Strain Discrimination Research Articles

PCR Based Technique for Identification of Bile Salt Hydrolase Positive Lactobacilli

In the burgeoning landscape of probiotic research, the intricate interplay between the gut microbiota and host health has underscored the significance of specific enzymatic activities, notably bile salt hydrolase (BSH) produced by Lactobacilli. Harnessing the potential health benefits associated with BSH activity necessitates a meticulous identification strategy. In response, this study introduces a sophisticated PCR-based technique tailored for the rapid and precise discrimination of lactobacilli strains boasting BSH enzymatic prowess. This PCR-based approach offers a valuable tool for researchers and industry professionals involved in the selection and characterization of probiotic strains with bile salt hydrolase activity. Its speed, accuracy, and adaptability make it a promising method for large-scale screenings and applications in diverse fields, ranging from food science to the development of probiotic supplements aimed at enhancing human health through the modulation of bile acid metabolism. robiotic supplementation, where the modulation of bile acid metabolism holds promise for promoting human health and well-being.

Distributed phase-matching measurement enabled dynamic temperature–strain discrimination using single chirped pulse probe

Distributed phase-matching measurement enabled dynamic temperature–strain discrimination using single chirped pulse probe

Multiplexed real-time PCR for the detection and differentiation of Klebsiella pneumoniae O-antigen serotypes.

Klebsiella pneumoniae has emerged as a global health threat due to its role in the spread of antimicrobial resistance and because it is a frequent cause of hospital-acquired infections and neonatal sepsis. Capsular and lipopolysaccharide (LPS) O-antigen polysaccharide surface antigens are major immunogens that are useful for strain classification and are candidates for vaccine development. We have developed real-time PCR reagents for molecular serotyping, subtyping, and quantitation of the most prevalent LPS O-antigen types (i.e., O1, O2, O3, and O5) of Klebsiella pneumoniae. We describe two applications for this O-typing assay: for screening culture isolates and for direct typing of Klebsiella pneumoniae present in stool samples. We find 100% concordance between the results of the O-typing assay and whole-genome sequencing of 81 culture isolates, and >90% agreement in O-typing performed directly on specimens of human stool, with disagreement arising primarily from a lack of sensitivity of the culture-based comparator method. Additionally, we find evidence for mixed O-type populations at varying levels of abundance in direct tests of stool from a hospitalized patient population. Taken together, these results demonstrate that this novel O-typing assay can be a useful tool for K. pneumoniae epidemiologic and vaccine studies.IMPORTANCEKlebsiella pneumoniae is an important opportunistic pathogen. The gastrointestinal (GI) tract is the primary reservoir of K. pneumoniae in humans, and GI carriage is believed to be a prerequisite for invasive infection. Knowledge about the dynamics and duration of GI carriage has been hampered by the lack of tools suitable for detection and strain discrimination. Real-time PCR is particularly suited to the higher-throughput workflows used in population-based studies, which are needed to improve our understanding of carriage dynamics and the factors influencing K. pneumoniae colonization.

Characterisation of European Field Goat Prion Isolates in Ovine PrP Overexpressing Transgenic Mice (Tgshp IX) Reveals Distinct Prion Strains.

After the detection of bovine spongiform encephalopathy (BSE), and a zoonotic transmissible spongiform encephalopathy (TSE) caused by the pathological prion protein (PrPSc) in two goats, the investigation of goat prions became of greater interest. Therefore, a broad collection of European goat TSE isolates, including atypical scrapie, CH1641 and goat BSE as reference prion strains were biochemically characterised and subsequently inoculated into seven rodent models for further analysis (already published results of this comprehensive study are reviewed here for comparative reasons). We report here the histopathological and immunohistochemical data of this goat TSE panel, obtained after the first passage in Tgshp IX (tg-shARQ) mice, which overexpress the ovine prion protein. In addition to the clear-cut discrimination of all reference prion strains from the classical scrapie (CS) isolates, we were further able to determine three categories of CS strains. The investigation further indicates the occurrence of sub-strains that slightly resemble distant TSE strains, such as BSE or CH1641, reinforcing the theory that CS is not a single strain but a mixture of sub-strains, existing at varying extents in one isolate. This study further proved that Tgshp IX is a potent and reliable tool for the in-depth characterisation of prion strains.

Rapid Classification and Differentiation of Sepsis-Related Pathogens Using FT-IR Spectroscopy.

Sepsis is a life-threatening condition arising from a dysregulated host immune response to infection, leading to a substantial global health burden. The accurate identification of bacterial pathogens in sepsis is essential for guiding effective antimicrobial therapy and optimising patient outcomes. Traditional culture-based bacterial typing methods present inherent limitations, necessitating the exploration of alternative diagnostic approaches. This study reports the successful application of Fourier-transform infrared (FT-IR) spectroscopy in combination with chemometrics as a potent tool for the classification and discrimination of microbial species and strains, primarily sourced from individuals with invasive infections. These samples were obtained from various children with suspected sepsis infections with bacteria and fungi originating at different sites. We conducted a comprehensive analysis utilising 212 isolates from 14 distinct genera, comprising 202 bacterial and 10 fungal isolates. With the spectral analysis taking several weeks, we present the incorporation of quality control samples to mitigate potential variations that may arise between different sample plates, especially when dealing with a large sample size. The results demonstrated a remarkable consistency in clustering patterns among 14 genera when subjected to principal component analysis (PCA). Particularly, Candida, a fungal genus, was distinctly recovered away from bacterial samples. Principal component discriminant function analysis (PC-DFA) allowed for distinct discrimination between different bacterial groups, particularly Gram-negative and Gram-positive bacteria. Clear differentiation was also observed between coagulase-negative staphylococci (CNS) and Staphylococcus aureus isolates, while methicillin-resistant S. aureus (MRSA) was also separated from methicillin-susceptible S. aureus (MSSA) isolates. Furthermore, highly accurate discrimination was achieved between Enterococcus and vancomycin-resistant enterococci isolates with 98.4% accuracy using partial least squares-discriminant analysis. The study also demonstrates the specificity of FT-IR, as it effectively discriminates between individual isolates of Streptococcus and Candida at their respective species levels. The findings of this study establish a strong groundwork for the broader implementation of FT-IR and chemometrics in clinical and microbiological applications. The potential of these techniques for enhanced microbial classification holds significant promise in the diagnosis and management of invasive bacterial infections, thereby contributing to improved patient outcomes.

Unmasking Bacterial Identities: Exploiting Silver Nanoparticle 'Masks' for Enhanced Raman Scattering in the Rapid Discrimination of Diverse Bacterial Species and Antibiotic-Resistant Strains.

Unraveling bacterial identity through Raman scattering techniques has been persistently challenging due to homogeneously amplified Raman signals across a wide variety of bacterial molecules, predominantly protein- or nucleic acid-mediated. In this study, we present an approach involving the use of silver nanoparticles to completely and uniformly "mask" adsorption on the surface of bacterial molecules through sodium borohydride and sodium chloride. This approach enables the acquisition of enhanced surface-enhanced Raman scattering (SERS) signals from all components on the bacterial surface, facilitating rapid, specific, and label-free bacterial identification. For the first time, we have characterized the identity of a bacterium, including its DNA, metabolites, and cell walls, enabling the accurate differentiation of various bacterial strains, even within the same species. In addition, we embarked on an exploration of the origin and variability patterns of the main characteristic peaks of Gram-positive and Gram-negative bacteria. Significantly, the SERS peak ratio was found to determine the inflection point of accelerated bacterial death upon treatment with antimicrobials. We further applied this platform to identify 15 unique clinical antibiotic-resistant bacterial strains, including five Escherichia coli strains in human urine, a first for Raman technology. This work has profound implications for prompt and accurate identification of bacteria, particularly antibiotic-resistant strains, thereby significantly enhancing clinical diagnostics and antimicrobial treatment strategies.

Versatile Platinum Nanoparticles-Decorated Phage Nanozyme Integrating Recognition, Bacteriolysis, and Catalysis Capabilities for On-Site Detection of Foodborne Pathogenic Strains Vitality Based on Bioluminescence/Pressure Dual-Mode Bioassay.

Sensitive and on-site discrimination of live and dead foodborne pathogenic strains remains a significant challenge due to the lack of appropriate assay and signal probes. In this work, a versatile platinum nanoparticle-decorated phage nanozyme (P2@PtNPs) that integrated recognition, bacteriolysis, and catalysis was designed to establish the bioluminescence/pressure dual-mode bioassay for on-site determination of the vitality of foodborne pathogenic strains. Benefiting from the bacterial strain-level specificity of phage, the target Salmonella typhimurium (S.T) was specially captured to form sandwich complexes with P2@PtNPs on another phage-modified glass microbead (GM@P1). As the other part of the P2@PtNPs nanozyme, the introduced PtNPs could not only catalyze the decomposition of hydrogen peroxide to generate a significant oxygen pressure signal but also produce hydroxyl radicals around the target bacteria to enhance the bacteriolysis of phage and adenosine triphosphate release. It significantly improved the bioluminescence signal. The two signals corresponded to the total and live target bacteria counts, so the dead target could be easily calculated from the difference between the total and live target bacteria counts. Meanwhile, the vitality of S.T was realized according to the ratio of live and total S.T. Under optimal conditions, the application range of this proposed bioassay for bacterial vitality was 102-107 CFU/mL, with a limit of detections for total and live S.T of 30 CFU/mL and 40 CFU/mL, respectively. This work provides an innovative and versatile nanozyme signal probe for the on-site determination of bacterial vitality for food safety.

Psychosocial stressors associated with frailty in community-dwelling older adults in the United States.

Frailty is multifactorial; however, psychosocial stressors contributing to frailty are poorly understood. This study aimed to examine whether gender, race/ ethnicity, and education are associated with differential exposure to psychosocial stressors, determine psychosocial stressors contributing to frailty, and explore the mediating psychosocial stressors pathway. This cross-sectional study involved 7679 community-dwelling older adults (≥65) from the Health and Retirement Study (2006 and 2008 waves). Psychosocial stressors such as loneliness, low subjective social status, financial strain, poor neighborhood cohesion, everyday discrimination, and traumatic life events were measured. Frailty was defined by the Fried phenotype measure. Multivariable logistic regressions were used to examine the association of gender, race/ethnicity, and education with psychosocial stressors, psychosocial stressors associated with frailty, and the mediating psychosocial stressors pathway. Females experienced greater financial strain but lower discrimination (both p < 0.05). Older adults who identified as Hispanic, Black, and racially or ethnically minoritized experienced low subjective social status, high financial strain, low neighborhood cohesion, and high discrimination than their White counterparts (all p < 0.05). Those with lower education experienced high loneliness, low subjective social status, high financial strain, low neighborhood cohesion but lower traumatic life events (all p < 0.05). Psychosocial stressors: High loneliness, low subjective social status, high financial strain, and low neighborhood cohesion (all p < 0.05) independently increased the odds of frailty. The mediating pathway of psychosocial stressors was not significant. CONCLUSION: Disparities exist in exposure to psychosocial stressors associated with frailty. Multilevel interventions are needed to reduce the influence of psychosocial stressors on frailty.

Species identification and strain discrimination of fermentation yeasts Saccharomyces cerevisiae and Saccharomyces uvarum using Raman spectroscopy and convolutional neural networks.

The use of S. cerevisiae and S. uvarum yeast starter cultures is a common practice in the alcoholic beverage fermentation industry. As yeast strains from different or the same species have variable fermentation properties, rapid and reliable typing of yeast strains plays an important role in the final quality of the product. In this study, Raman spectroscopy combined with CNN achieved accurate identification of S. cerevisiae and S. uvarum isolates at both the species and strain levels in a rapid, non-destructive, and easy-to-operate manner. This approach can be utilized to test the identity of commercialized dry yeast products and to monitor the diversity of yeast strains during fermentation. It provides great benefits as a high-throughput screening method for agri-food and the alcoholic beverage fermentation industry. This proposed method has the potential to be a powerful tool to discriminate S. cerevisiae and S. uvarum strains in taxonomic, ecological studies and fermentation applications.

Core-genome multilocus sequence typing and core-SNP analysis of Clostridium neonatale strains isolated in different spatio-temporal settings.

Clostridium neonatale was described as a new species within the Clostridium genus cluster I sensu stricto. It was recovered from the gut microbiota of infants and has been associated with necrotizing enterocolitis (NEC), a severe gastrointestinal disease affecting preterm neonates. In the absence of molecular typing methods, we developed a core (cg)-genome multilocus sequence typing (MLST) scheme based on 48 newly sequenced and 12 publicly available genomes of C. neonatale. Using the ChewBBACA algorithm, a stable MLST scheme consisting of 2,350 target genes (with genomes having ≥95% cgMLST targets) is proposed. The strains are distributed among five clades, and while some are clonality related within clades, the C. neonatale strain distribution is independent of geographic or temporal clustering. When considering strains isolated from patients with and without NEC, there were no observable differences in clustering. When compared to a core-single nucleotide polymorphism (SNP) analysis approach (31,248 positions), we showed that both cgMLST and cgSNP have comparable genetic discrimination of strains. In this study, we validated the use of cgMLST and cgSNP-based typing methods for the genetic comparison of clinical isolates of C. neonatale, which will allow for future surveillance and epidemiological clinical investigations of this potential opportunistic pathogen.IMPORTANCEClostridium neonatale has been isolated from the fecal samples of asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Taking advantage of a large collection of independent strains isolated from different spatio-temporal settings, we developed and established a cgMLST scheme for the molecular typing of C. neonatale. Both the cgMLST and cgSNP methods demonstrate comparable discrimination power. Results indicate geographic- and temporal- independent clustering of C. neonatale NEC-associated strains. No specific cgMLST clade of C. neonatale was genetically associated with NEC.

The Effects of Midlife Acute and Chronic Stressors on Black-White Differences in Cognitive Decline.

Midlife stressors may be particularly consequential for cognitive performance and disparities in cognitive decline. This study examined Black-White differences in trajectories of cognition among middle-aged adults and the effects of acute and chronic stressors on these trajectories. Data come from 4,011 cognitively healthy individuals aged 51-64 (620 Black and 3,391 White) who participated in the 2006-2018 waves of the Health and Retirement Study. Stressors included a count of recent life events and measures of financial strain and everyday discrimination. Global cognition was assessed using a modified version of the Telephone Interview for Cognitive Status. Linear mixed models with random slopes and intercepts assessed change in cognition over time. Race-by-time, race-by-stressor, time-by-stressor, and race-by-stressor-by-time interactions were assessed as were quadratic terms for time and each stressor. After adjusting for sociodemographic, health behaviors, and health-related factors, Black respondents had lower initial cognitive performance scores (b = -1.75, p < .001) but experienced earlier but slower decline in cognitive performance over time (Black × Time2 interaction: b = 0.02, p < .01). Financial strain, discrimination, and recent life events each had distinct associations with cognitive performance but did not influence racial differences in levels of or change in cognition over time. Middle-aged Black adults have lower initial cognition levels and experience earlier but less accelerated cognitive decline compared to White middle-aged adults. Midlife acute and chronic stressors influence baseline cognition but do so in different ways. Future research should examine the influence of other stressors on racial differences in cognitive decline at other points in the life course.

Near-full-length genome sequences of human norovirus GI.7[P7] and GII.4[P31] strains co-infecting a California patient in 2017.

Accurate identification of human noroviruses (HuNoVs) from outbreaks or sporadic cases is critical for source tracing and outbreak investigation. Whole-genome sequencing is a powerful tool for the detection, identification, and discrimination of HuNoV strains. We report here the nearly complete genome sequences of GI.7[P7] and GII.4[P31] strains detected in a Californian patient co-infected by both strains in 2017.

Molecular characterization of Glaesserella parasuis strains circulating in North American swine production systems

BackgroundGlaesserella parasuis is the causative agent of Glässer’s disease in pigs. Serotyping is the most common method used to type G. parasuis isolates. However, the high number of non-typables (NT) and low discriminatory power make serotyping problematic. In this study, 218 field clinical isolates and 15 G. parasuis reference strains were whole-genome sequenced (WGS). Multilocus sequence types (MLST), serotypes, core-genome phylogeny, antimicrobial resistance (AMR) genes, and putative virulence gene information was extracted.ResultsIn silico WGS serotyping identified 11 of 15 serotypes. The most frequently detected serotypes were 7, 13, 4, and 2. MLST identified 72 sequence types (STs), of which 66 were novel. The most predominant ST was ST454. Core-genome phylogeny depicted 3 primary lineages (LI, LII, and LIII), with LIIIA sublineage isolates lacking all vtaA genes, based on the structure of the phylogenetic tree and the number of virulence genes. At least one group 1 vtaA virulence genes were observed in most isolates (97.2%), except for serotype 8 (ST299 and ST406), 15 (ST408 and ST552) and NT (ST448). A few group 1 vtaA genes were significantly associated with certain serotypes or STs. The putative virulence gene lsgB, was detected in 8.3% of the isolates which were predominantly of serotype 5/12. While most isolates carried the bcr, ksgA, and bacA genes, the following antimicrobial resistant genes were detected in lower frequency; blaZ (6.9%), tetM (3.7%), spc (3.7%), tetB (2.8%), bla-ROB-1 (1.8%), ermA (1.8%), strA (1.4%), qnrB (0.5%), and aph3''Ia (0.5%). ConclusionThis study showed the use of WGS to type G. parasuis isolates and can be considered an alternative to the more labor-intensive and traditional serotyping and standard MLST. Core-genome phylogeny provided the best strain discrimination. These findings will lead to a better understanding of the molecular epidemiology and virulence in G. parasuis that can be applied to the future development of diagnostic tools, autogenous vaccines, evaluation of antibiotic use, prevention, and disease control.

A 3D Composited Flexible Sensor Based on Percolative Nanoparticle Arrays to Discriminate Coupled Pressure and Strain.

Flexible mechanical sensors based on nanomaterials operate on a deformation-response mechanism, making it challenging to discern different types of mechanical stimuli such as pressure and strain. Therefore, these sensors are susceptible to significant mechanical interference. Here, we introduce a multifunctional flexible sensor capable of discriminating coupled pressure and strain without cross-interference. Our design involves an elastic cantilever fixed on the pillar of the flexible main substrate, creating a three-dimensional (3D) substrate, and two percolative nanoparticle (NP) arrays are deposited on the cantilever and main substrate, respectively, as the sensing materials. The 3D flexible substrate could confine pressure/strain loading exclusively on the cantilever or main substrate, resulting in independent responses of the two nanoparticle arrays with no cross-interference. Benefitting from the quantum transport in nanoparticle arrays, our sensors demonstrate an exceptional sensitivity, enabling discrimination of subtle strains down to 1.34 × 10-4. Furthermore, the suspended cantilever with one movable end can enhance the pressure perception of the NP array, exhibiting a high sensitivity of -0.223 kPa-1 and an ultrahigh resolution of 4.24 Pa. This flexible sensor with multifunctional design will provide inspiration for the development of flexible mechanical sensors and the advancement of decoupling strategies.

Whole-Genome Sequencing of Shiga Toxin-Producing Escherichia coli for Characterization and Outbreak Investigation.

Shiga toxin-producing Escherichia coli (STEC) causes high frequencies of foodborne infections worldwide and has been linked to numerous outbreaks each year. Pulsed-field gel electrophoresis (PFGE) has been the gold standard for surveillance until the recent transition to whole-genome sequencing (WGS). To further understand the genetic diversity and relatedness of outbreak isolates, a retrospective analysis of 510 clinical STEC isolates was conducted. Among the 34 STEC serogroups represented, most (59.6%) belonged to the predominant six non-O157 serogroups. Core genome single nucleotide polymorphism (SNP) analysis differentiated clusters of isolates with similar PFGE patterns and multilocus sequence types (STs). One serogroup O26 outbreak strain and another non-typeable (NT) strain, for instance, were identical by PFGE and clustered together by MLST; however, both were distantly related in the SNP analysis. In contrast, six outbreak-associated serogroup O5 strains clustered with five ST-175 serogroup O5 isolates, which were not part of the same outbreak as determined by PFGE. The use of high-quality SNP analyses enhanced the discrimination of these O5 outbreak strains into a single cluster. In all, this study demonstrates how public health laboratories can more rapidly use WGS and phylogenetics to identify related strains during outbreak investigations while simultaneously uncovering important genetic attributes that can inform treatment practices.

Examining pandemic stress and mental health among a community-based sample of sexual minority women in Los Angeles County

ABSTRACT In partnership with community stakeholders, the present study aimed to gather descriptive data on pandemic-related stress and its association with mental health outcomes among a community sample of self-identified sexual minority women (SMW) in Los Angeles County (N = 84; Mage = 35.61). The sample was comprised solely of women (i.e. self-identified gender identity as woman, including cisgender and transgender women). Data were collected in April 2021, the ‘third wave’ of the COVID-19 global pandemic. Los Angeles experienced some of the highest COVID-19 incidence rates in the United States. Yet, there was a significant lack of COVID-19 data on SMW, thus making it difficult to address the specific needs of this community. Background data were collected on pre-existing health conditions and COVID-19 infection history. Data were specific to COVID-19 stressors in the areas of mental health, financial strain, social isolation and health and discrimination concerns. Findings revealed that mental health concern was strongly associated with financial strain (r = .63, p < .01), social isolation (r = .62, p < .01) and health and discrimination concerns (r = .63, p < .01), thus demonstrating the wide-ranging negative impact of pandemic stressors on the mental health of SMW in Los Angeles during the third wave of COVID-19. Recommendations for future research, practice and policy implications are discussed.

Radiation effects on Brillouin-based Sensors: Feasibility of temperature and strain discrimination using LEAF Single-Mode optical fiber

In this paper, we investigate the accessible performances in terms of strain and temperature discrimination using Brillouin Optical Time Domain Analysis (BOTDA) combined with the LEAF fiber (from Corning) when exposed to γ-rays up to doses of 1MGy(SiO2). This optical fiber offers a multipeak Brillouin Gain Spectrum (BGS) with unique dependencies of each of its peaks over temperature and strain. The discrimination capability evolution of LEAF is investigated at high γ doses and dose rates to evaluate the radiation effects on the sensing performances. Gamma irradiation leads to modifications of the LEAF BGS signature through two main effects. First, the Radiation Induced Attenuation (RIA) of this germanosilicate optical fiber limits the BGS amplitude, sensing range and capability for discrimination over BGS amplitude. Second, radiations modify the Brillouin scattering properties by slightly changing the refractive indices and the acoustic velocities of the fiber core and cladding leading to a small change in temperature and strain dependences as well as a limited Radiation Induced Brillouin Frequency Shift (RI-BFS) causing a direct error on the measured parameters. Results exhibit an overall decrease of temperature and strain uncertainties through discrimination process after 1MGy irradiation reaching 0.9°C and 29με for 0.1 MHz frequency uncertainty.

The pre-ejection period is a highly stress dependent parameter of paramount importance for pulse-wave-velocity based applications.

The pulse-wave-velocity, is used for indirect, cuff-less, continuous blood pressure estimation. It is commonly detected by measuring the time delay between a defined point in an ECG and the arrival of the peripheral pulse wave (e.g., oxygen saturation sensor). The period between electrical stimulation of the heart (ECG) and actual blood ejection from the heart is called the pre-ejection period (PEP). This study aims at characterizing the PEP under mental and physical stress with focus on its relations to other cardiovascular parameters such as heart rate and importance for blood pressure (BP) estimation. We measured the PEP in 71 young adults at rest, under mental (TSST) and physical stress (ergometer) via impedance-cardiography. The PEP is highly dependent on mental and physical load. It is strongly correlated with indicators of sympathetic strain (p < 0.001). At rest (mean 104.5 ms), the PEP shows a high interindividual variability but small intraindividual variability. Mental stress decreases the PEP by 16% (mean 90.0 ms) while physical stress halves PEP (mean 53.9 ms). The PEP does correlate differently with heart rate under differing circumstances (rest: R 2 0.06, mental stress: R 2 0.29, physical stress: R 2 0.65). Subsequently, using PEP and heart rate enables the discrimination of rest, mental and physical strain with a positive predictive value of 93%. The PEP is a cardiovascular parameter with large interindividual variability at rest and subject-depended dynamic under load which is of great importance for ECG-based pulse-wave-velocity (PWV) determination. Considering its variability and large impact on the pulse arrival time, PEP is a crucial factor in PWV based BP estimation.

Development of Spectral Nano-Flow Cytometry for High-Throughput Multiparameter Analysis of Individual Biological Nanoparticles

Correlated analysis of multiple biochemical parameters at the single-particle level and in a high-throughput manner is essential for insights into the diversity and functions of biological nanoparticles (BNPs), such as bacteria and subcellular organelles. To meet this challenge, we developed a highly sensitive spectral nano-flow cytometer (S-nFCM) by integrating a spectral recording module to a laboratory-built nFCM that is 4-6 orders of magnitude more sensitive in side scattering detection and 1-2 orders of magnitude more sensitive in fluorescence detection than conventional flow cytometers. An electron-multiplying charge-coupled device (EMCCD) was used to acquire the full fluorescence spectra of single BNPs upon holographic grating dispersion. Up to 10,000 spectra can be collected in 1 min with 2.1 nm resolution. The precision, linearity, and sensitivity were examined. Complete discernment of single influenza viruses against the background signal, discrimination of different strains of marine cyanobacteria in a mixed sample based on their spectral properties of natural fluorescence, classification of bacterial categories exhibiting different patterns of antigen expression, and multiparameter analysis of single mitochondria for drug discovery were successfully demonstrated.

Evaluation of Genomic Typing Methods in the Salmonella Reference Laboratory in Public Health, England, 2012–2020

We aim to provide an evidence-based evaluation of whole genome sequence (WGS) methods, employed at the Salmonella reference laboratory in England, in terms of its impact on public health and whether these methods remain a fit for purpose test under UKAS ISO 15189. The evaluation of the genomic methods were mapped against the value of detecting microbiological clusters to support the investigation of food-borne outbreaks of Salmonella in England between 2012-2020. The analysis of WGS with both SNP- and allelic-based methods provided an unprecedented level of strain discrimination and detection of additional clusters when comparing to all of the previous typing methods. The robustness of the routine genomic sequencing at the reference laboratory ensured confidence in the microbiological identifications, even in large outbreaks with complex international food distribution networks. There was evidence that the phylogeny derived from the WGS data can be used to inform the provenance of strains and support discrimination between domestic and non-domestic transmission events. Further insight on the evolutionary context of the emerging pathogenic strains was enabled with a deep dive of the phylogenetic data, including the detection of nested clusters. The public availability of the WGS data linked to the clinical, epidemiological and environmental context of the sequenced strains has improved the trace-back investigations during outbreaks. The global expansion in the use of WGS-based typing in reference laboratories has shown that the WGS methods are a fit for purpose test in public health as it has ensured the rapid implementation of interventions to protect public health, informed risk assessment and has facilitated the management of national and international food-borne outbreaks of Salmonella.