Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Inhibitors against New Delhi metallo-betalactamase-1 (NDM-1) and its variants endemic in Indian settings along with the laboratory functional gain mutant of NDM-1.

PURPOSE: The emergence of NDM-1 producing bacteria has become common in both hospital and community settings, but no inhibitor has yet been available for clinical treatment. Hence, demanding the urgent need of NDM-1 inhibitors, we initiated to screen broad spectrum inhibitors against NDM natural variants and laboratory mutant. METHODS: We used docking and molecular dynamics simulations, in silico pharmacokinetic investigations, and density functional theory calculation to characterize molecules. Furthermore, an in vitro study, including MIC, kinetics, and fluorescence study were carried out to confirm the efficacies of the selected compounds. RESULTS: According to the findings of the computational studies, three compounds were effective against NDM variants. Fourfold reduction in MIC of imipenem and meropenem was observed when combined with inhibitors (D2573, D2148, and D63) against blaNDM-1, blaNDM-4, blaNDM-6, and blaNDM-1Q123A, while twofold reduction in MIC of imipenem and meropenem was observed against blaNDM-5 and blaNDM-7. Similarly in the presence of inhibitors (D2573, D2148, and D63) the efficiency of nitrocefin hydrolysis by NDM-4, NDM-6, and Q123A decreases to much more extent as compared to NDM-5 and NDM-7. These results showed that the efficacy of these broad spectrum inhibitors decreases with increasing resistance of NDM variants. CONCLUSION: This is the first time inhibitors were tested against different NDM natural variants which are endemic in Indian settings. Moreover, a functional gain laboratory mutant was also checked for their efficacies. We may propose these molecules for the pre-clinical trial to further translate.

It's all relative: analyzing microbiome compositions, its significance, pathogenesis and microbiota derived biofilms: Challenges and opportunities for disease intervention.

Concept of microorganisms has largely been perceived from their pathogenic view point. Nevertheless, it is being gradually revisited in terms of its significance to human health and now appears to be the most dominant force that shapes the immune system of the human body and also determines an individual's predisposition to diseases. Human inhabits bacterial diversity (which is predominant among all microbial communities in human body) occupying 0.3% of body mass, known as microbiota. On birth, a part of microbiota that child obtains is essentially a mother's legacy. So, the review was initiated with this critical topic of microbiotal inheritance. Since, each body site has distinct physiological specifications; therefore, they contain discrete microbiome composition that has been separately discussed along with dysbiosis-induced pathologies originating in different body organs. Factors affecting microbiome composition and may cause dysbiosis like antibiotics, delivery, feeding method etc. as well as the strategies that immune system adopts to prevent dysbiosis have been highlighted. We also tried to bring into attention the topic of dysbiosis induced biofilms, that enables cohort to survive stresses, evolve, disseminate and infection resurgence that is still in dormancy. Eventually, we put spotlight on microbiome significance in medical therapeutics. We didn't merely confine article to gut microbiota, that is being studied more extensively. Numerous community forms at diverse body sites are inter-related, and being exposed to awfully variable perturbations appear to be challenging to evaluate perturbation risks holistically. All aspects have been elaborately discussed to achieve a global depiction of human microbiota in order to meet urgent necessity for protocol standardisation. Demonstrates that environmental challenges (antibiotic use, alterations in diet, stress, smoking etc.) might cause dysbiosis i.e. transition of healthy microbiome composition to the one in which pathogenic microorganisms become more abundant, and eventually results in an infected state.

Farm to table: colistin resistance hitchhiking through food.

Colistin is a high priority, last-resort antibiotic recklessly used in livestock and poultry farms. It is used as an antibiotic for treating multi-drug resistant Gram-negative bacterial infections as well as a growth promoter in poultry and animal farms. The sub-therapeutic doses of colistin exert a selection pressure on bacteria leading to the emergence of colistin resistance in the environment. Colistin resistance gene, mcr are mostly plasmid-mediated, amplifying the horizontal gene transfer. Food products such as chicken, meat, pork etc. disseminate colistin resistance to humans through zoonotic transfer. The antimicrobial residues used in livestock and poultry often leaches to soil and water through faeces. This review highlights the recent status of colistin use in food-producing animals, its association with colistin resistance adversely affecting public health. The underlying mechanism of colistin resistance has been explored. The prohibition of over-the-counter colistin sales and as growth promoters for animals and broilers has exhibited effective stewardship of colistin resistance in several countries.

Mechanistic Approach of Effective Combination of Antibiotics Against Clinical Bacterial Strains Having New Delhi Metallo-Beta-Lactamase Variants and Functional Gain Laboratory Mutant.

Antimicrobial resistance has emerged as a serious issue for physicians and health-care workers treating infections that could lead to the next pandemic. One of the key resistance mechanisms is beta-lactamases. Although several beta-lactamase inhibitors in combination with antibiotics have been created and are being utilized in clinical settings, resistance to these formulations has also been evolving in the bacterial population due to their distinct targets. In this study we used effective combination of antibiotic as an approach to inhibit multidrug resistance bacteria. We used four combinations and checked its efficacy against NDM (New Delhi Metallo-beta-lactamase) variants and functional gain laboratory mutant by employing FICI, enzyme kinetics, fluorescence and computational biology approaches (Docking and Molecular Dynamics Simulation). FICI values of all the combinations were either less than 0.5 or equal to 0.5. Binding features acquired by spectroscopic techniques showed important interaction and complex formation between drugs and enzymes with decreased ksv and kq values. In steady-state kinetics, a reduction in hydrolytic efficiency of enzymes was shown by cooperative binding behaviour when they were treated with different drugs. We have also tested functional gain laboratory mutant developed in our lab, keeping in view that if in future upcoming variants of this kind be emerged then these mutants could also be subsided by combinational therapy. This study identifies three other combinations better than fluoroquinolones effective against NDM variants and laboratory mutant.

CRISPR Interference (CRISPRi) Mediated Suppression of OmpR Gene in E. coli: An Alternative Approach to Inhibit Biofilm.

Biofilm plays an important role in the community and hospital-acquired infections. Especially E. coli biofilm that contributes towards the significant part of medical devices associated with microbial infections. OmpR/EnvZ, a two-component system, is one of the regulatory mechanisms involved in transcription regulation in response to environmental osmolarity changes. The main objective of this study was to elucidate the role of the OmpR/EnvZ two-component system in regulating the biofilm through curli and fimbriae (FimH gene), a contrary approach towards biofilm inhibition. In this study, the CRISPRi technique was used to suppress the expression of the OmpR gene. The RT-PCR assay was performed to quantify mRNA gene expression of curli and biofilm producing genes, and the data were further confirmed by different microscopic, spectroscopic and biofilm quantification assay (Crystal Violet). It is the first time we have shown downregulation of the OmpR gene in biofilm causing clinical isolates of E. coli, which further suppressed the FimH gene, leading to biofilm reduction. The crystal violet assay and microscopic studies also confirmed the biofilm reduction. We conclude that the OmpR gene of the OmpR/EnvZ two-component system could be one of the targets for biofilm mediated infection intervention. Our findings open new vistas to explore the pathways and targets to control biofilm mediated infections.

Exploring antibiofilm potential of bacitracin against streptococcus mutans.

Bacitracin has well familiar effects on growth and colonization of bacteria but its antibiofilm action on majority of bacteria is still not studied. Bacitracin is a bactericidal antibiotic that primarily acts on Gram positive bacteria by obstructing the process of cell wall synthesis. In this study, we have investigated antibiofilm potential and the mechanism of bacitracin against a cariogenic bacteria 'Streptococcus mutans' which has not been reported so far. Bacitracin has been found to affect propensity of S. mutans to form biofilm. On treatment with sub-MIC concentration of bacitracin resulted in significant reduction in bifilm formation as evaluated by crystal violet and congo red assays. The architecture of S. mutans biofilm was observed by scanning electron microscopy which revealed astonishing phenotype of biofilm. Deficient biofilm was found to be composed of abnormally elongated cells. Transmission electron microscopy showed multiple septa formation in each cell of biofilm thereby indicating, cell division defect as the most probable cause of cell elongation. To elucidate the effect of bacitracin on molecular level, expression profiling of genes critically important for cell division and biofilm formation was performed, which were found many folds downregulated. Bacitracin at very low concentration has been found to have potent antibiofilm activity, therefore is a potential antibiofilm agent to treat oral biofilms. It is being anticipated, this study will offer novel information to identify potential targets and effectively creates true innovation to understand the biofilm's basic biology. Besides, discovering new uses for currently marketed drugs makes commercial as well as research sense.

Generation of oxidative stress and induction of apoptotic like events in curcumin and thymoquinone treated adult Fasciola gigantica worms.

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds. The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms. In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions.

Synergistic fungicidal activity with low doses of eugenol and amphotericin B against Candida albicans.

Candida albicans frequently causes variety of superficial and invasive disseminated infections in HIV infected patients. Further, the emergence of non albicans species causing candidiasis predominantly in patients with advanced immune-suppression and drug resistance brings great apprehension. Hence, in this study we evaluate the capability of eugenol (EUG), a natural compound in combination with less toxic concentrations of amphotericin B (AmpB) for enhanced antifungal effects and reduced toxicity. Antifungal activity and time-kill assay were employed according to Clinical Laboratory Standard Institute (CLSI) guidelines with minor modifications on clinical isolates of Candida albicans. To confirm the synergistic interaction of EUG and AmpB, checkerboard experiments were employed. Interestingly, EUG-Amp B combination shows many fold higher anti-candida activity compared to single component treatment. Furthermore, our results depicts reactive oxygen species (ROS) driven killing and mitochondrial hyperpolarisation on treatment. Our data also suggests inhibition of calcium channel by EUG and predicts longer retainment of AmpB. Pronounced cellular damage was observed with combination treatment than to EUG and AmpB alone. Our finding is helpful for the removal of toxic concentrations of antifungal agents.

Identification of factors involved in Enterococcus faecalis biofilm under quercetin stress.

Enterococcus faecalis is a gram positive enteric commensal bacteria or opportunistic pathogen and its infection involves biofilm formation. Quercetin, a plant origin polyphenol was found to inhibit E. faecalis biofilm. Crystal violet assay, SEM and CLSM microscopy confirmed biofilm inhibition by quercetin. Proteomics was used to elucidate the changes occurred in bacterial cell by quercetin treatment. 2D-Electrophorosis and MALDI-TOF analysis revealed that nineteen proteins were differentially expressed in quercetin treated sample. Glycolytic pathways, protein translation-elongation pathways and protein folding pathways were under differential expression after treatment. Real Time-PCR (RT-PCR) validated the proteomic data at genomic level except for the translation elongation factor G which showed opposite data to proteomics. Protein-protein interaction networks constructed using STRING 10.0 demonstrated strong connection of translation-elongation proteins with many important proteins. The results of the comparative analysis indicate that quercetin exerts its inhibitory effect by disturbing glycolytic, protein translation-elongation and protein folding pathways. This disturbs bacterial physiology and stops transition of planktonic cells to biofilm state.

The role of conserved residues in the catalytic activity of NDM-1: an approach involving site directed mutagenesis and molecular dynamics.

The rise of New Delhi metallo-beta-lactamase-1 (NDM-1) producers is a major public health concern due to carbapenem resistance. Infections caused by carbapenem-resistant enterobacteria (CRE) are classified as a serious problem. To understand the structure and function of NDM-1, an amino acid replacement approach is considered as one of the methods to get structural insight. Therefore, we have generated novel mutations (N193A, S217A, G219A and T262A) near active sites and an omega-like loop to study the role of conserved residues of NDM-1. The minimum inhibitory concentrations (MICs) of ampicillin, imipenem, meropenem, cefotaxime, cefoxitin and ceftazidime for all mutants were found to be reduced 2 to 6 fold, compared to a wild type NDM-1 producing strain. The Km values increased while Kcat and Kcat/Km values were decreased compared to wild type. The affinity as well as the catalysis properties of these mutants were reduced considerably for imipenem, meropenem, cefotaxime, cefoxitin, and ceftazidimem compared to wild type, hence the catalytic efficiencies (Kcat/Km) of all mutant enzymes were reduced owing to the poor affinity of the enzyme. The IC50 values of these mutants with respect to each drug were reduced compared to wild type NDM-1. MD simulations and docking results from the mutant protein models, along with the wild type example, showed stable and consistent RMSD, RMSF and Rg behavior. The α-helix content values of all mutant proteins were reduced by 13%, 6%, 14% and 9% compared to NDM-1. Hence, this study revealed the impact role of active sites near residues on the enzyme catalytic activity of NDM-1.

Understanding the mode of binding mechanism of doripenem to human serum albumin: Spectroscopic and molecular docking approaches.

The infections caused by multidrug resistant bacteria are widely treated with carabapenem antibiotics as a drug of choice, and human serum albumin (HSA) plays a vital role in binding with drugs and affecting its rate of delivery and efficacy. So, we have initiated this study to characterize the mechanism of doripenem binding and to locate its site of binding on HSA by using spectroscopic and docking approaches. The binding of doripenem leads to alteration of the environment surrounding Trp-214 residue of HSA as observed by UV spectroscopic study. Fluorescence spectroscopic study revealed considerable interaction and complex formation of doripenem and HSA as indicated by Ksv and Kq values of the order of 104  M-1 and 1012  M-1  s-1 , respectively. Furthermore, doripenem quenches the fluorescence of HSA spontaneously on a single binding site with binding constant of the order of 103  M-1 , through an exothermic process. Van der Waals forces and hydrogen bonding are the major forces operating to stabilize HSA-doripenem complex. Circular dichroism spectroscopic study showed changes in the structure of HSA upon doripenem binding. Drug displacement and molecular docking studies revealed that the binding site of doripenem on HSA is located on subdomain IB and III A. This study concludes that, due to significant interaction of doripenem on either subdomain IB or IIIA of HSA, the availability of doripenem on the target site may be compromised. Hence, there is a possibility of unavailability of threshold amount of drug to be reached to the target; consequently, resistance may develop in the bacterial population.

Role of cell division protein divIVA in Enterococcus faecalis pathogenesis, biofilm and drug resistance: A future perspective by in silico approaches.

Antibiotics resistance is the major problem in clinical settings which leads to the emergence of drug resistant bacteria. Biofilm formation is one of the grounds for the emergence of antibiotics resistant strains of Enterococcus faecalis. Our group previously reported in a comparative proteomic study of biofilm and planktonic state of E. faecalis that cell division protein divIVA was two folds overexpressed in biofilm state as compared to planktonic one and suggested its involvement in biofilm formation and antibiotics resistance. In this in silico study molecular docking showed that DNA bind to the conserved amino acid residues of divIVA domain and suggested that divIVA possibly secretes DNA into extra polymeric substance (EPS) which is the part of biofilm. We also performed the STRING analysis of cell division protein divIVA and predicted their interactive partners {cell division proteins/divisome complex (ftsZ, ftsA, divIV, ftsL, & gpsB), hypothetical proteins (sepF, EF_0261, EF_1000, EF_0998, EF_1006 & EF_1040), isoleucyl-tRNA synthetase (ileS), septation ring formation regulator (ezrA), S4 domain-containing protein (EF_1001), rod shape-determining protein (mreC), UDP-N-acetylmuramoyl-L-alanyl-d-glutamate synthetase (murD), UDP-diphospho-muramoyl-pentapeptide beta-N- acetylglucosaminyltransferase (murG), Lipoprotein signal peptidase (lspA), adenylate kinase (adk) and DNA-binding response regulator (vicR)}. We suggest that cumulatively divIVA and its interactive partners might be directly or indirectly involved in E. faecalis cell division, growth, biofilm formation, virulence and resistance.

Retracted: Novel spiro-thiazolidin-4-one and thioether derivatives of benzylidene flavanones: New leads in cancer and microbial chemotherapy.

The above article from Archiv der Pharmazie, published online on 12 March 2018 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor-in-Chief, Prof. Holger Stark, and Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. The retraction has been agreed due to errors in the spectroscopic data of the investigated new compounds. REFERENCE TO RETRACTION: S. Mukhtar, M. A. Alsharif, M. I. Alahmdi, H. Parveen, A. U. Khan, Arch. Pharm. Chem. Life Sci. 2018;1-12. DOI: 10.1002/ardp.201700397.

Structure, Genetics and Worldwide Spread of New Delhi Metallo-ß-lactamase (NDM): a threat to public health.

BACKGROUND: The emergence of carbapenemase producing bacteria, especially New Delhi metallo-ß-lactamase (NDM-1) and its variants, worldwide, has raised amajor public health concern. NDM-1 hydrolyzes a wide range of ß-lactam antibiotics, including carbapenems, which are the last resort of antibiotics for the treatment of infections caused by resistant strain of bacteria. MAIN BODY: In this review, we have discussed bla NDM-1variants, its genetic analysis including type of specific mutation, origin of country and spread among several type of bacterial species. Wide members of enterobacteriaceae, most commonly Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and gram-negative non-fermenters Pseudomonas spp. and Acinetobacter baumannii were found to carry these markers. Moreover, at least seventeen variants of bla NDM-type gene differing into one or two residues of amino acids at distinct positions have been reported so far among different species of bacteria from different countries. The genetic and structural studies of these variants are important to understand the mechanism of antibiotic hydrolysis as well as to design new molecules with inhibitory activity against antibiotics. CONCLUSION: This review provides a comprehensive view of structural differences among NDM-1 variants, which are a driving force behind their spread across the globe.

Antibiofilm efficacy of green synthesized graphene oxide-silver nanocomposite using Lagerstroemia speciosa floral extract: A comparative study on inhibition of gram-positive and gram-negative biofilms.

Biofilm architecture provides bacteria with enhanced antibiotic resistance, thus raising the need to search for alternative therapies that can inhibit the bacterial colonization. In the present study, we synthesized graphene oxide-silver nanocomposite (GO-Ag) by non-toxic and eco-friendly route using a floral extract of Legistromia speciosa (L.) Pers. The gas chromatography-mass spectrometry (GC-MS) analysis of plant extract revealed the presence of compounds which can simultaneously act as reducing and capping agents. The sub-inhibitory concentrations of synthesized GO-Ag reduced the biofilm formation in both gram-negative (E. cloacae) and gram-positive (S. mutans) bacterial models. Growth curve assay, membrane integrity assay, scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) revealed different mechanisms of biofilm inhibition in E. cloacae and S. mutans. Moreover, quantitative RT-PCR (qRT-PCR) results suggested GO-Ag is acting on S. mutans biofilm formation cascade. Biofilm inhibitory concentrations GO-Ag were also found to be non-toxic against HEK-293 (human embryonic kidney cell line). The whole study highlights the therapeutic potential of GO-Ag to restrain the onset of biofilm formation in bacteria.

Nano-therapeutics: A revolution in infection control in post antibiotic era.

With the arrival of antibiotics 70 years ago, meant a paradigm shift in overcoming infectious diseases. For decades, drugs have been used to treat different infections. However, with time bacteria have become resistant to multiple antibiotics, making some diseases difficult to fight. Nanoparticles (NPs) as antibacterial agents appear to have potential to overcome such problems and to revolutionize the diagnosis and treatment of bacterial infections. Therefore, there is significant interest in the use of NPs to treat variety of infections, particularly caused by multidrug-resistant (MDR) strains. This review begins with illustration of types of NPs followed by the literature of current research addressing mechanisms of NPs antibacterial activity, steps involved in NP mediated drug delivery as well as areas where NPs use has potential to improve the treatment, like NP enabled vaccination. Besides, recently emerged innovative NP platforms have been highlighted and their progress made in each area has been reviewed.

Biofilms in Endodontics-Current Status and Future Directions.

Microbiota are found in highly organized and complex entities, known as biofilms, the characteristics of which are fundamentally different from microbes in planktonic suspensions. Root canal infections are biofilm mediated. The complexity and variability of the root canal system, together with the multi-species nature of biofilms, make disinfection of this system extremely challenging. Microbial persistence appears to be the most important factor for failure of root canal treatment and this could further have an impact on pain and quality of life. Biofilm removal is accomplished by a chemo-mechanical process, using specific instruments and disinfecting chemicals in the form of irrigants and/or intracanal medicaments. Endodontic research has focused on the characterization of root canal biofilms and the clinical methods to disrupt the biofilms in addition to achieving microbial killing. In this narrative review, we discuss the role of microbial biofilms in endodontics and review the literature on the role of root canal disinfectants and disinfectant-activating methods on biofilm removal.

Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-ß-Lactamase 1.

New Delhi metallo-ß-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defense mechanism to hydrolyze ß-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We cloned blaNDM-1 from an Enterobacter cloacae clinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4- to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (the Km was reduced, while the kcat was similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ΔGD (o) and Tm of Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the α-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1.

Protein translation machinery holds a key for transition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach.

Enterococcus faecalis is a member of human gut microflora causing nosocomial infection involving biofilm formation. Ethyl methyl sulfonate induced mutants were analysed using crystal violet assay, SEM and CLSM microscopy which confirmed AK-E12 as biofilm efficient and AK-F6 as biofilm deficient mutants. Growth curve pattern revealed AK-E12 was fast growing whereas, AK-F6 was found slow growing mutant. 2D-Electrophorosis and MALDI-TOF analysis revealed over and underexpression of many translation-elongation associated proteins in mutants compared to wild type. Protein translation elongation factor G, translation elongation factor Tu and ribosomal subunit interface proteins were underexpressed and UTP-glucose-1-phosphate uridylyl transferase and cell division protein divIVA were overexpressed in AK-E12 as compared to wild type. In AK-F6, except 10 kDa chaperonin which was over-expressed other selected proteins were found to be suppressed. RT-PCR confirmed proteomic data except for the translation elongation factor G which showed contradictory data of proteome expression in AK-E12. Protein-protein interaction networks were constructed using STRING 10.0 which demonstrated strong connection of translation-elongation proteins with other proteins. Hence, it concludes from the data that translation elongation factors are important in transition of planktonic cells to biofilm cells in Enterococcus faecalis.