Discovery of beta-lactamase CMY-10 inhibitors for combination therapy against multi-drug resistant Enterobacteriaceae.

ß-lactam antibiotics are the most widely used antimicrobial agents since the discovery of benzylpenicillin in the 1920s. Unfortunately, these life-saving antibiotics are vulnerable to inactivation by continuously evolving ß-lactamase enzymes that are primary resistance determinants in multi-drug resistant pathogens. The current study exploits the strategy of combination therapeutics and aims at identifying novel ß-lactamase inhibitors that can inactivate the ß-lactamase enzyme of the pathogen while allowing the ß-lactam antibiotic to act against its penicillin-binding protein target. Inhibitor discovery applied the Site-Identification by Ligand Competitive Saturation (SILCS) technology to map the functional group requirements of the ß-lactamase CMY-10 and generate pharmacophore models of active site. SILCS-MC, Ligand-grid Free Energy (LGFE) analysis and Machine-learning based random-forest (RF) scoring methods were then used to screen and filter a library of 700,000 compounds. From the computational screens 74 compounds were subjected to experimental validation in which ß-lactamase activity assay, in vitro susceptibility testing, and Scanning Electron Microscope (SEM) analysis were conducted to explore their antibacterial potential. Eleven compounds were identified as enhancers while 7 compounds were recognized as inhibitors of CMY-10. Of these, compound 11 showed promising activity in ß-lactamase activity assay, in vitro susceptibility testing against ATCC strains (E. coli, E. cloacae, E. agglomerans, E. alvei) and MDR clinical isolates (E. cloacae, E. alvei and E. agglomerans), with synergistic assay indicating its potential as a ß-lactam enhancer and ß-lactamase inhibitor. Structural similarity search against the active compound 11 yielded 28 more compounds. The majority of these compounds also exhibited ß-lactamase inhibition potential and antibacterial activity. The non-ß-lactam-based ß-lactamase inhibitors identified in the current study have the potential to be used in combination therapy with lactam-based antibiotics against MDR clinical isolates that have been found resistant against last-line antibiotics.

Applications of machine-learning methods for the discovery of NDM-1 inhibitors.

The emergence of New Delhi metal beta-lactamase (NDM-1)-producing bacteria and their worldwide spread pose great challenges for the treatment of drug-resistant bacterial infections. These bacteria can hydrolyze most ß-lactam antibacterials. Unfortunately, there are no clinically useful NDM-1 inhibitors. In the current work, we manually collected NDM-1 inhibitors reported in the past decade and established the first NDM-1 inhibitor database. Four machine-learning models were constructed using the structural and property characteristics of the collected compounds as input training set to discover potential NDM-1 inhibitors. In order to distinguish between high active inhibitors and putative positive drugs, a three-classification strategy was introduced in our study. In detail, the commonly used positive and negative divisions are converted into strongly active, weakly active, and inactive. The accuracy of the best prediction model designed based on this strategy reached 90.5%, compared with 69.14% achieved by the traditional docking-based virtual screening method. Consequently, the best model was used to virtually screen a natural product library. The safety of the selected compounds was analyzed by the ADMET prediction model based on machine learning. Seven novel NDM-1 inhibitors were identified, which will provide valuable clues for the discovery of NDM-1 inhibitors.

Epidemiology of colistin-resistant, carbapenemase-producing Enterobacteriaceae and Acinetobacter baumannii in Croatia.

Colistin is a last-resort antibiotic for the treatment of infections caused by multidrug and carbapenem-resistant Gram-negative bacteria. Colistin resistance has been emerging and multiple outbreaks have been reported in Europe and elsewhere. It has been most frequently reported in carbapenem-resistant K. pneumoniae. In this study, 24 multidrug and colistin-resistant clinical isolates (14 K. pneumoniae, one E. aerogenes, one E. cloacae, and eight A. baumannii) were collected from four hospitals in Croatia from 2013 to 2018, in order to analyse the molecular epidemiology and mechanisms of antibiotic resistance. ß-lactamase and carbapenemase genes were detected by PCR. Genotyping was done on selected isolates by rep-PCR. Whole genome sequencing (WGS) was performed to discover possible molecular mechanisms for the observed colistin resistance. All isolates, except two K. pneumoniae isolates, were extensively drug resistant. Ten out of 16 (63%) K. pneumoniae isolates possessed blaOXA-48, which is the most common carbapenem resistance gene in Croatia and in other parts of Europe. All A. baumannii isolates possessed the OXA-23-like carbapenem hydrolysing oxacillinase and five turned out to be pandrug-resistant. Colistin resistance was most likely chromosomally mediated. After sequence analysis, none of the isolates were found to possess any of the mcr gene variants. Several previously reported mutations were found in PmrB, PhoP, PhoQ, and MgrB, which are associated with colistin resistance. In the global phylogenetic analysis, DNA mutations causing mutations in the MgrB protein were present mostly in lineages comprising colistin resistant isolates, and the second most prevalent mutation (K3X) was also encountered in our isolates. In addition, based on genotyping by rep-PCR, the spread of colistin resistance is most likely to be clonal. Most importantly, the presence of colistin resistance together with carbapenemase genes in extensively drug resistant isolates poses real threats in the use of carbapenems and colistin to fight infections.

Dual-targeting potential of active constituents of Nigella sativa against FimH and CTX-M-15: A plausible therapeutic strategy against drug-resistant uropathogenic strains.

Uropathogenic strains belonging to the Enterobacteriaceae family are considered one of factors for urinary tract infections, and type 1 pilus fimbrial adhesin (FimH) and beta lactamase CTX-M-15 play crucial roles in their pathogenesis and resistance. Thus, a promising approach is to explore dual-targeting therapeutic agents that act against both FimH and CTX-M-15. In the present study, active constituents of Nigella sativa were selected on the basis of significant activity against UTIs. Molecular docking was used to target active constituents of Nigella sativa to the active sites of FimH and CTX-M-15; these included thymoquinone, dithymoquinone, carvacrol, p-cymene, thymol, thymohydroquinone and longifolene. Dithymoquinone was found to be the most potent dual inhibitor, with binding energy of -7.01 and -5.38kcal/mol against CTX-M-15 and FimH, respectively; In addition, Dithymoquinone exhibited superior activity compared to positive controls avibactam and heptyl α-D-mannopyranoside. Further molecular dynamic simulation studies were carried out to assess the stability of dithymoquinone-target protein complexes via RMSD, Rg, SASA, hydrogen bond number, and RMSF analysis. Both protein-ligand complexes were conserved and attained equilibrium at around 2.0 to 2.5 ns during 10 ns runs. These results suggest that active constituents of Nigella sativa, particularly dithymoquinone, might represent a plausible therapeutic strategy against resistant uropathogenic bacteria.

Investigation of common chemical components and inhibitory effect on GES-type ß-lactamase (GES22) in methanolic extracts of Algerian seaweeds.

This study aimed to investigate the total phenolic content (TPC), the identification of the common compounds by HPLC-ESI-MS and HPLC-ESI-MS-TOF and the inhibitory effects against class A-type ß-lactamase (GES-22 variant, produced recombinantly) in methanolic extracts (MEs) of four Algerian seaweeds [Ulva intestinalis, Codium tomentosum, Dictyota dichotoma and Halopteris scoparia]. The TPC varied among the four species, ranging between 0.93 ±â€¯0.65 and 2.66 ±â€¯1.33 mg GAEs/g DW. C.tomentosum had higher total phenol content than other seaweeds while, all of them inhibited uncompetitively GES-22 activity in a dose-dependent manner. Nitrocefin was used as chromogenic substrate to evaluate the inhibitory effect on GES-22. The methanolic extract of D.dichotoma exhibited significant inhibitory effect on GES-22 (IC50 = 13.01 ±â€¯0.046 µg/mL) more than clavulanate, sulbactam and tazobactam (classical ß-lactam inhibitors) (IC50 = 68.38 ±â€¯0.17 µg/mL, 52.68 ±â€¯0.64 µg/mL, and 29.94 ±â€¯0.01 µg/mL, respectively). IC50 of the other ME of U.intestinalis, C.tomentosum, and H.scoparia were 16.87 ±â€¯0.10 µg/mL, 16.54 ±â€¯0.048 µg/mL, and 25.72 ±â€¯0.15 µg/mL, respectively. Except H. scoparia, other three seaweed extracts showed almost two times or more inhibition on GES-22. Furthermore, four common compounds in these MEs were identified, α-linolenic acid (C18:3ω3), linoleic acid (C18:2ω6), oleic acid (C18:1ω9), the eicosanoid precursors ''arachidonic acid'' (C20:4ω6). Baicalein (C15H10O5) was identified in U.intestinalis and D.dichotoma seaweeds. The fact that all seaweed extracts inhibited the GES-22 better than commercial samples makes these seaweeds candidate for discovering new inhibitors against ß-lactamases. Besides that, they contain important components with potential health benefits.

Critical Review of Double-Carbapenem Therapy for the Treatment of Carbapenemase-Producing Klebsiella pneumoniae.

OBJECTIVE: To review the clinical data on the effectiveness and safety of double carbapenem therapy (DCT) in patients infected with carbapenemase-producing Klebsiella pneumoniae (CP-Kp). DATA SOURCES: A literature search was performed utilizing PubMed and EMBASE (from 1966 to May 2018); bibliographies of the retrieved articles were also searched. STUDY SELECTION AND DATA EXTRACTION: Articles were included if they evaluated patients with infections caused by CP-Kp and were treated with DCT. Meeting abstracts, editorials, and animal and in vitro studies were excluded. DATA SYNTHESIS: The search strategy revealed 8 case reports and 6 clinical studies (total of 171 patients) that evaluated the administration of ertapenem followed by prolonged infusions of meropenem or doripenem. Most patients were critically ill and commonly had infections in the blood, lungs, and urine. Clinical and microbiological success were reported in 70% of the patients and mortality in 24%. Adverse events, which included mostly seizures, sodium disorders, and gastrointestinal symptoms, were reported in 16 patients; none required interruption of treatment. Relevance to Patient Care and Clinical Practice: This review evaluated the clinical experience of DCT in the treatment of CP-Kp infections, based on case reports and clinical studies, for the potential role of DCT as a therapeutic option. CONCLUSION: Despite the limited studies, current data suggest that DCT may be an effective and safe strategy to treat CP-Kp. However, large randomized controlled trials are necessary to clearly define the role of DCT.

Use of Monte Carlo simulation to evaluate the efficacy of tigecycline and minocycline for the treatment of pneumonia due to carbapenemase-producing Klebsiella pneumoniae.

BACKGROUND: Pneumonia caused by carbapenemase-producing Klebsiella pneumoniae (CP-KP) are increasingly encountered in hospitals worldwide, causing high mortality due to lack of treatment options. The goal of this study was to assess the efficacy of tigecycline and minocycline for CP-KP hospital-acquired pneumonia (HAP) by using Monte Carlo simulation. METHODS: A total of 164 non-duplicated CP-KP strains were collected from sputum or blood in patients with HAP. The MICs for antimicrobials were determined by the agar dilution method. A 10,000-patient Monte Carlo Simulation based on a PK/PD model incorporating the MICs and population pharmacokinetic parameters were conducted to calculate probability of target attainment (PTA) at each MIC value and total cumulative fraction of response (CFR). RESULTS: The susceptibility rate of tigecycline and minocycline were 79.9% and 41.5%, respectively. At recommended doses, an optimal PTA of 90% was obtained for treating HAP caused by CP-KP with MICs of tigecycline ≤0.5 mg/L or minocycline ≤4 mg/L. The CFR of tigecycline at the recommended dose and double dose (100 mg q12h) were 71.2% and 90.2%, respectively. The CFR of minocycline at recommended dose and double dose (200 mg q12h) was 53.4% and 77.2%, respectively. CONCLUSIONS: The findings of this study suggest that the recommended dose of tigecycline was not effective in HAP caused by CP-KP, and a higher CFR indicating a better clinical efficacy can be gained by doubling the dose (100 mg q12h). minocycline (200 mg q12h) might be a potential alternative of tigecycline to against strains with MICs ≤ 8 mg/L.

ESBL E coli and P. aeruginosa Resistance to Ceftolozane-Tazobactam in a Patient with a Liver Abscess. The Search for an Omnipotent Antibiotic Goes On!

Multi drug resistant (MDR) Pseudomonas aeruginosa and Extended- Spectrum-lactamase (ESBL) Enterobacteriaceae are becoming an increasing difficult clinical problem. Immediate resistance to some of the new antimicrobials such as ceftolozane/tazobactam is unusual and is due to a variety of mechanisms such as hyper-production of inactivating enzymes and gene mutation. In addition, previous antimicrobial administration is a well-recognized risk factor to develop resistance. We present a patient with a liver abscess where the organism was resistant to ceftolozane/tazobactam resulting in a poor clinical outcome.

"Double carbapenem" and oral fosfomycin for the treatment of complicated urinary tract infections caused by blaNDM -harboring Enterobacteriaceae in kidney transplantation.

Infections with carbapenemase-producing carbapenem-resistant Enterobacteriaceae represent an emergent problem worldwide. Treatment of infections caused by New Delhi metallo-beta-lactamase (NDM)-harboring Enterobacteriaceae is particularly challenging as it frequently involves the use of nephrotoxic agents, which is problematic in kidney transplant recipients and non-renal transplant patients with marginal kidney function. We present two cases of urinary tract infections caused by NDM-harboring Enterobacteriaceae successfully treated with a combination of "double carbapenem" and oral fosfomycin.