Clinical and in vitro models identify distinct adaptations enhancing Staphylococcus aureus pathogenesis in human macrophages.

Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.

Contribution of the patient microbiome to surgical site infection and antibiotic prophylaxis failure in spine surgery.

Despite modern antiseptic techniques, surgical site infection (SSI) remains a leading complication of surgery. However, the origins of SSI and the high rates of antimicrobial resistance observed in these infections are poorly understood. Using instrumented spine surgery as a model of clean (class I) skin incision, we prospectively sampled preoperative microbiomes and postoperative SSI isolates in a cohort of 204 patients. Combining multiple forms of genomic analysis, we correlated the identity, anatomic distribution, and antimicrobial resistance profiles of SSI pathogens with those of preoperative strains obtained from the patient skin microbiome. We found that 86% of SSIs, comprising a broad range of bacterial species, originated endogenously from preoperative strains, with no evidence of common source infection among a superset of 1610 patients. Most SSI isolates (59%) were resistant to the prophylactic antibiotic administered during surgery, and their resistance phenotypes correlated with the patient's preoperative resistome (P = 0.0002). These findings indicate the need for SSI prevention strategies tailored to the preoperative microbiome and resistome present in individual patients.

Motor unit firing rates increase in prepubescent youth following linear periodization resistance exercise training.

PURPOSE: The purpose was to examine the effects of 8-weeks (3 days/week) of linear periodization resistance exercise training (RET) on neuromuscular function in prepubescent youth. METHODS: Twenty-five healthy prepubescent youth (11 males, 14 females, age = 9.1 ± 0.8 years) completed the RET (n = 17) or served as controls (CON, n = 8). Isometric maximal voluntary contractions (MVCs) and trapezoidal submaximal contractions at 35 and 60% MVC of the right leg extensors were performed with surface electromyography (EMG) recorded from the leg extensors [vastus lateralis (VL), rectus femoris, and vastus medialis] and flexors (biceps femoris and semitendinosus). EMG amplitude of the leg extensors and flexors were calculated during the MVCs. Motor unit (MU) action potential trains were decomposed from the surface EMG of the VL for the 35 and 60% MVCs. MU firing rates and action potential amplitudes were regressed against recruitment threshold with the y-intercepts and slopes calculated for each contraction. Total leg extensor muscle cross-sectional area (CSA) was collected using ultrasound images. ANOVA models were used to examine potential differences. RESULTS: Isometric strength increased post-RET (P = 0.006) with no changes in leg extensor and flexor EMG amplitude. Furthermore, there were no changes in total CSA or the MU action potential amplitude vs. recruitment threshold relationships. However, there were increases in the firing rates of the higher-threshold MUs post-RET as indicated with greater y-intercepts (P = 0.003) from the 60% MVC and less negative slope (P = 0.004) of the firing rates vs. recruitment threshold relationships at 35% MVC. CONCLUSIONS: MU adaptations contribute to strength increases following RET in prepubescent youth.

In Vitro Selection Identifies Staphylococcus aureus Genes Influencing Biofilm Formation.

Staphylococcus aureus generates biofilms during many chronic human infections, which contributes to its growth and persistence in the host. Multiple genes and pathways necessary for S. aureus biofilm production have been identified, but knowledge is incomplete, and little is known about spontaneous mutations that increase biofilm formation as infection progresses. Here, we performed in vitro selection of four S. aureus laboratory strains (ATCC 29213, JE2, N315, and Newman) to identify mutations associated with enhanced biofilm production. Biofilm formation increased in passaged isolates from all strains, exhibiting from 1.2- to 5-fold the capacity of parental lines. Whole-genome sequencing identified nonsynonymous mutations affecting 23 candidate genes and a genomic duplication encompassing sigB. Six candidate genes significantly impacted biofilm formation as isogenic transposon knockouts: three were previously reported to impact S. aureus biofilm formation (icaR, spdC, and codY), while the remaining three (manA, narH, and fruB) were newly implicated by this study. Plasmid-mediated genetic complementation of manA, narH, and fruB transposon mutants corrected biofilm deficiencies, with high-level expression of manA and fruB further enhancing biofilm formation over basal levels. This work recognizes genes not previously identified as contributing to biofilm formation in S. aureus and reveals genetic changes able to augment biofilm production by that organism.

Genome Capture Sequencing Selectively Enriches Bacterial DNA and Enables Genome-Wide Measurement of Intrastrain Genetic Diversity in Human Infections.

Within-host evolution produces genetic diversity in bacterial strains that cause chronic human infections. However, the lack of facile methods to measure bacterial allelic variation in clinical samples has limited understanding of intrastrain diversity's effects on disease. Here, we report a new method termed genome capture sequencing (GenCap-Seq) in which users inexpensively make hybridization probes from genomic DNA or PCR amplicons to selectively enrich and sequence targeted bacterial DNA from clinical samples containing abundant human or nontarget bacterial DNA. GenCap-Seq enables accurate measurement of allele frequencies over targeted regions and is scalable from specific genes to entire genomes, including the strain-specific accessory genome. The method is effective with samples in which target DNA is rare and inhibitory and DNA-degrading substances are abundant, including human sputum and feces. In proof-of-principle experiments, we used GenCap-Seq to investigate the responses of diversified Pseudomonas aeruginosa populations chronically infecting the lungs of people with cystic fibrosis to in vivo antibiotic exposure, and we found that treatment consistently reduced intrastrain genomic diversity. In addition, analysis of gene-level allele frequency changes suggested that some genes without conventional resistance functions may be important for bacterial fitness during in vivo antibiotic exposure. GenCap-Seq's ability to scalably enrich targeted bacterial DNA from complex samples will enable studies on the effects of intrastrain and intraspecies diversity in human infectious disease. IMPORTANCE Genetic diversity evolves in bacterial strains during human infections and could affect disease manifestations and treatment resistance. However, the extent of diversity present in vivo and its changes over time are difficult to measure by conventional methods. We developed a novel approach, GenCap-Seq, to enrich microbial DNA from complex human samples like sputum and feces for genome-wide measurements of bacterial allelic diversity. The approach is inexpensive, scalable to encompass entire targeted genomes, and works in the presence of abundant untargeted nucleic acids and inhibiting substances. We used GenCap-Seq to investigate in vivo responses of diversified bacterial strains to antibiotic treatment. This method will enable new ideas about the effects of intrastrain diversity on human infections to be tested.

Evolution of cefiderocol resistance in Stenotrophomonas maltophilia using in vitro serial passage techniques.

OBJECTIVES: Cefiderocol is a siderophore cephalosporin active against MDR Gram-negatives including Stenotrophomonas maltophilia. Cefiderocol resistance remains uncommon and incompletely understood. We selected for cefiderocol-resistant S. maltophilia in vitro and characterized the genetic mechanisms and potential for cross-resistance to other antimicrobials. METHODS: We selected cefiderocol resistance in three clinical strains of S. maltophilia by serial passage in escalating concentrations of cefiderocol. Emergent cefiderocol-resistant isolates were subjected to repeat susceptibility testing against a panel of relevant antimicrobials. Isolates with confirmed MIC changes were whole genome sequenced. RESULTS: Each parent strain was initially susceptible to cefiderocol (MICs of 0.03125, 0.03125 and 0.125 mg/L), and one initially tested susceptible to ceftazidime/avibactam (MIC 4 mg/L). We recovered evolved isolates achieving cefiderocol resistance at MICs of 8-32 mg/L from each parental strain. Some cefiderocol resistant isolates reverted following one to four drug-free passages. Ceftazidime/avibactam MICs of passaged isolates repeatedly increased to ≥256 mg/L, and while other MICs were largely unchanged, trimethoprim/sulfamethoxazole MICs declined 4-fold in two strains. WGS revealed one evolved isolate carrying six coding mutations, while four were isogenic mutants of tonB, tolQ, smf-1 and the smeT promoter. Mutation of the smeT promoter downregulated the smeDEF efflux pump and reduced susceptibility to penicillins but increased susceptibility to several other classes including sulphonamides. Other mutations occurred in genes putatively involved in iron metabolism including smlt1148 and cirA. CONCLUSIONS: S. maltophilia strains evolved cefiderocol resistance through different genetic pathways, but often involved iron transport. Future work is required to fully understand the role(s) of other genes in cefiderocol resistance.

Synergy Between Beta-Lactams and Lipo-, Glyco-, and Lipoglycopeptides, Is Independent of the Seesaw Effect in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant S. aureus (MRSA) are resistant to beta-lactams, but synergistic activity between beta-lactams and glycopeptides/lipopeptides is common. Many have attributed this synergy to the beta-lactam-glycopeptide seesaw effect; however, this association has not been rigorously tested. The objective of this study was to determine whether the seesaw effect is necessary for synergy and to measure the impact of beta-lactam exposure on lipid metabolism. We selected for three isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin by serial passaging the MRSA strain N315. We used whole genome sequencing to identify genetic variants that emerged and tested for synergy between vancomycin, daptomycin, or dalbavancin in combination with 6 beta-lactams with variable affinity for staphylococcal penicillin binding proteins (PBPs), including nafcillin, meropenem, ceftriaxone, ceftaroline, cephalexin, and cefoxitin, using time-kills. We observed that the seesaw effect with each beta-lactam was variable and the emergence of the seesaw effect for a particular beta-lactam was not necessary for synergy between that beta-lactam and vancomycin, daptomycin, or dalbavancin. Synergy was more commonly observed with vancomycin and daptomycin based combinations than dalbavancin in time-kills. Among the beta-lactams, cefoxitin and nafcillin were the most likely to exhibit synergy using the concentrations tested, while cephalexin was the least likely to exhibit synergy. Synergy was more common among the resistant mutants than the parent strain. Interestingly N315-D1 and N315-DAL0.5 both had mutations in vraTSR and walKR despite their differences in the seesaw effect. Lipidomic analysis of all strains exposed to individual beta-lactams at subinhibitory concentrations suggested that in general, the abundance of cardiolipins (CLs) and most free fatty acids (FFAs) positively correlated with the presence of synergistic effects while abundance of phosphatidylglycerols (PGs) and lysylPGs mostly negatively correlated with synergistic effects. In conclusion, the beta-lactam-glycopeptide seesaw effect and beta-lactam-glycopeptide synergy are distinct phenomena. This suggests that the emergence of the seesaw effect may not have clinical importance in terms of predicting synergy. Further work is warranted to characterize strains that don't exhibit beta-lactam synergy to identify which strains should be targeted with combination therapy and which ones cannot and to further investigate the potential role of CLs in mediating synergy.

Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing.

BACKGROUND: Urine cell-free DNA (cfDNA) is an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (MTB) infection, but has not been thoroughly characterized as a biomarker. METHODS: This study was performed to investigate the size and composition of urine cfDNA from tuberculosis (TB) patients with minimal bias using next-generation sequencing (NGS). A combination of DNA extraction and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments was employed. Urine cfDNA from 10 HIV-positive patients with pulmonary TB and two MTB-negative controls was examined. RESULTS: MTB-derived cfDNA was identifiable by NGS from all MTB-positive patients and was absent from negative controls. MTB cfDNA was significantly shorter than human cfDNA, with median fragment lengths of ≤19-52 bp and 42-92 bp, respectively. MTB cfDNA abundance increased exponentially with decreased fragment length, having a peak fragment length of ≤19 bp in most samples. In addition, we identified a larger fraction of short human genomic cfDNA, ranging from 29 to 53 bp, than previously reported. Urine cfDNA fragments spanned the MTB genome with relative uniformity, but nucleic acids derived from multicopy elements were proportionately over-represented. CONCLUSIONS: TB urine cfDNA is a potentially powerful biomarker but is highly fragmented, necessitating special procedures to maximize its recovery and detection.

Dalbavancin exposure in vitro selects for dalbavancin-non-susceptible and vancomycin-intermediate strains of methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Dalbavancin is a lipoglycopeptide active against methicillin-resistant Staphylococcus aureus (MRSA). Its long half-life (8.5-16 days) allows for once-weekly or single-dose treatments but could prolong the mutant selection window, promoting resistance and cross-resistance to related antimicrobials such as vancomycin. The objective of this study was to evaluate the capacity of post-distributional pharmacokinetic exposures of dalbavancin to select for resistance and cross-resistance in MRSA. METHODS: We simulated average, post-distributional exposures of single-dose (1500 mg) dalbavancin (fCmax 9.9 µg/mL, ß-elimination t1/2 204 h) in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model for 28 days (672 h) against five MRSA strains and one methicillin-susceptible strain (MSSA). Samples were collected at least daily, and surviving colonies were enumerated and screened for resistance on drug-free and dalbavancin-supplemented medium respectively. Isolates from resistance screening plates were subjected to whole-genome sequencing (WGS) and susceptibly testing against dalbavancin, vancomycin, daptomycin, and six ß-lactams with varying penicillin-binding protein (PBP) affinities. RESULTS: Dalbavancin was bactericidal against most strains for days 1-4 before regrowth of less susceptible subpopulations occurred. Isolates with eight-fold increases in dalbavancin MIC were detected as early as day 4 but increased 64-128-fold in all models by day 28. Vancomycin and daptomycin MICs increased 4-16-fold, exceeding the susceptibly breakpoints for both antibiotics; ß-lactam MICs generally decreased by two-to eight-fold, suggesting a dalbavancin-ß-lactam seesaw effect, but increased by eight-fold or more in certain isolates. Resistant isolates carried mutations in a variety of genes, most commonly walKR, apt, stp1, and atl. CONCLUSIONS: In our in vitro system, post-distributional dalbavancin exposures selected for stable mutants with reduced susceptibility to dalbavancin, vancomycin, and daptomycin, and generally increased susceptibility to ß-lactams in all strains of MRSA tested. The clinical significance of these findings remains unclear, but created an opportunity to genotype a unique collection of dalbavancin-resistant strains for the first time. Mutations involved genes previously associated with vancomycin intermediate susceptibility and daptomycin non-susceptibility, most commonly walKR-associated genes.

Cell free DNA from respiratory pathogens is detectable in the blood plasma of Cystic Fibrosis patients.

Diagnostically informative microbial cell-free DNA (cfDNA) can be detected from blood plasma during fulminant infections such as sepsis. However, the potential for DNA from airway pathogens to enter the circulation of cystic fibrosis (CF) patients during chronic infective states has not yet been evaluated. We assessed whether patient blood contained measurable quantities of cfDNA from CF respiratory microorganisms by sequencing plasma from 21 individuals with CF recruited from outpatient clinics and 12 healthy controls. To account for possible contamination with exogenous microbial nucleic acids, statistical significance of microbe-derived read counts from CF patients was determined relative to the healthy control population. In aggregate, relative abundance of microbial cfDNA was nearly an order of magnitude higher in CF patients than in healthy subjects (p = 8.0×10-3). 15 of 21 (71%) CF patients demonstrated cfDNA from one or more relevant organisms. In contrast, none of the healthy subjects evidenced significant microbial cfDNA for any of the organisms examined. Concordance of cfDNA with standard microbiological culture of contemporaneously collected patient sputum was variable. Our findings provide evidence that cfDNA from respiratory pathogens are present in the bloodstream of most CF patients, which could potentially be exploited for the purposes of noninvasive clinical diagnosis.

Occurrence of cross-resistance and ß-lactam seesaw effect in glycopeptide-, lipopeptide- and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels.

BACKGROUND: Glycopeptides (GPs), lipopeptides (LPs) and lipoglycopeptides (LGPs) are related antimicrobials important for the management of invasive MRSA infections. Cross-resistance among these antibiotics in MRSA is well documented, as is the observation that susceptibility of MRSA to ß-lactams increases as susceptibility to GPs and LPs decreases (i.e. the seesaw effect). Efforts to understand the relationship between GP/LP/LGP cross-resistance and the seesaw effect have focused on the PBPs, but the role of lipid metabolism has not been investigated. OBJECTIVES: Since the cell membrane is structurally and metabolically integrated with the cell wall and anchors associated proteins, including PBPs, we examined the relationship between membrane lipid composition and the phenomena of cross-resistance among GPs/LPs/LGPs and the ß-lactam seesaw effect. METHODS: We selected for daptomycin, vancomycin and dalbavancin resistance using the USA300 strain JE2 and evaluated the resulting mutants by WGS, MS-based lipidomics and antimicrobial susceptibility testing to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. RESULTS: We observed cross-resistance to GPs/LPs/LGPs among the selected strains and the seesaw effect against various ß-lactams, depending on the PBP targets of the particular ß-lactam. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that the abundance of most phosphatidylglycerols positively correlates with MICs of GPs/LPs/LGPs and negatively correlates with the MICs of ß-lactams. CONCLUSIONS: These studies demonstrate a major association between membrane remodelling, cross-resistance and the seesaw effect.

Genomic Analysis Identifies Novel Pseudomonas aeruginosa Resistance Genes under Selection during Inhaled Aztreonam Therapy In Vivo.

Inhaled aztreonam is increasingly used for chronic Pseudomonas aeruginosa suppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapy in vivo We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent being ftsI and ampC, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutant ampC alleles and performed artificial evolution of ampC for maximal activity against aztreonam. We found that naturally occurring ampC mutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other ß-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selection in vivo and in vitro, show that ampC has a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitness in vivo.

Artificial Selection for Pathogenicity Mutations in Staphylococcus aureus Identifies Novel Factors Relevant to Chronic Infection.

Adaptation of Staphylococcus aureus to host microenvironments during chronic infection involves spontaneous mutations, yet changes underlying adaptive phenotypes remain incompletely explored. Here, we employed artificial selection and whole-genome sequencing to better characterize spontaneous chromosomal mutations that alter two pathogenicity phenotypes relevant to chronic infection in S. aureus: intracellular invasiveness and intracellular cytotoxicity. We identified 23 genes whose alteration coincided with enhanced virulence, 11 that were previously known and 12 (52%) that had no previously described role in S. aureus pathogenicity. Using precision genome editing, transposon mutants, and gene complementation, we empirically assessed the contributions of individual genes to the two virulence phenotypes. We functionally validated 14 of 21 genes tested as measurably influencing invasion and/or cytotoxicity, including 8 newly implicated by this study. We identified inactivating mutations (murA, ndhC, and a hypothetical membrane protein) and gain-of-function mutations (aroE Thr182Ile, yhcF Thr74Ile, and Asp486Glu in a hypothetical peptidase) in previously unrecognized S. aureus virulence genes that enhance pathogenesis when introduced into a clean genetic background, as well as a novel activating mutation in the known virulence regulator gene saeS (Ala106Thr). Investigation of potentially epistatic interactions identified a tufA mutation (Ala271Val) that enhances virulence only in the context of purine operon repressor gene (purR) inactivation. This project reveals a functionally diverse range of genes affected by gain- or loss-of-function mutations that contribute to S. aureus adaptive virulence phenotypes. More generally, the work establishes artificial selection as a means to determine the genetic mechanisms underlying complex bacterial phenotypes relevant to adaptation during infection.

Efficient and Scalable Precision Genome Editing in Staphylococcus aureus through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection.

Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132, derived from Enterococcus faecalis, as being capable of integrating single-stranded DNA oligonucleotides into the S. aureus genome. We found that EF2132 can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some S. aureus strains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the S. aureus genome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in S. aureus which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds.IMPORTANCE Engineering genetic changes in bacteria is critical to understanding the function of particular genes or mutations but is currently a laborious and technically challenging process to perform for the important human pathogen Staphylococcus aureus In an effort to develop methods which are rapid, easy, scalable, versatile, and inexpensive, here we describe a system for incorporating synthetic, mutagenic DNA molecules into the S. aureus genome and for eliminating cells that lack the engineered mutation. This method allows efficient, precise, and high-throughput genetic engineering of S. aureus strains and will facilitate studies seeking to address a variety of issues about the function of particular genes and specific mutations.

Engineering of a miniaturized, robotic clinical laboratory.

The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.

Characterization of the Mechanisms of Daptomycin Resistance among Gram-Positive Bacterial Pathogens by Multidimensional Lipidomics.

Previous work suggests that altered lipid metabolism may be associated with daptomycin resistance in Gram-positive pathogens, but lipidomic changes underlying resistance are not fully understood. We performed untargeted lipidomics by using three-dimensional hydrophilic interaction liquid chromatography-ion mobility-mass spectrometry (HILIC-IM-MS) to characterize alterations in the lipidomes of daptomycin-susceptible and -resistant isogenic strain pairs of Enterococcus faecalis, Staphylococcus aureus, and Corynebacterium striatum. We first validated the HILIC-IM-MS method by replicating the expected alterations of phospholipid metabolism in the previously studied E. faecalis strain pairs, such as reduced phosphatidylglycerols (PGs), while also revealing additional changes in cardiolipins (CLs), lysyl-PGs, and glycolipids. Whole-genome sequencing of the S. aureus and C. striatum strains found that daptomycin resistance was associated with mutations in pgsA, which encodes phosphatidylglycerophosphate synthase, as well as mutations in genes affecting fatty acid biosynthesis and cell wall metabolism. Lipidomics revealed significantly decreased levels of PGs, CLs, and amino acid-modified PGs, as well as accumulation of lipids upstream of PGs, such as glycolipids and phosphatidic acids, in the resistant strains. Notably, the glycolipids, diglucosyldiacylglycerols, were significantly elevated in a fatty acid-dependent manner in the daptomycin-resistant S. aureus strain. In daptomycin-resistant C. striatum, which has a unique cell envelope architecture, the glycolipids, glucuronosyldiacylglycerols, and phosphatidylinositols were significantly elevated. These results demonstrate that alteration of lipid metabolism via mutations in pgsA is a common mechanism of daptomycin resistance in two distinct species of Gram-positive bacteria and point to the potential contribution of altered glycolipid and fatty acid compositions to daptomycin resistance. IMPORTANCE This work comprehensively characterizes lipidomic changes underlying daptomycin resistance in three Gram-positive bacterial species, E. faecalis, S. aureus, and C. striatum, by using a novel three-dimensional lipidomics methodology based on advanced mass spectrometry. We demonstrated a number of advantages of our method in comparison with other methods commonly used in the field, such as high molecular specificity, sensitivity, and throughput. Whole-genome sequencing of the S. aureus and C. striatum strains identified mutations in pgsA, which encodes phosphatidylglycerophosphate synthase, in both resistant strains. Lipidomics revealed significantly decreased levels of lipids downstream of PgsA, as well as accumulation of lipids upstream of PgsA in the resistant strains. Furthermore, we found that changes in individual molecular species of each lipid class depend on the their specific fatty acid compositions. The characteristic changes in individual lipid species could be used as biomarkers for identifying underlying resistance mechanisms and for evaluating potential therapies.

Variation in incidence of pediatric Crohn's disease in relation to latitude and ambient ultraviolet radiation: a systematic review and analysis.

BACKGROUND: Pediatric Crohn's disease (CD) is a lifelong, debilitating, and costly disease. In previous studies, CD incidence increased with higher geographic latitude in the Northern Hemisphere. This may indicate a role for lower vitamin D status as a risk factor for CD. Analysis of worldwide incidence of pediatric CD has not been previously reported. METHODS: We undertook a systematic review of population-based studies reporting incidence of pediatric CD and published between 2003 and 2013. Included studies had well-defined diagnostic criteria for CD, evidence of high case ascertainment, reported incidence according to age group, and provided a specific location. Average daily ambient ultraviolet radiation (UVR) for each location was derived from satellite data. Negative binomial regression was used to assess the association between pediatric CD incidence and latitude and ambient UVR, adjusting for the study year. RESULTS: Twenty-eight articles provided 39 incidence data points. Incidence of pediatric CD increased with higher latitude, and in association with a greater number of months where the average daily UVR was lower than a previously published threshold of 1.488 kJ/m. Incidence of pediatric CD increased over calendar time. CONCLUSIONS: After applying rigorous quality assessment criteria, and including only population-based studies, there was a modest increase in incidence of pediatric CD with higher latitude and greater number of months with low ambient UVR. Reporting using nonconsistent diagnostic criteria and age groups, with poorly defined geographic locations, makes it difficult to compare data across different studies.