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Objectives Aeromonads cause severe diseases in farmed aquatic organisms. Herein, we examined 28 isolates causing disease in farmed aquatic organisms from India (n = 24) and Taiwan (n = 4) to gain insight of their genotypic and phenotypic properties. Results API 20NE biochemical phenotyping showed ≥ 90% similarity classifying all isolates as Aeromonas hydrophila. 16S rRNA genotyping showed ≥ 98% homology among all isolates with A. sobria (NR119044.1ATCC), A. veronii (MK990549.1), A. caviae (NR029252.1) and A. hydrophila (MG984625.1ATCC) and other reference strains. In contrast, gyrB showed a higher intraspecies diversity (≥ 96%) than 16S rRNA delineating the 28 isolates into three groups. Group-I consisted of seven Indian isolates clustered with A. sobria (MK484163.1ATCC), group-II comprised of five Indian and two Taiwanese isolates clustered with A. veronii AF417626.1ATCC while group-III had 11 Indian and three Taiwanese isolates grouped with A. hydrophila (AY987520.1 and DQ519366.1) reference strains. None of our isolates clustered with A. caviae (AJ868400.1ATCC) reference strain. These findings suggest that A. sobria, A. veronii and A. hydrophila could be the etiological agents of diseases observed in farmed fish and soft-shelled turtles (Pelodiscus sinensis) examined in this study. Overall, our findings accentuate the importance of combining phenotyping with genotyping for correct taxonomic classification of Aeromonas spp. in Aquaculture.

Legionella is a fastidious organism that is difficult to culture in the lab but is widely distributed in environmental, domestic, and hospital settings. The clinical manifestations due to Legionella infections range from mild fever to fatal pneumonia and multiorgan pathologies. Legionella outbreaks though prevalent globally are not reported in developing countries due to difficulties in isolating this organism and the lack of simple diagnostic protocols. Here, we review the literature from across countries to present various methods used to detect Legionella from environmental and clinical samples. We compare the sensitivity and the specificity of the conventional culture-based assays with the recent methods and discuss approaches to develop better detection and diagnostic tests. With better cost-effective detection techniques and regular monitoring of the susceptible sites, which may harbor Legionella colonies, most of the Legionella infections can be prevented. As a result, considerable burden, caused by Legionella infections, on the healthcare system, in especially economically weaker countries, can be mitigated.

Bacterial cells adopt various strategies to adapt themselves in diverse environmental conditions. Salmonella is one such bacteria with diverse mechanisms to survive, replicate and infect in wide host range. This study aims at investigating the biofilm-forming ability of multidrug-resistant and sensitive Salmonella serovars on exposure to bile. Antibiogram of all the isolates was determined by disk diffusion method and their biofilm-forming ability in the presence or absence of bile was assessed by microtiter plate assay. Biofilm results were validated by calcofluor, Congo red plate and test tube method. Few isolates were selected for further study of their expression of biofilm related genes on exposure to bile using real time PCR. Among the 59 isolates of Salmonella isolated from seafood and poultry, 30 isolates were multi-drug resistant (MDR). Under control conditions, 57% (n = 25) of the serovars were able to form biofilm. While, 86% (n = 51) of the serovars produced biofilm in the presence of bile. The relative gene expression study of the selected serovars for 8 different genes showed a striking difference in the expression levels, supporting the hypothesis that the presence of bile triggers biofilm formation in food associated strains of non-typhoidal Salmonella by upregulation of genes involved in biofilm production.

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase (MBL) associated with Enterobacteriaceae constitutes an important growing public health threat. The present study aims to characterize the NDM-1 producing Gram-negative bacteria (GNB) from the effluents of two tertiary care hospitals in Mangalore, South India and to profile their antibiotic resistance pattern. A total of 134 GNB were isolated from 30 hospital wastewater samples (treated and untreated) and analyzed. High-level resistance among untreated effluent sample was found toward nalidixic acid (74.52%), followed by cefotaxime (72.64%) and ampicillin (66.03%). Among the treated effluent isolates, the high resistance was found toward ampicillin (85.71%) followed by cefotaxime (85.71%) and piperacillin-tazobactam (53.57%). From untreated effluent isolates, 9 were NDM-1 positive by PCR; no isolates from treated effluent samples harbored blaNDM-1 . Untreated hospital wastewater is found to be important reservoirs of antibiotic-resistant bacteria carrying blaNDM-1 , and the presence of such bacteria in the effluents is a matter of great concern because they can contribute the antibiotic resistance to the natural environment. However, the absence of NDM in treated effluents emphasizes the importance of effluent treatment in reducing the dissemination of antibiotic-resistant bacteria. PRACTITIONER POINTS: Hospital wastewater is the important reservoir of antibiotic-resistant bacteria especially metallo-β-lactamase producers (NDM-1). Wastewater treatment procedures in hospitals reduce the NDM isolates in the treated effluent. Thereby reduces the risk of resistance spread in the environment.

Cancer is of various kinds, so are the treatment modalities. Worldwide, cancer is the second leading cause of death, accounting for a whopping 9.6 million deaths in 2018. Globally, approximately one in six deaths is attributed to cancer. Photodynamic therapy (PDT) is a therapeutic strategy for the treatment of superficial lesions, warts, Barrett’s esophagus, premalignant lesions, malignant tumors, and ophthalmic diseases. The literature on PDT is approximately one-third of that in radiation therapy, yet the clinical implementation of PDT in cancer is relatively less. Despite substantial research, the clinical application of photodynamic strategy in cancer therapy is still in its infancy with only a limited number of case studies reported so far. The limitations of the photosensitizer and the shallow depth of penetration of light source are the key technical impediments. However, the use of nanomedicine in PDT can overcome these obstacles. Thus, it is necessary to gain knowledge on how nanomaterials can be merged with PDT and how it can be utilized in cancer theranostics. In this article, the focus is to understand how PDT works and how it can be utilized in improving the sensitivity of the existing diagnostic and therapeutic techniques. The article also addresses the current challenges for PDT and the future prospects of this technique, particularly in the area of diagnosis and treatment of cancer.

Purpose Burkholderia is a Gram-negative opportunistic bacterium capable of causing severe nosocomial infections. The aim of this study was to characterize Burkholderia cepacia complex and to compare different molecular methods used in its characterization. Methods In this study, 45 isolates of Burkholderia cepacia complex (Bcc) isolated from clinical cases were subjected to RAPD (Random amplified polymorphic DNA), recA-RFLP (Restriction fragment length polymorphism), 16SrDNA-RFLP, whole-cell protein analysis, recA DNA sequencing and biofilm assay. Results Of the 45 isolates tested, 97.7% were sensitive to ceftazidime, 82.2% were sensitive to Cotrimoxazole, 73.3% were sensitive to meropenem, 55.5% were sensitive to minocycline and 42.2% were sensitive to levofloxacin. Majority of the isolates harbored all the tested virulence genes except bpeA and cblA. The RAPD generated 11 groups (R1-R11), recA-RFLP 10 groups (A1-A10), 16SrRNA-RFLP 5 groups (S1–S5) and SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis) whole cell protein analysis revealed 12 groups (C1–C12). recA sequencing revealed that most of the isolates belonging to the genomovar III Burkholderia cenocepacia. Though all the methods are found to be efficient in differentiating Burkholderia spp., recA-RFLP was highly discriminatory at 96% similarity value. The study also identified a new strain Burkholderia pseudomultivorans for the first time in the country. Further, recA sequencing could identify the strains to species level. Majority of the multidrug-resistant strains also showed moderate to strong biofilm-forming ability, which further contributes to the virulence characteristics of the pathogens. Conclusions The study highlights the importance of combination of molecular methods to characterize Burkholderia cepacia complex. Molecular typing of these human pathogens yields important information for the clinicians in order to initiate the most appropriate therapy in the case of severe infections and to implement preventive measures for the effective control of transmission of Burkholderia spp.

Burkholderia cepacia complex is a Gram-negative opportunistic pathogen usually found in people with an immunocompromised condition such as cystic fibrosis (CF). In a tropical country like India, this organism has been associated with a number of hospital-acquired infections including sepsis. We present here a report of a case of Burkholderia vietnamiensis causing a non-lactational breast abscess in a non-CF patient. The pathogen was identified as B. cepacia using Vitek system and matrix-assisted laser desorption ionisation-time of flight. This was confirmed by polymerase chain reaction (PCR) using recA genus-specific gene and sequencing of the PCR amplicons. recA-restriction fragment length polymorphism and recA gene sequencing revealed that the isolate is B. vietnamiensis. This is the first description of B. vietnamiensis isolated from a clinical case from India.

In recent years, the concept of bacteria-mediated cancer therapy has gained significant attention as an alternative to conventional therapy. The focus has been on non-typhoidal Salmonella (NTS), particularly S. Typhimurium, for its anti-cancer properties, however, other NTS serovars such as Salmonella Oslo, which are associated with foodborne illnesses could potentially be effective anti-cancer agents. Here, we report the draft genome sequence of Salmonella Oslo isolated from seafood and its laboratory generated auxotrophic mutant.

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.

We report the use of next generation sequencing (NGS) for investigating an outbreak of 13 cases of Serratia marcescens blood stream infections in a non-Neonatal Intensive Care Unit (non-NICU) setting in a tertiary care hospital in India over 5 months. Thirteen cases of sepsis due to S. marcescens were identified in various Intensive Care Units (ICUs) over 5 months. Environmental surveillance identified isolates in the adult ICU (AICU). Antibiogram did not correlate with timeline. Sequencing libraries were prepared using Nextera XT chemistry (Illumina). Based on NGS, two clusters were identified. Cluster 1 had environmental and clinical isolates from the AICU and cluster 2 were isolates from the Coronary Care Unit (CCU). NICU and Paediatric ICU isolates did not belong to any cluster. Polyclonal outbreaks best identified by NGS can occur simultaneously. Good infection prevention practices like hand hygiene for compounded medicines and surface cleaning helped end the outbreak.

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.

Summary 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.

Background: Legionella have emerged as a pathogenic group due to increased use and poor maintenanceof artificial water environments. World Health Organization considers Legionella associated diseases tobe underestimated, especially in developing countries due to difficulty in detection. The aim of this studywas isolation and culture of Legionella spp. from artificial water systems and to test the importance of ironconcentration which can be developed as a reliable chemical marker.Method: Thirty samples each were collected from drinking water coolers, cooling towers and shower headsfitted in different houses of Mangaluru city. Isolation and identification of the bacteria were carried out as perthe standard protocols followed by determination of iron content prescribed by IS 3025 (Part 53).Conclusion: Our study shows that concentrations of iron, is an important factor that increases the likelihoodof Legionella spp. Statistically, a concentration of 300 mg Fe/L shows a positive correlation with Legionellapresence. Therefore, monitoring suspect sites for iron concentration and chelation of available iron, canbe developed as respective assays for identifying Legionella spp. Furthermore, we show that PCR baseddetection of Legionella is a more robust method than the classical 3-plate method, especially for virulentstrains.

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

Links between periodontitis and atherosclerosis can be predicted based on inflammatory mechanisms initiated by bacteria associated with periodontal lesions, which then influence the initiation or propagation of the atherosclerotic lesion. This study aimed to detect the presence of three periodontal pathogens, in atheromatous plaques of patients with coronary artery disease. Subgingival and atherosclerotic plaque samples were obtained from 80 patients scheduled for CABG or angioplasty. A nested PCR was done for the detection of the pathogens in the plaque samples. Porphyromonas gingivalis, Tanarella forsythia, and Treponema denticola were detected in 10%, 12.5%, and 1.3% of the atherosclerotic plaque samples respectively. It was also observed that patients whose atherosclerotic plaques tested positive for one or more of the pathogens had chronic periodontitis.

Objectives Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer health benefits to the host. Probiotics are currently being recommended and considered for many medical conditions. The Asia-Pacific region contributes to more than 40% of the global industry. Quality of commercial probiotics remains a challenge globally and has been a major concern in various countries in Europe, South Africa, Taiwan, India, Pakistan, and the USA. Research from these countries indicate that the contents do not correspond to the label information in terms of identity, viability, number of microorganisms or purity. The objective of this study is to assess the commercial probiotic bacterial contents and their label accuracy in India. No previous research has been done in this area in India, on commercial probiotics that are sold as "pharmaceuticals". Methods A random selection of the most prescribed probiotics for various clinical indications were chosen with a minimum shelf life of 12 months. The probiotics were single and multiple strains and these were evaluated by culture, viable plate count, DNA isolation and targeted metagenomics. Our study is the first step in scrutinizing probiotics in terms of quality and quantity analysis which are used across various age groups for multiple indications. Results Out of the 20 chosen probiotics eight products were single strain and 12 products were multiple strains. These probiotics showed very poor correlation between the declared contents on the pack and lab values in viable cell count colonies, the genus and species strain identification, presence of contaminants and these were confirmed with 16s RNA and next generation sequencing. Conclusion Poor correlation in the quality and quantity of probiotics proves that the label claim and actual claim of these "drugs" show exceptionally poor correlation and raises safety concerns in clinical use, especially in vulnerable age groups such as neonates, children and the elderly. Our study shows that "policing" of these probiotics is essential in protecting these patients who are at risk and ensuring quality control and helping clinicians making the right choice.

Pan genomic analysis of the global SARS-CoV-2 isolates has resulted in the identification of several regions of increased genetic variation but there is absence of research on its association with the clinical outcome. The present study fills the vacuum and does mutational analysis of genomic sequence of Indian SARS-CoV-2 isolates. Results reveal the existence of non-synonymous G614 spike protein mutation in 61.45% of the total study genome along with three other mutations. Further, temporal variation in the frequencies of G614 genotype in the country is observed. The examination of the probable association of G614 genotype with COVID-19 severity shows that CFR G614 genotype in India is positively and strongly correlated. It appears that the clinical outcome of the COVID-19 cases in India are significantly and adversely affected by the increasing trend in the G614 genotype; which needs to be addressed combining both laboratory experiments and epidemiological investigations.

Background & objectives: Infections caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli carrying blaCTX-M genes have been spreading globally, but there are geographical variations in the type of blaCTX-M genes prevalent and there are scanty data from India. This study was conducted to determine the CTX-M type ESBLs in E. coli isolates obtained from clinical specimens from patients with extra-intestinal infections attending a tertiary care hospital in south India. Methods: ESBL-producing E. coli isolated from patients with extra-intestinal infections were subjected to PCR using CTX-M group-specific primers. From a representative isolate, full-length CTX-M-15 gene was amplified and sequenced. An internal fragment of this gene was sequenced in 10 representative isolates. Results: Of the 300 isolates of E. coli tested, 88 per cent carried CTX-M genes and blaCTX-M-15 was the most dominant gene present in 90 per cent of the positive isolates. Most (91%) of the isolates positive for blaCTX-M were sensitive to meropenem. Interpretation & conclusions: Our findings showed blaCTX-M-15 to be the dominant gene. Based on the data on antimicrobial susceptibility, cefoperazone-sulbactum could be an antimicrobial of choice.

Background & objectives: Infection from fluoroquinolone-resistant extra-intestinal Escherichia coli is a global concern. In this study, isolation and characterization of fluoroquinolone-resistant extra-intestinal E. coli isolates obtained from hospital samples were undertaken to detect plasmid-mediated quinolone resistance (PMQR) genes. Methods: Forty three isolates of E. coli obtained from patients with extra-intestinal infections were subjected to antibiogram to detect fluoroquinolone resistance. The mechanism of fluoroquinolone resistance was determined by the detection of PMQR genes and mutations in quinolone resistance determining region (QRDR). Results: Of the 43 isolates, 36 were resistant to nalidixic acid (83.72%) and 28 to ciprofloxacin (65.11%). Eight E. coli isolates showed total resistance to both the antimicrobials without any minimum inhibitory concentration. The detection of PMQR genes with qnr primers showed the presence of qnrA in two, qnrB in six and qnrS in 21 isolates. The gene coding for quinolone efflux pump (qepA) was not detected in any of the isolates tested. The presence of some unexpressed PMQR genes in fluoroquinolone sensitive isolates was also observed. Interpretation & conclusions: The detection of silent PMQR genes as observed in the present study presents a risk of the transfer of the silent resistance genes to other microorganisms if present in conjugative plasmids, thus posing a therapeutic challenge to the physicians. Hence, frequent monitoring is to be done for all resistance determinants.

The resistance determinant blaCTX-M has many variants and has been the most commonly reported gene in clinical isolates of extended spectrum beta-lactamase producing Escherichia coli. Phages have been speculated as potential reservoirs of resistance genes and efficient vehicles for horizontal gene transfer. The objective of the study was to determine the prevalence and characterize bacteriophages that harbour the resistance determinant blaCTX-M . Escherichia coli specific bacteriophages were isolated from 15 samples including soil and water across Mangaluru, India using bacterial hosts that were sensitive to β-lactams. Phenotypic and genotypic characterization based on plaque morphology, host range, restriction fragment length polymorphism (RFLP), presence of blaCTX-M and electron microscopy was performed. Of 36 phages isolated, seven were positive for Group 1 of blaCTX-M . Based on host range and RFLP pattern, the seven phages were classified into four distinct groups, each harbouring a variant of blaCTX-M . Five phages were T4-like Myoviridae by electron microscopy which was further confirmed by polymerase chain reaction (PCR) for T4 specific gp14. Generalized transduction of the CTX-M gene from these phages was also observed. The high prevalence (20%) of this gene blaCTX-M in the phage pool confirms the significant role of Myoviridae members, specifically T4-like phages in the dissemination of this resistance gene. Significance and impact of the study The CTX-M gene that confers resistance to Beta-lactam class of drugs is widespread and diverse. Understanding mechanisms of antimicrobial resistance transfer is a key to devise methods for controlling it. Few studies indicate that bacteriophages are involved in the transfer of this gene but the type of phages involved and the degree of involvement remains to be explored. Our work has been able to identify the class of phages and the magnitude of involvement in the dissemination of this gene.

Thrombospondins (TSPs) are extracellular, calcium-binding glycoproteins that play an essential role in cell homeostasis and development, wound-healing, angiogenesis, connective tissue organization, immune response etc. and it conserves from sea sponges to mammals. However, their role in shrimp immunity is poorly understood. In the present study, the differential expression profiling of TSP transcripts in Penaeus monodon tissues such as gills, lymphoid organs, hepatopancreas, and hemolymph challenged with white spot syndrome virus (WSSV), were studied by quantitative real-time PCR. Further, shrimps fed with the immunostimulant (β-glucan) when challenged with WSSV showed significant upregulation of TSP expression in gills, hepatopancreas, and lymphoid organ at the early phase of WSSV infection. The results suggest that TSP may be an inducible acute phase response protein to WSSV infection. The possibility of differences in mRNA expression pattern seen in immunostimmulated shrimp after the viral challenge, possibility due to altered immune mechanisms getting triggered during immunostimulant administration and virus infections in the host.

The growing incidence of dengue outbreaks in the state of Karnataka prompted us to study the circulating dengue virus (DENV) and their proportion among the suspected cases of dengue patients during the disease outbreak at Mysuru district of Southern India. The presence of the DENV in a patient's serum sample was identified by RT-PCR using previously published primer pairs targeting CprM gene. DENV serotyping was carried out by semi-nested multiplex PCR using serotype-specific primers and nucleotide sequencing. Three hundred fifty-five samples of serum from suspected dengue cases were collected, and 203 samples (57.18%) were found positives. In 2016, DENV-4 (97.87%) was found to be the most dominant DENV serotype either alone or as co-infection, followed by DENV-2 (8.51%) and DENV-3 (4.25%). In 47 positive cases, co-infection with more than one serotype was detected in 4 cases (8.51%). The analysis of the dengue cases in 2017, DENV-4 was dominating serotype (33.97%), followed by the emergence of DENV-2 (32.05%), DENV-3 (25.64%), and DENV-1 (25.00%). Our study also reports the circulation of all four DENV serotypes in the Mysuru district of Southern India, with concurrent infections rate of 16.66% in 2017. The present study provides information regarding the genetic variation among the circulating DENV serotype in an Indian state of Karnataka. The need for the studying genetic diversity of DENV will be useful during the continuous monitoring for disease burden as well as the development of appropriate prophylactic measures to control the spread of dengue infection.

The south west coast of India has been showing a steady increase in shellfish cultivation both for local consumption and fishery export, over recent years. Perna viridis and Crassostrea madrasensis are two species of bivalve molluscs which grow in some selected regions of southern Karnataka, close to the city of Mangalore. In the early 1980s, shellfish consumers in the region were affected by intoxication from Paralytic Shellfish Poison present in local bivalves (clams and oysters) resulting in hospitalisation of many, including one fatality. Since then, there have been no further reports of serious shellfish intoxication and there is little awareness of the risks from natural toxins and no routine monitoring programme in place to protect shellfish consumers. This study presents the findings from the first ever systematic assessment of the presence of marine toxins in mussels and oysters grown in four different shellfish harvesting areas in the region. Shellfish were collected and subjected to analysis for ASP, PSP and lipophilic toxins, as well as a suite of non-EU regulated toxins such as tetrodotoxin and selected cyclic imines. Results revealed the presence of low levels of PSP toxins in oysters throughout the study period. Overall, total toxicities reached a maximum of 10% of the EU regulatory limit of 800 μg STX eq/kg. Toxin profiles were similar to those reported from the 1980 outbreak. No evidence was found for significant levels of ASP and lipophilic toxins, although some cyclic imines were detected, including gymnodimine. The results indicated that the risk to shellfish consumers during this specific study period would have been low. However, with historical evidence for extremely high levels of PSP toxins in molluscs, there is a strong need for routine surveillance of shellfish production areas for marine toxins, in order to mitigate against human health impacts resulting from unexpected harmful algal blooms, with potentially devastating socio-economic consequences.

Aquaculture is one of the fastest-growing food-producing sectors in the world. However, its growth is hampered by various disease problems due to infectious microorganisms, including Gram-negative bacteria in finfish aquaculture. Disease control in aquaculture by use of antibiotics is not recommended as it leads to antibiotic residues in the final product, selection, and spread of antibiotic resistance in the environment. Therefore, focus is on disease prevention by vaccination. All Gram-negative bacteria possess surface-associated outer membrane proteins (OMPs), some of which have long been recognized as potential vaccine candidates. OMPs are essential for maintaining the integrity and selective permeability of the bacterial membrane and play a key role in adaptive responses of bacteria such as solute and ion uptake, iron acquisition, antimicrobial resistance, serum resistance, and bile salt resistance and some adhesins have virulence attributes. Antigenic diversity among bacterial strains even within the same bacterial species has constrained vaccine developments, but OMPs that are conserved across serotypes could be used as potential candidates in vaccine development, and several studies have demonstrated their efficacy and potential as vaccine candidates. In this review, we will look into the application of OMPs for the design of vaccines based on recombinant proteins, subunit vaccines, chimeric proteins, and DNA vaccines as new-generation vaccine candidates for major bacterial pathogens of fish for sustainable aquaculture.

Background & objectives: The resistance to antibiotics in pathogenic bacteria has increased at an alarming rate in recent years due to the indiscriminate use of antibiotics in healthcare, livestock and aquaculture. In this context, it is necessary to monitor the antibiotic resistance patterns of bacteria isolated from the environmental samples. This study was conducted to determine the phenotypic and genotypic profile of antimicrobial resistance in Gram-negative bacteria isolated from environmental samples. Methods: Two hundred and fifty samples were collected from different sources, viz. fish and fishery products (99), livestock wastes (81) and aquaculture systems (70), in and around Mangaluru, India. Isolation, identification and antimicrobial profiling were carried out as per standard protocols. The isolates were screened for the presence of resistance genes using PCR. Results: A total of 519 Gram-negative bacteria comprising Escherichia coli (116), Salmonella spp. (14), Vibrio spp. (258), Pseudomonas spp. (56), Citrobacter spp. (26) and Proteus spp. (49) were isolated and characterized from 250 samples obtained from different sources. A total of 12 antibiotics were checked for their effectiveness against the isolates. While 31.6 per cent of the isolates were sensitive to all the antibiotics used, 68.4 per cent of the isolates showed resistance to at least one of the antibiotics used. One-third of the isolates showed multidrug resistance. Maximum resistance was observed for ampicillin (43.4%), followed by nitrofurantoin (20.8%). Least resistance was seen for carbapenems and chloramphenicol. PCR profiling of the resistant isolates confirmed the presence of resistance genes corresponding to their antibiotic profile. Interpretation & conclusions: This study results showed high rate of occurrence of antimicrobial resistance and their determinants in Gram-negative bacteria isolated from different environmental sources.

Use of bacteria in cancer therapy, despite being considered as a potent strategy, has not really picked up the way other methods of cancer therapies have evolved. However, in recent years, the interest on use of bacteria to kill cancer cells has renewed considerably. The standard and widely followed strategies of cancer treatment often fail either due to the complexity of tumour biology or because of the accompanying side effects. In contrast, these limitations can be easily overcome in a bacteria-mediated approach. Salmonella is a bacterium, which is known for its ability to colonize solid or semisolid tumours more efficiently than any other bacteria. Among more than 2500 serovars of Salmonella, S. Typhimurium has been widely studied for its antagonistic effects on cancer cells. Here in, we review the current status of the preclinical and the clinical studies with a focus on the mechanisms that attribute the anticancer properties to nontyphoidal Salmonella.

Purpose: Salmonella is a significant pathogen affecting wider range of animals and one of the main causes of diarroeal diseases leading to millions of human cases globally. Pathogenicity islands of Salmonella (SPI) are imperative in invasion of host cell and pathogenesis within the cell. It is found that SPI from 1 to 5 are present in every serovars of S. enterica and these are laterally acquired virulence regions. Research Method: This study investigated the presence of some virulence genes that belong to the SPI1, SPI2, SPI3 and SPI5 within 23 Salmonella isolates from broiler chicken meat in Sri Lanka using PCR method and further some virulence genes (gene encoding the invasion-associated protein, structural gene for attachment and invasion, regulatory gene for invasion and like nucleoid structuring gene) were quantified using RT- PCR. Findings: The study revealed the presence of Pathogenicity Island 1 genes such as hilA, invH, invF, invA and hns in all the isolates. Among the SPI 2 genes investigated, all the isolates of Salmonella showed the presence of ssaO, ssaQ, ssaP and ssaS (genes encoding for T3SS apparatus proteins), sscB and sscA (gene for secretion system chaperon proteins), sseF, sseD (genes coding for secreted eff ector proteins). In all isolates, SPI 3 genes, responsible in magenisum transport (mgtB and mgtC) were found. The rhuM gene (that supposed to code for a cytoplasmic protein) was absent in four isolates whereas the gene cigR (which predicted as codes for membrane protein) was absent in two isolates. The SPI 5 genes pipB was present in all the isolates except in one isolate whereas sopB was absent in one isolate. Originality/Value: This study found that some SPI genes are conserved in most of the Salmonella isolates from fresh broiler chicken meat in Sri Lanka and the expression of diff erent virulence genes vary with the isolate. This molecular basic will pave the way to explore more on local isolates of Salmonella, detection methods and control methods.

Purpose : Salmonella infections continue to be a global problem with millions of humans and animal cases occurring annually. Broiler chicken plays a significant role causing Salmonella infections in Sri Lanka. Consumption of food contaminated with antimicrobial resistant Salmonella aggravates the problem. This study isolated, identified, and serotype the Salmonella spp. from broiler chicken meat in Sri Lanka and examined their antimicrobial susceptibility to be used in establishment of control measures. Research Method : Isolation of Salmonella species from broiler chicken meat was done by conventional method of isolation followed by polymerase chain reaction (PCR) confirmation. All PCR confirmed isolates of Salmonella were serotype and then, isolates were tested for antibiotic susceptibility using disc diffusion assay followed by the detection of antibiotic resistance genes using PCR. Findings : Broiler chicken meat in Sri Lanka is contaminated with Salmonella spp. at the prevalence of 11.6% and 8.9% of them carried hns and invA specific genes. Isolates were serotyped as Salmonella Typhimurium (47.8%), Salmonella Enteritidis (26.1%) and non typable (26.1%). Three isolates were resistant to ampicillin. Intermediate resistance was shown to three antibiotics and all the isolates were sensitive to nine antibiotics. Majority (56.5%) of Salmonella were sensitive to all the tested antibiotics. Prevalence of resistant genes for tetracycline, sulfonamides and aminoglycosides were within 4%-26%. None of the isolates except one (4%) carried chloramphenicol resistance genes. Originality / Value : Steps must be taken to minimize contamination of broiler chicken meat with Salmonella spp in Sri Lanka. Although, there is a low prevalence of antibiotic resistant isolates, its mere presence in broiler chicken is a warning signal of possibility of emergence of multidrug resistant strains.

Bacteriophage therapy is a viable proposition for controlling luminous vibriosis caused by Vibrio harveyi in shrimp aquaculture. However, environmental factors influence the growth and activity of phage and affect its efficiency in controlling bacterial diseases. An essential problem in the use of vibrio phage as a therapeutic agent was the development of resistance to phage attachment, rendering them resistant to the lytic action of phage. This problem could be overcome by applying a cocktail of phages. This study aimed to evaluate the effect of salinity and pH on the phage activity and also to study the role of recombinant shrimp lysozyme on the performance of the V. harveyi phage. Out of three different levels of salinity (20, 25 and 30 ppt) and pH (6, 7 and 8) tested, optimum phage activity was observed at a salinity of 25 ppt and at neutral pH. Application of recombinant shrimp lysozyme in combination with V. harveyi phage significantly improved the activity of phage in in vitro assay as well as in microcosm study using seawater. The application of phage along with lysozyme can be a useful approach to overcome the inability of phage to enter the bacteria and thus eliminate or reduce fish/shrimp pathogenic bacteria in aquaculture.

Fluoroquinolones are the drug of choice for most of the infections caused by Escherichia coli, and their indiscriminate use has resulted in increased selective pressure for antibiotic resistance. At present, sequencing is the only reliable and direct technique to detect mutations in the quinolone resistance determining region (QRDR). In this study, a rapid and reliable mismatch amplification mutation assay (MAMA) PCR to detect mutations in the QRDR was evaluated and compared to PCR-restriction fragment length polymorphism (PCR-RFLP). One hundred one clinical isolates of E. coli were subjected to MAMA-PCR and PCR-RFLP to detect QRDR mutations. Overall, 92 (91.08%) resistant isolates harbored a point mutation of S83L in gyrA. Double mutations in gyrA were also detected in 45 (44.55%) isolates. Similarly, 41 (40.59%) isolates possessed a point mutation at parC 80, and 25 (24.75%) isolates possessed a point mutation at parC 84. Additionally, MAMA-PCR-the first of its kind-was also standardized to detect mutations in regions gyrB 447 and parE 416, although no mutations were detected in these regions. The rapid and sensitive MAMA-PCR method evaluated in this study would be helpful in exploring the underlying mechanism of fluoroquinolone resistance to enhance control strategies.

Emergence of multidrug resistant nontyphoidal Salmonella is a major health concern worldwide due to the predominant occurrence of Salmonella enterica sub-species enterica serovar Typhimurium phage type 104 (DT104) conferring resistance to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline. Apart from antibiotic resistance, the identification and genotypic characterization of pathogens is essential for epidemiological surveillance and outbreak investigations. In this study 39 isolates of Salmonella obtained from seafood samples were examined for their susceptibility to various antibiotics and subjected to PFGE analysis using the restriction enzyme Xba1. The highest percentage resistance was for erythromycin (100%) followed by nalidixic acid (15.38%), co-trimoxazole (15.38%), chloramphenicol (12.82%), ampicillin (12.82%) and tetracycline (10.25%). Six (15.38%) of the 39 isolates were multidrug resistant. The XbaI digested chromosomal DNA generated 7 clusters suggesting the presence of diverse Salmonella strains in seafood. The Discriminatory Index for PFGE obtained by XbaI restriction enzyme was 0.91. The PFGE has been found highly discriminatory for subtyping S. Weltevreden and S. Newport. The XbaI PFGE was not only discriminatory but could also distinguish multidrug-resistant strains from the sensitive ones as the two groups they belonged to different pulsotypes. The study also demonstrated multiple clones of S. Weltevreden, S. Newport and S. Oslo present in seafood from the south west coast of India. Genetic diversity among the similar seafood sources suggests the presence of different clones of Salmonella which further increases the risk of seafood being a potential source of highly pathogenic bacteria like Salmonella.

The Toll-like receptors (TLR), being pattern recognition molecules, are a powerful first line of defense in response to pathogen invasion. They are known to play a crucial role in detecting and binding to the microbial molecule and triggering a non-specific immune response. Quantitative real time PCR (qPCR) expression of the TLR gene was studied in healthy and Vibrio harveyi infected black tiger shrimp ( Penaeus monodon ). Lymphoid tissue expression of TLR in V. harveyi infected animals 24 h post injection showed statistically significant up regulation of the gene as compared to the control animals sham injected with phosphate buffered saline (PBS). The qPCR expression pattern of TLR at different time points in shrimp administered with the immunostimulant glucan for 6 days by oral feeding followed by challenge with V. harveyi showed statistically significant level at 48 h post bacterial challenge as compared to the control (immunostimulant treated) animals sham injected with PBS. The novelty of the study is that it elicits the role of TLRs as important response proteins of the innate immune system in the shrimp.

Vibrio vulnificus is a Gram-negative, opportunistic human pathogen capable of causing life-threatening septicaemia, wound infections, and gastroenteritis, especially in immunocompromised individuals. Two cases of V. vulnificus-associated wound infection occurring in diabetic patients are reported here. The pathogen was detected by PCR targeting species-specific marker gyrB and virulence markers, including repeats in toxin (rtxA) and hemolysin (vvhA), but the causative agent could not be cultured. Genotyping based on the virulence-correlated gene revealed that the V. vulnificus detected in this study belonged to the vcg-C type, which is commonly associated with clinical cases. This report highlights the clinical applicability of PCR-based methods in the detection of V. vulnificus in culture-negative cases. Such methods may add a very useful clinical dimension to currently used diagnostic practices. Keywords: Vibrio vulnificus, Wound infection, Polymerase chain reaction, Diagnosis

Antimicrobial-resistant salmonellosis is a significant public health concern globally. A study was conducted to screen for Salmonella species from a total of 120 samples, of which 50 were retail meat samples purchased from five randomly selected sales outlets in the city of Mangalore, India. Twenty poultry fecal materials freshly voided before slaughter were obtained with sterile spatula and placed in sterile sealable polythene envelopes, and 20 clams were purchased from the estuaries of Nethravathi and Kankarnady market. In addition, 30 clinical isolates from Nigeria suspected to be Salmonella by only cultural characterization were also included in the study. In all, 30 samples-6 poultry, 8 seafood, and 16 Salmonella isolates from clinical samples-were confirmed positive by PCR and used in this study. The disk-diffusion test was performed to determine the zone of inhibition, and detection of resistance genes was tested by PCR targeting various antimicrobial genes. Resistance to tetracycline (TET), cotrimoxazole, nalidixic acid, nitrofurantion, and piperacillin/tazobactin was found in 66.7%, 60%, 53.3%, 50% and 50% of the isolates, respectively. About 60-100% of MDR isolates possessed antibiotic-resistant genes, of the tetracyclines resistant isolates, 20 (100%) 6 (30%), 7 (35%), and 10 (50%) carried tetA, tetB, tetC, and tetG genes, respectively. Of 18 cotrimoxazole-resistant strains, 18 (100%), 14 (77.7%), and 4 (22.2%) had sul1, sul2, and sul3 genes, respectively. Of the 14 multidrug-resistant isolates tested, 8 (61%) and 9 (69%) were positive for cmlA and cmlB genes, respectively, 10 (1.4%) tested positive for aph(3)11a genes, 8 (57%) tested positive for aac(3)lla, while none was positive for the aac6 gene. The results show the presence of antibiotic-resistant Salmonella spp. in food samples from India and in human samples from Nigeria.

The outer membrane protein K, OmpK first identified in Vibrio parahaemolyticus has been shown to be a receptor for a broad host range vibriophage KVP40 infecting members of the Vibrionaceae. In the study, the effect of culture conditions on the expression of ompK in V. anguillarum was studied using real-time PCR. The expression increased significantly in the presence of bile salts and iron chelating agent 2, 2' bipyridine, suggesting a role for this protein in bile resistance and also in iron acquisition by V. anguillarum. OmpK induction by iron limitation and the presence of bile salts was reconfirmed by western blot technique after growing the cells in trypticase soy broth supplemented with bile salts, blood and 2, 2' bipyridine. We surmise that the expression of OmpK protein of V. anguillarum is bile salt and iron chelating agent-dependent.

Salmonella enterica serovars are virulent pathogens of humans and animals with many strains possessing multiple drug resistance traits. They have been found to carry resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT-resistant). A rapid and sensitive multiplex PCR (mPCR)-based assay was developed for the detection of Salmonella serovars from seafood. Six sets of primers which are one primer pair targeting Salmonella specific gene invA (284 bp), two Salmonella pathogenicity island 2 (SPI-2) genes ssaT (780 bp) and sseF (888 bp) and three antibiotic resistance genes floR (198 bp), sul1 (425 bp), tetG (550 bp) were used for the study. The specificity and sensitivity of the assay were tested by spiking shrimp/fish/clam homogenate with viable cells of Salmonella. This assay allows for the cost effective and reliable detection of pathogenic Salmonella enterica from seafood. The mPCR developed in the present study proved to be a potent analytical tool for the rapid identification of multidrug-resistant Salmonella serovars from seafood.

Dentures are inert and nonshading surfaces and therefore get easily colonized by Candida species. Subsequent biofilm produced by them lead to denture stomatitis and candidiasis. This study was aimed to understand the prevalence of Candida species among healthy denture and nondenture wearers with respect to their age and hygiene status. Swabs were collected from 50 complete dentures and 50 non-denture wearers and processed on Sabouraud′s dextrose agar. Identification of Candida species was done by staining and a battery of biochemical tests. Data obtained was correlated with age & oral hygiene and statistical analysis was performed. Candida was isolated from both denture and nondenture wearers. Prevalence of different Candida species was significantly higher in denture wearers and found predominated by C. albicans, C. tropicalis, C. dubliensis and C. glabrata. Among nondenture wearers, C. albicans and C. tropicalis were isolated. Prevalence of Candida increased with increasing age among denture wearers. Men presented declining denture hygiene compared to women with increasing age. In comparison to nondenture wearers, multispecies of Candida colonized the dentures thus presenting higher risk of candidiasis especially with increasing age.

The family Nodaviridae include the genera Alphanodavirus and the Betanodavirus which are non-enveloped, single stranded RNA viruses. Alphanodavirus include the insect viruses while betanodavirus include species that are responsible for causing disease outbreaks in hatchery-reared larvae and juveniles of a wide variety of marine and freshwater fish throughout the world and has impacted fish culture over the last decade. According to International Committee on Taxonomy of Viruses, the genus Betanodavirus comprises four recognized species viz barfin flounder nervous necrosis virus, red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus and tiger puffer nervous necrosis virus with the RGNNV being the most common. The viruses are distributed worldwide having been recorded in Southeast Asia, Mediterranean countries, United Kingdom, North America and Australia. The disease has been reported by different names such as viral nervous necrosis, fish encephalitis, viral encephalopathy and retinopathy by various investigators. The virus is composed of two segments designated RNA1 and RNA2 and sometimes possesses an additional segment designated RNA3. However, genome arrangement of the virus can vary from strain to strain. The virus is diagnosed by microscopy and other rapid and sensitive molecular methods as well as immunological assays. Several cell lines have been developed for the virus propagation and study of infection mechanism. Control of nodavirus infection is a serious issue in aquaculture industry since it is responsible for huge economic losses. In combination with other management practices, vaccination of fish would be a useful strategy to control the disease.

Background: Diarrheal diseases are a major cause of morbidity and mortality in resource-limited countries. Among the bacterial pathogens, diarrheagenic E. coli (DEC) are most frequently implicated in cases of epidemic and endemic diarrhea worldwide. The objective of this study was to determine the prevalence of DEC in stool specimens from patients with acute diarrhea using polymerase chain reaction (PCR). Materials and Methods: Escherichia coli stool samples were collected from 115 hospitalized children and adults with acute diarrhea in Mangalore, a coastal city, in southern India. PCR amplification of eae, bfp, stx, ehx genes were used for detection of enteropathogenic (EPEC) and shigatoxigenic E. coli (STEC), lt and st genes were used for enterotoxigenic E. coli (ETEC) and astA gene for enteroaggregative E. coli (EAEC). Results: During the 24 month study period, of the 115 stool samples, DEC type was detected in 20 (17.4%) using the PCR method. The most prevalent DEC was atypical EPEC accounting for 12 (10.4%) cases followed by 4 cases of EAEC (3.4%) and 4 of STEC (3.4%). No ETEC strains were isolated from any of the examined stool samples. Conclusion: This study suggests that the atypical EPEC are the newly emerging group among DEC stains in Southern India. Further studies are needed to evaluate the epidemiology and virulence properties of atypical EPEC strains.