Jundishapur Journal of Microbiology
Volume:13 Issue: 10, Oct 2020

 Infections caused by metallo-β-lactamases (MβLs)-producing antibiotic-resistant bacteria pose a severe threat to public health. The synergistic use of current antibiotics in combination with MβL inhibitors is a promising therapeutic mode against these antibiotic-resistant bacteria.

 The study aimed to probe the inhibition of MβLs and obtain the active component, P1, in the degradation product after imipenem was hydrolyzed by ImiS.

 The hydrolysis of two carbapenems with MβL ImiS was monitored by UV-Vis in real-time, and the degradation product from the leaving group produced after imipenem was hydrolyzed (but not for faropenem) was purified by HPLC to give one component, P1.

 Kinetic assays revealed that P1 exhibited a broad-spectrum inhibition against VIM-2, NDM-1, ImiS, and L1, from three sub-classes of MβLs, with IC50 values of 8 - 32, 13.8 - 29.3, and 14.2 - 19.2 µM, using imipenem, cefazolin, and nitrocefin as substrates, respectively. Also, P1 showed synergistic antibacterial efficacy against drug-resistant Escherichia coli producing VIM-2, NDM-1, ImiS, and L1, in combination with antibiotics, restoring 16 to 32-fold and 32 to 128-fold efficacies of imipenem and cefazolin, respectively. Spectroscopic and Ellman's reagent analyses suggested that P1, a mercaptoethyl-form imidamide, is a mechanism-based inhibitor, while faropenem has no substrate inhibition, due to the lack of a leaving group.

 This work reveals that the hydrolysate of imipenem, a carbapenem with a good leaving group, can be used in screening for broad-spectrum inhibitors of MβLs.

 Vulvovaginal candidiasis (VVC) is a significant health issue due to Candida spp. Although Candida albicans is considered a major causative agent of vaginal candidiasis, non-albicans species have increased during previous decades.

 This research aimed at molecular identification and assessing antifungal susceptibility of VVC isolated Candida spp.

 A professional physician examined two hundred and ninety-five suspected females with vaginitis. The specimens were collected by sterile cotton swabs. Swabs were inoculated on Sabouraud dextrose agar plates and then incubated for 48 - 72 hours at 35°C. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect all Candida species. Broth microdilution, according to the M27-A3 and M27-S4 CLSI documents, were employed for determining the antifungal susceptibility tests of caspofungin (CAS), voriconazole (VRC), itraconazole (ITC), fluconazole (FLU), clotrimazole (CLO), ketoconazole (KTO), amphotericin B (AMB), and nystatin (NYS).

 A total of 295 females suspected of vulvovaginal candidiasis were examined. The culture results were positive in 50.5% (149 of 295) of specimens. According to molecular identification techniques, C. albicans 133/149 (89.2%), C. glabrata 8/149 (5.4%), and C. kefyr 2/149 (1.4%) were the main species. A mixed infection of C. albicans and C. glabrata 6/149 (4 %) was detected. The geometric mean values to all Candida strains were in increasing order as the following: CAS, 0.075 µg/mL; VRC, 0.091 µg/mL; ITC, 0.15 µg/mL; AMB, 0.22 µg/mL; CLO, 0.23 µg/mL; KTO, 0.28 µg/mL; NYS, 0.88 µg/mL; FLU, 1.48 µg/mL. Further, the MIC ranges of all Candida isolates to the tested antifungal agents were in increasing order as follows: CAS: 0.031 - 0.25 µg/mL, KTO and ITC: 0.031 - 2 µg/mL, VRC: 0.031 - 4 µg/mL, CLO and AMB: 0.031 - 8 µg/mL, NYS: 0.06 - 4 µg/mL, and FLU: 0.12 - 128 µg/mL.

 We reported 1 (7.2 %) C. glabrata isolate resistance to FLU and 2 (14.3%) C. glabrata isolates susceptible-dose-dependent (SDD) to CAS. We also reported 6 (4.5%), 5 (3.8%), and 2 (1.5%) C. albicans resistance to ITC, FLU, and AMB, respectively, but 100% C. albicans susceptible to CAS and VRC.

 Fusarium sp. and Rhizoctonia sp. fungi have been always threats to short-term crops. In Vietnam, corn and soybean suffer serious losses annually. Therefore, it is necessary to utilize an environmentally friendly antifungal compound that is highly effective against phytopathogenic fungi. Pseudomonas sp. is a popular soil bacterial strain and well known for its high antifungal activity.

 This study was carried out to evaluate and assess the antifungal activity of a local bacterial strain namely DA3.1 that was later identified as Pseudomonas aeruginosa. This would be strong scientific evidence to develop an environmentally friendly biocide from a local microorganism strain for commercial use.

 The antifungal compound was purified from ethyl acetate extraction of deproteinized cell culture broth by a silica gel column (CH2Cl2/MeOH (0% - 10% MeOH)). The purity of the isolated compound was determined by HPLC, and its molecular structure was elucidated using spectroscopic experiments including one-dimensional (1D) (1H NMR, 13C NMR, DEPT) and two-dimensional (2D) (HMBC and HSQC) spectra. The activity of the purified compound against Fusarium sp. and Rhizoctonia sp. fungi was measured using the PDA-disk diffusion method, and its growth-promoting ability was evaluated using the seed germination test of corn and soybean.

 The results showed that the antifungal compound produced by Pseudomonas aeruginosa DA3.1 had a retention factor (Rf) of 0.86 on thin layer chromatography (TLC). Based on the evidence of spectral data including proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR), distortionless enhancement by polarization transfer (DEPT), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum coherence (HSQC), the chemical structure was elucidated as phenazine-1-carboxylic. The purified compound showed inhibitory activity against F. oxysporum and R. solani and exhibited the ability of the germination of corn and soybean seeds. The results revealed the benefit of native P. aeruginosa DA3.1 and phenazine-1-carboxylic acid for use as a biocontrol agent, as well as a plant growth promoter.

 The antifungal compound isolated from local Pseudomonas DA3.1 was identified as phenazine-1-carboxylic acid that posed high antifungal activity and was a plant germination booster.

 The West Nile Virus (WNV), discovered in New York, USA in 1999 after it was first isolated in Uganda in 1937, has since spread not only in the United States but also around the world. Africa, Eurasia, Australia, and the Middle East have sporadic cases of the disease.

 We aimed to find real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to be more sensitive than conventional RT-PCR, and more rapid and efficient than conventional RT-PCR and real-time RT-PCR for WNV detection.

 A total of 32 genomic sequences from different strains of WNV were analyzed to identify conserved nucleotide sequence regions. Six WNV specific RT-LAMP primers targeting the E gene were designed.

 The novel primer for the real-time RT-LAMP assay can detect WNV with high specificity. The efficiency of the real-time RT-LAMP assay is higher than the conventional RT-PCR and real-time RT-PCR. Real-time RT-PCR and conventional PCR require at least 30 – 40 min and 2 h, respectively, to yield results, whereas real-time RT-LAMP provides positive results in only 10 – 20 min.

 The novel primers were developed by analyzing of 32 genomic sequences of WNV strains. The primers were designed from the most conserved region of the E gene for real-time RT-LAMP. The LAMP assay is a rapid, efficient, highly sensitive, and specific tool for the identification of WNV.

 Bloodstream infection (BSI) has been one of the biggest headaches for clinicians, as it not only aggravates symptoms but also increases the length of stay, the cost of hospitalization, and the side effects caused by antibiotics. It is an urgent need for clinicians to develop timely and accurate methods to find microorganisms. Currently, the gold standard for diagnosing BSI is blood culture, but it takes three to eight days to produce results, and its positive rate is extremely low. Next-generation sequencing (NGS) has emerged as a better technology desperately needed by doctors and patients to diagnose BSI.

 This study compared NGS and blood culture methods in clinical patients with BSI.

 In this study, blood culture and NGS were used to analyze the blood of patients with BSI in different departments of the First Affiliated Hospital of Kunming Medical University.

 Next-generation sequencing detected 60 pathogens in 63 blood samples, while blood culture detected 15 pathogens in 336 blood samples from 63 patients who were clinically considered to be infected. Pathogens detected by NGS included bacteria, fungi, and viruses, while blood culture only found bacteria and fungi. The positive rates of blood culture diagnosis and NGS diagnosis in BSI patients were 23.8% (15/63) (CI: 13.3% - 34.3%) and 95% (60/63) (CI: 90% - 100%), respectively.

 Our results showed that NGS creates a new diagnostic platform for patients with BSI. Its wide detection range, high positive rate, and characteristics of rapid detection will benefit patients with BSI.

 The refractory infection induced by multidrug-resistant (MDR) Pseudomonas aeruginosa has become one of the most urgent problems in hospitals. The biofilms formed by P. aeruginosa increase its resistance to antibiotics. A simulated microgravity (SMG) environment provides a platform to understand the factors affecting biofilm formation in bacteria.

 This study aimed to investigate the SMG effects on MDR P. aeruginosa biofilm formation and explore the relevant mechanisms.

 In this study, a clinostat was used to simulate a microgravity (MG) environment. The motility and biofilm formation ability of MDR P. aeruginosa were observed using the swimming test and the crystal violet staining method, respectively. The underlying mechanism of phenotypic changes was further investigated by comparative transcriptomic analysis.

 Multidrug-resistant P. aeruginosa grown under the SMG condition exhibited decreased swimming motility and biofilm formation ability compared to those under the normal gravity (NG) condition. Further analysis revealed that the decreased swimming motility and biofilm formation ability could be attributed to the downregulated expression of genes responsible for flagellar synthesis (flhB, fliQ, and fliR) and type IV pili biogenesis (pilDEXY1Y2VW).

 This is the first study to perform experiments on MDR P. aeruginosa under the SMG condition. It will be beneficial to understand the mechanism of MDR P. aeruginosa biofilm formation and develop new treatment strategies for infectious diseases induced by MDR P. aeruginosa in the future.
 

 Enterococci are one of the opportunistic pathogenic microorganisms that can cause significant problems for human and animal health. Enterococcus faecium seems to be more resistant to antibiotics than E. faecalis. It is thought that pathogenic E. faecium can develop antibiotic resistance very quickly, and the ability to transfer this feature is considered to be an important health risk.

 This study aimed to determine the prevalence, biotypes, and in vitro antimicrobial susceptibility of E. faecalis and E. faecium strains isolated from 267 routine urine and stool samples that were brought to the microbiology laboratory of Regional Training and Research Hospital of Van, with permission of the patients.

 In the present study, enterococci using species-specific primers to examine E. faecalis and E. faecium multiplex PCR technique was applied. Biotyping of the isolates was used to identify them as E. faecalis and E. faecium by molecular techniques, and antibiotic susceptibility of all samples was examined, as well.

 The isolates were identified by multiplex PCR using species-specific primers for E. faecalis and E. faecium. Biotyping based on 13 biochemical tests showed that 72.5%, 12.5%, and 15% of E. faecalis strains were of biotypes I, II, and III, respectively, whereas E. faecium strains could be divided into biotype I (10%), biotype II (12.5%), biotype III (27.5%), and biotype IV (50%). Additionally, all E. faecalis strains were found to be susceptible to penicillin G and imipenem. On the other hand, 95% of the E. faecalis strains were found to be resistant to clindamycin, 77.5% to tetracycline and trimethoprim/sulfamethoxazole, 42.5% to erythromycin, 32.5% to gentamicin, and 17.5% to ciprofloxacin. Of E. faecium strains, 37.5% were found to be resistant to clindamycin, 32.5% to penicillin G, 27.5% to erythromycin and imipenem, 20% to ciprofloxacin, 17.5% to tetracycline and trimethoprim/sulfamethoxazole, 15% to gentamicin, and 5% to vancomycin.

 In conclusion, the identification of E. faecalis and E. faecium strains by PCR is reliable and faster than biochemical tests. Additionally, the results of antimicrobial susceptibility tests may provide important contributions to the clinical approach.