Comparison of the relative efficacy of ß-lactam/ß-lactamase inhibitors and carbapenems in the treatment of complicated urinary tract infections caused by ceftriaxone-non-susceptible Enterobacterales: a multicentre retrospective observational cohort study.

BACKGROUND: Treating complicated urinary tract infections (cUTIs) caused by ESBL-producing Enterobacterales represents a significant clinical challenge. The present study was thus developed to explore the relative efficacy of ß-lactam/ß-lactamase inhibitors (BLBLIs) and carbapenems for the treatment of hospitalized patients suffering from cUTIs caused by BLBLI-susceptible ceftriaxone-non-susceptible Enterobacterales. METHODS: Data from 557 patients from four Chinese teaching hospitals diagnosed with cUTIs caused by ceftriaxone-non-susceptible Enterobacterales from January 2017 to May 2022 were retrospectively assessed. RESULT: The 30 day rate of treatment failure, defined by unresolved symptoms or mortality, was 10.4% (58/557). Independent predictors of 30 day treatment failure included immunocompromised status, bacteraemia, septic shock, lack of infection source control and appropriate empirical treatment. When data were controlled for potential confounding variables, BLBLI treatment exhibited a comparable risk of 14 day (OR 1.61, 95% CI 0.86-3.00, P = 0.133) and 30 day treatment failure (OR 1.45, 95% CI 0.66-3.15, P = 0.354) relative to carbapenem treatment for the overall cohort of patients. In contrast, BLBLI treatment in immunocompromised patients was associated with an elevated risk of both 14 day (OR 3.18, 95% CI 1.43-7.10, P = 0.005) and 30 day treatment failure (OR 3.06, 95% CI 1.07-8.80, P = 0.038) relative to carbapenem treatment. CONCLUSIONS: These results suggested that carbapenem treatment may be superior to BLBLI treatment for immunocompromised patients suffering from cUTIs caused by ceftriaxone-non-susceptible Enterobacterales species. However, these results will need to be validated in appropriately constructed randomized controlled trials to ensure appropriate patient treatment.

CYP4G100 contributes to desiccation resistance by mediating cuticular hydrocarbon synthesis in Bactrocera dorsalis.

The oriental fruit fly Bactrocera dorsalis (Hendel) is expanding its distribution to higher latitudes. Our goal in this study was to understand how B. dorsalis adapts to higher latitude environments that are more arid than tropical regions. Cuticular hydrocarbons (CHCs) on the surface of the epicuticle in insects act as a hydrophobic barrier against water loss. The essential decarbonylation reaction in CHC synthesis is catalysed by CYP4G, a cytochrome P450 subfamily protein. Hence, in B. dorsalis it is necessary to clarify the function of the CYP4G gene and its role in desiccation resistance. CYP4G100 was identified in the B. dorsalis genome. The complete open reading frame (ORF) encodes a CYP4 family protein (552 amino acid residues) that has the CYP4G-specific insertion. This CYP4G gene was highly expressed in adults, especially in the oenocyte-rich peripheral fat body. The gene can be induced by desiccation treatment, suggesting its role in CHC synthesis and waterproofing. Silencing of CYP4G100 resulted in a decrease of CHC levels and the accumulation of triglycerides. It also increased water loss and resulted in higher desiccation susceptibility. CYP4G100 is involved in hydrocarbon synthesis and contributes to cuticle waterproofing to help B. dorsalis resist desiccation in arid environments.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment.

BACKGROUND: Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. RESULTS: The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a ß-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. CONCLUSION: KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

NADPH-cytochrome P450 reductase mediates the susceptibility of Asian citrus psyllid Diaphorina citri to imidacloprid and thiamethoxam.

BACKGROUND: The Asian citrus psyllid Diaphorina citri has developed high levels of resistance to many insecticides, and understanding its resistance mechanism will aid in the chemical control of this species. Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is crucial in cytochrome P450 function, and in some insects CPR knockdown has increased their susceptibility to insecticides. However, the CPR from D. citri has not been characterized and its function is undescribed. RESULTS: The CPR gene of D. citri (DcCPR) was cloned and sequenced. The expression level of DcCPR, determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis, was highest in the midgut and in nymphs. After feeding on double-stranded RNA for 72 h, the DcCPR messenger RNA level in D. citri adults decreased by 68.4%, and the susceptibility of D. citri to imidacloprid and thiamethoxam significantly increased. Meanwhile, after DcCPR silencing, the specific activities of DcCPR protein and P450s were significantly reduced by 41.6% and 44.7%, respectively. The subsequent western blot analysis and quantification of band intensity also showed that DcCPR content significantly decreased, consistent with the results of the specific activity test. In a eukaryotic expression assay, the viability of cells expressing DcCPR was significantly higher than the viability of cells expressing green fluorescent protein (GFP) when cells were exposed to imidacloprid or thiamethoxam. CONCLUSION: These results indicate that DcCPR contributes to D. citri susceptibility to imidacloprid and thiamethoxam.

Odorant binding protein 2 reduces imidacloprid susceptibility of Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is the principal vector of Huanglongbing pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe economic losses to citrus industry worldwide. Use of broad-spectrum insecticides to control D. citri has resulted in considerable resistance development. Specific chemosensory proteins such as odorant binding proteins (OBPs) are potentially involved in reduced insecticide susceptibility. However, functional data on the contribution of OBPs to reduced susceptibility of D. citri are unavailable. We found that DcitOBP2 was stably expressed in different developmental stages and highly expressed in the legs, head and cuticle of D. citri. Expression of DcitOBP2 was significantly induced by 12 to 48 h of imidacloprid exposure and ranged from a 1.34- to 2.44-fold increase. RNAi of DcitOBP2 increased the susceptibility of D. citri adults to imidacloprid. The purified recombinant protein of DcitOBP2 expressed in Escherichia coli showed strong in vitro binding activity (Kd = 62.39 nM) to imidacloprid using microscale thermophoresis technology (MST). DcitOBP2 also had strong binding ability to thiamethoxam and dinotefuran but it had no response to abamectin, fenpropathrin and chlorpyrifos. The results showed that DcitOBP2 can interact with several neonicotinoid insecticides. This suggests that DcitOBP2 is involved in the decreased susceptibility of D. citri to imidacloprid. Our data reveal a new function of insect OBPs as a buffering protein that helps insects survive insecticide exposure. Our investigation may also aid in the development of new methods for resistance management of D. citri.