Acid fast bacillus smear, mycobacterial culture and Xpert MTB/RIF assay for the diagnosis of tuberculous peritonitis in patients with and without end stage renal failure.

Introduction: Diagnosis of tuberculous peritonitis (TBP) requires a high index of suspicion. Hypothesis /gap statement: Information about the diagnostic features of TBP among patients with end-stage renal failure (ESRF) from India is limited. Aim: To assess the utility of the Gene Xpert MTB/RIF assay in the diagnosis of TBP in patients with end-stage renal failure (ESRF), compared with those without ESRF. Methodology: This prospective observational single centre cohort study was performed at a tertiary care centre in Northern India. Ascitic fluid and/or whole continuous ambulatory peritoneal dialysis (CAPD) bag with effluent from 300 clinically suspected cases of TBP were included in the study. Diagnosis was based on detection of Mycobacteria on smear, Xpert MTB/RIF assay and/or culture. Cell counting was done in a Neubauer chamber. Cell predominance was seen by Giemsa stain. Line probe assay (LPA) for drug susceptibility testing was performed on all positive cultures. Results: TBP was diagnosed in 168 cases. Diabetes mellitus was a significant risk factor for developing TBP in patients with ESRF (P value<0.01). Lymphocytic predominance was seen in 21 patients without ESRF (P value 0.033) while majority of the patients in both groups had neutrophils in their ascitic and peritoneal fluids (138/168; P value 0.033). We recovered 15 cases of laboratory diagnosed TBP (11 without ESRF and four with ESRF). Microscopy was positive in two cases while ten isolates were recovered on culture. The Xpert MTB/RIF assay was positive in seven ascitic fluid samples out of which three were rifampicin resistant. All these were patients without renal failure (P value 0.010). Eight culture positive samples tested by the line probe assay did not detect any resistance to either rifampicin or isoniazid. Conclusion: The GeneXpert MTB/RIF assay has a limited value in the diagnosis of TBP in patients with ESRF.

Emerging frontiers of antibiotics use and their impacts on the human gut microbiome.

Antibiotics, the primary drugs used to cure bacterial diseases, are increasingly becoming ineffective due to the emergence of multiple drug resistance (MDR) leading to recurrence of previously sensitive pathogens. Human gut microbiome (GM), known to play an important role in various physiological processes, consists of pool of diverse microbes. Indiscriminate use of antibiotics during the life span of an individual may lead to development of resistant microbes e.g. Vibrio, Acinetobacter, Escherichia, Klebsiella, Clostridia, etc. in the human GM. Transmission of antibiotic resistant genes (ARGs) between pathogenic and commensal bacteria occurs more frequently in microbiome communities wherein bacteria communicate and exchange cellular constituents both among themselves and with the host. Additionally, co-factors like 'early vs. late' exposure, type of antibiotics and duration of treatment modulate the adverse effects of antibiotics on GM maturation. Furthermore, factors like mode of birth, ethnicity, malnutrition, demography, diet, lifestyle, etc., which influence GM composition, can also indirectly alter the host response to antibiotics. Currently, advanced 'omics' and culturomics approaches are revealing novel avenues to study the interplay between antibiotics and the microbiome and to identify resistant genes in these bacterial communities. Here, we discuss the recent developments that have given insights into the effects of antibiotics on the homeostatic balance of the gut microbiome and thus on human health.

Factors impacting behavioural intentions to adopt the electronic marketplace: findings from small businesses in India.

The pandemic has accelerated e-commerce adoption for both consumers and sellers. This study aims to identify factors critical to the adoption of electronic markets (EM) during the pandemic, from the perspective of small sellers in non-metro cities. The research design utilizes core dimensions of the UTAUT model and selected constructs from protection motivation theory; since business closure vulnerability also triggers electronic market adoption. A questionnaire survey method was used to collect data from 150 sellers from tier-II/III cities of India. Study results identified performance expectancy, effort expectancy, social influence and perceived vulnerability as significant determinants of behavioural intention towards adoption of EM. The findings also explain the moderating impact of sellers' awareness of information technology and merchants' age on behavioural outcomes. Given the growing demands from such cities, the research offers insights for marketers to understand the bottlenecks and ways to motivate small sellers to get associated with EMs.

Evaluating the efficacy of stool sample on Xpert MTB/RIF Ultra and its comparison with other sample types by meta-analysis for TB diagnostics.

Precise and timely detection of tuberculosis (TB) is crucial to reduce transmission. This study aims to assess the accuracy of Xpert MTB/RIF Ultra on stool samples and systematically review the performance of Xpert MTB/RIF Ultra with different sample types by meta-analysis. Stool samples of smear-negative pulmonary TB (PTB), cervical lymph node TB, and abdominal TB patients were tested on the Xpert MTB/RIF Ultra system. Meta-analysis was performed on a set of 44 studies. Data were grouped by sample type, and the pooled sensitivity and specificity of Xpert MTB/RIF Ultra were calculated. The sensitivity of Xpert MTB/RIF Ultra with stool samples was 100% for smear-negative PTB, 27.27% for cervical lymph node TB, and 50% for abdominal TB patients, with 100% specificity for all included TB groups. The summary estimate for all PTB samples showed 84.2% sensitivity and 94.5% specificity, and EPTB samples showed 88.6% sensitivity and 96.4% specificity. Among all sample types included in our meta-analysis, urine showed the best performance for EPTB diagnosis. This pilot study supports the use of stool as an alternative non-invasive sample on Xpert MTB/RIF Ultra for rapid testing, suitable for both PTB and EPTB diagnosis.

A review emphasizing on utility of heptad repeat sequence as a tool to design pharmacologically safe peptide-based antibiotics.

Extensive usage of antibiotics has created an unprecedented scenario of the rapid emergence of many drug-resistant bacteria, which has become an alarming public health concern around the globe. Search for better alternatives that are as efficacious as antibiotics led to the discovery of antimicrobial peptides (AMPs). These small cationic amphiphilic peptides have emerged as a promising option as antimicrobial agents, owing to their multifaceted implications against varied pathogens. Recent years have witnessed tremendous growth in research on AMPs resulting in them being tested in clinical trials of which six got approved for topical application. The relatively less successful outcome has been attributed to the poor cell selectivity shown by most of the naturally occurring AMPs. This drawback needs to be circumvented by identifying strategies to design safe and effective peptides. In the present review, we have emphasized the importance of heptad repeat sequence (leucine and/or phenylalanine zipper motif) as a tool that has shown great promise in remodeling the toxic AMPs to safe antimicrobial agents.

Assessment of burden of drug-resistant tuberculosis at a tertiary care centre in northern India: a prospective single centre cohort study.

OBJECTIVES: We aim to define the burden of rifampicin monoresistant tuberculosis (TB) at a tertiary care centre in northern India as well as determine the second-line drug susceptibilities (SL-DST) in a subset of patients. METHODS: A total of 3045 pulmonary (n=1883) and extrapulmonary (n=1162) samples from likely patients with TB were subjected to microscopy, culture and the Xpert MTB/RIF assay from March 2017 to June 2019. SL-DST testing by line probe assay version 2 for fluoroquinolones (FQs) and second-line injectable drugs were performed on 62 samples. RESULTS: Out of 3045 samples processed in our laboratory during the study period, 36.1% (1101/3045) were positive for Mycobacterium tuberculosis complex (MTBC) and 21.6% were rifampicin monoresistant (223/1032). The rate of rifampicin resistance in pulmonary samples was 23.5% (166/706) and in extrapulmonary cases, it was 17.4% (57/326). Out of 62 cases included for second-line testing, 48 were resistant to FQs (77.4%) while 11 were extensively drug resistant. CONCLUSIONS: India urgently needs to arrest an emerging multidrug-resistant TB epidemic with associated resistance to FQs. A robust surveillance system is needed to execute the National Strategic Plan for 2017-2025.

Evaluation of ventilator associated events in critically ill patients with invasive mechanical ventilation: A prospective cohort study at a resource limited setting in Northern India.

PURPOSE: The primary aim of this study was to identify the modifiable risk factors for acquiring ventilator associated events (VAE). Secondary aims were to investigate the intensive care unit (ICU) course and impact of VAE on patient outcome. METHODS: This prospective, observational single center cohort study included 247 patients on mechanical ventilation for 4 calendar days at a 20-bed ICU between January 2018-June 2019. RESULTS: VAE occurred in 59 episodes (rate 11.3 per 1000 ventilator-days). The Ventilator Utilization Ratio (VUR) was 0.57. The median time to onset of VAE was 6 days. Sepsis was the most common reason for initiating patients on invasive mechanical ventilation (IMV). Cumulative fluid balance ≥2 l (Odds Ratio 30.92; 95% CI 9.82-97.37) and greater number of days with vasopressor support (Odds Ratio 1.92; 95% CI 1.57-2.36) within 7 days of initiating IMV were significant risk factors for acquiring VAE (p < 0.001). VAE cases were ventilated for significantly more days (20 vs 14 days, p = 0.001, had longer days of ICU stay (29 vs 18 days; p = 0.002) and higher hospital mortality (p = 0.02). Klebsiella pneumoniae was the most common isolate (N = 28) and 32.1% were colistin resistant. CONCLUSIONS: Prospective intervention studies are needed to determine if targeting these risk factors can lower VAE rates in our setting.

Diagnostic yield of endoscopic ultrasound-guided fine-needle aspiration of tubercular lymphadenitis using combination of cytology and Gene Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) genes.

Diagnostic yield of an automated molecular test, Gene Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF), was evaluated in this study to simultaneously detect the MTB gene and resistance to rifampicin (RIF) on cytology samples acquired via endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNAC) in suspected tubercular lymphadenitis. Microscopy, cytology, Gene Xpert MTB/RIF assay data on Acid-fast bacillus (AFB), and traditional culture of lymph nodes were retrospectively analyzed. Thirty-one patients (median age 33.5 years, inter-quartile range [IQR] 21-66, 18, 58% female) presented with fever (28, 90%), dysphagia (2, 7%), and recurrent subacute intestinal obstruction (1, 3%). Gene Xpert showed higher sensitivity (30, 97%) compared to the other tests: cytology (23, 77%; odds ratio [OR] 8.8, 95% confidence interval [CI] 1.0-76.9; p = 0.05), AFB smears (12, 39%; OR 50, 95% CI 5.9-420.4; p = 0.00001), and conventional culture (4, 13%; OR 188.5, 95% CI 19.7-1796.3; p = 0.0000). We conclude that Gene Xpert MTB/RIF test on EUS-guided FNAC samples is very useful to diagnose tubercular lymphadenitis.

Comparative Genomic Analysis of Mycobacteriaceae Reveals Horizontal Gene Transfer-Mediated Evolution of the CRISPR-Cas System in the Mycobacterium tuberculosis Complex.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are conserved genetic elements in many prokaryotes, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Although knowledge of CRISPR locus variability has been utilized in M. tuberculosis strain genotyping, its evolutionary path in Mycobacteriaceae is not well understood. In this study, we have performed a comparative analysis of 141 mycobacterial genomes and identified the exclusive presence of the CRISPR-Cas type III-A system in M. tuberculosis complex (MTBC). Our global phylogenetic analysis of CRISPR repeats and Cas10 proteins offers evidence of horizontal gene transfer (HGT) of the CRISPR-Cas module in the last common ancestor of MTBC and Mycobacterium canettii from a Streptococcus-like environmental bacterium. Additionally, our results show that the variation of CRISPR-Cas organization in M. tuberculosis lineages, especially in the Beijing sublineage of lineage 2, is due to the transposition of insertion sequence IS6110 The direct repeat (DR) region of the CRISPR-Cas locus acts as a hot spot for IS6110 insertion. We show in M. tuberculosis H37Rv that the repeat at the 5' end of CRISPR1 of the forward strand is an atypical repeat made up partly of IS-terminal inverted repeat and partly CRISPR DR. By tracing an undetectable spacer sequence in the DR region, the two CRISPR loci could theoretically be joined to reconstruct the ancestral single CRISPR-Cas locus organization, as seen in M. canettii This study retracing the evolutionary events of HGT and IS6110-driven genomic deletions helps us to better understand the strain-specific variations in M. tuberculosis lineages.IMPORTANCE Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is M. tuberculosis, the leading cause of bacterial infection worldwide. Recent evidence on the functionality of the CRISPR-Cas system in M. tuberculosis has brought back focus on these conserved genetic elements, present in many prokaryotes. Our study advances understanding of mycobacterial CRISPR-Cas origin and its diversity among the different species. We provide phylogenetic evidence of acquisition of CRISPR-Cas type III-A in the last common ancestor shared between MTBC and M. canettii, by HGT-mediated events. The most likely source of HGT was an environmental Firmicutes bacterium. Genomic mapping of the CRISPR loci showed the IS6110 transposition-driven variations in M. tuberculosis strains. Thus, this study offers insights into events related to the evolution of CRISPR-Cas in M. tuberculosis lineages.

Methylation of two-component response regulator MtrA in mycobacteria negatively modulates its DNA binding and transcriptional activation.

Post-translational modifications such as phosphorylation, nitrosylation, and pupylation modulate multiple cellular processes in Mycobacterium tuberculosis. While protein methylation at lysine and arginine residues is widespread in eukaryotes, to date only two methylated proteins in Mtb have been identified. Here, we report the identification of methylation at lysine and/or arginine residues in nine mycobacterial proteins. Among the proteins identified, we chose MtrA, an essential response regulator of a two-component signaling system, which gets methylated on multiple lysine and arginine residues to examine the functional consequences of methylation. While methylation of K207 confers a marginal decrease in the DNA-binding ability of MtrA, methylation of R122 or K204 significantly reduces the interaction with the DNA. Overexpression of S-adenosyl homocysteine hydrolase (SahH), an enzyme that modulates the levels of S-adenosyl methionine in mycobacteria decreases the extent of MtrA methylation. Most importantly, we show that decreased MtrA methylation results in transcriptional activation of mtrA and sahH promoters. Collectively, we identify novel methylated proteins, expand the list of modifications in mycobacteria by adding arginine methylation, and show that methylation regulates MtrA activity. We propose that protein methylation could be a more prevalent modification in mycobacterial proteins.

Diagnostic performance of non-invasive, stool-based molecular assays in patients with paucibacillary tuberculosis.

Timely diagnosis of paucibacillary tuberculosis (TB) which includes smear-negative pulmonary TB (PTB) and extra-pulmonary TB (EPTB) remains a challenge. This study was performed to assess the diagnostic utility of stool as a specimen of choice for detection of mycobacterial DNA in paucibacillary TB patients in a TB-endemic setting. Stool samples were collected from 246 subjects including 129 TB patients (62 PTB and 67 EPTB) recruited at TB hospital in Delhi, India. Diagnostic efficacy of stool IS6110 PCR (n = 228) was measured, using microbiologically/clinically confirmed TB as the reference standard. The clinical sensitivity of stool PCR was 97.22% (95% confidence interval (CI), 85.47-99.93) for detection of Mycobacterium tuberculosis in stool samples of smear-positive PTB patients and 76.92% (CI, 56.35-91.03) in samples from smear-negative PTB patients. Overall sensitivity of PCR for EPTB was 68.66% (CI, 56.16-79.44), with the highest sensitivity for stool samples from patients with lymph node TB (73.5%), followed by abdominal TB (66.7%) and pleural effusion (56.3%). Stool PCR presented a specificity of 95.12%. The receiver operating characteristic curve also indicated the diagnostic utility of stool PCR in TB detection (AUC: 0.882). The performance characteristic of the molecular assay suggests that stool DNA testing has clinical value in detection of TB.

Comparison of DNA Extraction Methods for Optimal Recovery of Metagenomic DNA from Human and Environmental Samples.

Metagenomics is the study of gene pool of an entire community in a particular niche. This provides valuable information about the functionality of host-microbe interaction in a biological ecosystem. Efficient metagenomic DNA extraction is a critical pre-requisite for a successful sequencing run in a metagenomic study. Although isolation of human stool metagenomic DNA is fairly standardized, the same protocol does not work as efficiently in fecal DNA from other organisms. In this study, we report a comparison of manual and commercial DNA extraction methods for diverse samples such as human stool, fish gut and soil. Fishes are known to have variable microbial diversity based on their food habits, so the study included two different varieties of fishes. A modified protocol for effective isolation of metagenomic DNA from human milk samples is also reported, highlighting critical precautions. Recent studies have emphasized the importance of studying functionality of human milk metagenome to understand its influence on infants' health. While manual method works well with most samples and therefore can be a method of choice for testing new samples, broad-range commercial kit offers advantage of high purity and quality. DNA extraction of different samples would go a long way in unraveling the unexplored association between microbes and host in a biological system.

The Ser/Thr protein kinase PrkC imprints phenotypic memory in Bacillus anthracis spores by phosphorylating the glycolytic enzyme enolase.

Bacillus anthracis is the causative agent of anthrax in humans, bovine, and other animals. B. anthracis pathogenesis requires differentiation of dormant spores into vegetative cells. The spores inherit cellular components as phenotypic memory from the parent cell, and this memory plays a critical role in facilitating the spores' revival. Because metabolism initiates at the beginning of spore germination, here we metabolically reprogrammed B. anthracis cells to understand the role of glycolytic enzymes in this process. We show that increased expression of enolase (Eno) in the sporulating mother cell decreases germination efficiency. Eno is phosphorylated by the conserved Ser/Thr protein kinase PrkC which decreases the catalytic activity of Eno. We found that phosphorylation also regulates Eno expression and localization, thereby controlling the overall spore germination process. Using MS analysis, we identified the sites of phosphorylation in Eno, and substitution(s) of selected phosphorylation sites helped establish the functional correlation between phosphorylation and Eno activity. We propose that PrkC-mediated regulation of Eno may help sporulating B. anthracis cells in adapting to nutrient deprivation. In summary, to the best of our knowledge, our study provides the first evidence that in sporulating B. anthracis, PrkC imprints phenotypic memory that facilitates the germination process.

Tuning the Mycobacterium tuberculosis Alternative Sigma Factor SigF through the Multidomain Regulator Rv1364c and Osmosensory Kinase Protein Kinase D.

Bacterial alternative sigma factors are mostly regulated by a partner-switching mechanism. Regulation of the virulence-associated alternative sigma factor SigF of Mycobacterium tuberculosis has been an area of intrigue, with SigF having more predicted regulators than other sigma factors in this organism. Rv1364c is one such predicted regulator, the mechanism of which is confounded by the presence of both anti-sigma factor and anti-sigma factor antagonist functions in a single polypeptide. Using protein binding and phosphorylation assays, we demonstrate that the anti-sigma factor domain of Rv1364c mediates autophosphorylation of its antagonist domain and binds efficiently to SigF. Furthermore, we identified a direct role for the osmosensor serine/threonine kinase PknD in regulating the SigF-Rv1364c interaction, adding to the current understanding about the intersection of these discrete signaling networks. Phosphorylation of SigF also showed functional implications in its DNA binding ability, which may help in activation of the regulon. In M. tuberculosis, osmotic stress-dependent induction of espA, a SigF target involved in maintaining cell wall integrity, is curtailed upon overexpression of Rv1364c, showing its role as an anti-SigF factor. Overexpression of Rv1364c led to induction of another target, pks6, involved in lipid metabolism. This induction was, however, curtailed in the presence of osmotic stress conditions, suggesting modulation of SigF target gene expression via Rv1364c. These data provide evidence that Rv1364c acts an independent SigF regulator that is sensitive to the osmosensory signal, mediating the cross talk of PknD with the SigF regulon.IMPORTANCEMycobacterium tuberculosis, capable of latently infecting the host and causing aggressive tissue damage during active tuberculosis, is endowed with a complex regulatory capacity built of several sigma factors, protein kinases, and phosphatases. These proteins regulate expression of genes that allow the bacteria to adapt to various host-derived stresses, like nutrient starvation, acidic pH, and hypoxia. The cross talk between these systems is not well understood. SigF is one such regulator of gene expression that helps M. tuberculosis to adapt to stresses and imparts virulence. This work provides evidence for its inhibition by the multidomain regulator Rv1364c and activation by the kinase PknD. The coexistence of negative and positive regulators of SigF in pathogenic bacteria reveals an underlying requirement for tight control of virulence factor expression.

Asymptomatic filariasis and leprosy co-morbidity in a patient with suspected Guillain-Barrè syndrome: the first case report of an incidental finding in a slit-skin smear.

INTRODUCTION: Lymphatic filariasis (LF) and leprosy are both endemic in India. These diseases are on the World Health Organization (WHO) list of neglected tropical diseases (NTDs), as they affect the most marginalized communities in the world, resulting in deformities and functional limitation. We report the first case of asymptomatic filariasis and leprosy co-morbidity in a patient with suspected Guillain-Barré syndrome. CASE PRESENTATION: A 55-year-old male who was a farmer by occupation presented to the Neurology Outpatient Department (OPD) of our institute with complaints of weakness in all four limbs for the last 15 days. After admission, a detailed history revealed that the patient had been taking multi-drug therapy (MDT) for leprosy for the previous 6 months. After symptomatic management of the presenting complaints, the patient was sent to the Department of Microbiology for a consultation and six-site slit-skin sampling. The initial screening of Ziehl-Neelsen (ZN)-stained smears under a 10× objective led to the incidental finding of sheathed structures resembling microfilaria (Mf) on the smear made from ear lobules. In addition, short acid-fast bacilli (AFB) were also observed under the oil-immersion objective. CONCLUSION: We emphasize that a high index of suspicion and thorough screening of smears by a microbiologist is essential in specimens obtained from any body site.

Validation of a method for diosgenin extraction from fenugreek (Trigonella foenum-graecum L.).

BACKGROUND: Diosgenin is a very important plant secondary metabolite and raw material for the drug indus- try. Plant sources rich in diosgenin include yam (Dioscorea spp.) and fenugreek (Trigonella foenum-graecum L.). A method for diosgenin extraction from yam extracts has previously been validated, but its extraction from fenugreek plants still requires validation. In addition, all available methods require time-consuming additional purification steps. The present study was aimed at developing a low cost, less time-consuming single-step method for diosgenin extraction from fenugreek. METHODS: This study represents a method developed for diosgenin extraction from fenugreek plants without any additional/supportive purification methods such as chromatography or thin-layer chroma- tography. Diosgenin yield estimation and purity analysis by HPLC method, along with accuracy and preci- sion analysis, is presented. RESULTS: Five different fenugreek varieties were subjected to a newly developed diosgenin extraction method, and an HPLC chromatogram showed a single peak corresponding to diosgenin. Yield was determined by the standard curve method. Limit of detection (LOD) and limit of quantification (LOQ) for the assay were found to be 0.0312 and 0.102 μg, respectively; tcalculated for slope and other statistical parameters were found to be significant (P value < 0.001) for this method. CONCLUSIONS: We have developed a fast, accurate and low cost method for diosgenin extraction from fenu- greek. Although the authors have studied this method only in fenugreek plants, it could be applied to the extraction of a few other plant secondary metabolites, which will help researchers to save time and effort.

Gut microbiome contributes to impairment of immunity in pulmonary tuberculosis patients by alteration of butyrate and propionate producers.

Tuberculosis (TB) is primarily associated with decline in immune health status. As gut microbiome (GM) is implicated in the regulation of host immunity and metabolism, here we investigate GM alteration in TB patients by 16S rRNA gene and whole-genome shotgun sequencing. The study group constituted of patients with pulmonary TB and their healthy household contacts as controls (HCs). Significant alteration of microbial taxonomic and functional capacity was observed in patients with active TB as compared to the HCs. We observed that Prevotella and Bifidobacterium abundance were associated with HCs, whereas butyrate and propionate-producing bacteria like Faecalibacterium, Roseburia, Eubacterium and Phascolarctobacterium were significantly enriched in TB patients. Functional analysis showed reduced biosynthesis of vitamins and amino acids in favour of enriched metabolism of butyrate and propionate in TB subjects. The TB subjects were also investigated during the course of treatment, to analyse the variation of GM. Although perturbation in microbial composition was still evident after a month's administration of anti-TB drugs, significant changes were observed in metagenome gene pool that pointed towards recovery in functional capacity. Therefore, the findings from this pilot study suggest that microbial dysbiosis may contribute to pathophysiology of TB by enhancing the anti-inflammatory milieu in the host.

Multi-Drug Resistant Surgical Site Infection Due to Mycobacterium abscessus Complex.

M. abscessus complex is notoriously resistant to most antimicrobial agents. The complex is differentiated into 3 subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Skin and soft tissue infections due to this organism can be acquired by direct contact with contaminated material through traumatic injury, surgical wound and environmental exposure or by secondary involvement of skin/soft tissue during disseminated disease. We report a case of Mycobacterium abscessus infection recovered from a post-operative mid-line abdominal wound to illustrate the diagnostic and management difficulties encountered in such patients.

Ser/Thr protein kinase PrkC-mediated regulation of GroEL is critical for biofilm formation in Bacillus anthracis.

PrkC is a conserved Ser/Thr protein kinase encoded in Bacillus anthracis genome. PrkC is shown to be important for B. anthracis pathogenesis, but little is known about its other functions and phosphorylated substrates. Systemic analyses indicate the compelling role of PrkC in phosphorylating multiple substrates, including the essential chaperone GroEL. Through mass spectrometry, we identified that PrkC phosphorylates GroEL on six threonine residues that are distributed in three canonical regions. Phosphorylation facilitates the oligomerization of GroEL to the physiologically active tetradecameric state and increases its affinity toward the co-chaperone GroES. Deletion of prkC in B. anthracis abrogates its ability to form biofilm. Overexpression of native GroEL recovers the biofilm-forming ability of prkC deletion strain. Similar overexpression of GroEL phosphorylation site mutants (Thr to Ala) does not augment biofilm formation. Further analyses indicate the phosphorylation of GroEL in diverse bacterial species. Thus, our results suggest that PrkC regulates biofilm formation by modulating the GroEL activity in a phosphorylation-dependent manner. The study deciphers the molecular signaling events that are important for biofilm formation in B. anthracis.

Exploring the Carbamazepine Interaction with Human Pregnane X Receptor and Effect on ABCC2 Using in Vitro and in Silico Approach.

PURPOSE: Over expression of ATP-binding cassette transporters is considered one of the major reasons for non-responsiveness to antiepileptic drugs. Carbamazepine (CBZ), one of first line antiepileptic drug is known to influence ABCC2 expression but its exact molecular mechanism is unknown. METHODS: We investigated the effect of CBZ on expression of ABCC2 and pregnane X receptor (PXR) in HepG2 cell line and compared with hyperforin (agonist of PXR) and ketoconazole (antagonist of PXR) through realtime PCR and western blot assay. Involvement of PXR was demonstrated through nuclear translocation and RNA interference and related effect of CBZ on ABCC2 through functional activity assay. Molecular docking and dynamic simulation approach was used to understand the interaction of CBZ with PXR. RESULTS: CBZ and hyperforin increased the PXR and ABCC2 expression whereas reversed when present it in combination with ketoconazole. Experiments confirmed CBZ induced ABCC2 expression is PXR dependent. Molecular dynamic (MD) simulation and in vitro experiment indicated possibility of CBZ to be PXR agonist and PXR residue Gln285 to be important for CBZ-PXR interaction. CONCLUSIONS: CBZ alters the functional activity of ABCC2 through PXR, which in turn can interfere with therapy. Mutational analysis of residues revealed the importance of Gln285 in ligand interaction.