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Soil copper (Cu) pollution is a serious environmental risk in the Panax notoginseng planting area. However, the effect of Cu on soil microbial metabolism and nutrient cycling in this area remains unknown. Therefore, Biolog ECO-plate and enzyme stoichiometry methods were utilized in this study to investigate the impact of exogenous Cu (control: 0 mg·kg−1; Cu100: 100 mg·kg−1; Cu400: 400 mg·kg−1; and Cu600: 600 mg·kg−1) on the metabolic function of soil microbial and nutrient limitation in the P. notoginseng soil. The results indicated that Cu100 significantly increased soil organic carbon (SOC), total phosphorus (TP), soil C:N, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) 9.89%, 15.65%, 17.91%, 61.87%, and 90.56% higher than the control, respectively. Moreover, the carbon source utilization ratio of carbohydrates, amino acids, and amphiphilic compounds of Cu100 also increased by 7.16%, 25.47%, and 84.68%, respectively, compared with the control. The activities of β-1,4-glucosidase, cellobiohyrolase, leucine amino peptidase, β-1,4-N-acetylglucosaminidase, and phosphatase significantly decreased with increasing Cu concentration. Soil enzyme stoichiometry showed that all treatments were limited by nitrogen (vector angle < 45°; 19.045–22.081). Cu600 led to the lowest carbon limitation (1.798) and highest carbon use efficiency (CUE:0.267). The PLS-SEM model also showed that MBC, MBN, MBP, and microbial diversity positively affected carbon and nitrogen limitation (0.654 and 0.424). Soil carbon, nitrogen, phosphorus, stoichiometric ratio, MBC, MBN, and MBP positively affected CUE (0.527 and 0.589). The microbial diversity index significantly negatively affected CUE (−1.490). Multiple linear stepwise regression analyses showed that CUE was mainly influenced by MBC, AP, C:P, and LAP. Thus, P. notoginseng soil can benefit soil microbial carbon and nitrogen limitations at low Cu concentrations. Clarifying the metabolic activity and nutritional status of microorganisms under Cu stress can provide some theoretical basis for realizing China's comprehensive and effective management and control policies for environmental risks from metals by 2035.

The objective of this study is to optimize the ultrasonic-assisted extraction process of Ku Shen (Sophorae Flavescentis Radix) extracts (KSE) against Vibrio parahaemolyticus and explore their anti-biofilm activity and mechanism of action. The ultrasonic-assisted extraction process of KSE optimized by single factor experiment, Box–Behnken design and response surface methodology was as follows: 93% ethanol as solvent, liquid/material ratio of 30 mL/g, ultrasonic power of 500 W, extraction temperature of 80°C and time of 30 min. Under these conditions, the diameter of inhibition circle of KSE was 15.60 ± 0.17 mm, which had no significant difference with the predicted value. The yield of dried KSE is 32.32 ± 0.57% and the content of total flavonoids in KSE was 57.02 ± 5.54%. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of KSE against V. parahaemolyticus were 0.25 and 0.5 mg/mL, respectively. Crystal violet staining, Congo red plate, spectrophotometry, CCK-8 and scanning electron microscopy were used to investigate the activity and mechanism of action of KSE against V. parahaemolyticus biofilm. The results showed that the sub-MIC of KSE could significantly inhibit biofilm formation, reduce the synthesis of polysaccharide intercellular adhesin (PIA) and the secretion of extracellular DNA. In addition, the inhibition rate of biofilm formation and clearance rate of mature biofilm of 1.0 mg/mL KSE were 85.32 and 74.04%, and the reduction rate of metabolic activity of developing and mature biofilm were 77.98 and 74.46%, respectively. These results were confirmed by visual images obtained by scanning electron microscopy. Therefore, KSE has the potential to further isolate the anti-biofilm agent and evaluate it for the preservation process of aquatic products.

IntroductionThis study aimed to investigate the digestive function, urea utilization ability, and bacterial composition changes in rumen microbiota under high urea (5% urea in diet) over 23 days of continuous batch culture in vitro.MethodsThe gas production, dry matter digestibility, and bacterial counts were determined for the continuously batch-cultured rumen fluid (CRF). The changes in fermentation parameters, NH3-N utilization efficiency, and microbial taxa were analyzed in CRF and were compared with that of fresh rumen fluid (RF), frozen rumen fluid (FRF, frozen rumen fluid at −80°C for 1 month), and the mixed rumen fluid (MRF, 3/4 RF mixed with 1/4 CRF) with in vitro rumen fermentation.ResultsThe results showed that the dry matter digestibility remained stable while both the microbial counts and diversity significantly decreased over the 23 days of continuous batch culture. However, the NH3-N utilization efficiency of the CRF group was significantly higher than that of RF, FRF, and MRF groups (p

Many studies have reported obvious seasonal differences in the intestinal flora of rats, and this stable distribution of the seasonal flora helps in maintaining the normal physiological function of the host. However, the mechanism underlying these seasonal differences in intestinal flora remains unclear. To explore the correlation among seasonal factors and intestinal water metabolism and intestinal flora, 20 Sprague Dawley (SD) rats were divided into spring, summer, autumn, and winter groups. The environment for the four seasons was simulated using the Balanced Temperature and Humidity Control system. The intestinal water metabolism was evaluated by determining the intestinal transmission function, fecal water content, water content of colonic tissue, and the colonic expression levels of AQP3, AQP4, and AQP8. The composition and relative abundance of intestinal microflora in rats in each season were assessed through 16S rDNA amplifier sequencing, and the relationship between the dominant flora and intestinal water metabolism in each season was analyzed using Spearman correlation analysis. The high temperature and humidity season could lead to an increase in intestinal water metabolism and intestinal water content in rats, whereas the low temperature and humidity season could lead to a decrease, which was closely related to the change in microflora. To explore the molecular mechanism of seasonal changes in intestinal water metabolism, the concentration of colonic 5-HT, VIP, cAMP, and PKA associated with intestinal water metabolism in rats were also examined. Seasonal changes could affect the concentration of colonic 5-HT and VIP in rats, and then regulate AQPs through cAMP/PKA pathway to affect the intestinal water metabolism. These results suggest that seasonal factors affect the level of intestinal water metabolism in rats and result in seasonal differences in intestinal flora.

Candida duobushaemulonii, type II Candida haemulonii complex, is closely related to Candida auris and capable of causing invasive and non-invasive infections in humans. Eleven strains of C. duobushaemulonii were collected from China Hospital Invasive Fungal Surveillance Net (CHIF-NET) and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), VITEK 2 Yeast Identification Card (YST), and internal transcribed spacer (ITS) sequencing. Whole genome sequencing of C. duobushaemulonii was done to determine their genotypes. Furthermore, C. duobushaemulonii strains were tested by Sensititre YeastOne™ and Clinical and Laboratory Institute (CLSI) broth microdilution panel for antifungal susceptibility. Three C. duobushaemulonii could not be identified by VITEK 2. All 11 isolates had high minimum inhibitory concentrations (MICs) to amphotericin B more than 2 μg/ml. One isolate showed a high MIC value of ≥64 μg/ml to 5-flucytosine. All isolates were wild type (WT) for triazoles and echinocandins. FUR1 variation may result in C. duobushaemulonii with high MIC to 5-flucytosine. Candida duobushaemulonii mainly infects patients with weakened immunity, and the amphotericin B resistance of these isolates might represent a challenge to clinical treatment.

Lacticaseibacillus paracasei SMN-LBK, which was isolated in Xinjiang, has been shown to be a probiotic strain and used as the auxiliary starter for dairy fermentation. Comparative genomic analysis was performed to investigate the metabolic preference and ethanol tolerance mechanisms of L. paracasei SMN-LBK. The results of comparative genomics showed that L. paracasei strains had high conservation and genetic diversity. SMN-LBK encoded various genes related to carbohydrate and amino acid metabolism pathways, which endow this strain with good fermentation potential. In addition, 6 CRISPR sequences and 8 cas proteins were found in SMN-LBK, and these could play vital roles in the immune system. Furthermore, a unique cluster of potential secondary metabolism genes related to bacteriocins was detected in the genome of SMN-LBK, and this could be important for the preservation of fermented foods. Multiple genes related to alcohol tolerance were also identified. In conclusion, our study explained the traits that were previously demonstrated for SMN-LBK as phenotypes and provided a theoretical basis for the application of SMN-LBK in the food industry.

ObjectiveTo investigate the bacterial distribution and antimicrobial resistance profile of clinical isolates from Gram-negative bacteria bloodstream infections (GNBSI) in China.MethodsThe clinical bacterial strains isolated from blood culture were collected during April 2019 to December 2021 in 21 member hospitals of China Bloodstream Gram-negative Pathogens Antimicrobial Resistance and Virulence Surveillance Network (CARVIS-NET). Antibiotic susceptibility test was conducted by broth microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI, United States). WHONET 2021 and SPSS 22.0 were used to analyze data.ResultsDuring the study period, 1939 Gram-negative bacteria were collected from 21 hospitals, among which 1,724 (88.9%) were Enterobacteriaceae, 207 (10.7%) were non-fermenting Gram-negative bacteria and 8 (0.4%) were others. The top five bacterial species were Escherichia coli (46.2%), Klebsiella pneumoniae (31.6%), Pseudomonas aeruginosa (4.9%), Acinetobacter baumannii (4.2%) and Enterobacter cloacae (3.0%). For K. pneumoniae, antibiotic resistance was mainly prevalent in hospital-associated bloodstream infections, while for A. baumannii, antibiotic resistance was mainly prevalent in community-associated bloodstream infections. It is worth mentioning that 94.1% of the 1939 Gram-negative isolates were susceptible to polymyxin B. The sensitivity of the strains involved in our investigation to polymyxin B is highly correlated with their sensitivity to colistin.ConclusionThe surveillance results in CARVIS-NET-2021 showed that the main pathogens of GNBSI in China were Enterobacteriaceae, while E. coli was the most common pathogen. The resistance rates of K. pneumonia, P. aeruginosa, A. baumannii, and E. cloacae to multiple antibiotics kept on a high level. In many cases, polymyxin B and colistin has become the last-resort agents to combat bloodstream infections caused by multidrug-resistant (MDR) Gram-negative bacteria.

Crude sophorolipids (SLs) have been proven to perform varying degrees of inhibitory effects on different pathogenic bacteria. However, systematic comparative studies of pure lactonic sophorolipid (LSL) among different types of bacteria are few. In this study, the antibacterial effects and mechanisms of LSL on pathogenic bacteria of Staphylococcus aureus, Lactobacillus sp., Pseudomonas aeruginosa, and Escherichia coli were investigated. Bacteriostatic circle, antibacterial rate, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of LSL on different pathogenic bacteria were measured. Then, the antibacterial mechanisms of LSL on S. aureus and P. aeruginosa were explored using ultrastructural observation, cell membrane permeability analysis, intracellular ATP content determination, and extracellular UV absorption detection. With the minimum MIC and MBC values of 0.05 and 0.20 mg/ml, LSL exhibited the best inhibitory effect against S. aureus, followed by P. aeruginosa. LSL showed no significant inhibitory effect on E. coli and Lactobacillus sp. For both S. aureus and P. aeruginosa, LSL achieved bacteriostatic and bactericidal effects by destroying the cell wall, increasing the permeability of the cell membrane and leading to the flow out of intracellular contents. However, the action mode and action intensity of LSL on the cell wall and membrane of these two bacteria were significantly different. LSL had a greater influence on the cell membrane of S. aureus by “leaking,” while it exhibited a stronger effect on the cell wall of P. aeruginosa by “blasting.” These results contributed to a better understanding of the relationship between LSL and different bacterial cell structures, further suggesting the conclusion that LSL might be used for the targeted treatment of special pathogenic bacteria.

Soil salinity has adverse effects on soil microbial activity and nutrient cycles and therefore limits crop growth and yield. Amendments with halotolerant phosphate-solubilizing bacteria (PSB) and rock phosphate (RP) may improve properties of saline soil. In this study, we investigated the effects of RP either alone or in combination with PSB (Providencia rettgeri strain TPM23) on peanut growth and soil quality in a saline soil. With the combined application of RP and PSB, plant length and biomass (roots and shoots) and uptake of phosphorus (P), nitrogen (N), and potassium (K) increased significantly. Soil Na+ and Cl– contents decreased in the PR alone or PR combined with PSB treatment groups. There were strongly synergistic effects of RP and PSB on soil quality, including a decrease in pH. The soil available N, P, and K contents were significantly affected by the PSB treatments. In addition, the alkaline phosphomonoesterases, urease, and dehydrogenase activities increased significantly compared with the untreated group; highest alkaline phosphomonoesterases activity was observed in the RP and PSB treatment groups. The composition of rhizosphere soil bacterial communities was determined using 454-pyrosequencing of the 16S rRNA gene. In the PR alone or PR combined with PSB treatment groups, the structure of the soil bacterial community improved with increasing richness and diversity. With PSB inoculation, the relative abundance of Acidobacteria, Chloroflexi, and Planctomycetes increased. The three phyla were also positively correlated with soil available N and root dry weight. These results suggested microbiological mechanisms by which the combined use of RP and PSB improved saline soil and promoted plant growth. Overall, the study indicates the combined use of RP and PSB can be an economical and sustainable strategy to increase plant growth in P-deficient and salt-affected soils.

Objectives: Ceftazidime–avibactam is a novel synthetic beta-lactam + beta-lactamase inhibitor combination. We evaluated the performance of the gradient diffusion strip method and the disk diffusion method for the determination of ceftazidime–avibactam against Enterobacterales and Pseudomonas aeruginosa.Methods: Antimicrobial susceptibility testing of 302 clinical Enterobacterales and Pseudomonas aeruginosa isolates from two centers were conducted by broth microdilution (BMD), gradient diffusion strip method, and disk diffusion method for ceftazidime–avibactam. Using BMD as a gold standard, essential agreement (EA), categorical agreement (CA), major error (ME), and very major error (VME) were determined according to CLSI guidelines. CA and EA rate > 90%, ME rate < 3%, and VME rate < 1.5% were considered as acceptable criteria. Polymerase chain reaction and Sanger sequencing were performed to determine the carbapenem resistance genes of all 302 isolates.Results: A total of 302 strains were enrolled, among which 182 strains were from center 1 and 120 strains were from center 2. A percentage of 18.21% (55/302) of the enrolled isolates were resistant to ceftazidime–avibactam. The CA rates of the gradient diffusion strip method for Enterobacterales and P. aeruginosa were 100% and 98.65% (73/74), respectively, and the EA rates were 97.37% (222/228) and 98.65% (73/74), respectively. The CA rates of the disk diffusion method for Enterobacterales and P. aeruginosa were 100% and 95.95% (71/74), respectively. No VMEs were found by using the gradient diffusion strip method, while the ME rate was 0.40% (1/247). No MEs were found by using the disk diffusion method, but the VME rate was 5.45% (3/55). Therefore, all the parameters of the gradient diffusion strip method were in line with acceptable criteria. For 31 blaKPC, 33 blaNDM, 7 blaIMP, and 2 blaVIM positive isolates, both CA and EA rates were 100%; no MEs or VMEs were detected by either method. For 15 carbapenemase-non-producing resistant isolates, the CA and EA rates of the gradient diffusion strips method were 100%. Whereas the CA rate of the disk diffusion method was 80.00% (12/15), the VME rate was 20.00% (3/15).Conclusion: The gradient diffusion strip method can meet the needs of clinical microbiological laboratories for testing the susceptibility of ceftazidime–avibactam drugs. However, the VME rate > 1.5% (5.45%) by the disk diffusion method. By comparison, the performance of the gradient diffusion strip method was better than that of the disk diffusion method.

ObjectivesThe performance of mainstream commercial antimicrobial susceptibility testing systems on polymyxins has not been well evaluated in China. In this study, three antimicrobial susceptibility testing systems were evaluated for polymyxin B and colistin.MethodsThe MICs of 257 Gram-negative strains collected from clinical cases and livestock were determined and analyzed. Using Broth Microdilution as the gold standard, the performance of VITEK 2® COMPACT, PhoenixTM M50, and Bio-kont AST System were evaluated. Essential agreement (EA), category agreement (CA), very major error (VME), and major error (ME) were calculated for comparison. The results of mcr-1 positive strains were separately discussed.ResultsThe EA, CA, VME, and ME to polymyxin B for Bio-kont were 83.5, 95.6, 13.1, and 0.6%, respectively. The EAs, CAs, VMEs, and MEs to colistin were as follows: Bio-kont, 86.7%/96.5%/7.2%/1.7%; Vitek 2, 64.2%/86.8%/41.0%/0%, and Phoenix M50, 92.9%/92.9%/21.7%/0%. The performance of Bio-kont to polymyxin B and colistin for Pseudomonas spp. (EA, CA < 90%, VME > 1.5%, ME = 5.6%/10%) and Enterobacter spp. (EA, CA < 90%, VME > 1.5% and ME = 0%), Vitek to colistin for most genera, and Phoenix to colistin for Enterobacter spp. (EA, CA < 90%, VME > 1.5%, ME = 0%) were unsatisfactory compared with other genera. The performance of Bio-kont to polymyxins for Escherichia spp. and Phoenix to colistin for Citrobacter spp., Escherichia spp., and Klebsiella spp., which all met the CLSI standard, were satisfactory. When the susceptibility of mcr-1 positive E. coli was tested, Bio-kont and Phoenix M50 presented excellent performance with no category errors, while Vitek 2 performed a high VME (25.5%).ConclusionWith relatively more accurate results for polymyxin B and colistin and lower VME, Bio-kont has an advantage in polymyxin antimicrobial susceptibility testing, especially for Escherichia spp., Klebsiella spp., Citrobacter spp. and Acinetobacter spp.

Infections by Pseudomonas aeruginosa are difficult to cure due to its high intrinsic and acquired antibiotic resistance. Once colonized the human host, and thanks to antibiotic treatment pressure, P. aeruginosa usually acquires genetic mutations which provide bacteria with antibiotic resistance as well as ability to better adapt to the host environment. Deciphering the evolutionary traits may provide important insights into the development of effective combinatory antibiotic therapy to treat P. aeruginosa infections. In this study, we investigated the molecular mechanisms by which a clinical isolate (ISP50) yields a carbapenem-resistant derivative (IRP41). RNAseq and genomic DNA reference mapping were conducted to compare the transcriptional profiles and in vivo evolutionary trajectories between the two isolates. Our results demonstrated that oprD mutation together with ampC hyper-expression contributed to the increased resistance to carbapenem in the isolate IRP41. Furthermore, a ldcA (PA5198) gene, encoding murein tetrapeptide carboxypeptidase, has been demonstrated for the first time to negatively influence the ampC expression in P. aeruginosa.

Long non-coding RNAs are involved in many infectious diseases. Our previous studies showed that lncRNA-ENST00000421645 expression is increased in T lymphocytes of neurosyphilis patients compared to healthy controls. However, whether lncRNA-ENST00000421645 has biological functions remains unclear. The current study was undertaken to understand the mechanism of lncRNA-ENST00000421645 in T lymphocyte function in neurosyphilis patients. The lncRNA-ENST00000421645 pull-down assay showed that lncRNA-ENST00000421645 acted on the acetylase NAT10. The chromatin immunoprecipitation (ChIP)-PCR results showed that lncRNA-ENST00000421645 promoted the acetylation of histone H3K27 adjacent to the Kank1 promoter, thereby promoting Kank1 protein expression. Kank1 promotes 14-3-3 protein expression, inhibits NF-kB activation, inhibits IFN-γ secretion by T lymphocytes, and promotes T lymphocyte apoptosis. Taken together, our findings suggest a novel mechanism that LncRNA-ENST00000421645 upregulates Kank1 to inhibit IFN-γ expression and promote T cell apoptosis in neurosyphilis.