Improved antioxidant and anti-tubercular potential of liquiritigenin grafted on low molecular weight chitosan from gladius of Sepioteuthis lessoniana.

Chitosan (CH) is natural abundant biopolymer present on earth after cellulose. CH can be functionalized by numerous functional groups such as amino and carboxyl groups, potential biologically active compounds. The functionalization of CH with polyphenols had a greater biological than non-grafted CH. In the present study, the polyphenolic compound liquiritigenin (LTG) is chemically functionalized on the low molecular weight chitosan (LMW-CH) (693.09 Da). This was extracted and irradiated with gamma radiation from the gladius of Sepioteuthis lessoniana. The grafted compound was to in vitro anti-oxidant employing physicochemical methods and characterization was made by spectroscopic methods. The degree of deacetylation (DDA) of the LMW-CH was detected in 74 % of the samples, and at higher concentrations (100 g/mL). LMW-CH grafted with LTG had improved water solubility (5 mg/mL), and was thermally stable upto 143.58 °C. Its molecular weight was 855.1 Da. In conclusion the in vitro antioxidant and the anti-tuberculosis (anti-TB) properties of the grafted samples were significantly (P < 0.001) increased compared to the unconjugated LMW-CH and LTG. Overall, functionalization of LTG with LMW-CH improved the anti-tuberculosis activity. Further studies are needed to explore the possibilities of its use in vivo models.

Challenges and mitigation strategies associated with Burkholderia cepacia complex contamination in pharmaceutical manufacturing.

Burkholderia cepacia complex (BCC) is a Gram-negative, non-spore-forming bacterium with more than 20 opportunistic pathogenic species, most commonly found in soil and water. Due to their rapid mutation rates, these organisms are adaptable and possess high genomic plasticity. BCC can cause life-threatening infections in immunocompromised individuals, such as those with cystic fibrosis, chronic granulomatous disease, and neonates. BCC contamination is a significant concern in pharmaceutical manufacturing, frequently causing non-sterile product recalls. BCC has been found in purified water, cosmetics, household items, and even ultrasound gel used in veterinary practices. Pharmaceuticals, personal care products, and cleaning solutions have been implicated in numerous outbreaks worldwide, highlighting the risks associated with intrinsic manufacturing site contamination. Regulatory compliance, product safety, and human health protection depend on testing for BCC in pharmaceutical manufacturing. Identification challenges exist, with BCC often misidentified as other bacteria like non-lactose fermenting Escherichia coli or Pseudomonas spp., particularly in developing countries where reporting BCC in pharmaceuticals remains limited. This review comprehensively aims to address the organisms causing BCC contamination, genetic diversity, identification challenges, regulatory requirements, and mitigation strategies. Recommendations are proposed to aid pharmaceutical chemists in managing BCC-associated risks and implementing prevention strategies within manufacturing processes.

Antioxidant and Teratogenic Activities of Formulated Agar Extracted from Brown Seaweed Turbinaria conoides against Zebrafish Larvae.

This study examines the antioxidant and teratogenic effects of two different type's methods of formulating agar from Turbinaria conoides (T. conoides) using a zebrafish model. The agar was extracted using the aqueous extraction method and developed in two different formulations using separate procedures. Formulated agar1 (FA1) used a higher concentration of the ingredients while formulated agar 2 (FA2) had a lesser concentration. The two unique formulated agars (FAs) were studied using biochemical composition, Fourier infrared (FT-IR) spectroscopy, gas chromatography-mass spectroscopy (GC-MS), and scanning electron microscopy (SEM). The antioxidant activities of both FAs in vitro were shown to be significantly different (P < 0.05) at various concentrations (60-180 µl/ml) in the study. The toxicity of the FAs was dose-dependent, with FA1 having the least teratogenic activity when compared to FA2. In comparison to FA2, FA1 was found to have higher antioxidant activity. At various concentrations (0.5, 0.25, and 0.125 µg/ml), the teratogenic activity of two FAs was examined in zebrafish embryos (ZFE) at 24, 48, 72, and 96 hours post fertilization (hpf). Both FAs exhibit dose-dependent toxicity and increased antioxidant activity, and this can be utilized as an alternative for standard antioxidants, according to this study.

Adaptations of Vibrio parahaemolyticus to Stress During Environmental Survival, Host Colonization, and Infection.

Vibrio parahaemolyticus (Vp) is an aquatic Gram-negative bacterium that may infect humans and cause gastroenteritis and wound infections. The first pandemic of Vp associated infection was caused by the serovar O3:K6 and epidemics caused by the other serovars are increasingly reported. The two major virulence factors, thermostable direct hemolysin (TDH) and/or TDH-related hemolysin (TRH), are associated with hemolysis and cytotoxicity. Vp strains lacking tdh and/or trh are avirulent and able to colonize in the human gut and cause infection using other unknown factors. This pathogen is well adapted to survive in the environment and human host using several genetic mechanisms. The presence of prophages in Vp contributes to the emergence of pathogenic strains from the marine environment. Vp has two putative type-III and type-VI secretion systems (T3SS and T6SS, respectively) located on both the chromosomes. T3SS play a crucial role during the infection process by causing cytotoxicity and enterotoxicity. T6SS contribute to adhesion, virulence associated with interbacterial competition in the gut milieu. Due to differential expression, type III secretion system 2 (encoded on chromosome-2, T3SS2) and other genes are activated and transcribed by interaction with bile salts within the host. Chromosome-1 encoded T6SS1 has been predominantly identified in clinical isolates. Acquisition of genomic islands by horizontal gene transfer provides enhanced tolerance of Vp toward several antibiotics and heavy metals. Vp consists of evolutionarily conserved targets of GTPases and kinases. Expression of these genes is responsible for the survival of Vp in the host and biochemical changes during its survival. Advanced genomic analysis has revealed that various genes are encoded in Vp pathogenicity island that control and expression of virulence in the host. In the environment, the biofilm gene expression has been positively correlated to tolerance toward aerobic, anaerobic, and micro-aerobic conditions. The genetic similarity analysis of toxin/antitoxin systems of Escherichia coli with VP genome has shown a function that could induce a viable non-culturable state by preventing cell division. A better interpretation of the Vp virulence and other mechanisms that support its environmental fitness are important for diagnosis, treatment, prevention and spread of infections. This review identifies some of the common regulatory pathways of Vp in response to different stresses that influence its survival, gut colonization and virulence.

Chitosan grafted butein: A metal-free transducer for electrochemical genosensing of exosomal CD24.

Metal-free cost-efficient biocompatible molecules are beneficial for opto-electrochemical bioassays. Herein, chitosan (CS) conjugated butein is prepared via graft polymerization. Structural integrity between radical active sites of CS and its probable conjugation routes with reactive OH group of butein during grafting were comprehensively studied using optical absorbance/emission property, NMR, FT-IR and XPS analysis. Fluorescence emission of CS-conjugated butein (CSB) was studied in dried flaky state as well as in drop casted form. Cyclic voltammetric study of CSB modified glassy carbon electrode exhibits 2e-/2H+ transfer reaction in phosphate buffered saline electrolyte following a surface-confined process with a correlation coefficient of 0.99. Unlike pristine butein, CSB modified electrode display a highly reversible redox behavior under various pH ranging from 4 to 9. For the proof-of-concept CSB-modified flexible screen printed electrodes were processed for electrochemical biosensing of exosomal CD24 specific nucleic acid at an ultralow sample concentration, promising for ovarian cancer diagnosis.

Pathogenic Vibrio parahaemolyticus indiarrhoeal patients, fish and aquatic environments and their potential for inter-source transmission.

The role of Vibrio parahaemolyticus in causing diarrhoeal disease is well known. However, phenotypic and genetic traits of this pathogen isolated from diverse sources have not been investigated in detail. In this study, we have screened samples from diarrhoeal cases (2603), brackish water fish (301) and aquatic environments (115) and identified V. parahaemolyticus in 29 (1.1%), 171 (56.8%) and 43 (37.4%) samples, respectively. Incidence of pathogenic V. parahaemolyticuswith virulence encoding thermostable-direct haemolysin gene (tdh) was detected mostly in fishes (19.3%) and waters (15.6%) than clinical samples (1.04%). The pandemic strain marker genes (toxRS and PGS-sequences) have been detected relatively more in water (6%) and fish (5%) samples than in clinical samples (0.7%). Majority of the V. parahaemolyticus isolates from clinical cases and fish samples (26.3%) belonged to classical pandemic serovars (O3:K6). In addition, several newly recognised pandemic serovars have also been identified. Pulsed field-gel electrophoresis (PFGE) analysis showed clonal relatedness (60-85%) of V. parahaemolyticus from different sources. The study observation revealed that the brackish water fishes and water bodies may act as a reservoir of pathogenic V. parahaemolyticus. Emergence of several new serovars of pandemic V. parahaemolyticussignifies the changing phenotypic characteristics of the pathogen.

Chemically synthesized butein and butin: Optical, structure and electrochemical redox functionality at electrode interface.

Progress in the development of phytochemistry has delivered advancement in materials functionality for range of inter/trans-disciplinary application. Here, we investigated the structural functionality of chemically synthesized phytoconstitutent, chalcone (butein) and flavanone (butin). Photoactive and electroactive behavior of butein and butin were comprehensively studied using UV-vis absorbance, photoluminescence and cyclic voltammetric techniques. Surface morphology of the butein and butin powders was characterized from scanning electron microscope at an operating voltage of 10 kV. Significant ultraviolet absorbance property are observed from butein and butin due to the distribution of π → π* and n → π* transitions. Photoluminescence emission spectra of the prepared materials are well resolved at visible region via keto-enol tautomerization and can be influenced by solvent pH. Cyclic voltammetric studies on the prepared materials enabled a direct electron-transfer reaction at gold-screen printed electrode, indicating the feasibility for analytical validation in herbal industries. Existence of multiple electroactive hydroxyl groups makes butein and butin a redox-functional species at electrode interface. Dispersion ability in aqueous and organic solvents makes butein and butin suitable for variety of photochemical applications. This phytochemical material offers new degrees of optical and redox functionality similar to inorganic nanostructures, in addition to inherent bioactivity, that may be advantageous for further biomedical function.

Molecular characterization of locus of enterocyte effacement pathogenicity island in shigatoxic Escherichia coli isolated from human & cattle in West Bengal, India.

BACKGROUND & OBJECTIVES: Shigatoxic Escherichia coli (STEC) recovered from dairy animals of Kolkata, India, harboured the putative virulence genes; however, the animals did not exhibit clinical symptoms. Similarly, human isolates in this locality also showed variations in degree of symptoms. Hence, this study was designed to know the presence of recognized gene(s) in the locus of enterocyte effacement (LEE) pathogenicity island in these STEC isolates and functional status of the cardinal gene (eae) related to pathogenicity. METHODS: Genes were characterized using polymerase chain reaction (PCR) assays, and functional status of cardinal gene (eae) was evaluated by fluorescent actin staining (FAS) assay. Variation in eae gene was determined by intimin PCR. RESULTS: Cattle STEC isolates carried 22 genes in LEE pathogenicity island in different frequencies ranging from 5.63 to 47.88 per cent of the isolates. In human isolates, the genes namely ler, escRSTU, orf 2, esc C, esc V, orf 3 and tir that are associated with secretory function, were found to be absent and rest of the genes were present in lower frequency. Further, the cardinal gene (eae) responsible for initiation of pathogenesis was in a very low frequency in human (n=2; 10.5%) and cattle (n=11; 15.5%) isolates. None of theseeae+ STEC isolates from human and cattle revealed positivity in FAS assay. INTERPRETATION & CONCLUSIONS: Majority of human STEC isolates lacked the cardinal virulence gene (eae), and genes for secretory function that are essential for facilitating pathogenesis. This may partially be attributed to low occurrence of STEC in human clinical diarrhoea in this area. Although a few isolates (11 of 71) from cattle had eae gene, they did not express phenotypically. This could be one of the reasons for not appearing of clinical symptoms in the hosts.

Carbapenem Resistance in Clonally Distinct Clinical Strains of Vibrio fluvialis Isolated from Diarrheal Samples.

Carbapenems have been used for many years to treat severe nosocomial Enterobacteriaceae infections. The spread of resistance to these drugs among other bacterial families is an emerging problem worldwide, mostly caused by New Delhi metallo-ß-lactamase (NDM-1). We screened for the prevalence of NDM-1-expressing enteric pathogens from hospitalized patients with acute diarrhea in Kolkata, India, and identified 27 Vibrio fluvialis-harboring blaNDM-1 (NDM-VF) strains. These isolates were also resistant to all the tested antimicrobial drugs except doxycycline. The large plasmid of V. fluvialis harboring blaNDM-1 could be easily transferred to other enteric pathogens. Genes flanking the blaNDM-1 were found to be identical to the reported sequence from an Escherichia coli isolate. Analyses showed that the V. fluvialis possessing the NDM-VF region belonged to different clones. The pathogenicity of V. fluvialis to humans and its ubiquitous presence in the environment call for constant monitoring of this species for emerging antimicrobial drug resistance.

Unique Clones of Vibrio cholerae O1 El Tor with Haitian Type ctxB Allele Implicated in the Recent Cholera Epidemics from Nigeria, Africa.

BACKGROUND AND OBJECTIVES: The antimicrobial susceptibility patterns and genetic characteristics of Vibrio cholerae O1, which is responsible for several cholera epidemics in Nigeria, are not reported in detail since 2007. In this study, we screened V. cholerae O1 El Tor biotype isolates from cholera cases and water samples from different states to investigate their phenotypic and genetic attributes with special reference to their clonality. RESULTS: All the V. cholerae O1 biotype El Tor isolates isolated during 2007-2013 were susceptible to fluoroquinolones and tetracycline, the drugs currently used in the treatment of cholera cases in Nigeria. Emergence of CT genotype 7 (Haitian type of ctxB allele) was predominantly seen among Ogawa serotype and the CT genotype 1 (classical ctxB allele) was mostly found in Inaba serotype. Overall, V. cholerae O1 from clinical and water samples were found to be closely related as determined by the pulsed-field gel electrophoresis. V. cholerae isolates from Abia, Kano and Bauchi were found to be genetically distinct from the other states of Nigeria. CONCLUSION: Fecal contamination of the water sources may be the possible source of the cholera infection. Combined prevalence of Haitian and classical ctxB alleles were detected in Ogawa and Inaba serotypes, respectively. This study further demonstrated that V. cholerae O1 with the ctxB has been emerged similar to the isolates reported in Haiti. Our findings suggest that the use of fluoroquinolones or tetracycline/doxycycline may help in the effective management of acute cholera in the affected Nigerian states. In addition, strengthening the existing surveillance in the hospitals of all the states and supply of clean drinking water may control cholera outbreaks in the future.

Draft Genome Sequence of an Environmental trh+ Vibrio parahaemolyticus K23 Strain Isolated from Kerala, India.

Vibrio parahaemolyticusis the leading cause of seafood-related gastroenteritis. Here, we report the draft genome sequence of atrh(+)strain,V. parahaemolyticusK23, isolated from seafood. The sequence will be useful for comparative analysis between environmental and clinical isolates ofV. parahaemolyticus.

Vibrio cholerae O1 El Tor variant and emergence of Haitian ctxB variant in the strains isolated from South India.

Cholera still continues to be an important cause of human infection, especially in developing countries that lack access to safe drinking water and proper sanitation. In the present study, we report the emergence of new variant form of V. cholerae O1 El Tor biotype with a novel mutation in ctxB in strains isolated from various outbreaks during 2010-2014 in Belgaum situated in north-west Karnataka, India. A total of 14 occurrences of cholera were documented from Belgaum Division of North Karnataka during the 4-year period from 2010 to 2014. All the V. cholerae O1 isolates were subjected to DAMA PCR to detect the three different allelic subtypes of ctxB and PCR-based detection of virulent genes, and subsequently, 14 strains (one strain from each outbreak or sporadic case) were subjected to ctxB gene sequence and pulsed-field gel electrophoresis (PFGE) analysis. A total of 54 V. cholerae O1 strains were obtained of which 21 strains isolated during 2010-2011 had classical ctxB and remaining 33 strains isolated during 2012-2014 belonged to Haitian variant. In the cluster analysis, the PFGE profiles were divided into clades A with and B. Clade A contained eight strains with 94 % similarity and Haitian type of ctxB. Clade B contained six strains and had Haitian type of ctxB except one with classical ctxB. To the best of our knowledge, this is the first report of the Haitian variant of V. cholerae O1 Ogawa causing outbreaks and sporadic cases of cholera in South India.

Phenotypic and genetic characterization of Vibrio cholerae O1 isolated from various regions of Kenya between 2007 and 2010.

INTRODUCTION: Cholera, a disease caused by Vibrio cholerae O1 and O139 remains an important public health problem globally. In the last decade, Kenya has experienced a steady increase of cholera cases. In 2009 alone, 11,769 cases were reported to the Ministry of Public Health and Sanitation. This study sought to describe the phenotypic characteristics of the isolated V. cholerae isolates. METHODS: This was a laboratory based cross-sectional study that involved isolates from different cholera outbreaks. Seventy six Vibrio cholerae O1 strains from different geographical areas were used to represent 2007 to 2010 cholera epidemics in Kenya, and were characterized by serotyping, biotyping, polymerase chain r(PCR), pulsed-field gel electrophoresis (PFGE) and ribotyping along with antimicrobial susceptibility testing. RESULTS: Seventy six Vibrio cholerae O1 strains from different geographical areas were used to represent 2007 to 2010 cholera epidemics in Kenya. Serotype Inaba was dominant (88.2%) compared to Ogawa. The isolates showed varying levels of antibiotic resistance ranging from 100% susceptible to tetracycline, doxycycline, ofloxacin, azithromycin, norfloxacin and ceftriaxone to 100% resistant to furazolidone, trimethoprim-sulfamethoxazole, polymyxin-B and streptomycin. The isolates were positive for ctxA, tcpA (El Tor), rtxC genes and were biotype El Tor variant harboring classical ctxB gene. All the isolates were classified as cholera toxin (CT) genotype 1 as they had mutation in the ctxB at positions 39 and 68. All the isolates had genetically similar NotI PFGE and BglI ribotype patterns. The absence of any observed variation is consistent with a clonal origin for all of the isolates. CONCLUSION: Kenya experienced cholera numerous outbreak from 2007-2010. The clinical Vibrio cholerae O1 isolates from the recent cholera epidemic were serotypes Inaba and Ogawa, Inaba being the predominant serotype. The Vibrio cholerae O1 strains were biotype El Tor variants that produce cholera toxin B (ctx B) of the classical type and were positive for ctxA, tcpA El Tor and rtxC genes.

Transferable plasmid-mediated quinolone resistance in association with extended-spectrum ß-lactamases and fluoroquinolone-acetylating aminoglycoside-6'-N-acetyltransferase in clinical isolates of Vibrio fluvialis.

Vibrio fluvialis, which causes cholera-like diarrhoea in humans, is one of the aetiological agents of acute diarrhoea in Kolkata, India, and is resistant to many antimicrobial agents. Two V. fluvialis isolates resistant to fluoroquinolones and ß-lactam antimicrobials were found to have mutations in the quinolone resistance-determining regions (QRDRs) of GyrA at position 83 and of ParC at position 85 as well as carrying a 150 kb plasmid harbouring the quinolone resistance gene qnrA1, the ciprofloxacin-modifying enzyme-encoding gene aac(6')-Ib-cr and genes encoding for extended-spectrum ß-lactamases such as bla(SHV) and bla(CTX-M-3). When this large plasmid was transferred to Escherichia coli by conjugation, the transconjugants showed a 10-75-fold increase in the minimum inhibitory concentrations of ciprofloxacin and norfloxacin. The qnrA1 gene was identified in a complex sul1-type integron in a plasmid of the transconjugants. Southern hybridisation and sequence analysis of qnrA1 and its flanking regions confirmed the presence of aac(6')-Ib-cr and bla(CTX-M-3) but these were not associated with the sul1-type integron. Pulsed-field gel electrophoresis (PFGE) revealed that the two V. fluvialis isolates belonged to different clones. Although the presence of many qnr alleles has been reported amongst enteric bacteria in Asian countries, this is the first report on the emergence of qnrA1 in India. qnrA1 along with aac(6')-Ib-cr and bla(CTX-M-3) genes on a mobile plasmid may spread to other bacterial species that are under the selective pressure of fluoroquinolones and ß-lactam antimicrobials in this region.

Molecular characterization of multidrug-resistant Shigella species isolated from epidemic and endemic cases of shigellosis in India.

Shigella species represent one of the growing numbers of antimicrobial-resistant bacteria in developing countries. Fluoroquinolone-resistant strains of Shigella dysenteriae type 1 and Shigella flexneri type 2a emerged in India during 2002 and 2003, respectively. Sixty strains of Shigella from different parts of India were analysed for antimicrobial susceptibility, the presence of the qnr plasmid, mutations in the quinolone resistance determining regions (QRDRs), fluoroquinolone accumulation, and the presence of other genes encoding resistance to various antimicrobials. Fluoroquinolone-resistant strains had mutations in gyrA and parC genes and had an active efflux system. They were also resistant to several other antimicrobials but were susceptible to azithromycin and ceftriaxone. The majority of the strains harboured genes encoding resistance to ampicillin (97 %), tetracycline (95 %), streptomycin (95 %) and chloramphenicol (94 %). PFGE analysis revealed clonality among strains of S. dysenteriae types 1 and 5, S. flexneri type 2a and Shigella boydii type 12.

Phenotypic and molecular characteristics of Escherichia coli isolated from aquatic environment of Bangladesh.

Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype-based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid pro-filing showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.

Clonal multidrug-resistant Shigella dysenteriae type 1 strains associated with epidemic and sporadic dysenteries in eastern India.

Multidrug-resistant strains of Shigella dysenteriae type 1 were implicated in three outbreaks and sporadic cases of dysentery in eastern India in 2002 and 2003. After a hiatus of 14 years, this pathogen reemerged with an altered antibiotic resistance pattern. In addition to ampicillin, co-trimoxazole, tetracycline, chloramphenicol, and nalidixic acid, all the recent strains were resistant to norfloxacin, lomefloxacin, pefloxacin, and ofloxacin and showed reduced susceptibility to ciprofloxacin. Pulsed-field gel electrophoresis identified a new clone of S. dysenteriae type 1 that was associated with the recent outbreaks and sporadic cases. Based on the spatial and temporal spread of multidrug-resistant S. dysenteriae type 1, we predict that this clonal type may spread further in this region.