Genomic characterization of carbapenem-non-susceptible Pseudomonas aeruginosa in Singapore.

Pseudomonas aeruginosa is a clinically important pathogen implicated in many hospital-acquired infections. Its propensity to acquire broad-spectrum resistance has earned the organism its status as a severe public health threat requiring urgent control measures. While whole-genome sequencing-based genomic surveillance provides a means to track antimicrobial resistance, its use in molecular epidemiological surveys of P. aeruginosa remains limited, especially in the Southeast Asian region. We sequenced the whole genomes of 222 carbapenem-non-susceptible P. aeruginosa (CNPA) isolates collected in 2006-2020 at the largest public acute care hospital in Singapore. Antimicrobial susceptibilities were determined using broth microdilution. Clonal relatedness, multi-locus sequence types, and antimicrobial resistance determinants (acquired and chromosomal) were determined. In this study, CNPA exhibited broad-spectrum resistance (87.8% multi-drug resistance), retaining susceptibility only to polymyxin B (95.0%) and amikacin (55.0%). Carbapenemases were detected in 51.4% of the isolates, where IMP and NDM metallo-ß-lactamases were the most frequent. Carbapenem resistance was also likely associated with OprD alterations or efflux mechanisms (ArmZ/NalD mutations), which occurred in strains with or without carbapenemases. The population of CNPA in the hospital was diverse; the 222 isolates grouped into 68 sequence types (ST), which included various high-risk clones. We detected an emerging clone, the NDM-1-producing ST308, in addition to the global high-risk ST235 clone which was the predominant clone in our population. Our results thus provide a "snapshot" of the circulating lineages of CNPA locally and the prevailing genetic mechanisms contributing to carbapenem resistance. This database also serves as the baseline for future prospective surveillance studies.

Ceftolozane/Tazobactam Resistance and Mechanisms in Carbapenem-Nonsusceptible Pseudomonas aeruginosa.

This study established the in vitro activity of ceftolozane/tazobactam (C/T) and its genotypic resistance mechanisms by whole-genome sequencing (WGS) in 195 carbapenem-nonsusceptible Pseudomonas aeruginosa (CNSPA) clinical isolates recovered from Singapore between 2009 and 2020. C/T susceptibility rates were low, at 37.9%. Cross-resistance to ceftazidime/avibactam was observed, although susceptibility to the agent was slightly higher, at 41.0%. Whole-genome sequencing revealed that C/T resistance was largely mediated by the presence of horizontally acquired ß-lactamases, especially metallo-ß-lactamases. These were primarily disseminated in well-recognized high-risk clones belonging to sequence types (ST) 235, 308, and 179. C/T resistance was also observed in several non-carbapenemase-producing isolates, in which resistance was likely mediated by ß-lactamases and, to a smaller extent, mutations in AmpC-related genes. There was no obvious mechanism of resistance observed in five isolates. The high C/T resistance highlights the limited utility of the agent as an empirical agent in our setting. Knowledge of local molecular epidemiology is crucial in determining the potential of therapy with novel agents.IMPORTANCEPseudomonas aeruginosa infection is one of the most difficult health care-associated infections to treat due to the ability of the organism to acquire a multitude of resistance mechanisms and express the multidrug resistance phenotype. Ceftolozane/tazobactam (C/T), a novel ß-lactam/ß-lactamase inhibitor combination, addresses an unmet medical need in patients with these multidrug-resistant P. aeruginosa infections. Our findings demonstrate geographical variation in C/T susceptibility owing to the distinct local molecular epidemiology. This study adds on to the growing knowledge of C/T resistance, particularly mutational resistance, and will aid in the design of future ß-lactams and ß-lactamase inhibitors. WGS proved to be a useful tool to understand the P. aeruginosa resistome and its contribution to emerging resistance in novel antimicrobial agents.

Epigoitrin, an Alkaloid From Isatis indigotica, Reduces H1N1 Infection in Stress-Induced Susceptible Model in vivo and in vitro.

Stress has been proven to modulate an individual's immune system through the release of pituitary and adrenal hormones such as the catecholamines, growth hormone, and glucocorticoids. These signal molecules can significantly alter the host immune system and make it susceptible to viral infection. In this study, we investigate whether epigoitrin, a natural alkaloid from Isatis indigotica, provides protection against influenza infection by reducing the host's susceptibility to influenza virus under stress and its underlying mechanism. To support it, the mouse restraint stress model and the corticosterone-induced stress model were employed. Our results demonstrated that epigoitrin significantly decreased the susceptibility of restraint mice to influenza virus, evidenced by lowered mortality, attenuated inflammation, and decreased viral replications in lungs. Further results revealed that epigoitrin reduced the protein expression of mitofusin-2 (MFN2), which elevated mitochondria antiviral signaling (MAVS) protein expression and subsequently increased the production of IFN-ß and interferon inducible transmembrane 3 (IFITM3), thereby helping to fight viral infections. In conclusion, our study indicated that epigoitrin could reduce the susceptibility to influenza virus via mitochondrial antiviral signaling.

Indirubin, a bisindole alkaloid from Isatis indigotica, reduces H1N1 susceptibility in stressed mice by regulating MAVS signaling.

Isatis indigotica has a long history in treating virus infection and related symptoms in China. Nevertheless, its antivirus evidence in animal studies is not satisfactory, which might be due to the lack of appropriate animal model. Previously, we had utilized restraint stress to establish mouse H1N1 susceptibility model which was helpful in evaluating the anti-virus effect of medicines targeting host factors, such as type I interferon production. In this study, this model was employed to investigate the effect and mechanism of indirubin, a natural bisindole alkaloid from Isatis indigotica, on influenza A virus susceptibility. In the in vitro study, the stress hormone corticosterone was used to simulate restraint stress. Our results demonstrated that indirubin decreased the susceptibility to influenza virus with lowered mortality and alleviated lung damage in restraint-stressed mice model. Moreover, indirubin promoted the expression of interferon-ß and interferon inducible transmembrane 3. In addition, indirubin maintained the morphology and function of mitochondria following influenza A virus infection. Further study revealed that indirubin promoted interferon-ß production through promoting mitochondrial antiviral signaling pathway. Our study indicated that indirubin could be a candidate for the therapy of influenza.

New insights into the effects and mechanism of a classic traditional Chinese medicinal formula on influenza prevention.

BACKGROUND: KangBingDu (KBD) is a classic traditional Chinese medicinal formula widely used to treat influenza. However, little information is available from controlled studies regarding the anti-influenza pharmacological activities of KBD and its underlying mechanisms, at least partly due to the lack of appropriate study models. PURPOSE: We hypothesized that KBD might provide a protection against influenza infection by reducing the host's susceptibility to viruses. To prove it, mouse restraint stress model was employed. METHODS: Mice were restricted and infected with influenza virus. KBD (13 and 26mg/kg/d) was orally administrated to mice from the first day of restraint stress and lasted for 7 days (twice a day). Mice were monitored daily for morbidity, symptom severity, and mortality for 21 days. The histopathologic changes were examined. For the study of mechanisms of action, we investigated whether KBD could promote mitochondria antiviral signaling protein (MAVS)-mediated antiviral signal and inhibit nuclear factor-kappa B (NF-κB)-mediated inflammation response. RESULTS: KBD significantly decreased the susceptibility of restraint mice to influenza virus, as evidenced by lowered mortality, attenuated inflammation and reduced viral replications in lungs. Further results revealed that KBD elevated the protein expression of MAVS, which subsequently increased the IFN-ß and IFITM3 protein levels, thereby helping to fight viral infections. Finally, we identified that (R,S)-goitrin, mangiferin, forsythin and forsythoside A were effective components in KBD against influenza viral infections. CONCLUSION: KBD can reduce the susceptibility to influenza virus via mitochondrial antiviral signaling.

[Heterogeneous reactions on the surface of atmospheric particles: a transmission FTIR spectroscopy study].

Heterogeneous reactions of trace gases on the surface of particles in the atmosphere play a important role in the key atmospheric processes, and have been of increasingly interest in atmospheric sciences. However, these reactions, especially the corresponding detailed kinetics and mechanisms, have not been well understood, due to the deficiencies in the current experimental methods. The authors developed a novel experimental method for the study of heterogeneous reactions, based on transmission FTIR spectroscopy (T-FTIR), providing an understanding of the detailed physical chemistry that occurs on the surface of particles at the molecular level. In this method, the particles were evenly coated on a metal grid, and the gaseous reactant and the infrared beam were introduced to pass through the grid in the reactor. This method was used to explore the heterogeneous reaction of methacrolein (MAC) on the surface of SiO2 particles, a model heterogeneous reaction, under simulated atmospheric conditions. The result demonstrated that T-FTIR is an applicable and powerful technique for the qualitative and quantitative analysis of such a heterogeneous reaction, due to its excellent spectral signal. In addition, the particle sample will not be scathed due to the low energy of infrared light, thus the in situ investigation of the reaction on particles could be carried out. Compared with the HPLC analysis results, the repeatability and accuracy of the qualitative and quantitative analysis of T-FTIR have proven satisfactory, even though the reactions ran in the humid air. The authors' method can provide an effective tool for the laboratory simulation of heterogeneous reactions under the atmospheric conditions.