The bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To ensure consistent product quality of the therapeutic proteins, it is imperative to have a controlled production process. Regulatory agencies and the biotechnology industry consider cell line "clonal origin" an important aspect of maintaining process control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in the past few years, and the industry has improved methods and devised standards to increase the probability and/or assurance of clonal derivation. However, older production cell lines developed before the implementation of these methods, herein referred to as "legacy cell lines," may not meet current regulatory expectations for demonstration of clonal derivation. In this article, the members of the IQ Consortium Working Group on Clonality present our position that the demonstration of process consistency and product comparability of critical quality attributes throughout the development life cycle should be sufficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines that may not meet current day clonal derivation standards. With this commentary, we discuss advantages and limitations of genetic testing methods to support clonal derivation of legacy cell lines and wish to promote a mutual understanding with the regulatory authorities regarding their optional use during early drug development, subsequent to Investigational New Drug (IND) application and before demonstration of product and process consistency at Biologics License Applications (BLA) submission.

This technology review, written by a small group of pharmaceutical microbiologists experienced in cell therapies, discussed a risk-based approach to microbiological contamination detection and control during gene and cell therapy production. Topics discussed include a brief overview of cell therapies, a risk analysis related to donor selection, cell collection and infectious agent testing, cell transformation and expansion, packaging, storage, and administration, and cell therapy microbial contamination testing and release.

Capture bioprocessing unit operations were previously shown to clear or kill several log₁₀ of a model mycoplasma Acholeplasma laidlawii in lab-scale spike/removal studies. Here, we confirm this observation with two additional mollicute species relevant to biotechnology products for human use: Mycoplasma orale and Mycoplasma arginini. Clearance of M. orale and M. arginini from protein A column purification was similar to that seen with A. laidlawii, though some between cycle carryover was evident, especially for M. orale. However, on-resin growth studies for all three species revealed that residual mycoplasma in a column slowly die off over time rather than expanding further. Solvent/detergent exposure completely inactivated M. arginini though detectable levels of M. orale remained. A small-scale model of a commercial low-pH hold step did inactivate live M. orale, but this inactivation required a lower pH set point and occurred with slower kinetics than previously seen with A. laidlawii. Additionally, ultraviolet-C irradiation was shown to be effective for A. laidlawii and M. orale inactivation whereas virus-retentive filters for upstream and downstream processes, as expected, cleared A. laidlawii. These data argue that M. orale and M. arginini overall would be largely cleared by early bioprocessing steps as shown previously for A. laidlawii, and that barrier technologies can effectively reduce the risk from media components. For some unit operations, M. orale and M. arginini may be hardier, and require more stringent processing or equipment cleaning conditions to assure effective mycoplasma reduction. By exploring how some of the failure modes in commercial antibody manufacturing processes can still eliminate mycoplasma burden, we demonstrate that required best practices assure biotechnology products will be safe for patients.

Technologies allowing genetic sequencing of the human microbiome are opening new realms to discovery. The host microbiota substantially impacts immune responses both in immune-mediated inflammatory diseases (IMIDs) and in tumors affecting tissues beyond skin and mucosae. However, a mechanistic link between host microbiota and cancer or IMIDs has not been well established. Here, we propose T helper 17 (T_H17) lymphocytes as the connecting factor between host microbiota and rheumatoid or psoriatic arthritides, multiple sclerosis, breast or ovarian cancer, and multiple myeloma. We theorize that similar mechanisms favor the expansion of gut-borne T_H17 cells and their deployment at the site of inflammation in extraborder IMIDs and tumors, where T_H17 cells are driving forces. Thus, from a pathogenic standpoint, tumors may share mechanistic routes with IMIDs. A review of similarities and divergences in microbiota-T_H17 cell interactions in IMIDs and cancer sheds light on previously ignored pathways in either one of the two groups of pathologies and identifies novel therapeutic avenues.

Although enteroviruses are associated with a wide variety of diseases and conditions, their mode of replication is well conserved. Their genome is carried as a single, positive-sense RNA strand. At the 5' end of the strand is an approximately 90-nucleotide self-complementary region called the 5' cloverleaf, or the oriL. This noncoding region serves as a platform upon which host and virus proteins, including the 3B, 3C, and 3D virus proteins, assemble in order to initiate replication of a negative-sense RNA strand. The negative strand in turn serves as a template for synthesis of multiple positive-sense RNA strands. Building on structural studies of individual RNA stem-loops, the structure of the intact 5' cloverleaf from rhinovirus has recently been determined via nuclear magnetic resonance/small-angle X-ray scattering (NMR/SAXS)-based methods, while structures have also been determined for enterovirus 3A, 3B, 3C, and 3D proteins. Analysis of these structures, together with structural and modeling studies of interactions between host and virus proteins and RNA, has begun to provide insight into the enterovirus replication mechanism and the potential to inhibit replication by blocking these interactions.

The substantial discrepancy between the strong effects of functional foods and various drugs, especially traditional Chinese medicines (TCMs), and the poor bioavailability of these substances remains a perplexing problem. Understanding the gut microbiota, which acts as an effective bioreactor in the human intestinal tract, provides an opportunity for the redefinition of bioavailability. Here, we discuss four different pathways associated with the role of the gut microbiota in the transformation of parent compounds to beneficial or detrimental small molecules, which can enter the body’s circulatory system and be available to target cells, tissues, and organs. We further describe and propose effective strategies for improving bioavailability and alleviating side effects with the help of the gut microbiota. This review also broadens our perspectives for the discovery of new medicinal components.

Assemblages of upper lower through upper Miocene Discoaster spp. have been quantified from Integrated Ocean Drilling Program (IODP) Site U1338 in the eastern equatorial Pacific Ocean. These assemblages can be grouped into five broad morphological categories: six-rayed with bifurcated ray tips, six-rayed with large central areas, six-rayed with pointed ray tips, five-rayed with bifurcated ray tips and five-rayed with pointed ray tips. Discoaster deflandrei dominates the assemblages prior to 15.8 Ma. The decline in abundance of D. deflandrei close to the early–middle Miocene boundary occurs together with the evolution of the D. variabilis group, including D. signus and D. exilis. Six-rayed discoasters having large central areas become a prominent member of the assemblages for a 400 ka interval in the late middle Miocene. Five- and six-rayed forms having pointed tips become prominent in the early late Miocene and show a strong antiphasing relationship with the D. variabilis group. Discoaster bellus completely dominates the Discoaster assemblages for a 400 ka interval in the middle late Miocene. Abundances of all discoasters, or discoasters at the species level, show only (surprisingly) weak correlations to carbonate contents or oxygen and carbon isotopes of bulk sediment when calculated over the entire sample interval.

OBJECTIVE

The effect of early-life antibiotic treatment on the risk of type 1 diabetes is debated. This study assessed this question, applying a register-based design in children up to age 10 years including a large sibling-control analysis.

RESEARCH DESIGN AND METHODS

All singleton children (n = 797,318) born in Sweden between 1 July 2005 and 30 September 2013 were included and monitored to 31 December 2014. Cox proportional hazards models, adjusted for parental and perinatal characteristics, were applied, and stratified models were used to account for unmeasured confounders shared by siblings.

RESULTS

Type 1 diabetes developed in 1,297 children during the follow-up (median 4.0 years [range 0–8.3]). Prescribed antibiotics in the 1st year of life (23.8%) were associated with an increased risk of type 1 diabetes (adjusted hazard ratio [HR] 1.19 [95% CI 1.05–1.36]), with larger effect estimates among children delivered by cesarean section (P for interaction = 0.016). The association was driven by exposure to antibiotics primarily used for acute otitis media and respiratory tract infections. Further, we found an association of antibiotic prescriptions in pregnancy (22.5%) with type 1 diabetes (adjusted HR 1.15 [95% CI 1.00–1.32]). In general, sibling analysis supported these results, albeit often with statistically nonsignificant associations.

CONCLUSIONS

Dispensed prescription of antibiotics, mainly for acute otitis media and respiratory tract infections, in the 1st year of life is associated with an increased risk of type 1 diabetes before age 10 years, most prominently in children delivered by cesarean section.

Patients with systemic immunoglobulin light chain amyloidosis (AL) with no evidence of cardiac involvement by consensus criteria have excellent survival, but 20% will die within 5 years of diagnosis and prognostic factors remain poorly characterised. We report the outcomes of 378 prospectively followed Mayo stage I patients (N-terminal pro b-type natriuretic peptide <332 ng/L, high sensitivity cardiac troponin <55 ng/L). The median presenting N-terminal pro b-type natriuretic peptide was 161 ng/L, high sensitivity cardiac troponin 10 ng/L, creatinine 76 μmol/L and mean left ventricular septal wall thickness, 10 mm. Median follow up was 42 (1-117 months), with 71 deaths; median overall survival was not reached (78% survival at 5 years). Although no patients had cardiac involvement by echocardiogram, a proportion (n=25/90, 28%) had cardiac involvement by cardiac magnetic resonance imaging. Age, autonomic nervous system involvement, N-terminal pro b-type natriuretic peptide >152 ng/L, high sensitivity cardiac troponin >10 ng/L and cardiac involvement by magnetic resonance imaging were predictive for survival; on multivariate analysis only N-terminal pro b-type natriuretic peptide >152 ng/L (P<0.008, hazard ratio [HR] 3.180, confidence interval [CI]: 1.349-7.495) and cardiac involvement on magnetic resonance imaging (P=0.026, HR=5.360, CI: 1.219-23.574) were prognostic. At 5 years, 70% of patients with N-terminal pro b-type natriuretic peptide >152 ng/L were alive. In conclusion, N-terminal pro b-type natriuretic peptide is prognostic for survival in patients with no cardiac involvement by consensus criteria and cardiac involvement is detected by magnetic resonance imaging in such cases. This suggests that N-terminal pro b-type natriuretic peptide thresholds for cardiac involvement in AL may need to be redefined.

Advances in small RNA sequencing have revealed the enormous diversity of small noncoding RNA (sRNA) classes in mammalian cells. At this point, most investigators in diabetes are aware of the success of microRNA (miRNA) research and appreciate the importance of posttranscriptional gene regulation in glycemic control. Nevertheless, miRNAs are just one of multiple classes of sRNAs and likely represent only a minor fraction of sRNA sequences in a given cell. Despite the widespread appreciation of sRNAs, very little research into non-miRNA sRNA function has been completed, likely due to some major barriers that present unique challenges for study. To emphasize the importance of sRNA research in cardiometabolic diseases, we highlight the success of miRNAs and competitive endogenous RNAs in cholesterol and glucose metabolism. Moreover, we argue that sequencing studies have demonstrated that miRNAs are just the tip of the iceberg for sRNAs. We are likely standing at the precipice of immense discovery for novel sRNA-mediated gene regulation in cardiometabolic diseases. To realize this potential, we must first address critical barriers with an open mind and refrain from viewing non-miRNA sRNA function through the lens of miRNAs, as they likely have their own set of distinct regulatory factors and functional mechanisms.

The kidney is a complex organ responsible for maintaining multiple aspects of homeostasis in the human body. The combination of distinct, yet interrelated, molecular functions across different cell types make the delineation of factors associated with loss or decline in kidney function challenging. Consequently, there has been a paucity of new diagnostic markers and treatment options becoming available to clinicians and patients in managing kidney diseases. A systems biology approach to understanding the kidney leverages recent advances in computational technology and methods to integrate diverse sets of data. It has the potential to unravel the interplay of multiple genes, proteins, and molecular mechanisms that drive key functions in kidney health and disease. The emergence of large, detailed, multilevel biologic and clinical data from national databases, cohort studies, and trials now provide the critical pieces needed for meaningful application of systems biology approaches in nephrology. The purpose of this review is to provide an overview of the current state in the evolution of the field. Recent successes of systems biology to identify targeted therapies linked to mechanistic biomarkers in the kidney are described to emphasize the relevance to clinical care and the outlook for improving outcomes for patients with kidney diseases.

Background and objectives

Bioelectrical impedance analysis (BIA) devices can help assess volume overload in patients receiving maintenance peritoneal dialysis. However, the effects of BIA on the short-term hard end points of peritoneal dialysis lack consistency. This study aimed to test whether BIA-guided fluid management could improve short-term outcomes in patients on peritoneal dialysis.

Design, setting, participants, & measurements

A single-center, open-labeled, randomized, controlled trial was conducted. Patients on prevalent peritoneal dialysis with volume overload were recruited from July 1, 2013 to March 30, 2014 and followed for 1 year in the initial protocol. All participants with volume overload were 1:1 randomized to the BIA-guided arm (BIA and traditional clinical methods) and control arm (only traditional clinical methods). The primary end point was all-cause mortality and secondary end points were cardiovascular disease mortality and technique survival.

Results

A total of 240 patients (mean age, 49 years; men, 51%; diabetic, 21%, 120 per group) were enrolled. After 1-year follow-up, 11(5%) patients died (three in BIA versus eight in control) and 21 patients were permanently transferred to hemodialysis (eight in BIA versus 13 in control). The rate of extracellular water/total body water decline in the BIA group was significantly higher than that in the control group. The 1-year patient survival rates were 96% and 92% in BIA and control groups, respectively. No significant statistical differences were found between patients randomized to the BIA-guided or control arm in terms of patient survival, cardiovascular disease mortality, and technique survival (P>0.05).

Conclusions

Although BIA-guided fluid management improved the fluid overload status better than the traditional clinical method, no significant effect was found on 1-year patient survival and technique survival in patients on peritoneal dialysis.

Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5, and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Both the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to a 50% reduction in nitrogenase activity, and double deletion of fnr1 and fnr3 resulted to a 90% reduction in activity. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic conditions. Fnr1 and Fnr3 inhibited expression of the genes involved in the aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.

IMPORTANCE The members of the nitrogen-fixing Paenibacillus spp. have great potential to be used as a bacterial fertilizer in agriculture. However, the functions of the fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we found that in P. polymyxa WLY78, Fnr1 and Fnr3 were responsible for regulation of numerous genes in response to changes in oxygen levels, but Fnr5 and Fnr7 exhibited little effect. Fnr1 and Fnr3 indirectly or directly regulated many types of important metabolism, such as nitrogen fixation, Fe uptake, respiration, and electron transport. This study not only reveals the function of the fnr genes of P. polymyxa WLY78 in nitrogen fixation and other metabolisms but also will provide insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

This study investigated the effects of long-term soil fertilization on the composition and potential for phosphorus (P) and nitrogen (N) cycling of bacterial communities associated with hyphae of the P-solubilizing fungus Penicillium canescens. Using a baiting approach, hyphosphere bacterial communities were recovered from three soils that had received long-term amendment in the field with mineral or mineral plus organic fertilizers. P. canescens hyphae recruited bacterial communities with a decreased diversity and an increased abundance of Proteobacteria relative to what was observed in soil communities. As core bacterial taxa, Delftia and Pseudomonas spp. were present in all hyphosphere samples irrespective of soil fertilization. However, the type of fertilization showed significant impacts on the diversity, composition, and distinctive taxa/operational taxonomic units (OTUs) of hyphosphere communities. The soil factors P (Olsen method), exchangeable Mg, exchangeable K, and pH were important for shaping soil and hyphosphere bacterial community compositions. An increased relative abundance of organic P metabolism genes was found in hyphosphere communities from soil that had not received P fertilizers, which could indicate P limitation near the fungal hyphae. Additionally, P. canescens hyphae recruited bacterial communities with a higher abundance of N fixation genes than found in soil communities, which might imply a role of hyphosphere communities for fungal N nutrition. Furthermore, the relative abundances of denitrification genes were greater in several hyphosphere communities, indicating an at least partly anoxic microenvironment with a high carbon-to-N ratio around the hyphae. In conclusion, soil fertilization legacy shapes P. canescens hyphosphere microbiomes and their functional potential related to P and N cycling.

IMPORTANCE P-solubilizing Penicillium strains are introduced as biofertilizers to agricultural soils to improve plant P nutrition. Currently, little is known about the ecology of these biofertilizers, including their interactions with other soil microorganisms. This study shows that communities dominated by Betaproteobacteria and Gammaproteobacteria colonize P. canescens hyphae in soil and that the compositions of these communities depend on the soil conditions. The potential of these communities for N and organic P cycling is generally higher than that of soil communities. The high potential for organic P metabolism might complement the ability of the fungus to solubilize inorganic P, and it points to the hyphosphere as a hot spot for P metabolism. Furthermore, the high potential for N fixation could indicate that P. canescens recruits bacteria that are able to improve its N nutrition. Hence, this community study identifies functional groups relevant for the future optimization of next-generation biofertilizer consortia for applications in soil.

Resistance to the "last-resort" antibiotics, such as carbapenems, has led to very few antibiotics being left to treat infections by multidrug-resistant bacteria. Spread of carbapenem resistance (CR) has been well characterized for the clinical environment. However, there is a lack of information about its environmental distribution. Our study reveals that CR is present in a wide range of Gram-negative bacteria in the coastal seawater environment, including four phyla, eight classes, and 30 genera. These bacteria were likely introduced into seawater via stormwater flows. Some CR isolates found here, such as Acinetobacter junii, Acinetobacter johnsonii, Brevundimonas vesicularis, Enterococcus durans, Pseudomonas monteilii, Pseudomonas fulva, and Stenotrophomonas maltophilia, are further relevant to human health. We also describe a novel metallo-β-lactamase (MBL) for marine Rheinheimera isolates with CR, which has likely been horizontally transferred to Citrobacter freundii or Enterobacter cloacae. In contrast, another MBL of the New Delhi type was likely acquired by environmental Variovorax isolates from Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii utilizing a plasmid. Our findings add to the growing body of evidence that the aquatic environment is both a reservoir and a vector for novel CR genes.

IMPORTANCE Resistance against the "last-resort" antibiotics of the carbapenem family is often based on the production of carbapenemases, and this has been frequently observed in clinical samples. However, the dissemination of carbapenem resistance (CR) in the environment has been less well explored. Our study shows that CR is commonly found in a range of bacterial taxa in the coastal aquatic environment and can involve the exchange of novel metallo-β-lactamases from typical environmental bacteria to potential human pathogens or vice versa. The outcomes of this study contribute to a better understanding of how aquatic and marine bacteria can act as reservoirs and vectors for CR outside the clinical setting.

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.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a K_m of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes.

IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β' linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

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.

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:

Unnecessary antibiotic use in the community in Canada is not well defined. Our objective was to quantify unnecessary antibiotic prescribing in a Canadian primary care setting.

Methods:

We performed a descriptive analysis in Ontario from April 2011 to March 2016 using the Electronic Medical Records Primary Care database linked to other health administrative data sets at ICES. We determined antibiotic prescribing rates (per 100 patient–physician encounters) for 23 common conditions and estimated rates of unnecessary prescribing using predefined expected prescribing rates, both stratified by condition and patient age group.

Results:

The study included 341 physicians, 204 313 patients and 499 570 encounters. The rate of unnecessary antibiotic prescribing for included conditions was 15.4% overall and was 17.6% for those less than 2 years of age, 18.6% for those aged 2–18, 14.5% for those aged 19–64 and 13.0% for those aged 65 or more. The highest unnecessary prescribing rates were observed for acute bronchitis (52.6%), acute sinusitis (48.4%) and acute otitis media (39.3%). The common cold, acute bronchitis, acute sinusitis and miscellaneous nonbacterial infections were responsible for 80% of the unnecessary antibiotic prescriptions. Of all antibiotics prescribed, 12.0% were for conditions for which they are never indicated, and 12.3% for conditions for which they are rarely indicated. In children, 25% of antibiotics were for conditions for which they are never indicated (e.g., common cold).

Interpretation:

Antibiotics were prescribed unnecessarily for 15.4% of included encounters in a Canadian primary care setting. Almost one-quarter of antibiotics were prescribed for conditions for which they are rarely or never indicated. These findings should guide safe reductions in the use of antibiotics for the common cold, bronchitis and sinusitis.

Clostridioides difficileDiagnosticsMetabolomicsMicrobiome

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.

The aim of this study was to assess the association of high-sensitivity cardiac troponin (hs-cTnT) and other cardiac, kidney, hyperglycemia, and inflammatory biomarkers with peripheral neuropathy (PN) in a community-based population.We conducted a cross-sectional analysis of 3056 black and white participants in the Atherosclerosis Risk in Communities (ARIC) study who underwent standardized monofilament PN testing and had measures of cardiac function (hs-cTnT, N-terminal pro–B-type natriuretic peptide [NT-proBNP], and growth differentiation factor 15 [GDF15]), kidney function (serum creatinine, cystatin C, β-2 microglobulin, urine albumin-to-creatinine ratio), hyperglycemia (fasting glucose, hemoglobin A_1c [Hb A_1c], fructosamine, glycated albumin, 1,5-anhydroglucitol), and inflammation (C-reactive protein) assessed at visit 6 (2016–2017; age 71–94 years). We used logistic regression to assess the associations of these biomarkers (modeled in diabetes-specific tertiles) with PN in older adults with and without diabetes after adjusting for traditional risk factors.In total, 33.5% of participants had PN (37.3% with diabetes and 31.9% without diabetes). There was an independent association of hs-cTnT with PN regardless of diabetes status (diabetes T3 vs. T1: odds ratio [OR], 2.15 [95% CI, 1.44–3.22]; no diabetes: OR, 2.31 [95%CI, 1.76–3.03]; P = 0.72 for interaction). Among participants without diabetes, there were also significant associations of NT-proBNP (OR, 1.40 [95% CI, 1.08–1.81]) and urine albumin-to-creatinine ratio (OR, 1.55 [95% CI, 1.22–1.97]) with PN. Associations of hyperglycemia biomarkers including Hb A_1c (OR, 1.76 [95% CI, 1.22–2.54]), fructosamine (OR, 1.71 [95% CI, 1.19–2.46]), and glycated albumin (OR, 1.45 [95% CI, 1.03–2.03]) with PN were significant only among participants with diabetes.Overall, hs-cTnT appears to be a global marker of end organ damage, including PN. Laboratory biomarkers may be able to help us identify those individuals with PN.

Peripheral neuropathy (PN) is a condition affecting up to 20% of the general population. The symptoms range from mild to disabling, depending on the types of nerve fiber affected and the type and severity of damage.

Recognizing that guideline-directed histologic grading of endomyocardial biopsy tissue samples for rejection surveillance has limited diagnostic accuracy, quantitative, in situ characterization was performed of several important immune cell types in a retrospective cohort of clinical endomyocardial tissue samples. Differences between cases were identified and were grouped by histologic grade versus clinical rejection trajectory, with significantly increased programmed death ligand 1+, forkhead box P3+, and cluster of differentiation 68+ cells suppressed in clinically evident rejections, especially cases with marked clinical-histologic discordance. Programmed death ligand 1+, forkhead box P3+, and cluster of differentiation 68+ cell proportions are also significantly higher in "never-rejection" when compared with "future-rejection." These findings suggest that in situ immune modulators regulate the severity of cardiac allograft rejection.

Purpose:

Thymidine kinase 1 (TK1) is downstream to the CDK4/6 pathway, and TK activity (TKa) measured in blood is a dynamic marker of outcome in patients with advanced breast cancer (ABC). This study explores TK1 as a biomarker of palbociclib response, both in vitro and in patients with ABC.

Experimental Design:

Modulation of TK1 levels and activity by palbociclib were studied in seven estrogen receptor–positive breast cancer cell lines: sensitive (PDS) and with palbociclib acquired resistance (PDR). TKa was assayed in plasma obtained at baseline (T0), after one cycle (T1), and at disease progression on palbociclib (T2) in patients enrolled in the "To Reverse ENDocrine Resistance" (TREnd) trial (n = 46).

Results:

Among E2F-dependent genes, TK1 was significantly downregulated after short-term palbociclib. Early TKa reduction by palbociclib occurred in PDS but not in PDR cells. In patients, median TKa (mTKa) at T0 was 75 DiviTum units per liter (Du/L), with baseline TKa not proving prognostic. At T1, mTKa decreased to 35 Du/L, with a minority of patients (n = 8) showing an increase—correlating with a worse outcome than those with decreased/stable TKa (n = 33; mPFS 3.0 vs 9.0 months; P = 0.002). At T2, mTKa was 251 Du/L; patients with TKa above the median had worse outcomes on post-study treatment compared with those with lower TKa (2.9 vs 8.7 months; P = 0.05).

Conclusions:

TK is a dynamic marker of resistance to palbociclib which may lead to early identification of patients in whom treatment escalation may be feasible. In addition, TKa may stratify prognosis in patients with acquired resistance to palbociclib.

New drugs or therapeutic combinations are urgently needed against Mycobacterium abscessus. Previously, we demonstrated the potent activity of indole-2-carboxamides 6 and 12 against M. abscessus. We show here that these compounds act synergistically with imipenem and cefoxitin in vitro and increase the bactericidal activity of the β-lactams against M. abscessus. In addition, compound 12 also displays synergism with imipenem and cefoxitin within infected macrophages. The clinical potential of these new drug combinations requires further evaluation.

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.

Antibiotics revolutionized the treatment of infectious diseases; however, it is now clear that broad-spectrum antibiotics alter the composition and function of the host’s microbiome. The microbiome plays a key role in human health, and its perturbation is increasingly recognized as contributing to many human diseases. Widespread broad-spectrum antibiotic use has also resulted in the emergence of multidrug-resistant pathogens, spurring the development of pathogen-specific strategies such as monoclonal antibodies (MAbs) to combat bacterial infection. Not only are pathogen-specific approaches not expected to induce resistance in nontargeted bacteria, but they are hypothesized to have minimal impact on the gut microbiome. Here, we compare the effects of antibiotics, pathogen-specific MAbs, and their controls (saline or control IgG [c-IgG]) on the gut microbiome of 7-week-old, female, C57BL/6 mice. The magnitude of change in taxonomic abundance, bacterial diversity, and bacterial metabolites, including short-chain fatty acids (SCFA) and bile acids in the fecal pellets from mice treated with pathogen-specific MAbs, was no different from that with animals treated with saline or an IgG control. Conversely, dramatic changes were observed in the relative abundance, as well as alpha and beta diversity, of the fecal microbiome and bacterial metabolites in the feces of all antibiotic-treated mice. Taken together, these results indicate that pathogen-specific MAbs do not alter the fecal microbiome like broad-spectrum antibiotics and may represent a safer, more-targeted approach to antibacterial therapy.

Advances in synthetic biology have enabled the production of a variety of compounds using bacteria as a vehicle for complex compound biosynthesis. Violacein, a naturally occurring indole pigment with antibiotic properties, can be biosynthetically engineered in Escherichia coli expressing its nonnative synthesis pathway. To explore whether this synthetic biosynthesis platform could be used for drug discovery, here we have screened bacterially derived violacein against the main causative agent of human malaria, Plasmodium falciparum. We show the antiparasitic activity of bacterially derived violacein against the P. falciparum 3D7 laboratory reference strain as well as drug-sensitive and -resistant patient isolates, confirming the potential utility of this drug as an antimalarial agent. We then screen a biosynthetic series of violacein derivatives against P. falciparum growth. The varied activity of each derivative against asexual parasite growth points to the need to further develop violacein as an antimalarial. Towards defining its mode of action, we show that biosynthetic violacein affects the parasite actin cytoskeleton, resulting in an accumulation of actin signal that is independent of actin polymerization. This activity points to a target that modulates actin behavior in the cell either in terms of its regulation or its folding. More broadly, our data show that bacterial synthetic biosynthesis could become a suitable platform for antimalarial drug discovery, with potential applications in future high-throughput drug screening with otherwise chemically intractable natural products.

Enterococcus faecium strains are commonly resistant to vancomycin and β-lactams. In addition, E. faecium often causes biofilm-associated infections and these infections are difficult to treat. In this context, we investigated the activity of dosing regimens using daptomycin (DAP) (8, 10, 12, and 14 mg/kg of body weight/day) alone and in combination with ceftaroline (CPT), ampicillin (AMP), ertapenem (ERT), and rifampin (RIF) against 2 clinical strains of biofilm-producing vancomycin-resistant Enterococcus faecium (VREfm), namely, strains S447 and HOU503, in an in vitro biofilm model. HOU503 harbors common LiaS and LiaR substitutions, whereas S447 lacks mutations associated with the LiaFSR pathway. MIC results demonstrated that both strains were susceptible to DAP and resistant to CPT, AMP, ERT, and RIF. The 168-h pharmacokinetic/pharmacodynamic (PK/PD) CDC biofilm reactor models (simulating human antibiotic exposures) were used with titanium and polyurethane coupons to evaluate the efficacy of antibiotic combinations. DAP 12 and 14 achieved bactericidal activity against S447 but lacked such effect against HOU503. Addition of ERT and RIF enhanced DAP activity, allowing DAP 8 and 10 plus ERT or RIF to produce bactericidal activity against both strains at 168 h. While DAP 8 and 10 plus CPT improved killing, they did not reach bactericidal reduction against S447. Combination of AMP, CPT, ERT, or RIF resulted in enhanced and bactericidal activity for DAP against HOU503 at 168 h. Our data provide further support for the use of combinations of DAP with AMP, ERT, CPT, and RIF in infections caused by biofilm producing VREfm. Further research involving DAP combinations against biofilm-producing enterococci is warranted.

Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a nondividing, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against nondividing Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and division proteins, confirming its potential as a treatment for S. aureus biofilm infections.

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.

The pks⁺ E. coli metabolite colibactin caused a unique mutational signature in intestinal organoids.

A large international study found that the composition of the intestinal microbiome can predict clinical outcomes in patients undergoing allogenic hematopoietic-cell transplant (HCT) for blood cancers. The findings may help assess patients' transplantation-related mortality risk and aid in developing interventions to prevent or mitigate microbiome changes that affect HCT outcomes.

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