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.

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.

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.

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:

The negative effect of adverse childhood experiences (ACEs) on physical and mental health has led to calls for routine screening for ACEs in primary care settings. We aimed to examine the association between maternal ACEs and children’s behaviour problems (externalizing and internalizing) at age 5 in the context of other known predictors.

Methods:

We analyzed data from mother-and-child dyads participating in the All Our Families cohort in Calgary, Canada, between 2011 and 2017. Data were collected for factors related to the individual child (sex, age, temperament and behaviour), the mother (adverse childhood experiences, mental health, personality and parenting) and sociodemographic characteristics (family income, ethnicity and family structure) when the children were 3 and 5 years of age. We used logistic regression models to estimate crude and adjusted associations between maternal ACEs and children’s externalizing (hyperactivity and aggression) and internalizing (anxiety, depression and somatization) behaviours.

Results:

Data were available for 1688 mother-and-child dyads. In the crude models, the presence of 4 or more maternal ACEs was associated with children’s externalizing and internalizing behaviours at age 5. However, these associations were attenuated with adjustment. Persistent maternal mental health symptoms were associated with both externalizing and internalizing behaviours at age 5 (adjusted odds ratio [OR] 4.20, 95% confidence interval [CI] 2.50–7.05, and adjusted OR 2.52, 95% CI 1.66–3.81, respectively). High levels of ineffective parenting behaviours were also associated with both externalizing and internalizing behaviours at age 5 (adjusted OR 6.27, 95% CI 4.30–9.14, and adjusted OR 1.43, 95% CI 1.03–1.99, respectively).

Interpretation:

The association between maternal ACEs and children’s behaviour at age 5 was weakened in the presence of other maternal and family-level factors. Assessments of maternal mental health and parenting behaviours may be better targets for identifying children at risk of behavioural problems.

Background:

Incentive payments for chronic diseases in British Columbia were intended to support primary care physicians in providing more comprehensive care, but research shows that not all physicians bill incentives and not all eligible patients have them billed on their behalf. We investigated patient and physician characteristics associated with billing incentives for chronic diseases in BC.

Methods:

We conducted a retrospective cohort analysis using linked administrative health data to examine community-based primary care physicians and patients with eligible chronic conditions in BC during 2010–2013. Descriptive analyses of patients and physicians compared 3 groups: no incentives in any of the 4 years, incentives in all 4 years, and incentives in any of the study years. We used hierarchical logistic regression models to identify the patient- and physician-level characteristics associated with billing incentives.

Results:

Of 428 770 eligible patients, 142 475 (33.2%) had an incentive billed on their behalf in all 4 years, and 152 686 (35.6%) never did. Of 3936 physicians, 2625 (66.7%) billed at least 1 incentive in each of the 4 years, and 740 (18.8%) billed no incentives during the study period. The strongest predictors of having an incentive billed were the number of physician contacts a patient had (odds ratio [OR] for > 48 contacts 134.77, 95% confidence interval [CI] 112.27–161.78) and whether a physician had a large number of patients in his or her practice for whom incentives were billed (OR 42.38 [95% CI 34.55–52.00] for quartile 4 v. quartile 1).

Interpretation:

The findings suggest that primary care physicians bill incentives for patients based on whom they see most often rather than using a population health management approach to their practice.

Background:

Drugs are the fastest growing cost in the Canadian health care system, owing to the increasing number of high-cost drugs. The objective of this study was to examine the characteristics of high–drug-cost beneficiaries of public drug plans across Canada relative to other beneficiaries.

Methods:

We conducted a cross-sectional study among public drug plan beneficiaries residing in all provinces except Quebec. We used the Canadian Institute for Health Information’s National Prescription Drug Utilization Information System to identify all drugs dispensed to beneficiaries of public drug programs in 2016/17. We stratified the cohort into 2 groups: high–drug-cost beneficiaries (top 5% of beneficiaries based on annual costs) and other beneficiaries (remaining 95%). For each group, we reported total drug costs, prevalence of high-cost claims (> $1000), median number of drugs, proportion of beneficiaries aged 65 or more, the 10 most costly reimbursed medications and the 10 medications most commonly reimbursed. We reported estimates overall and by province.

Results:

High–drug-cost beneficiaries accounted for nearly half (46.5%) of annual spending, with an average annual spend of $14 610 per beneficiary, compared to $1570 among other beneficiaries. The median number of drugs dispensed was higher among high–drug-cost beneficiaries than among other beneficiaries (13 [interquartile range (IQR) 7–19] v. 8 [IQR 4–13]), and a much larger proportion of high–drug-cost beneficiaries than other beneficiaries received at least 1 high-cost claim (40.9% v. 0.6%). Long-term medications were the most commonly used medications for both groups, whereas biologics and antivirals were the most costly medications for high–drug-cost beneficiaries.

Interpretation:

High–drug-cost beneficiaries were characterized by the use of expensive medications and polypharmacy relative to other beneficiaries. Interventions and policies to help reduce spending need to consider both of these factors.

Background:

Patients with multimorbidity often require services across different health care settings, yet team processes among settings are rarely implemented. We explored perceptions of specialists and family physicians collaborating in a telemedicine interprofessional consultation for patients with multimorbidity to better understand the value of bringing physicians together across the boundaries of health care settings.

Methods:

This was a descriptive qualitative, interview-based study. Physicians who had previously participated in the Telemedicine Interprofessional Model of Practice for Aging and Complex Treatments (Telemedicine IMPACT Plus [TIP] Program) were invited to participate and asked to describe their experience of being a member of the program. Interviews were conducted from March to May 2016. We conducted an iterative and interpretive process using both individual and team analysis to identify themes.

Results:

There were 15 participants, 9 specialists and 6 family physicians. Three themes emerged in the analysis: creating new perspectives on care for patients with multimorbidity by sharing knowledge, skills and attitudes; the shift from a consultant model to an interprofessional team model (allowing a window into the community, extending discussions beyond the medical model and focusing on the patient’s health in context); and opportunities for learners, including learning about interprofessional collaboration and gaining exposure to a real-world model for caring for people with multimorbidity in outpatient settings.

Interpretation:

Family physicians and specialists participating in a TIP Program believed the program improved their knowledge and skills, while also serving as an effective care delivery strategy. The findings also support that learners require more exposure to nontraditional consultant models in order to care for patients with multimorbidity effectively.

Recent advances in the study and clinical applications of circulating tumor DNA (ctDNA) are limited by practical considerations of sample collection. Whole-genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations and fragmentation patterns. We hypothesized that low-depth/shallow WGS (sWGS) data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may remove contaminating genomic DNA from small blood volumes. Dried blood spots have practical advantages for sample collection, may facilitate serial sampling, and could support novel study designs in humans and animal models.We developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots using filter paper cards and bead-based size selection. DNA extracted and size-selected from dried spots was analyzed using sWGS and polymerase chain reaction (PCR).Analyzing a 50 μL dried blood spot from frozen whole blood of a patient with melanoma, we identified ctDNA based on the presence of tumor-specific somatic copy-number alterations, and found a fragment-size profile similar to that observed in plasma DNA. We found alterations in different chromosomes in blood spots from 2 patients with high-grade serous ovarian carcinoma. Extending this approach to serial dried blood spots from mouse xenograft models, we detect tumor-derived cell-free DNA and identified ctDNA from the originally grafted ascites.Our data suggest that ctDNA can be detected and monitored in dried blood spots from archived and fresh blood samples, enabling new approaches for sample collection and novel study/trial designs for both patients and in vivo models.

S100A8/A9 is implicated in inflammation mechanisms related to atherosclerosis and plaque vulnerability, but it remains unclear whether S100A8/A9 is associated with the prognosis of ischemic stroke. The aim of this study was to investigate these associations in 2 independent multicenter cohorts.Plasma S100A8/A9 concentrations at baseline were measured among 4785 patients with ischemic stroke from 2 independent cohorts: Infectious Factors, Inflammatory Markers, and Prognosis of Acute Ischemic Stroke (IIPAIS) and China Antihypertensive Trial in Acute Ischemic Stroke (CATIS). The primary outcome was a composite outcome of death or major disability at 3 months after ischemic stroke. Secondary outcomes were major disability, death, and a composite outcome of death or vascular events.Among the combined participants of IIPAIS and CATIS, the adjusted odds ratios associated with the highest quartile of plasma S100A8/A9 were 2.11 (95% CI, 1.66–2.68) for the primary outcome and 1.62 (95% CI, 1.27–2.07) for the secondary outcome of major disability; adjusted hazard ratios were 4.14 (95% CI, 2.10–8.15) for the secondary outcome of death and 2.08 (95% CI, 1.38–3.13) for the composite outcome of death or vascular events. Each SD increase of log-transformed S100A8/A9 was associated with 28% (95% CI, 18%–39%; P < 0.001) increased risk of the primary outcome. Multivariable-adjusted spline regression analyses showed a linear association between plasma S100A8/A9 concentrations and primary outcome (P < 0.001 for linearity). Subgroup analyses further confirmed these associations.High plasma S100A8/A9 concentrations at baseline were independently associated with increased risks of adverse clinical outcomes at 3 months after ischemic stroke, suggesting that S100A8/A9 might have a role as a prognostic marker of ischemic stroke.

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.

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Dramatically rising costs in drug development are in large part because of the high failure rates in clinical phase trials. The poor correlation of animal studies to human toxicity and efficacy have led many developers to question the value of requiring animal studies in determining which drugs should enter in-human trials. Part 1 of this 2-part series examined some of the data regarding the lack of concordance between animal toxicity studies and human trials, as well as some of the potential reasons behind it. This second part of the series focuses on some alternatives to animal trials (hereafter referred to as animal research) as well as current regulatory discussions and developments regarding such alternatives.

BACKGROUND:

Prescribing of levothyroxine and rates of thyroid function testing may be sensitive to minor changes in the upper limit of the reference range for thyroid-stimulating hormone (TSH) that increase the proportion of abnormal results. We evaluated the population-level change in levothyroxine prescribing and TSH testing after a minor planned decrease in the upper limit of the reference range for TSH in a large urban centre with a single medical laboratory.

METHODS:

Using provincial administrative data, we compared predicted volumes of TSH tests with actual TSH test volumes before and after a planned change in the TSH reference range. We also determined the number of new levothyroxine prescriptions for previously untreated patients and the rate of changes to the prescribed dose for those on previously stable, long-term levothyroxine therapy before and after the change in the TSH reference range.

RESULTS:

Before the change in the TSH reference range, actual and predicted monthly volumes of TSH testing followed an identical course. After the change, actual test volumes exceeded predicted test volumes by 7.3% (95% confidence interval [CI] 5.3%–9.3%) or about 3000 to 5000 extra tests per month. The proportion of patients with newly "abnormal" TSH results almost tripled, from 3.3% (95% CI 3.2%–3.4%) to 9.1% (95% CI 9.0%–9.2%). The rate of new levothyroxine prescriptions increased from 3.24 (95% CI 3.15–3.33) per 1000 population in 2013 to 4.06 (95% CI 3.96–4.15) per 1000 population in 2014. Among patients with preexisting stable levothyroxine therapy, there was a significant increase in the number of dose escalations (p < 0.001) and a total increase of 500 new prescriptions per month.

INTERPRETATION:

Our findings suggest that clinicians may have responded to mildly elevated TSH results with new or increased levothyroxine prescriptions and more TSH testing. Knowledge translation efforts may be useful to accompany minor changes in reference ranges.

Caseum, the central necrotic material of tuberculous lesions, is a reservoir of drug-recalcitrant persisting mycobacteria. Caseum is found in closed nodules and in open cavities connecting with an airway. Several commonly accepted characteristics of caseum were established during the preantibiotic era, when autopsies of deceased tuberculosis (TB) patients were common but methodologies were limited. These pioneering studies generated concepts such as acidic pH, low oxygen tension, and paucity of nutrients being the drivers of nonreplication and persistence in caseum. Here we review widely accepted beliefs about the caseum-specific stress factors thought to trigger the shift of Mycobacterium tuberculosis to drug tolerance. Our current state of knowledge reveals that M. tuberculosis is faced with a lipid-rich diet rather than nutrient deprivation in caseum. Variable caseum pH is seen across lesions, possibly transiently acidic in young lesions but overall near neutral in most mature lesions. Oxygen tension is low in the avascular caseum of closed nodules and high at the cavity surface, and a gradient of decreasing oxygen tension likely forms toward the cavity wall. Since caseum is largely made of infected and necrotized macrophages filled with lipid droplets, the microenvironmental conditions encountered by M. tuberculosis in foamy macrophages and in caseum bear many similarities. While there remain a few knowledge gaps, these findings constitute a solid starting point to develop high-throughput drug discovery assays that combine the right balance of oxygen tension, pH, lipid abundance, and lipid species to model the profound drug tolerance of M. tuberculosis in caseum.

Burkholderia cepacia (formerly Pseudomonas cepacia) was once thought to be a single bacterial species but has expanded to the Burkholderia cepacia complex (Bcc), comprising 24 closely related opportunistic pathogenic species. These bacteria have a widespread environmental distribution, an extraordinary metabolic versatility, a complex genome with three chromosomes, and a high capacity for rapid mutation and adaptation. Additionally, they present an inherent resistance to antibiotics and antiseptics, as well as the abilities to survive under nutrient-limited conditions and to metabolize the organic matter present in oligotrophic aquatic environments, even using certain antimicrobials as carbon sources. These traits constitute the reason that Bcc bacteria are considered feared contaminants of aqueous pharmaceutical and personal care products and the frequent reason behind nonsterile product recalls. Contamination with Bcc has caused numerous nosocomial outbreaks in health care facilities, presenting a health threat, particularly for patients with cystic fibrosis and chronic granulomatous disease and for immunocompromised individuals. This review addresses the role of Bcc bacteria as a potential public health problem, the mechanisms behind their success as contaminants of pharmaceutical products, particularly in the presence of biocides, the difficulties encountered in their detection, and the preventive measures applied during manufacturing processes to control contamination with these objectionable microorganisms. A summary of Bcc-related outbreaks in different clinical settings, due to contamination of diverse types of pharmaceutical products, is provided.

Intrathecal administration of anti-infectives is indicated in central nervous system infections by multiresistant pathogens when drugs that can reach adequate cerebrospinal fluid (CSF) concentrations by systemic therapy are not available. Antibiotics that readily pass the blood-brain and blood-CSF barriers and/or that have low toxicity allowing an increase in the daily dosage should not be used for intrathecal therapy. Intrathecal therapy is accompanied by systemic treatment. Antibacterials indispensable for intrathecal therapy include aminoglycosides, colistin, daptomycin, tigecycline, and vancomycin. Limited experience suggests the utility of the antifungals amphotericin B and caspofungin. Intraventricular administration ensures distribution throughout the CSF compartment, whereas intralumbar dosing often fails to attain adequate antibiotic concentrations in the ventricles. The individual dose is determined by the estimated size of the CSF space and by the estimated clearance from CSF. For moderately lipophilic anti-infectives with a molecular weight above approximately 1,000 g/mol, as well as for hydrophilic drugs with a molecular weight above approximately 400 g/mol, one daily dose is normally adequate. The ventricular drain should be clamped for 15 to 120 min to facilitate the distribution of the anti-infective in the CSF space. Therapeutic drug monitoring of the trough levels is necessary only in cases of therapeutic failure.

Purpose:

Unleashing the immune system by PD-1 and/or CTLA-4 blockade can cause severe immune-related toxicity necessitating immunosuppressive treatment. Whether immunosuppression for toxicity impacts survival is largely unknown.

Experimental Design:

Using data from the prospective nationwide Dutch Melanoma Treatment Registry (DMTR), we analyzed the association between severe toxicity and overall survival (OS) in 1,250 patients with advanced melanoma who were treated with immune checkpoint inhibitors (ICI) in first line between 2012 and 2017. Furthermore, we analyzed whether toxicity management affected survival in these patients.

Results:

A total of 1,250 patients were included, of whom 589 received anti-PD1 monotherapy, 576 ipilimumab, and 85 combination therapy. A total of 312 patients (25%) developed severe (grade ≥3) toxicity. Patients experiencing severe ICI toxicity had a significantly prolonged survival with a median OS of 23 months compared with 15 months for patients without severe toxicity [hazard ratio (HR_adj) = 0.77; 95% confidence interval (CI), 0.63–0.93]. Among patients experiencing severe toxicity, survival was significantly decreased in patients who received anti-TNF ± steroids for steroid-refractory toxicity compared with patients who were managed with steroids only (HR_adj = 1.61; 95% CI, 1.03–2.51), with a median OS of 17 and 27 months, respectively.

Conclusions:

Patients experiencing severe ICI toxicity have a prolonged OS. However, this survival advantage is abrogated when anti-TNF is administered for steroid-refractory toxicity. Further prospective studies are needed to assess the effect of different immunosuppressive regimens on checkpoint inhibitor efficacy.

See related commentary by Weber and Postow, p. 2085

Purpose:

Preclinical data demonstrating androgen receptor (AR)–positive (AR⁺) triple-negative breast cancer (TNBC) cells are sensitive to AR antagonists, and PI3K inhibition catalyzed an investigator-initiated, multi-institutional phase Ib/II study TBCRC032. The trial investigated the safety and efficacy of the AR-antagonist enzalutamide alone or in combination with the PI3K inhibitor taselisib in patients with metastatic AR⁺ (≥10%) breast cancer.

Patients and Methods:

Phase Ib patients [estrogen receptor positive (ER⁺) or TNBC] with AR⁺ breast cancer received 160 mg enzalutamide in combination with taselisib to determine dose-limiting toxicities and the maximum tolerated dose (MTD). Phase II TNBC patients were randomized to receive either enzalutamide alone or in combination with 4 mg taselisib until disease progression. Primary endpoint was clinical benefit rate (CBR) at 16 weeks.

Results:

The combination was tolerated, and the MTD was not reached. The adverse events were hyperglycemia and skin rash. Overall, CBR for evaluable patients receiving the combination was 35.7%, and median progression-free survival (PFS) was 3.4 months. Luminal AR (LAR) TNBC subtype patients trended toward better response compared with non-LAR (75.0% vs. 12.5%, P = 0.06), and increased PFS (4.6 vs. 2.0 months, P = 0.082). Genomic analyses revealed subtype-specific treatment response, and novel FGFR2 fusions and AR splice variants.

Conclusions:

The combination of enzalutamide and taselisib increased CBR in TNBC patients with AR⁺ tumors. Correlative analyses suggest AR protein expression alone is insufficient for identifying patients with AR-dependent tumors and knowledge of tumor LAR subtype and AR splice variants may identify patients more or less likely to benefit from AR antagonists.

A strain of extensively drug-resistant (XDR) Salmonella enterica serovar Typhi has caused a large ongoing outbreak in Pakistan since 2016. In Ontario, Canada, 10 cases of mainly bloodstream infections (n = 9) were identified in patients who traveled to Pakistan. Whole-genome sequencing showed that Canadian cases were genetically related to the Pakistan outbreak strain. The appearance of XDR typhoid cases in Ontario prompted a provincial wide alert to physicians to recommend treatment with carbapenems or azithromycin in suspected typhoid cases with travel history to Pakistan.

In this retrospective study, whole-genome sequencing (WGS) data generated on an Ion Torrent platform was used to predict phenotypic drug resistance profiles for first- and second-line drugs among Swedish clinical Mycobacterium tuberculosis isolates from 2016 to 2018. The accuracy was ~99% for all first-line drugs and 100% for four second-line drugs. Our analysis supports the introduction of WGS into routine diagnostics, which might, at least in Sweden, replace phenotypic drug susceptibility testing in the future.

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.

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.

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.

Plazomicin was active against 97.0% of 8,783 Enterobacterales isolates collected in the United States (2016 and 2017), and only 6 isolates carried 16S rRNA methyltransferases conferring resistance to virtually all aminoglycosides. Plazomicin (89.2% to 95.9% susceptible) displayed greater activity than amikacin (72.5% to 78.6%), gentamicin (30.4% to 45.9%), and tobramycin (7.8% to 22.4%) against carbapenem-resistant and extensively drug-resistant isolates. The discrepancies among the susceptibility rates for these agents was greater when applying breakpoints generated using the same stringent contemporary methods applied to determine plazomicin breakpoints.

Treating malaria in HIV-coinfected individuals should consider potential drug-drug interactions. Artemether-lumefantrine is the most widely recommended treatment for uncomplicated malaria globally. Lumefantrine is metabolized by CYP3A4, an enzyme that commonly used antiretrovirals often induce or inhibit. A population pharmacokinetic meta-analysis was conducted using individual participant data from 10 studies with 6,100 lumefantrine concentrations from 793 nonpregnant adult participants (41% HIV-malaria-coinfected, 36% malaria-infected, 20% HIV-infected, and 3% healthy volunteers). Lumefantrine exposure increased 3.4-fold with coadministration of lopinavir-ritonavir-based antiretroviral therapy (ART), while it decreased by 47% with efavirenz-based ART and by 59% in the patients with rifampin-based antituberculosis treatment. Nevirapine- or dolutegravir-based ART and malaria or HIV infection were not associated with significant effects. Monte Carlo simulations showed that those on concomitant efavirenz or rifampin have 49% and 80% probability of day 7 concentrations <200 ng/ml, respectively, a threshold associated with an increased risk of treatment failure. The risk of achieving subtherapeutic concentrations increases with larger body weight. An extended 5-day and 6-day artemether-lumefantrine regimen is predicted to overcome these drug-drug interactions with efavirenz and rifampin, respectively.

Bacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance toward antibiotics. Persisters have been linked to the recalcitrance of biofilm-related infections; hence, a complete understanding of their physiology can lead to improvement of therapeutic strategies for such infections. Mechanisms pertaining to persister formation are thought to be associated with stress response pathways triggered by intra- or extracellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH-induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, we studied the effects of various osmolytes and pH conditions on Escherichia coli persistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite, and acidic pH, significantly reduced persister formation in E. coli compared to no-osmolyte/no-buffer controls, this reduction in persister levels was less pronounced in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

KBP-7072 is a novel third-generation tetracycline (aminomethylcycline) antibacterial that overcomes common efflux and ribosomal protection resistance mechanisms that cause resistance in older-generation tetracyclines. KBP-7072 completed phase 1 clinical development studies for safety, tolerability, and pharmacokinetics (ClinicalTrials.gov identifier NCT02454361) and multiple ascending doses in healthy subjects (ClinicalTrials.gov identifier NCT02654626) in December 2015. Both oral and intravenous formulations of KBP-7072 are being developed. In this study, we evaluated the in vitro activities of KBP-7072 and comparator agents by CLSI document M07 (2018) broth microdilution against 531 recent geographically diverse and/or molecularly characterized Acinetobacter baumannii-A. calcoaceticus species complex (A. baumannii) isolates from the United States, Europe, Asia-Pacific (excluding China), and Latin America. A. baumannii isolates included carbapenem-resistant, colistin-resistant, tetracycline-resistant, and extended-spectrum-β-lactamase (ESBL)- and metallo-β-lactamase (MBL)-producing isolates. Overall, KBP-7072 (MIC_50/90, 0.25/1 mg/liter) was comparable in activity to colistin (92.8%/92.8% susceptible [S] [CLSI/EUCAST]) against A. baumannii isolates, inhibiting 99.2% of isolates at ≤2 mg/liter and 97.6% of isolates at ≤1 mg/liter. KBP-7072 was equally active against A. baumannii isolates, including carbapenem-resistant, colistin-resistant, and tetracycline-resistant isolates, regardless of geographic location, and maintained activity against ESBL- and MBL-producing isolates. KBP-7072 outperformed comparator agents, including ceftazidime (40.3% S [CLSI]), gentamicin (48.2%/48.2% S [CLSI/EUCAST]), levofloxacin (39.5%/37.9% S [CLSI/EUCAST]), meropenem (42.0%/42.0% S [CLSI/EUCAST]), piperacillin-tazobactam (33.3% S [CLSI]), and all tetracycline-class comparator agents, which include doxycycline (67.3% S [CLSI]), minocycline (73.8% S [CLSI]), tetracycline (37.2% S [CLSI]), and tigecycline (79.5% inhibited by ≤2 mg/liter). The potent in vitro activity of KBP-7072 against recent geographically diverse, molecularly characterized, and drug-resistant A. baumannii isolates supports continued clinical development for the treatment of serious infections, including those caused by A. baumannii.

Infections with nontuberculous mycobacteria (NTM) have a poor prognosis in patients with underlying respiratory diseases. Clofazimine (CFZ) showed both experimental and clinical promising results against clinically relevant NTM. However, there are no data on CFZ in combination with the current recommended treatment; therefore, we aimed to study its in vivo activity in an aerosol mouse model of Mycobacterium avium. In an aerosol infection BALB/c mouse model using M. avium strain Chester, we treated 58 mice with four combinations of rifampin (RIF) at 10 mg/kg, CFZ at 25 mg/kg, and clarithromycin (CLR) and ethambutol (EMB) at 100 mg/kg. Treatment efficacy was assessed on the basis of lung CFU counts after 2 (M2) and 4 (M4) months of treatment. At M2, CLR-RIF-EMB was slightly but significantly more efficient than CFZ-RIF-EMB (3.02 ± 0.12 versus 3.55 ± 0.28, respectively, P < 0.01), whereas CLR-CFZ-EMB and CLR-CFZ-RIF-EMB dramatically decreased lung CFU counts by 4.32 and 4.47 log₁₀, respectively, compared to untreated group. At M4, CLR-RIF-EMB was significantly more efficient than CFZ-RIF-EMB (2 ± 0.53 versus 2.66 ± 0.22, respectively, P = 0.01). The addition of CLZ to CLR dramatically decreased the lung CFU count, with CFU counts 5.41 and 5.79 log₁₀ lower in the CLR-CFZ-EMB and CLR-CFZ-RIF-EMB groups, respectively, than in the untreated group. The addition of CFZ to CLR seems to improve the efficacy of CLR as early as M2 and was confirmed at M4. CFZ, in addition to RIF and EMB, on the other hand, is less effective than CLR-RIF-EMB. These results need to be confirmed by similar studies along with CFZ potential for shortening treatment.

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.

The comparative efficacy of ceftazidime-avibactam and meropenem-vaborbactam for treatment of carbapenem-resistant Enterobacteriaceae (CRE) infections remains unknown. This was a multicenter, retrospective cohort study of adults with CRE infections who received ceftazidime-avibactam or meropenem-vaborbactam for ≥72 hours from February 2015 to October 2018. Patients with a localized urinary tract infection and repeat study drug exposures after the first episode were excluded. The primary endpoint was clinical success compared between treatment groups. Secondary endpoints included 30- and 90-day mortality, adverse events (AE), 90-day CRE infection recurrence, and development of resistance in patients with recurrent infection. A post hoc subgroup analysis was completed comparing patients who received ceftazidime-avibactam monotherapy, ceftazidime-avibactam combination therapy, and meropenem-vaborbactam monotherapy. A total of 131 patients were included (ceftazidime-avibactam, n = 105; meropenem-vaborbactam, n = 26), 40% of whom had bacteremia. No significant difference in clinical success was observed between groups (62% versus 69%; P = 0.49). Patients in the ceftazidime-avibactam arm received combination therapy more often than patients in the meropenem-vaborbactam arm (61% versus 15%; P < 0.01). No difference in 30- and 90-day mortality resulted, and rates of AE were similar between groups. In patients with recurrent infection, development of resistance occurred in three patients that received ceftazidime-avibactam monotherapy and in no patients in the meropenem-vaborbactam arm. Clinical success was similar between patients receiving ceftazidime-avibactam and meropenem-vaborbactam for treatment of CRE infections, despite ceftazidime-avibactam being used more often as a combination therapy. Development of resistance was more common with ceftazidime-avibactam monotherapy.

Tedizolid (TZD) and daptomycin (DAP) were assessed in a rat endocarditis model against Enterococcus faecalis, Enterococcus faecium (resistant to vancomycin and ampicillin), and Staphylococcus aureus. As a monotherapy, TZD for 5 days was not effective in a comparison with no-treatment controls, while DAP for 5 days was significantly effective against these bacteria. Step-down therapy (DAP for 3 days followed by TZD for 2 days) was as effective as DAP for 5 days and was comparable to 3 days of DAP plus ceftriaxone against all bacteria and to 3 days of DAP plus gentamicin against E. faecalis OG1RF.

Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 μg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus. Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

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.

Animal models of bacterial infection have been widely used to explore the in vivo activity of antibacterial drugs. These data are often submitted to the U.S. Food and Drug Administration to support human use in an investigational new drug application (IND). To better understand the range and scientific use of animal models in regulatory submissions, a database was created surveying recent pneumonia models submitted as part of IND application packages. The IND studies were compared to animal models of bacterial pneumonia published in the scientific literature over the same period of time. In this review, we analyze the key experimental design elements, such as animal species, immune status, pathogens selected, and route of administration, and study endpoints.

Nacubactam is a novel β-lactamase inhibitor with dual mechanisms of action as an inhibitor of serine β-lactamases (classes A and C and some class D) and an inhibitor of penicillin binding protein 2 in Enterobacteriaceae. The safety, tolerability, and pharmacokinetics of intravenous nacubactam were evaluated in single- and multiple-ascending-dose, placebo-controlled studies. Healthy participants received single ascending doses of nacubactam of 50 to 8,000 mg, multiple ascending doses of nacubactam of 1,000 to 4,000 mg every 8 h (q8h) for up to 7 days, or nacubactam of 2,000 mg plus meropenem of 2,000 mg q8h for 6 days after a 3-day lead-in period. Nacubactam was generally well tolerated, with the most frequently reported adverse events (AEs) being mild to moderate complications associated with intravenous access and headache. There was no apparent relationship between drug dose and the pattern, incidence, or severity of AEs. No clinically relevant dose-related trends were observed in laboratory safety test results. No serious AEs, dose-limiting AEs, or deaths were reported. After single or multiple doses, nacubactam pharmacokinetics appeared linear, and exposure increased in an approximately dose-proportional manner across the dose range investigated. Nacubactam was excreted largely unchanged into urine. Coadministration of nacubactam with meropenem did not significantly alter the pharmacokinetics of either drug. These findings support the continued clinical development of nacubactam and demonstrate the suitability of meropenem as a potential β-lactam partner for nacubactam. (The studies described in this paper have been registered at ClinicalTrials.gov under NCT02134834 [single ascending dose study] and NCT02972255 [multiple ascending dose study].)