Therapeutic Potential of a Novel Lytic Phage, vB_EclM_ECLFM1, against Carbapenem-Resistant Enterobacter cloacae.

The global rise of multidrug-resistant Enterobacter cloacae strains, especially those that are resistant to carbapenems and produce metallo-ß-lactamases, poses a critical challenge in clinical settings owing to limited treatment options. While bacteriophages show promise in treating these infections, their use is hindered by scarce resources and insufficient genomic data. In this study, we isolated ECLFM1, a novel E. cloacae phage, from sewage water using a carbapenem-resistant clinical strain as the host. ECLFM1 exhibited rapid adsorption and a 15-min latent period, with a burst size of approximately 75 PFU/infected cell. Its genome, spanning 172,036 bp, was characterized and identified as a member of Karamvirus. In therapeutic applications, owing to a high multiplicity of infection, ECLFM1 showed increased survival in zebrafish infected with E. cloacae. This study highlights ECLFM1's potential as a candidate for controlling clinical E. cloacae infections, which would help address challenges in treating multidrug-resistant strains and contribute to the development of alternative treatments.

Characterization of a novel and active temperate phage vB_AbaM_ABMM1 with antibacterial activity against Acinetobacter baumannii infection.

Acinetobacter baumannii is an opportunistic pathogen that significantly causes hospital-acquired infections. Due to its multidrug resistance, treating infections caused by this pathogen is challenging. Recently, phages have gained attention as a potential alternative to antibiotics in treating bacterial infections. While lytic phages are preferred in therapy, the use of temperate phages for this purpose has received less attention. This study characterized a novel temperate phage vB_AbaM_ABMM1 (ABMM1) with antibacterial activity toward A. baumannii. ABMM1 adsorbs quickly, has short latent periods, and is relatively stable at various temperatures and neutral pH. ABMM1 has an icosahedral head and a contractile tail. It has a 75,731 kb circular permuted dsDNA genome containing 86 gene products with 37.3% G + C content and a mosaic arrangement typical of temperate phages. Genomic analysis confirmed that ABMM1 does not have antibiotic-resistance genes or virulence-related factors. The packaging strategy was predicted in silico, suggesting that ABMM1 represents a headful phage. Only truncated ABMM1 prophage was detected and has similarity in the genome of several A. baumannii strains. Despite its ability to integrate into the host chromosome, the high MOI of ABMM1 (MOI 10) effectively killed the host bacterial cells and reduced the fatality rate of bacterial infection in the zebrafish model. These findings indicate that ABMM1 can be an alternative treatment for A. baumannii infection.

Therapeutic effect and anti-biofilm ability assessment of a novel phage, phiPA1-3, against carbapenem-resistant Pseudomonas aeruginosa.

Multiple drug-resistant (MDR) Pseudomonas aeruginosa commonly causes severe hospital-acquired infections. The gradual emergence of carbapenem-resistant P. aeruginosa has recently gained attention. A wide array of P. aeruginosa-mediated pathogenic mechanisms, including its biofilm-forming ability, limits the use of effective antimicrobial treatments against it. In the present study, we isolated and characterized the phenotypic, biological, and genomic characteristics of a bacteriophage, vB_PaP_phiPA1-3 (phiPA1-3). Biofilm eradication and phage rescue from bacterial infections were assessed to demonstrate the efficacy of the application potential. Host range spectrum analysis revealed that phiPA1-3 is a moderate host range phage that infects 20% of the clinically isolated strains of P. aeruginosa tested, including carbapenem-resistant P. aeruginosa (CRPA). The phage exhibited stability at pH 7.0 and 9.0, with significantly reduced viability below pH 5.0 and beyond pH 9.0. phiPA1-3 is a lytic phage with a burst size of 619 plaque-forming units/infected cell at 37 °C and can effectively lyse bacteria in a multiplicity of infection-dependent manner. The genome size of phiPA1-3 was found to be 73,402 bp, with a G+C content of 54.7%, containing 93 open reading frames, of which 62 were annotated as hypothetical proteins and the remaining 31 had known functions. The phage possesses several proteins similar to those found in N4-like phages, including three types of RNA polymerases. This study concluded that phiPA1-3 belongs to the N4-like Schitoviridae family, can potentially eradicate P. aeruginosa biofilms, and thus, serve as a valuable tool for controlling CRPA infections.

Isolation and Characterization of a Novel Siphoviridae Phage, vB_AbaS_TCUP2199, Infecting Multidrug-Resistant Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii (MDRAB) is a pathogen recognized as antimicrobial-resistant bacteria involved in healthcare-associated infections. Resistance to antibiotics has made alternative therapies necessary. Bacteriophage therapy is considered a potential solution to treat MDRAB. In this study, we isolated and characterized the phage vB_AbaS_TCUP2199 (TCUP2199), which can infect MDRAB. Morphological analysis revealed that TCUP2199 belongs to the Siphoviridae family. TCUP2199 has a wide host range, can adsorb rapidly (68.28% in 2 min), and has a burst size of 196 PFU/cell. At least 16 distinct structural proteins were visualized by SDS polyacrylamide gel electrophoresis. A stability test showed that TCUP2199 was stable at 37 °C and pH 7. Genome analysis of TCUP2199 showed that it consists of a double-stranded DNA genome of 79,572 bp with a G+C content of 40.39%, which contains 98 putative open reading frames, none of which is closely related to the bacteriophage genome sequence that was found in the public database. TCUP2199 shows similarity in genomic organization and putative packaging mechanism with Achromobacter phage JWF and Pseudoalteromonas phage KB12-38 based on protein BLAST and phylogenetic analysis. Because of those unique characteristics, we consider TCUP2199 to be a novel phage that is suitable for inclusion in a phage cocktail to treat A. baumannii infection.

Preoptimized phage cocktail for use in aerosols against nosocomial transmission of carbapenem-resistant Acinetobacter baumannii: A 3-year prospective intervention study.

Using bacteriophages (phages) as environmental sanitizers has been recognized as a potential alternative method to remove bacterial contamination in vitro; however, very few studies are available on the application of phages for infection control in hospitals. Here, we performed a 3-year prospective intervention study using aerosolized phage cocktails as biocontrol agents against carbapenem-resistant Acinetobacter baumannii (CRAB) infection in the hospital. When a CRAB-infected patient was identified in an intensive care unit (ICU), their surrounding environment was chosen for phage aerosol decontamination. Before decontamination, 501 clinical specimens from the patients were subjected to antibiotic resistance analysis and phage typing. The optimal phage cocktails were a combination of different phage families or were constructed by next-evolutionary phage typing with the highest score for the host lysis zone to prevent the development of environmental CRAB phage resistance. The phage infection percentage of the antibiotic-resistant A. baumannii strains was 97.1%, whereas the infection percentage in the antibiotic-susceptible strains was 79.3%. During the phage decontamination periods from 2017 to 2019, the percentage of carbapenem-resistant A. baumannii in test ICUs decreased significantly from 65.3% to 55%. The rate of new acquisitions of CRAB infection over the three years was 4.4 per 1000 patient-days, which was significantly lower than that in the control wards (8.9 per 1000 patient-days) where phage decontamination had never been performed. In conclusion, our results support the potential of phage cocktails to decrease CRAB infection rates, and the aerosol generation process may make this approach more comprehensive and time-saving.

Trends in Fruit Quality Improvement From 15 Years of Selection in the Apple Breeding Program of Washington State University.

Washington State University's apple breeding program (WABP) was initiated in 1994 to select new apple cultivars with improved eating quality, appearance, and storability that are suitable for production in the main growing regions of the state. Fruit quality is phenotyped using various instrumental measures, such as penetrometers (texture), titrator (acidity), and refractometer (soluble solids concentration; SSC), as well as sensory assessment. The selection regime of WABP occurs in three sequential phases: phase one (P1)-single, unreplicated seedlings at one site, phase two (P2)-replicated selections at three geographically diverse sites, and phase three (P3)-highly replicated elite selections at one to two grower sites. Most of the data collection of WABP occurs in P2. Knowledge of trends/changes associated with advancing selections is essential for understanding the selection criteria and progress of WABP throughout the changing compositions of advancing and culling selections. For each post-harvest trait, P2 data from harvest years 2005 to 2019 were split across sites, and between selections and reference cultivars (e.g., Cripps Pink, Gala, and Honeycrisp). Means of instrumental crispness (Cn) and inner cortex firmness for the advancing selections increased gradually over this period and were significantly higher than those for cultivars. Means of outer cortex firmness measurements were stable for selections but significantly higher than those for cultivars. The average fruit acidity of selections increased marginally over this period and was higher than that of the cultivars. Meanwhile, the average fruit SSCs of selections and cultivars were statistically indistinguishable. These 15-year trends indicate that WABP has been selecting apples with improved eating quality and storability through increased crispness and inner cortex firmness, respectively.

Structural and vibrational characterization of HCO+ and Rg-HCO+, Rg = {He, Ne, Ar, Kr, and Xe}.

The structures of the formyl ion (HCO+) and its rare gas tagged counterparts (Rg-HCO+, Rg = He, Ne, Ar, Kr, and Xe) were studied at the coupled-cluster singles, doubles, and perturbative triples [CCSD(T)]/aug-cc-pVTZ level of theory and basis set. A linear structure for these tagged complexes was predicted. The Rg binding energies for Rg-HCO+ are also examined at the CCSD(T) level. It was found that the binding interaction increases from He-HCO+ to Xe-HCO+. A multilevel potential energy surface built at the CCSD(T) and second-order Møller-Plesset perturbation levels of theory were used to study these species' vibrational spectra. By changing the Rg in the first-solvation shell for HCO+, the Fermi resonance interaction between the first H+ bend overtone and the asymmetric and symmetric H-C-O stretches can be modulated. This Fermi resonance modulation is demonstrated by examining a series of rare gas solvated HCO+.

Size of the hydrogen bond network in liquid methanol: a quantum cluster equilibrium model with extensive structure search.

Studies have debated what is a favorable cluster size in liquid methanol. Applications of the quantum cluster equilibrium (QCE) model on a limited set of cluster structures have demonstrated the dominance of cyclic hexamers in liquid methanol. In this study, we examined the aforementioned question by integrating our implementation of QCE with a molecular-dynamics-based structural searching scheme. QCE simulations were performed using a database comprising extensively searched stable conformers of (MeOH)n for n = 2-14, which were optimized by B3LYP/6-31+G(d,p) with and without the dispersion correction. Our analysis indicated that an octamer structure can contribute significantly to cluster probability. By reoptimizing selected conformers with high probability at the MP2 level, we found that the aforementioned octamer became the dominant species due to favorable vibrational free energy, which was attributed to modes of intermolecular vibration.

Contributions of Reduced Susceptibility Alleles in Breeding Apple Cultivars with Durable Resistance to Fire Blight.

Breeding apple cultivars with durable genetic resistance is a potential long-term solution to fire blight, a devastating bacterial disease caused by Erwinia amylovora. However, phenotyping resistance/susceptibility to fire blight is challenging due to E. amylovora strain virulence, differential host × strain interactions, quantitative host resistance, environmental influences on disease, and impacts of tree vigor on susceptibility. Inheritance of resistance/susceptibility to fire blight is complex and phenotypic information alone is insufficient to guide breeding decisions targeting resistance. Several quantitative trait loci (QTLs) associated with resistance/susceptibility to fire blight have been detected throughout the apple genome. Most resistance alleles at fire blight QTLs have been identified in wild Malus germplasm with poor fruit quality, which limits their breeding utility. Several QTLs have been identified in populations derived from cultivars and reduced-susceptibility alleles have been characterized in multiple important breeding parents. Although resistance to fire blight is an attractive target for DNA-informed breeding, relatively few trait-predictive DNA tests for breeding relevant fire blight QTLs are available. Here we discuss (1) considerations and challenges associated with phenotyping resistance/susceptibility to fire blight; (2) sources of resistance that have been identified for use as parents; and (3) our perspective on short and long-term strategies to breed apple cultivars with durable resistance to fire blight with emphasis on the potential contributions of reduced susceptibility alleles to achieve this goal.

Fire blight QTL analysis in a multi-family apple population identifies a reduced-susceptibility allele in 'Honeycrisp'.

Breeding apple cultivars with resistance offers a potential solution to fire blight, a damaging bacterial disease caused by Erwinia amylovora. Most resistance alleles at quantitative trait loci (QTLs) were previously characterized in diverse Malus germplasm with poor fruit quality, which reduces breeding utility. This study utilized a pedigree-based QTL analysis approach to elucidate the genetic basis of resistance/susceptibility to fire blight from multiple genetic sources in germplasm relevant to U.S. apple breeding programs. Twenty-seven important breeding parents (IBPs) were represented by 314 offspring from 32 full-sib families, with 'Honeycrisp' being the most highly represented IBP. Analyzing resistance/susceptibility data from a two-year replicated field inoculation study and previously curated genome-wide single nucleotide polymorphism data, QTLs were consistently mapped on chromosomes (Chrs.) 6, 7, and 15. These QTLs together explained ~28% of phenotypic variation. The Chr. 6 and Chr. 15 QTLs colocalized with previously reported QTLs, while the Chr. 7 QTL is possibly novel. 'Honeycrisp' inherited a rare reduced-susceptibility allele at the Chr. 6 QTL from its grandparent 'Frostbite'. The highly resistant IBP 'Enterprise' had at least one putative reduced-susceptibility allele at all three QTLs. In general, lower susceptibility was observed for individuals with higher numbers of reduced-susceptibility alleles across QTLs. This study highlighted QTL mapping and allele characterization of resistance/susceptibility to fire blight in complex pedigree-connected apple breeding germplasm. Knowledge gained will enable more informed parental selection and development of trait-predictive DNA tests for pyramiding favorable alleles and selection of superior apple cultivars with resistance to fire blight.

A cohort study: The Association Between Autoimmune Disorders and Leptospirosis.

There are limited studies on the association between systemic autoimmune rheumatic diseases (SARDs) and leptospirosis. Therefore, this study aims to identify the effects of leptospirosis on the risks of developing SARDs with a nationwide retrospective cohort study. Patients with leptospirosis who did not have a diagnosis of SARDs before the index date were enrolled from the Taiwan National Health Insurance Research Database between 2000 and 2010, as the leptospirosis cohort. For each patient with leptospirosis, one control without a history of leptospirosis and SARDs was randomly selected (non-leptospirosis cohort). Cox proportional hazards regression models were used to analyze the risk of SARDs according to sex, age, and comorbidities. Among the 23 million people in the cohort, 3,393 patients with leptospirosis (68.91% men, mean age 52.65 years) and 33,930 controls were followed for 18,778 and 232,999 person-years, respectively. The incidence of SARDs was higher in the leptospirosis cohort than in the non-leptospirosis cohort (1.38 vs 0.33 per 1000 person-years), with a hazard ratio (HR) of 4.42 (95% confidence interval [CI] = 2.82-6.92). The risk of developing SARDs was highest for leptospirosis patients aged ≥65 years (HR = 2.81% CI = 1.07-7.36) compared with patients aged ≤39 years. Patients with leptospirosis have a 4.42-fold higher risk of SARDs than that in the general population. Further research is warranted to investigate the mechanism underlying this association.

Genetic analysis of stilbenoid profiles in grapevine stems reveals a major mQTL hotspot on chromosome 18 associated with disease-resistance motifs.

Grapevine (Vitis spp.) contains a wealth of phytochemicals that have received considerable attention due to health-promoting properties and biological activities as phytoalexins. To date, the genetic basis of the quantitative variations for these potentially beneficial compounds has been limited. Here, metabolic quantitative trait locus (mQTL) mapping was conducted using grapevine stems of a segregating F2 population. Metabolic profiling of grapevine stems was performed using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), resulting in the detection of 1317 ions/features. In total, 19 of these features matched with literature-reported stilbenoid masses and were genetically mapped using a 1449-SNP linkage map and R/qtl software, resulting in the identification of four mQTLs. Two large-effect mQTLs that corresponded to a stilbenoid dimer and a trimer were mapped on chromosome 18, accounting for phenotypic variances of 29.0% and 38.4%. Functional annotations of these large-effect mQTLs on the VitisNet network database revealed a major hotspot of disease-resistance motifs on chromosome 18. This 2.8-Mbp region contains 48 genes with R-gene motifs, including variants of TIR, NBS, and LRR, that might potentially confer resistance to powdery mildew, downy mildew, or other pathogens. The locus also encompasses genes associated with flavonoid and biosynthetic pathways that are likely involved in the production of secondary metabolites, including phytoalexins. In addition, haplotype dosage effects of the five mQTLs further characterized the genomic regions for differential production of stilbenoids that can be applied in resistance breeding through manipulation of stilbenoid production in planta.

Elucidation of the 'Honeycrisp' pedigree through haplotype analysis with a multi-family integrated SNP linkage map and a large apple (Malus×domestica) pedigree-connected SNP data set.

The apple (Malus×domestica) cultivar Honeycrisp has become important economically and as a breeding parent. An earlier study with SSR markers indicated the original recorded pedigree of 'Honeycrisp' was incorrect and 'Keepsake' was identified as one putative parent, the other being unknown. The objective of this study was to verify 'Keepsake' as a parent and identify and genetically describe the unknown parent and its grandparents. A multi-family based dense and high-quality integrated SNP map was created using the apple 8 K Illumina Infinium SNP array. This map was used alongside a large pedigree-connected data set from the RosBREED project to build extended SNP haplotypes and to identify pedigree relationships. 'Keepsake' was verified as one parent of 'Honeycrisp' and 'Duchess of Oldenburg' and 'Golden Delicious' were identified as grandparents through the unknown parent. Following this finding, siblings of 'Honeycrisp' were identified using the SNP data. Breeding records from several of these siblings suggested that the previously unreported parent is a University of Minnesota selection, MN1627. This selection is no longer available, but now is genetically described through imputed SNP haplotypes. We also present the mosaic grandparental composition of 'Honeycrisp' for each of its 17 chromosome pairs. This new pedigree and genetic information will be useful in future pedigree-based genetic studies to connect 'Honeycrisp' with other cultivars used widely in apple breeding programs. The created SNP linkage map will benefit future research using the data from the Illumina apple 8 and 20 K and Affymetrix 480 K SNP arrays.

Genetic dissection of powdery mildew resistance in interspecific half-sib grapevine families using SNP-based maps.

Quantitative trait locus (QTL) identification in perennial fruit crops is impeded largely by their lengthy generation time, resulting in costly and labor-intensive maintenance of breeding programs. In a grapevine (genus Vitis) breeding program, although experimental families are typically unreplicated, the genetic backgrounds may contain similar progenitors previously selected due to their contribution of favorable alleles. In this study, we investigated the utility of joint QTL identification provided by analyzing half-sib families. The genetic control of powdery mildew was studied using two half-sib F1 families, namely GE0711/1009 (MN1264 × MN1214; N = 147) and GE1025 (MN1264 × MN1246; N = 125) with multiple species in their ancestry. Maternal genetic maps consisting of 1077 and 1641 single nucleotide polymorphism (SNP) markers, respectively, were constructed using a pseudo-testcross strategy. Ratings of field resistance to powdery mildew were obtained based on whole-plant evaluation of disease severity. This 2-year analysis uncovered two QTLs that were validated on a consensus map in these half-sib families with improved precision relative to the parental maps. Examination of haplotype combinations based on the two QTL regions identified strong association of haplotypes inherited from 'Seyval blanc', through MN1264, with powdery mildew resistance. This investigation also encompassed the use of microsatellite markers to establish a correlation between 206-bp (UDV-015b) and 357-bp (VViv67) fragment sizes with resistance-carrying haplotypes. Our work is one of the first reports in grapevine demonstrating the use of SNP-based maps and haplotypes for QTL identification and tagging of powdery mildew resistance in half-sib families.

Uncovering the Genetic Architecture of Seed Weight and Size in Intermediate Wheatgrass through Linkage and Association Mapping.

Intermediate wheatgrass [IWG; (Host) Barkworth & D.R. Dewey subsp. ] is being developed as a new perennial grain crop that has a large allohexaploid genome similar to that of wheat ( L.). Breeding for increased seed weight is one of the primary goals for improving grain yield of IWG. As a new crop, however, the genetic architecture of seed weight and size has not been characterized, and selective breeding of IWG may be more intricate than wheat because of its self-incompatible mating system and perennial growth habit. Here, seed weight, seed area size, seed width, and seed length were evaluated across multiple years, in a heterogeneous breeding population comprised of 1126 genets and two clonally replicated biparental populations comprised of 172 and 265 genets. Among 10,171 DNA markers discovered using genotyping-by-sequencing (GBS) in the breeding population, 4731 markers were present in a consensus genetic map previously constructed using seven full-sib populations. Thirty-three quantitative trait loci (QTL) associated with seed weight and size were identified using association mapping (AM), of which 23 were verified using linkage mapping in the biparental populations. About 37.6% of seed weight variation in the breeding population was explained by 15 QTL, 12 of which also contributed to either seed length or seed width. When performing either phenotypic selection or genomic selection for seed weight, we observed the frequency of favorable QTL alleles were increased to >46%. Thus, by combining AM and genomic selection, we can effectively select the favorable QTL alleles for seed weight and size in IWG breeding populations.

Management and outcome of adults with skin and soft tissue infection caused by methicillin-resistant Staphylococcus aureus in a tertiary hospital in central Taiwan.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has been increasingly causing skin and soft tissue infections (SSTIs). Only limited studies have made comparisons between incision and drainage (I&D) alone and I&D with adjunctive antibiotic therapy for treatment effects, and most of the studies were conducted before the emergence of MRSA. This study was to evaluate whether antibiotics provide added benefit to I&D alone for purulent MRSA SSTIs. METHODS: This retrospective study collected data on SSTI patients, including patient demographics, treatment strategies, antibiotic susceptibilities of the infecting MRSA isolates, and clinical outcomes over the course of 24 months. RESULTS: Antimicrobial drug susceptibility rate were 100% for vancomycin, teicoplanin, and linezolid. Among the 211 patients, 7.6% were treated solely with I&D (Group A), 62.6% were treated via I&D with adjunctive antibiotic (Group B), and 29.8% patients received only antibiotics (Group C). The cure rate was highest in Group A (93.8%), followed by Group B (90.9%) and Group C (77.8%). Combining Group B and Group C, patients who were treated appropriately demonstrated a higher cute rate (91.3% vs. 75.4%, p = 0.005). Multivariate analysis showed that Group B was more likely to be successfully treated compared to Group C (odds ratio = 2.51, 95% confidence interval = 1.01-6.25, p = 0.047), whereas no difference between Group A and Group B was found (odds ratio = 2.09, 95% confidence interval = 0.20-22.34, p = 0.542, data not shown). CONCLUSION: Surgical intervention is the definitive therapy for purulent SSTIs. Adjunctive antibiotic therapy increased the cure rate and appropriateness of prescription is influential.