P-type ATPase zinc transporter Rv3270 of Mycobacterium tuberculosis enhances multi-drug efflux activity.

Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn2+ and Zn2+ metal ion toxicity in Mycobacterium tuberculosis, in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of Escherichia coli and Mycobacterium smegmatis. Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn2+ resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.

Septal venous channel perforation during left bundle branch area pacing: a prospective study.

AIMS: To characterize the diagnosis, frequency, and procedural implications of septal venous channel perforation during left bundle branch area pacing (LBBAP). METHODS AND RESULTS: All consecutive patients undergoing LBBAP over an 8-month period were prospectively studied. During lead placement, obligatory septal contrast injection was performed twice, at initiation (implant entry zone) and at completion (fixation zone). An intuitive fluoroscopic schema using orthogonal views (left anterior oblique/right anterior oblique) and familiar landmarks is described. Using this, we resolved zonal distribution (I-VI) of lead position on the ventricular septum and its angulation (post-fixation angle θ). Subjects with and without septal venous channel perforation were compared. Sixty-one patients {male 57.3%, median age [interquartile range (IQR)] 69.5 [62.5-74.5] years} were enrolled. Septal venous channel perforation was observed in eight (13.1%) patients [male 28.5%, median age (IQR) 64 (50-75) years]. They had higher frequency of (i) right-sided implant (25% vs. 1.9%, P = 0.04), (ii) fixation in zone III at the mid-superior septum (75% vs. 28.3%, P = 0.04), (iii) steeper angle of fixation-median θ (IQR) [19 (10-30)° vs. 5 (4-19)°, P = 0.01], and (iv) longer median penetrated-lead length (IQR) [13 (10-14.8) vs. 10 (8.5-12.5) mm, P = 0.03]. Coronary sinus drainage of contrast was noted in five (62.5%) patients. Abnormal impedance drops during implantation (12.5% vs. 5.7%, P = NS) were not significantly different. CONCLUSION: When evaluated systematically, septal venous channel perforation may be encountered commonly after LBBAP. The fiducial reference framework described using fluoroscopic imaging identified salient associated findings. This may be addressed with lead repositioning to a more inferior location and is not associated with adverse consequence acutely or in early follow-up.

Budd-Chiari syndrome in children: Radiological intervention and role of shear wave elastography in monitoring response.

OBJECTIVES: Radiological intervention (RI) is the preferred treatment in children with Budd-Chiari syndrome (BCS). We studied the comparative long-term outcome of BCS children, with and without RI and utility of liver and splenic stiffness measurement (LSM, SSM) by 2-dimensional shear wave elastography (2D-SWE) in assessing response. METHODS: Sixty children (40 boys, median age 10.5 [6.5-15.25] years) with BCS (29 newly diagnosed, 31 follow-up) were evaluated. LSM and SSM by 2D-SWE and vascular patency were monitored pre- and postprocedure (≥ 6 months postprocedure) in those undergoing RI. Medical therapy without anticoagulation and monitoring was done in subjects without RI. The RI and no-RI groups were compared. RESULTS: Ascites (54,90%), hepatomegaly (56,93%) and prominent abdominal-veins (42,70%), were the commonest features. The majority (46,78%) had isolated hepatic vein block. 44 (73%) cases underwent RI, while 16 (27%) were managed conservatively. Both groups were similar at baseline. Post-RI subjects showed significant improvement in clinical findings, liver functions and portal hypertension. LSM [33 (32-34.5) to 19.2 (18-20.67) kPa] and SSM [54.5 (52.3-57.6) to 28.9 (27.6-30.25) kPa] showed a significant decline from baseline value over a follow-up of 12 (6-13) months. Gradual reduction occurred in the LSM and SSM over 1-5 years, with near-normal LSM [10.2 (9.2-11.5) kPa] and SSM [22.3 (20.5-24.3) kPa] values in patients (n-16) with > 5 years follow-up. Patients without RI showed worsening in LSM and SSM. Hepatopulmonary syndrome and hepatocellular carcinoma developed in 4 (8%) and 1 (1.7%) cases respectively. CONCLUSION: RI leads to clinical recovery and reduction with near normalization of LSM and SSM over long-term follow-up in children with BCS. 2D-SWE is a promising tool to monitor outcomes.

Open window mapping for redo accessory pathway ablation in Ebstein anomaly.

Accessory pathway ablation in Ebstein anomaly can be significantly more challenging than in structurally normal hearts. An alternative to the conventional approach to mapping APs is to detect points with a high-density mapping catheter based on an automated detection algorithm using open window mapping. It detects the sharpest signal at each point with high-density mapping rather than relying on the origin of the local electrogram to localize the pathway and determine a site for successful ablation. We herein report the first case in literature of a redo-accessory pathway ablation in Ebstein anomaly using this technique.

The MSMEG_1586 of M. smegmatis Is a Penicillin-Interactive Enzyme That Can Potentially Hydrolyse Aztreonam and Cephalosporins.

Mycobacteria are intrinsically resistant to beta-lactams as they possess several putative penicillin-interactive enzymes (PIEs), some of those are with dual-activity, namely DD-carboxypeptidase and beta-lactamase. Here, with help of molecular approaches, we elucidated the nature of one such putative PIE, MSMEG_1586, in Mycobacterium smegmatis. The in vivo expression of the membrane-bound form of MSMEG_1586 enhanced the beta-lactam resistance of a beta-lactamase deleted host E. coli strain (AM1OC), particularly for aztreonam (eight-fold) and cephalosporins (8-16 fold). To understand the reason for such elevation of resistance, soluble-form of MSMEG_1586 (sMSMEG_1586) was created by removing signal peptides and partially eliminating the amphipathic helix, and finally, expressed and purified. The purified sMSMEG_1586 was active and manifested a strong penicillin-binding affinity as shown by its ability to bind to fluorescent penicillin (Bocillin-FL). Interestingly, the steady-state kinetics apparently confirmed the hydrolytic ability of sMSMEG_1586 towards cefotaxime and aztreonam where hydrolysing aztreonam is a unique and rare behaviour among the beta-lactamases. However, sMSMEG_1586 was devoid of exerting DD-carboxypeptidase like activity. Finally, in silico analysis of MSMEG_1586 revealed a special folding that resembles class C beta-lactamase, except for the absence of a characteristic R2 loop. Overall, MSMEG_1586 could be categorized as a cephalosporinase with the ability to hydrolyse aztreonam.

Deciphering the role of residues in the loops nearing the active site of OXA-58 in imparting beta-lactamase activity.

The existence of OXA-58 carbapenemase alone or in combination with other beta-lactam resistance factors poses significant beta-lactam resistance. The exact mechanism of action of OXA type beta-lactamases is debatable due to the involvement of multiple residues within or outside the active site. In the present work, we have elucidated the relative role of residues present in the putative omega (W169, L170, K171) and ß6-ß7 (A226 and D228) loops on the activity of OXA-58 by substituting into alanine (and aspartate for A226) through site-directed mutagenesis. E. coli cells harbouring OXA-58, substituted at the putative omega loop, manifest a significant decrease in the beta-lactam resistance profile than that of the cells expressing OXA-58. Further, a reduction in the catalytic efficiency is observed for the purified variants of OXA-58 carrying individual substitutions in the putative omega loop than that of OXA-58. However, the addition of NaHCO3 (for carbamylation of K86) increases catalytic efficiency of the individual protein as revealed by nitrocefin hydrolysis assay and steady state kinetics. Moreover, W169A and K171A substitutions show significant effects on the thermal stability of OXA-58. Therefore, we conclude that the putative omega loop residues W169, L170 and K171, individually, have significant role in the activity and stability of OXA-58, mostly by stabilising carbamylated lysine of active site.

A NiCoT family metal transporter of Mycobacterium tuberculosis (Rv2856/NicT) behaves as a drug efflux pump that facilitates cross-resistance to antibiotics.

Metals often act as a facilitator in the proliferation and persistence of antibiotic resistance. Efflux pumps play key roles in the co-selection of metal and antibiotic resistance. Here, we report the ability of a putative nickel/cobalt transporter (NiCoT family), Rv2856 or NicT of Mycobacterium tuberculosis (Mtb), to transport metal and antibiotics and identified some key amino acid residues that are important for its function. Ectopic expression of NicT in Escherichia coli CS109 resulted in the increase of intracellular nickel uptake. Additionally, enhanced tolerance towards several antibiotics (norfloxacin, sparfloxacin, ofloxacin, gentamicin, nalidixic acid and isoniazid) was observed with NicT overexpression in E. coli and Mycobacterium smegmatis. A comparatively lower intracellular accumulation of norfloxacin upon NicT overexpression than that of the cells without NicT indicated the involvement of NicT in an active efflux process. Although expression of NicT did not alter the sensitivity towards kanamycin, doxycycline, tetracycline, apramycin, neomycin and ethambutol, the presence of a sub-inhibitory dose of Ni2+ resulted in the manifestation of low-level tolerance towards these drugs. Further, substitution of four residues (H77I, D82I, H83L and D227I) in the conserved regions of NicT by isoleucine and leucine resulted in reduced to nearly complete loss of the transport function for both metals and antimicrobials. Therefore, the study suggests that nickel transporter Rv2856/NicT may actively export different drugs and the presence of nickel might drive the cross-resistance to some of the antibiotics.

The Putative Major Facilitator Superfamily (MFS) Protein Named Rv1877 in Mycobacterium tuberculosis Behaves as a Multidrug Efflux Pump.

Efflux pumps are one of the major contributors in the intrinsic multidrug resistance of Mycobacterium tuberculosis. These active transporters, localized in the cytoplasmic membrane, often carry an array of unrelated substances, from toxic substances to metabolites and maintain cellular homeostasis. Rv1877, a putative Major Facilitator Superfamily efflux pump from M. tuberculosis, was investigated in this study. Expression of Rv1877 in Escherichia coli resulted in elevated resistance towards antibiotics of various families. A reversal of this resistance was observed in the presence of sub-inhibitory concentration of the uncoupler carbonyl cyanide-m-chlorophenylhydrazone, indicating its dependence on proton motive force (pmf). Lower intracellular accumulation of the fluoroquinolones ofloxacin and levofloxacin in E. coli cells harbouring Rv1877 implied an active efflux of the drugs. Interestingly, real time, energy-dependent efflux was demonstrated by cells expressing Rv1877 with a lipophilic dye Nile Red. In addition, expression of Rv1877 in trans increased the biofilm formation by the host E. coli cells. Moreover, in silico docking analysis of the molecular interactions between Rv1877 and antibiotics corroborated the experimental observations. Based on the in vivo analyses of Rv1877 in E. coli, it could be designated as a pmf-dependent multidrug transporter with the ability of extruding structurally unrelated antibiotics, preferably some of the fluoroquinolones, and a facilitator of biofilm formation.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation.

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

The unusual case of a Topsy-Turvy ECG with a pseudo-infarct pattern.

Unique electrocardiogram (ECG) patterns can be generated by inadvertent lead misplacements. The commonest of these is left arm-right arm (LA-RA) reversal leading to inverted lead I, right axis deviation and an upright aVR. Changes in placement of the neutral (right leg: RL) electrode with one of the LA or RA cables may produce more atypical patterns, which often mimic myocardial ischemia. We describe an unusual case wherein the ECG suggested an ectopic atrial rhythm and ischemic changes in the inferior leads. Closer scrutiny revealed a bilateral LA-LL and RA-RL (left arm- left leg and right arm- right leg) reversal. Familiarity and quick recognition of such ECG patterns shall help clinicians in avoiding diagnostic and therapeutic errors.

MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and ß-lactamase activities.

Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. dd-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal d-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG_2432 of Mycobacterium smegmatis, by showing that it exhibits both dd-CPase and ß-lactamase activities. Like conventional dd-CPase (PBP5 from E. coli), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli, MSMEG_2432 has manifested its ability to restore ~75 % of the cell population to their normal rod shape. Further, in vitrodd-CPase assay has confirmed its ability to release terminal d-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with d-Ala-d-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG_2432 suggests the presence of ß-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of its in vivo and in vitrodd-CPase and ß-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both dd-CPase and ß-lactamase activities.

Absence of the glycosyltransferase WcaJ in Klebsiella pneumoniae ATCC13883 affects biofilm formation, increases polymyxin resistance and reduces murine macrophage activation.

Multidrug-resistant Klebsiella pneumoniae has emerged as one of the deadliest opportunistic nosocomial pathogens that forms biofilm for the establishment of chronic K. pneumoniae infections. Herein, we made an attempt to identify the genes involved in biofilm formation in the strain K. pneumoniae ATCC13883. To achieve this, we constructed mini-Tn5 transposon insertion mutants and screened them for biofilm production. We observed that the biofilm formation was enhanced in the mutant where the wcaJ gene was disrupted. WcaJ is the initiating enzyme of colanic acid synthesis and loads the first sugar (glucose-1-P) on the lipid carrier undecaprenyl phosphate. The absence of this glycosyltransferase results in the absence of colanic acid, which renders a non-mucoid phenotype to the mutant. Further, to determine the effect of mucoidy on antibiotic susceptibility, we tested the sensitivity of the strains towards different groups of antibiotics. Unlike the mucoid strains, the resistance of the non-mucoid cells was greater for polymyxins, but less for quinolones. Capsular polysaccharides are known to have a protective effect against phagocytosis, therefore we assessed the role of colanic acid in virulence by conducting infection studies on murine macrophages. Surprisingly, the ΔwcaJ strain was less efficient in macrophage activation and was not readily phagocytosed. Thus, the presence of colanic acid appeared to increase the immunogenicity of K. pneumoniae. Overall, the results indicate that the presence of colanic acid increases the vulnerability of K. pneumoniae towards both polymyxins and macrophages, implying that the mucoid strains are less threatening as compared to their high biofilm forming non-mucoid counterparts.

Selective Detection of Human Serum Albumin by Near Infrared Emissive Fluorophores: Insights into Structure-property Relationship.

Two donor-acceptor fluorophores were prepared and tested for quantitative determination of HSA in aqueous samples. Fluorophores were non-emissive in polar solvents due to energy loss via non-radiative decays. Complexation of the fluorophores with HSA resulted multi-fold enhancement of emission in the red-near infrared (NIR) region. The emission intensity was linearly correlated to the amount of protein in the solution, which enabled us to develop calibration graphs for quantitative estimation of HSA in synthetic urine samples. Between the two fluorophores, the methoxy substituted fluorophore 1 selectively recognized HSA. It exhibited remarkable fluorescence enhancement with HSA over bovine serum albumin (BSA) and other globular proteins. The selective sensing aptitude of 1 was attributed to its restricted motions in the protein's microenvironment due to multiple non-covalent interactions, preventing energy loss by radiationless decay. The different recognition properties of the fluorophores were estimated by the steady-state fluorescence and molecular docking studies. These findings indicate that this class of fluorophores can be useful for quantitative estimation of HSA in biological urine and blood samples in clinical practice.

Two dd-Carboxypeptidases from Mycobacterium smegmatis Affect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids.

During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (between d-Ala and meso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. The dd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used by ld-transpeptidases as substrates to form 3-3 cross-links. Therefore, dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of dd-CPases in mycobacteria is relatively unexplored. In this study, we deleted two dd-CPase genes, msmeg_2433 and msmeg_2432, both individually and in combination, from Mycobacterium smegmatis mc2155. Though the single dd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant, viz, a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to ß-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of the dd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCE The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The dd-CPases generate tetrapeptides by acting on the pentapeptides, and ld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of two dd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, including Mycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology of dd-CPases might help us design antimycobacterial therapeutics in the future.