Drug Repurposing for Influenza Virus Polymerase Acidic (PA) Endonuclease Inhibitor

Drug repurposing can quickly and effectively identify novel drug repurposing opportunities. The PA endonuclease catalytic site has recently become regarded as an attractive target for the screening of anti-influenza drugs. PA N-terminal (PAN) inhibitor can inhibit the entire PA endonuclease activity. In this study, we screened the effectivity of PAN inhibitors from the FDA database through in silico methods and in vitro experiments. PAN and mutant PAN-I38T were chosen as virtual screening targets for overcoming drug resistance. Gel-based PA endonuclease analysis determined that the drug lifitegrast can effectively inhibit PAN and PAN-I38T, when the IC50 is 32.82 ± 1.34 μM and 26.81 ± 1.2 μM, respectively. Molecular docking calculation showed that lifitegrast interacted with the residues around PA or PA-I38 T’s active site, occupying the catalytic site pocket. Both PAN/PAN-I38T and lifitegrast can acquire good equilibrium in 100 ns molecular dynamic simulation. Because of these properties, lifitegrast, which can effectively inhibit PA endonuclease activity, was screened through in silico and in vitro research. This new research will be of significance in developing more effective and selective drugs for anti-influenza therapy.

Characterization of Clostridioides difficile isolates recovered from two Phase 3 surotomycin treatment trials by restriction endonuclease analysis, PCR ribotyping and antimicrobial susceptibilities

Objectives To investigate the molecular epidemiology and antimicrobial susceptibility of Clostridioides difficile isolates from patients with C. difficile infection (CDI) from two Phase 3 clinical trials of surotomycin. Methods In both trials [Protocol MK-4261-005 (NCT01597505) conducted across Europe, North America and Israel; and Protocol MK-4261-006 (NCT01598311) conducted across North America, Asia-Pacific and South America], patients with CDI were randomized (1:1) to receive oral surotomycin (250 mg twice daily) or oral vancomycin (125 mg four times per day) for 10 days. Stool samples were collected at baseline and C. difficile isolates were characterized by restriction endonuclease analysis (REA) and PCR ribotyping. Susceptibility testing was performed by agar dilution, according to CLSI recommendations. Results In total, 1147 patients were included in the microbiological modified ITT population. Of 992 recovered isolates, 922 (92.9%) were typed. There was a high association between REA groups and their corresponding predominant PCR ribotype (RT) for BI, DH, G and CF strains. REA group A showed more diverse PCR RTs. Overall, the most common strain was BI/RT027 (20.3%) followed by Y/RT014/020 (15.0%) and DH/RT106 (7.2%). The BI/RT027 strain was particularly prevalent in Europe (29.9%) and Canada (23.6%), with lower prevalence in the USA (16.8%) and Australia/New Zealand (3.4%). Resistance was most prevalent in the BI/RT027 strain, particularly to metronidazole, vancomycin and moxifloxacin. Conclusions A wide variation in C. difficile strains, both within and across different geographical regions, was documented by both REA and ribotyping, which showed overall good correlation.

Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin-based serotyping of Pasteurella multocida

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in the serotyping and genotyping of Pasteurella multocida. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis has become the gold standard for other organisms, offering higher resolution than previously available methods. We compared WGS to REA and GDPT on 163 isolates of P. multocida to determine if WGS produced more precise results. The isolates used represented the 16 reference serovars, isolates with REA profiles matching an attenuated fowl cholera vaccine strain, and isolates from 10 different animal species. Isolates originated from across the United States and from Chile. Identical REA profiles clustered together in the phylogenetic tree. REA profiles that differed by only a few bands had fewer SNP differences than REA profiles with more differences, as expected. The GDPT results were diverse but it was common to see a single serovar show up repeatedly within clusters. Several errors were found when examining the REA profiles. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. Also, results of WGS and SNP analysis correlated more closely with the epidemiologic data than GDPT. In silico results were also compared to a lipopolysaccharide rapid multiplex PCR test. From the data produced in our study, WGS and SNP analysis was superior to REA and GDPT and highlighted some of the issues with the older tests.

Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms.

A significant number of DNA-based techniques has been introduced into the field of microorganisms' characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before DNA amplification by PCR, known as Ligation-Mediated PCR methods (LM PCR), have been successfully applied for the typing of microorganisms below the species level. These molecular methods include: Amplified Fragment Length Polymorphism (AFLP), Amplification of DNA fragments Surrounding Rare Restriction Sites (ADSRRS), PCR Melting Profiles (PCR MP), Ligation Mediated PCR/Shifter (LM PCR/Shifter), Infrequent-Restriction-Site Amplification (IRS PCR), double digestion Ligation Mediated Suppression PCR (ddLMS PCR). These techniques are now applied more and more often because they involve less time, are comparably inexpensive, and require only standard lab equipment. Here, we present a general review of this group of methods showing their possibilities and limitations. We also identify questions and propose solutions which may be helpful in choosing a particular LM PCR method for the achievement of the required goal.

Fluoroquinolone and Macrolide Exposure Predict Clostridium difficile Infection with the Highly Fluoroquinolone- and Macrolide-Resistant Epidemic C. difficile Strain BI/NAP1/027

ABSTRACTAntibiotics have been shown to influence the risk of infection with specificClostridium difficilestrains as well as the risk ofC. difficileinfection (CDI). We performed a retrospective case-control study of patients infected with the epidemic BI/NAP1/027 strain in a U.S. hospital following recognition of increased CDI severity and culture of stools positive byC. difficiletoxin immunoassay. Between 2005 and 2007, 72% (103/143) of patients with first-episode CDIs were infected with the BI strain by restriction endonuclease analysis (REA) typing. Most patients received multiple antibiotics within 6 weeks of CDI onset (median of 3 antibiotic classes). By multivariate analysis, fluoroquinolone and macrolide exposure was more frequent among BI cases than among non-BI-infected controls (odds ratio [OR] for fluoroquinolones, 3.2; 95% confidence interval [CI], 1.3 to 7.5; (P< 0.001; OR for macrolides, 5.2; 95% CI, 1.1 to 24.0;P= 0.04)). In contrast, clindamycin use was less frequent among the BI cases than among the controls (OR, 0.1; 95% CI, 0.03 to 0.4;P= 0.001). High-level resistance to moxifloxacin and azithromycin was more frequent among BI strains (moxifloxacin, 49/102 [48%] BI versus 0/40 non-BI,P= 0.0001; azithromycin, 100/102 [98%] BI versus 22/40 [55%] non-BI,P= 0.0001). High-level resistance to clindamycin was more frequent among non-BI strains (22/40 [55%] non-BI versus 7/102 [7%] BI,P= 0.0001). Fluoroquinolone use, macrolide use, andC. difficileresistance to these antibiotic classes were associated with infection by the epidemic BI strain ofC. difficilein a U.S. hospital during a time when CDI rates were increasing nationally due to the highly fluoroquinolone-resistant BI/NAP1/027 strain.