In this review work, we highlight the origin of morphological structures such as nanofibers/nanorods in case of various materials in nano as well as bulk form. In addition, a discussion on different cations of different ionic radii and other intrinsic factors is provided. The materials  exhibiting the nanofibers/nanorods like morphological structures are tabulated. Furthermore, the significance of nanofibers/nanorods obtained from distinct materials is elucidated in multiple scientific and technological fields. At the end, the device applications of these morphological species are also described in the current technology. To identify the nano ordered species within a material, the surface morphology of the same is a well-suited study. In addition, the morphology of different materials can be considered as an important parameter for achieving advanced properties. In view of this, ample of nanostructures such as nanospheres, nanoplates, nanorods, nanofibers, nanowires, nanoflowers, nanoleaves, nanotubes, nanocages, nanofilms, nanoparticles, nanosheets, nanochains, nanofoam, nanoholes, nanomesh, nanopillar, nanorings, nanoribbons, nanoshells, nanocubes, quantum wells, quantum dots etc., are evolved because of synthesizing nanomaterials via different synthesis techniques  as the coordination position. Moreover, these ligands are normally considered as the electron donors attracted O the Fe  at the center of the complex system.The nanofibers look to be flexible in nature while the nanorods will have rigidity in nature. Normally, the nanofibers/nanorods can be formed by the generation and growth of the nuclei of the material in vertical/horizontal direction. Besides, the heterogeneous nucleation will be reinforced at the solid interface owing to the deposition of catalytic clusters. During this kind of mechanism, the formed uniform nanopores can act as the templates for the nanofibers. The filled nanopores produce nanorods. This indicates that the nanorods will have apparent high dense structure rather than the nanofibers. The schematic representation for the formation of α-MnOElectrospinning is a synthesis technique of nanofibers with nano/micrometer size, wherein the electrostatic forces will be produced from the polymer-based material solution. For the preparation of nanofibers, the conventional spinning process is used as shown in Fig. 12O19 nanorods are prepared via the hydrothermal process as. Herein, the nitrate precursors are selected as the starting materials. After dissolving the precursors in the distilled water, the NaOH solution is added drop by drop to maintain the pH value. The stirred solution is transferred to 300 ml autoclave and kept in an oven to perform the hydrothermal reaction at 150 °C for 8 h. In this method, the reaction temperature can be varied between 120 and 200 °C based on the kind of material compound. During the reaction, owing to the pressure and temperature developed in the autoclave, the nucleation process will be taken place. This will be occurred due to the presence of high and small ionic radii elements in the compound. In addition, different valence position of cations is also responsible for this kind of behavior. Once, the reaction is over, the sample is removed from the autoclave and then, it is washed multiple numbers times to keep the pH value neutral. Then, the powder sample is heated at 60 °C to remove the moisture content. The FESEM picture of BaSrLaFe12O19 reveals the pure nanorods (NRs) as shown in Fig. 2/Mo2C/C nanofibers prepared via the hydrothermal method is provided in Fig. 12O19 nanorods like the crystal structure, bonding preferences, kind of ions, pressure, temperature, surfactant, and pH as reported by Sunaina et al. [0.8-yLayCo0.2TiO3 (y = 0.2–0.8)), and PCLT (Pb0.8Co0.2-zLazTiO3 (z = 0.05–0.2)) samples via the microwave processed sol-gel method. The obtained results exhibited the nanofibers-like structures in the morphology. Similarly, the BCLF (Ba0.2Cu0.8-xLaxFe2O4 (x = 0.2–0.6)), and BCF (BaxCux1-Fe2O4 (x = 0.2–0.8)) samples prepared via the hydrothermal method reveal the nanorod/nanofibers in the morphology as reported by Naresh et al. [+3 cations, whereas the BCF samples perform the same nature at high percentage of Ba+2 cations of high ionic radii. The similar observations are noticed by Prakash et al. [3 nanorods prepared using the hydrothermal method.The hydrothermal method is also preferable to prepare the nanofibers/nanorods. For this, several scientists considered the nitrate materials as the precursors. For instance, the BaSrLaFea et al. . In the h et al. , 11. BVSh et al. , synthesh et al. , in caseIt is a known fact that the materials are classified into several families based on the adapted structure. In view of this, the perovskites, spinels, nanocomposites, polymer composites, oxides, etc., are the popular materials achieving the nanofibers/nanorods like structures in the morphology. In Table2, ethanol, CO, and CH4 with UV radiation assistance at the room temperature. The mere zinc oxide (ZnO) is not up to the mark to sense the gas at room temperature as the smaller number of electron carrier’s generation on the surface of the ZnO. Therefore, the silver (Ag) nanoparticles have been incorporated into ZnO structures to defeat the drawbacks so that to exhibit sensor application at the room temperature [2O3/CNFs which are synthesized by hydrothermal process and gives raise to rhombohedral structures with 20–40 nm of diameter. The iron oxides used in these batteries are nontoxic and eco-friendly in nature. In many research papers reported that the gravimetric and volumetric capacities 1000 mAh/g and 5300 mA h ml−1, respectively which are higher than the graphite electrodes. Lithium-ion batteries are the rechargeable and highest energy density batteries used in hybrid; plug in hybrid and electronic transport vehicles [3 and BiVO4/bi-phase TiO2 nanofibers exhibit photoelectrochemical and photo-electrocatalytic applications. In the two-step technique Pt/WO3 nanofibers could be synthesized with a monolithic structure with 70 nm diameter. The tungsten oxide is an n-type semiconductor with very narrow bandgap about 2.6 to 2.8 range and considered as a good photocatalyst. From the tungsten oxide nanorods, nanofibers and nanoplates can be prepared but the nanofibers are the most preferable because of high length to diameter ratio. The BiVO4/bi-phase TiO2 nanofibers prepared by the electrospinning combined with hydrothermal process gives tetragonal/monoclinic with a range of 8–200 nm diameters. The TiO2 based materials are eco-friendly and applied to decompose organic contaminants, reduction of carbon dioxide, and split water molecule into hydrogen [6Fe2Ti3O18 and TiO2 nanofibers are prepared by the electrospinning method. The diameters of the Bi6Fe2Ti3O18 and TiO2 nanofibers are 100-150 nm and 100 nm, respectively. The load of Au nanoparticles into Bi6Fe2Ti3O18 increases the photocatalytic activity used in the ferroelectric devices like multi-state memory devices, field sensing and catalytic activities. From the TiO2 nanoparticles we can prepare nanorods, nanofibers, nanobelts and nanosheets. Among these nanostructures the nanofibers are being preferred because of high surface to volume ratio, excellent electron transfer, and charge transferability. The famous applications of the TiO2 nanofibers are in the top-gate field-effect transistor. If the cadmium nanoparticles are doped with the TiO2, then its morphological structure becomes tetragonal with the diameter size 200–330 nm. These nanocomposites are used in the dye-sensitized solar cells [2O2) plays important role in the biological systems, the required or balanced quantity of hydrogen peroxide is to be maintained. Hence to identify the hydrogen peroxide in the biological system we need to undergo electrochemical methods. In this method electrochemical sensors have been employed or adopted to analyze H2O2. The α-MnO2 nanorods grown on the NGNFs (nitrogen-doped graphite nanofibers) prepared by one-pot hydrothermal process with 130–170 nm diameter of nanorods and tetragonal structure are efficient electrochemical detectors of H2O2 [14 kg which is very low percentage (~0.17 mg/L) compared to other elements such as Na, Ca, Mg, and K. The recent studies and research have been concentrated on inorganic good adsorbent like highly porous hydrogen manganese oxide (HMO) nanofibers prepared via combining electrospinning, calcination, and ion exchanges. The HMOs will have cubic structure with diameter of 2–80 nm which have high capacity to absorb the lithium ions [The varieties of nanorods or nanofibers have been prepared for different applications via different techniques. The ZnO nanorods and Ag nanofibers with hexagonal structures have been prepared on the chip via soft solution and polyol process. These nanomaterials are used as an optoelectronic gas sensor to identify the gases in surroundings such gas is commonly NOperature . The oneperature . The recvehicles . The Pt/hydrogen , 22. Thear cells –21. The ar cells . The hyd of H2O2 . The porium ions  (Table1)2, and ZnO nanoparticles are being used in the antibacterial agents and among these ZnO is the most effective nanomaterial in this connection. The ZnO is also used in the dye removal process effectively because of excellent adsorption coefficient by virtue of its surface morphology and volume to surface ratio. Hence the zinc oxide nanoparticles attached to polyacrylonitrile nanofibers with hinokitiol as gluing agent for synergistic antibacterial activities and effective dye removal [0.2La0.8TiO3 are the new trends in the field of photocatalytic applications. These nanocomposites are prepared by the hydrothermal process and depending on the composition the structure can be changed which is observed based on the X-ray diffraction analysis. This compound (1−x) (Al0.2La0.8TiO3) + (x) (BaTiO3) (x = 0.2–0.8) exhibits very good electrical properties and several advantages over other composites such as X-ray density (~11.210 g/cm3), surface area (~62.29 m2/g) electrical conductivity (~4.21E − 05 S/cm at 1 MHz) and dielectric constant of 62,768 at 1 kHz [2 nanofibers are prepared by electrospinning used in dye-sensitized solar cells as an anode material. The TiO2 based materials are cost-effective and eco-friendly in nature [Environmental hazardous are being increased in the globe due to the manmade and natural calamities. The chemical effluents are one of the major challenges to modern society and governments in the present context. Hence, the wastewater management is essential to scale down the hazardous outbursts. Some of the methods have been followed such as chemical oxidation, adsorption, photocatalysis, and membrane separation. Among these methods all are not cost effective and eco-friendly but photocatalysis is the cost-effective and greener technology with lower consumption energy. In the photocatalysis, the photocatalysts are the semiconductors with dopants or defect-induced or hybrid nanomaterials which can give higher photocatalytic action due to synergistic and plasmonic effect. The ZnO is n type semiconductor with non-toxicity, higher thermal and chemical stability with unique optical and photoelectrochemical properties. The ZnO has other important properties such as s high quantum yield and inherent surface defects which can sensitize under visible light. The most preferable nanostructure of ZnO is the nanofibers in this context as it provides high aspect ratio and a defined flow pattern for enhanced photocatalytic property. These nanofibers possess 800 nm of diameter with hexagonal structure prepared by electrospinning . In the  removal . And the removal , 30. Theat 1 kHz . The TiOn nature .2H5OH, carbon monoxide, nitrogen dioxide, and methane after subjecting them to UV radiation. Based on the surface morphological behavior of these two samples, the gas sensing behavior was identified. That is, as indicated in the FESEM pictures of ZnO nanorods  nanofiber composites and SiC (SC) nanofibers were performed along with electrochemical kinetics as indicated in Fig. RSEI) while the mid frequency arcs were connected to the interphase charge transfer impedance (RCT). It was also clear that the RSEI of STTC and SC nanofibers was almost constant at high frequency arcs for the first discharge cycle, whereas after the first discharge, the RSEI  was computed with the help of DFT and molecular dynamics model. The mean square displacement against the time (t) plots  analysis of Si/TiOSEI Fig.  of STTC SEI Fig.  approachtability . In addiications . To undets Figs. , 12 indi12 cm2/s . In addi domains . Fig. 112S3nanorods was depicted [2S3 nanorods for a step scan of 0.1 mV/s. It was found that the three reduction peaks were noticed at four voltages as indicated in Fig. 2S3 [2S3 nanorods (at specific density = 100 mA/g) showed two plateaus indicating the reductive and alloying reactions [2S3 nanorods offered low-rate capabilities as compared to RGO decorated nanorods. The discharge capacity was decreased to 214 mAh/g at 2 × 103 mA/g current density [In Fig. depicted . EspeciaFig. 2S3 . Similareactions . In addi density . This ev density . This al density .In Fig. The nanofibers and nanorods are one dimensional nanomaterial which will have variety of applications in the medical or medicine field such as drug delivery, Cell patterning, cell differentiation, cell cluster formation, and biosensor. The structure itself is supplementary to the transportation in tubular structures of nanorods.2) nanofibers can be used in the bone tissue engineering [2 is one of the best nanomaterials in connection with biocompatibility due to its geometry, high aspect ratio and preserving the specific surface area on the level of reasonable porosity. In the bone, predominant inorganic material is the CaCO3 and calcium phosphate compounds. Due to emergence of 3D printing technology, which is coupled with computer-based CAD program, the artificial bones can be prepared. In the bone printing process, the mere calcium phosphate compounds do not give much mechanical strength as they are very low brittle in nature hence, we must add some nanoceramics into slurry to obtain high mechanical strength. The common ceramic materials are alumina, zirconia, and titanium. The titanium-based nanocomposite is superior for joint prostheses due to their appropriate compressive strength and biocompatibility than other ceramics. In terms of nano topographic effects on bone cell behaviors, TiO2 materials with various nanostructures display improved effects on development of bone tissue and bone regenerating capability. And the TiO2 nanotubes are expected to exhibit bone regeneration in medicine field and due to its porosity interaction between tissue and bone can be attained and achieve the transportation of the biomolecule between bone and tissue. Hence, the TiO2 supported hollow porous nanotubes functionalized with bioactive calcium salt might be a promising biomaterial for hard tissue replacement. The fabricated TiO2 immobilized cellulose acetate electro spun fibers prepared by electrospinning can be used in the photocatalytic degradation of textile effluents like methylene blue. The higher photocatalytic activity of fibers was discerned with high TiO2 content as CA composite ultrafine fibers with 5 wt% TiO2 were as high as 90% after 4 h of degradation [2) was noticed from 1 to 5 days. In addition, the sulphonated titanium dioxide (STiO2) achieves the minimum absorbance for 5 days. However, the HTiO2 performed the good cell response at the end of incubation period. Further, it was also clear that the toxicity was completely reduced. Figure 2 nanofibers possessing the rounded cells. On looking at the performance of carbonated titanium dioxide (CTiO2) nanofibers, it was evidenced that these nanofibers revealed the significant cell spread rather than the STiO2 nanofibers. Moreover, comparatively, for HTiO2 nanofibers, the cell spread was improved with some dead cells on cell culture. The HTiO2 nanofibers showed good biocompatibility by providing the high cell spread due to the porous and hollow structure. Therefore, it can be well understood that the above strategies can improve the bone regeneration as well.High porous electro spun hollow titania  and micro-patterned areas . Conventionally the micro patterns have been prepared by the photo or soft lithography. The micro-pattern and Electro spun nanofibers extensively have been used in the drug delivery system. Micro-structured and electro spun materials have large surface area which reduces the toxicity, enhanced drug encapsulation and increases the efficiency of delivery to the target. However, the behavior of drug delivery depends on the nature of the structure and reported that sustainable drug release can be achieved in nanofiber alignment and patterned fibrous scaffold. The release rate of fenbufen is slower in aligned Polylactic-co-glycolic acid/chitosan nanofibrous scaffolds compared to random fibers. The decrease of pore size in the aligned nanofibers reduces the delivery or diffusion rate than that of random fibers. Micro-structured cellulose acetate nanofibers by nylon mesh with 50–100 µm dimension openings showed a zero-order sustained drug release up to 12 h compared to nonpatterned scaffolds. The relevant reason is that minimization of solid-liquid interface and higher angle of contact controls the drug release. The hydrophobicity (depends on the nanofiber density and distribution pattern) of the substrate also alters the drug release. Polyvinyl butyral mesh patterns showed slower drug release due to the even distribution of nanofibers and higher contact angle. Multicompartmental fiber matrix and hydrogel microparticle were evaluated for the sustainable growth factor release .The cell differentiation is one of the biological processes which depend on the many factors such as diffusible molecules, cell–cell interactions, cell–matrix interaction etc. The cell fate is affected by many factors including surface topography, surface chemistry, etc. The nanofiber orientation and micropatterned structure plays important role in the tissue engineering. The micropatterned polyethylene glycol hydrogel with gelatin fiber was considered for osteogenic differentiation. Fiber attached by MSC will be differentiated into osteogenesis due to two reasons such as sustained release of basic fibroblast growth factor and bone morphogenetic protein 2 up to 4 days and 4 weeks from the fiber and PEG hydrogel. The staining plays the crucial role in the osteogenesis differentiation due to the presence of alkaline phosphatase and alizarin red. Su-8 micropatterned nanotube of 20 µm dimension and grooved can induce self-early osteo differentiation without any external agencies. The cells rotate and align along the patterns with the respect to time. The three-dimensional nanofibers surface fabricated by cylindrical collector is twice greater than that of two-dimensional surfaces. Due to higher pore size in the three-dimensional nanofiber the cell infiltration and migration are higher. In addition, this scaffold showed osteogenic differentiation, which is correlated with the cell adherence, flattening, and spread. The patterned, Polycaprolactone nanofiber mesh which is from conducting wire net collector is reported as one of the best osteogenic differentiations in human bone marrow stromal cell , 124. InThese fibers will have different applications which are obtained from bio-macromolecules from botanical sources, polysaccharides from seaweeds, and polysaccharides from animal sources. The cellulose, starch and pectin are the bio-macromolecules and from these electrospinning patterns can be prepared by Electrospinning method. The cellulose is one of the good renewable biomaterial ecofriendly good mechanical strength and biocompatibility. And it has excellent ecological credentials with potential medical, pharmaceutical, paper production, sanitary products, rayon, cellophane derivatives, textile fibers, medical fabrics, and industrial applications. The major cellulose sources are the cotton, wood pulp and other pulps. The cellulose extracted from both plants and bacteria in the form of nanomaterials will have uniqueness over the microstructures vowing to excellent physical, mechanical strength, chemical reactivity and coupled with advanced technology like medical, catalytic, environmental, biosensory, and energy. In most of the cases the synthetic approach employed is top–down approach in the materials synthesis. The cellulose after undergoing electrospinning and obtaining the spun pattern of derivatives exhibits immobilization of biological active molecules or drug and vitamins such as alkannin, amoxicillin, curcumin, ibuprofen, indomethacin, naproxen, shikonin, sulindac, vitamin A, and vitamin E and these can improve the cell adhesion, proliferation and cell differentiation in the biological applications that too in the drug delivery and tissue engineering. In the tissue engineering, embedded gentamicin sulfate  and halloysite clay  into cellulose ether-PVA nanofiber mats to deliver therapeutics to the skin tissue and scaffold composite heals the wound of in-vitro and in vivo. Similarly, polyaniline nanorods are preferred to transfer electro spun carboxymethyl cellulose and these are cylindrical and 3D surface materials which provides excellent electroactive surface which envisages electron transfer and mass transportation. Amino based cellulose synthesized by 6-deoxy-6-trisaminoethyl-amino (TEAE) cellulose and polyvinyl alcohol (PVA) blended solutions in aqueous media, and this can be used to stop the growth of S. aureus and this nanomaterial used to cure pneumonia which is caused by Gram-negative K. pneumoniae bacteria. Sodium doped cellulose with tungsten precursor on calcinations exhibits excellent gas (hydrogen disulfide) sensor applications. Starch is obtained from the higher plants used as a storage material and used in the food and nonfood applications. Starch consisting of complex polysaccharides α-D-glucopyranosyl units such as amylose and amylopectin found in wheat, maize, rice, potato, sago, tapioca/cassava, rye, barley, and oats. The starch with nanographene enhances electro spinnability, reduced diameter, improved hydrophilicity, and thermal stability. The starch derived nanotubes coupled with carbon nanotubes as an electrode are used in the rechargeable batteries which give rise outstanding performance. Pectin is obtained by the plants and it is hetero polysaccharide, and major sources are citrus peel, apple pomace, and sugar beet pulp. The pectin is used in the food industries as a thickening, gelling, and stabilizing agent and possesses biomedical applications , 124. MoThe materials which are in nanoscale exhibit various structures like nanospheres, nanoplates, nanorods, nanofibers, nanowires, nanoflowers, nanotubes, nanocages, nanofilms, nanoparticles, nanosheets, nanochains, nanofoam, nanoholes, nanopillar, nanoribbons, quantum wells, quantum dots etc. Amongst, nanofibers/nanorods exhibit different characteristics. Owing to this, we have focused on recent advancements in nanofibers/nanorods. In this connection, this article provides the brief knowledge on the origin along with synthesis of nanofibers/nanorods by electrospinning and hydrothermal techniques. Further, various materials showed the nanofiber/nanorod like morphology along with their synthesis techniques, crystal structure and applications are tabulated. Finally, the device applications of nanofibers/nanorods such as nanogenerator melt spun filament and the role of nanofibers in the treatment of tumors are also discussed.


Rapid molecular diagnostic tests to investigate the microbial aetiology of pneumonias may improve treatment and antimicrobial stewardship in intensive care units (ICUs). Clinicians’ endorsement and uptake of these tests is crucial to maximise engagement; however, adoption may be impeded if users harbour unaddressed concerns or if device usage is incompatible with local practice. Accordingly, we strove to identify ICU clinicians’ beliefs about molecular diagnostic tests for pneumonias before implementation at the point-of-care.We conducted semi-structured interviews with 35 critical care doctors working in four ICUs in the United Kingdom. A clinical vignette depicting a fictitious patient with signs of pneumonia was used to explore clinicians’ beliefs about the importance of molecular diagnostics and their concerns. Data were analysed thematically.Clinicians’ beliefs about molecular tests could be grouped into two categories: perceived potential of molecular diagnostics to improve antibiotic prescribing (Molecular Diagnostic Necessity) and concerns about how the test results could be implemented into practice (Molecular Diagnostic Concerns). Molecular Diagnostic Necessity stemmed from beliefs that positive results would facilitate targeted antimicrobial therapy; that negative results would signal the absence of a pathogen, and consequently that having the molecular diagnostic results would bolster clinicians’ prescribing confidence. Molecular Diagnostic Concerns included unfamiliarity with the device’s capabilities, worry that it would detect non-pathogenic bacteria, uncertainty whether it would fail to detect pathogens, and discomfort with withholding antibiotics until receiving molecular test results.Clinicians believed rapid molecular diagnostics for pneumonias were potentially important and were open to using them; however, they harboured concerns about the tests’ capabilities and integration into clinical practice. Implementation strategies should bolster users’ necessity beliefs while reducing their concerns; this can be accomplished by publicising the tests’ purpose and benefits, identifying and addressing clinicians’ misconceptions, establishing a trial period for first-hand familiarisation, and emphasising that, with a swift  test, antibiotics can be started and refined after molecular diagnostic results become available.The online version contains supplementary material available at 10.1186/s13756-021-00961-4. Rapid molecular diagnostic tests for pneumonia pathogens may improve antimicrobial stewardship (AMS). Results become available within 1 to 6 h, with accurate detection of multiple respiratory bacteria, viruses, and antimicrobial resistance genes directly from respiratory secretions without culture [Clostridium difficile infection [Such tests may be particularly useful in intensive care units (ICUs), where patients with pneumonia are frequent , 3, thernfection , (b) empnfection , and (c)Molecular diagnostics can identify pathogens and their resistance genes within hours rather than days, potentially directing early tailored antimicrobial therapy . RandomiClinicians’ endorsement and uptake of molecular tests is crucial to maximise engagement in the context of such a RCT and for any subsequent deployment. Yet, adoption may be impeded if users harbour unaddressed concerns or if device usage is incompatible with local practice . For insClinicians’ beliefs about molecular diagnostics remain largely unknown, with the UK Department of Health and Social Care identifying a lack of understanding frontline needs as a potential delayer to the clinical adoption of molecular tests . AccordiThis study is reported following Standards for Reporting Qualitative Research guidelines . It emplInterviews occurred in four UK ICUs that varied in patient population and prevalence of multi-drug resistant organisms. One was a large district general hospital in Norfolk; the remainder were London-based and comprised a tertiary referral hospital, a paediatric hospital, and a private hospital with an international patient population. None had molecular diagnostic tests for pneumonia available at the time of this research.All intensivists practicing at the four participating ICUs were eligible to participate. Clinicians who could spare time from clinical duties were recruited via local promotion after ward rounds.This research received Health Research Authority approval before data collection; all participants gave written informed consent.Semi-structured VBIs explored clinicians’ antibiotic decision-making processes. Participants read a vignette depicting a hypothetical patient exhibiting signs of pneumonia , NB, and YJ verified transcription accuracy. Data were analysed by AMP and NB using thematic analysis, following Braun and Clarke’s recommendations .patients’ treatments. ‘Molecular Diagnostic Necessity’ refers to clinicians’ perceived importance of molecular diagnostic results in practice while ‘Molecular Diagnostic Concerns’ are their beliefs about the consequences associated with test adoption [We adopted an inductive approach whereby data were coded to capture themes that represented a pattern of responses across transcripts. Themes were then deductively mapped onto the Necessity-Concerns Framework (NCF). NCF proposes that patients’ adherence to recommended treatment plans is influenced by their beliefs about the importance of their treatment and their treatment concerns . Consistadoption .Interviews occurred between August and December 2018, were between seven and 20 min in length, and enrolment continued until data saturation [Participants comprised seven early-career trainees, sixteen middle-grade trainees, and eleven consultants. Eleven participants were employed at Hospital 1, ten at Hospital 2, seven at Hospital 3, and six at Hospital 4. All intensivists practicing at Hospital 2 treated children and neonates; remaining participants treated adult patients. Interviews were individual except in one case where one early-career trainee and one middle-grade trainee were interviewed together.We describe clinicians’ perceived importance of molecular diagnostics followed by their concerns. Tables Positive molecular diagnostic results  were generally believed to facilitate antibiotic choice by rapidly identifying the organism(s) and predicting their antibiotic susceptibilities (Quotes 1–3). Currently, this information would be available approximately 72 h after the initial prescribing decision, leading to lengthy courses of potentially sub-optimal therapy. Many participants felt that molecular test results would encourage either starting appropriate antibiotics at the outset or swiftly de-escalating empirical therapy to narrow-spectrum antibiotics.lower their threshold for starting antibiotics. For example, one trainee likened molecular diagnostics to other infection indicators  which give “proof” of an infection but should be combined with the clinical context before making an antimicrobial decision (Quote 4). This decision was frequently described as reflecting a combination of clinical factors, including prospectively, the molecular test result. None of our participants said that they would start antibiotics solely based on positive results (Quote 5).In addition to improving antibiotic choice, ten clinicians asserted that positive results would appropriate antibiotics because their decision to start antibiotics would only be based on the clinical context (Quote 7).Some doctors stated that molecular diagnostics would not influence their threshold to start antimicrobial therapy (Quote 6). These clinicians believed that they would exclusively use this test to choose Negative results  would generally be interpreted to indicate that a respiratory infection was unlikely. Some clinicians believed that a negative result in a clinically stable patient would encourage withholding or stopping antibiotics (Quotes 8–9), whereas a negative result in a deteriorating patient would be interpreted to indicate a non-respiratory source of infection (Quote 9).angst” and “panic” (Quotes 10–11). They described uncertainty regarding whether their prescribing was appropriate and worried that it contributed to AMR (Quotes 10–11). This reflects the present difficulty of verifying the appropriateness of antibiotic decisions until laboratory culture results become available.When currently making antibiotic decisions, some participants reported experiencing negative emotions like “happier” and more “confident” in their prescribing (Quotes 10–11). They believed that their prescribing decisions would be beneficial to their patient and society and felt that access to these tests would encourage AMS practices.Such participants believed that the information provided by the molecular test would make them “caveat emptor—as future users of the test’s results, these clinicians believed that familiarisation was essential to understand the test’s capabilities and to make an informed decision about its usefulness in practice (Quotes 13–14). These participants would not adopt the test if, after familiarisation, they concluded that it did not meet their standards or would negatively impact patient care.Many clinicians stated that they needed to familiarise themselves with the molecular test before using its results in practice. First, they wanted more information about the test, including its sensitivity, specificity, and its place in the diagnostic process (Quote 12). Several raised misconceptions about the test’s capabilities . Second, they wanted first-hand experience to verify the test’s suitability (Quote 13). These participants felt a sense of Concerns were common that the molecular test would detect organisms present, but of no consequence to the patient (Quote 15). Many clinicians worried that the test would detect non-pathogenic bacteria, and that, paradoxically, this might encourage unnecessary antibiotic prescriptions (Quote 16). To mitigate against antibiotic over-treatment based on detection of colonisation rather than infection, some participants stressed that they would not automatically prescribe based on the test result. They would combine the results with the clinical context to determine the likelihood that the bacteria detected were causing current disease before making a decision (Quote 17).Some clinicians reported that negative results would not be reassuring enough to withhold or stop antibiotics. These doctors were uncertain whether the test was able to detect all possible respiratory pathogens (Quote 18). They consequently believed that negative results should be overridden by other evidence of respiratory infection , which would lead them to prescribe or continue antibiotics (Quote 19).Many early-career and middle-grade trainees assumed that they would need to withhold antibiotics until molecular test results become available; no consultants took this view. Consequently, some junior doctors expressed discomfort with the prospect of withholding antibiotics for up to 6 h. They worried that both the vignette and their actual patients would significantly deteriorate within this period (Quotes 20–21), believing that withholding antibiotics would be indefensible if the patient was exhibiting signs of sepsis (Quote 22).Other trainees felt more comfortable with withholding antibiotics until receiving molecular test results. These participants believed that the patient was stable enough to ‘wait and watch’ and valued using the results to aid their ultimate prescription decision (Quotes 23–24).This is the first study exploring UK intensivists’ salient beliefs influencing molecular diagnostic test uptake. Consistent with the NCF, facilitators related to clinicians’ beliefs that molecular tests would improve AMS whereas barriers related to their concerns about integrating these tests into clinical practice .Many participants were open to using the molecular test because they believed that its results would improve antibiotic prescription practices. They saw positive test results, with early specific identification of a pathogen, as facilitating targeted antimicrobial therapy. Similarly, they viewed negative results to signal the likely absence of respiratory infection and thought that these results would provide reassurance that withholding an antibiotic prescription was appropriate. Our findings are consistent with arguments that molecular diagnostics would aid the optimisation of antimicrobial therapy , 9, and Despite clinicians recognising the potential of molecular tests, concerns about tests’ integration into clinical practice may impede uptake. Consistent with previous assertions , 21, parIndividual and environmental factors may also influence prescribers’ acceptance of molecular diagnostics. Individual clinicians’ trust in molecular diagnostics may affect its usage; for example, prescribers with low levels of trust in the machine likely would harbour concerns while having low beliefs about the device’s utility. Similarly, the prescribing environment may affect machine uptake. For instance, unit-specific norms may encourage continuing empiric antibiotics for a minimum course; as such, receiving rapid test results may not impact antibiotic de-escalation or stopping. Thought should also be given to machine placement, whether at the point-of-care or in the laboratory.Our findings show the importance of understanding clinicians’ beliefs about molecular diagnostic tests to ensure that they understand their rationale, and to sufficiently address their concerns. To encourage molecular diagnostic uptake in ICU—whether for RCTs or for permanent use—implementation strategies should bolster users’ necessity beliefs while reducing concerns; this may be achieved with relatively simple solutions (summarised in Table This study has limitations. Firstly, our interviews were conducted before the molecular diagnostic test was deployed for the INHALE trial. Different beliefs may emerge after implementation and should be explored. Secondly, we interviewed ICU intensivists as the INHALE RCT runs with the molecular test located in ICUs; however some clinicians have recommended collaborating with microbiologists during test implementation , 24. FutThis study is the first to characterise prescribers’ beliefs about molecular diagnostics as facilitators and barriers to applying it to antibiotic prescribing for patients with suspected pneumonia. Our findings demonstrate the importance of prescribers’ beliefs as determinants of molecular diagnostic uptake. If this technology proves successful in improving antibiotic prescribing and stewardship, interventions should understand and address these beliefs to ensure the optimal application of molecular diagnostics in clinical practice.Additional file 1. Interview guide and vignettes.


This effect is explained in terms of destructiveQI (DQI) for the meta-connected pyrrole and shiftedDQI for the para-connected isomer. These resultsdemonstrate modulation of antiresonances by molecular design and verifythe validity of ECARs as a simple “pen-and-paper” methodfor predicting QI behavior. The principles offer new fundamental insightsinto structure–property relationships in molecular junctionsand can now be exploited in a range of different heterocycles formolecular electronic applications, such as switches based on externalgating, or in thermoelectric devices.Controlling chargetransport through molecular wires by utilizingquantum interference (QI) is a growing topic in single-molecular electronics.In this article, scanning tunneling microscopy-break junction techniquesand density functional theory calculations are employed to investigatethe single-molecule conductance properties of four molecules thathave been specifically designed to test extended curly arrow rules(ECARs) for predicting QI in molecular junctions. Specifically, fortwo new isomeric 1-phenylpyrrole derivatives, the conductance pathwaybetween the gold electrodes must pass through a nitrogen atom: thisnovel feature is designed to maximize the influence of the heteroatomon conductance properties and has not been the subject of prior investigationsof QI. It is shown, experimentally and computationally, that the presenceof a nitrogen atom in the conductance pathway increases the effectof changing the position of the anchoring group on the phenyl ringfrom  By combiningthese techniques with the power of organic synthesis, it has beenwidely demonstrated that substantial variation in the conductanceof molecular wires can be achieved by small structural modifications,such as structural isomerism and/or the presence of heteroatoms.13 Particularly, in the case of π-conjugated systems, much ofthis behavior can be attributed to quantum interference (QI) effects,16 which are readily visualized in transmission functions derived fromcharge-transport simulations.20Single-moleculeconductance values have been determined for a diversearray of molecular wires trapped between metal electrodes since thedevelopment of specialized measurement techniques in the late 1990sand early 2000s.T(E) of a molecular junction is a plot of the probability of electronswith energy E passing from one electrode to the otherthrough the molecule and is proportional to molecular conductance. E is usually considered relative to the system’sFermi energy, EF. Calculated transmissionfunctions from first-principles simulations reliably show qualitativeagreement with experimental conductance studies.2 Quantitative agreement is more challenging due to difficultiessuch as the accurate determination of EF using density functional theory (DFT).21 Sharp resonances coincident with the energies of molecular orbitals are key features of a typical transmissionfunction. In low-bias conductance studies, EF usually lies near the center of the HOMO–LUMO gap.Furthermore, the low-bias QI behavior of a molecular junction relatesto the characteristics of the transmission function in the HOMO–LUMOgap. QI can be constructive (CQI) or destructive (DQI). Where CQIoccurs, a smooth, featureless transmission curve is usually seen betweenthe HOMO and LUMO resonances. A characteristic feature of DQI is asharp antiresonance in the transmission curve where T(E) approaches zero.The transmission function EF, and significantly reducedlow-bias conductance would be expected relative to a similar systemwithout an antiresonance. Shifted DQI (SDQI) refers to systems wherean antiresonance occurs in the transmission function but does notlie close to EF and so the conductanceof the junction remains high in the low-bias regime.20 Where SDQI occurs, an antiresonance can even be shifted beyond theHOMO–LUMO gap,20 meaning that SDQIis not always readily distinguishable from CQI.This work considers twosubcategories of DQI, based on the energyat which an antiresonance appears in the transmission function ofa molecular junction. DQI refers to cases where an antiresonance occursclose to 24 and graphical or topological methods.26 Other methods require a mathematical or computationalinput, such as orbital symmetry approaches,28 QI maps,29 and magic ratio rules.33 These more straightforward methods necessarily have limitationsto their scope compared to charge-transport simulations. They generallywork well for bipartite hydrocarbon lattices but can be less accuratefor molecular wires that incorporate more elaborate structural features,such as (i) deviation from a framework of fused six-membered rings;(ii) the inclusion of heteroatoms, either as substituents or withinthe lattice; or (iii) cross-conjugation.In additionto computationally demanding charge-transport simulations,many simpler methods exist to predict and rationalize the QI behaviorof molecular wires. Some methods are based on structural considerationsalone, such as “curly arrow” rules (CARs)24 This was in part inspired by work from anothertwo of the present authors which showed that simple CARs as widelyapplied in molecular electronics22 “brokedown” when applied to cross-conjugated anthraquinone derivatives.8 ECARs are a “pen-and-paper” methodthat can predict whether a given molecule will exhibit CQI, DQI, orSDQI. ECARs account for previously reported QI behavior of molecularwires containing heteroatoms, nonbipartite structures, and cross-conjugation.24 However, ECARs cannot predict the relative conductanceof wires with respect to one another. Despite this, the conductanceof structurally similar materials would usually be expected to followthe trend CQI ≥ SDQI > DQI. The rules24 are as follows:Two of the presentauthors recently presented an extension to predictiveCARs for QI behavior [extended curly arrow rules (ECARs)].Identify the two anchoringunits of a molecularwire and replace one with a donor group (D) and the other with anacceptor group (A). If the D lone pair can be delocalized onto A usingcurly arrows, CQI is expected; if not, DQI is expected.EF.If DQI is expected, identify any electron-withdrawinggroups (EWGs) or electron-donating groups (EDGs) present in the molecularwire. If EWGs are present, replace each anchor with D. If a lone pairfrom each D can be independently delocalized to the same EWG, SDQIis expected. If EDGs are present, replace each contact with A. Ifa lone pair (or negative charge) from the same EDG can be independentlydelocalized to each A, SDQI is expected. Otherwise, DQI is expectedaround 24 To our knowledge, this is the first study ofQI effects in organic molecules where an all-carbon conductance pathwayis not available between the anchoring groups. The predictions madeby ECARs for these new wires have been tested experimentally usingthe STM-BJ technique and investigated computationally by calculatingtransmission functions using a simple tight-binding method and DFT-basedmaterial-specific charge-transport simulations.To further test the validityof ECARs, we have designed and synthesized new heteroatom-containingmolecular wires that differ structurally from those considered inthe development of the rules. Specifically, the novel feature of thesemolecules is that when they are held between gold electrodes, theconductance pathway through these molecules must pass through a nitrogenatom. In contrast, a pathway comprising only carbon atoms existedin all of the examples used in the conception of ECARs.1–4 are given in the Supporting Information. In brief, the 1-phenylpyrrole derivatives 1 and 2 were prepared from 3-bromo-1-(triisopropylsilyl)pyrrole.The thiomethyl anchor was first installed through lithiation followedby treatment with dimethyl disulfide.34 The TIPS protecting group was then removed before forming the aryl–aryl C–N bond via Ullmann coupling35 with the appropriate bromothioanisole. The biphenylspecies 3 and 4 were prepared based on areported synthesis of 4. All experiments were carried out in a solutionof the target molecules (0.1 mM) in mesitylene under ambient conditionswith a 0.1 V bias voltage. Logarithmically binned one-dimensional(1D) conductance histograms and two-dimensional (2D) conductance-displacementhistograms were plotted by compiling at least 2000 molecular conductance-displacementtraces. Statistical analysis was performed using the methods we reportedpreviously.37Molecular conductancemeasurements were performed using the lab-builtSTM-BJ technique, which has been reported in previous publications.1–4 was investigated computationallyusing DFT combined with quantumtransport calculations.21 From the optimizedgeometry of each molecule in the gas phase and between two gold electrodes,we obtained a ground-state Hamiltonian from the Siesta38 implementation of DFT and combined it with theGollum39 transport code to obtain a transmissioncoefficient T(E) for electrons withenergy E passing from one electrode to the other. The low-bias electrical conductance was thencalculated from the Landauer formula G = G0T(EF), where G0 is the conductance quantumand EF is the Fermi energy of the electrodes.The room temperature electrical conductance was obtained from thethermal averaging of T(E) .The molecular conductancebehavior of molecules N-phenylpyrrole)derivatives 1 and 2  or meta (2) to the pyrrole ring. As shown in 1, itis possible to independently delocalize the nitrogen lone pair toan A group in either anchoring position, so ECARs predict SDQI. For 2, it is only possible to delocalize the nitrogen lone pairto an A group in the pyrrole-anchoring position ; therefore, DQI is expected.Each 1-phenylpyrrole isomer has a thiomethyl anchoringgroup inthe pyrrole 3-position and a second thiomethyl anchor on the benzenering, either 3 and 4  and biphenyl (3 and 4) species could be complicated by differences in the torsionalangle (θ) between the connected rings. The angle θ wasnot expected to vary significantly within each isomer pair, as thesteric environment around the aryl–aryl bond remains the same.We, therefore, reasoned that any influence of θ would be overshadowedby comparing the relative effect of changing the position of the secondanchoring group (i.e. that on the right of the structures in para to meta for the two isomeric pairs. If the predictionof ECARs is correct and 1 shows SDQI (and therefore higherlow-bias conductance), while the other three species show DQI, then,the following relationship between molecular conductances GX (where X is the molecule number) should hold around EF:para- to meta-position. In practice, the DFT-minimized conformationsof 1–4  showed thatθ was similar for all four species in the gas phase. In theDFT-minimized molecular junction conformations, θ differed by6° in the 1-phenylpyrrole isomer pair and 8° in the biphenylpair. We emphasize that these values relate only to the energy-minimizedjunction conformation. Experimental conductance measurements samplea broad range of conformations, where θ is likely to vary similarlyfor a given isomer pair. As the barrier to rotation is low at roomtemperature, we, therefore, anticipated that the proposed conductancerelationship would remain valid.It was anticipatedthat direct comparison between the 1-phenylpyrrole(37 was used to investigate the molecularconductance of the four molecules(see Supporting Information). 1D conductance histograms are shown in Supporting Information and exampleconductance traces in 1 (10–3.05G0) > 3 (10–3.50G0) > 2 (10–3.85G0) > 4 (10–4.05G0). The broader conductance histogramsobserved for 2 and 4 (and to a lesser extent 3) in comparison to 1 can be related to a widerrange of possible junction configurations.45 A small shoulder is visible in the 1D histogram of 4 , whereas that between 3 and 4 is only 100.55 (ca. 3.5). Changing the position of thesecond anchoring group from para to meta has nearly double the effect for the 1-phenylpyrrole species 1 and 2 than it has for the biphenyl wires 3 and 4. The higher conductance of 1 relative to 3 (and 2 relative to 4) indicates that the 1-substituted 3-(methylthio)pyrroleunit affords improved conductance relative to a meta-linked benzene ring. Indeed, the conductance of 1 iscomparable to that of the para-linked biphenyl species4,4′-bis(methylsulfide)biphenyl,375, which was determined to be 10–3.10G0 under the same experimental conditionsused for 1–4 .The hypothesized relationshipbetween the molecular conductancesbased on ECARs holds: the ratio between the most probable conductancesof molecules 2, 3, and 4 arecompared. Each hasa meta-anchored benzene bound to a second aromaticsystem, respectively, 3-(methylthio)pyrrole (via the pyrrole 1-position), para-(methylthio)benzene, or meta-(methylthio)benzene.As molecular conductance increases in the sequence 4 < 2 < 3, 1-linked 3-(methylthio)pyrrole canbe considered an intermediate between para- and meta-(methylthio)benzene. This trend in relative conductanceis notably compatible with the QI behavior that ECARs predict forwires comprising only a meta-benzene (DQI), 1,3-difunctionalisedpyrrole (SDQI), or para-benzene (CQI).A similar trend is observed if the relative conductancesof molecules 1–4 between gold electrodes based onDFT material-specific Hamiltonians. The conductance of 1 is higher than 2 for a wide range of EF around the DFT Fermi energy (EF = 0 eV), and the conductance of 3 is higherthan that of 4 around EF =0 eV. This is in qualitative agreement with the experimentally determinedconductance values (EF = 0 eV (see Table S2 in the Supporting Information), which decreases in the sequence 1 > 3 > 2 > 4. . Furthermore, EF <ca. 0.25 eV, G1 > G2 and G3 is similar to or< G4. This agrees with the ECARs-predictedrelationshipbetween the four molecular conductances. Taking the values at EF = 0 eV as an example, the ratio of the conductancesof 1 and 2 is 100.57 (ca. 3.7)and that between 3 and 4 is 100.24 (ca. 1.7). Similar to the trend observed in the STM-BJ data, theeffect of switching the second anchoring group from para to meta for the 1-phenylpyrrole species is aroundtwice as large as for the biphenyl wires. However, the DQI-mediatedantiresonance feature near EF that waspredicted using ECARs is not clearly visible in the transmission functionof 2, 3, or 4. We attributethis to the effect of σ-orbitals on transport.51 Molecules 1–4 are short, and thereforethe contribution to transport from σ-orbitals is higher thanthat from π-orbitals at energies around the antiresonance feature,causing it to be masked.e values 2, as is 2, 3, and 4 but not for 1. Note that additional sharp features can be seen outside the HOMO–LUMOgap, at E ≈ ± 1 eV for 2–4 and E ≈ −0.6 and +1.6 eV for 1 and 2. The origin of these features was investigatedby calculating the tight-binding energy levels and corresponding wavefunctionsfor the 1-phenylpyrrole molecular core of 1 and 2 . A transmissionresonance is usually expected close to the energy levels of a moleculein a junction. As exemplified in E = −1 eV for the molecular core . The width of a transmission resonance is proportionalto the sum of the density of states  at the connection points toelectrodes.21 When both LDOSs are zero,a resonance with zero width (a “vanishing resonance”)is expected, as seen for 1 in 2 in 2 close to E = −1 eV is, therefore, a Fanoresonance (a resonance attached to an antiresonance), but the amplitudeof the resonance is small because of a large asymmetry in the self-energiesdue to the coupling to the left and right electrodes.To illustrate this effect, we note that transmission functionscalculated using a simple tight-binding method with a single π-orbitalper atom 3c,dshow 1 and 2 S14. A tr 1 and 2 S14. A tr 1 and 2 S14. A tr 1 and 2 S14. A tr 1 and 2 S14. A tr51 the electrical conductance of extended analoguesof molecules 1 and 2  was calculated. Phenylacetylene units were addedbetween the molecular core and the anchoring groups to lengthen theconductance pathway and weaken the effect of σ-channels on totaltransport. The resulting calculations  show that the antiresonancefeature predicted by ECARs is present for 7 , whereas noantiresonance feature is observed in the HOMO–LUMO gap for 6. The observed QI behavior agrees with the predictions ofECARs and the simple tight-binding study.To furtherdemonstrate that the absence of antiresonance featuresin the DFT calculations is due to conduction through the σ-orbitals,1 and 2, based on 1-phenylpyrrole,were designed and synthesized to test recently reported ECARs forpredicting QI behavior.24 By comparisonwith analogous biphenyl wires 3 and 4, itwas shown using STM-BJ studies that the presence of an unavoidablenitrogen atom in the through-bond conductance pathway increases theeffect of changing the position of the anchoring group on the phenylring from para (1) to meta (2). This agrees with ECARs which predict DQI near EF for 2–4 (i.e. a low low-biasconductance) and SDQI for 1 (i.e. a higher low-bias conductancedue to a shifted antiresonance). The experimental results are supportedby charge-transport simulations of the measured molecules and extendedanalogues. This work verifies the validity of ECARs as a “pen-and-paper”method for predicting QI behavior and will, therefore, have an impacton the design criteria of new molecular wires.Molecularwires 1 is comparable to that of linearly conjugated 4,4′-bis(methylsulfide)biphenyl, 5. 1,3-Disubstituted pyrroles therefore represent a prototypeof heteroaromatic units that can be used to add SDQI behavior to amolecular wire without significantly reducing low-bias conductance.These results offer new fundamental understanding of structure–propertyrelationships in molecular junctions which can now be exploited ina range of molecular electronic applications such as switches basedon external gating or in thermoelectric devices.Despite the absenceof an alternating pathway of single and doublebonds between the anchoring units, the conductance of


Optimal management of haemodynamically significant patent ductus arteriosus (HsPDA) in premature babies remains controversial. Our aim is to compare death and/or adverse neurodevelopmental outcomes in extremely low birth weight (ELBW) infants with HsPDA who were managed with conservative [C], medical [M] and/or surgical [S] treatment, with secondary aim to examine short-term morbidities among [S] and [C] groups. The study also compared outcomes in very low birth weight (VLBW) infants with HsPDA and non-HsPDA.A retrospective study of VLBW preterm infants born before 29 weeks in Singapore from 2007 to 2016 was conducted.p < 0.05), but not global developmental delay (GDD). No significant differences in neurodevelopmental outcomes such as hearing loss, cerebral palsy (CP) and speech delay were found. [M + S] infants were at a higher risk of developing chronic lung disease (CLD)  and short-term growth failure compared to [C] infants. They were significantly shorter and had a smaller head circumference at discharge (p < 0.05). [M + S] infants also had elevated creatinine compared to those in group [C] .A total of 474 VLBW infants were admitted in NUH from 2007 to 2016. Infants aged between 24 + 0 and 28 + 6 weeks of gestation, weighing ≤1500 g and diagnosed with patent ductus arteriosus (PDA) were included in the study, of which 172 infants (124 HsPDA and 48 non-HsPDA) were analyzed. Among infants with HsPDA, 17 infants were managed with [C], 83 with [M] and 24 with [S]. Mortality was not increased regardless of the presence of HsPDA or treatment received. Infants with non-HsPDA were less likely to have isolated speech delay (Compared to conservative management, infants requiring [M + S] treatment for HsPDA were more likely to have short-term complications such as CLD, elevated creatinine, and poorer growth. Despite a more turbulent postnatal course, death and/or adverse neurodevelopmental outcomes were not worse in infants managed with [M + S]. PDA treatment options range from conservative approach to medical and surgical interventions.Optimal management is controversial.CLD incidence in infant managed medical and/or surgical more common than those managed conservatively.Elevated creatinine and growth failure are more common in infants managed medically and/or surgically.Death and adverse neurodevelopment outcomes in ELBWs were comparable at 18 months.In term infants, the patent ductus arteriosus (PDA) normally constricts after birth and closes within 24–48 h of life . In pretIn infants with PDA, blood flows from the left to right circulation, i.e., from the aorta into the pulmonary arteries, increasing the blood flow in the pulmonary circulation. This “ductal steal” phenomenon may result in pulmonary over-circulation and diversion of blood from the systemic circulation . DependiProlonged exposure to PDA is also associated with neonatal mortality and multiple neonatal morbidities such as chronic lung disease (CLD), necrotising enterocolitis (NEC), retinopathy of prematurity (ROP), intraventricular haemorrhage (IVH), periventricular leukomalacia (PVL) and adverse long-term neurodevelopmental outcomes . The manTo date, the optimal management of HsPDA remains controversial and is largely guided by professional opinions , 10. TheThe decision to treat HsPDA with medical treatment or surgery has its risks. Treatment with COX inhibitors is associated with renal impairment, intestinal perforation and altered cerebrovascular regulation . SurgicaAs the managment option of HsPDA remains controversial, further information such as short-term morbidities and long-term outcomes is important to help guide the phyisicians and parents to determine the most suitable option. The primary objective of this study is to compare the death and/or adverse neurodevelopmental outcomes in early childhood among extremely low birth weight (ELBW) infants with HsPDA who were managed with conservative [C], medical [M] and/or surgical [S] treatment. The secondary objectives are to examine baseline demographics predisposing to [S] treatment and compare neonatal short-term morbidities among the [S] and [C] infants. The study also compared outcomes in very low birth weight (VLBW) infants with HsPDA and non-HsPDA.This is a retrospective study of VLBW infants (BW ≤ 1500 g) born at National University Hospital (NUH), Singapore from 2007 to 2016 and admitted to the neonatal intensive care unit (NICU). Ethics approval was given by the National Health Group Domain Specific Review Board (NHG DSRB ref. 2020/00001).The inclusion criteria for comparing outcomes of management in HsPDA infants were: (1) between 24 + 0 and 28 + 6 weeks of gestation, (2) VLBW and 3) diagnosed with PDA (either HsPDA or non-HsPDA). Infants with duct dependent cardiac lesions, those with HsPDA who died before the initiation of treatment for PDA closure, those with non-HsPDA who died between day 0–1  and those lost to follow up were excluded. We limited the study to infants < 29 weeks as more mature infants tend to have spontaneous closure of PDA  diagnose.We defined HsPDA based on combination of echocardiographic and clinical parameters. Echocardiography criteria were: PDA diameter ≥ 1.5 mm, left atrium: aortic root (LA:Ao) ratio ≥ 1.5:1, absent or reverse diastolic flow in the post-ductal descending aorta or organ arteries , 6, 15. Infants were grouped to have either HsPDA or non-HsPDA. Infants with HsPDA were further divided based on their management: conservative [C], medical [M] or surgical [S] treatment. The primary composite outcome was 1) death and 2) adverse neurodevelopment outcomes up to 5 years. Neurodevelopmental outcomes studied include hearing loss requiring implants/hearing aids, cerebral palsy (CP) and developmental delay. CP was defined if the child had either diplegia, hemiplegia or quadriplegia between 18 and 24 months of age. Developmental delay was assessed from 18 months corrected age and was defined as global developmental delay (GDD) or isolated speech delay upon confirmation by the independent developmental paediatrician and after appropriate psychological testing for age. The secondary outcomes were baseline demographics predisposing to treatment complications and short-term neonatal morbidities. General demographics, antenatal and neonatal data were collected from electronic medical records and include gender, mode of delivery, GA, birth and discharge anthropometric measures. We used the Fenton’s growth chart  to derivt-test with conservative arm as the comparator. Categorical data were presented as frequency and analysed using Chi Square test with 2 × 2 tables. Odds ratio (OR) was determined by comparing the conservative and treatment arm. Multinomial logistic regression was used for multivariate analysis. Further analysis was conducted for the complications related to surgical ligation. A confidence interval of 95% is provided and a P-value of < 0.05 is considered significant.Data were analysed using SPSS version 25 . Continuous normally distributed data were presented as mean with standard deviations unless stated otherwise and analysed using Student n = 28, 5.9%) to follow up.A total of 474 VLBW infants were admitted in NUH from 2007 to 2016; of which 172 infants (124 HsPDA and 48 non-HsPDA) met study criteria  infants were managed conservatively, 83 (66.9%) infants were managed medically and 24 (19.4%) infants were managed with surgical ligation after failure of medical management. Of the 24 infants, 13 underwent surgical treatment before 30 days of age (early) and 11 underwent after 30 days of age (late). Of those managed conservatively, HsPDA was closed in all cases spontaneously before discharge. Of those managed medically alone, all had closure prior to discharge and did not require surgery. All infants with HsPDA that failed to close after medical treatment were managed with surgical ligation.Of note, there were 13 deaths after treatment was initiated  and treatment complications between infants managed [C] and surgically [S].Table p = 0.046) and had more severe RDS (p = 0.04). More than 29% of infants in group [M + S] had a CRIB II score exceeding 12 compared to only 6.3% in those in group [C] (p = 0.046) and had more severe RDS (p = 0.04). The mean days to surgical ligation was 33.4 (SD 9.4) days. The incidence of CLD in the group [M + S] infants was statistically significant (p = 0.005). The OR of group [M + S] infants developing CLD was also significant (OR 6.83 (95% CI 1.49–31.3)) compared to group [C] infants. At discharge, group [M + S] infants were significantly shorter (p = 0.017) and had a smaller head circumference (p = 0.039) than group [C] infants. However, the weight at discharge z-score between the group [M + S] infants and [C] infants was not statistically significant (p = 0.17).Table p = 0.000).In the analysis of treatment specific complications Table , the onlp = 0.105)).Table p = 0.007) as measured by CRIB II, but presence of severe RDS was not statistically significant (p = 0.21). The incidence of CLD in group [S] was higher than [C] group (p = 0.000). Group [S] infants were more likely than group [C] infants to develop CLD. The OR of group [S] infants developing CLD was 22.50  when compared to group [C] infants. This is especially so for those who were operated after 30 days of life. There were notable differences in the birth and discharge anthropometric data between the treatment groups. Group [S] infants were significantly younger by 1 week (p = 0.004) and lighter by 130 g (p = 0.007) as compared to group [C] infants. At discharge, group [S] infants continued to have poorer growth with lower z-scores in length (p = 0.001) and head circumference (p = 0.026) as compared to group [C].Table p = 0.004) infants than in group [C] infants. The incidence of vocal cord palsy was 25% in group [S] (p = 0.026). The OR of developing vocal cord palsy for group [S] as compared to group [C] was 1.94  (Table The highest measured creatinine was significantly higher in group [S] (p < 0.01)  and [S] were sicker at birth compared to infants in group [C] with a higher CRIB II score > 12, predicting a greater risk of neonatal mortality among this group of infants . Base exThe effects of medical or surgical treatment of PDA on neurodevelopmental outcomes has become a topic of increasing interest. Yet, there has been no well-established relationship between the two. Our results found no significant differences in neurodevelopmental outcomes at 18 months of corrected age between infants managed with [M + S] compared to [C]. Furthermore, multivariate analysis found no statistical significance of treatment type received and death and/or adverse neurodevelopmental outcomes except for the duration of hospitalization and the gestational age at discharge. Our findings are consistent with Chorne et al.  who founThe association between medical treatment and CLD is also not well defined. Our findings suggest that there is a higher incidence and OR of group [M + S] infants developing CLD than group [C] infants, assuming that infants who died prior to 36 weeks had CLD. Prolonged exposure to PDA has been associated with CLD , 35. OurIn our study, we were not able to compare with gestation stratified infants below 24 weeks requiring conservative treatment. This lends further support to earlier studies that found the relationship of the rate of ductal closure with GA and BW , 39 to bThis study has several limitations due to its retrospective nature. Firstly, there are no definite criteria for the diagnosis of HsPDA as bedside measurement of the PDA can be subjective and the decision for treatment is not standardised due to a lack of universal guidelines. However, as a small unit, we were able to minimise care variations through repeated and consensus discussions. Secondly, there were variations of treatment practices over the years with increasing trend towards conservative treatment. It has only been in the recent years that the trend of PDA treatment has shifted towards a less aggressive approach . As suchWe showed in our cohort of Asian ELBW infants requiring treatment for HsPDA, especially delayed surgical treatment, had more neonatal complications of CLD, elevated creatinine and poorer growth. However, despite a more turbulent postnatal course, they did not have higher incidence of death or adverse neurodevelopmental outcomes at 18-months corrected age.


Vibrio vulnificus is a gram-negative, opportunistic human pathogen associated with life-threatening wound infections and is commonly found in warm coastal marine water environments, globally. In this study, two fishing harbors and three tributaries of the river basin were analyzed for the prevalence of V. vulnificus in the water bodies and shellfish that are under the pressure of external pollutions. The average detection rate of V. vulnificus in the river basins and fishing harbors was 8.3% and 4.2%, respectively, in all seasons. A total of nine strains of V. vulnificus were isolated in pure cultures from 160 samples belonging to river basins and fishing harbors to analyze the antibiotic susceptibility, virulence gene profiles, and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) fingerprinting. All isolates were susceptible to 10 tested antibiotics. The genotypic characterization revealed that 11.1% (n = 1/9) strain was nonvirulent, whereas 88.9% (n = 8/9) isolates were virulent strains, which possessed the four most prevalent toxin genes such as vcgC (88.9%), 16S B (88.9%), vvhA (88.9%), and manIIA (88.9%), followed by nanA (77.8%), CPS1 (66.7), and PRXII (44.4%). Additionally, ERIC-PCR fingerprinting grouped these nine isolates into two main clusters, among which the river basin isolates showed genetically diverse profiles, suggesting multiple sources of V. vulnificus. Ultimately, this study highlighted the virulent strains of V. vulnificus in the coastal aquatic environments of Taiwan, harboring a potential risk of infection to human health through water-borne transmission. Vibrio vulnificus is a halophilic, gram-negative, motile, curved, and rod-shaped pathogenic bacterium that belongs to the family Vibrionaceae, which naturally occurs in the coastal and estuarine environment, especially in warm temperatures (22–30 °C) and moderate saline zones  worldwide [V. vulnificus turns into a viable but not culturable (VBNC) state, which makes it more resistant to various lethal environmental stress factors, as compared with the culturable cells of these bacteria [V. vulnificus. Shellfish, in turn, may become potential reservoirs for the entrance of these pathogenic bacteria into the food chain, becoming a source of wound infections in humans [V. vulnificus has been frequently observed in various geographical areas in the world. Recent studies indicate that due to the global climate change, which resulted in increased surface water temperatures, enabled the global distribution and spread of V. vulnificus [eratures 2–30 °C abacteria ,5. Coastn humans ,7,8,9. Vlnificus ,12,13,14V. vulnificus has been classified into three biotypes, on the basis of biochemical and pathogenic characterization [V. vulnificus lethal illnesses caused by septicemia, with high fever and chills, and wound infections resulting in tissue necrosis and severe bacteremia, with a fatality rate of >50%, especially in immuno-incompetent individuals [V. vulnificus has a high variation rate in the strains’ virulence potential, which makes it difficult to differentiate rapidly, posing a threat to public health [vvhA), which also serves as a primary feature to distinguish between clinical (C-) genotypes and environmental (E-) genotypes, have been developed [rization . Among trization ,16,17. Tividuals ,20,21,22c health . To overeveloped .V. vulnificus strains are generally susceptible to most veterinary and clinically used antibiotics [V. vulnificus, whereby these life-threatening pathogens are transmitted to the food chain and human body surface, becoming a cause of V. vulnificus infection [ibiotics ,25. Howeibiotics ,27,28. Sibiotics ,29,30. Cnfection .methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii, Salmonella [V. vulnificus in aquatic environments was approximately two decades ago [V. vulnificus, by analyzing the antibiotic susceptibility profile and the virulence gene pattern based on ERIC-PCR fingerprinting for conducting a detailed investigation of V. vulnificus in the fishing harbors and nearby river basins. Therefore, this study will also be carried out in order to provide the possible contamination sources and drug-resistant situations related to this pathogenic micro-organism.The Puzih River has livestock wastewater contamination, and our previous studies show many species of bacteria with severe drug-resistant problems, such as lmonella ,34,35,36ades ago . This stV. vulnificus. In spring and autumn, no V. vulnificus was detected from all the PR basins (V. vulnificus was 25% in the estuary area (area C), 12.5% in the middle part (area B) in the vicinity to the urban area, and 0% in the upper section (area A), with a total detection rate of 12.5% in all PR basins. Area A of PR is the upstream confluence region, which is the farthest from the estuary. Similarly, in winter, the detection rate was as follows: 37.5% (area B), 25% (area C), and 0% (area A), with a total detection rate of 20.8% in the PR regions. From the water samples collected from fishing harbor areas (DS and BD), V. vulnificus was detected only in the autumn season (16.7%), with a total detection rate of 4.2% in all seasons. However, none of the isolates were detected from shellfish samples. The overall detection rate in the PR basin was 8.3% and 4.2% in the fishing harbor, irrespective of the seasons. During the study, nine isolates were successfully purified from eight positive sampling sites. These isolated strains were confirmed to be positive for vvhA (V. vulnificus targeting gene) by PCR detection.A total of 96 river samples, 24 seawaters, and 40 shellfish samples were collected from the Puzih river basin and two fishing harbors (DS and BD) for the detection of R basins . HoweverV. vulnificus isolates were subjected to an antibiotic susceptibility test against 10 antimicrobial categories. All of these isolates were susceptible to the employed categories of antibiotics, irrespective of the sampling locations, as shown in A total of nine V. vulnificus in aquatic environments was checked by targeting a combination of virulence and nonvirulence genes to confirm the toxicity threat to humans, as shown in n = 8/9) strains showed the four most prevalent virulence genes in similar distribution , which included vcgC , 16S B , vvhA (encoding V. vulnificus cytolysin⁄ haemolysin protien), and manIIA (encoding enzyme IIA for mannitol fermentation operon), followed by three other toxin genes, which are: nanA ; CPS1 (66.7), which serves as a capsular polysaccharide operon, regulating the production of polysaccharides; and PRXII . Additionally, 56% of the strains possessed both 16S A  and 16S B genes , whereas one strain (10%) possessed both the vcgC  and vcgE genes . In this study, the strain (10%) that belonged to the PR area B showed only non-virulence genes , e.g., vcgE, 16S A, CPS2, and vvhA.The status of virulent and nonvirulent strains of V. vulnificus strains. The ERIC-PCR fingerprinting successfully categorized the nine strains isolated from river basins and fishing harbors into two major clusters exhibiting a less than 40% Pearson similarity coefficient based on reference strain, sampling sites, and phylogenetic diversity (n = 6/9) of the strains collected from river basins belonging to Area B  and Area C . Cluster 1 was further subdivided into two sub-clusters, assigned as clusters 1.1 and 1.2. In the sub-cluster 1.1, the nonvirulent strain primarily belonging to the river basin Area B was separated, exhibiting a less than 57% Pearson similarity in genotypic profile, compared with the virulent strains of sub-cluster 1.2. Additionally, sub-cluster 1.2, containing virulent strains, was further subdivided into A and B clusters. The virulent strains S02PR2911 and S02PR311, primarily from the river basin (Area A) and grouped into cluster A, showed 100% similarity in genotypic profile. Similarly, the strains S04PR2211 and S04PR2311, which belonged to the same sampling site (Area B) and was grouped into cluster B, exhibited almost a 90% similarity in genotypic profile. Cluster 2 contained the remaining 44.4% strains, belonging to the river basin Area B , Area C , and fishing harbors (25%); n = 1/4). This cluster’s strains showed a less than 62% Pearson similarity, which were further subdivided into two sub-clusters, assigned as 2.1 and 2.2, respectively. The sub-cluster 2.1 was further subdivided into two clusters, A and B. In cluster A, the strains BD-FH W211 and S04PR2711 were isolated from fishing harbors and the river basin (Area C), and were grouped into cluster A, exhibiting an 85% similarity in genotypic profile. Additionally, the strains S04PR2511 and S04PR3111, primarily from the river basin Area B and C, were grouped into profile B, showing almost a 90% similarity in genotypic profile. In contrast, the reference strain showing a less than 75% similarity was grouped separately.We combined ERIC-PCR typing and genetic diversity results with genotypic data to better interpret the origin/source and genetic variation of iversity . ClusterV. vulnificus in different water bodies [V. vulnificus varies from 6% to 69% in different seasons, globally [V. vulnificus in the river basins was higher in the winter season (20.8%) than in the summer season (12.5%), which is unique and different from what was shown in most of the previous studies [V. vulnificus, where, as previously highlighted, the optimal growth temperature ranges from 20–30 °C [V. vulnificus during four seasons was 8.3% , which is higher than what was shown in a recent study on V. vulnificus from a river in China [V. vulnificus from five major harbors in Taiwan [V. vulnificus (in the score year) was approximately 5% in Taiwan’s harbor environments.The differences in various environmental constraints such as temperature, salinity, precipitation, geographical locations, and pollution contents greatly influence the population and growth rate of r bodies ,37,38,39globally ,41,42. I studies . The riv20–30 °C ,44,45,46n Taiwan . These fV. vulnificus in the surrounding waters, compared with waters containing oysters, shellfish, and mussels, whose filter-feeding ability when obtaining food causes a higher concentration of V. vulnificus in their intestines [V. vulnificus cases were isolated from the 40 shellfish samples associated with fishing harbors in this study. The highest detection rate of V. vulnificus from the estuary area (Area C) and urban residential area (Area B) of the river basin might be associated with the mixing of domestic waste and organic matters along with the supporting salinity parameters.The previous reports highlighted the relatively lower concentration of testines ,8. InterV. vulnificus is considered to be susceptible to most antibiotics used for human and animal treatment [V. vulnificus showed susceptibility against all of the tested antibiotics. The V. vulnificus strains’ resistance to some antibiotics has been reported; however, most of the strains were also susceptible to several antibiotics [E. coli, Salmonella, etc.), Enterococcaceae, and Staphylococcaceae, among others [V. vulnificus populations with high drug-resistance were isolated from the shellfish [V. vulnificus against a wide range of antibiotics, based on the environmental parameters or host [Usually, reatment . In thisibiotics . The aqug others ,49,50,51hellfish ,48. A co or host ,53, whicV. vulnificus consist of heterogenic bacterial species that display variations in virulence and pathogenicity factors at strain level [V. vulnificus infections. Previous studies have emphasized the targeting of multiple virulence genes through PCR amplification for identification, differentiation, and typing of V. vulnificus at strain level [vcgC, 16S B, CPS1, and vvhA-1 to differentiate virulent strains of V. vulnificus from non-virulent strains. Previous study has also indicated that these virulence genes of V. vulnificus are highly specific to the virulent strains [nanA, manIIA, and PRXII, were also included for risk assessment purposes, as previously suggested [V. vulnificus carried virulence genes, which were associated with both the river basins and fishing harbors. Our result is in accordance with the previous study, where the prevalence rate of virulent strains, isolated from Ariake Sea, Japan, was 90%; whereas, in Mikawa and Ise Bay, the prevalence rate was 70% [V. vulnificus was only 10%. Several studies have used vcg (vcgC and vcgE), 16S rRNA (16S A and 16S B), and CPS (CPS1 and CPS2) to differentiate between the clinical (virulent) and environmental strains (nonvirulent) of V. vulnificus. Rosche et al. (2005) demonstrated that 90% of clinical strains carried the vcgC gene, whereas the detection rate of vcgE was 87% in environmental strains. In this study, the detection rate of vcgC was associated with 89.6% isolated strains, whereas the vcgE gene detection rate was 22.2%. However, one strain showed the PCR amplification of both vcgC and vcgE genes associated with the river basin. This is in accordance with the report of Warner and Oliver (2008a), where the V. vulnificus strain isolated from water and oysters was found to be both vcgC- and vcgE-positive. However, in a previous report, 26% of the clinical strains isolated from infected patients exhibited the vcgE gene [16S B, and environmental strains possessed 15% 16S A [16S B and 16S A gene was 89.8% and 66.7%; whereas, 56% of strains were found to possess both 16S B and A, simultaneously. This latter phenomenon has been reported frequently; some environmental strains of V. vulnificus, isolated from aquatic environments and oyster, possessed both 16S B and A simultaneously [V. vulnificus under different environmental conditions [CPS allele 1 and 2 differentiation between virulent  and non-virulent strains, only one strain showed CPS2 , 66.7% (n = 6/9) possessed CPS1, and 22% (n = 2/9) of the strains did not show any of these two alleles. Additionally, none of the strains possessed both alleles simultaneously. However, the absence of both alleles has been reported in V. vulnificus strains isolated from different environments [CPS plays an important role in virulence associated with clinical strains, whereas in environmental strain, it is mostly involved in survival mechanisms [V. vulnificus genotyping in this study’s virulence factors, the vcgC, 16S B, vvhA, manIIA, and GPS 2 genotypes were more appropriate for distinguishing virulent and non-virulent strains. This finding suggested that most V. vulnificus wild types are also a human health concern.Populations of in level ,55. Not in level ,56. In t strains . Additiouggested . In this was 70% . Similar was 70% . HowevercgE gene . Previou5% 16S A . In thisaneously , probablnditions . Based oronments ,62. It hchanisms . Based oV. vulnificus from different environmental niches, using the ERIC-PCR method, which is more accurate as compared with REP-PCR [V. vulnificus with ERIC-PCR, which grouped these isolates into two main clusters based on their respective sampling sites, reference strain, and genotypic profiles. This is consistent with the result of the previous study, where the V. vulnificus strains isolated from the Baltic Sea region were classified into two main clusters using multi-locus sequence typing (MLST) [V. vulnificus isolated from the river basin showed diverse heterogeneity in genotypic profiles, indicating unique sources of this pathogenic strain and implying an uneven distribution of genetic differences across each sampling site. The genetically divergent strains may also be associated with geographical distribution, invertebrate host preferences, environmental stress, and changing estuarine conditions [V. vulnificus strains from source tracking and the differences between their distinct environments.Previous studies have demonstrated the higher genetic diversity among the isolates of  REP-PCR . In thisg (MLST) . The strnditions . HoweverIn this study, three sampling sites were investigated around the neighboring Puzih River (PR), Dongshi fishing harbor (DS), and Budai fishing harbor (BD). The PR region was divided into three sections according to the distance to the estuary, as shown in g for 30 min. In the case of shellfish samples, 10–20 g of shellfish meat was suspended in a 50 mL centrifuge tube along with 1S PBS, with a total volume of 40 mL. Finally, homogenization was carried out twice using an ultra-turrax tube drive  at a maximum rotating speed of 30 s.About 300 mL of water was filtered through a 47 mm sterilized filter membrane with a pore size of 0.45 um  to get the high concentration of bacteria. This concentrated membrane was eluted in 25 mL phosphate-buffered saline (PBS) and centrifuged at 2600× V. vulnificus ATCC27562, which served as a control for subsequent experimental analysis. The DNA extraction from overnight culture was centrifuged at 10,000× g for 5 min and removed from the supernatant. DNA from concentrated pellet was extracted for molecular analysis using ZP02006 MagPurix automatic DNA extraction system  provided with the bacterial DNA extraction kit, following the procedure of the manufacturer’s instructions, with the final elution in 100 µL. This eluent was then used for PCR experiments with a total reaction volume of 25 µL by adding the appropriate concentration of template, primers, and master mix, respectively, as shown in vvhA gene in PCR, which is a common gene marker for the identification of V. vulnificus species. Subsequently, the detected five major virulence-associated genes, including V. vulnificus cytolysin⁄hemolysin gene (vvhA), viral correlated gene (vcg), capsular polysaccharide operon (CPS), pathogenicity region XII (PRXII), Sial acid catastrophe region (nanA), and enzyme IIA of mannitol fermentation operon (manIIA) were used for phylogenetic analysis and strain typing, by means of ERIC-PCR fingerprinting and with the aid of commercial software BioNumerics . Finally, the cluster analysis was performed using curve-based Pearson correlation, and the resulting dendrogram was generated based on the unweighted pair group method with arithmetic mean (UPGMA).The pre-treated water sample and 1 mL shellfish homogenized liquid sample was transferred into 10 mL Alkaline Peptone Water , followed by incubation at 30 °C, for 24 h, for enrichment. The next day, a loopful of broth from the pre-enrichment step was streaked on CHROMagar Vibrio  plates and Thiosulfate Citrate Bile Salt Sucrose  agar plate, followed by incubating at 37 °C, for 24 h. The next day, a single colony from agar plate was picked with the help of a toothpick and inoculated into an APW tube and then incubated at 30 °C, for 24 h. After incubation, 300 µL culture containing candidate isolates were mixed with 700 µL 33% glycerol into a 1.5 mL centrifuge tube and stored at −20 °C. The reference strain source in this study was V. vulnificus was carried out using the disc diffusion method, following the guidelines of Clinical and Laboratory Standards Institute instructions . The isolated strains of V. vulnificus were cultured in 5 mL Cation Adjusted Mueller Hinton Broth , for 16–25 h, at 35 °C. The bacterial suspension was adjusted to 0.5 McFarland and evenly streaked on Mueller Hinton Agar (MHA). Subsequently, we aseptically placed the selected antibiotic paper disc on evenly streaked MHA and incubated at 34 °C, for 16–20 h. Finally, the antibiotic susceptibility of different strains was observed by measuring the size of the zone of inhibition (ZOI). The criteria for MDR was defined as non–susceptibility to at least one agent in three or more antimicrobial categories [The antibiotic susceptibility testing of all isolates of tegories .V. vulnificus strains was higher in downstream of the river basins adjacent to the residential area followed by the estuary area and fishing harbors. Additionally, none of the isolates were purified from the upper section of the river basin and the shellfish samples of these fishing harbors. Furthermore, we could isolate only one strain from the water sample of fishing harbors. These data suggest that the virulent strains of V. vulnificus might be enriched and spread from the surrounding urban, residential areas, most probably through domestic waste discharge mixing into the river basin. Consequently, following the water flow, this might also lead to the contamination of downflow areas, including the estuary area of the river basin and the fishing harbors. Notably, 88.9% isolated strain of V. vulnificus exhibited multiple virulence factors. The comparison of V. vulnificus genotyping based on this study’s virulence factors, the vcgC, 16S B, vvhA, manIIA, and GPS 2 genotype were more appropriate for distinguishing virulent and non-virulent strains. Fortunately, all V. vulnificus isolates in this study were susceptible to all tested antibiotics. The ERIC-PCR fingerprinting revealed heterogeneity among the isolated strains even at a single sampling site, the river basin. This defined the broad distribution of genetic differences across sampling sites and respective isolated strains, indicating multiple sources of these strains. Even at individual strain-level, these virulent strains of V. vulnificus exhibit multiple toxigenic profiles in the aquatic environments, which is a significant threat to human health. Therefore, a continuous monitoring of these coastal aquatic environments and their adjacent waste discharge areas is warranted in order to timely prevent the further spread of V. vulnificus.The detection rate of