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Neurological complications occurring due to Salmonella infection in some typhoid patients remain a relatively unexplored serious complication. This study firstly aimed to explore whether disseminative ability of Salmonella from gut to brain is strain specific or not and on the basis of bacterial load, histopathology, and behavioral changes, it was observed that Salmonella enterica serovar Typhimurium NCTC 74 did not cause brain infection in murine model in contrast to Salmonella Typhimurium SL1344. Simultaneously, alarming escalation in antimicrobial resistance, making the existing antibiotics treatment inefficacious, prompted us to evaluate other bio-compatible strategies as a potential treatment option. In this context, the role of gut microbiota in influencing behavior, brain neurochemistry, and physiology by modulating key molecules associated with gut-brain axis has captured the interest of the scientific community. Followed by in vitro screening of potential probiotic strains for beneficial attributes, efficacy of the selected strain was systematically evaluated at various levels of gut-brain axis against Salmonella induced brain infection. Analysis of behavioral (depression, anxiety, and locomotor), neurochemical [gamma amino butyric acid and acetylcholinesterase (AChE)], neuropathological (brain and intestinal histology; bacterial burden), and immunohistochemical studies (tight junction proteins expression) revealed its role in preventing serious manifestations and proving its potential as “psychobiotic.” To the best of our knowledge, this is the first report elaborating strain specificity of Salmonella in causing post-typhoidal neurological manifestations and simultaneous use of probiotic in managing the same by influencing the pathophysiology at gut-brain axis.

Introduction: Hypertensive disorder complicates the pregnancy. The exact aetiology of hypertensive disorders of the pregnancy is still unknown and management is controversial. Aim: To evaluate the role of urinary Calcium Creatinine Ratio (CCR) as a marker in early diagnosis of pre-eclampsia. Materials and Methods: Urinary calcium creatinine ratio was determined in a random sample of urine in 100 patients of pre-eclampsia (Study group) and 100 normotensive pregnant (Control group) patients of gestational age 20- 36 weeks. Cut off value for CCR was taken as ≤ 0.04. Comparative study was done by using chi-square test. Results: In study group, 89% of the cases had urinary CCR ≤ 0.04. Amongst the controls only 6% of the cases had CCR ≤ 0.04. Statistically, CCR as high risk factor for early diagnosis of pre-eclampsia was significant (p

Haskap (Lonicera caerulea L.) berries have long been used for their health promoting properties against chronic conditions. The current study investigated the effect of Canadian haskap berry extracts on pro-inflammatory cytokines using a human monocytic cell line THP-1 derived macrophages stimulated by lipopolysaccharide. Methanol extracts of haskap from different growing locations in Canada were prepared and characterized for their total phenolic profile using colorimetric assays and liquid chromatography—Mass spectrometry (UPLC-MS/MS). Human THP-1 monocytes were seeded in 24-well plates (5 × 105/well) and treated with phorbol 12-myristate 13-acetate (PMA, 0.1 μg/mL) for 48 h to induce macrophage differentiation. After 48 h, the differentiated macrophages were washed with Hank’s buffer and treated with various concentrations of test compounds for 4 h, followed by the lipopolysaccharide (LPS)-stimulation (18 h). Borealis cultivar showed the highest phenolic content, flavonoid content and anthocyanin content (p < 0.05). A negative correlation existed between the polyphenol concentration of the extracts and pro-inflammatory cytokines: Interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), prostaglandin (PGE2), and cyclooxygenase-2 (COX-2) enzyme. Borealis exhibited comparable anti-inflammatory effects to COX inhibitory drug, diclofenac. The results showed that haskap berry polyphenols has the potential to act as an effective inflammation inhibitor.

IntroductionTraditional fermented foods have long been recognized for their numerous health benefits along with their potential to aid in the treatment of gastrointestinal disorders. These fermented foods have been shown to promote gut health and contribute to a longer, healthier life.MethodsThe high-throughput sequencing using the Illumina MiSeq platform was employed to investigate the microbiome communities of rice-based fermented beverages consumed by ethnic tribes in Southern Assam, namely Zeme Naga, Dimasa Kachari, Hmar, Karbi and Tea tribes.ResultsThe fermented rice-based beverages were highly predominated by Firmicutes, Bacteroides, Proteobacteria, and Actinobacteria exhibiting the highest relative abundance across all tribes. At genus level, significant abundance of pediococcus, lactobacillus, bacillus, leuconostoc, acetobacter, staphylococcus, delftia, erwinia, klebsiella and chrysebacterium were found amongst these ethnic tribes.DiscussionUnderstanding the fermented food microbiome will help to know the relationships between microbial communities and their effect on health of humans amongst the tribes. Furthermore, the use of these fermented products could provide enhanced health benefits to southern Assam region of India.

Calcium is an important second messenger that is involved in almost all cellular processes. Disruptions in the regulation of intracellular Ca2+ levels ([Ca2+]i) adversely impact normal physiological function and can contribute to various diseased conditions. STIM and Orai proteins play important roles in maintaining [Ca2+]i through store-operated Ca2+ entry (SOCE), with STIM being the primary regulatory protein that governs the function of Orai channels. STIM1 and STIM2 are single-pass ER-transmembrane proteins with their N- and C-termini located in the ER lumen and cytoplasm, respectively. The N-terminal EF-SAM domain of STIMs senses [Ca2+]ER changes, while the C-terminus mediates clustering in ER-PM junctions and gating of Orai1. ER-Ca2+ store depletion triggers activation of the STIM proteins, which involves their multimerization and clustering in ER-PM junctions, where they recruit and activate Orai1 channels. In this review, we will discuss the structure, organization, and function of EF-hand motifs and the SAM domain of STIM proteins in relation to those of other eukaryotic proteins.

ABSTRACT Noncanonical secondary structures in nucleic acids have been studied intensively in recent years. Important biological roles of cruciform structures formed by inverted repeats (IRs) have been demonstrated in diverse organisms, including humans. Using Palindrome analyser, we analyzed IRs in all accessible bacterial genome sequences to determine their frequencies, lengths, and localizations. IR sequences were identified in all species, but their frequencies differed significantly across various evolutionary groups. We detected 242,373,717 IRs in all 1,565 bacterial genomes. The highest mean IR frequency was detected in the Tenericutes (61.89 IRs/kbp) and the lowest mean frequency was found in the Alphaproteobacteria (27.08 IRs/kbp). IRs were abundant near genes and around regulatory, tRNA, transfer-messenger RNA (tmRNA), and rRNA regions, pointing to the importance of IRs in such basic cellular processes as genome maintenance, DNA replication, and transcription. Moreover, we found that organisms with high IR frequencies were more likely to be endosymbiotic, antibiotic producing, or pathogenic. On the other hand, those with low IR frequencies were far more likely to be thermophilic. This first comprehensive analysis of IRs in all available bacterial genomes demonstrates their genomic ubiquity, nonrandom distribution, and enrichment in genomic regulatory regions. IMPORTANCE Our manuscript reports for the first time a complete analysis of inverted repeats in all fully sequenced bacterial genomes. Thanks to the availability of unique computational resources, we were able to statistically evaluate the presence and localization of these important regulatory sequences in bacterial genomes. This work revealed a strong abundance of these sequences in regulatory regions and provides researchers with a valuable tool for their manipulation.

Through the use of smart sensors to monitor and regulate plant conditions, smart home gardening management systems can maximize resource utilisation and minimize human intervention. This study offers a new system that remotely controls the water supply to ensure optimal plant growth without the need for personal presence. The system uses the Blynk IoT platform to monitor soil moisture and water levels. A Raspberry Pi is used in conjunction with several sensors, such as a soil moisture sensor and a DHT11 sensor for temperature and humidity readings. The technology activates a motor to provide water to the plants automatically when the soil moisture level falls below a certain threshold. Users can remotely monitor and manage the system from their cell phones thanks to integration with the Blynk platform. The suggested method is an affordable and effective way to garden in your home, and it’s simply customizable to fit the requirements of different users.

Introduction: Urinary Tract Infection (UTI) is the most common medical problems associated with pregnancy occurring in about 5-10% of pregnant women. It is attributed by various micro- organisms. As the uropathogens is usually Multidrug resistance so, timely identification of these uropathogens and their resistance pattern detection is essential. If UTI is not treated promptly, it can lead to various complications and ultimately can affect health of infant or the pregnant mother. Aim: To estimate the frequency of UTI and different micro- organisms in pregnant women as well as their antibiotic resistant pattern among them. Materials and Methods: Present study was conducted in a tertiary care hospital in rural district of Chhattisgarh. During the study period from August 2021 to October 2021, 130 urine samples were collected and processed as per standard protocol. With significant growth on culture ≥105 Colony Forming Unit (CFU)/mL of single morphotype bacteria from a clean catch midstream urine sample UTI diagnosis was established. The bacteria isolated were identified up to species level by standard biochemical tests. Results: In this study, UTI was found in n=69.2% of pregnant women. The most frequently isolated organism was E. coli (53.3%), followed by Klebsiella spp.(18.9%) and Pseudomonas spp. (3.3%). Highest prevalence rate (85.7%) was observed in the age group 21 to 25 years; most of these (77.8%) were in their 2nd trimester. The isolated pathogens mostly showed multiple drug resistance to two or more antibacterial agents tested. Conclusion: There was higher incidence of UTI in pregnancy and the most predominant organism isolated was E. coli; in 2nd trimester UTI was more common and high level of multidrug resistance was observed. In pregnancy, continuous monitoring of uropathogens including their resistance pattern is required.

IntroductionDiabetes is a potent risk factor for the activation of latent tuberculosis and worsens the tuberculosis (TB) treatment outcome. The major reason for mortality and morbidity in diabetic patients is due to their increased susceptibility to TB. Thus, the study was conducted to understand the crosstalk between M. tuberculosis and its host upon latent tuberculosis infection and under hyperglycemic conditions or diabetes.MethodsAn animal model was employed to study the relationship between latent tuberculosis and diabetes. BCG immunization was done in mice before infection with M. tuberculosis, and latency was confirmed by bacillary load, histopathological changes in the lungs and gene expression of hspX, tgs1, tgs3 and tgs5. Diabetes was then induced by a single high dose of streptozotocin (150 mg/kg body weight). Host factors, like various cytokines and MMPs (Matrix metalloproteinases), which play an important role in the containment of mycobacterial infection were studied in vivo and in vitro.ResultsA murine model of latent TB was developed, which was confirmed by CFU counts (200 mg/dl. An in vitro study employing a THP-1 macrophage model of latent and active tuberculosis under normal and high glucose conditions showed that dormant bacilli were better contained in the presence of 5.5 mM glucose concentration as compared with active bacilli. However, the killing and restriction efficiency of macrophages decreased, and CFU counts increased significantly with an increase in glucose concentration.DiscussionThe decreased levels of MCP-1, decreased expression of mmp-9, and increased expression of mmp-1 in the latent group at high glucose concentrations could explain the failure of granuloma formation at high glucose conditions.

Malaria, a disease caused by the protozoan parasites Plasmodium spp., is still causing serious problems in endemic regions in the world. Although the WHO recommends artemisinin combination therapies for the treatment of malaria patients, the emergence of artemisinin-resistant parasites has become a serious issue and underscores the need for the development of new antimalarial drugs. On the other hand, new and re-emergences of infectious diseases, such as the influenza pandemic, Ebola virus disease, and COVID-19, are urging the world to develop effective chemotherapeutic agents against the causative viruses, which are not achieved to the desired level yet. In this review article, we describe existing drugs which are active against both Plasmodium spp. and microorganisms including viruses, bacteria, and fungi. We also focus on the current knowledge about the mechanism of actions of these drugs. Our major aims of this article are to describe examples of drugs that kill both Plasmodium parasites and other microbes and to provide valuable information to help find new ideas for developing novel drugs, rather than merely augmenting already existing drug repurposing efforts.

Abstract Background Some of the spore-forming strains of Bacillus probiotics are marketed commercially as they survive harsh gastrointestinal conditions and bestow health benefits to the host. Results We report the composite genome of Bacillus clausii ENTPro from a commercially available probiotic Enterogermina® and compare it with the genomes of other Bacillus probiotics. We find that the members of B. clausii species harbor high heterogeneity at the species as well as genus level. The genes conferring resistance to chloramphenicol, streptomycin, rifampicin, and tetracycline in the B. clausii ENTPro strain could be identified. The genes coding for the bacteriocin gallidermin, which prevents biofilm formation in the pathogens Staphylococcus aureus and S. epidermidis, were also identified. KEGG Pathway analysis suggested that the folate biosynthesis pathway, which depicts one of the important roles of probiotics in the host, is conserved completely in B. subtilis and minimally in B. clausii and other probiotics. Conclusions We identified various antibiotic resistance, bacteriocins, stress-related, and adhesion-related domains, and industrially-relevant pathways, in the genomes of these probiotic bacteria that are likely to help them survive in the harsh gastrointestinal tract, facilitating adhesion to host epithelial cells, persistence during antibiotic treatment and combating bacterial infections.

Background/Objective: Although microRNA (miRNA)-directed regulation of bovine tuberculosis (bTB) has already been reported, very little is known about the incited pathways and genes. We profiled bTB-upregulated miRNAs through an in silico methodology. Methods: The data of upregulated miRNAs in bTB versus healthy controls were collected and clustered into three groups by their tissue specificity as follows: G1 (mammary gland-specific): bta-miR-146a; G2 (peripheral blood mononuclear cell-specific): bta-miR-155; and G3 (alveolar macrophage-specific): bta-miR-146a, bta-miR-155, bta-miR-142-5p, bta-miR-423-3p, bta-miR-21-5p, bta-miR-27a-3p, bta-miR-99b, bta-miR-147, bta-miR-223, and bta-let-7i. The miRNA–mRNA interaction network was defined by TargetScan. The gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways of these transcripts were examined. Results: The results illustrate the induction of pathways in cancer, highly enriched, and unanimous to all three gene sets (G1, G2, and G3). Mitogen-activated protein kinase and PI3K-Akt signaling were specific to G2 and G3 with fibroblast growth factors formed the key factors. Conclusion: The inferred cancer cascades denote a probable modulation of innate immune response in an infectious state. These baseline pictures could lay the ground for further substantive studies.

ER-PM junctions are membrane contact sites formed by the endoplasmic reticulum (ER) and plasma membrane (PM) in close apposition together. The formation and stability of these junctions are dependent on constitutive and dynamic enrichment of proteins, which either contribute to junctional stability or modulate the lipid levels of both ER and plasma membranes. The ER-PM junctions have come under much scrutiny recently as they serve as hubs for assembling the Ca2+ signaling complexes. This review summarizes: (1) key findings that underlie the abilities of STIM proteins to accumulate in ER-PM junctions; (2) the modulation of Orai/STIM complexes by other components found within the same junction; and (3) how Orai1 channel activation is coordinated and coupled with downstream signaling pathways.

The drug resistance developed by bacteria during antibiotic treatment has been a call to action for researchers and scientists across the globe, as bacteria and fungi develop ever increasing resistance to current drugs. Innovative antimicrobial/antibacterial materials and coatings to combat such infections have become a priority, as many infections are caused by indwelling implants (e.g., catheters) as well as improving postsurgical function and outcomes. Pathogenic microorganisms that can exist either in planktonic form or as biofilms in water-carrying pipelines are one of the sources responsible for causing water-borne infections. To combat this, researchers have developed nanotextured surfaces with bactericidal properties mirroring the topographical features of some natural antibacterial materials. Protein-based adhesives, secreted by marine mussels, contain a catecholic amino acid, 3,4-dihydroxyphenylalanine (DOPA), which, in the presence of lysine amino acid, empowers with the ability to anchor them to various surfaces in both wet and saline habitats. Inspired by these features, a novel coating material derived from a catechol derivative, dopamine, known as polydopamine (PDA), has been designed and developed with the ability to adhere to almost all kinds of substrates. Looking at the immense potential of PDA, this review article offers an overview of the recent growth in the field of PDA and its derivatives, especially focusing the promising applications as antibacterial nanocoatings and discussing various antimicrobial mechanisms including reactive oxygen species-mediated antimicrobial properties.

Aim: Microsatellite instability (MSI) pathway is known to be implicated in carcinogenesis of 15% colorectal carcinomas (CRC), including 2%–3% of cases of Lynch syndrome, as per western literature. MSI status has important prognostic and therapeutic implications. The prevalence of MSI in Indian CRC patients is unknown. We aimed to determine the prevalence by studying 231 consecutive unselected cases of CRC. Methods: Tissue microarrays using duplicate cores per case for 141 cases, and whole tissue sections for 90 cases, were used. Immunohistochemistry with four mismatch repair (MMR) markers – MLH1, MSH2, MSH6, and PMS2 was performed. Molecular analysis for MSI status was performed in 18 randomly selected cases. Correlation with various clinical and histopathological features was done using univariate and multivariate analysis. Results: Loss of MMR immunohistochemical (IHC) was seen in 53/231 cases, i.e. 22.94% (95% confidence interval 17.52%–28.36%). MLH1-PMS2 dual loss comprised 13.9%, MSH2-MSH6 7.4%, and isolated PMS2 loss in 1.73% of cases. Univariate analysis showed significant association with age (

Background: The prevalence of multidrug-resistant-tuberculosis (MDR-TB) among new and previously treated cases is increasing worldwide as well as in India. Rapid detection of MDR-TB allows the establishment of an effective treatment regimen; minimizes the risk of further resistance, and limits the spread of drug-resistant strains. Early diagnosis of MDR-TB is the need of the hour in high-TB burden countries like India, and GenotypeMTBDRplus is quite sensitive and specific in determining the molecular resistance in drugs such as rifampicin and isoniazid. Methods: The present study was done for molecular detection of rifampicin and isoniazid resistance and resistance patterns among MDR-TB suspects and comparison of resistance patterns among new and previously treated cases by GenoType® MTBDRplus Line Probe Assay. A total of 1268 sputum samples of MDR-TB suspects were subjected to fluorescent microscopy. Fluorescent microscopy positive samples were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay. Results: MDR-TB was detected 11.02%, 20.03% in new and previously treated cases. Among MDR-TB patients S531 L was the most common mutation detected in rpoB gene; 71.43% in new, and 72.17% in previously treated cases. S315T1 was the most common mutation noted in katG gene; 100% in new and 81.74% in previously treated. While in hA gene, it was C15T (7.8%) among previously treated cases. Conclusion: MDR-TB has high prevalence in the western part of Uttar Pradesh, India. Previously treated cases have even more high rate of MDR-TB than new TB cases. The most dominant gene mutations associated with resistance to INH and RIF were observed in codon 315 of the katG gene and codon 531 of the rpoB gene. While comparing the mutation patterns by Genotype MTBDRplus assay, previously treated cases showed more diversity of mutations and had greater number of unknown mutations.

Context: There is heterogeneity in the clinical behavior of breast carcinoma patients with node negativity. Studies have analyzed different factors influencing the outcome in such patients. It is suggested that the presence of nodal micrometastasis can act as a tool in predicting the aggressiveness of these tumors. Aims: The aim of this study is to assess the yield of micrometastasis/isolated tumor cell (ITC) by ultrastaging the morphologically negative axillary nodes and staining them with immunohistochemistry for epithelial membrane antigen. The association of such metastasis with the clinical outcome is determined. Settings and Design: This was a retrospective analytical study. One hundred cases of node-negative breast carcinoma patients who underwent surgery along with axillary lymph node dissection were selected. Materials and Methods: The largest node from the axillary dissection was selected and subjected to ultrastaging and immunohistochemical staining (as sentinel node dissection was not a routine practice at that time), to look for occult metastasis in the form of micrometastasis or ITCs. Statistical Analysis: Occurrence of events in the form of recurrence or death was noted. Association of the parameters was analyzed using Fisher's exact test. Results: Among the 100 cases, 79 patients were followed up for a minimum period of 5 years. Two cases had micromets in one node each. These two patients were among the eight, who developed events subsequently (25%). Hence, a statistically significant association was found between the presence of micromets with events. Conclusions: There is a statistically significant association between the presence of micromets and disease recurrence. Hence, we suggest that ultrastaging of the negative axillary node (now sentinel node, as it is being routinely done) might prove effective in predicting the events/prognosis in clinically and morphologically node-negative breast carcinoma patients

Context: Mycoplasma pneumoniae (M. pneumoniae) causes up to 40% of community-acquired pneumonia in children. It is impossible to identify M. pneumoniae infection on the basis of clinical signs, symptoms, and radiological features. Therefore, correct etiological diagnosis strongly depends on laboratory diagnosis. Aims: This study aims to investigate the role of M. pneumonia e in pediatric lower respiratory tract infections (LRTIs) employing enzyme-linked immunosorbent assays (ELISA) and particle agglutination (PA) test. Settings and Design: Two hundred and eighty children, age 6 months to 12 years with community-acquired LRTIs were investigated for M. pneumoniae etiology. Materials and Methods: We investigated 280 children hospitalized for community-acquired LRTIs, using ELISA and PA test for detecting M. pneumoniae immunoglobulin M (IgM) and immunoglobulin G antibodies. Statistical Analysis Used: The difference of proportion between the qualitative variables was tested using the Chi-square test and Fischer exact test. P ≤ 0.05 was considered as statistically significant. Kappa value was used to assess agreement between ELISA and PA test. Results: M. pneumoniae was positive in 51 (23.2%) 5 years of age.

Background: Scrub typhus is lesser known cause of fever of unknown origin in India. Even if there have been reports documenting the prevalence of scrub typhus in different parts of India, it is still an unknown entity, and clinicians usually do not consider it as differential diagnosis. The present study was performed to document the prevalence of scrub typhus among febrile patients in western part of Uttar Pradesh and to assess the clinical profile of infected patients on the one hand and knowledge, attitude, and practices among clinicians on the other. Materials and Methods: A total of 357 adult patients with fever of more than 5-day duration were recruited. All patients underwent complete physical examination, and detailed clinical history was elicited as per predesigned pro forma. After primary screening to rule out malaria, enteric fever, and leptospirosis infection, secondary screening for scrub typhus was done by rapid screen test and IgM ELISA. Results: Scrub typhus infection was positive in 91 (25.5%) cases. The most common symptoms among the patients were fever (100%), pain in abdomen (79.1%), pedal edema 56 (61.5%), rash 44 (48.3%), headache 44 (48.3%), vomiting 42 (46.1%), constipation 33 (36.2%), cough 28 (30.7%), and lymphadenopathy 20 (21.9%). The median values of interleukin-8, interferon-gamma, and tumor necrosis factor-alpha in healthy controls were 15.54 pg/ml, 7.77 pg/ml, and 54.1 pg/ml, respectively, while the median values of these cytokines in scrub typhus-positive patients were 21.04 pg/ml, 8.74 pg/ml, and 73.8 pg/ml, respectively. Conclusion: Our results highlight that scrub typhus infection is an important cause of pyrexia of unknown origin, and active surveillance is necessary to assess the exact magnitude and distribution of the disease.

Abstract Background Diarrheagenic Escherichia coli are associated with infantile diarrhea in the developing countries. The present study was conducted to determine the occurrence and antimicrobial resistance pattern of enteropathogenic and enteroinvasive E. coli associated with diarrhoea among the paediatric patients. Methods A total of 262 stool samples were collected from children with and without diarrhea from Mizoram, Northeast India. E. coli were isolated and subjected to multiplex PCR to detect virulent genes of EPEC (eaeA and bfpA) and EIEC (ial). Isolates were subjected to antimicrobial sensitivity assay using disc diffusion method. Selected eaeA genes were sequenced for identification and genetic relationship. Results A total of 334 E. coli was isolated, of which 17.37% were carrying at least one virulent gene. Altogether, 14.97 and 2.40% isolates were categorized as EPEC and EIEC, respectively. Among the DEC isolates, 4.79% were EPEC and 7.78% were EIEC. A total of 8 (2.40%) isolates were EIEC (ial+), of which 6 (1.80%) and 2 (0.60%) were from diarrhoeic and non-diarrhoeic patients, respectively. A total of 24 (41.40%) DEC isolates were MDR (resistance against ≥5 antimicrobials). Conclusions A high frequency of EPEC pathotypes associated with paediatric diarrhea was observed in Mizoram, Northeast India and majority of the isolates are resistant to antibiotics with a high frequency of MDR, which is a matter of concern to the public health. This also raises an alarm to the world communities to monitor the resistance pattern and analyse in a global scale to combat the problems of resistance development.

The genus Rhodobacter is taxonomically well studied, and some members are model organisms. However, this genus is comprised of a heterogeneous group of members. 16S rRNA gene-based phylogeny of the genus Rhodobacter indicates a motley assemblage of anoxygenic phototrophic bacteria (genus Rhodobacter) with interspersing members of other genera (chemotrophs) making the genus polyphyletic. Taxogenomics was performed to resolve the taxonomic conflicts of the genus Rhodobacter using twelve type strains. The phylogenomic analysis showed that Rhodobacter spp. can be grouped into four monophyletic clusters with interspersing chemotrophs. Genomic indices (ANI and dDDH) confirmed that all the current species are well defined, except Rhodobacter megalophilus. The average amino acid identity values between the monophyletic clusters of Rhodobacter members, as well as with the chemotrophic genera, are less than 80% whereas the percentage of conserved proteins values were below 70%, which has been observed among several genera related to Rhodobacter. The pan-genome analysis has shown that there are only 1239 core genes shared between the 12 species of the genus Rhodobacter. The polyphasic taxonomic analysis supports the phylogenomic and genomic studies in distinguishing the four Rhodobacter clusters. Each cluster is comprised of one to seven species according to the current Rhodobacter taxonomy. Therefore, to address this taxonomic discrepancy we propose to reclassify the members of the genus Rhodobacter into three new genera, Luteovulum gen. nov., Phaeovulum gen. nov. and Fuscovulum gen. nov., and provide an emended description of the genus Rhodobacter sensu stricto. Also, we propose reclassification of Rhodobacter megalophilus as a sub-species of Rhodobacter sphaeroides.

Campylobacter jejuni is the leading cause of human foodborne illness globally, and is strongly linked with the consumption of contaminated poultry products. Several studies have shown that C. jejuni can form sanitizer tolerant biofilm leading to product contamination, however, limited research has been conducted to develop effective control strategies against C. jejuni biofilms. This study investigated the efficacy of three generally recognized as safe status phytochemicals namely, trans-cinnamaldehyde (TC), eugenol (EG), or carvacrol (CR) in inhibiting C. jejuni biofilm formation and inactivating mature biofilm on common food contact surfaces at 20 and 37°C. In addition, the effect of phytochemicals on biofilm architecture and expression of genes and proteins essential for biofilm formation was evaluated. For the inhibition study, C. jejuni was allowed to form biofilms either in the presence or absence of sub-inhibitory concentrations of TC (0.75 mM), EG (0.61 mM), or CR (0.13 mM) for 48 h and the biofilm formation was quantified at 24-h interval. For the inactivation study, C. jejuni biofilms developed at 20 or 37°C for 48 h were exposed to the phytochemicals for 1, 5, or 10 min and surviving C. jejuni in the biofilm were enumerated. All phytochemicals reduced C. jejuni biofilm formation as well as inactivated mature biofilm on polystyrene and steel surface at both temperatures (P < 0.05). The highest dose of TC (75.64 mM), EG (60.9 mM) and CR (66.56 mM) inactivated (>7 log reduction) biofilm developed on steel (20°C) within 5 min. The genes encoding for motility systems (flaA, flaB, and flgA) were downregulated by all phytochemicals (P < 0.05). The expression of stress response (cosR, ahpC) and cell surface modifying genes (waaF) was reduced by EG. LC-MS/MS based proteomic analysis revealed that TC, EG, and CR significantly downregulated the expression of NapA protein required for oxidative stress response. The expression of chaperone protein DnaK and bacterioferritin required for biofilm formation was reduced by TC and CR. Scanning electron microscopy revealed disruption of biofilm architecture and loss of extracellular polymeric substances after treatment. Results suggest that TC, EG, and CR could be used as a natural disinfectant for controlling C. jejuni biofilms in processing areas.

Campylobacter jejuni, a leading cause of foodborne disease in humans, associate primarily with consumption of contaminated poultry and poultry products. Intervention strategies aimed at reducing C. jejuni contamination on poultry products could significantly reduce C. jejuni infection in humans. This study evaluated the efficacy of gum arabic (GA) and chitosan (CH) fortified with carvacrol (CR) as an antimicrobial coating treatment for reducing C. jejuni on chicken wingettes. Aforementioned compounds are generally recognized as safe status compounds obtained from gum arabic tree, crustaceans and oregano oil respectively. A total of four separate trials were conducted in which wingettes were randomly assigned to baseline, saline control (wingettes washed with saline), GA (10%), CH (2%), CR (0.25, 0.5, or 1%) or their combinations. Each wingette was inoculated with a cocktail of four wild-type strains of C. jejuni (∼7.5 log10 cfu/sample). Following 1 min of coating in aforementioned treatments, wingettes were air dried (1 h) and sampled at 0, 1, 3, 5, and 7 days of refrigerated storage for C. jejuni and total aerobic counts (n = 5 wingettes/treatment/day). In addition, the effect of treatments on wingette color was measured using a Minolta colorimeter. Furthermore, the effect of treatments on the expression of C. jejuni survival/virulence genes was evaluated using real-time quantitative PCR. Results showed that all three doses of CR, CH or GA-based coating fortified with CR reduced C. jejuni from day 0 through 7 by up to 3.0 log10 cfu/sample (P < 0.05). The antimicrobial efficacy of GA was improved by CR and the coatings reduced C. jejuni by ∼1 to 2 log10 cfu/sample at day 7. Moreover, CH + CR coatings reduced total aerobic counts when compared with non-coated samples for a majority of the storage times. No significant difference in the color of chicken wingettes was observed between treatments. Exposure of pathogen to sublethal concentrations of CR, CH or combination significantly modulated select genes encoding for energy taxis (cetB), motility (motA), binding (cadF), and attachment (jlpA). The results suggest that GA or CH-based coating with CR could potentially be used as a natural antimicrobial to control C. jejuni in postharvest poultry products.

Background: Intraocular tuberculosis (IOTB), an extrapulmonary manifestation of tuberculosis of the eye, has unique and varied clinical presentations with poorly understood pathogenesis. As it is a significant cause of inflammation and visual morbidity, particularly in TB endemic countries, it is essential to study the pathogenesis of IOTB. Clinical and histopathologic studies suggest the presence of Mycobacterium tuberculosis in retinal pigment epithelium (RPE) cells.Methods: A human retinal pigment epithelium (ARPE-19) cell line was infected with a virulent strain of M. tuberculosis (H37Rv). Electron microscopy and colony forming units (CFU) assay were performed to monitor the M. tuberculosis adherence, invasion, and intracellular replication, whereas confocal microscopy was done to study its intracellular fate in the RPE cells. To understand the pathogenesis, the transcriptional profile of M. tuberculosis in ARPE-19 cells was studied by whole genome microarray. Three upregulated M. tuberculosis transcripts were also examined in human IOTB vitreous samples.Results: Scanning electron micrographs of the infected ARPE-19 cells indicated adherence of bacilli, which were further observed to be internalized as monitored by transmission electron microscopy. The CFU assay showed that 22.7 and 8.4% of the initial inoculum of bacilli adhered and invaded the ARPE-19 cells, respectively, with an increase in fold CFU from 1 dpi (0.84) to 5dpi (6.58). The intracellular bacilli were co-localized with lysosomal-associated membrane protein-1 (LAMP-1) and LAMP-2 in ARPE-19 cells. The transcriptome study of intracellular bacilli showed that most of the upregulated transcripts correspond to the genes encoding the proteins involved in the processes such as adherence (e.g., Rv1759c and Rv1026), invasion (e.g., Rv1971 and Rv0169), virulence (e.g., Rv2844 and Rv0775), and intracellular survival (e.g., Rv1884c and Rv2450c) as well as regulators of various metabolic pathways. Two of the upregulated transcripts (Rv1971, Rv1230c) were also present in the vitreous samples of the IOTB patients.Conclusions:M. tuberculosis is phagocytosed by RPE cells and utilizes these cells for intracellular multiplication with the involvement of late endosomal/lysosomal compartments and alters its transcriptional profile plausibly for its intracellular adaptation and survival. The findings of the present study could be important to understanding the molecular pathogenesis of IOTB with a potential role in the development of diagnostics and therapeutics for IOTB.

The blast crisis of chronic myelogenous leukemia (CML) can be hematological or extramedullary. About 25% of these cases fulfill the criteria for mixed phenotype acute leukemia. We here report a case of a second blast crisis of CML which was extramedullary and was immunophenotypically bilineage T/myeloid.

This study investigated the effect of carvacrol (CR), a phytophenolic compound on antibiotic-associated gut dysbiosis and C. difficile infection in a mouse model. Five to six-week-old C57BL/6 mice were randomly divided into seven treatment groups (challenge and control) of eight mice each. Mice were fed with irradiated feed supplemented with CR (0, 0.05, and 0.1%); the challenge groups were made susceptible to C. difficile by orally administering an antibiotic cocktail in water and an intra-peritoneal injection of clindamycin. Both challenge and control groups were infected with 105CFU/ml of hypervirulent C. difficile (ATCC 1870) spores or PBS, and observed for clinical signs for 10 days. Respective control groups for CR, antibiotics, and their combination were included for investigating their effect on mouse enteric microflora. Mouse body weight and clinical and diarrhea scores were recorded daily post infection. Fecal samples were collected for microbiome analysis using rRNA sequencing in MiSeq platform. Carvacrol supplementation significantly reduced the incidence of diarrhea and improved the clinical and diarrhea scores in mice (p < 0.05). Microbiome analysis revealed a significant increase in Proteobacteria and reduction in the abundance of protective bacterial flora in antibiotic-treated and C. difficile-infected mice compared to controls (p < 0.05). However, CR supplementation positively altered the microbiome composition, as revealed by an increased abundance of beneficial bacteria, including Firmicutes, and significantly reduced the proportion of detrimental flora such as Proteobacteria, without significantly affecting the gut microbiome diversity compared to control. Results suggest that CR could potentially be used to control gut dysbiosis and reduce C. difficile infection.

Campylobacter jejuni is a major foodborne pathogen that causes severe gastroenteritis in humans characterized by fever, diarrhea, and abdominal cramps. In the human gut, Campylobacter adheres and invades the intestinal epithelium followed by cytolethal distending toxin mediated cell death, and enteritis. Reducing the attachment and invasion of Campylobacter to intestinal epithelium and expression of its virulence factors such as motility and cytolethal distending toxin (CDT) production could potentially reduce infection in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentration not inhibiting bacterial growth) of three GRAS (generally recognized as safe) status phytochemicals namely trans-cinnamaldehyde (TC; 0.005, 0.01%), carvacrol (CR; 0.001, 0.002%), and eugenol (EG; 0.005, 0.01%) in reducing the attachment, invasion, and translocation of C. jejuni on human intestinal epithelial cells (Caco-2). Additionally, the effect of these phytochemicals on Campylobacter motility and CDT production was studied using standard bioassays and gene expression analysis. All experiments had duplicate samples and were replicated three times on three strains (wild type S-8, NCTC 11168, 81–176) of C. jejuni. Data were analyzed using ANOVA with GraphPad ver. 6. Differences between the means were considered significantly different at P < 0.05. The majority of phytochemical treatments reduced C. jejuni adhesion, invasion, and translocation of Caco-2 cells (P < 0.05). In addition, the phytochemicals reduced pathogen motility and production of CDT in S-8 and NCTC 11168 (P < 0.05). Real-time quantitative PCR revealed that phytochemicals reduced the transcription of select C. jejuni genes critical for infection in humans (P < 0.05). Results suggest that TC, CR, and EG could potentially be used to control C. jejuni infection in humans.

Background: Etiology of nearly 30% cases of chronic viral hepatitis remains undetected. Occult HBV infection (OBI) has emerged as an important clinical entity in this scenario. Apart from prevalence and clinical outcome of OBI patients genotype was determined in northern region of India. Materials and Methods: A total of 847 patients with chronic liver disease (CLD) were screened for common viral etiologies and others serological markers of HBV. Amplification of surface, precore and polymerase genes of HBV was performed in patients negative for other etiologies. Genotyping and sequencing of the precore region was performed for OBI cases. Results: Twenty-nine (7.61%) cases of OBI were identifiedof which 9 had chronic liver disease (CHD), 11 liver cirrhosis (LC) and 9 hepatocellular carcinoma (HCC). Majority of OBI cases were detected by amplification of surface gene 26 (89.6%), followed by pre-core gene 12 (41.3%). Their liver functions tests were significantly deranged in comparison to overt HBV cases. IgG anti HBc was present in 8 (27.6%) OBI cases. Mutation was observed in 8 (32%) in pre-core region at nt. 1896 of overt HBV cases. Genotype D was the predominant genotype. In conclusion: OBI in our study was characterized by predominance of genotype D and more severe clinical and biochemical profile in comparison to overt HBV. IgG anti HBc positivity could be utilized as a marker of OBI. We recommend use of sensitive nested PCR for diagnosis of OBI, amplifying at least surface and precore gene.

Context: Bacterial isolates from intra-abdominal infections, in particular, peritonitis and their unpredictable antimicrobial resistance patterns, continue to be a matter of concern not only globally but regionally too. Aim: An attempt in the present study was made to study the patterns of drug resistance in bacterial isolates, especially gram negative bacilli in intra-abdominal infections (IAI) in our hospital. Materials and Methods: From 100 cases of peritonitis, identification of isolates was done as per recommended methods. Antimicrobial susceptibility and extended-spectrum beta-lactamase (ESBL) testing were performed following the CLSI guidelines. Results: A total of 133 clinical isolates were obtained, of which 108 were aerobes and 22 anaerobes. Fungal isolates were recovered in only three cases. Escherichia coli (47/108) emerged as the most predominant pathogen followed by Klebsiella spp. (27/108), while Bacteroides fragilis emerged as the predominant anaerobe (12/22). Among coliforms, 61.7% E. coli and 74.1% Klebsiella spp. were ESBL positive. A high level of resistance was observed for beta lactams, ciprofloxacin, amikacin, and ertapenem. Ertapenem resistance (30-41%) seen in coliforms, appears as an important issue. Imipenem, tigecycline, and colistin were the most consistently active agents tested against ESBL producers. Conclusion: Drug resistance continues to be a major concern in isolates from intra-abdominal infections. Treatment with appropriate antibiotics preceded by antimicrobial resistance testing aided by early diagnosis, adequate surgical management, and knowledge of antibiotic - resistant organisms appears effective in reducing morbidity and mortality in IAI cases.

The intervening sequence (IVS) of Coxiella burnetii, the agent of Q fever, is a 428-nt selfish genetic element located in helix 45 of the precursor 23S rRNA. The IVS element, in turn, contains an ORF that encodes a hypothetical ribosomal S23 protein (S23p). Although S23p can be synthesized in vitro in the presence of an engineered E. coli promoter and ribosome binding site, results suggest that the protein is not synthesized in vivo. In spite of a high degree of IVS conservation among different strains of C. burnetii, the region immediately upstream of the S23p start codon is prone to change, and the S23p-encoding ORF is evidently undergoing reductive evolution. We determined that IVS excision from 23S rRNA was mediated by RNase III, and IVS RNA was rapidly degraded, thereafter. Levels of the resulting 23S rRNA fragments that flank the IVS, F1 (~1.2 kb) and F2 (~1.7 kb), were quantified over C. burnetii’s logarithmic growth phase (1-5d). Results showed that 23S F1 quantities were consistently higher than those of F2 and 16S rRNA. The disparity between levels of the two 23S rRNA fragments following excision of IVS is an interesting phenomenon of unknown significance. Based upon phylogenetic analyses, IVS was acquired through horizontal transfer after C. burnetii’s divergence from an ancestral bacterium and has been subsequently maintained by vertical transfer. The widespread occurrence, maintenance and conservation of the IVS in C. burnetii imply that it plays an adaptive role or has a neutral effect on fitness.

Escherichia coli O157: H7 (EHEC) is a major foodborne pathogen largely transmitted to humans through the consumption of undercooked ground beef. This study investigated the efficacy of two food-grade, plant-derived antimicrobials, namely rutin (RT), and resveratrol (RV) with or without chitosan (CH) in enhancing EHEC inactivation in undercooked hamburger patties. Further, the effect of aforementioned treatments on beef color and lipid oxidation was analyzed. Additionally, the deleterious effects of these antimicrobial treatments on EHEC was determined using scanning electron microscopy (SEM).Ground beef was inoculated with a five-strain mixture of EHEC (7.0 log CFU/g), followed by the addition of RT (0.05%, 0.1% w/w) or RV (0.1, 0.2% w/w) with or without CH (0.01% w/w). The meat was formed into patties (25 g) and stored at 4°C for 5 days. On days 1, 3 and 5, the patties were cooked (65°C, medium rare) and surviving EHEC was enumerated. The effect of these treatments on meat color and lipid oxidation during storage was also determined as per American Meat Science Association guidelines. The study was repeated three times with duplicate samples of each treatment.Both RT and RV enhanced the thermal destruction of EHEC, and reduced the pathogen load by at least 3 log CFU/g compared to control (P < 0.05). The combination of RT or RV with CH was found to be more effective, and reduced EHEC by 5 log CFU/g (P < 0.05). EHEC counts in uncooked patties did not decline during storage for 5 days (P > 0.05). Moreover, patties treated with RV plus chitosan were more color stable with higher a* values (P < 0.05). SEM results revealed that heat treatment with antimicrobials (CH+RV 0.2%) caused a complete destruction of EHEC cells with pore formation and extrusion of intracellular contents. Results suggest that the aforementioned antimicrobials could be used for enhancing the thermal inactivation of EHEC in undercooked patties; however, detailed sensory studies are warranted.

A new subspecies of Staphylococcus hominis described by Kloos et al. in 1998 and named S. hominis subsp. novobiosepticus (SHN) has been implicated in nosocomial outbreaks. Multidrug resistance, including resistance to novobiocin and oxacillin, is a particularly important feature of SHN. In our institute, we encountered 13 cases of S. hominis subsp. hominis in cancer patients with septicemia, of which seven were methicillin resistant. The isolates were identified by VITEK ® 2 compact automated system, using GP REF 21342 identification card and antimicrobial susceptibility testing card P-628. The biochemical reactions and antibiotic susceptibility pattern of the seven methicillin-resistant isolates were re-analyzed and patient details were re-checked to finally identify them as SHN. The increasing number of cases reporting isolation of SHN from biological specimens point to potential virulence and clinical importance of this bacterium.

Objective: Typhoid fever is a major public health problem. A test which is simple, reliable and can be carried out in small laboratories is the need of the hour. We prospectively evaluated typhidot M and Diazo tests vis-à-vis blood culture and Widal test in children. Methods: Patients aged 6 months to 12 years, having fever of more than four days duration with clinical suspicion of typhoid fever were enrolled. Patients in whom other diagnosis was made served as control. The tests under scrutiny were validated against blood culture and then all the four tests were evaluated among patients who presented in the first week of illness. Results: Blood culture was positive in only 27.3% of the cases. Among these culture positive cases, typhidot M test had the highest sensitivity, specificity, PPV and NPV of 90% (95% CI = 74.4–96.5), 100% (95% CI = 90.1–100), 100% (95% CI = 87.5–100), and 92.1% (95% CI = 79.2–97.3) respectively. Diazo test ranked next with sensitivity, specificity, PPV and NPV of 86.7% (95% CI = 70.3–94.7), 85.7% (95% CI = 70.6–93.7), 83.9% (95% CI = 67.4–92.9), 88.2% (95% CI = 73.4–95.3) respectively. Among clinically suspected typhoid cases, the overall sensitivity, of blood culture, Widal, typhidot M, Diazo was 27.3% (95% CI = 19.8–36.3), 64.6% (95% CI = 55.3–72.9), 89.1% (95% CI = 81.9–93.7), 80.9% (95% CI = 72.6–87.2) respectively. In the first week of illness, typhidot M showed the best sensitivity [86.2% (95% CI = 69.4–94.5)] followed by Diazo [79% (95% CI = 61.6–90.2)], Widal [41.4% (95% CI = 25.5–59.3)] and blood culture [31% (95% CI = 17.3–49.2)]. Conclusion: Both Typhidot M and Diazo are good screening tests for the diagnosis of typhoid fever. Typhidot M is superior to Diazo but the latter is more suitable to resource poor settings being economic and easy to perform. Keywords: Typhidot test, Diazo test, Widal test, Typhoid fever

Objective: The subunit vaccine strategies and development of various diagnostic reagents for Mycobacterium avium infection relies on the presence of secreted, species-specific mycobacterial antigens. The M. avium 35 kDa protein has been suggested as a candidate for vaccine/diagnostic reagent, specifically for M. avium infection. The present study was conducted to evaluate the diagnostic specificity of the M. avium 35 kDa protein in the Indian population. Materials and Methods: Culture filtrate proteins were isolated by growing the bacilli in modified Youman′s medium. The 35 kDa protein was purified by high-resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blast search was carried out. Western blotting was performed with either monoclonal antibody CS-38 or serum samples of tuberculosis (TB) patients. The 35 kDa-specific immunoglobulin G antibody titer was estimated in the sera of TB patients and healthy individuals by indirect enzyme-linked immunosorbent assay (ELISA). Results: Despite the absence of gene for the 35 kDa protein, the sera of TB patients and TB patient′s contacts nonspecifically recognize it. Of 109 TB patients tested, the sera of 84 patients in ELISA (percentage recognition = 87.5%) and 27 of 29 TB patients tested in western immunoblotting (percentage recognition = 93.10%) recognized the M. avium 35 kDa protein, while with sera of TB patient′s contacts, the recognition was 50%. Conclusion: Contrary to Western studies, the M. avium 35 kDa protein does not seem to be a good candidate for the specific diagnosis of M. avium infection in the Indian population.

ABSTRACT Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by 13C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets. IMPORTANCE To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.

The diagnosis of clear cell renal cell carcinoma may sometimes pose challenges because of the presence of uncharacteristic morphology, varied immunophenotypic patterns and due to lack of molecular or genetic determinants. More often, the morphological variations can be easily overlooked in routine practice and a more common diagnosis is usually put forward. Solid, acinar and alveolar are the common patterns described in the literature. We report a recently described pattern of clear cell renal cell carcinoma which has hemangioblastoma-like morphology and an unusual immunoprofile. In our case, the tumor showed a diffuse hemangioblastoma-like pattern and diffuse positivity for Alpha-inhibin on immunohistochemistry. A thorough literature search, extensive sampling and an expanded immunohistochemistry panel revealed a clear cell renal cell carcinoma component. Presence of renal vein thrombosis and focal necrosis were other helpful features in discerning the malignant nature of tumor.

Background and Objectives: Food contamination by aflatoxins is an important food safety concern for agricultural products. In order to identify and develop novel antifungal agents, several plant extracts and isolated compounds have been evaluated for their bioactivities. Anti-infectious activity of Piper betle used in traditional medicine of Malaysia has been reported previously. Materials and Methods: Crude methanol extract from P. betel powdered leaves was partitioned between chloroform and water. The fractions were tested against A. flavus UPMC 89, a strong aflatoxin producing strain. Inhibition of mycelial growth and aflatoxin biosynthesis were tested by disk diffusion and macrodillution techniques respectively. The presence of aflatoxin was determined by thin-layer chromatography (TLC) and fluorescence spectroscopy techniques using AFB1 standard. The chloroform soluble compounds were identified using HPLC-Tandem mass spectrometry technique. Results: The results, evaluated by measuring the mycelial growth and quantification of aflatoxin B1(AFLB1) production in broth medium revealed that chloroform soluble compounds extract from P. betle dried leaves was able to block the aflatoxin biosynthesis pathway at concentration of 500µg/ml without a significant effect on mycelium growth. In analyzing of this effective fractions using HPLC-MS2 with ESI ionization technique, 11 phenolic compounds were identified. Conclusion: The results showed that the certain phenolic compounds are able to decline the aflatoxin production in A. flavus with no significant effect on the fungus mycelia growth. The result also suggested P. betle could be used as potential antitoxin product.

Introduction. HBV genotypes and subtypes are useful clinical and epidemiological markers. In this study prevalent HBV genotypes were assessed in relation to serological profile and clinical status. Material & Methods. 107 cases of HBV were genotyped. Detailed clinical history was elicited from them. HBsAg, HBeAg, anti-HBs, anti-HBe, and anti-HBc-IgM were assessed. HBV genotyping was performed using Kirschberg's type specific primers (TSP-PCR), heminested PCR, and Naito's monoplex PCR. Nucleotide sequencing was performed. Results. A total of 97 (91%) were genotyped following the methods of Kirschberg et al./Naito et al. Genotype D was by far the most prevalent genotype 91 (85.04%) in this region. A surprising finding was the detection of genotype F in 5 (4.67%) of our patients. Genotype A strangely was observed only in one case. In 85.7% genotype D was associated with moderate to severe liver disease, 43.9% HBeAg, and 18.7% anti-HBc-IgM positivity. Majority of genotype F (80%) was seen in mild to moderate liver disease. It was strongly associated with HBeAg 60% and 20% anti-HBc-IgM positivity. Conclusion. Emergence of genotype F in India merits further study regarding its clinical implications and treatment modalities. Knowledge about HBV genotypes can direct a clinician towards more informed management of HBV patients.

Abstract Background Mycoplasma gallisepticum is a major poultry pathogen and causes severe economic loss to the poultry industry. In mycoplasmas lipoproteins are abundant on the membrane surface and play a critical role in interactions with the host, but tools for exploring their molecular biology are limited. Results In this study we examined whether the alkaline phosphatase gene (phoA ) from Escherichia coli could be used as a reporter in mycoplasmas. The promoter region from the gene for elongation factor Tu (ltuf) and the signal and acylation sequences from the vlhA 1.1 gene, both from Mycoplasma gallisepticum , together with the coding region of phoA , were assembled in the transposon-containing plasmid pISM2062.2 (pTAP) to enable expression of alkaline phosphatase (AP) as a recombinant lipoprotein. The transposon was used to transform M. gallisepticum strain S6. As a control, a plasmid containing a similar construct, but lacking the signal and acylation sequences, was also produced (pTP) and also introduced into M. gallisepticum . Using a colorimetric substrate for detection of alkaline phosphatase activity, it was possible to detect transformed M. gallisepticum . The level of transcription of phoA in organisms transformed with pTP was lower than in those transformed with pTAP, and alkaline phosphatase was not detected by immunoblotting or enzymatic assays in pTP transformants, eventhough alkaline phosphatase expression could be readily detected by both assays in pTAP transformants. Alkaline phosphatase was shown to be located in the hydrophobic fraction of transformed mycoplasmas following Triton X-114 partitioning and in the membrane fraction after differential fractionation. Trypsin proteolysis confirmed its surface exposure. The inclusion of the VlhA lipoprotein signal sequence in pTAP enabled translocation of PhoA and acylation of the amino terminal cysteine moiety, as confirmed by the effect of treatment with globomycin and radiolabelling studies with [14 C]palmitate. PhoA could be identified by mass-spectrometry after separation by two-dimensional electrophoresis. Conclusion This is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas.

OBJECTIVE: Typhoid fever is a major public health problem. A test which is simple, reliable and can be carried out in small laboratories is the need of the hour. We prospectively evaluated typhidot M and Diazo tests vis-à-vis blood culture and Widal test in children. METHODS: Patients aged 6 months to 12 years, having fever of more than four days duration with clinical suspicion of typhoid fever were enrolled. Patients in whom other diagnosis was made served as control. The tests under scrutiny were validated against blood culture and then all the four tests were evaluated among patients who presented in the first week of illness. RESULTS: Blood culture was positive in only 27.3% of the cases. Among these culture positive cases, typhidot M test had the highest sensitivity, specificity, PPV and NPV of 90% (95% CI = 74.4-96.5), 100% (95% CI = 90.1-100), 100% (95% CI = 87.5-100), and 92.1% (95% CI = 79.2-97.3) respectively. Diazo test ranked next with sensitivity, specificity, PPV and NPV of 86.7% (95% CI = 70.3-94.7), 85.7% (95% CI = 70.6-93.7), 83.9% (95% CI = 67.4-92.9), 88.2% (95% CI = 73.4-95.3) respectively. Among clinically suspected typhoid cases, the overall sensitivity, of blood culture, Widal, typhidot M, Diazo was 27.3% (95% CI = 19.8- 36.3), 64.6% (95% CI = 55.3-72.9), 89.1% (95% CI = 81.9-93.7), 80.9% (95% CI = 72.6-87.2) respectively. In the first week of illness, typhidot M showed the best sensitivity [86.2% (95% CI = 69.4-94.5)] followed by Diazo [79% (95% CI = 61.6-90.2)], Widal [41.4% (95% CI = 25.5-59.3)] and blood culture [31% (95% CI = 17.3-49.2)]. CONCLUSION: Both Typhidot M and Diazo are good screening tests for the diagnosis of typhoid fever. Typhidot M is superior to Diazo but the latter is more suitable to resource poor settings being economic and easy to perform.