Transcending the challenge of evolving resistance mechanisms in Pseudomonas aeruginosa through ß-lactam-enhancer-mechanism-based cefepime/zidebactam.

Multi-drug resistant (MDR) Pseudomonas aeruginosa harbor a complex array of ß-lactamases and non-enzymatic resistance mechanisms. In this study, the activity of a ß-lactam/ß-lactam-enhancer, cefepime/zidebactam, and novel ß-lactam/ß-lactamase inhibitor combinations was determined against an MDR phenotype-enriched, challenge panel of P. aeruginosa (n = 108). Isolates were multi-clonal as they belonged to at least 29 distinct sequence types (STs) and harbored metallo-ß-lactamases, serine ß-lactamases, penicillin binding protein (PBP) mutations, and other non-enzymatic resistance mechanisms. Ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/relebactam, and cefepime/taniborbactam demonstrated MIC90s of >128 mg/L, while cefepime/zidebactam MIC90 was 16 mg/L. In a neutropenic-murine lung infection model, a cefepime/zidebactam human epithelial-lining fluid-simulated regimen achieved or exceeded a translational end point of 1-log10 kill for the isolates with elevated cefepime/zidebactam MICs (16-32 mg/L), harboring VIM-2 or KPC-2 and alterations in PBP2 and PBP3. In the same model, to assess the impact of zidebactam on the pharmacodynamic (PD) requirement of cefepime, dose-fractionation studies were undertaken employing cefepime-susceptible P. aeruginosa isolates. Administered alone, cefepime required 47%-68% fT >MIC for stasis to ~1 log10 kill effect, while cefepime in the presence of zidebactam required just 8%-16% for >2 log10 kill effect, thus, providing the pharmacokinetic/PD basis for in vivo efficacy of cefepime/zidebactam against isolates with MICs up to 32 mg/L. Unlike ß-lactam/ß-lactamase inhibitors, ß-lactam enhancer mechanism-based cefepime/zidebactam shows a potential to transcend the challenge of ever-evolving resistance mechanisms by targeting multiple PBPs and overcoming diverse ß-lactamases including carbapenemases in P. aeruginosa.IMPORTANCECompared 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 ß-lactam and ß-lactamase inhibitor combinations. This study shows that the novel ß-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 ß-lactam enhancer mechanism is a promising alternative to the conventional ß-lactam/ß-lactamase inhibitor approach in combating ever-evolving MDR P. aeruginosa.

Investigations on In Vivo Pharmacokinetic/Pharmacodynamic Determinants of Fosfomycin in Murine Thigh and Kidney Infection Models.

Background: Amidst the era of widespread resistance, there has been a renewed interest in older antibiotics such as fosfomycin, owing to its activity against certain resistant Gram-negative pathogens, including multidrug-resistant variants expressing extended spectrum ß-lactamases or carbapenemases. The goal of the study was to investigate pharmacokinetic/pharmacodynamic (PK/PD) index and PK/PD targets of fosfomycin in murine thigh and kidney infection models, employing clinical isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Methods: Seven isolates of E. coli (one wild-type and six clinical isolates) and five isolates of K. pneumoniae (one wild-type and four clinical isolates) were utilized for in vivo PK/PD studies. Single-dose plasma PK studies were conducted in infected mice by subcutaneous route. PD index was determined from exposure-response analysis employing 24-hr dose fractionation studies in neutropenic murine thigh infection model, while pharmacodynamic targets (PDTs) were derived from both thigh and kidney infection models. Results: Dose fractionation studies demonstrated that in vivo efficacy of fosfomycin best correlated with AUC/MIC for E. coli (R2 = 0.9227) and K. pneumoniae (R2 = 0.8693). The median AUC/MIC linked to 1 log10 kill effects were 346.2 and 745.2 in thigh infection model and 244.1 and 425.4 in kidney infection model for E. coli and K. pneumoniae, respectively. The mice plasma protein binding of fosfomycin was estimated to be 5.4%. Conclusions: The in vivo efficacy of fosfomycin against Enterobacterales was best described by AUC/MIC. The PDTs derived from this study may help define the coverage potential of fosfomycin at the clinical doses approved.

WCK 4034: A promising oxazolidinone for treating gram positive infections.

Increased resistance to gram positive infections have highlighted the limitations of currently available drug treatments including penicillins, macrolides and glycopeptides. As an alternative to address these challenges; Linezolid, the first antibiotic from oxazolidinone class, have shown the promising activities against such infections, although associated toxicological issues limiting the use of linezolid for prolonged treatments. In order to circumvent disadvantages allied with the marketed drugs, we herein reporting the synthesis of WCK 4034, an oxazolidinone antibiotic through our structure activity relationship (SAR) program. Through this exercise, WCK 4034, has shown competitive MIC values against Methicillin Sensitive S. aureus (MSSA, Sta-001), Methicillin Resistant S. aureus (MRSA, Sta-032), S. pneumoniae ATCC 49619 and H. influenza ATCC 35054 species as like linezolid. Although with an additional advantage; WCK 4034 has been found superior during dog PK studies as compare to Linezolid. With the preliminary studies in our hand, we herein assuming these improved pharmacokinetic values would be helpful. Moreover, WCK 4034 has successfully completed pre-clinical studies and ready to enter the clinical space, and paved the way for in house development of other oxazolidinone NCEs.

WCK 1152, WCK 1153: Discovery and structure activity relationship for the treatment of resistant pneumococcal and staphylococcal respiratory infections.

Novel antibacterial agents needed constantly to counter the ever emergent resistance development to commercially available drugs; one of the effective synthetic antibacterial classes is fluoroquinolone (FQ). This study includes structure activity relationship based design and synthesis of novel fluoroquinolone molecules active against resistant pathogens bearing mutations of DNA gyrase and/or topoisomerase IV which also express efflux pumps. Here, series of compounds were prepared by treating 1-cyclopropyl-6,7-difluoro-8-methoxy-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid as a core with various 4-substituted-3,3-dialkyl piperidines as side chains, through conventional synthetic approaches. Subsequently, antibacterial activities of these fluoroquinolones were examined against Streptococcus pneumoniae, SPN 5844 (Moxi resistant DNA gyrase and topo IV mutant) and SPN 706 (FQ efflux positive). The current manuscript covers >50 examples of fluoroquinolone NCEs, amongst 20 NCEs have shown MIC in the range of (0.4 to >6.25 µg/ml) for SPN 5844 and (0.1-12.5 µg/ml) for SPN 706 strains. During the course of this study; WCK 919, comprising two chiral isomers; WCK 1152 and WCK 1153 were emerged as lead among the different series synthesized. Advance studies suggested either WCK 1152 or WCK 1153 are the worthy candidates for further clinical developments for respiratory infections caused by resistant pneumococci and staphylococci. However, on the basis of in house preclinical work, WCK 1152 had been selected for phase-1 domestic clinical trials.

Ensuring robustness in the quality of antibiotic susceptibility test discs for four novel antibiotics.

Antibiotic susceptibility test (AST) discs are used as an in-vitro diagnostic tool to select the appropriate antibiotic to treat an infection. Generally, the concentration of the drug loaded on to the AST discs is measured by studying its activity against quality control organisms. This methodology has several limitations-it is time consuming, requires trained manpower, has a wider acceptance criteria of zone of inhibitions-causing ambiguity in judging smaller variations in drug concentration. To overcome these issues, we have developed and validated high-performance liquid chromatographic (HPLC) methods for the determination of strength of AST discs for in-house researched antibiotics, namely Levonadifloxacin/WCK 771, Nafithromycin/WCK 4873, Cefepime-Tazobactam/WCK 4282, and Cefepime-Zidebactam/WCK 5222. The drugs were extracted from the AST discs using an appropriate solvent. The developed methods are simple, accurate, precise, reproducible, rugged, and robust. They are efficient in terms of time, and can be easily conducted in a quality control laboratory during release as well as stability evaluation of AST disc. Application of HPLC methods for the determination of strength of AST discs ensures flawless quality and, consequently, a better selection of drugs to treat bacterial infections in clinics.

Design and synthesis of an oral prodrug alalevonadifloxacin for the treatment of MRSA infection.

Levonadifloxacin is a parenteral anti-MRSA benzoquinolizine antibacterial drug recently launched as, EMROK in India to treat acute bacterial skin and skin structure infections (ABSSSI) in hospitalized patients. As a step down therapy an oral form of levonadifloxacin with comparable PK/PD was needed because the levonadifloxacin exhibits very poor oral absorption. To improve the drugability in terms of oral absorption a pro-drug approach was evaluated. Structurally levonadifloxacin provides two sites amenable for ester or amide formation, a carboxyl function of benzoquinolizine pharmacophore and hydroxyl group on piperidine side chain. Several aliphatic, aromatic and amino acid esters of C-2 carboxylic acid, C-4-hydroxyl piperidine and double esters at both C-2, C-4 positions were synthesized. The cleavage of prodrugs was studied in vitro as well as in animal models to access their suitability as prodrug function. Among C-2 carboxylic ester prodrugs, daloxate (WCK 2320) showed highest cleavage in serum as well as in liver enzyme; however its stability in aqueous solution was unfavorable. In contrast, most of the esters at the hydroxyl group like propionyl ester (WCK 2305) and amino acid esters such as l-alanine (WCK 2349), l-valine (WCK 2630) were cleaved readily releasing active drug. Thus, indicating C-4-hydroxyl piperidine was amenable site for enzymatic cleavage over esters of C-2 carboxylic acid. Additionally, amino acid esters provided an opportunity to make salt, facilitating improved aqueous solubility. Methanesulfonate salt of l-alanine ester of levonadifloxacin (WCK 2349) was successfully developed and launched as oral prodrug alalevonadifloxacin (EMROK-O).

In vitro evolution of cefepime/zidebactam (WCK 5222) resistance in Pseudomonas aeruginosa: dynamics, mechanisms, fitness trade-off and impact on in vivo efficacy.

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.

Activity of ß-lactam plus ß-lactam-enhancer combination cefepime/zidebactam against Klebsiella pneumoniae harbouring defective OmpK35/36 porins and carbapenemases.

Cefepime/zidebactam is a ß-lactam/ß-lactam-enhancer based novel antibiotic which is in clinical development for treating infections caused by multidrug-resistant Gram-negative bacteria. Here, in vitro activity of cefepime/zidebactam was determined against multicentre Klebsiella pneumoniae clinical isolates co-expressing serine and/or metallo-carbapenemases and defective OmpK35 and OmpK36 porins. The MICs were determined using the reference broth microdilution method. Outer membrane protein expression was assessed using SDS-PAGE and mutations in the genes encoding OmpK35 and OmpK36 were identified by DNA sequencing. Among 34 isolates studied, carbapenemase genes, blaKPC and blaOXA-48-like, were present in 18 and 11 isolates, respectively; 5 isolates harboured both blaOXA-48-like and blaNDM. Point mutations, insertions, and duplications in OmpK35 and OmpK36, which are known to impact the activity of carbapenems, were detected. Against these isolates, cefepime/zidebactam (1:1) showed a consistent activity (MICs ≤4 mg/L). In conclusion, cefepime/zidebactam overcomes enzymatic, and non-enzymatic carbapenem-impacting resistance mechanisms concurrently expressed in K. pneumoniae.

In Vitro Activity of Fosfomycin and Nitrofurantoin Against Contemporary Enterobacterales Pathogens Isolated from Indian Tertiary Care Hospitals.

Background: In India, multidrug resistance in community and hospital associated Gram-negative pathogens has increased sharply over the past few years. In the absence of novel oral multidrug resistant-pathogen active therapies, the therapeutic situation with regard to community infections is even more challenging. Hence, the focus is now shifting toward potentially expanding the utility of older antibiotics such as fosfomycin and nitrofurantoin beyond their approved pathogen coverage. The current study was undertaken to assess the activity of fosfomycin and nitrofurantoin against Enterobacterales pathogens through minimum inhibitory concentration (MIC) determination to facilitate monitoring future shifts in susceptibility to these agents. Materials and Methods: The present study used 1,350 Enterobacterales, recently collected from various Indian tertiary care hospitals and preserved at Wockhardt Strain Repository. The MIC50/90 for fosfomycin and nitrofurantoin and the comparator antibiotics was determined for Escherichia coli (N = 470), Klebsiella pneumoniae (N = 429), Enterobacter spp., (N = 144), Proteus spp. (N = 262), and Citrobacter spp. (N = 45), using Clinical and Laboratory Standards Institute recommended agar dilution method. Results: Applying E. coli breakpoints, the susceptibility rates of fosfomycin for E. coli, K. pneumoniae, Enterobacter spp., Proteus spp., and Citrobacter spp., were 95.5%, 53.2%, 71.5%, 76.7%, and 91.1%, respectively. Applying respective breakpoints, the susceptibility rates of comparator drugs, including meropenem, were lower than fosfomycin. Susceptibility of nitrofurantoin for E. coli and Citrobacter isolate was 83%, while limited coverage (<13.2% susceptibility) was observed for other genera. Conclusion: Amidst widespread resistance, a > 70% fosfomycin susceptibility observed for clinical isolates, including strains expressing carbapenemases, is encouraging and supports conducting additional susceptibility and pharmacokinetic/pharmacodynamic studies to explore its potential for expanded therapeutic use. Nitrofurantoin activity spectrum was restricted to E. coli and Citrobacter spp. and, therefore, offers a relatively limited therapeutic scope.

In vitro activity of cefepime/zidebactam (WCK 5222) against recent Gram-negative isolates collected from high resistance settings of Greek hospitals.

Cefepime/zidebactam is in clinical development for the treatment of carbapenem-resistant Gram-negative infections. MICs of cefepime/zidebactam (1:1) and comparators against Enterobacterales (n = 563), Pseudomonas (n = 172) and Acinetobacter baumannii (n =181) collected from 15 Greek hospitals (2014-2018) were determined by reference broth microdilution method. The isolates exhibited high carbapenem resistance rates [(Enterobacterales (75%), Pseudomonas (75%) and A. baumannii (98.3%)]. Cefepime/zidebactam showed MIC50/90 of 0.5/2 mg/L, against Enterobacterales including metallo-ß-lactamases (MBL)-producers. Reduced susceptibility rates to tigecycline (16.8%), colistin (47.4%), ceftazidime/avibactam (59.8%), and imipenem/relebactam (61%) indicated high prevalence of multi-drug resistance among Greek Enterobacterales. Cefepime/zidebactam exhibited MIC50/90 of 8/16 mg/L against Pseudomonas including MBL-producers. The MIC50/90 of ceftazidime/avibactam and imipenem/relebactam were high (≥32 mg/L). Cefepime/zidebactam showed MIC90 of 64 mg/L against A. baumannii which is within its therapeutic scope. Other antibiotics including colistin showed limited activity against A. baumannii. The activity of cefepime/zidebactam against multi-drug-resistant isolates is attributable to zidebactam mediated novel ß-lactam-enhancer mechanism.

Structural Characterization of Diazabicyclooctane ß-Lactam "Enhancers" in Complex with Penicillin-Binding Proteins PBP2 and PBP3 of Pseudomonas aeruginosa.

Multidrug-resistant (MDR) pathogens pose a significant public health threat. A major mechanism of resistance expressed by MDR pathogens is ß-lactamase-mediated degradation of ß-lactam antibiotics. The diazabicyclooctane (DBO) compounds zidebactam and WCK 5153, recognized as ß-lactam "enhancers" due to inhibition of Pseudomonas aeruginosa penicillin-binding protein 2 (PBP2), are also class A and C ß-lactamase inhibitors. To structurally probe their mode of PBP2 inhibition as well as investigate why P. aeruginosa PBP2 is less susceptible to inhibition by ß-lactam antibiotics compared to the Escherichia coli PBP2, we determined the crystal structure of P. aeruginosa PBP2 in complex with WCK 5153. WCK 5153 forms an inhibitory covalent bond with the catalytic S327 of PBP2. The structure suggests a significant role for the diacylhydrazide moiety of WCK 5153 in interacting with the aspartate in the S-X-N/D PBP motif. Modeling of zidebactam in the active site of PBP2 reveals a similar binding mode. Both DBOs increase the melting temperature of PBP2, affirming their stabilizing interactions. To aid in the design of DBOs that can inhibit multiple PBPs, the ability of three DBOs to interact with P. aeruginosa PBP3 was explored crystallographically. Even though the DBOs show covalent binding to PBP3, they destabilized PBP3. Overall, the studies provide insights into zidebactam and WCK 5153 inhibition of PBP2 compared to their inhibition of PBP3 and the evolutionarily related KPC-2 ß-lactamase. These molecular insights into the dual-target DBOs advance our knowledge regarding further DBO optimization efforts to develop novel potent ß-lactamase-resistant, non-ß-lactam PBP inhibitors.IMPORTANCE Antibiotic resistance is a significant clinical problem. Developing novel antibiotics that overcome known resistance mechanisms is highly desired. Diazabicyclooctane inhibitors such as zidebactam possess this potential as they readily inactivate penicillin-binding proteins, yet cannot be degraded by ß-lactamases. In this study, we characterized the inhibition by diazabicyclooctanes of penicillin-binding proteins PBP2 and PBP3 from Pseudomonas aeruginosa using protein crystallography and biophysical analyses. These structures and analyses help define the antibiotic properties of these inhibitors, explain the decreased susceptibility of P. aeruginosa PBP2 to be inhibited by ß-lactam antibiotics, and provide insights that could be used for further antibiotic development.

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of nafithromycin, a next generation lactone ketolide antibiotic.

Nafithromycin is a next generation lactone ketolide antibiotic slated to enter phase III clinical development in India for the treatment of CABP as a shorter 800 mg-OD X3 day therapeutic regimen. Nafithromycin exhibits potent activity against MDR Streptococcus pneumoniae including erythromycin and telithromycin-resistant resistant strains. Older macrolides/ketolides are reported to be potent inhibitors of CYP3A4/5. To facilitate comparative assessment of drug-drug interaction potential, CYP inhibitory activities of nafithromycin was evaluated in comparison with clarithromycin, telithromycin, cethromycin and solithromycin. CYP inhibitory activities were assessed against key CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and CYP3A4/5) using human liver microsomes. Additionally, time-dependent inhibition (TDI), metabolism-based inhibition (MBI) and k inact /K I activities were also investigated for CYP3A4/5. Nafithromycin did not inhibit key CYP enzymes and was found to be a weak inhibitor of CYP3A4/5. Comparator antibiotics were found to be potent inhibitors with 2- to 50-fold leftward shifts in CYP3A4/5 IC50 values, while such shift was not noted for nafithromycin. k inact /K I ratio of nafithromycin was 3- to 153-fold lower than comparator drugs, further substantiating its lower affinity for CYP3A4/5. In sum, weaker inhibition and lower k inact /K I ratio for CYP3A4/5, points towards nafithromycin's lower propensities towards clinical drug-drug interactions as compared to other macrolides/ketolides antibiotics.

Activity of cefepime/zidebactam against MDR Escherichia coli isolates harbouring a novel mechanism of resistance based on four-amino-acid inserts in PBP3.

BACKGROUND: Recent reports reveal the emergence of Escherichia coli isolates harbouring a novel resistance mechanism based on four-amino-acid inserts in PBP3. These organisms concomitantly expressed ESBLs or/and serine-/metallo-carbapenemases and were phenotypically detected by elevated aztreonam/avibactam MICs. OBJECTIVES: The in vitro activities of the investigational antibiotic cefepime/zidebactam and approved antibiotics (ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/relebactam and others) were determined against E. coli isolates harbouring four-amino-acid inserts in PBP3. METHODS: Whole-genome sequenced E. coli isolates (n = 89) collected from a large tertiary care hospital in Southern India (n = 64) and from 12 tertiary care hospitals located across India (n = 25) during 2016-18, showing aztreonam/avibactam MICs ≥1 mg/L (≥4 times the aztreonam epidemiological cut-off) were included in this study. The MICs of antibiotics were determined using the reference broth microdilution method. RESULTS: Four-amino-acid inserts [YRIK (n = 30) and YRIN (n = 53)] were found in 83/89 isolates. Among 83 isolates, 65 carried carbapenemase genes [blaNDM (n = 39), blaOXA-48-like (n = 11) and blaNDM + blaOXA-48-like (n = 15)] and 18 isolates produced ESBLs/class C ß-lactamases only. At least 16 unique STs were noted. Cefepime/zidebactam demonstrated potent activity, with all isolates inhibited at ≤1 mg/L. Comparator antibiotics including ceftazidime/avibactam and imipenem/relebactam showed limited activities. CONCLUSIONS: E. coli isolates concurrently harbouring four-amino-acid inserts in PBP3 and NDM are an emerging therapeutic challenge. Assisted by the PBP2-binding action of zidebactam, the cefepime/zidebactam combination overcomes both target modification (PBP3 insert)- and carbapenemase (NDM)-mediated resistance mechanisms in E. coli.

Assessment of in vitro inhibitory effects of novel anti MRSA benzoquinolizine fluoroquinolone WCK 771 (levonadifloxacin) and its metabolite on human liver cytochrome P450 enzymes.

WCK 771 (INN: levonadifloxacin) is a novel antibacterial agent belonging to benzoquinolizine subclass of fluoroquinolones which is under clinical development as a parenteral formulation and its prodrug WCK 2349 (INN: alalevonadifloxacin) as an oral option. Both the drugs have been approved recently in India based on phase III trial completed for ABSSSI.In vitro CYP inhibition potential of levonadifloxacin and its sulfate metabolite (WCK 2146) were assessed in this study. The inhibitory effects of levonadifloxacin and its sulfate metabolite were assessed for seven key human liver CYP isoforms 1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 using human liver microsome (HLM) employing validated LC-MS/MS method.The results showed that levonadifloxacin and its metabolite did not inhibit enzyme activity of any of the key CYP isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) even at supra therapeutic concentrations (12-24X, Clinical Cmax: 25-35µg/mL).These in vitro CYP inhibition studies of levonadifloxacin and its sulfate metabolite indicate lack of potential for pharmacokinetic drug interactions of levonadifloxacin when co-administered with drugs which are substrate of these isoforms. Therefore, further clinical studies evaluating CYP mediated drug-drug interactions are not warranted for levonadifloxacin and alalevonadifloxacin.

Pretreatment of nafithromycin attenuates inflammatory response in murine lipopolysaccharide induced acute lung injury.

Acute respiratory distress syndrome following an acute lung injury (ALI) is a life threatening inflammatory condition predominantly characterized by vascular protein leakage, neutrophil recruitment and overexpression of proinflammatory cytokines. Pulmonary and systemic bacterial infections are the major cause of ALI wherein the bacterial cell components play a crucial role. Macrolide/ketolide antibiotics are reported to possess immunomodulatory activity; as a result improved survival has been noted in pneumonia patients. Hence immunomodulatory activity of nafithromycin, a novel lactone ketolide antibacterial agent was assessed in the murine LPS induced ALI model. Vehicle, nafithromycin (100 mg/kg), azithromycin (600 mg/kg) and dexamethasone (20 mg/kg) were administered orally, 1 h prior to LPS challenge and bronchoalveolar lavage (BAL) fluid was collected thereafter at 18, 24 and 48 h to determine the total cell count, total protein, myeloperoxidase (MPO), tumor necrosis factor (TNF)-α and interleukin (IL)-6. Results from the current study showed that pretreatment with nafithromycin significantly reduced the total cell count, total protein, MPO, TNF-α and IL-6 levels in BAL fluid compared to LPS control group. Histopathological evaluations also suggest significant reduction in neutrophil infiltration by nafithromycin. Dexamethasone, a positive reference standard as expected exhibited potent anti-inflammatory activity. The immunomodulatory effect of nafithromycin at dose of 100 mg/kg was comparable to azithromycin dosed at 600 mg/kg. As a result of immunomodulatory activity, nafithromycin is expected to provide additional clinical benefits by resolving the secondary complications associated with severe pneumonia and thereby improving survival in such patients.

Levonadifloxacin, a Novel Benzoquinolizine Fluoroquinolone, Modulates Lipopolysaccharide-Induced Inflammatory Responses in Human Whole-Blood Assay and Murine Acute Lung Injury Model.

Fluoroquinolones are reported to possess immunomodulatory activity; hence, a novel benzoquinolizine fluoroquinolone, levonadifloxacin, was evaluated in lipopolysaccharide-stimulated human whole-blood (HWB) and mouse acute lung injury (ALI) models. Levonadifloxacin significantly mitigated the inflammatory responses in an HWB assay through inhibition of proinflammatory cytokines and in the ALI model by lowering lung total white blood cell count, myeloperoxidase, and cytokine levels. The immunomodulatory effect of levonadifloxacin, along with promising antibacterial activity, is expected to provide clinical benefits in the treatment of infections.

Effective inhibition of PBPs by cefepime and zidebactam in the presence of VIM-1 drives potent bactericidal activity against MBL-expressing Pseudomonas aeruginosa.

OBJECTIVES: The combination of cefepime and the novel ß-lactam enhancer zidebactam (WCK 5222) is under development for the treatment of difficult-to-treat Gram-negative infections. Against MBL-producing pathogens, cefepime and zidebactam induce cell elongation and spheroplast formation, indicating PBP3 and PBP2 dysfunction, respectively, having a potent bactericidal effect as a combination. The objective of the present study was to determine the mechanistic basis of the bactericidal effect of cefepime/zidebactam on MBL-expressing pathogens. METHODS: Pseudomonal PBP-binding affinities of cefepime, zidebactam and imipenem were assessed at different timepoints and also in the presence of purified VIM-1 using a Bocillin FL competition assay. The antibacterial activity of cefepime/zidebactam against three VIM-expressing Pseudomonas aeruginosa isolates was assessed by time-kill and neutropenic mouse lung/thigh infection studies. RESULTS: Amidst cefepime-hydrolysing concentrations of VIM-1, substantial cefepime binding to target PBPs was observed. High-affinity binding of zidebactam to PBP2 remained unaltered in the presence of VIM-1; however, MBL addition significantly affected imipenem PBP2 binding. Furthermore, the rate of cefepime binding to the primary target PBP3 was found to be higher compared with the imipenem PBP2 binding rate. Finally, complementary PBP inhibition by cefepime/zidebactam resulted in enhanced bactericidal activity in time-kill and neutropenic mouse lung/thigh infection studies against VIM-6-, VIM-10- and VIM-11-expressing P. aeruginosa, thus revealing the mechanistic basis of ß-lactam enhancer action. CONCLUSIONS: For the first time ever (to the best of our knowledge), this study demonstrates that in the presence of VIM-1 MBL, ß-lactamase-labile cefepime and ß-lactamase-stable zidebactam produce effective inhibition of respective target PBPs. For cefepime, this seems to be a result of a faster rate of PBP binding, which helps it overcome ß-lactamase-mediated hydrolysis.

In vitro activity of a novel antibacterial agent, levonadifloxacin, against clinical isolates collected in a prospective, multicentre surveillance study in India during 2016-18.

BACKGROUND: Levonadifloxacin is a novel antibiotic belonging to the benzoquinolizine subclass of fluoroquinolones with potent activity against MRSA and quinolone-resistant Staphylococcus aureus. IV levonadifloxacin and its oral prodrug alalevonadifloxacin have recently been approved in India for the treatment of acute bacterial skin and skin structure infections (ABSSSIs) including diabetic foot infections. OBJECTIVES: To investigate the in vitro activity of levonadifloxacin against contemporary clinical isolates collected from multiple tertiary care hospitals across India in the Antimicrobial Susceptibility Profiling of Indian Resistotypes (ASPIRE) surveillance study. METHODS: A total of 1376 clinical isolates, consisting of staphylococci (n = 677), streptococci (n = 178), Enterobacterales (n = 320), Pseudomonas aeruginosa (n = 140) and Acinetobacter baumannii (n = 61), collected (2016-18) from 16 tertiary hospitals located across 12 states in India, were included in the study. The MICs of levonadifloxacin and comparator antibiotics were determined using the reference agar dilution method and broth microdilution method. RESULTS: Levonadifloxacin exhibited potent activity against MSSA (MIC50/90: 0.5/1 mg/L), MRSA (MIC50/90: 0.5/1 mg/L) and levofloxacin-resistant S. aureus (MIC50/90: 1/1 mg/L) isolates. Similarly, potent activity of levonadifloxacin was also observed against CoNS including MDR isolates (MIC50/90: 1/2 mg/L). Against Streptococcus pneumoniae, levonadifloxacin (MIC50/90: 0.5/0.5 mg/L) showed superior activity compared with levofloxacin (MIC50/90: 1/2 mg/L). Among levofloxacin-susceptible Enterobacterales, 80.6% of isolates were inhibited at ≤2 mg/L levonadifloxacin. CONCLUSIONS: Levonadifloxacin displayed potent activity against contemporary MRSA and fluoroquinolone-resistant staphylococcal isolates, thus offering a valuable IV as well as an oral therapeutic option for the treatment of ABSSSIs. Furthermore, levonadifloxacin exhibited a broad-spectrum activity profile as evident from its activity against streptococci and levofloxacin-susceptible Gram-negative isolates.