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Sachin S Bhagwat,1 Manohar Nandanwar,2 Atul Kansagara,2 Anusuya Patel,3 Swapna Takalkar,1 Rajesh Chavan,4 Hariharan Periasamy,1 Ravindra Yeole,5 Prasad K Deshpande,6 Satish Bhavsar,6 Ashima Bhatia,7 Jaishid Ahdal,8 Rishi Jain,8 Mahesh Patel9 1Department of Microbiology, Wockhardt Research Centre, Aurangabad, India; 2Department of Toxicology, Wockhardt Research Centre, Aurangabad, India; 3Department of Safety Pharmacology, Wockhardt Research Centre, Aurangabad, India; 4Department of Drug Metabolism and Pharmacokinetics, Wockhardt Research Centre, Aurangabad, India; 5Department of Analytical Chemistry, Wockhardt Research Centre, Aurangabad, India; 6Department of Medicinal Chemistry, Wockhardt Research Centre, Aurangabad, India; 7Global Clinical Operations, Wockhardt Ltd, Mumbai, India; 8Department of Medical Affairs, Wockhardt Ltd, Mumbai, India; 9Drug Discovery Research, Wockhardt Research Centre, Aurangabad, IndiaCorrespondence: Sachin S BhagwatWockhardt Research Centre, Aurangabad 431006, IndiaTel +91240-669-4185Email sbhagwat@wockhardt.comAbstract: Levonadifloxacin and its prodrug alalevonadifloxacin are novel broad-spectrum anti-MRSA agents belonging to the benzoquinolizine subclass of quinolone, formulated for intravenous and oral administration, respectively. Various in vitro and in vivo studies have established their antimicrobial spectrum against clinically significant Gram-positive, Gram-negative, atypical, and anaerobic pathogens. The potent activity of levonadifloxacin against MRSA, quinolone-resistant Staphylococcus aureus, and hetero-vancomycin-intermediate strains is an outcome of its well-differentiated mechanism of action involving preferential targeting to DNA gyrase. Potent anti-staphylococcal activity of levonadifloxacin was also observed in clinically relevant experimental conditions such as acidic pH, the intracellular environment, and biofilms, suggesting that the drug is bestowed with enabling features for the treatment of difficult-to-treat MRSA infections. Levonadifloxacin also retains clinically relevant activity against resistant respiratory pathogens such as macrolide- and penicillin-resistant Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and Moraxella catarrhalis and, in conjunction with clinically established best-in-class human epithelial lung fluid concentration, has promising potential in the management of recalcitrant respiratory infections. Attractive features, such as resistance to NorA efflux, divergent mechanism of action in S. aureus, cidality against high-inoculum cultures, and low mutant prevention concentration, are likely to confer favorable resistance-suppression features to both agents. In vivo studies have shown promising efficacy in models of acute bacterial skin and skin structure infection, respiratory infections, pyelonephritis, and peritonitis at human-equivalent mouse doses. Both formulations were well tolerated in multiple phase I studies and overall showed a dose-dependent exposure. In particular, oral alalevonadifloxacin showed excellent bioavailability (∼90%), almost mirroring the pharmacokinetic profile of intravenous levonadifloxacin, indicating the prodrug’s seamless absorption and efficient cleavage to release the active parent drug. Hepatic impairment studies showed that clinical doses of levonadifloxacin/alalevonadifloxacin are not required to be adjusted for various degrees of hepatic impairment. With the successful completion of phase II and phase III studies for both levonadifloxacin and alalevonadifloxacin, they represent clinically attractive therapeutic options for the treatment of infections caused by multi-drug-resistant Gram-positive organisms. Herein, we review the current evidence on therapeutically appealing attributes of levonadifloxacin and alalevonadifloxacin, which are based on a range of non-clinical in vitro and in vivo investigations and clinical studies.Keywords: levonadifloxacin, WCK 771, alalevonadifloxacin, WCK 2349, broad-spectrum antibiotic, MRSA

Compared to other genera of Gram-negative pathogens, Pseudomonas is adept in acquiring complex non-enzymatic and enzymatic resistance mechanisms thus remaining a challenge to even novel antibiotics including recently developed β-lactam and β-lactamase inhibitor combinations. This study shows that the novel β-lactam enhancer approach enables cefepime/zidebactam to overcome both non-enzymatic and enzymatic resistance mechanisms associated with a challenging panel of P. aeruginosa . This study highlights that the β-lactam enhancer mechanism is a promising alternative to the conventional β-lactam/β-lactamase inhibitor approach in combating ever-evolving MDR P. aeruginosa ., Competing Interests: Some of the coauthors are employees of Wockhardt. R.A.B. receives grant funding from Wockhardt.

Objectives: To study the dynamics, mechanisms and fitness cost of resistance selection to cefepime, zidebactam and cefepime/zidebactam in Pseudomonas aeruginosa., Methods: WT P. aeruginosa PAO1 and its ΔmutS derivative (PAOMS) were exposed to stepwise increasing concentrations of cefepime, zidebactam and cefepime/zidebactam. Selected mutants were characterized for change in susceptibility profiles, acquired mutations, fitness, virulence and in vivo susceptibility to cefepime/zidebactam. Mutations were identified through WGS. In vitro fitness was assessed by measuring growth in minimal medium and human serum-supplemented Mueller-Hinton broth. Virulence was determined in Caenorhabditis elegans and neutropenic mice lung infection models. In vivo susceptibility to a human-simulated regimen (HSR) of cefepime/zidebactam was studied in neutropenic mice lung infection., Results: Resistance development was lower for the cefepime/zidebactam combination than for the individual components and high-level resistance was only achieved for PAOMS. Cefepime resistance development was associated with mutations leading to the hyperexpression of AmpC or MexXY-OprM, combined with PBP3 mutations and/or large chromosomal deletions involving galU. Zidebactam resistance was mainly associated with mutations in PBP2. On the other hand, resistance to cefepime/zidebactam required multiple mutations in genes encoding MexAB-OprM and its regulators, as well as PBP2 and PBP3. Cumulatively, these mutations inflicted significant fitness cost and cefepime/zidebactam-resistant mutants (MIC = 16-64 mg/L) remained susceptible in vivo to the HSR., Conclusions: Development of cefepime/zidebactam resistance in P. aeruginosa required multiple simultaneous mutations that were associated with a significant impairment of fitness and virulence., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Authors report on a carbon nanotube (CNT)-based lateral flow immunoassay (LFI) for ultrasensitive detection of proteins. Shortened multiwalled CNTs were used as a colored (black) tag. The detection antibody was covalently immobilized on the CNT surface via diimide-activated conjugation between the carboxyl groups on the CNT surface and amino groups of antibodies. The assay involved the capture of target protein in a sandwich-type format between an immobilized capture antibody on the test zone of LFI and a CNT-labelled detection antibody. CNTs were thus captured on the test zone of the LFI and gave a black colored line to enable visual detection of protein. Quantitative results were obtained by reading the test line intensities with a portable strip reader. Rabbit IgG was used as a model target to demonstrate the proof-of-concept. Combining the advantages of lateral flow assay with the unique physical properties of CNT (color, high aspect-to-size ratio and ease of surface modification), the optimized LFI can detect of 1.3 pg mL -1 of rabbit IgG (S/N = 3). This is three orders lower than that of gold nanoparticle-based LFI. Rabbit IgG spiked into human plasma samples was successfully detected with this LFI. Conceivably, this method can be extended to various other proteins for which adequate antibodies do exist. Graphical abstract Carbon nanotubes are used as black tags in an ultrasensitive lateral flow immunoassay (LFI). The LFI was applied to the determination of rabbit IgG. The detection limit is more than 3 orders of magnitude lower than that of the conventional gold nanopaticle-based LFI.

Zidebactam and WCK 5153 are novel bicyclo-acyl hydrazide (BCH) agents that have previously been shown to act as β-lactam enhancer (BLE) antibiotics in Pseudomonas aeruginosa and Acinetobacter baumannii The objectives of this work were to identify the molecular targets of these BCHs in Klebsiella pneumoniae and to investigate their potential BLE activity for cefepime and aztreonam against metallo-β-lactamase (MBL)-producing strains in vitro and in vivo Penicillin binding protein (PBP) binding profiles were determined by Bocillin FL assay, and 50% inhibitory concentrations (IC 50 s) were determined using ImageQuant TL software. MICs and kill kinetics for zidebactam, WCK 5153, and cefepime or aztreonam, alone and in combination, were determined against clinical K. pneumoniae isolates producing MBLs VIM-1 or NDM-1 (plus ESBLs and class C β-lactamases) to assess the in vitro enhancer effect of BCH compounds in conjunction with β-lactams. Additionally, murine systemic and thigh infection studies were conducted to evaluate BLE effects in vivo Zidebactam and WCK 5153 showed specific, high PBP2 affinity in K. pneumoniae The MICs of BLEs were >64 μg/ml for all MBL-producing strains. Time-kill studies showed that a combination of these BLEs with either cefepime or aztreonam provided 1 to >3 log 10 kill against MBL-producing K. pneumoniae strains. Furthermore, the bactericidal synergy observed for these BLE-β-lactam combinations translated well into in vivo efficacy even in the absence of MBL inhibition by BLEs, a characteristic feature of the β-lactam enhancer mechanism of action. Zidebactam and WCK 5153 are potent PBP2 inhibitors and display in vitro and in vivo BLE effects against multidrug-resistant (MDR) K. pneumoniae clinical isolates producing MBLs., (Copyright © 2019 American Society for Microbiology.)

We report a fluorescent carbon nanoparticle (FCN)-based lateral flow biosensor for ultrasensitive detection of DNA. Fluorescent carbon nanoparticle with a diameter of around 15nm was used as a tag to label a detection DNA probe, which was complementary with the part of target DNA. A capture DNA probe was immobilized on the test zone of the lateral flow biosensor. Sandwich-type hybridization reactions among the FCN-labeled DNA probe, target DNA and capture DNA probe were performed on the lateral flow biosensor. In the presence of target DNA, FCNs were captured on the test zone of the biosensor and the fluorescent intensity of the captured FCNs was measured with a portable fluorescent reader. After systematic optimizations of experimental parameters (the components of running buffers, the concentration of detection DNA probe used in the preparation of FCN-DNA conjugates, the amount of FCN-DNA dispensed on the conjugate pad and the dispensing cycles of the capture DNA probes on the test-zone), the biosensor could detect a minimum concentration of 0.4 fM DNA. This study provides a rapid and low-cost approach for DNA detection with high sensitivity, showing great promise for clinical application and biomedical diagnosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A quantitative immunochromatographic assay (QIA) was developed by using gold nanoparticle (GNP)-based lateral flow strip biosensor (LFSB) and a portable strip reader for rapid and sensitive quantitation of Carbohydrate Antigen 19-9 (CA 19-9) in human plasma. CA 19-9 is a biomarker that has been associated with cancers (such as pancreatic and colorectal cancers) and various non-cancerous diseases. The principle is based on sandwich-type immunoreactions between gold nanoparticle (GNP)-labelled detection antibody, anti-CA 19-9 capture antibody and CA 19-9to capture the GNPs on the test zone of LFSB. The accumulation of GNPs on the test zone gave a red line whose intensity was read with a portable strip reader to quantify the concentration of CA 19-9. Assay parameters including the membrane type, antibody concentration, amount of GNP-anti-CA 19-9 conjugates and the components of the running buffer were optimized to obtain the best sensitivity and reproducibility of the assay. The detection limit of the assay was determined to be 5UmL -1 (S/N=3) with a linear range of 5UmL -1 -100UmL -1 . CA 19-9 concentrations in healthy human and pancreatic cancer patient plasma samples were successfully evaluated using the developed quantitative immunochromatographic assay (QIA), and the results were in accordance with that obtained with enzyme linked immunosorbent assay (ELISA). The developed assay shows great promise for clinical application and biomedical diagnosis, particularly in limited resource settings., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The authors describe a magnetized carbon nanotube (MCNT)-based lateral flow strip biosensor for visual detection of proteins directly in whole blood avoiding complex purification and sample pre-treatments. MCNT were synthesized by coating Fe 3 O 4 nanoparticles on the shortened multiwalled carbon nanotube (CNT) surface via co-precipitation of ferric and ferrous ions within a dispersion of shorten multiwalled CNTs. The antibody-modified MCNTs were used to capture target protein in whole blood; the formed MCNT-antibody-target protein complexes were applied to the lateral flow strip biosensor, in which a capture antibody was immobilized on the test zone of the biosensor. The captured MCNTs on the test zone and control zone were producing characteristic brown/black bands, and this enabled target protein to be visually detected. Quantification was accomplished by reading the intensities of the bands with a portable strip reader. Rabbit IgG was used as a model target to demonstrate the proof-of-concept. After systematic optimizations of assay parameters, the detection limit of the assay in whole blood was determined to be 10 ng mL -1  (S/N = 3) with a linear dynamic range of 10-200 ng mL -1 . This study provides a rapid and low-cost approach for detecting proteins in blood, showing great promise for clinical application and biomedical diagnosis, particularly in limited resource settings., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The authors describe a rapid, low-cost and sensitive approach for the determination of carbohydrate antigen 19-9 (CA 19-9) in whole blood by using magnetized carbon nanotube (MCNT) and lateral flow strip biosensor (LFSB). MCNTs were synthesized by depositing magnetite (Fe 3 O 4 ) nanoparticles on multiwalled carbon nanotube (CNT) via co-precipitation of ferric and ferrous ions within a dispersion of shortened multiwalled CNTs. Antibody against CA 19-9 (Ab 1 ) was covalently immobilized on the MCNTs and were used to capture CA 19-9 in blood. After magnetic separation, the formed MCNT-Ab 1 -CA 19-9 complexes are applied to the LFSB, in which a capture antibody (Ab 2 ) and a secondary antibody (Ab 3 ) are immobilized on the test zone and control zone of the LFSB, respectively. The captured MCNTs on the test zone and control zone are producing characteristic brown bands, and this enables CA 19-9 to be visually detected. Quantitation is accomplished by reading the intensities of the bands with a portable strip reader. Under optimized conditions, the assay has a detection limit as low as 30 U⋅mL -1 of CA19-9 in blood. This is below the cutoff value (37 U mL -1 ) of CA 19-9. The assay duration for blood samples is 35 min. In our perception, the assay represents a rapid and low-cost tool for rapid determination of CA19-9 in blood that holds promise for clinical applications, particularly in limited resource settings.

We present a rapid and highly sensitive approach for visual detection of microRNA (miRNA) using a gold nanoparticles coated silica nanorod label and lateral flow strip biosensor. Gold nanoparticles were decorated on the silica nanorod surface by a seeding and growth procedure. A single strand DNA probe was immobilized on the gold nanoparticles-silica nanorod surface by a self-assembling process, and the formed DNA-gold nanoparticles-silica nanorod conjugate was used to construct the lateral flow nucleic acid biosensor for detecting miRNA. The captured gold nanoparticles-silica nanorods by sandwich-type hybridization reactions (DNA-RNA-DNA) on the test zone of the lateral flow nucleic acid biosensor produced the characteristic color bands, enabling visual detection of miRNA. After systematic optimization, the new lateral flow nucleic acid biosensor was capable of detecting 10 pM of the miRNA target without instrumentation, which is six times lower than that obtained with the gold nanoparticle-based lateral flow nucleic acid biosensor. The gold nanoparticles coated silica nanorod thus provides a new and sensitive nanolabel for visual detection of biological molecules on the lateral flow biosensor.

In this article, we describe a carbon nanotube (CNT)-based lateral flow biosensor (LFB) for rapid and sensitive detection of DNA sequence. Amine-modified DNA detection probe was covalently immobilized on the shortened multi-walled carbon nanotubes (MWCNTs) via diimide-activated amidation between the carboxyl groups on the CNT surface and amine groups on the detection DNA probes. Sandwich-type DNA hybridization reactions were performed on the LFB and the captured MWCNTs on test zone and control zone of LFB produced the characteristic black bands, enabling visual detection of DNA sequences. Combining the advantages of lateral flow chromatographic separation with unique physical properties of MWCNT (large surface area), the optimized LFB was capable of detecting of 0.1 nM target DNA without instrumentation. Quantitative detection could be realized by recording the intensity of the test line with the Image J software, and the detection limit of 40 pM was obtained. This detection limit is 12.5 times lower than that of gold nanoparticle (GNP)-based LFB (0.5 nM, Mao et al. Anal. Chem. 2009, 81, 1660-1668). Another important feature is that the preparation of MWCNT-DNA conjugates was robust and the use of MWCNT labels avoided the aggregation of conjugates and tedious preparation time, which were often met in the traditional GNP-based nucleic acid LFB. The applications of MWCNT-based LFB can be extended to visually detect protein biomarkers using MWCNT-antibody conjugates. The MWCNT-based LFB thus open a new door to prepare a new generation of LFB, and shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report a rapid and highly sensitive approach based on gold-nanoparticle-decorated silica nanorods (GNP-SiNRs) label and lateral-flow strip biosensor (LFSB) for visually detecting proteins. Owing to its biocompatibility and convenient surface modification, SiNRs were used as carriers to load numerous GNPs, and the GNP-SiNRs were used as labels for the lateral-flow assay. The LFSB detection limit was lowered 50 times compared to the traditional GNP-based lateral-flow assay. Rabbit IgG was used as a model target to demonstrate the proof-of-concept. Sandwich-type immunoreactions were performed on the immunochromatographic strips, and the accumulation of GNP-SiNRs on the test zone produced the characteristic colored bands, enabling visual detection of proteins without instrumentation. The quantitative detection was performed by reading the intensities of the colored bands with a portable strip reader. The response of the optimized device was highly linear for the range of 0.05-2 ng mL(-1), and the detection limit was estimated to be 0.01 ng mL(-1). The GNP-SiNR-based LFSB, thus, offered an ultrasensitive method for rapidly detecting trace amounts of proteins. This method has a potential application with point-of-care screening for clinical diagnostics and biomedical research.

The Upflow Anaerobic Sludge Blanket (UASB) Reactor is widely applied anaerobic wastewater treatment method all over the world. Uniform distribution of wastewater at reactor bottom is necessary to establish proper contact between sludge and wastewater. In addition, proper functioning of Gas-Liquid-Solid (GLS) separator is crucial to ensure maximum sludge retention in the reactor and to achieve maximum COD removal rate in the reactor. Hence, proper design of reactor is necessary for appropriate functioning of various components for a given wastewater flow rate and COD concentration. The design procedure for UASB reactor taking due consideration to the GLS design and design of inlet arrangement is discussed in this paper for various wastewater strength and flow rates. A software is developed to make economical design of UASB reactor for different type of wastewater by adopting maximum loading conditions, based on literature recommendations, and at the same time to satisfy all design recommendation, as far as possible.