Unveiling genetic diversity and forensic utility of SureID® human DNA identification kits: a comprehensive analysis of 44 autosomal STR loci in English and Irish populations.

Background: Human identification and kinship testing in forensic science rely on Short Tandem Repeat (STR) multiplex kits, typically containing loci recommended by standard sets. However, complementary kits with additional STR loci can be valuable in complex cases. Allele frequency databases specific to the population are essential for accurate forensic analysis.Aim: This study aimed to generate allele frequencies and population genetic data for 44 autosomal STR loci from SureID® PanGlobal and 27comp kits in English and Irish populations for forensic casework, human identification, and kinship testing.Subjects and methods: Buccal swab samples from 645 White Caucasians (365 English, 280 Irish) were collected. DNA was extracted and amplified using the mentioned kits. Quality control, statistical analysis, and genetic distance calculations were performed.Results: Both kits demonstrated robustness with no significant deviations from Hardy-Weinberg Equilibrium (HWE). Variant alleles and minor discordances between kits were observed. Syntenic STR pairs were identified but showed no significant linkage. A close genetic relationship was found between English and Irish populations, allowing for combined databases.Conclusions: The SureID® PanGlobal and 27comp kits showed high discriminatory power and reliability in the English and Irish populations. Care is needed when handling variant alleles, discordances, and syntenic loci. Combining data from both populations is feasible for a comprehensive database. Further studies are required to explore their effectiveness in diverse populations.

Phosphonopeptides Revisited, in an Era of Increasing Antimicrobial Resistance.

Given the increase in resistance to antibacterial agents, there is an urgent need for the development of new agents with novel modes of action. As an interim solution, it is also prudent to reinvestigate old or abandoned antibacterial compounds to assess their efficacy in the context of widespread resistance to conventional agents. In the 1970s, much work was performed on the development of peptide mimetics, exemplified by the phosphonopeptide, alafosfalin. We investigated the activity of alafosfalin, di-alanyl fosfalin and ß-chloro-L-alanyl-ß-chloro-L-alanine against 297 bacterial isolates, including carbapenemase-producing Enterobacterales (CPE) (n = 128), methicillin-resistant Staphylococcus aureus (MRSA) (n = 37) and glycopeptide-resistant enterococci (GRE) (n = 43). The interaction of alafosfalin with meropenem was also examined against 20 isolates of CPE. The MIC50 and MIC90 of alafosfalin for CPE were 1 mg/L and 4 mg/L, respectively and alafosfalin acted synergistically when combined with meropenem against 16 of 20 isolates of CPE. Di-alanyl fosfalin showed potent activity against glycopeptide-resistant isolates of Enterococcus faecalis (MIC90; 0.5 mg/L) and Enterococcus faecium (MIC90; 2 mg/L). Alafosfalin was only moderately active against MRSA (MIC90; 8 mg/L), whereas ß-chloro-L-alanyl-ß-chloro-L-alanine was slightly more active (MIC90; 4 mg/L). This study shows that phosphonopeptides, including alafosfalin, may have a therapeutic role to play in an era of increasing antibacterial resistance.

Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients.

RATIONALE: Aspiration of infective subglottic secretions causes ventilator-associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP. OBJECTIVES: To characterize subglottic host defense dysfunction in mechanically ventilated patients in the ICU; to determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth. METHODS: Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 min); isolation and culture of primary subglottic epithelial cells from control patients; laboratory analysis of host innate immune defenses. MEASUREMENTS AND MAIN RESULTS: Twenty-four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in the ICU was characterized by neutrophilic inflammation, significantly increased proinflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic impairment was reversible on treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air-liquid interface model of primary human subglottic epithelium. CONCLUSIONS: Mechanical ventilation in the ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil function and promotes bacterial growth. Mucolytic agents reverse mucin-mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage.

Evaluation of Various Culture Media for Detection of Rapidly Growing Mycobacteria from Patients with Cystic Fibrosis.

Isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF) is challenging due to overgrowth by rapidly growing species that colonize the lungs of patients with CF. Extended incubation on Burkholderia cepacia selective agar (BCSA) has been recommended as an expedient culture method for the isolation of rapidly growing NTM in this setting. The aim of this study was to assess five selective media designed for the isolation of Burkholderia cepacia complex, along with two media designed for the isolation of mycobacteria (rapidly growing mycobacteria [RGM] medium and Middlebrook 7H11 agar), for their abilities to isolate NTM. All seven media were challenged with 147 isolates of rapidly growing mycobacteria and 185 isolates belonging to other species. RGM medium was then compared with the most selective brand of BCSA for the isolation of NTM from 224 sputum samples from patients with CF. Different agars designed for the isolation of B. cepacia complex varied considerably in their inhibition of other bacteria and fungi. RGM medium supported the growth of all isolates of mycobacteria and was more selective than any other medium. NTM were recovered from 17 of 224 sputum samples using RGM medium, compared with only 7 samples using the most selective brand of BCSA (P = 0.023). RGM medium offers a superior option, compared to other selective agars, for the isolation of rapidly growing mycobacteria from the sputum of patients with CF. Furthermore, the convenience of using RGM medium enables routine screening for rapidly growing NTM in all submitted sputum samples from patients with CF.

Metabolomic and proteomic analysis of serum from preterm infants with necrotising entercolitis and late-onset sepsis.

BACKGROUND: Necrotising enterocolitis (NEC) and late-onset sepsis (LOS) are the leading causes of death among preterm infants in the developed world. This study aimed to explore the serum proteome and metabolome longitudinally in preterm infants with NEC or LOS, matched to controls. METHODS: Nineteen patients (10 cases, 9 controls) were included. A sample 14 d prior to and following, as well as at disease diagnosis, was included for cases. Controls had serum matched at diagnosis for corresponding case. All samples (n = 39) underwent shotgun proteomic analysis, and 37 samples also underwent metabolomics analysis using ultra performance liquid chromatography-tandem mass spectrometry. RESULTS: The proteomic and metabolomic profiles of serum were comparable between all infants. Eight proteins were associated with NEC and four proteins were associated with LOS. C-reactive protein was increased in all NEC patients at diagnosis. CONCLUSION: No single protein or metabolite was detected in all NEC or LOS cases which was absent from controls; however, several proteins were identified which were associated with disease status. The differing expression of these proteins between diseased infants potentially relates to differing pathophysiology of disease. Thus, it is unlikely a single biomarker exists for NEC and/or LOS.

Cytomegalovirus and other common enteric viruses are not commonly associated with NEC.

AIM: Changes in gut microbiota may contribute to NEC, but most studies focus on bacteria. Case reports suggest a link between cytomegalovirus (CMV) or other enteric viruses and NEC, but there are few case series systematically looking at common potential viral causes. We aimed to assess the presence of candidate viruses in blood or stool of a case series of infants with NEC managed in one surgical centre. METHODS: We identified 22 infants diagnosed with NEC (from November 2011 to March 2014): 17 had suitable blood stored, of whom 14 also had suitable stool samples stored. Blood was analysed with polymerase chain reaction (PCR) for CMV, Epstein-Barr virus (EBV) and adenovirus, and stool by PCR for norovirus, sappovirus, astrovirus, adenovirus and rotavirus. RESULTS: All samples were negative. CONCLUSION: Although case reports indicate an episodic association of enteric viruses in NEC, the inability to detect any of these viruses in our 17 NEC infants suggests that a viral aetiology is unlikely to be causative for most sporadic forms of NEC.

Inpatient aggression in community hospitals.

Physical violence is a frequent occurrence in acute community psychiatry units worldwide. Violent acts by patients cause many direct injuries and significantly degrade quality of care. The most accurate tools for predicting near-term violence on acute units rely on current clinical features rather than demographic risk factors. The efficacy of risk assessment strategies to lower incidence of violence on acute units is unknown. A range of behavioral and psychopharmacologic treatments have been shown to reduce violence among psychiatric inpatients.

The neonatal bowel microbiome in health and infection.

PURPOSE OF REVIEW: In newborns, interactions between the host and the microbiome operate synergistically, modulating host immune function and shaping the microbiome. Next generation molecular sequencing methodologies in tandem with modeling complex communities allow insights into the role of the microbiome in health and disease states. Infection-related disease states in which dysbiosis is integral include late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), which still cause deaths and morbidity. Understanding microbiomic interactions may lead to alternative prevention, monitoring or treatment strategies, and modulation of long-term health outcomes especially in the preterm population. Recent studies have advanced understanding of the microbiome in NEC and LOS. RECENT FINDINGS: Mechanisms of host-microbiome interaction have been demonstrated. Patterns of microbiomic change in association with NEC and LOS have been observed, with community changes dominated by Proteobacteria and Firmicutes appearing to precede NEC, and very early microbiomic signatures influencing LOS. Data on viral and fungal elements are emerging. SUMMARY: Greater understanding of the neonatal bowel microbiome may allow tailored clinical practice and therapeutic intervention. Data handling and interpretation is challenging. Mechanistic studies of clinical interventions that affect the gut microbiome are important next steps.

Polymicrobial airway bacterial communities in adult bronchiectasis patients.

BACKGROUND: Chronic airway infection contributes to the underlying pathogenesis of non-cystic fibrosis bronchiectasis (NCFBr). In contrast to other chronic airway infections, associated with COPD and CF bronchiectasis, where polymicrobial communities have been implicated in lung damage due to the vicious circle of recurrent bacterial infections and inflammation, there is sparse information on the composition of bacterial communities in NCFBr. Seventy consecutive patients were recruited from an outpatient adult NCFBr clinic. Bacterial communities in sputum samples were analysed by culture and pyrosequencing approaches. Bacterial sequences were analysed using partial least square discrimination analyses to investigate trends in community composition and identify those taxa that contribute most to community variation. RESULTS: The lower airway in NCFBr is dominated by three bacterial taxa Pasteurellaceae, Streptococcaceae and Pseudomonadaceae. Moreover, the bacterial community is much more diverse than indicated by culture and contains significant numbers of other genera including anaerobic Prevotellaceae, Veillonellaceae and Actinomycetaceae. We found particular taxa are correlated with different clinical states, 27 taxa were associated with acute exacerbations, whereas 11 taxa correlated with stable clinical states. We were unable to demonstrate a significant effect of antibiotic therapy, gender, or lung function on the diversity of the bacterial community. However, presence of clinically significant culturable taxa; particularly Pseudomonas aeruginosa and Haemophilus influenzae correlated with a significant change in the diversity of the bacterial community in the lung. CONCLUSIONS: We have demonstrated that acute exacerbations, the frequency of exacerbation and episodes of clinical stability are correlated, in some patients, with a significantly different bacterial community structure, that are associated with a presence of particular taxa in the NCFBr lung. Moreover, there appears to be an inverse relationship between the abundance of P. aeruginosa and that of of H. influenzae within the NCFBr lung bacterial community. This interaction requires further exploration.

Proportionate reduction in uncertainty of late onset infection in pre-term infants by neutrophil CD64 measurement.

OBJECTIVE: Establish how neutrophil CD64 performs as a marker of definite infection in pre-term infants in comparison to C reactive protein (CRP) and procalcitonin (PCT). METHODS: A total of 38 pre-term infants with suspected late onset infection had CD64 measured by flow cytometry. Proportionate reduction in uncertainty (PRU) curves were generated for CD64 counts at various threshold values. RESULTS: PRU curves reduced the residual uncertainty of the presence of infection by up to 64%. CONCLUSIONS: The CD64 appears to be a useful point of care test (POCT) for further defining the likelihood of infection and performs better than CRP or PCT at helping to rule in infection.

Distinct Effects of Respiratory Viral Infection Models on miR-149-5p, IL-6 and p63 Expression in BEAS-2B and A549 Epithelial Cells.

Respiratory viruses cause airway inflammation, resulting in epithelial injury and repair. miRNAs, including miR-149-5p, regulate different pathological conditions. We aimed to determine how miR-149-5p functions in regulating pro-inflammatory IL-6 and p63, key regulators of airway epithelial wound repair, in response to viral proteins in bronchial (BEAS-2B) and alveolar (A549) epithelial cells. BEAS-2B or A549 cells were incubated with poly (I:C, 0.5 µg/mL) for 48 h or SARS-CoV-2 spike protein-1 or 2 subunit (S1 or S2, 1 µg/mL) for 24 h. miR-149-5p was suppressed in BEAS-2B challenged with poly (I:C), correlating with IL-6 and p63 upregulation. miR-149-5p was down-regulated in A549 stimulated with poly (I:C); IL-6 expression increased, but p63 protein levels were undetectable. miR-149-5p remained unchanged in cells exposed to S1 or S2, while S1 transfection increased IL-6 expression in BEAS-2B cells. Ectopic over-expression of miR-149-5p in BEAS-2B cells suppressed IL-6 and p63 mRNA levels and inhibited poly (I:C)-induced IL-6 and p63 mRNA expressions. miR-149-5p directly suppressed IL-6 mRNA in BEAS-2B cells. Hence, BEAS-2B cells respond differently to poly (I:C), S1 or S2 compared to A549 cells. Thus, miR-149-5p dysregulation may be involved in poly (I:C)-stimulated but not S1- or S2-stimulated increased IL-6 production and p63 expression in BEAS-2B cells.

Use of faropenem as an indicator of carbapenemase activity in the Enterobacteriaceae.

The aim of this study was to determine the ability of a disc susceptibility test using faropenem (10 µg) to predict carbapenemase activity in Enterobacteriaceae. A collection of 166 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 82 isolates of Enterobacteriaceae that produced other ß-lactamases was compiled from diverse sources. Disc susceptibility testing was performed using the CLSI/EUCAST methodology with discs of faropenem (10 µg), temocillin (30 µg), and four carbapenems (each 10 µg). A further prospective evaluation of the faropenem disc susceptibility test was performed using 205 consecutive isolates referred to a United Kingdom reference laboratory in parallel with molecular methods for carbapenemase detection. Of 166 isolates of CPE, 99% showed growth up to the edge of a 10-µg faropenem disc compared with only 6% of other ß-lactamase producers (sensitivity, 99%; specificity, 94%). A "double zone" around 10-µg faropenem discs was frequently associated with OXA-48 producers. Of the carbapenems, the most useful agent was imipenem, where a zone diameter of ≤ 23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. The presence of no zone of inhibition around a 30-µg temocillin disc was a consistent feature of strains producing OXA-48 carbapenemase. For 205 isolates of Enterobacteriaceae referred to a United Kingdom reference laboratory, growth up to a 10-µg faropenem disc correctly identified 84 of 86 carbapenemase producers (98% sensitivity), with a specificity of 87%. Disc susceptibility testing using faropenem (10 µg) is a simple, convenient, and highly predictive screening test for carbapenemase-producing Enterobacteriaceae.

Nanostructures in water-in-CO2 microemulsions stabilized by double-chain fluorocarbon solubilizers.

High-pressure small-angle neutron scattering (HP-SANS) studies were conducted to investigate nanostructures and interfacial properties of water-in-supercritical CO2 (W/CO2) microemulsions with double-fluorocarbon-tail anionic surfactants, having different fluorocarbon chain lengths and linking groups (glutarate or succinate). At constant pressure and temperature, the microemulsion aqueous cores were found to swell with an increase in water-to-surfactant ratio, W0, until their solubilizing capacities were reached. Surfactants with fluorocarbon chain lengths of n = 4, 6, and 8 formed spherical reversed micelles in supercritical CO2 even at W0 over the solubilizing powers as determined by phase behavior studies, suggesting formation of Winsor-IV W/CO2 microemulsions and then Winsor-II W/CO2 microemulsions. On the other hand, a short C2 chain fluorocarbon surfactant analogue displayed a transition from Winsor-IV microemulsions to lamellar liquid crystals at W0 = 25. Critical packing parameters and aggregation numbers were calculated by using area per headgroup, shell thickness, the core/shell radii determined from SANS data analysis: these parameters were used to help understand differences in aggregation behavior and solubilizing power in CO2. Increasing the microemulsion water loading led the critical packing parameter to decrease to ~1.3 and the aggregation number to increase to >90. Although these parameters were comparable between glutarate and succinate surfactants with the same fluorocarbon chain, decreasing the fluorocarbon chain length n reduced the critical packing parameter. At the same time, reducing chain length to 2 reduced negative interfacial curvature, favoring planar structures, as demonstrated by generation of lamellar liquid crystal phases.

Determining the influence of environmental and patient specific factors on the polymicrobial communities of the cystic fibrosis airway.

The aim of this study was to investigate the polymicrobial communities in an adult Cystic Fibrosis population stratified by gender and the most common CFTR mutation, F508del. In this pilot study, DNA was extracted from sputum samples of 29 adult patients (16 male: 13 female) with an F508del mutation in a stable clinical state. Universal primers were used to amplify DNA from bacterial and fungal communities and the resulting fragments were analysed by denaturing gradient gel electrophoresis. Bacterial profiles showed a significant effect of gender (P = 0.046) and P. aeruginosa carriage (P = 0.034) on community structure. Bacterial communities were found to be randomly assembled. Fungal community analysis found that F508del homozygous patients had a greater diversity than heterozygous patients (P = 0.007). This study indicates that the bacterial lung communities of adult CF patients are randomly assembled but have distinct gender based differences. Furthermore, the fungal communities colonising the CF lung are more diverse in F508 homozygotes. This is the first paper to identify a reduced bacterial diversity in female patients with CF and to implicate more severe CFTR genotypes with increased risk of infection with multiple fungal species.

Mechanisms affecting the gut of preterm infants in enteral feeding trials: a nested cohort within a randomised controlled trial of lactoferrin.

OBJECTIVE: To determine the impact of supplemental bovine lactoferrin on the gut microbiome and metabolome of preterm infants. DESIGN: Cohort study nested within a randomised controlled trial (RCT). Infants across different trial arms were matched on several clinical variables. Bacteria and metabolite compositions of longitudinal stool and urine samples were analysed to investigate the impact of lactoferrin supplementation. SETTING: Thirteen UK hospitals participating in a RCT of lactoferrin. PATIENTS: 479 infants born <32 weeks' gestation between June 2016 and September 2017. RESULTS: 10 990 stool and 22 341 urine samples were collected. Analyses of gut microbiome (1304 stools, 201 infants), metabolites (171 stools, 83 infants; 225 urines, 90 infants) and volatile organic compounds (314 stools, 117 infants) were performed. Gut microbiome Shannon diversity at 34 weeks corrected age was not significantly different between infants in the lactoferrin (mean=1.24) or placebo (mean=1.06) groups (p=0.11). Lactoferrin receipt explained less than 1% variance in microbiome compositions between groups. Metabolomic analysis identified six discriminative features between trial groups. Hospital site (16%) and postnatal age (6%) explained the greatest variation in microbiome composition. CONCLUSIONS: This multiomic study identified minimal impacts of lactoferrin but much larger impacts of hospital site and postnatal age. This may be due to the specific lactoferrin product used, but more likely supports the findings of the RCT in which this study was nested, which showed no impact of lactoferrin on reducing rates of sepsis. Multisite mechanistic studies nested within RCTs are feasible and help inform trial interpretation and future trial design.

Comparison of four chromogenic culture media for carbapenemase-producing Enterobacteriaceae.

Four chromogenic media for carbapenemase-producing Enterobacteriaceae (CPE) and two selective broths were challenged with a collection of Enterobacteriaceae with well-defined ß-lactamases and 100 stool samples. With low inocula of 130 isolates of CPE, the sensitivities of the four chromogenic media were as follows: Brilliance CRE, 78%; chromID Carba, 91%; chromID ESBL, 96%; and Colorex KPC, 56%. The corresponding sensitivities of Trypticase soy broth plus ertapenem or meropenem were 78% and 47%, respectively.

Effective and efficient surfactant for CO2 having only short fluorocarbon chains.

A previous study (Langmuir2011, 27, 5772) found the fluorinated double-tail sulfogulutarate 8FG(EO)(2) to act as a superefficient solubilizer for water in supercritical CO(2) (W/CO(2)) microemulsions. To explore more economic CO(2)-philic surfactants with high solubilizing power as well as rapid solubilization rates, the effects of fluorocarbon chain length and linking group were examined with sodium 1,5-bis(1H,1H,2H,2H-perfluoroalkyloxy)-1,5-dioxopentane-2-sulfonates (nFG(EO)(2), fluorocarbon chain length n = 4, 6, 8) and sodium 1,4-bis(1H,1H,2H,2H-perfluoroalkyloxy)-1,4-dioxobutane-2-sulfonate (nFS(EO)(2), n = 4, 8). Visual observation and UV-vis spectral measurements with methyl orange as a reporter dye indicated a maximum water-to-surfactant molar ratio (W(0)) in the microemulsions, which was 60-80 for nFG(EO)(2) and 40-50 for nFG(EO)(2). Although it is normally expected that high solubilizing power requires long fluorocarbon surfactant chains, the shortest fluorocarbon 4FG(EO)(2) interestingly achieved the highest W(0) (80) transparent single-phase W/CO(2) microemulsion. In addition, a very rapid solubilization of loaded water into CO(2) was observed for 4FG(EO)(2) even at a high W(0) of ~80.

Key role of dysregulated airway epithelium in response to respiratory viral infections in asthma.

A differentiated air-liquid interface model shows that the airway epithelium plays a key role in response to respiratory viral infections in people with asthma https://bit.ly/3yDgiX1.

Formalin-Fixed Paraffin-Embedded (FFPE) samples are not a beneficial replacement for frozen tissues in fetal membrane microbiota research.

Formalin-Fixed Paraffin-Embedded (FFPE) tissues are routinely collected, archived, and used for clinical diagnosis, including maternal and neonatal health. Applying FFPE samples to microbiota research would be beneficial to reduce preparation, storage and costs associated with limited available frozen samples. This research aims to understand if FFPE fetal membrane samples are comparable to frozen tissues, which are the current gold standard for DNA microbiota analysis. Extracted DNA from nine matched paired patients were sequenced by Illumina sequencing of the V4 16S rRNA gene region. This included duplicate frozen amnion and chorion fetal membrane rolls or FFPE combined amniochorionic samples. Negative controls of surrounding wax blocks and DNA extraction reagents were processed alongside samples using identical methods. DNA quality and quantity was assessed by NanoDrop, agarose gel electrophoresis and Bioanalyzer. Decontam and SourceTracker were integrated into microbiota analysis to identify the presence of contaminating sources. The bacterial profile and nine genera differed between FFPE and frozen fetal membranes. There were no differences in bacterial profiles between FFPE samples and corresponding wax negative controls, with 49% of bacteria in FFPE fetal membrane samples matched to the source origin of paraffin wax, and 40% originating from DNA extraction reagent sources. FFPE samples displayed high fragmentation and low quantity of extracted DNA compared to frozen samples. The microbiota of FFPE fetal membrane samples is influenced by processing methods, with the inability to differentiate between the microbiota of the tissue sample and the surrounding wax block. Illumina sequencing results of FFPE and frozen fetal membrane samples should not be compared using the methods employed here. Variation could be influenced by limitations including storage time, DNA extraction and purification methods. To utilise FFPE fetal membrane samples in microbiota research then contamination prevention and detection methods must be included into optimised and standardised protocols, with recommendations presented here.