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.

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.

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.

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.

"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.

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.

Reversal of resistance in bacteria underlies synergistic effect of essential oils with conventional antibiotics.

The pervasive of bacterial resistance earnestly threaten the prevention and the treatment of infectious diseases. Therefore, scientific communities take precedence over development of new antimicrobial agents. The aim of the study was to determine antimicrobial potency of three North-African essential oils Pituranthos chloranthus, Teucruim ramosissimum and Pistacia lentiscus individually, and in combination with antibiotics, to inhibit the growth of highly resistant clinical pathogen. Bacteria clinically isolated from patients, subsequently, challenged to a panel of drugs to determine the antibiotic-resistance profiles. Drugs displaying clinically irrelevant CMI were subjected to further studies in order to rescue antibiotic actions. Singular activity of essential oils and activity when combined with an antibiotic was hence elucidated. The results obtained highlighted the occurrence of strong antibacterial potential of essential oils when administrated alone. In the interactive experiment essential oils were found highly effective in reducing the resistance of Methicillin-resistant Staphylococcus aureus to amoxicillin, tetracycline, piperacillin, ofloxacin and oxacillin and resistance of Acinetobacter baumannii to amoxicillin and to ofloxacin in interactive manner. Furthermore, the results proved synergism among essential oils and both antibiotics ofloxacin and novobiocin against the Extended-Spectrum Beta-Lactamase producing E. coli (ESBL). Time kill kinetics was performed with a combination of sub-inhibitory concentrations to confirm the efficiency and killing rate of the combination over time. Further, the hypothetical toxicity of essential oils against human keratinocytes HaCat and murine spleenocytes were examined. The chemical composition of essential oils was assessed by GC/MS analysis and the major constituents found were sabinene, limonene, terpinen-4-ol, and ß-eudesmol.

Single and joint antibacterial activity of aqueous garlic extract and Manuka honey on extended-spectrum beta-lactamase-producing Escherichia coli.

Background: Multidrug resistance and recent technological advances have renewed interest in natural product drug discovery from ancient remedies such as Allium sativum (garlic) and honey. This study assessed antibacterial activity of aqueous garlic extract (AGE) and Manuka honey on extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. Methods: Thirty clinical isolates of E. coli were collected and screened for ESBL production by double-disc synergy test. Single and joint antibacterial activity of AGE and Manuka honey against ESBL-producing E. coli were determined by agar well dilution and checkerboard methods, respectively. Results: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AGE ranged from 125-250 mg/mL and 250-500 mg/mL, respectively. MIC and MBC of Manuka honey ranged from 12.5-25% v/v and 25-50% v/v, respectively. The combination of AGE and Manuka honey exhibited different effects on selected ESBL-producing E. coli; synergism (1/4H+1/16G), additive (1/8H+1/2G, 1/2H+1/16G), indifference (1/16H+MICG, MICH+1/16G, 2MICH+1/32G) and antagonism (4MICH+1/32G). Conclusions: Manuka honey exhibited greater antibacterial activity against ESBL-producing E. coli than AGE. Antibacterial activity, and the interaction of AGE and Manuka honey against ESBL-producing E. coli are dependent on their concentration. Studies assessing antibacterial activity of potent phytochemicals in AGE and honey would provide insights to mechanisms of interaction for development of new drug leads.

Empiric Antibiotic Therapy of Nosocomial Bacterial Infections.

Broad-spectrum antibiotics are commonly used by physicians to treat various infections. The source of infection and causative organisms are not always apparent during the initial evaluation of the patient, and antibiotics are often given empirically to patients with suspected sepsis. Fear of attempting cephalosporins and carbapenems in penicillin-allergic septic patients may result in significant decrease in the spectrum of antimicrobial coverage. Empiric antibiotic therapy should sufficiently cover all the suspected pathogens, guided by the bacteriologic susceptibilities of the medical center. It is important to understand the major pharmacokinetic properties of antibacterial agents for proper use and to minimize the development of resistance. In several septic patients, negative cultures do not exclude active infection and positive cultures may not represent the actual infection. This article will review the important differences in the spectrum of commonly used antibiotics for nosocomial bacterial infections with a particular emphasis on culture-negative sepsis and colonization.

The ongoing battle against multi-resistant strains: in-vitro inhibition of hospital-acquired MRSA, VRE, Pseudomonas, ESBL E. coli and Klebsiella species in the presence of plant-derived antiseptic oils.

The fight against hospital-acquired infections involving antibiotic-resistant microorganisms has become of critical concern to surgeons worldwide. In addition to the development of new effective antibiotic chemotherapy, exploration of 'forgotten' topical antibacterial agents from the pre-antibiotic era has recently gained new attention. We report the promising efficacy of plant-derived antiseptic oils used in traditional aboriginal and south-east Asian treatments such as Lemongrass, Eucalyptus and Tea Tree Oil in the inhibition of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), multi-resistant Pseudomonas aeruginosa, ESBL-producing Escherichia coli and Klebsiella pneumoniae in the in-vitro setting. Large consistent zones of inhibition were observed for all three plant-derived oils tested in an agar diffusion test. The commonly used antibacterial agents chlorhexidine 0.1%, and ethanol (70%), and standard olive oil consistently demonstrated notably lower or no efficacy in regard to growth inhibition of strains. Notably, Lemongrass oil proved to be particularly active against gram-positive bacteria, while Tea Tree oil showed superior inhibition of gram-negative microorganisms. As proven in vitro, plant-derived antiseptic oils may represent a promising and affordable topical agent to support surgical treatment against multi-resistant and hospital-acquired infections.

CTX-M-15 extended-spectrum beta-lactamases in Enterobacteriaceae in the intensive care unit of Tlemcen Hospital, Algeria.

The aim of this study was to detect extended-spectrum beta-lactamases (ESBL) in Enterobacteriaceae isolates in the intensive care unit (ICU) of Tlemcen hospital in north-western Algeria. Antimicrobial susceptibility testing, molecular typing, characterization of ESBL-encoding genes and the genetic environment, conjugation experiments and plasmid analysis were carried out. In all, 28 Enterobacteriaceae isolates were isolated from specimens recovered from patients in the ICU and 2 from surfaces of the unit. Of these, 11 isolates (4 Escherichia coli, 5 Klebsiella pneumoniae and 2 Enterobacter cloacae) produced ESBL of the CT-X-M-15 type. Molecular typing of the isolates showed the clonal nature of 4 K. pneumoniae isolates. The bla(CTXM-15) gene was genetically linked to insertion sequence lSEcp1B and was transferable by conjugation from 3 isolates. Regular monitoring of resistance mechanisms, the establishment of a prevention strategy, and more rational and appropriate use of antibiotics are needed.

[NDM-1: the superbug?]. / NDM-1: il superbug?

A novel type of carbapenemase, New Delhi metallo beta-lactamase 1 (NDM 1), was first identified in 2008 in two Enterobacteriacea isolates, both recovered from a Swedish patient transferred from India. The emergence of NDM 1 is now reported from all continents, often in patients with a history of travel or hospitalization in the Indian subcontinent. The NDM 1 producing Gram-negative bacteria are mainly Enterobacteriaceae, which can cause colonization or fatal infections, with worrying antimicrobial susceptibility profiles: some isolates have developed resistance to practically all available antibiotics. Is the NDM-1 the super-bug? Are we in the post-antibiotic era? This review is a summary of currently available knowledge of NDM-1 that draws attention to future antimicrobial resistance scenarios.

Antimicrobial treatment of Capnocytophaga infections.

Capnocytophaga spp. are normal inhabitants of the oropharyngeal flora. They are also involved in periodontal diseases or animal bites, complicated by septicaemia with dissemination to a great variety of sites, both in immunocompetent and immunocompromised hosts. This review will focus on their pathogenesis, spectrum of clinical infections and susceptibility to disinfectants and antibiotics. The spread of beta-lactamase-producing strains limits the use of beta-lactams as first-line treatments, underlying the necessity to test the in vitro susceptibility of clinical strains. Many antimicrobial treatments have been used, despite an absence of randomised studies and guidelines regarding the duration of treatment according to infected sites. Imipenem/cilastatin, clindamycin or beta-lactamase inhibitor combinations are always effective and their use can be recommended in all infections.

[High relationship between ciprofloxacin use and prevalence of ESBL Klebsiella pneumoniae]. / Alta correlación entre el consumo de ciprofloxacina y la prevalencia de Klebsiella pneumoniae productora de beta-lactamasas de espectro extendido.

BACKGROUND: To assess the relationship between ciprofloxacin use and the prevalence of extended spectrum betalactamases (ESBL) Klebsiella pneumoniae. PATIENTS AND METHODS: Semestral mean values regarding use of antibiotic and prevalence of ESBL Kp were compared during 9 semesters using linear regression and coefficient of correlation. RESULTS: The only statistically significant correlation was ciprofloxacin use and ESBL(+) K. pneumoniae prevalence, with a coefficient of correlation of 0.86 and p = 0.0027 using linear regression. CONCLUSIONS: Ciprofloxacin use must be taking into account when considering infection control programs due to high prevalence rates of ESBL(+) K. pneumoniae in the hospital setting.

Alta correlación entre el consumo de ciprofloxacina y la prevalencia de Klebsiella pneumoniae productora de ß-lactamasas de espectro extendido / High relationship between ciprofloxacin use and prevalence of ESBL Klebsiella pneumoniae

Background: To assess the relationship between ciprofloxacin use and the prevalence of extended spectrum betalactamases (ESBL) Klebsiella pneumoniae. Patients and Methods: Semestral mean values regarding use of antibiotic and prevalence of ESBL Kp were compared during 9 semesters using linear regression and coefficient of correlation. Results: The only statistically significant correlation was ciprofloxacin use and ESBL(+) K. pneumoniae prevalence, with a coefficient of correlation of 0.86 and p = 0.0027 using linear regression. Conclusions: Ciprofloxacin use must be taking into account when considering infection control programs due to high prevalence rates of ESBL(+) K. pneumoniae in the hospital setting.

Fundamento: Evaluar la correlación entre el consumo de cefalosporinas de tercera generación y ciprofloxacina con la prevalencia de cepas de Klebsiella pneumoniae productoras de ß-lactamasas de espectro extendido (BLEE). Pacientes y Métodos: Los valores promedios semestrales, correspondientes a consumo y prevalencia se compararon durante 9 semestres, usando coeficiente de correlación y regresión lineal. Resultados: La única asociación que resultó estadísticamente significativa, fue la correspondiente al consumo de ciprofloxacina y K. pneumoniae BLEE (+), con un coeficiente de correlación de 0,86 y una p de 0,0027, en el análisis de regresión lineal. Conclusiones: El consumo de ciprofloxacina debe ser tenido en cuenta al momento de establecer programas de control de infecciones frente a elevadas tasas de prevalencia de K. pneumoniae productoras de BLEE en un hospital.