Novel 2,4-disubstituted quinazoline analogs as antibacterial agents with improved cytotoxicity profile: Optimization of the 2,4-substituents.

The emergence of bacterial resistance has triggered a multitude of efforts to develop new antibacterial agents. There are many compounds in literature that were reported as potent antibacterial agents, however, they lacked the required safety to mammalian cells or no clear picture about their toxicity profile was presented. Inspired by discovered hit from our in-house library and by previously reported 2,4-diaminosubstituted quinazolines, we describe the design and synthesis of novel 2,4-disubstituted-thioquinazolines (3-13 and 36), 2-thio-4-amino substituted quinazolines (14-33) and 6-substituted 2,4-diamonsubstituted quinazolines (37-39). The synthesized compounds showed potent antibacterial activity against a panel of Gram-positive, efflux deficient E.coli and Mycobacterium smegmatis. The panel also involved resistant strains including methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis and vancomycin-resistant Enterococcus faecium, in addition to Mycobacterium smegmatis. The newly synthesized compounds revealed MIC values against the tested strains ranging from 1 to 64 µg/mL with a good safety profile. Most of the 2-thio-4-amino substituted-quinazolines showed significant antimycobacterial activity with the variations at position 2 and 4 offering additional antibacterial activity against the different strains. Compared to previously reported 2,4-diaminosubstituted quinazolines, the bioisosteric replacement of the 2-amino with sulfur offered a successful approach to keep the high antibacterial potency while substantially improving safety profile as indicated by the reduced activity on different cell lines and a lack of hemolytic activity.

Molecular characterization, toxin detection and resistance testing of human clinical Clostridium difficile isolates from Lebanon.

Clostridium (Clostridioides) difficile is the main cause for nosocomial diarrhoea in industrialised nations. Epidemiologic data on the pathogen's occurrence in other world regions are still scarce. In this context we characterized with phenotypic and molecular genetic methods C. difficile isolates stemming from hospitalised patients with diarrhoea in Lebanon. From 129 stool samples of symptomatic patients at a tertiary care University hospital in Lebanon, a total of 107 C. difficile strains were cultivated and underwent ribotyping, toxin gene detection and antibiotic resistance testing. Ribotype 014 (RT014, 16.8%) predominated, followed by RT002 (9.3%), RT106 (8.4%) and RT070 (6.5%). Binary toxin gene-positive isolates (RT023, RT078 and RT126) were rarely detected and RT027 was absent. Interestingly, within one isolate only the toxin A gene (tcdA) was detected. Multiple-locus variable-number tandem repeat analysis (MLVA) revealed strong strain diversity in most RTs. The isolates were sensitive to metronidazole and vancomycin, and only a small proportion of strains displayed resistance against moxifloxacin, rifampicin, and clarithromycin (5.6%, 1.9%, and 2.8%), respectively. The data indicate that the genetic strain composition of Lebanese strains differs markedly from the situation seen in Europe and North America. Especially the epidemic RTs seen in the latter regions were almost absent in Lebanon. Interestingly, most strains showed almost no resistance to commonly used antibiotics that are suspected to play a major role in the development of C. difficile infection, despite frequent use of these antibiotics in Lebanon. Thus, the role of antimicrobial resistance as a major driving force for infection development remains uncertain in this area.

Expanding the knowledge around antitubercular 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides: Hit-to-lead optimization and release of a novel antitubercular chemotype via scaffold derivatization.

Tuberculosis is one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extensively drug-resistant strains is a reason for concern. We have previously reported a series of substituted 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides with growth inhibitory activity against Mycobacterium tuberculosis strains and low propensity to be substrate of efflux pumps. Encouraged by these preliminary results, we have undertaken a medicinal chemistry campaign to determine the metabolic fate of these compounds and to delineate a reliable body of Structure-Activity Relationships. Keeping intact the (thiazol-4-yl)isoxazole-3-carboxamide core, as it is deemed to be the pharmacophore of the molecule, we have extensively explored the structural modifications able to confer good activity and avoid rapid clearance. Also, a small set of analogues based on isostere manipulation of the 2-aminothiazole were prepared and tested, with the aim to disclose novel antitubercular chemotypes. These studies, combined, were instrumental in designing improved compounds such as 42g and 42l, escaping metabolic degradation by human liver microsomes and, at the same time, maintaining good antitubercular activity against both drug-susceptible and drug-resistant strains.

Natural products and polysorbates: Potential Inhibitors of biofilm formation in Pseudomonas aeruginosa.

INTRODUCTION: With all the challenges super bugs are imposing, biofilm formation opens the door against various more complicated challenges. Such issue may be highlighted with the ability of the latter to render the antibiotics hardly accessible to bacterial cells and sheds the light on the importance of finding antibiofilm formers. Therefore, we assessed the inhibitory effect of natural product extracts (ginger, wild blueberry) and polysorbates (PS20, PS80) on biofilm formation at the molecular level. METHODOLOGY: Growth inhibition assay was performed to test the effect of ginger (Zingiber Officinale), wild blueberry (Vaccinium Angustifolium), and polysorbates on Pseudomonas aeruginosa (PAN14) growth. Transcription levels of biofilm exopolysaccharides encoding genes (ndvB, pelC, algC) and quorum sensing genes (lasI, lasR, rhlI, rhlR) for LasI/LasR and RhlI/ RhlR systems were evaluated by RT qPCR. RESULTS: The polysorbates and the extracts of both ginger and wild blueberry had no effect on the growth of P. aeruginosa. Biofilms' examination has unraveled the effectiveness of treatments used in reducing its formation. Moreover, a significant reduction in the expression of all genes tested for biofilm exopolysaccharides and its quorum sensing system was observed. CONCLUSION: The decrease in the relative gene expression of the exopolysaccharides and quorum sensing encoding genes sheds the light on the mechanism of action of ginger and wild blueberry's constituents as well as polysorbates 20 and 80 on P. aeruginosa biofilm formation. Future studies need to assess the antibiofilm effect of each fraction of herbal extracts separately.

Incidence, outcome, and risk factors for recurrence of nosocomial Clostridioides difficile infection in adults: A prospective cohort study.

BACKGROUND: Nosocomial Clostridioides difficile infection (CDI) complicates up to 1% of all hospital admissions and is associated with considerable health burden. AIMS: To determine the incidence and outcomes of nosocomial CDI at a major University Medical Center. METHODS: Consecutive adult nosocomial CDI cases were prospectively identified. Stool samples were collected for ribotyping and antibiotic resistance testing. Patients were followed for eight weeks after discharge for relapse. RESULTS: Over a 2-year period, 215 patients developed nosocomial CDI (incidence 2:1000) and 200 (mean age 62.2±19.6 years) gave informed consent. Mean hospital stay was 23.3±28.9 days (range 0-278). Infection was diagnosed within 7 days of admission (range 0-95) in 129 patients (64.5%). More than two-thirds (69.0%) were previously hospitalized within 12 weeks of the index hospitalization. Twenty five percent received prior antibiotics within eight weeks. Fifty-two patients (26.0%) did not receive antibiotics prior to diagnosis. Considerable comorbidities (Charlson Comorbidity Index ≥8) were noted in 33.5% of patients. Recurrence occurred in 43 patients (21.5%). On multivariate logistic regression, fluoroquinolone exposure was the only predictor of recurrence (OR=2.9, 95%CI 1.1-7.7). Overall mortality was 14.0% and CCI ≥8 was the only predictor on multivariate analysis (p=0.004). Genotyping did not identify any known hypervirulent strains and all isolates were susceptible to metronidazole and vancomycin. CONCLUSION: Antibiotic exposure, comorbidities, and prior hospitalization constitute the major risk factors for nosocomial CDI. Recurrence is common and is associated with fluoroquinolones exposure. High baseline comorbidity score was the only predictor of increased mortality in this prospective cohort.

Evaluating the Efficacies of Carbapenem/ß-Lactamase Inhibitors Against Carbapenem-Resistant Gram-Negative Bacteria in vitro and in vivo.

BACKGROUND: Carbapenem-resistant Gram-negative bacteria are a major clinical concern as they cause virtually untreatable infections since carbapenems are among the last-resort antimicrobial agents. ß-Lactamases implicated in carbapenem resistance include KPC, NDM, and OXA-type carbapenemases. Antimicrobial combination therapy is the current treatment approach against carbapenem resistance in order to limit the excessive use of colistin; however, its advantages over monotherapy remain debatable. An alternative treatment strategy would be the use of carbapenem/ß-lactamase inhibitor (ßLI) combinations. In this study, we assessed the in vitro and in vivo phenotypic and molecular efficacies of three ßLIs when combined with different carbapenems against carbapenem-resistant Gram-negative clinical isolates. The chosen ßLIs were (1) Avibactam, against OXA-type carbapenemases, (2) calcium-EDTA, against NDM-1, and (3) Relebactam, against KPC-2. METHODS: Six Acinetobacter baumannii clinical isolates were screened for bla OXA-23-like, bla OXA-24/40, bla OXA-51-like, bla OXA-58, and bla OXA-143-like, and eight Enterobacteriaceae clinical isolates were screened for bla OXA-48, bla NDM-1, and bla KPC-2. The minimal inhibitory concentrations of Imipenem (IPM), Ertapenem (ETP), and Meropenem (MEM) with corresponding ßLIs for each isolate were determined. The efficacy of the most suitable in vitro treatment option against each of bla OXA-48, bla NDM-1, and bla KPC-2 was assessed via survival studies in a BALB/c murine infection model. Finally, RT-qPCR was performed to assess the molecular response of the genes of resistance to the carbapenem/ßLI combinations used under both in vitro and in vivo settings. RESULTS: Combining MEM, IPM, and ETP with the corresponding ßLIs restored the isolates' susceptibilities to those antimicrobial agents in 66.7%, 57.1%, and 30.8% of the samples, respectively. Survival studies in mice revealed 100% survival rates when MEM was combined with either Avibactam or Relebactam against bla OXA-48 and bla KPC-2, respectively. RT-qPCR demonstrated the consistent overexpression of bla OXA-48 upon treatment, without hindering Avibactam's activity, while bla NDM-1 and bla KPC-2 experienced variable expression levels upon treatment under in vitro and in vivo settings despite their effective phenotypic results. CONCLUSION: New carbapenem/ßLI combinations may be viable alternatives to antimicrobial combination therapy as they displayed high efficacy in vitro and in vivo. Meropenem/Avibactam and Meropenem/Relebactam should be tested on larger sample sizes with different carbapenemases before progressing further in its preclinical development.

Assessing the effect of micafungin on Pseudomonas aeruginosa biofilm formation using confocal microscopy and gene expression.

INTRODUCTION: 1,3-ß-D-glucan of the fungal cell wall and extracellular matrix (ECM) of Candida biofilm is also present as a periplasmic glucan and within the ECM of P. aeruginosa biofilm. Micafungin inhibits the synthesis of ß-D-glucans. This project evaluates the effect of micafungin on P. aeruginosa biofilm formation, by determining transcription levels of biofilm formation encoding genes and measuring the thickness of biofilms in treated and untreated samples from BALB/c mice. METHODOLOGY: Relative gene transcription levels of P. aeruginosa biofilm-encoding pelC, algC, and ndvB genes were assessed by RT-qPCR on treated and untreated samples. Thickness calculation by Z-stacking of treated and untreated biofilms obtained from in vitro and in vivo samples was determined by confocal scanning laser microscopy (CSLM). RESULTS: Samples from micafungin-treated mice showed decreased pelC, ndvB, and algC transcription levels with values of 260, 74, and 2-fold decreases, respectively. Reduction in biofilms thickness was confirmed with Z-stacking using CSLM that revealed a 16.8% drop in the thickness of biofilms after treatment with micafungin in vitro, and a 64% reduction in thickness post treatment with micafungin in vivo. CONCLUSION: Micafungin inhibits biofilm formation as measured by decrease in transcription levels of biofilm encoding genes and confocal microscopy. This reflects the events occurring in the course of an acute infection with P. aeruginosa, whereby the administration of micafungin would inhibit subsequent slime production, thus eliminating such barrier that could prevent antibacterial delivery to the core planktonic cells in biofilms.

Increase of blaOXA-23-like in Acinetobacter baumannii at a tertiary care center in Lebanon between 2007 and 2013.

INTRODUCTION: The multi-drug resistant nature of Acinetobacter baumannii isolates have rendered many broad-spectrum antimicrobial agents ineffective against them. The purpose of this retrospective study is to define and compare the molecular characteristics of A. baumannii isolates from patients at a tertiary care center in Lebanon from two outbreaks, the first in 2007-2008, as part of a case-controlled study involving A. baumannii cases admitted to the ICU, and the second in 2013. METHODOLOGY: A total of 148 A. baumannii clinical isolates were collected from various clinical specimens during 2007-2008 and 2013. All A. baumannii isolates were screened for blaOXA-23-like and blaOXA-51-like genes of carbapenem resistance. Additionally, in an effort to assess the degree of the isolates' genomic relatedness, random amplification of polymorphic DNA (RAPD) was performed. RESULTS: There was an increase in the prevalence of blaOXA-23-like and blaOXA-51-like genes between the two time periods; however, only 22% isolate genomic relatedness was calculated between 2007-2008 and 2013. Taking 80% as a margin of compatibility, 31 distinct clusters containing 2 to 11 strains were observed when both time periods were analyzed. CONCLUSION: The presence of numerous clusters accompanied by a predominant increase in the prevalence of blaOXA-23-like between 2007 and 2013 suggests a horizontal transmission of the gene within various strains of the species, contributing to the persistent increase in carbapenem resistance over the years. Therefore, infection control measures are required with compliance among all healthcare workers.

Increased blaOXA-23-like prevalence in Acinetobacter baumannii at a tertiary care center in Lebanon (2007-2013).

INTRODUCTION: Acinetobacter baumannii has become one of the most feared organisms in hospital-acquired infections during the past decades. Their multi-drug resistant profiles have rendered many broad-spectrum antibiotics ineffective. The purpose of this retrospective study is to describe and compare molecular characteristics of A. baumannii isolated from patients at a tertiary care center in Lebanon from two outbreaks, the first in 2007-2008 as part of a case-controlled study involving Acinetobacter baumannii cases admitted to the ICU and the second in 2013. METHODOLOGY: A total of 148 A. baumannii clinical isolates were collected from various clinical specimens during 2007-2008 and 2013. All A. baumannii isolates were subjected to PCR amplification of blaOXA-23-like and blaOXA-51-like genes of carbapenem resistance. Random amplification of polymorphic DNA (RAPD) was also performed to assess their genomic relatedness. RESULTS: There was an increase in the prevalence of blaOXA-23-like and blaOXA-51-like between the two time periods; however, only with 22% genomic relatedness between 2007-2008 and 2013 isolates. Taking 80% as margin of compatibility, 31 distinct clusters containing 2 to 11 strains were observed in both time periods. CONCLUSION: The presence of numerous clusters accompanied by a predominant increase in the prevalence of blaOXA-23-like gene between 2007 and 2013 suggests a horizontal transmission of the gene within various strains of the species, constituting a primary factor in the continued increase of carbapenem resistance over the years. As such, infection control measures ought to be taken with the highest priority and compliance among all involved healthcare workers is of utmost importance.