Identification of circulating miRNA biomarkers in Leptospirosis for early detection: A Promising Diagnostic Approach.

Leptospirosis is a zoonotic disease of global significance, contributing to morbidity and mortality worldwide. It is endemic to tropical regions, with outbreaks during monsoons. The disease manifestations are similar to that of other febrile illness such as dengue, malaria hence often misdiagnosed and underreported. The zoonoses if undetected, progresses to cause severe life-threatening complications also known as Weil's disease. Routine diagnostic tests are based on the detection of antibodies in patient serum and are not accurate during the initial phase of the infection. Therefore, it is necessary to detect novel biomarkers that can be used in early detection of leptospirosis. Circulating miRNAs are known to be promising biomarkers for various diseases including cancer, tuberculosis, influenza; hence in this study the potential of miRNAs as biomarkers for leptospirosis was evaluated. A total of 30 leptospirosis cases were screened for the differential expression of 10 miRNA by RT-qPCR assay. The differential expression was calculated by relative quantification using healthy individuals as controls. Among the 10 miRNA,3 miRNA, miR-28-5p, miR-302c-3p and miR-302a-3p were reported to exhibit a significant trend of upregulation. Further their role in immune pathways and biological processes was investigated by KEGG analysis and Gene Ontology. The 3 miRNAs were observed to target various immune response pathways, thus confirming their role in host immune response. Based on the results obtained in this study, miR-28-5p, miR-302c-3p and miR-302a-3p can be considered as potential biomarkers for the detection of leptospirosis.

Oral administration of recombinant outer membrane protein A-based nanovaccine affords protection against Aeromonas hydrophila in zebrafish.

Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio.

Non-clinical isolates of Vibrio parahaemolyticus harbouring traits of potential pathogenicity and fitness: A molecular analysis.

Here we investigated the distribution of virulence and fitness attributes V. parahaemolyticus isolated from marine environment (n = 105). We discovered ∼1% of isolates positive for tdh, 8.57% for trh, and 4.76% had tdh and trh genes. More than 50% of the isolates had pathogenicity islands specific to pandemic clones and secretion systems which are detected partially or entirely. VPaI-1 found in 59.04%; VPaI-4 in 60%; VPaI-5 in 34.28%; VPaI-2 in 99.04%; VPaI-3 in 91.42% and VPaI-6 in 99.04% isolates. Also, 34.28% of the isolates harboured T3SS2 encoding VPaI 7; T3SS1 in 98.09%; T6SS2 in 99.04% isolates and T6SS1 in 60.95% isolates. The cytotoxicity analysis showed a significant effect by causing when infected with trh+ environmental isolates. The expression of the trh, VopC, and VopA genes during infection showed a significant upregulation. This suggests the presence of virulence traits among V. parahaemolyticus that could threaten public health.

In vitro determination of probiotic efficacy of Bacillus subtilis TLDK301120C24 isolated from tilapia against warm water fish pathogens and in vivo validation using gnotobiotic zebrafish model.

Eco-friendly alternatives such as probiotics are needed to prevent economically relevant infectious diseases for a successful disease-free harvest in aquaculture. The use of antibiotics has been the favored practice, but its empirical and indiscriminate use has led to antibiotic resistance in the aquatic environment and residues in the food fish. With this rationale, a probiotic was isolated from tilapia, a commercially important cultured fish worldwide. The characteristics of the probiotic were checked against common bacterial pathogens affecting aquaculture. In vitro tests demonstrated the inhibitory effects of the isolated probiotic on the growth of Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum, and V. alginolyticus. The candidate probiotic, referred to as TLDK301120C24, was identified as Bacillus subtilis by a battery of biochemical tests and genotypic confirmation by 16S rDNA sequencing. The in vitro results revealed the ability of the probiotic to withstand the gut conditions that included pH range of 3-9, salt concentration of 0.5-6%, and bile salt concentration of up to 6%. The isolate could hydrolyze starch (12-14 mm clearance zone), protein (20-22 mm clearance zone), and cellulose (22-24 mm clearance zone). Further, the inhibitory ability of the probiotic against aquatic pathogens was determined in vivo using gnotobiotic zebrafish by employing a novel approach that involved tagging the probiotic with a red fluorescent protein and the pathogens with a green fluorescent protein, respectively. The colonizing ability of probiotics and its inhibitory effects against the pathogens were evaluated by fluorescence microscopy, PCR, and estimation of viable counts in LBA + Amp plates. Finally, the competitive inhibition and exclusion of fish pathogens A. hydrophila and E. tarda by B. subtilis was confirmed semi-quantitatively, through challenge experiments. This study shows the potential of B. subtilis as a probiotic and its excellent ability to inhibit major fish pathogens in vivo and in vitro. It also shows promise as a potent substitute for antibiotics.

Isothermal amplification-based assays for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2: Opportunities and recent developments.

The coronavirus disease 2019 (COVID-19) is a global pandemic caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, the virus has been detected in 219 countries of the world. Therefore, managing the disease becomes the priority, in which detecting the presence of the virus is a crucial step. Presently, real-time RT polymerase chain reaction (RT-qPCR) is considered a gold standard nucleic acid amplification test (NAAT). The test protocol of RT-qPCR is complicated, places high demands on equipment, testing reagents, research personnel skills and is expensive. Therefore, simpler point-of-care (POC) tests are needed to accelerate clinical decision-making and take some of the workload from centralized test laboratories. Various isothermal amplification-based assays have been developed for the sensitive detection of different microorganisms, and recently some of them have been applied for detection of SARS-CoV-2. These do not require any programable thermocycler, can produce the results in a single temperature, and therefore, are considered simple. Unlike RT-qPCR, these methods are highly sensitive, specific, less time-consuming, simple and affordable, and can be used as POC diagnostic kit for COVID-19. In this review, we have discussed the potential of isothermal amplification-based assays as an alternative to RT-qPCR for the detection of SARS-CoV-2.

Metagenomic Analysis to Uncover the Subgingival and Atherosclerotic Plaque Microbiota in Patients with Coronary Artery Disease.

The role of periodontal pathogens in the initiation and progression of atherosclerosis has been extensively researched, yet a precise causal mechanism has not been established. The subgingival microbiota may be a source of dissemination and may contribute to the development of atherosclerosis; hence this study attempted to characterize and compare the subgingival and atherosclerotic plaques. Plaque samples were subjected to 16S rRNA-based metagenomics to study microbiota associated with subgingival and atherosclerotic plaques collected from patients with coronary artery disease. The PCoA analysis showed that the microbiomes of subgingival plaques were highly scattered and showed a diverse microbial composition, unlike the atherosclerotic plaques that did not show evident variability in the microbial composition and formed a close distinct group. The abundance of various genera in the subgingival plaques revealed Fusobacterium (11%), Acinetobacter (13%), Veillonella (9%), and Prevotella (11%) among the top ten genera. The atherosclerotic plaques contained Acinetobacter (39%), Chryseobacterium (9%), Rhizobium (5%), and Staphylococcus (4%). All the patients examined in this study had either generalized or localized periodontitis with varying degrees of severity. The community microbiota analysis revealed that 22 bacterial genera were shared between two different plaques, with Acinetobacter being dominant. Based on the Human Oral Microbiome Database, 55% of the shared microbiota in this study have been listed as periodontal microbiota, with some of them found in increased proportions in patients with periodontitis suggesting the translocation of bacteria from the periodontal pockets into the circulation. This study provides valuable insights into the possible relationship between periodontal pathogens and atherosclerotic cardiovascular disease.

Characterisation of broad-spectrum phiKZ like jumbo phage and its utilisation in controlling multidrug-resistant Pseudomonas aeruginosa isolates.

The emergence of highly virulent multidrug-resistant P. aeruginosa has become increasingly evident among hospital-acquired infections and has raised the need for alternative therapies. Phage therapy can be one such alternative to antibiotic therapy to combat multidrug-resistant pathogenic bacteria, but this requires the availability of phages with a broad host range. In this study, isolation and molecular characterisation of P. aeruginosa specific phages were carried out. A total of 17 phages isolated showed different spectra of activity and efficiency of lysis against 82 isolates of P. aeruginosa obtained from clinical samples (n = 13), hospital effluent (n = 46) and fish processing plant effluent (n = 23). Antibiotic susceptibility test results revealed multi-drug resistance in 61 of the total 82 isolates. Three new jumbo lytic P. aeruginosa specific broad host range phages were isolated and characterised in this present study belonged to the family Myoviridae (order Caudovirales). The genetic analysis of ɸU5 revealed that phage has a genome size of 282.6 kbp with 373 putative open reading frames (ORFs), and its genetic architecture is similar to phiKZ like jumbo phages infecting P. aeruginosa. The bacteriophages isolated in this study had lytic ability against biofilm-forming and multidrug-resistant P. aeruginosa and could be candidates for further studies towards phage therapy.

Effect of NaCl, high iron, iron chelator and antibiotics on growth, virulence gene expression and drug susceptibility in non-typhoidal Salmonella: an in vitro fitness study.

Salmonella is one among the most versatile and resilient enteric pathogens that is known to have developed various survival strategies within the host system. The ability of the bacteria to circumvent the physiological parameters as well as dodge the antimicrobial stress environment within the host is one of the most crucial steps in establishing an infection. With an alarming rise in multi-drug resistant serovars of non-typhoidal Salmonella and lack of vaccine for combatting the infections, behaviour of the bacteria in the presence of host physiological conditions (NaCl, high and low iron) and antibiotics will help in understanding the survival strategies as well as mechanisms of resistance. Two multi-drug resistant and two sensitive serovars of Salmonella Weltevreden and Salmonella Newport isolated from poultry and seafood were used for growth kinetics and virulence gene expression study. The results obtained revealed that despite similar resistance pattern, effect of individual class of antibiotics on the growth of serovars varied. On the contrary, no significant difference was observed in growth pattern on exposure to these in vitro experimental conditions. Nevertheless, coupling these conditions with antibiotics drastically reduced the minimum inhibitory concentration (MIC) of antibiotics in resistant strains. A first of its kind study that draws attention on the significant effect of antibiotics and physiological conditions on MIC between resistant and sensitive non-typhoidal Salmonella serovars and expression of virulence genes from Salmonella pathogenicity island (SPI) 1 and 2 (invA, hilC, fliC2, sseA and ssrB).

Effect of ciprofloxacin and in vitro gut conditions on biofilm of Escherichia coli isolated from clinical and environmental sources.

AIM: This study aimed at characterizing the biofilm-forming ability of drug-resistant and sensitive Escherichia coli under in vitro gut conditions and in the presence of ciprofloxacin. METHODS AND RESULTS: 153 E. coli isolates comprising 80 from clinical and 73 from environment source were studied for their ability to form biofilm under control and in vitro simulated gut conditions. The integrity of preformed biofilm on exposure to ciprofloxacin was assessed. Expression of biofilm-associated genes was analysed using qPCR. A high degree of resistance was observed in clinical isolates with a concomitant prevalence of blaTEM . Bile, pH and low temperature enabled the E. coli biofilm to resist the effect of ciprofloxacin. Clinical isolates of E. coli formed strong biofilms in in vitro gut conditions following exposure to high concentration of ciprofloxacin. The expression of biofilm genes varied between different gut conditions viz., presence of bile, pH and low temperature, included in this study. CONCLUSIONS: This study demonstrates the importance of papC and csgA for maintaining the biofilm integrity upon antibiotic exposure. Escherichia coli form biofilm as a survival strategy to adapt to the conditions in their environment irrespective of their drug resistance status. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides an understanding of the effect of different parameters of the gut conditions during infection and the effect of antibiotic on survival and biofilm-forming ability of clinical and environmental E. coli isolates. It further suggests that bacteria resort to biofilm formation as one of the mechanisms to adjust to alterations in gut conditions and once the biofilm is formed, it requires high concentration of ciprofloxacin to eradicate it.

Association of exopolysaccharide genes in biofilm developing antibiotic-resistant Pseudomonas aeruginosa from hospital wastewater.

The study aimed to examine the relationship between antibiotic resistance, biofilm formation and genes responsible for biofilm formation. Sixty-six Pseudomonas aeruginosa isolates were obtained from hospital wastewater and analyzed for their antibiotic resistance. Biofilm production among the isolates was tested by indirect quantification method crystal violet assay. Biofilm-associated genes among these isolates psl, alg, and pel were also checked. The maximum resistance was observed for ampicillins (88.24%) followed by nalidixic (83.82%), and nitrofurantoin (64.71%), respectively. Biofilm phenotypes are distributed in the following categories: high 39.39% (n = 26); moderate 57.57% (n = 38), and weak 3.0% (n = 2). Among the total isolates, biofilm-associated genes were detected in 84.84% (n = 56) of isolates and the remaining isolates 15.15% (n = 10) did not harbor any genes. In this study, pslB was the most predominant gene observed (71.21%, n = 47) followed by pslA (57.57%, n = 38), pelA (45.45%, n = 30), algD (43.93%, n = 29), and pelD (27.27%, n = 18), respectively. The present study reveals that the majority of the isolates are multidrug resistant being moderate and high biofilm formers. The study implies that biofilm acts as a machinery for bacteria to survive in the hospital effluent which is an antibiotic stress environment.

Biofilm Formation and Associated Gene Expression in Multidrug-Resistant Klebsiella pneumoniae Isolated from Clinical Specimens.

Biofilms reduce the bacterial growth rate, inhibit antibiotic penetration, lead to the development of persister cells and facilitate genetic exchange. The biofilm-associated Klebsiella pneumoniae infections have not been well studied, and their implications in overcoming the effects of antimicrobial therapy are yet to be fully understood. Hence this study evaluated the antibiotic resistance pattern, antibiotic resistance determinants of extended-spectrum beta-lactamase (ESBL) family. Biofilm-forming ability of seventy multidrug-resistant clinical isolates of K. pneumoniae and the biofilm-associated genes of representative biofilm formers from a tertiary care hospital were also assessed. The K. pneumoniae isolated from urine exhibited resistance towards ceftazidime, nalidixic acid and meropenem. Isolates from blood were resistant to cefuroxime. Higher rates of resistance were observed towards cefuroxime, nalidixic acid, and meropenem for the isolates from the endotracheal aspirate. Extended spectrum beta-lactamase production by CLSI's disc diffusion-based confirmation test revealed all the K. pneumoniae to be as ESBL producers. Most of the isolates harboured the bla gene variants, blaSHV and blaTEM. Majority of the isolates were colistin sensitive. 97.1% of the K. pneumoniae produced biofilm. K. pneumoniae isolated from pus and blood produced fully established biofilms. Strong biofilm formers were sensitive to co-trimoxazole and ciprofloxacin. Moderate biofilm formers exhibited sensitivity towards meropenem and imipenem. Expression of the fimH gene was increased, while mrkD showed reduced expression among the strong biofilm formers. Moderate biofilm formers showed variable expression of the genes associated with the biofilm formation. The weak and non-biofilm formers showed reduced expression of both the fimbrial genes. Multidrug-resistant isolates produced ESBLs and formed well-established biofilms.

Genome analysis of clinical isolate of Campylobacter fetus subspecies fetus MMM01 from India reveals genetic determinants of pathogenesis and adaptation.

In this study we report the whole genome sequencing (WGS) based analysis of blood-borne Campylobacter fetus subsp. fetus MMM01 isolated from a diabetic patient to obtain deeper insights in to the virulence and host adaptability. The sequenced genome of C. fetus subsp. fetus MMM01 along with reference genomes retrieved from NCBI was subjected to various in-silico analysis including JSpecies, MLST server, PATRIC server, VFanalyzer, CARD, PHASTER to understand their phylogenetic relation, virulence and antimicrobial resistance profile. The genome had a size of 1,788,790 bp, with a GC content of 33.09%, nearly identical to the reference strain C. fetus subsp. fetus 82-40. The MLST based phylogenetic tree constructed revealed the polyphyletic branching and MMM01 (ST25) was found to be closely related to ST11, both belong to the sap-A serotype which are more common in human infections. VFanalyzer identified 88 protein-coding genes coding for several virulence factors including Campylobacter adhesion to fibronectin, flagellar apparatus, cytolethal distending toxin operons and Campylobacter invasion antigen proteins which enhance the virulence of bacteria along with resistance genes against antibiotics including fluoroquinolone, chloramphenicol, tetracycline, and aminoglycoside in MMM01, which points to enhanced survival and pathogenicity of this zoonotic pathogen. It was interesting to find that MMM01 lacked FGI-II island found in most of the clinical isolates, which encoded CRISPR Cas and prophage II regions. More details about the complexity and evolution of this zoonotic pathogen could be learned from future studies that concentrate on comparative genome analysis using larger genome datasets.

Phenotypic characterization of auxotrophic mutant of nontyphoidal Salmonella and determination of its cytotoxicity, tumor inhibiting cytokine gene expression in cell line models.

An auxotrophic mutant of nontyphoidal Salmonella (NTS) strain (Salmonella Oslo) was phenotypically characterized in this study. The characterization was based on phenotype, morphology, motility, biofilm forming ability, growth kinetics, etc. The phenotypic results from the above experiments determined that the mutant showed variation in phenotypic characters from that of wild-type strain. Subsequently, mutant and wild-type NTS were subjected to epithelial cell invasion and intracellular replication assays. The real-time PCR analysis was also performed to analyse expression of tumor inhibiting cytokine genes and virulence genes post-bacterial infection in cell lines. The mutant showed highest invasion potential than wild-type NTS whereas the replication of mutant was slower in both the cell lines. Similar to the wild-type strain, the mutant also retained the cytotoxic potential when analysed in vitro. Furthermore, the expression of proinflammatory cytokine genes such as TNF-α and IL-1ß was upsurged with the downregulation of anti-inflammatory cytokine genes like TGF-ß, IL-6 and IL-10 post-infection of the mutant strain in cell lines. In addition, virulence genes of Salmonella pathogenicity island one and two of mutant were downregulated in vitro except invA in HeLa cell line. Therefore, the auxotrophic mutant showed positive attributes of a potential antitumor agent in terms of expressing tumor inhibiting cytokine genes when assessed in vitro. Though the study did not check the tumor inhibitory effect of NTS strain directly, findings of the study emphasizes on the development of a novel strain of NTS with less virulence and more immunogenic traits to inhibit tumor cells.

Evaluation of loop-mediated isothermal amplification assay along with conventional and real-time PCR assay for sensitive detection of pathogenic Vibrio parahaemolyticus from seafood sample without enrichment.

The primary reason for foodborne illness is improper seafood safety testing, and hence, an appropriate tool for testing is the key to control the outbreaks. The current study aimed to develop a loop-mediated isothermal amplification (LAMP) assay to detect pathogenic Vibrio parahaemolyticus, important foodborne pathogen, targeting tdh, and trh genes. The specificity of the LAMP assay was good without any false-positive and false-negative results. The assay was highly sensitive and could detect the pathogenic V. parahaemolyticus as low as 1 CFU/reaction in spiked seafood samples and 1 pg of extracted DNA. Out of 62 seafood samples from India's southwest coastal region tested with LAMP assay, eight (12.9%) were positive for trh, and seven (11.29%) samples were positive tdh gene. LAMP-based on tdh and trh was found to be significantly more sensitive (p < 0.05) than conventional PCR and nearly equal sensitive as real-time PCR (RT-PCR) for the detection of pathogenic V. parahaemolyticus. Our study shows that LAMP assay can be a better approach as a point-of-care (POC) diagnostic tool and could detect pathogenic V. parahaemolyticus on seafood samples directly without enrichment and isolation. The high sensitivity and simplicity make LAMP assay a better alternative method than the conventional method and RT-PCR for the detection of pathogens. LAMP assay can be considered as a good alternative to PCR for the routine detection of pathogenic V. parahaemolyticus in seafood.

Detection technologies and recent developments in the diagnosis of COVID-19 infection.

COVID-19 is a disease caused by SARS-CoV-2 capable of causing mild to severe infections in humans. Since its first appearance in China in December 2019, the pandemic has spread rapidly throughout the world. Despite considerable efforts made to contain the disease, the virus has continued its prevalence in many countries with varying degrees of clinical manifestations. To contain this pandemic, collaborative approach involving accurate diagnosis, epidemiology, surveillance, and prophylaxis is essential. However, proper diagnosis using rapid technologies plays a crucial role. With increasing incidence of COVID-19 cases, the accurate and early detection of the SARS-CoV-2 is need of the hour for effective prevention and management of COVID-19 cases as well as to curb its spread. RT-qPCR assay is considered to be the gold standard for the early detection of virus, but this protocol has limited application to use as bedside test because of its technical complexity. To address these challenges, several POC assays have been developed to facilitate the COVID-19 diagnosis outside the centralized testing laboratories as well to accelerate the clinical decision making with a least turnaround time. Hence, in this report, we review different nucleic acid-based and serological techniques available for the diagnosis and effective prevention of COVID-19. KEY POINTS : • Provides comprehensive information on the different diagnostic tools available for COVID-19 • Nucleic acid based tests or antigen detection tests are used for diagnostic purpose • Accurate diagnosis is essential for the efficient management of COVID-19.

Recombinant viral proteins delivered orally through inactivated bacterial cells induce protection in Macrobrachium rosenbergii (de Man) against White Tail Disease.

White tail disease (WTD) is a disease of Macrobrachium rosenbergii caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) with the potential to devastate the aquaculture industry. The present study aimed to explore the possible protection of M. rosenbergii against the disease by oral administration of bacterially expressed recombinant capsid proteins of MrNV and XSV. Juvenile M. rosenbergii were fed with the feed coated with inactivated bacteria encapsulated expressed recombinant viral proteins either individually or in combination for 7 days. Challenge studies using WTD causing agents were carried out after 3 (group I), 10 (group II) and 20 (group III) days post-feeding of viral proteins. Recombinant capsid protein of MrNV showed better protection when compared to other treatments with relative per cent survival of 62.5% (group I), 57.9% (group II) and 39.5% (group III). Treatment controls of groups I, II and III showed 100%, 95% and 95% mortality, respectively. The study demonstrates that oral administration of recombinant capsid proteins of MrNV and XSV provides effective protection against WTD in freshwater prawn.

Occurrence of antibiotic resistance among Gram negative bacteria isolated from effluents of fish processing plants in and around Mangalore.

The presence of antibiotic-resistant bacteria in seafood not only poses a serious health risk for the consumers but also contributes to the spread of these antibiotic-resistant bacteria in the natural environments through the effluents discharged from the fish processing plants. The aims of this study were to isolate Gram-negative bacteria from the effluents of fish processing plants in and around Mangalore, India and to profile their antibiotic resistance pattern. Maximum resistance was seen for ampicillin (40.78%) followed by tetracycline (40.22%) and nitrofurantoin (29.05%). Further, the detection of genes that contribute to antibiotic resistance revealed the presence of sulfonamide resistance genes (sul1 and sul2) and extended spectrum ß-lactamase genes (bla CTX-M, bla TEM) in a few isolates. The presence of such bacteria in fish processing effluents is a matter of great concern because they can contribute significantly to the antibiotic resistance in the natural environment. It is imperative that seafood processing plants follow the safe disposal of effluents in order to reduce or eliminate the antibiotic resistance menace.

Rapid visual detection of Vibrio parahaemolyticus in seafood samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

The detection and monitoring of Vibrio parahaemolyticus pathogen in aquatic foods have become essential for preventing outbreaks. In this study, loop-mediated isothermal amplification (LAMP) assay with the azo dye, hydroxynaphthol blue (HNB) was developed targeting species-specific tlh gene. The assay was carried out on 62 seafood samples that included clam and shrimp and compared with conventional LAMP assay performed with the commonly used fluorescent dye, conventional PCR, and real-time PCR (RT-PCR). Of 62 samples studied for tlh gene, 32 (51.61%) gave positive by HNB-LAMP, which comprised 22 (70.96%) clam samples and 10 (32.25%) shrimp samples. The HNB-LAMP assay was found to be highly sensitive, specific, and superior to conventional PCR (p > 0.05). RT-PCR presented higher sensitivity than HNB-LAMP; however, it has the limitation of being cost-intensive and requiring technical expertise to perform. HNB-LAMP is affordable, rapid, simple, and easy to perform, allowing naked eye visualization.

Phages amid antimicrobial resistance.

Increasing levels of resistance to antimicrobial agents have created chaos in the health sector, with several infections not responding to antibiotic treatments. Search for alternative strategies has looked at bacteriophages as potential therapeutics and in the last couple of years. There are reports of phages being successfully used to treat life-threatening infections. Phages are also mobile elements that exchange genes between and within different bacterial species and account significantly for strain differences across and within a species. A gap in metagenomics analysis and conservative methods of detection have failed to give an accurate account of the role of bacteriophages in antimicrobial resistance. Recent studies have focussed on the role of bacteriophages in the adaptation of pathogens to new hosts and the emergence of multidrug-resistance, which are a significant concern against phage therapy. This article presents a comprehensive account of weighing the odds of phage therapy verses phage-mediated antimicrobial resistance.

Unveiling the acid stress response of clinical genotype Vibrio vulnificus isolated from the marine environments of Mangaluru coast, India.

Gastric acidity is one of the earliest host defences faced by ingested organisms, and successful pathogens need to overcome this hurdle. The objective of this study was the systematic assessment of acid-stress response of Vibrio vulnificus isolated from coastal regions of Mangaluru. Acid-shock experiments were carried out at pH 4.0 and pH 4.5, with different experimental conditions expected to produce a varied acid response. Exposure to mild acid before the acid shock was favourable to the bacteria but was dependent on cell population and pH of the media and was independent of the strains tested. Lysine-dependent acid response was demonstrated with reference to the previously identified lysine decarboxylase system. Additionally, the results showed that inoculation into oysters provided some level of protection against acid stress. Increased expression of lysine/cadaverine genes was observed upon the addition of ground oyster and was confirmed by quantitative real-time PCR. The potential role of ornithine was analyzed with regard to acid stress, but no change in the survival pattern was observed. These findings highlight the physiology of bacteria in acid stress.