem functioning, and our results on the potential recovery of diazotrophs in secondary forests imply the possible restoration of ecosystem functions in secondary forests.

Microbial mat communities are associated with extensive (~700 km²) and morphologically variable carbonate structures, termed microbialites, in the hypersaline Great Salt Lake (GSL), Utah. However, whether the composition of GSL mat communities covaries with microbialite morphology and lake environment is unknown. Moreover, the potential adaptations that allow the establishment of these extensive mat communities at high salinity (14% to 17% total salts) are poorly understood. To address these questions, microbial mats were sampled from seven locations in the south arm of GSL representing different lake environments and microbialite morphologies. Despite the morphological differences, microbialite-associated mats were taxonomically similar and were dominated by the cyanobacterium Euhalothece and several heterotrophic bacteria. Metagenomic sequencing of a representative mat revealed Euhalothece and subdominant Thiohalocapsa populations that harbor the Calvin cycle and nitrogenase, suggesting they supply fixed carbon and nitrogen to heterotrophic bacteria. Fifteen of the next sixteen most abundant taxa are inferred to be aerobic heterotrophs and, surprisingly, harbor reaction center, rhodopsin, and/or bacteriochlorophyll biosynthesis proteins, suggesting aerobic photoheterotrophic (APH) capabilities. Importantly, proteins involved in APH are enriched in the GSL community relative to that in microbialite mat communities from lower salinity environments. These findings indicate that the ability to integrate light into energy metabolism is a key adaptation allowing for robust mat development in the hypersaline GSL.

IMPORTANCE The earliest evidence of life on Earth is from organosedimentary structures, termed microbialites, preserved in 3.481-billion-year-old (Ga) rocks. Phototrophic microbial mats form in association with an ~700-km² expanse of morphologically diverse microbialites in the hypersaline Great Salt Lake (GSL), Utah. Here, we show taxonomically similar microbial mat communities are associated with morphologically diverse microbialites across the lake. Metagenomic sequencing reveals an abundance and diversity of autotrophic and heterotrophic taxa capable of harvesting light energy to drive metabolism. The unexpected abundance of and diversity in the mechanisms of harvesting light energy observed in GSL mat populations likely function to minimize niche overlap among coinhabiting taxa, provide a mechanism(s) to increase energy yield and osmotic balance during salt stress, and enhance fitness. Together, these physiological benefits promote the formation of robust mats that, in turn, influence the formation of morphologically diverse microbialite structures that can be imprinted in the rock record.

Most freshwater bacterial communities are characterized by a few dominant taxa that are often ubiquitous across freshwater biomes worldwide. Our understanding of the genomic diversity within these taxonomic groups is limited to a subset of taxa. Here, we investigated the genomic diversity that enables Limnohabitans, a freshwater genus key in funneling carbon from primary producers to higher trophic levels, to achieve abundance and ubiquity. We reconstructed eight putative Limnohabitans metagenome-assembled genomes (MAGs) from stations located along broad environmental gradients existing in Lake Michigan, part of Earth’s largest surface freshwater system. De novo strain inference analysis resolved a total of 23 strains from these MAGs, which strongly partitioned into two habitat-specific clusters with cooccurring strains from different lineages. The largest number of strains belonged to the abundant LimB lineage, for which robust in situ strain delineation had not previously been achieved. Our data show that temperature and nutrient levels may be important environmental parameters associated with microdiversification within the Limnohabitans genus. In addition, strains predominant in low- and high-phosphorus conditions had larger genomic divergence than strains abundant under different temperatures. Comparative genomics and gene expression analysis yielded evidence for the ability of LimB populations to exhibit cellular motility and chemotaxis, a phenotype not yet associated with available Limnohabitans isolates. Our findings broaden historical marker gene-based surveys of Limnohabitans microdiversification and provide in situ evidence of genome diversity and its functional implications across freshwater gradients.

IMPORTANCE Limnohabitans is an important bacterial taxonomic group for cycling carbon in freshwater ecosystems worldwide. Here, we examined the genomic diversity of different Limnohabitans lineages. We focused on the LimB lineage of this genus, which is globally distributed and often abundant, and its abundance has shown to be largely invariant to environmental change. Our data show that the LimB lineage is actually comprised of multiple cooccurring populations for which the composition and genomic characteristics are associated with variations in temperature and nutrient levels. The gene expression profiles of this lineage suggest the importance of chemotaxis and motility, traits that had not yet been associated with the Limnohabitans genus, in adapting to environmental conditions.

Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2. Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX. Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria.

IMPORTANCE Vibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria. Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.

Biofilm formation by Pseudomonas aeruginosa contributes to its survival on surfaces and represents a major clinical threat because of the increased tolerance of biofilms to disinfecting agents. This study aimed to investigate the efficacy of 405-nm light-emitting diode (LED) illumination in eliminating P. aeruginosa biofilms formed on stainless steel coupons under different temperatures. Time-dependent killing assays using planktonic and biofilm cells were used to determine the antimicrobial and antibiofilm activities of LED illumination. We also evaluated the effects of LED illumination on the disinfectant susceptibility, biofilm structure, extracellular polymeric substance (EPS) structure and composition, and biofilm-related gene expression of P. aeruginosa biofilm cells. Results showed that the abundance of planktonic P. aeruginosa cells was reduced by 0.88, 0.53, and 0.85 log CFU/ml following LED treatment for 2 h compared with untreated controls at 4, 10, and 25°C, respectively. For cells in biofilms, significant reductions (1.73, 1.59, and 1.68 log CFU/cm²) were observed following LED illumination for 2 h at 4, 10, and 25°C, respectively. Moreover, illuminated P. aeruginosa biofilm cells were more sensitive to benzalkonium chloride or chlorhexidine than untreated cells. Scanning electron microscopy and confocal laser scanning microscopic observation indicated that both the biofilm structure and EPS structure were disrupted by LED illumination. Further, reverse transcription-quantitative PCR revealed that LED illumination downregulated the transcription of several genes associated with biofilm formation. These findings suggest that LED illumination has the potential to be developed as an alternative method for prevention and control of P. aeruginosa biofilm contamination.

IMPORTANCE Pseudomonas aeruginosa can form biofilms on medical implants, industrial equipment, and domestic surfaces, contributing to high morbidity and mortality rates. This study examined the antibiofilm activity of 405-nm light-emitting diode (LED) illumination against mature biofilms formed on stainless steel coupons. We found that the disinfectant susceptibility, biofilm structure, and extracellular polymeric substance structure and composition were disrupted by LED illumination. We then investigated the transcription of several critical P. aeruginosa biofilm-related genes and analyzed the effect of illumination temperature on the above characteristics. Our results confirmed that LED illumination could be developed into an effective and safe method to counter P. aeruginosa biofilm contamination. Further research will be focused on the efficacy and application of LED illumination for elimination of complicated biofilms in the environment.

The response to iron limitation of the Gram-positive soil bacterium Corynebacterium glutamicum was analyzed with respect to secreted metabolites, the transcriptome, and the proteome. During growth in glucose minimal medium, iron limitation caused a shift from lactate to pyruvate as the major secreted organic acid complemented by l-alanine and 2-oxoglutarate. Transcriptome and proteome analyses revealed that a pronounced iron starvation response governed by the transcriptional regulators DtxR and RipA was detectable in the late, but not in the early, exponential-growth phase. A link between iron starvation and thiamine pyrophosphate (TPP) biosynthesis was uncovered by the strong upregulation of thiC. As phosphomethylpyrimidine synthase (ThiC) contains an iron-sulfur cluster, limiting activities of the TPP-dependent pyruvate–2-oxoglutarate dehydrogenase supercomplex probably cause the excretion of pyruvate and 2-oxoglutarate. In line with this explanation, thiamine supplementation could strongly diminish the secretion of these acids. The upregulation of thiC and other genes involved in thiamine biosynthesis and transport is presumably due to TPP riboswitches present at the 5' end of the corresponding operons. The results obtained in this study provide new insights into iron homeostasis in C. glutamicum and demonstrate that the metabolic consequences of iron limitation can be due to the iron dependency of coenzyme biosynthesis.

IMPORTANCE Iron is an essential element for most organisms but causes problems due to poor solubility under oxic conditions and due to toxicity by catalyzing the formation of reactive oxygen species (ROS). Therefore, bacteria have evolved complex regulatory networks for iron homeostasis aiming at a sufficient iron supply while minimizing ROS formation. In our study, the responses of the actinobacterium Corynebacterium glutamicum to iron limitation were analyzed, resulting in a detailed view on the processes involved in iron homeostasis in this model organism. In particular, we provide evidence that iron limitation causes TPP deficiency, presumably due to insufficient activity of the iron-dependent phosphomethylpyrimidine synthase (ThiC). TPP deficiency was deduced from the upregulation of genes controlled by a TPP riboswitch and secretion of metabolites caused by insufficient activity of the TPP-dependent enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. To our knowledge, the link between iron starvation and thiamine synthesis has not been elaborated previously.

Background:

Care services have not been sufficiently adapted to meet the comprehensive care needs of women living with HIV. Our study objective was to engage patients and providers in codesigning care recommendations to improve care for this population in the province of Quebec.

Methods:

We conducted a 5-hour deliberative dialogue workshop in April 2019 in Montréal as the final phase of a mixed-methods study investigating comprehensive care for women living with HIV. The study drew on data from the Canadian HIV Women’s Sexual and Reproductive Health Cohort Study (CHIWOS). Recruitment was guided by a purposive maximum-variation sampling strategy to ensure an appropriate mix of participants and was facilitated by our existing CHIWOS networks. Participants included patients (women living with HIV) and HIV care providers (doctors, nurses, pharmacists). The workshop was facilitated professionally and included a synthesis of the evidence, small- and large-group deliberations, and voting on care improvements.

Results:

Eight patients and 8 HIV care providers participated. Drawing on identified care priorities, the participants identified 4 relatively rapid care improvements and 3 longer-term improvements. The rapid care improvements included delegating medical acts to members of multidisciplinary care teams; greater involvement of HIV community members within care settings and health care decision-making; creating a women’s health information booklet; and increasing HIV education among all health care providers and raising awareness of women’s care needs beyond HIV-specific care among HIV care providers. The longer-term care improvements included advocating for complete financial coverage of antiretroviral therapy within the government-sponsored Medicare program, facilitating access to allied care providers (e.g., physiotherapists and psychologists) and launching a population-wide campaign to increase awareness about the Undetectable = Untransmittable (U=U) initiative and other HIV advances.

Interpretation:

The deliberative dialogue workshop yielded evidence-based, stakeholder-driven recommendations to improve the comprehensive care of women living with HIV in Quebec.

With increased interest in lipoprotein(a) (Lp[a]) concentration as a target for risk reduction and growing clinical evidence of its impact on cardiovascular disease (CVD) risk, rigorous analytical performance specifications (APS) and accuracy targets for Lp(a) are required. We investigated the biological variation (BV) of Lp(a), and 2 other major biomarkers of CVD, apolipoprotein A-I (apoA-I) and apolipoprotein B-100 (apoB), in the European Biological Variation Study population.Serum samples were drawn from 91 healthy individuals for 10 consecutive weeks at 6 European laboratories and analyzed in duplicate on a Roche Cobas 8000 c702. Outlier, homogeneity, and trend analysis were performed, followed by CV-ANOVA to determine BV estimates and their 95% CIs. These estimates were used to calculate APS and reference change values. For Lp(a), BV estimates were determined on normalized concentration quintiles.Within-subject BV estimates were significantly different between sexes for Lp(a) and between women aged <50 and >50 years for apoA-I and apoB. Lp(a) APS was constant across concentration quintiles and, overall, lower than APS based on currently published data, whereas results were similar for apoA-I and apoB.Using a fully Biological Variation Data Critical Appraisal Checklist (BIVAC)–compliant protocol, our study data confirm BV estimates of Lp(a) listed in the European Federation of Clinical Chemistry and Laboratory Medicine database and reinforce concerns expressed in recent articles regarding the suitability of older APS recommendations for Lp(a) measurements. Given the heterogeneity of Lp(a), more BIVAC-compliant studies on large numbers of individuals of different ethnic groups would be desirable.

Tigecycline serves as one of the antibiotics of last resort to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanism, Tet(X), challenges the clinical efficacy of this class of antibiotics. In this study, we detected 180 tet(X)-harboring Acinetobacter isolates (8.9%, n = 180) from 2,018 samples collected from avian farms and adjacent environments in China. Eighteen tet(X)-harboring isolates (10.0%) were found to cocarry the carbapenemase gene bla_NDM-1, mostly from waterfowl samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, tet(X) and bla_NDM-1 were found to colocalize on the same plasmids. Moreover, whole-genome sequencing (WGS) revealed a novel orthologue of tet(X) in the six isolates coharboring tet(X) and bla_NDM-1. Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be cotransferred. Sequence comparison between six tet(X)- and bla_NDM-1-coharboring plasmids showed that they shared a highly homologous plasmid backbone even though they were isolated from different Acinetobacter species (three from Acinetobacter indicus, two from Acinetobacter schindleri, and one from Acinetobacter lwoffii) from various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X)- and bla_NDM-1-coharboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

Omadacycline is a broad-spectrum aminomethylcycline approved in October 2018 by the U.S. Food and Drug Administration for treating acute bacterial skin and skin structure infections and community-acquired pneumonia as both an oral and intravenous once-daily formulation. In this report, the activities of omadacycline and comparators were tested against 49,000 nonduplicate bacterial isolates collected prospectively during 2016 to 2018 from medical centers in Europe (24,500 isolates, 40 medical centers [19 countries]) and the United States (24,500 isolates, 33 medical centers [23 states and all 9 U.S. census divisions]). Omadacycline was tested by broth microdilution following the methods in Clinical and Laboratory Standards Institute document M07 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 11th ed., 2018). Omadacycline (MIC_50/90, 0.12/0.25 mg/liter) inhibited 98.6% of Staphylococcus aureus isolates at ≤0.5 mg/liter, including 96.3% of methicillin-resistant S. aureus isolates and 99.8% of methicillin-susceptible S. aureus isolates. Omadacycline potency was comparable for Streptococcus pneumoniae (MIC_50/90, 0.06/0.12 mg/liter), viridans group streptococci (MIC_50/90, 0.06/0.12 mg/liter), and beta-hemolytic streptococci (MIC_50/90, 0.12/0.25 mg/liter), regardless of species and susceptibility to penicillin, macrolides, or tetracycline. Omadacycline was active against all Enterobacterales tested (MIC_50/90, 1/8 mg/liter; 87.5% of isolates were inhibited at ≤4 mg/liter) except Proteus mirabilis (MIC_50/90, 16/>32 mg/liter) and indole-positive Proteus spp. (MIC_50/90, 8/32 mg/liter) and was most active against Escherichia coli (MIC_50/90, 0.5/2 mg/liter), Klebsiella oxytoca (MIC_50/90, 1/2 mg/liter), and Citrobacter spp. (MIC_50/90, 1/4 mg/liter). Omadacycline inhibited 92.4% of Enterobacter cloacae species complex and 88.5% of Klebsiella pneumoniae isolates at ≤4 mg/liter. Omadacycline was active against Haemophilus influenzae (MIC_50/90, 0.5/1 mg/liter), regardless of β-lactamase status, and against Moraxella catarrhalis (MIC_50/90, ≤0.12/0.25 mg/liter). The potent activity of omadacycline against Gram-positive and -negative bacteria indicates that omadacycline merits further study in serious infections in which multidrug resistance and mixed Gram-positive and Gram-negative bacterial infections may be a concern.

Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of M. tuberculosis target genes in vitro. Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured in vitro. We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress tet promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of tet promoters in infected tissues of mice and rabbits.

In 2016, the proportion of Neisseria gonorrhoeae isolates with reduced susceptibility to azithromycin rose to 3.6%. A phylogenetic analysis of 334 N. gonorrhoeae isolates collected in 2016 revealed a single, geographically diverse lineage of isolates with MICs of 2 to 16 μg/ml that carried a mosaic-like mtr locus, whereas the majority of isolates with MICs of ≥16 μg/ml appeared sporadically and carried 23S rRNA mutations. Continued molecular surveillance of N. gonorrhoeae isolates will identify new resistance mechanisms.

The rising rates of antibiotic resistance increasingly compromise empirical treatment. Knowing the antibiotic susceptibility of a pathogen’s close genetic relative(s) may improve empirical antibiotic selection. Using genomic and phenotypic data for Escherichia coli isolates from three separate clinically derived databases, we evaluated multiple genomic methods and statistical models for predicting antibiotic susceptibility, focusing on potentially rapidly available information, such as lineage or genetic distance from archived isolates. We applied these methods to derive and validate the prediction of antibiotic susceptibility to common antibiotics. We evaluated 968 separate episodes of suspected and confirmed infection with Escherichia coli from three geographically and temporally separated databases in Ontario, Canada, from 2010 to 2018. Across all approaches, model performance (area under the curve [AUC]) ranges for predicting antibiotic susceptibility were the greatest for ciprofloxacin (AUC, 0.76 to 0.97) and the lowest for trimethoprim-sulfamethoxazole (AUC, 0.51 to 0.80). When a model predicted that an isolate was susceptible, the resulting (posttest) probabilities of susceptibility were sufficient to warrant empirical therapy for most antibiotics (mean, 92%). An approach combining multiple models could permit the use of narrower-spectrum oral agents in 2 out of every 3 patients while maintaining high treatment adequacy (~90%). Methods based on genetic relatedness to archived samples of E. coli could be used to predict antibiotic resistance and improve antibiotic selection.

Imipenem-relebactam (I-R) is a recently developed carbapenem–beta-lactamase inhibitor combination agent that can overcome carbapenem resistance, which has now emerged in Escherichia coli, including sequence type 131 (ST131) and its fluoroquinolone-resistant H30R subclone, the leading cause of extraintestinal E. coli infections globally. To clarify the likely utility of I-R for carbapenem-resistant (CR) E. coli infections in the United States, we characterized 203 recent CR clinical E. coli isolates from across the United States (years 2002 to 2017) for phylogroup, clonal group (including ST131, H30R, and the CTX-M-15-associated H30Rx subset within H30R), relevant beta-lactamase genes, and broth microdilution MICs for I-R and 11 comparator agents. Overall, I-R was highly active (89% susceptible), more so than all comparators except tigecycline and colistin (both 99% susceptible). I-R’s activity varied significantly in relation to phylogroup, clonal background, resistance genotype, and region. It was greatest among phylogroup B2, ST131-H30R, H30Rx, Klebsiella pneumoniae carbapenemase (KPC)-positive, and northeast U.S. isolates and lowest among phylogroup C, New Delhi metallo-β-lactamase (NDM)-positive, and southeast U.S. isolates. Relebactam improved imipenem’s activity against CR isolates within each phylogroup—especially groups A, B1, and B2—and particularly against isolates containing KPC. I-R remained substantially active against isolates coresistant to comparator agents, albeit somewhat less so than against the corresponding susceptible isolates. These findings suggest that I-R should be useful for treating most CR E. coli infections in the United States, largely independent of coresistance, although this likely will vary in relation to the local prevalence of specific E. coli lineages and carbapenem resistance mechanisms.

We investigated dose-fractionated polymyxin B (PB) on acute kidney injury (AKI). PB at 12 mg of drug/kg of body weight per day (once, twice, and thrice daily) was administered in rats over 72 h. The thrice-daily group demonstrated the highest KIM-1 increase (P = 0.018) versus that of the controls (P = 0.99) and histopathological damage (P = 0.013). A three-compartment model best described the data (bias, 0.129 mg/liter; imprecision, 0.729 mg²/liter²; R², 0.652,). Area under the concentration-time curve at 24 h (AUC₂₄) values were similar (P = 0.87). The thrice-daily dosing scheme resulted in the most PB-associated AKI in a rat model.

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the bla_KPC family) is one of the most common transmissible carbapenem resistance mechanisms worldwide. The dissemination of bla_KPC historically has been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the United Kingdom, bla_KPC has represented a large-scale, persistent management challenge for some hospitals, particularly in North West England. The dissemination of bla_KPC has evolved to be polyclonal and polyspecies, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole-genome sequencing of 604 bla_KPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterization. We observed the dissemination of bla_KPC (predominantly bla_KPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 [97%] isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), bla_KPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments among plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates), and IncR (252/604 isolates) replicons. The subset of reconstructed plasmid sequences (21 isolates, 77 plasmids) also highlighted modular exchange among non-bla_KPC and bla_KPC plasmids and the common presence of multiple replicons within bla_KPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales for the implementation of adequate surveillance approaches and for control.

Dalbavancin offers a possible treatment option for infectious peritonitis associated with peritoneal dialysis (PD) due to its coverage of Gram-positive bacteria and pharmacokinetic properties. We aimed to evaluate the clinical pharmacokinetics (PK) and pharmacodynamics of dalbavancin in a prospective, randomized, open-label, crossover PK study of adult patients with end-stage renal disease ESRD who were receiving PD. Sampling occurred prior to a single 30-min infusion of dalbavancin at 1,500 mg and at 1, 2, 3, 4, and 6 h and 7 and 14 days postadministration. Concentration-time data were analyzed via noncompartmental analysis. Pharmacodynamic parameters against common infectious peritonitis-causing pathogens were evaluated. Ten patients were enrolled. Patients were a median of 55 years old and had a median weight of 78.2 kg, 50% were female, and 70% were Caucasian. The terminal plasma half-life of dalbavancin was 181.4 ± 35.5 h. The day 0 to day 14 dalbavancin mean area under the curve (AUC) was 40,573.2 ± 9,800.3 mg·h/liter. The terminal-phase half-life of dalbavancin within the peritoneal fluid was 4.309 x 10⁸ ± 1.140 x 10⁹ h. The day 0 to day 14 dalbavancin mean peritoneal fluid AUC was 2,125.0 ± 1,794.3 mg·h/liter. The target plasma AUC/MIC was attained with the intravenous dose in all 10 patients for all Staphylococcus and Streptococcus species at the recommended MIC breakpoints. The intraperitoneal arm of the study was stopped early, because the first 3 patients experienced moderate to severe pain and bloating within 1 h following the administration of dalbavancin. Dalbavancin at 1,500 mg administered intravenously can be utilized without dose adjustment in peritoneal dialysis patients and will likely achieve the necessary peritoneal fluid concentrations to treat peritonitis caused by typical Gram-positive pathogens.

The effects of multiple-dose administration of tenofovir disoproxil fumarate (TDF) on the pharmacokinetics of morinidazole (MOR) were compared in healthy subjects. MOR exposure was similar, with an area under the curve from 0 h to infinity (AUC_0-) treatment ratio for MOR+TDF/MOR of 1.01 (90% confidence interval, 0.97 to 1.06). No relevant differences were observed regarding plasma exposure of metabolites. Renal clearances of MOR and its metabolites were not affected by TDF. No unexpected safety or tolerability issues were observed.

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with bla_CMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.

Plasmodium vivax relapse is one of the major causes of sustained global malaria transmission. Primaquine (PQ) is the only commercial drug available to prevent relapses, and its efficacy is dependent on metabolic activation by cytochrome P450 2D6 (CYP2D6). Impaired CYP2D6 function, caused by allelic polymorphisms, leads to the therapeutic failure of PQ as a radical cure for P. vivax malaria. Here, we hypothesized that the host immune response to malaria parasites modulates susceptibility to P. vivax recurrences in association with CYP2D6 activity. We performed a 10-year retrospective study by genotyping CYP2D6 polymorphisms in 261 malaria-exposed individuals from the Brazilian Amazon. The immune responses against a panel of P. vivax blood-stage antigens were evaluated by serological assays. We confirmed our previous findings, which indicated an association between impaired CYP2D6 activity and a higher risk of multiple episodes of P. vivax recurrence (risk ratio, 1.75; 95% confidence interval [CI], 1.2 to 2.6; P = 0.0035). An important finding was a reduction of 3% in the risk of recurrence (risk ratio, 0.97; 95% CI, 0.96 to 0.98; P < 0.0001) per year of malaria exposure, which was observed for individuals with both reduced and normal CYP2D6 activity. Accordingly, subjects with long-term malaria exposure and persistent antibody responses to various antigens showed fewer episodes of malaria recurrence. Our findings have direct implications for malaria control, since it was shown that nonimmune individuals who do not respond adequately to treatment due to reduced CYP2D6 activity may present a significant challenge for sustainable progress toward P. vivax malaria elimination.

The new complexes Zn(ITZ)₂Cl₂ (1) and Zn(ITZ)₂(OH)₂ (2) were synthetized by a reaction of itraconazole with their respective zinc salts under reflux. These Zn-ITZ complexes were characterized by elemental analyses, molar conductivity, mass spectrometry, ¹H and ¹³C{¹H} nuclear magnetic resonance, and UV-vis and infrared spectroscopies. The antiparasitic and antifungal activity of Zn-ITZ complexes was evaluated against three protozoans of medical importance, namely, Leishmania amazonensis, Trypanosoma cruzi, and Toxoplasma gondii, and two fungi, namely, Sporothrix brasiliensis and Sporothrix schenckii. The Zn-ITZ complexes exhibited a broad spectrum of action, with antiparasitic and antifungal activity in low concentrations. The strategy of combining zinc with ITZ was efficient to enhance ITZ activity since Zn-ITZ-complexes were more active than the azole alone. This study opens perspectives for future applications of these Zn-ITZ complexes in the treatment of parasitic diseases and sporotrichosis.

In children requiring lopinavir coformulated with ritonavir in a 4:1 ratio (lopinavir-ritonavir-4:1) and rifampin, adding ritonavir to achieve a 4:4 ratio with lopinavir (LPV/r-4:4) overcomes the drug-drug interaction. Possible drug-drug interactions within this regimen may affect abacavir concentrations, but this has never been studied. Children weighing <15 kg needing rifampin and LPV/r-4:4 were enrolled in a pharmacokinetic study and underwent intensive pharmacokinetic sampling on 3 visits: (i) during the intensive and (ii) continuation phases of antituberculosis treatment with LPV/r-4:4 and (iii) 1 month after antituberculosis treatment completion on LPV/r-4:1. Pharmacometric modeling and simulation were used to compare exposures across weight bands with adult target exposures. Eighty-seven children with a median (interquartile range) age and weight of 19 (4 to 64) months and 8.7 (3.9 to 14.9) kg, respectively, were included in the abacavir analysis. Abacavir pharmacokinetics were best described by a two-compartment model with first-order elimination and transit compartment absorption. After allometric scaling adjusted for the effect of body size, maturation could be identified: clearance was predicted to be fully mature at about 2 years of age and to reach half of this mature value at about 2 months of age. Abacavir bioavailability decreased 36% during treatment with rifampin and LPV/r-4:4 but remained within the median adult recommended exposure, except for children in the 3- to 4.9-kg weight band, in which the exposures were higher. The observed predose morning trough concentrations were higher than the evening values. Though abacavir exposure significantly decreased during concomitant administration of rifampin and LPV/r-4:4, it remained within acceptable ranges. (This study is registered in ClinicalTrials.gov under identifier NCT02348177.)

Antituberculosis (anti-TB) drug development is dependent on informative trials to secure the development of new antibiotics and combination regimens. Clofazimine (CLO) and pyrazinamide (PZA) are important components of recommended standard multidrug treatments of TB. Paradoxically, in a phase IIa trial aiming to define the early bactericidal activity (EBA) of CLO and PZA monotherapy over the first 14 days of treatment, no significant drug effect was demonstrated for the two drugs using traditional statistical analysis. Using a model-based analysis, we characterized the statistically significant exposure-response relationships for both drugs that could explain the original findings of an increase in the numbers of CFU with CLO treatment and no effect with PZA. Sensitive analyses are crucial for exploring drug effects in early clinical trials to make the right decisions for advancement to further development. We propose that this quantitative semimechanistic approach provides a rational framework for analyzing phase IIa EBA studies and can accelerate anti-TB drug development.

A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying bla_NDM-1. Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. bla_NDM-1 was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains. A resistance gene island carrying bla_PER-7 and other resistance determinants was found on a plasmid in some A. baumannii strains.

Hypervirulent Klebsiella pneumoniae strains are the major cause of liver abscesses throughout East Asia, and these strains are usually antibiotic susceptible. Recently, multidrug-resistant and hypervirulent (MDR-HV) K. pneumoniae strains have emerged due to hypervirulent strains acquiring antimicrobial resistance determinants or the transfer of a virulence plasmid into a classic MDR strain. In this study, we characterized the clinical and microbiological properties of K. pneumoniae liver abscess (KPLA) caused by MDR-HV strains in Taiwan. Patients with community onset KPLA were retrospectively identified at Taipei Veterans General Hospital during January 2013 to May 2018. Antimicrobial resistance mechanisms, capsular types, and sequence types were determined. MDR-HV strains and their parental antimicrobial-susceptible strains further underwent whole-genome sequencing (WGS) and in vivo mice lethality tests. Thirteen MDR-HV strains were identified from a total of 218 KPLA episodes. MDR-HV strains resulted in similar outcomes to antimicrobial-susceptible strains. All MDR-HV strains were traditional hypervirulent clones carrying virulence capsular types. The major resistance mechanisms were the overexpression of efflux pumps and/or the acquisition of ESBL or AmpC β-lactamase genes. WGS revealed that two hypervirulent strains had evolved to an MDR phenotype due to mutation in the ramR gene and the acquisition of an SHV-12-bearing plasmid, respectively. Both these MDR-HV strains retained high virulence compared to their parental strains. The spread of MDR-HV K. pneumoniae strains in the community raises significant public concerns, and measures should be taken to prevent the further acquisition of carbapenemase and other resistance genes among these strains in order to avoid the occurrence of untreatable KPLA.

Fluoroquinolones are reported to possess immunomodulatory activity; hence, a novel benzoquinolizine fluoroquinolone, levonadifloxacin, was evaluated in lipopolysaccharide-stimulated human whole-blood (HWB) and mouse acute lung injury (ALI) models. Levonadifloxacin significantly mitigated the inflammatory responses in an HWB assay through inhibition of proinflammatory cytokines and in the ALI model by lowering lung total white blood cell count, myeloperoxidase, and cytokine levels. The immunomodulatory effect of levonadifloxacin, along with promising antibacterial activity, is expected to provide clinical benefits in the treatment of infections.

In patients with renal impairment (n = 22 of 39), the median serum concentrations of linezolid, PNU-142300, and PNU-142586 were 1.6-, 3.3-, 2.8-fold higher, respectively, than in patients without renal impairment. Metabolite concentrations in paired samples were poorly correlated with linezolid concentrations (r² = 0.26 for PNU-142300 and 0.06 for PNU-142586). Linezolid and its metabolites share potential toxicophores that deserve characterization to mitigate higher myelosuppression risk in patients with renal impairment.

A cluster of dendritic cells (termed mregDCs), observed in humans and mice, restricted antitumor immunity.

Monoubiquitinated FANCI and FANCD2 constitute the ID complex, which forms a sliding clamp on DNA.

A chemotherapy-induced shift to ICOSL⁺ B cells in breast tumors correlated with better survival.

Tumor-derived retinoic acid promotes monocyte differentiation into immunosuppressive macrophages.

T cell–expressed PD-L1 exerts tolerogenic effects on tumor immunity in pancreatic cancer.

A chambered heart is common to all vertebrates, but reptiles show unparalleled variation in ventricular septation, ranging from almost absent in tuataras to full in crocodilians. Because mammals and birds evolved independently from reptile lineages, studies on reptile development may yield insight into the evolution and development of the full ventricular septum. Compared with reptiles, mammals and birds have evolved several other adaptations, including compact chamber walls and a specialized conduction system. These adaptations appear to have evolved from precursor structures that can be studied in present-day reptiles. The increase in the number of studies on reptile heart development has been greatly facilitated by sequencing of several genomes and the availability of good staging systems. Here, we place reptiles in their phylogenetic context with a focus on features that are primitive when compared with the homologous features of mammals. Further, an outline of major developmental events is given, and variation between reptile species is discussed.

Understanding how coronary blood vessels form and regenerate during development and progression of cardiac diseases will shed light on the development of new treatment options targeting coronary artery diseases. Recent studies with the state-of-the-art technologies have identified novel origins of, as well as new, cellular and molecular mechanisms underlying the formation of coronary vessels in the postnatal heart, including collateral artery formation, endocardial-to-endothelial differentiation and mesenchymal-to-endothelial transition. These new mechanisms of coronary vessel formation and regeneration open up new possibilities targeting neovascularization for promoting cardiac repair and regeneration. Here, we highlight some recent studies on cellular mechanisms of coronary vessel formation, and discuss the potential impact and significance of the findings on basic research and clinical application for treating ischemic heart disease.

Inflammasomes assemble in the cytosol of myeloid and epithelial cells on sensing of cellular stress and pathogen-associated molecular patterns and serve as scaffolds for recruitment and activation of inflammatory caspases. Inflammasomes play beneficial roles in host and immune responses against diverse pathogens but may also promote inflammatory tissue damage if uncontrolled. Gasdermin D (GSDMD) is a recently identified substrate of murine caspase-1 and caspase-11, and human caspases-1, -4, and -5 that mediates a regulated lytic cell death mode termed pyroptosis. Recent studies have identified pyroptosis as a critical inflammasome effector mechanism that controls inflammasome-dependent cytokine secretion and contributes to antimicrobial defense and inflammasome-mediated autoinflammatory diseases. Here, we review recent developments on inflammasome-associated effector functions with an emphasis on the emerging roles of gasdermin pores and pyroptosis.

Cell death is an invariant feature throughout our life span, starting with extensive scheduled cell death during morphogenesis and continuing with death under homeostasis in adult tissues. Additionally, cells become victims of accidental, unscheduled death following injury and infection. Cell death in each of these occasions triggers specific and specialized responses in the living cells that surround them or are attracted to the dying/dead cells. These responses sculpt tissues during morphogenesis, replenish lost cells in homeostasis to maintain tissue/system function, and repair damaged tissues after injury. Wherein lies the information that sets in motion the cascade of effector responses culminating in remodeling, renewal, or repair? Here, we attempt to provide a framework for thinking about cell death in terms of the specific effector responses that accompanies various modalities of cell death. We also propose an integrated threefold "cell death code" consisting of information intrinsic to the dying/dead cell, the surroundings of the dying cell, and the identity of the responder.

To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses.

Maintenance of the proteome, ensuring the proper locations, proper conformations, appropriate concentrations, etc., is essential to preserve the health of an organism in the face of environmental insults, infectious diseases, and the challenges associated with aging. Maintaining the proteome is even more difficult in the background of inherited mutations that render a given protein and others handled by the same proteostasis machinery misfolding prone and/or aggregation prone. Maintenance of the proteome or maintaining proteostasis requires the orchestration of protein synthesis, folding, trafficking, and degradation by way of highly conserved, interacting, and competitive proteostasis pathways. Each subcellular compartment has a unique proteostasis network compromising common and specialized proteostasis maintenance pathways. Stress-responsive signaling pathways detect the misfolding and/or aggregation of proteins in specific subcellular compartments using stress sensors and respond by generating an active transcription factor. Subsequent transcriptional programs up-regulate proteostasis network capacity (i.e., ability to fold and degrade proteins in that compartment). Stress-responsive signaling pathways can also be linked by way of signaling cascades to nontranscriptional means to reestablish proteostasis (e.g., by translational attenuation). Proteostasis is also strongly influenced by the inherent kinetics and thermodynamics of the folding, misfolding, and aggregation of individual proteins, and these sequence-based attributes in combination with proteostasis network capacity together influence proteostasis. In this review, we will focus on the growing body of evidence that proteostasis deficits leading to human pathology can be reversed by pharmacologic adaptation of proteostasis network capacity through stress-responsive signaling pathway activation. The power of this approach will be exemplified by focusing on the ATF6 arm of the unfolded protein response stress responsive-signaling pathway that regulates proteostasis network capacity of the secretory pathway.