Distinct epigenetic signatures of classical and hypervirulent Klebsiella pneumoniae.

Emergence and spread of the hypervirulent pathotype of Klebsiella pneumoniae have significantly increased infection rates in community as well as healthcare settings. There is an increasing interest to identify discriminating features between classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) to facilitate our understanding of the rapid emergence and dissemination of the hypervirulent pathotype. Here, we sought to identify unique epigenetic signatures of hvKp pathotype that differ from its classical counterpart using single-base resolution methylome analysis of native DNA sequencing on the Oxford Nanopore Technologies platform. The overall global adenine methylation in GATC motifs (i.e., Dam methylation motif) and cytosine methylation in CCWGG motifs (i.e., Dcm methylation motif) were significantly higher in hvKp isolates compared to that in cKp isolates, irrespective of their position in chromosomes or putative extra-chromosomal genetic elements. Notably, we observed significant enrichment of hypermethylated GATC and CCWGG motifs in the virulome of hvKp compared to hvKp genes not directly associated with virulence. We also observed increased methylation of GATC and CCWGG motifs in the capsule synthesis locus of hvKp isolates compared to cKp isolates. Furthermore, we identified several differentially methylated genes (DMGs) between the two pathotypes; interestingly, these DMGs include metal ion transporters, multidrug efflux pumps, transcriptional regulators of stress response, and genes associated with biofilm formation. Our results highlight hypermethylation of GATC and CCWGG motifs as unique epigenetic signatures of hvKp isolates.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a more virulent and rapidly evolving hypermucoviscous pathotype of classical K. pneumoniae (cKp). The hypervirulent pathotype is a major public health concern and is associated with high infection rates in community as well as hospital settings. With the recent emergence of multidrug-resistant hvKp, it has become imperative to investigate non-classical mechanisms such as epigenetics in addition to canonical biochemical and genetic mechanisms that delineate and differentiate the hypervirulent pathotype from its classical counterpart. Here, we identify genome-wide differences in adenine and cytosine methylation marks at well-characterized motifs between the two pathotypes. Overall, significantly higher levels of methylation were observed across chromosomal DNA and extrachromosomal elements in hvKp compared to cKp. Among hvKp isolates, the genes associated with virulence are particularly enriched for methylation marks. Our findings shed light on how epigenetic signatures may help distinguish the pathogenic potential of bacteria.

A tough nut to crack? Proficiency in coconut processing of the Nicobar long-tailed macaque Macaca fascicularis umbrosus.

The ability to manipulate objects enables macaques to utilize resources well. Coconut (Cocos nucifera) is one such food that has high energy and nutrient value but requires complex motor skills for extraction. In this study, we examined the proficiency of a single group of Macaca fascicularis umbrosus from Campbell Bay, India, in feeding on different types of coconuts. The group comprised 16 individuals with 3 adult males, 6 adult females, 3 sub-adult males, and 4 infants. We observed individuals feeding on tender, mature, dry, and dry-dehusked coconuts in 90 feeding bouts between August 2013 and December 2015. Their feeding behaviour was divided into four types of behavioural acts: pluck, process, drink, and feed. We prepared an ethogram consisting of 32 behavioural acts associated with feeding on coconuts, and recorded the occurrence of these acts during focal animal sampling. We used NMDS to examine differences among age/sex classes in the occurrence of behavioural acts associated with feeding on coconuts. Although females and males showed no significant separation in these behaviours, adults and young individuals formed distinct groups. However, only 3 out of 32 acts were exclusive to age classes. Using Discrete Time Markov Chain analysis, we found that adults tended to transition back to processing behaviours after drinking and feeding behaviours more often than young individuals. An almost similar repertoire of behaviours among age and sex classes indicates that there is no single model behaviour. Further, this study indicates that among different age/sex classes, adults tend to maximise the nutritional gains during single feeding bouts than younger individuals.

Paper Sensor Modified with MoS2 for Detection of Dopamine Using a Machine-Intelligent Web App Interface.

The sensing behavior of a MoS2-functionalized paper sensor towards dopamine was explored through a combinatorial approach of theoretical analysis, subsequent experimental validation, and machine-learning-driven predictive modeling of the measured electrochemical outputs. The suitability of the chosen 2D material for efficient detection of dopamine was confirmed using density functional theory. The physisorption behavior along with electrostatic interaction due to the incorporation of dopamine on MoS2 was unraveled under the purview of theoretically estimated noncovalent interaction and charge density difference plot. The theoretical Löwdin population analysis elucidates the alteration in oxidation potential of dopamine, as observed in electrochemical experiments. The electrochemical responses of the developed sensor with the spiked serum samples showed an average accuracy of more than 96% with a limit of detection of 10 nM. Furthermore, implementation of a machine-intelligent interactive web app interface improved the resolution of the sensing platform significantly with an enhanced accuracy of nearly 99%.

SERS Nanowire Chip and Machine Learning-Enabled Classification of Wild-Type and Antibiotic-Resistant Bacteria at Species and Strain Levels.

Antimicrobial resistance (AMR) is a major health threat worldwide and the culture-based bacterial detection methods are slow. Surface-enhanced Raman spectroscopy (SERS) can be used to identify target analytes in real time with sensitivity down to the single-molecule level, providing a promising solution for the culture-free bacterial detection. We report the fabrication of SERS substrates having tightly packed silver (Ag) nanoparticles loaded onto long silicon nanowires (Si NWs) grown by the metal-assisted chemical etching (MACE) method for the detection of bacteria. The optimized SERS chips exhibited sensitivity down to 10-12 M concentration of R6G molecules and detected reproducible Raman spectra of bacteria down to a concentration of 100 colony forming units (CFU)/mL, which is a thousand times lower than the clinical threshold of bacterial infections like UTI (105 CFU/mL). A Siamese neural network model was used to classify SERS spectra from bacteria specimens. The trained model identified 12 different bacterial species, including those which are causative agents for tuberculosis and urinary tract infection (UTI). Next, the SERS chips and another Siamese neural network model were used to differentiate AMR strains from susceptible strains of Escherichia coli (E. coli). The enhancement offered by SERS chip-enabled acquisitions of Raman spectra of bacteria directly in the synthetic urine by spiking the sample with only 103 CFU/mL E. coli. Thus, the present study lays the ground for the identification and quantification of bacteria on SERS chips, thereby offering a potential future use for rapid, reproducible, label-free, and low limit detection of clinical pathogens.

Long-term treatment outcome and mutational analysis of patients on third-line antiretroviral therapy in programmatic conditions.

BACKGROUND: In low- and middle-income countries where most patients receive standardized third-line ART through national programmes, real-world data are scarce. This study was done to assess the long-term survival, and virological and mutational outcomes of people living with HIV receiving third-line ART between July 2016 and December 2019 in an ART centre in India. METHODS: Eighty-five patients were started on third-line ART. Genotypic resistance testing to identify drug resistance mutations in the integrase, reverse transcriptase and protease genes was done at the start of third-line therapy, as well as in those who did not attain virological suppression after 12 months of therapy. RESULTS: Survival was 85% (72/85) at 12 months and 72% (61/85) at the end of follow-up in March 2022. Virological suppression was present in 82% (59/72) and 88% (59/67) at 12 months and at the end of follow-up, respectively. Five out of 13 patients who had virological failure at 12 months showed virological suppression at the end of the study. At the start of third-line therapy, 35% (14/40) and 45% (17/38) of patients had major integrase- and protease-associated mutations, respectively, even though they had never been on integrase inhibitor-based regimens. At 1 year follow-up, among those failing third-line therapy, 33% (4/12) of patients had major integrase mutations, but none had major protease mutations. CONCLUSIONS: This study demonstrates good long-term outcome in patients on standardized third-line ART in programmatic conditions with very few mutations in those failing the therapy.

Machine learning based urinary pH sensing using polyaniline deposited paper device and integration of smart web app interface: Theory to application.

The present study employs density functional theory-based first principle calculation to investigate the electron transport properties of polyaniline following exposure to acidic and alkaline pH. In-situ deposited polyaniline-based paper device maintains emeraldine salt form while it is exposed to acidic pH and converts to emeraldine base when it is subjected to alkaline pH solutions. These structural changes at acidic and alkaline pH are validated experimentally by Raman spectra. Furthermore, the Raman spectra computed from density functional theory are validated with the experimental spectra. The changes in the theoretical energy band gap of polyaniline obtained from first principle calculations were correlated with the changes in the experimental impedimetric response of the sensor after exposure to acidic and alkaline solutions. Finally, the impedimetric responses were used to predict urine pH through a machine learning based smart and interactive web application. Different machine learning based regression models were implemented to acquire the best possible outcome. Gradient Boosting Regressor with least square loss model was selected as it showed lowest mean square, mean absolute, and root mean square error than other models. The smart sensing platform successfully predicts the unknown pH of urine samples with an average accuracy of more than 98%. The locally deployed smart web app can be accessed within a local area network by the end-user, which holds promise towards effective detection of urinary pH.

Immunological profiling and development of a sensing device for detection of IL-13 in COPD and asthma.

Chronic obstructive pulmonary disease (COPD) and asthma are the two most common obstructive lung diseases which affects millions worldwide and impose an enormous burden on global healthcare. The overlapping features shared by these two diseases often make differential diagnosis difficult to achieve, leading to misdiagnosis of these patients. Both asthma and COPD are associated with chronic inflammation of the airways which is perpetuated by the interplay between immunological mediators. The crucial role played by these mediators make them attractive targets for disease diagnosis. The present study investigates the immunological mediator profile in these patients as compared with controls. Further, a potential biomarker for the development of a sensing platform is identified. Multiplexed analysis of 8 commonly studied immunological markers (IL-4, IL-5, IL-6, IL-13, TGF-ß, IFN-γ, MCP-1 and NGAL) in serum showed distinct dysregulation pattern, with IL-13 showing the highest potential for differential diagnosis. An impedimetric self-assembled monolayer (SAM) based sensor for detecting IL-13 is developed to distinguish between asthma and COPD. The device shows reliable output with high accuracy and sensitivity towards the detection of IL-13.

Blowing the lid off! Bottle-directed, extractive foraging strategies in synurbic bonnet macaques Macaca radiata in southern India.

Nonhuman individuals and groups, living in anthropogenic landscapes, often adopt adaptive foraging strategies, mediated by their day-to-day interactions with humans and their artefacts. Exploring such novel behavioral manifestations, especially in the Anthropocene, offers us insights into behavioral innovations and their transmission in such rapidly changing ecologies. In this study, employing field experiments, we investigated an example of human-induced, extractive foraging behavior - the extraction of liquid contents from plastic bottles - in a synurbic bonnet macaque Macaca radiata population. The main aims of the study were to examine the distribution, diversity, inter-individual variability and intra-individual flexibility of bottle-directed manipulative behaviors, and to explore the social and environmental factors driving this behavioral practice. We video-recorded the manipulation of partially filled plastic bottles and the extraction of liquid across four groups of bonnet macaques in southern India. Two socio-demographic factors - age class and group membership - and one environmental factor - food provisioning - were identified as major determinants of inter-individual variation in the performance of sophisticated manipulative techniques and in bottle-opening success. Our results also suggest that age-related physical maturation, experiential trial-and-error learning, and possibly social learning contributed to the acquisition of foraging competence in this task. These findings illuminate the mechanisms underlying inter-individual behavioral variability and intra-individual behavioral flexibility amongst free-ranging individuals of a cercopithecine primate species, traditionally known for its ecological adaptability and behavioral plasticity. Finally, this study documents how the presence of humans, their artefacts and their activities facilitate the development of certain behavioral traditions in free-ranging nonhuman populations, thus providing valuable insights into how human-alloprimate relations can be restructured within the increasingly resource-competitive environments of the Anthropocene.

A Novel Bioimpedance-Based Detection of Miltefosine Susceptibility Among Clinical Leishmania donovani Isolates of the Indian Subcontinent Exhibiting Resistance to Multiple Drugs.

The extent of susceptibility towards miltefosine (Mil), amphotericin B (AmpB), and paromomycin (Paro) was measured among 19 clinical isolates of Leishmania donovani (LD). Thirteen of these clinical isolates were reported to exhibit low susceptibility towards sodium stibogluconate (SSG-R), while six of them were highly susceptible (SSG-S). The degree of clearance of amastigotes (EC50) for these predefined SSG-R- and SSG-S-infected macrophages was determined against Mil, AmpB, and Paro. Two out of the 13 SSG-R isolates (BHU575 and BHU814) showed low susceptibility towards all three drugs studied, while the rest of the 11 SSG-R isolates showed varying degrees of susceptibility either towards none or only towards individual drugs. Interestingly, all the SSG-S isolates showed high susceptibility towards Mil/AmpB/Paro. The total intracellular non-protein thiol content of the LD promastigotes, which have been previously reported to be positively co-related with EC50 towards SSG, was found to be independent from the degree of susceptibility towards Mil/AmpB/Paro. Impedance spectra analysis, which quantifies membrane resistance, revealed lower impedimetric values for all those isolates exhibiting low efficacy to Mil (Mil-R). Our analysis points out that while non-protein thiol content can be an attribute of SSG-R, lower impedimetric values can be linked with lower Mil susceptibility, although neither of these parameters seems to get influenced by the degree of susceptibility towards AmpB/Paro. Finally, a correlation analysis with established biological methods suggests that impedance spectral analysis can be used for the accurate determination of lower Mil susceptibility among LD isolates, which is further validated in the LD-infected in vivo hamster model.

Structure-based functional fitness analyses of carbapenemase variants identified among pathogenic carbapenem-resistant Gram-negative bacteria.

Carbapenemase-mediated carbapenem resistance is a major public health concerns worldwide. In the present study, prevalence of circulating carbapenemases was estimated among carbapenem-resistant clinical isolates using PCR and sequencing. Diameters of zone of inhibition (ZDs) were compared for imipenem, meropenem and ertapenem among single carbapenemase producing isolates. Structure-based functional fitness of those carbapenemases was predicted through several in silico analyses. Approximately, 63.76% isolates demonstrated carbapenem resistance, of which 39.13% harboured carbapenemases like blaNDM-1 (33.23%), blaNDM-1-like (0.31%), blaVIM-2 (4.35%), blaKPC-2 (4.04%), blaOXA-181 (6.85%), blaOXA-23 (16.50%), blaOXA-69 (3.88%), blaOXA-66 (2.91%) and blaOXA-104 (1.94%). Omega values indicated selection pressure over blaOXA-69, blaOXA-66 and blaOXA-104. Protein structural dynamics predicted NDM-1 and KPC-2 to have the highest and least flexibility, indicating differences in ß-lactam binding and catalytic efficiency. Increased requirement of free folding energy, improved solvent accessibility and decreased melting temperatures among NDM-1-like, OXA-181, OXA-66, OXA-69 and OXA-104 predicted functional improvement over their ancestral variants. NDM-1-like carbapenemases demonstrated improvement in binding stability, affinity and catalysis of meropenem than that of NDM-1. Catalytic activity of imipenem was predicted to improve among OXA-181, which could be correlated with more than 1.5 folds smaller ZDs around imipenem disc, than that of meropenem/ertapenem, among OXA-181 producing isolates. However, OXA-66 indicated greater binding stability and affinity for imipenem and meropenem. This study indicated structural/functional convergence as well as divergence among several carbapenemase variants and provided useful insights into carbapenemase-mediated carbapenem resistance that might help in identifying appropriate treatment regimen for bacterial infections.

Demonstration of bactericidal and synergistic activity of quercetin with meropenem among pathogenic carbapenem resistant Escherichia coli and Klebsiella pneumoniae.

Rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE) is considered to be a global issue. Quercetin is a well known antimicrobial agent. Hence, it would be important to investigate bactericidal and synergistic interaction of quercetin with meropenem and elucidate effects of quercetin alone and quercetin-meropenem combination on blaNDM, blaVIM,acrB, ompC, ompF, ompk35 and ompk36 expressions, cellular morphology and cell-wall/membrane integrity. MIC, Time-kill and Baclight assays were performed to determine antibacterial/bactericidal activity of quercetin. Synergism with meropenem was evaluated by checkerboard assay followed by dose-response, isobologram analysis and FIC index, combination index calculation. Effects of meropenem, quercetin and their combinations on blaNDM, blaVIM,acrB, ompC, ompF, ompk35 and ompk36 expressions were evaluated by qRT-PCR. SEM was performed to evaluate effects of aforesaid combinations on cellular morphology. Quercetin alone exhibited at least four-fold reduced MIC value (16-256 µg/mL) than that of meropenem against CRE. It exhibited synergism with meropenem against 89.25% CRE. Again, only 128 µg/mL quercetin killed upto 99.95% bacteria within 4-6 h of dosing, which increased further to 99.99% in MIC combination of meropenem-quercetin. Thus, effective bactericidal activity of quercetin-meropenem combination might have been achieved through alteration of blaVIM, ompC expression and cellular morphology of bacteria. Quercetin exhibited bactericidal and synergistic activity with meropenem.

Do males bond? A study of male-male relationships in Nicobar long-tailed macaques Macaca fascicularis umbrosus.

In primates, males compete for a mate, which is a non-sharable resource. This makes the conditions less conducive for males to have stable relationships. One such special kind of relationship is a bond where the interactions are reciprocated, equitable and differentiated. Bonds in macaque societies are based on the degree of within-group contest competition for mates which is dependent on the synchronization of female fertile phase and reliability of fertility signals. Species of the Fascicularis group, including Nicobar subspecies, show intermediate reliability in the signals with mild peaks, and studies have shown reciprocity but no differentiation. We conducted a study on a group of wild Nicobar long-tailed macaques Macaca fascicularis umbrosus to understand the existing patterns of male-male relationships. We examined whether there is reciprocity in affiliation among the individuals and whether the rate of affiliation is balanced. We also measured the dominance linearity and steepness in the group to understand the monopolizability of females. We used social network analysis to understand whether the relations are differentiated based on hierarchical position and whether the high-ranking individuals are the most central individuals in the distribution of grooming in the group. We found that there is reciprocity among the males although that is not equitable. There was no rankrelated differentiation of affiliation among the males of the group. Instead, the identities of individuals influenced affiliation patterns. Our results correspond to the existent strong relationships but lack of social bond otherwise found in the Fascicularis group of macaques.

Does rank rule? Rank-related grooming patterns in Nicobar long-tailed macaques Macaca fascicularis umbrosus.

Primates maintain social bonds with specific individuals in the group by directing grooming toward them. Social grooming is often targeted toward individuals with whom the most benefits can be exchanged, which are usually the high-ranking individuals. We used the Seyfarth model to investigate whether dominance rank alters the distribution of grooming in a group of Macaca fascicularis umbrosus at Great Nicobar Island. We investigated whether dominance rank predicted grooming rate in both males and females. We used social network analysis to examine whether high-ranking animals maintain a central position in the grooming network. We also investigated whether adult individuals exchange grooming for social tolerance, as indicated by physical proximity. We found that more social grooming was directed toward high-ranking females, but not high-ranking males. Social network analysis suggested that high-ranking animals are not central individuals in the distribution of grooming; rather, middle-ranking animals were major contributors to the distribution of grooming. There was no relation between the grooming rate and proximity in both males and females. Overall, our findings suggest that females prefer to direct grooming toward high-ranking partners, although the dominance hierarchy is less steep than expected for Macaca fascicularis. Thus rank-related grooming patterns may be a relatively conserved trait in this isolated island population.

Processing two environmental chemical signals with a synthetic genetic IMPLY gate, a 2-input-2-output integrated logic circuit, and a process pipeline to optimize its systems chemistry in Escherichia coli.

Synthetic genetic devices can perform molecular computation in living bacteria, which may sense more than one environmental chemical signal, perform complex signal processing in a human-designed way, and respond in a logical manner. IMPLY is one of the four fundamental logic functions and unlike others, it is an "IF-THEN" constraint-based logic. By adopting physical hierarchy of electronics in the realm of in-cell systems chemistry, a full-spectrum transcriptional cascaded synthetic genetic IMPLY gate, which senses and integrates two environmental chemical signals, is designed, fabricated, and optimized in a single Escherichia coli cell. This IMPLY gate is successfully integrated into a 2-input-2-output integrated logic circuit and showed higher signal-decoding efficiency. Further, we showed simple application of those devices by integrating them with an inherent cellular process, where we controlled the cell morphology and color in a logical manner. To fabricate and optimize the genetic devices, a new process pipeline named NETWORK Brick is developed. This pipeline allows fast parallel kinetic optimization and reduction in the unwanted kinetic influence of one DNA module over another. A mathematical model is developed and it shows that response of the genetic devices are digital-like and are mathematically predictable. This single-cell IMPLY gate provides the fundamental constraint-based logic and completes the in-cell molecular logic processing toolbox. The work has significance in the smart biosensor, artificial in-cell molecular computation, synthetic biology, and microbiorobotics.

Toxicological and behavioral study of two potential antibacterial agents:4-chloromercuribenzoic acid and quercetin on Swiss-albino mice.

Global dissemination of carbapenem resistant-Gram negative bacteria (CR-GNB) is supposed to be clinically alarming because it extremely delimits the treatment options against serious infections. 4-Chloromercuribenzoic acid (pCMB) is an efficient metallo-enzyme inhibitor, and quercetin is known for antioxidant, antiviral, anticancer, antimicrobial, and anti-inflammatory activities. These two compounds could be considered as potential candidates for the treatment of CR-GNB mediated infections. Hence, in this study, antibacterial activity of pCMB and quercetin was evaluated against CR-GNB through minimum inhibitory concentration (MIC) determination. Toxicity of pCMB and quercetin was evaluated by LC50 calculation through brine shrimp test (BST) and by investigating hematological, serum biochemical, and histopathological parameters in Swiss-albino mice. Moreover, aggressive-depressive-cognitive behavioral effects of pCMB and quercetin on murine model were evaluated. All the carbapenem resistant isolates (CR-GNB) exhibited MIC values in the range of 4-256 µg/ml, 16-256 µg/ml, and 64-1024 µg/ml for pCMB, quercetin, and meropenem, respectively. BST determined LC50 of pCMB and quercetin at 91.57 ± 0.35 mg/L and 448.45 ± 0.46 mg/L, respectively. Oral administration of low dose of pCMB and quercetin did not induce any significant changes in morphological, behavioral, hematological, serum biochemical, and histopathological parameters among Swiss-albino mice. But, a high dose of pCMB and quercetin exhibited slight toxicity. However, no death was reported for any dosage of pCMB and quercetin. Therefore, pCMB and quercetin might be considered for further investigations on alternative therapeutics to combat against CR-GNB.

4-Chloromercuribenzoic acid enhances carbapenem sensitivity among pathogenic Gram negative bacteria by altering blaVIM, adeB and ompC expression.

BACKGROUND: Rapid global dissemination of carbapenem resistant Gram negative bacteria (CRGNB) is supposed to be clinically most alarming. Since, p-chloromercuribenzoic acid (pCMB) is a well known metallo-beta-lactamase inhibitor; evaluation of its bactericidal and carbapenem resistance reversing potential would be important. METHODS: In this study, bactericidal and meropenem resistance reversing potential of pCMB was investigated against CRGNB by MIC determination, checkerboard assay, time-kill assay and cellular viability assay. Effect of pCMB on cellular morphology was visualized by Scanning Electron Microscopy. Further, quantitative Real Time-PCR was performed to evaluate effects of pCMB on clinically relevant metallo-beta-lactamases, major efflux pumps and outer membrane proteins expression. RESULTS: pCMB exhibited at least four fold reduced MIC value (2-256µg/ml) than that of meropenem against CRGNB. Moreover, pCMB exhibited synergism with meropenem against 86.06% of CRGNB. MIC of pCMB (16-32µg/ml) could kill upto 99.96% bacteria within 6-8h of dosing. pCMB exerted bactericidal activity by severely disrupting cell wall integrity. Reversal of carbapenemase property of CRGNB by pCMB might have developed through alteration of blaVIM, acrB, mexB and ompk36 expression. CONCLUSIONS: Hence, the current study identified pCMB as a potential bactericidal agent which enhanced meropenem sensitivity by altering blaVIM, acrB, mexB and ompk36 expression.

Quercetin inhibits carbapenemase and efflux pump activities among carbapenem-resistant Gram-negative bacteria.

Rapid dissemination of carbapenem-resistant Gram-negative bacteria (CRGNB) is a global threat. Quercetin is known for its antimicrobial activity. In this study, carbapenemase and efflux pump inhibitory activities of quercetin were demonstrated against carbapenem-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Further, molecular docking was performed to elucidate molecular mechanisms of such inhibition. CRGNB, expressing one of the carbapenemases, demonstrated significant inhibition of carbapenemase activity when pre-incubated with 64 µg/ml quercetin. Moreover, acrB overexpressing enterobacterial isolates exhibited significant inhibition of efflux activity upon quercetin treatment. Molecular docking studies revealed stability of quercetin-carbapenemase complexes. (i) Virtual superimposition of quercetin onto meropenem, (ii) proximity of quercetin to attacking nucleophile and (iii) involvement of same amino acids that stabilize both meropenem and quercetin - indicated competition between quercetin and meropenem for ligand binding. Although quercetin and PAßN, a standard efflux pump inhibitor, docked at both central cavity and periplasmic drug binding sites of AcrB, they did not virtually superimpose on each other. However, sufficient release of Gibb's free energy and involvement of same set of amino acids in PAßN and quercetin stability predicted quercetin's efflux pump inhibitory potential. Hence, quercetin could be potential adjuvant therapeutics for CRGNB-mediated infections.

Contribution of acrB upregulation & OmpC/Ompk36 loss over the presence of blaNDM towards carbapenem resistance development among pathogenic Escherichia coli & Klebsiella spp.

Background & objectives: The global spread of carbapenem-resistant Enterobacteriaceae (CRE) is an emerging clinical problem. Hence, in this study, the plausible role of extended-spectrum beta-lactamases (ESBLs)/carbapenemases, OmpC/Ompk36, acrB and their combinations was explored among CRE. Methods: The minimum inhibitory concentration (MIC) of meropenem, enzyme-phenotypes (ESBLs/IR and metallo-beta-lactamase (MBL)/non-MBL carbapenemase), genotypes (blaTEM, blaSHV and blaCTX-M; blaNDM and blaVIM; blaKPC and blaOXA-48-like variants), acrB and outer membrane protein (OMP) expressions were analyzed with a total of 101 non-duplicate clinical isolates, obtained from various samples of patients visiting two tertiary care units of Eastern India during May 2013 - October 2016. This included Escherichia coli (n=36) and Klebsiella pneumoniae (n=65), categorized into two groups, namely Group I (resistant to all carbapenems; n=93; E. coli=34 and Klebsiella spp.=59) and Group II (non-resistant to all the carbapenems; n=8; E. coli=2 and Klebsiella spp.=6). Results: Though 88.17 per cent of Group I isolates exhibited ESBL property, the presence of carbapenemase activity (70.96%) and that of blaNDM gene (42/66: 63.63%) indicated their contributions towards the emergence of CRE. Further, porin loss and/or efflux pump activation among ESBL/carbapenemase-producing isolates heightened the MIC of meropenem from 64 to 256 mg/l (range exhibited by only ESBL/carbapenemase-producing isolates) to >256 mg/l. Interpretation & conclusions: These findings implied the major contribution of porin loss and/or efflux pump activation over the presence of ESBLs/carbapenemases in imparting carbapenem resistance in pathogenic bacteria.

Prevalence of gastrointestinal parasites in bonnet macaque and possible consequences of their unmanaged relocations.

Relocation is one of the mitigating measures taken by either local people or related officers to reduce the human-bonnet macaque Macaca radiata conflict in India. The review on relocations of primates in India indicates that monkeys are unscreened for diseases or gastrointestinal parasites (henceforth endoparasites) before relocation. We collected 161 spatial samples from 20 groups of bonnet macaque across their distribution range in south India and 205 temporal samples from a group in Chiksuli in the central Western Ghats. The isolation of endoparasite eggs/cysts from the fecal samples was by the centrifugation flotation and sedimentation method. All the sampled groups, except one, had an infection of at least one endoparasite taxa, and a total of 21 endoparasite taxon were recorded. The number of helminth taxon (16) were more than protozoan (5), further, among helminths, nematodes (11) were more common than cestodes (5). Although the prevalence of Ascaris sp. (26.0%), Strongyloides sp. (13.0%), and Coccidia sp. (13.0%) were greater, the load of Entamoeba coli, Giardia sp., Dipylidium caninum and Diphyllobothrium sp. were very high. Distant groups had more similarity in composition of endoparasites taxon than closely located groups. Among all the variables, the degree of provisioning was the topmost determinant factor for the endoparasite taxon richness and their load. Temporal sampling indicates that the endoparasite infection remains continuous throughout the year. Monthly rainfall and average maximum temperature in the month did not influence the endoparasite richness. A total of 17 taxon of helminths and four-taxon of protozoan were recorded. The prevalence of Oesophagostomum sp., and Strongyloides sp., and mean egg load of Spirurids and Trichuris sp. was higher than other endoparasite taxon. The overall endoparasite load and helminth load was higher in immatures than adults, where, adult females had the highest protozoan load in the monsoon. The findings indicate that relocation of commensal bonnet macaque to wild habitat can possible to lead transmission of novel endoparasites that can affect their population. Thus, we suggest avoidance of such relocations, however, if inevitable the captured animals need to be screened and treated for diseases and endoparasites before relocations.