Antibacterial Efficacy and Mechanisms of Action of a Novel Beta-Defensin from Snakehead Murrel, Channa striata.

Beta-defensins, identified from fishes, constitute a crucial category of antimicrobial peptides important in combating bacterial fish pathogens. The present investigation centers on the molecular and functional characterization of CsDef, a 63-amino acid beta-defensin antimicrobial peptide derived from snakehead murrel (Channa striata). The physicochemical attributes of CsDef align with the distinctive characteristics observed in AMPs. CsDef was recombinantly produced, and the recombinant peptide, rCsDef, exhibited notable antibacterial efficacy against bacterial fish pathogens with an MIC of 16 µM for V. proteolyticus. A. hydrophila exhibited 91% inhibition, E. tarda 92%, and V. harveyi 53% at 32 µM of rCsDef. The rCsDef exhibited a multifaceted mechanism of action against bacteria, i.e., through membrane depolarization, membrane permeabilization, and generation of ROS. The rCsDef was non-hemolytic to hRBCs and non-cytotoxic to normal mammalian cell line CHO-K1. However, it exhibited anticancer properties in MCF-7. rCsDef demonstrated notable stability with respect to pH, temperature, salt, metal ions, and proteases. These findings suggest it is a potential candidate molecule for prospective applications in aquaculture.

Coxiella burnetii-containing vacuoles interact with host recycling endosomal proteins Rab11a and Rab35 for vacuolar expansion and bacterial growth.

Introduction: Coxiella burnetii is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for Coxiella in the U.S. warrants further research into Coxiella pathogenesis. Within the host cells, Coxiella replicates in an acidic phagolysosome-like vacuole termed Coxiella-containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for Coxiella survival and that the Coxiella Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the 'immature' endosomes in Coxiella- infected cells remained unclear. Methods: We transfected HeLa cells with GFP-tagged Rab proteins and subsequently infected them with mCherry-Coxiella to visualize Rab protein localization. Infected cells were immunostained with anti-Rab antibodies to confirm the Rab localization to the CCV, to quantitate Rab11a and Rab35- positive CCVs, and to quantitate total recycling endosome content of infected cells. A dual-hit siRNA mediated knockdown combined with either immunofluorescent assay or an agarose-based colony-forming unit assay were used to measure the effects of Rab11a and Rab35 knockdown on CCV area and Coxiella intracellular growth. Results: The CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. Quantitation of Rab11a/Rab35-positive CCVs revealed that while Rab11a interacts with the CCV more at 3 dpi, Rab35 is significantly more prevalent at CCVs at 6 dpi, suggesting that the CCV preferentially interacts with Rab11a and Rab35 depending on the stage of infection. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in Coxiella-infected cells compared to mock, suggesting that Coxiella increases the recycling endosome content in infected cells. Finally, siRNA-mediated knockdown of Rab11a and Rab35 resulted in significantly smaller CCVs and reduced Coxiella intracellular growth, suggesting that recycling endosomal Rab proteins are essential for CCV expansion and bacterial multiplication. Discussion: Our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for Coxiella intracellular survival and potentially uncovers novel host cell factors essential for Coxiella pathogenesis.

Multiplexed bacterial pathogen detection and clinical characteristics of orthopedic infection in hospitalized patients.

Introduction: Accurate identification of the etiology of orthopedic infection is very important for correct and timely clinical management, but it has been poorly studied. In the current study we explored the association of multiple bacterial pathogens with orthopedic infection. Methods: Hospitalized orthopedic patients were enrolled in a rural hospital in Qingdao, China. Wound or exudate swab samples were collected and tested for twelve bacterial pathogens with both culture and multiplex real time PCR. Results and discussion: A total of 349 hospitalized orthopedic patients were enrolled including 193 cases presenting infection manifestations upon admission and 156 with no sign of infection. Orthopedic infection patients were mainly male (72.5%) with more lengthy hospital stay (median 15 days). At least one pathogen was detected in 42.5% (82/193) of patients with infection while 7.1% (11/156) in the patients without infection (P < 0.001). S. aureus was the most prevalent causative pathogen (15.5%). Quantity dependent pathogen association with infection was observed, particularly for P. aeruginosa and K. pneumoniae, possibly indicating subclinical infection. Most of the patients with detected pathogens had a previous history of orthopedic surgery (odds ratio 2.8, P = 0.038). Pathogen specific clinical manifestations were characterized. Multiplex qPCR, because of its high sensitivity, superior specificity, and powerful quantification could be utilized in combination with culture to guide antimicrobial therapy and track the progression of orthopedic infection during treatment.

Novel induction of broad-spectrum antibiotics by the human pathogen Legionella.

The majority of antibiotics are natural products, with microorganism-generated molecules and their derivatives being the most prevalent source of drugs to treat infections. Thus, identifying natural products remains the most valuable resource for novel therapeutics. Here, we report the discovery of a series of dormant bacteria in honey that have bactericidal activity toward Legionella, a bacterial pathogen that causes respiratory disease in humans. We show that, in response to bacterial products secreted by Legionella, the honey bacteria release diffusible antimicrobial molecules. Remarkably, the honey bacteria only produce these molecules in response to Legionella spp., when compared to a panel of 24 bacterial pathogens from different genera. However, the molecules induced by Legionella have broad activity against several clinically important pathogens, including many high-priority pathogens. Thus, Legionella spp. are potent drivers of antimicrobial molecule production by uncharacterized bacteria isolated from honey, providing access to new antimicrobial products and an unprecedented strategy for discovering novel antibiotics. IMPORTANCE: Natural products generated by microorganisms remain the most viable and abundant source of new antibiotics. However, their discovery depends on the ability to isolate and culture the producing organisms and to identify conditions that promote antibiotic production. Here, we identify a series of previously undescribed bacteria isolated from raw honey and specific culture conditions that induce the production of antimicrobial molecules that are active against a wide variety of pathogenic bacteria.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection.

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R. solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

Antimicrobial susceptibility and resistome of Actinobacillus pleuropneumoniae in Taiwan: a next-generation sequencing analysis.

Actinobacillus pleuropneumoniae infection causes a high mortality rate in porcine animals. Antimicrobial resistance poses global threats to public health. The current study aimed to determine the antimicrobial susceptibilities and probe the resistome of A. pleuropneumoniae in Taiwan. Herein, 133 isolates were retrospectively collected; upon initial screening, 38 samples were subjected to next-generation sequencing (NGS). Over the period 2017-2022, the lowest frequencies of resistant isolates were found for ceftiofur, cephalexin, cephalothin, and enrofloxacin, while the highest frequencies of resistant isolates were found for oxytetracycline, streptomycin, doxycycline, ampicillin, amoxicillin, kanamycin, and florfenicol. Furthermore, most isolates (71.4%) showed multiple drug resistance. NGS-based resistome analysis revealed aminoglycoside- and tetracycline-related genes at the highest prevalence, followed by genes related to beta-lactam, sulfamethoxazole, florphenicol, and macrolide. A plasmid replicon (repUS47) and insertion sequences (IS10R and ISVAp11) were identified in resistant isolates. Notably, the multiple resistance roles of the insertion sequence IS10R were widely proposed in human medicine; however, this is the first time IS10R has been reported in veterinary medicine. Concordance analysis revealed a high consistency of phenotypic and genotypic susceptibility to florphenicol, tilmicosin, doxycycline, and oxytetracycline. The current study reports the antimicrobial characterization of A. pleuropneumoniae for the first time in Taiwan using NGS.

Epidemiological insights into seasonal, sex­specific and age­related distribution of bacterial pathogens in urinary tract infections.

Urinary tract infections (UTIs) are prevalent and recurrent bacterial infections that affect individuals worldwide, posing a significant burden on healthcare systems. The present study aimed to explore the epidemiology of UTIs, investigating the seasonal, gender-specific and age-related bacterial pathogen distribution to guide clinical diagnosis. Data were retrospectively collected from electronic medical records and laboratory reports of 926 UTIs diagnosed in Fuding Hospital (Fujian University of Traditional Chinese Medicine, Fuding, China). Bacterial isolates were identified using standard microbiological techniques. χ2 tests were performed to assess associations between pathogens and the seasons, sex and age groups. Significant associations were found between bacterial species and seasons. Enterococcus faecium exhibited a substantial prevalence in spring (χ2, 12.824; P=0.005), while Acinetobacter baumannii demonstrated increased prevalence in autumn (χ2, 16.404; P=0.001). Female patients showed a higher incidence of UTIs. Gram-positive bacteria were more prevalent in males, with Staphylococcus aureus showing significant male predominance (χ2, 14.607; P<0.001). E. faecium displayed an age-related increase in prevalence (χ2, 17.775; P<0.001), whereas Escherichia coli tended to be more prevalent in younger patients (χ2, 12.813; P=0.005). These findings highlight the complex nature of UTIs and offer insights for tailored diagnostic and preventive strategies, potentially enhancing healthcare outcomes.

Characterization of the diversity of type IV secretion system-encoding plasmids in Acinetobacter.

The multi-drug resistant pathogen Acinetobacter baumannii has gained global attention as an important clinical challenge. Owing to its ability to survive on surfaces, its capacity for horizontal gene transfer, and its resistance to front-line antibiotics, A. baumannii has established itself as a successful pathogen. Bacterial conjugation is a central mechanism for pathogen evolution. The epidemic multidrug-resistant A. baumannii ACICU harbours a plasmid encoding a Type IV Secretion System (T4SS) with homology to the E. coli F-plasmid, and plasmids with homologous gene clusters have been identified in several A. baumannii sequence types. However the genetic and host strain diversity, global distribution, and functional ability of this group of plasmids is not fully understood. Using systematic analysis, we show that pACICU2 belongs to a group of almost 120 T4SS-encoding plasmids within four different species of Acinetobacter and one strain of Klebsiella pneumoniae from human and environmental origin, and globally distributed across 20 countries spanning 4 continents. Genetic diversity was observed both outside and within the T4SS-encoding cluster, and 47% of plasmids harboured resistance determinants, with two plasmids harbouring eleven. Conjugation studies with an extensively drug-resistant (XDR) strain showed that the XDR plasmid could be successfully transferred to a more divergent A. baumanii, and transconjugants exhibited the resistance phenotype of the plasmid. Collectively, this demonstrates that these T4SS-encoding plasmids are globally distributed and more widespread among Acinetobacter than previously thought, and that they represent an important potential reservoir for future clinical concern.

Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity.

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6-16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes.

mRNA vaccine platforms to prevent bacterial infections.

Bacterial infections are an urgent public health priority. The application of mRNA vaccine technology to prevent bacterial infections is a promising therapeutic strategy undergoing active development. This article discusses recent advances and limitations of mRNA vaccines to prevent bacterial diseases and provides perspectives on future research directions.

Pathogenic Rickettsia spp. as emerging models for bacterial biology.

Our understanding of free-living bacterial models like Escherichia coli far outpaces that of obligate intracellular bacteria, which cannot be cultured axenically. All obligate intracellular bacteria are host-associated, and many cause serious human diseases. Their constant exposure to the distinct biochemical niche of the host has driven the evolution of numerous specialized bacteriological and genetic adaptations, as well as innovative molecular mechanisms of infection. Here, we review the history and use of pathogenic Rickettsia species, which cause an array of vector-borne vascular illnesses, as model systems to probe microbial biology. Although many challenges remain in our studies of these organisms, the rich pathogenic and biological diversity of Rickettsia spp. constitutes a unique backdrop to investigate how microbes survive and thrive in host and vector cells. We take a bacterial-focused perspective and highlight emerging insights that relate to new host-pathogen interactions, bacterial physiology, and evolution. The transformation of Rickettsia spp. from pathogens to models demonstrates how recalcitrant microbes may be leveraged in the lab to tap unmined bacterial diversity for new discoveries. Rickettsia spp. hold great promise as model systems not only to understand other obligate intracellular pathogens but also to discover new biology across and beyond bacteria.

Ceramide-1-phosphate is a regulator of Golgi structure and is co-opted by the obligate intracellular bacterial pathogen Anaplasma phagocytophilum.

Many intracellular pathogens structurally disrupt the Golgi apparatus as an evolutionarily conserved promicrobial strategy. Yet, the host factors and signaling processes involved are often poorly understood, particularly for Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We found that A. phagocytophilum elevated cellular levels of the bioactive sphingolipid, ceramide-1-phosphate (C1P), to promote Golgi fragmentation that enables bacterial proliferation, conversion from its non-infectious to infectious form, and productive infection. A. phagocytophilum poorly infected mice deficient in ceramide kinase, the Golgi-localized enzyme responsible for C1P biosynthesis. C1P regulated Golgi morphology via activation of a PKCα/Cdc42/JNK signaling axis that culminates in phosphorylation of Golgi structural proteins, GRASP55 and GRASP65. siRNA-mediated depletion of Cdc42 blocked A. phagocytophilum from altering Golgi morphology, which impaired anterograde trafficking of trans-Golgi vesicles into and maturation of the pathogen-occupied vacuole. Cells overexpressing phosphorylation-resistant versions of GRASP55 and GRASP65 presented with suppressed C1P- and A. phagocytophilum-induced Golgi fragmentation and poorly supported infection by the bacterium. By studying A. phagocytophilum, we identify C1P as a regulator of Golgi structure and a host factor that is relevant to disease progression associated with Golgi fragmentation.IMPORTANCECeramide-1-phosphate (C1P), a bioactive sphingolipid that regulates diverse processes vital to mammalian physiology, is linked to disease states such as cancer, inflammation, and wound healing. By studying the obligate intracellular bacterium Anaplasma phagocytophilum, we discovered that C1P is a major regulator of Golgi morphology. A. phagocytophilum elevated C1P levels to induce signaling events that promote Golgi fragmentation and increase vesicular traffic into the pathogen-occupied vacuole that the bacterium parasitizes. As several intracellular microbial pathogens destabilize the Golgi to drive their infection cycles and changes in Golgi morphology is also linked to cancer and neurodegenerative disorder progression, this study identifies C1P as a potential broad-spectrum therapeutic target for infectious and non-infectious diseases.

A mathematical model of biofilm growth and spread within plant xylem: Case study of Xylella fastidiosa in olive trees.

Xylem-limited bacterial pathogens cause some of the most destructive plant diseases. Though imposed measures to control these pathogens are generally ineffective, even among susceptible taxa, some hosts can limit bacterial loads and symptom expression. Mechanisms by which this resistance is achieved are poorly understood. In particular, it is still unknown how differences in vascular structure may influence biofilm growth and spread within a host. To address this, we developed a novel theoretical framework to describe biofilm behaviour within xylem vessels, adopting a polymer-based modelling approach. We then parameterised the model to investigate the relevance of xylem vessel diameters on Xylella fastidiosa resistance among olive cultivars. The functionality of all vessels was severely reduced under infection, with hydraulic flow reductions of 2-3 orders of magnitude. However, results suggest wider vessels act as biofilm incubators; allowing biofilms to develop over a long time while still transporting them through the vasculature. By contrast, thinner vessels become blocked much earlier, limiting biofilm spread. Using experimental data on vessel diameter distributions, we were able to determine that a mechanism of resistance in the olive cultivar Leccino is a relatively low abundance of the widest vessels, limiting X. fastidiosa spread.

Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for naked-eye detection of Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions. RESULTS: In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min. SIGNIFICANCE: We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.

CRISPR genotyping methods: Tracing the evolution from spoligotyping to machine learning.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with adaptive immunity defenses against foreign genetic invaders. The identification of CRISPR-Cas function is among the most impactful discoveries of recent decades that have shaped the development of genome editing in various organisms paving the way for a plethora of promising applications in biotechnology and health. Even before the discovery of CRISPR-Cas biological role, the particular structure of CRISPR loci has been explored for epidemiological genotyping of bacterial pathogens. CRISPR-Cas loci are arranged in CRISPR arrays of mostly identical direct repeats intercalated with invader-derived spacers and an operon of cas genes encoding the Cas protein components. Each small CRISPR RNA (crRNA) encoded within the CRISPR array constitutes a key functional unit of this RNA-based CRISPR-Cas defense system guiding the Cas effector proteins toward the foreign nucleic acids for their destruction. The information acquired from prior invader encounters and stored within CRISPR arrays turns out to be extremely valuable in tracing the microevolution and epidemiology of major bacterial pathogens. We review here the history of CRISPR-based typing strategies highlighting the first PCR-based methods that have set the stage for recent developments of high-throughput sequencing and machine learning-based approaches. A great amount of whole genome sequencing and metagenomic data accumulated in recent years opens up new avenues for combining experimental and computational approaches of high-resolution CRISPR-based typing.

Microbial organic fertilizer prepared by co-composting of Trichoderma dregs mitigates dissemination of resistance, virulence genes, and bacterial pathogens in soil and rhizosphere.

The use of manure, mycelium dregs and other waste as organic fertilizer is the main source of antibiotic resistance genes (ARGs) and pathogens in farmland. Composting of waste may effectively remove ARGs and pathogens. However, the profiles and drivers of changes in metal resistance genes (MRGs), biocide resistance genes (BRGs), and virulence genes (VGs) in soil-crop rhizosphere systems after compost application remain largely unknown. Here, we prepared two kinds of microbial organic fertilizers (MOF) by using Trichoderma dregs (TDs) and organic fertilizer mixing method (MOF1) and TDs co-composting method (MOF2). The effects of different types and doses of MOF on resistance genes, VGs and pathogens in soil-rhizosphere system and their potential mechanisms were studied. The results showed that co-composting of TDs promoted the decomposition of organic carbon and decreased the absolute abundance of ARGs and mobile genetic elements (MGEs) by 53.4-65.0%. MOF1 application significantly increased the abundance and diversity of soil ARGs, BRGs, and VGs, while low and medium doses of MOF2 significantly decreased their abundance and diversity in soil and rhizosphere. Patterns of positive co-occurrence between MGEs and VGs/MRGs/BRGs/ARGs were observed through statistical analysis and gene arrangements. ARGs/MRGs reductions in MOF2 soil were directly driven by weakened horizontal gene transfer triggered by MGEs. Furthermore, MOF2 reduced soil BRGs/VGs levels by shifting bacterial communities (e.g., reduced bacterial host) or improving soil property. Our study provided new insights into the rational use of waste to minimize the spread of resistomes and VGs in soil.

Principles, Methods, and Real-Time Applications of Bacteriophage-Based Pathogen Detection.

Bacterial pathogens in water, food, and the environment are spreading diseases around the world. According to a World Health Organization (WHO) report, waterborne pathogens pose the most significant global health risks to living organisms, including humans and animals. Conventional bacterial detection approaches such as colony counting, microscopic analysis, biochemical analysis, and molecular analysis are expensive, time-consuming, less sensitive, and require a pre-enrichment step. However, the bacteriophage-based detection of pathogenic bacteria is a robust approach that utilizes bacteriophages, which are viruses that specifically target and infect bacteria, for rapid and accurate detection of targets. This review shed light on cutting-edge technologies about the novel structure of phages and the immobilization process on the surface of electrodes to detect targeted bacterial cells. Similarly, the purpose of this study was to provide a comprehensive assessment of bacteriophage-based biosensors utilized for pathogen detection, as well as their trends, outcomes, and problems. This review article summaries current phage-based pathogen detection strategies for the development of low-cost lab-on-chip (LOC) and point-of-care (POC) devices using electrochemical and optical methods such as surface-enhanced Raman spectroscopy (SERS).

High-throughput functional trait testing for bacterial pathogens.

Functional traits are characteristics that affect the fitness and metabolic function of a microorganism. There is growing interest in using high-throughput methods to characterize bacterial pathogens based on functional virulence traits. Traditional methods that phenotype a single organism for a single virulence trait can be time consuming and labor intensive. Alternatively, machine learning of whole-genome sequences (WGS) has shown some success in predicting virulence. However, relying solely on WGS can miss functional traits, particularly for organisms lacking classical virulence factors. We propose that high-throughput assays for functional virulence trait identification should become a prominent method of characterizing bacterial pathogens on a population scale. This work is critical as we move from compiling lists of bacterial species associated with disease to pathogen-agnostic approaches capable of detecting novel microbes. We discuss six key areas of functional trait testing and how advancing high-throughput methods could provide a greater understanding of pathogens.