Characteristics and pathogenicity of Vibrio alginolyticus SWS causing high mortality in mud crab (Scylla serrata) aquaculture in Hong Kong.

Introduction: Vibrio alginolyticus is a Gram-negative, rod-shaped bacterium belonging to the family of Vibrionaceae, a common pathogen in aquaculture animals, However, studies on its impact on Scylla serrata (mud crabs) are limited. In this study, we isolated V. alginolyticus SWS from dead mud crab during a disease outbreak in a Hong Kong aquaculture farm, which caused up to 70% mortality during summer. Methods: Experimental infection and histopathology were used to investigate the pathogenicity of V. alginolyticus SWS in S. serrata and validate Koch's postulates. Comprehensive whole-genome analysis and phylogenetic analysis antimicrobial susceptibility testing, and biochemical characterization were also performed. Results: Our findings showed that V. alginolyticus SWS caused high mortality (75%) in S. serrata with infected individuals exhibiting inactivity, loss of appetite, decolored and darkened hepatopancreas, gills, and opaque muscle in the claw. Histopathological analysis revealed tissue damage and degeneration in the hepatopancreas, gills, and claw muscle suggesting direct and indirect impacts of V. alginolyticus SWS infection. Conclusions: This study provides a comprehensive characterization of V. alginolyticus SWS as an emerging pathogen in S. serrata aquaculture. Our findings underscore the importance of ongoing surveillance, early detection, and the development of targeted disease management strategies to mitigate the economic impact of vibriosis outbreaks in mud crab aquaculture.

Transmission Patterns of Co-Circulation of Omicron Sub-Lineages in Hong Kong SAR, China, a City with Rigorous Social Distancing Measures, in 2022.

OBJECTIVE: This study aimed to characterize the changing landscape of circulating SARS-CoV-2 lineages in the local community of Hong Kong throughout 2022. We examined how adjustments to quarantine arrangements influenced the transmission pattern of Omicron variants in a city with relatively rigorous social distancing measures at that time. METHODS: In 2022, a total of 4684 local SARS-CoV-2 genomes were sequenced using the Oxford Nanopore GridION sequencer. SARS-CoV-2 consensus genomes were generated by MAFFT, and the maximum likelihood phylogeny of these genomes was determined using IQ-TREE. The dynamic changes in lineages were depicted in a time tree created by Nextstrain. Statistical analysis was conducted to assess the correlation between changes in the number of lineages and adjustments to quarantine arrangements. RESULTS: By the end of 2022, a total of 83 SARS-CoV-2 lineages were identified in the community. The increase in the number of new lineages was significantly associated with the relaxation of quarantine arrangements (One-way ANOVA, F(5, 47) = 18.233, p < 0.001)). Over time, Omicron BA.5 sub-lineages replaced BA.2.2 and became the predominant Omicron variants in Hong Kong. The influx of new lineages reshaped the dynamics of Omicron variants in the community without fluctuating the death rate and hospitalization rate (One-way ANOVA, F(5, 47) = 2.037, p = 0.091). CONCLUSION: This study revealed that even with an extended mandatory quarantine period for incoming travelers, it may not be feasible to completely prevent the introduction and subsequent community spread of highly contagious Omicron variants. Ongoing molecular surveillance of COVID-19 remains essential to monitor the emergence of new recombinant variants.

Advancements in magnetic nanoparticle-based biosensors for point-of-care testing.

The significance of point-of-care testing (POCT) in early clinical diagnosis and personalized patient care is increasingly recognized as a crucial tool in reducing disease outbreaks and improving patient survival rates. Within the realm of POCT, biosensors utilizing magnetic nanoparticles (MNPs) have emerged as a subject of substantial interest. This review aims to provide a comprehensive evaluation of the current landscape of POCT, emphasizing its growing significance within clinical practice. Subsequently, the current status of the combination of MNPs in the Biological detection has been presented. Furthermore, it delves into the specific domain of MNP-based biosensors, assessing their potential impact on POCT. By combining existing research and spotlighting pivotal discoveries, this review enhances our comprehension of the advancements and promising prospects offered by MNP-based biosensors in the context of POCT. It seeks to facilitate informed decision-making among healthcare professionals and researchers while also promoting further exploration in this promising field of study.

Comprehensive identification of pathogenic microbes and antimicrobial resistance genes in food products using nanopore sequencing-based metagenomics.

Foodborne pathogens, particularly antimicrobial-resistant (AMR) bacteria, remain a significant threat to global health. Given the limitations of conventional culture-based approaches, which are limited in scope and time-consuming, metagenomic sequencing of food products emerges as a promising solution. This method provides a fast and comprehensive way to detect the presence of pathogenic microbes and antimicrobial resistance genes (ARGs). Notably, nanopore long-read sequencing provides more accurate bacterial taxonomic classification in comparison to short-read sequencing. Here, we revealed the impact of food types and attributes (origin, retail place, and food processing methods) on microbial communities and the AMR profile using nanopore metagenomic sequencing. We analyzed a total of 260 food products, including raw meat, sashimi, and ready-to-eat (RTE) vegetables. Clostridium botulinum, Acinetobacter baumannii, and Vibrio parahaemolyticus were identified as the top three foodborne pathogens in raw meat and sashimi. Importantly, even with low pathogen abundance, higher percentages of samples containing carbapenem and cephalosporin resistance genes were identified in chicken and RTE vegetables, respectively. In parallel, our results demonstrated that fresh, peeled, and minced foods exhibited higher levels of pathogenic bacteria. In conclusion, this comprehensive study offers invaluable data that can contribute to food safety assessments and serve as a basis for quality indicators.

Medulla Tetrapanacis water extract alleviates inflammation and infection by regulating macrophage polarization through MAPK signaling pathway.

Medulla Tetrapanacis (MT) is a commonly used herb to promote lactation and manage mastitis in lactating mothers. However, its anti-inflammatory and anti-bacterial effects are currently unknown. We hypothesized that MT water extract possesses anti-inflammatory and anti-bacterial effects by modulating macrophage polarization to reduce the release of inflammatory mediators and phagocytosis via inactivation of MAPKs pathways. The chemical composition of the MT water extract was analyzed by UPLC-Orbitrap-mass spectrometry. The anti-inflammatory and anti-bacterial properties of the MT water extract were examined using LPS-stimulated inflammation and Staphylococcus aureus infection model in RAW 264.7 cells, respectively. The underlying mechanism of action of the MT water extract was also investigated. We identified eight compounds by UPLC-Orbitrap-mass spectrometry that are abundant within the MT water extract. MT water extract significantly suppressed LPS-induced nitric oxide, TNF-α and IL-6 secretion in RAW 264.7 cells which was accompanied by the promotion of macrophage polarization from pro-inflammatory towards anti-inflammatory phenotypes. MT water extract significantly suppressed the LPS-induced MAPK activation. Finally, MT water extract decreased the phagocytic capacity of the RAW 264.7 cells against S. aureus infection. MT water extract could suppress LPS-induced inflammation by promoting macrophages towards an anti-inflammatory phenotype. In addition, MT also inhibited the growth of S. aureus.

Successful Management of Fetal Torsades de Pointes and Long QT Syndrome by a Cardio-Obstetrical Team.

A 32-week fetus with tachycardia and bradycardia, diagnosed with torsades de pointes, atrioventricular block, and sinus bradycardia due to a de novo KCNH2 mutation was successfully managed by a cardio-obstetrical team. Maternal/fetal pharmacogenomic testing resulted in appropriate drug dosing without toxicity and delivery of a term infant in sinus rhythm.

A sensitive and simple RT-LAMP assay for sarbecovirus screening in bats.

IMPORTANCE: We report the application of a colorimetric and fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to facilitate mass screening for sarbecoviruses in bats. The assay was evaluated using a total of 838 oral and alimentary samples from bats and demonstrated comparable sensitivity and specificity to quantitative reverse transcription PCR (qRT-PCR), with a simple setup. The addition of SYTO9, a fluorescent nucleic acid stain, also allows for quantitative analysis. The scalability and simplicity of the assay are believed to contribute to improving preparedness for detecting emerging coronaviruses by applying it to field studies and surveillance.

Role of Legionella pneumophila outer membrane vesicles in host-pathogen interaction.

Legionella pneumophila is an opportunistic intracellular pathogen that inhabits artificial water systems and can be transmitted to human hosts by contaminated aerosols. Upon inhalation, it colonizes and grows inside the alveolar macrophages and causes Legionnaires' disease. To effectively control and manage Legionnaires' disease, a deep understanding of the host-pathogen interaction is crucial. Bacterial extracellular vesicles, particularly outer membrane vesicles (OMVs) have emerged as mediators of intercellular communication between bacteria and host cells. These OMVs carry a diverse cargo, including proteins, toxins, virulence factors, and nucleic acids. OMVs play a pivotal role in disease pathogenesis by helping bacteria in colonization, delivering virulence factors into host cells, and modulating host immune responses. This review highlights the role of OMVs in the context of host-pathogen interaction shedding light on the pathogenesis of L. pneumophila. Understanding the functions of OMVs and their cargo provides valuable insights into potential therapeutic targets and interventions for combating Legionnaires' disease.

Extracellular Vesicles in Microbes, Pathogens, and Infectious Diseases.

Extracellular vesicles (EVs) are nanosized lipid bilayer particles that are produced by all kinds of organisms, including both pathogenic and non-pathogenic archaea, bacteria, fungi, and parasites [...].

Impairment of novel non-coding small RNA00203 inhibits biofilm formation and reduces biofilm-specific antibiotic resistance in Acinetobacter baumannii.

Small RNAs (sRNAs) are post-transcriptional regulators of many biological processes in bacteria, including biofilm formation and antibiotic resistance. The mechanisms by which sRNA regulates the biofilm-specific antibiotic resistance in Acinetobacter baumannii have not been reported to date. This study aimed to investigate the influence of sRNA00203 (53 nucleotides) on biofilm formation, antibiotic susceptibility, and expression of genes associated with biofilm formation and antibiotic resistance. The results showed that deletion of the sRNA00203-encoding gene decreased the biomass of biofilm by 85%. Deletion of the sRNA00203-encoding gene also reduced the minimum biofilm inhibitory concentrations for imipenem and ciprofloxacin 1024- and 128-fold, respectively. Knocking out of sRNA00203 significantly downregulated genes involved in biofilm matrix synthesis (pgaB), efflux pump production (novel00738), lipopolysaccharide biosynthesis (novel00626), preprotein translocase subunit (secA) and the CRP transcriptional regulator. Overall, the suppression of sRNA00203 in an A. baumannii ST1894 strain impaired biofilm formation and sensitized the biofilm cells to imipenem and ciprofloxacin. As sRNA00203 was found to be conserved in A. baumannii, a therapeutic strategy targeting sRNA00203 may be a potential solution for the treatment of biofilm-associated infections caused by A. baumannii. To the best of the authors' knowledge, this is the first study to show the impact of sRNA00203 on biofilm formation and biofilm-specific antibiotic resistance in A. baumannii.

Probable airborne transmission of Burkholderia pseudomallei causing an urban outbreak of melioidosis during typhoon season in Hong Kong, China.

Between January 2015 and October 2022, 38 patients with culture-confirmed melioidosis were identified in the Kowloon West (KW) Region, Hong Kong. Notably, 30 of them were clustered in the Sham Shui Po (SSP) district, which covers an estimated area of 2.5 km2. Between August and October 2022, 18 patients were identified in this district after heavy rainfall and typhoons. The sudden upsurge in cases prompted an environmental investigation, which involved collecting 20 air samples and 72 soil samples from residential areas near the patients. A viable isolate of Burkholderia pseudomallei was obtained from an air sample collected at a building site five days after a typhoon. B. pseudomallei DNA was also detected in 21 soil samples collected from the building site and adjacent gardening areas using full-length 16S rRNA gene sequencing, suggesting that B. psuedomallei is widely distributed in the soil environment surrounding the district. Core genome-multilocus sequence typing showed that the air sample isolate was phylogenetically clustered with the outbreak isolates in KW Region. Multispectral satellite imagery revealed a continuous reduction in vegetation region in SSP district by 162,255 m2 from 2016 to 2022, supporting the hypothesis of inhalation of aerosols from the contaminated soil as the transmission route of melioidosis during extreme weather events. This is because the bacteria in unvegetated soil are more easily spread by winds. In consistent with inhalational melioidosis, 24 (63.2%) patients had pneumonia. Clinicians should be aware of melioidosis during typhoon season and initiate appropriate investigation and treatment for patients with compatible symptoms.

Enzyme-Assisted Nucleic Acid Amplification in Molecular Diagnosis: A Review.

Infectious diseases and tumors have become the biggest medical challenges in the 21st century. They are driven by multiple factors such as population growth, aging, climate change, genetic predispositions and more. Nucleic acid amplification technologies (NAATs) are used for rapid and accurate diagnostic testing, providing critical information in order to facilitate better follow-up treatment and prognosis. NAATs are widely used due their high sensitivity, specificity, rapid amplification and detection. It should be noted that different NAATs can be selected according to different environments and research fields; for example, isothermal amplification with a simple operation can be preferred in developing countries or resource-poor areas. In the field of translational medicine, CRISPR has shown great prospects. The core component of NAAT lies in the activity of different enzymes. As the most critical material of nucleic acid amplification, the key role of the enzyme is self-evident, playing the upmost important role in molecular diagnosis. In this review, several common enzymes used in NAATs are compared and described in detail. Furthermore, we summarize both the advances and common issues of NAATs in clinical application.

Genomics: Infectious Disease and Host-Pathogen Interaction.

Infectious diseases, which are caused by pathogens such as bacteria, viruses, fungi, and parasites, pose a serious threat to humans, animals, and plants [...].

Evolution of postoperative care: marked reduction of opioid consumption when ERAC pathway added to wound soaker therapy for cesarean delivery.

OBJECTIVE: Achieving functional recovery after cesarean delivery is critical to a parturient's ability to care for herself and her newborn. Adequate pain control is vital, and without it, many other aspects of the recovery process may be delayed. Reducing opioid consumption without compromising analgesia is of paramount importance, and enhanced recovery pathways have generated considerable interest given their ability to facilitate this. Our group's process for reducing opioid consumption for cesarean delivery patients evolved over time. We first demonstrated that providing additional incisional pain control with continuous bupivacaine infusions through wound catheters, with the concurrent use of neuraxial morphine, reduced postoperative opioid use. Iterations of an enhanced recovery after cesarean (ERAC) delivery pathway were then implemented after the Society for Obstetric Anesthesia and Perinatology's consensus statement for ERAC was issued to eliminate variability in both hospital course and in the treatment of postoperative pain. In this retrospective cohort analysis, we sought to identify whether adding ERAC protocols to our existing combination of neuraxial morphine and wound soaker catheters further reduced opioid consumption after cesarean delivery. METHODS: A retrospective cohort analysis of cesarean deliveries from 2015 through 2020 was performed. Deliveries were divided by analgesic pathway into four time-periods - time-point 1 [January 2015-April 2016, previous standard of care (control, N = 61)]: neuraxial morphine in addition to as needed opioid and non-opioid analgesics; time-point 2 [May 2016-May 2019, introduction of wound soaker (wound-soaker, N = 40)]: continuous wound catheter infusions of local anesthetic, neuraxial morphine in addition to as needed opioid and non-opioid analgesics; time-point 3 [May 2019-December 2019, wound soaker + early ERAC pathway (early ERAC, N = 78)]: continuous wound catheter infusion of local anesthetic, neuraxial morphine, in addition to scheduled non-opioid analgesics (acetaminophen and ibuprofen) every 6 h, alternating in relation to one another so that one is given every 3 h; time-point 4 [January 2020-July 2020, wound soaker + late ERAC pathway (late ERAC, N = 57)]: continuous wound catheter infusion of local anesthetic, neuraxial morphine in addition to non-opioid analgesics scheduled together every 6 h (to facilitate periods of uninterrupted rest). Cumulative and average daily opioid use for postoperative days (POD) 1-4 were analyzed using ANOVA and a mixed effect model, respectively. RESULTS: Average daily opioid consumption and total cumulative opioid consumption POD 1-4 (morphine milligram equivalents) for both early and late ERAC groups (23.9 ± 31.1 and 29.4 ± 35.1) were significantly reduced compared to control and wound soaker groups (185.1 ± 93.7 and 134.8 ± 77.1) (p < .001). CONCLUSION: The addition of ERAC protocols to our standardized multimodal analgesic regimen (local anesthetic wound infusion catheters and neuraxial morphine) for cesarean delivery significantly reduced postoperative opioid consumption.

The clinical utility of Nanopore 16S rRNA gene sequencing for direct bacterial identification in normally sterile body fluids.

The prolonged incubation period of traditional culture methods leads to a delay in diagnosing invasive infections. Nanopore 16S rRNA gene sequencing (Nanopore 16S) offers a potential rapid diagnostic approach for directly identifying bacteria in infected body fluids. To evaluate the clinical utility of Nanopore 16S, we conducted a study involving the collection and sequencing of 128 monomicrobial samples, 65 polymicrobial samples, and 20 culture-negative body fluids. To minimize classification bias, taxonomic classification was performed using 3 analysis pipelines: Epi2me, Emu, and NanoCLUST. The result was compared to the culture references. The limit of detection of Nanopore 16S was also determined using simulated bacteremic blood samples. Among the three classifiers, Emu demonstrated the highest concordance with the culture results. It correctly identified the taxon of 125 (97.7%) of the 128 monomicrobial samples, compared to 109 (85.2%) for Epi2me and 102 (79.7%) for NanoCLUST. For the 230 cultured species in the 65 polymicrobial samples, Emu correctly identified 188 (81.7%) cultured species, compared to 174 (75.7%) for Epi2me and 125 (54.3%) for NanoCLUST. Through ROC analysis on the monomicrobial samples, we determined a threshold of relative abundance at 0.058 for distinguishing potential pathogens from background in Nanopore 16S. Applying this threshold resulted in the identification of 107 (83.6%), 117 (91.4%), and 114 (91.2%) correctly detected samples for Epi2me, Emu, and NanoCLUST, respectively, in the monomicrobial samples. Nanopore 16S coupled with Epi2me could provide preliminary results within 6 h. However, the ROC analysis of polymicrobial samples exhibited a random-like performance, making it difficult to establish a threshold. The overall limit of detection for Nanopore 16S was found to be about 90 CFU/ml.

Effects of Sub-Minimum Inhibitory Concentrations of Imipenem and Colistin on Expression of Biofilm-Specific Antibiotic Resistance and Virulence Genes in Acinetobacter baumannii Sequence Type 1894.

Antibiotics at suboptimal doses promote biofilm formation and the development of antibiotic resistance. The underlying molecular mechanisms, however, were not investigated. Here, we report the effects of sub-minimum inhibitory concentrations (sub-MICs) of imipenem and colistin on genes associated with biofilm formation and biofilm-specific antibiotic resistance in a multidrug-tolerant clinical strain of Acinetobacter baumannii Sequence Type (ST) 1894. Comparative transcriptome analysis was performed in untreated biofilm and biofilm treated with sub-MIC doses of imipenem and colistin. RNA sequencing data showed that 78 and 285 genes were differentially expressed in imipenem and colistin-treated biofilm cells, respectively. Among the differentially expressed genes (DEGs), 48 and 197 genes were upregulated exclusively in imipenem and colistin-treated biofilm cells, respectively. The upregulated genes included those encoding matrix synthesis (pgaB), multidrug efflux pump (novel00738), fimbrial proteins, and homoserine lactone synthase (AbaI). Upregulation of biofilm-associated genes might enhance biofilm formation when treated with sub-MICs of antibiotics. The downregulated genes include those encoding DNA gyrase (novel00171), 30S ribosomal protein S20 (novel00584), and ribosome releasing factor (RRF) were downregulated when the biofilm cells were treated with imipenem and colistin. Downregulation of these genes affects protein synthesis, which in turn slows down cell metabolism and makes biofilm cells more tolerant to antibiotics. In this investigation, we also found that 5 of 138 small RNAs (sRNAs) were differentially expressed in biofilm regardless of antibiotic treatment or not. Of these, sRNA00203 showed the highest expression levels in biofilm. sRNAs regulate gene expression and are associated with biofilm formation, which may in turn affect the expression of biofilm-specific antibiotic resistance. In summary, when biofilm cells were exposed to sub-MIC doses of colistin and imipenem, coordinated gene responses result in increased biofilm production, multidrug efflux pump expression, and the slowdown of metabolism, which leads to drug tolerance in biofilm. Targeting antibiotic-induced or repressed biofilm-specific genes represents a new strategy for the development of innovative and effective treatments for biofilm-associated infections caused by A. baumannii.

Development of a high specificity typing method for the detection of herpes simplex virus.

Herpes disease is caused by Herpes simplex virus (HSV). It has become one of the global health problems. This paper reports a method for HSV type testing. First specific primers sequence for HSV-1 and HSV-2 were selected, designed, and synthesized. Then, these amplification products were proved by sequencing and analysis. Lastly, we optimized the reaction system and PCR reaction program by orthogonal design and sensitivity testing. Results showed that the lowest concentration in HSV-type testing is about 6.67 × 106 copies/ml. Moreover, the specificity of detection was very high. So, this method has very great potentials for HSV type testing in clinical practice.

Induction of amphotericin B resistance in susceptible Candida auris by extracellular vesicles.

Drug resistance derived from extracellular vesicles (EVs) is an increasingly important research area but has seldom been described regarding fungal pathogens. Here, we characterized EVs derived from a triazole-resistant but amphotericin B-susceptible strain of Candida auris. Nano- to microgram concentrations of C. auris EVs prepared from both broth and solid agar cultures could robustly increase the yeast's survival against both pure and clinical amphotericin B formulations in a dose-dependent manner, resulting in up to 16-fold changes of minimum inhibitory concentration. Meanwhile, this effect was not observed upon addition of these EVs to C. albicans, nor upon addition of C. albicans EVs to C. auris. No change in susceptibilities was observed upon EV treatment for fluconazole, voriconazole, micafungin, and flucytosine. Mass spectrometry indicated the presence of immunogenic-/drug resistance-implicated proteins in C. auris EVs, including alcohol dehydrogenase 1 as well as C. albicans Mp65-like and Xog1-like proteins in high quantities. Based on these observations, we propose a potential species-specific role for EVs in amphotericin B resistance in C. auris. These observations may provide critical insights into treatment of multidrug-resistant C. auris.

Recent Advances of Human Leukocyte Antigen (HLA) Typing Technology Based on High-Throughput Sequencing.

The major histocompatibility complex (MHC) in humans is a genetic region consisting of cell surface proteins located on the short arm of chromosome 6. This is also known as the human leukocyte antigen (HLA) region. The HLA region consists of genes that exhibit complex genetic polymorphisms, and are extensively involved in immune responses. Each individual has a unique set of HLAs. Donor-recipient HLA allele matching is an important factor for organ transplantation. Therefore, an established rapid and accurate HLA typing technology is instrumental to preventing graft-verses-host disease (GVHD) in organ recipients. As of recent, high-throughput sequencing has allowed for an increase read length and higher accuracy and throughput, thus achieving complete and high-resolution full-length typing. With more advanced nanotechnology used in high-throughput sequencing, HLA typing is more widely used in third-generation single-molecule sequencing. This review article summarizes some of the most widely used sequencing typing platforms and evaluates the latest developments in HLA typing kits and their clinical applications.

A Sensitive and Specific Competitive Enzyme-Linked Immunosorbent Assay for Serodiagnosis of COVID-19 in Animals.

In addition to human cases, cases of COVID-19 in captive animals and pets are increasingly reported. This raises the concern for two-way COVID-19 transmission between humans and animals. Here, we developed a SARS-CoV-2 nucleocapsid protein-based competitive enzyme-linked immunosorbent assay (cELISA) for serodiagnosis of COVID-19 which can theoretically be used in virtually all kinds of animals. We used 187 serum samples from patients with/without COVID-19, laboratory animals immunized with inactive SARS-CoV-2 virions, COVID-19-negative animals, and animals seropositive to other betacoronaviruses. A cut-off percent inhibition value of 22.345% was determined and the analytical sensitivity and specificity were found to be 1:64-1:256 and 93.9%, respectively. Evaluation on its diagnostic performance using 155 serum samples from COVID-19-negative animals and COVID-19 human patients showed a diagnostic sensitivity and specificity of 80.8% and 100%, respectively. The cELISA can be incorporated into routine blood testing of farmed/captive animals for COVID-19 surveillance.