Metagenomic next-generation sequencing for the identification of infections caused by Gram-negative pathogens and the prediction of antimicrobial resistance.

OBJECTIVE: The aim of this study was to evaluate the efficacy of metagenomic next-generation sequencing (mNGS) for the identification of Gram-negative bacteria (GNB) infections and the prediction of antimicrobial resistance. METHODS: A retrospective analysis was conducted on 182 patients with diagnosis of GNB infections who underwent mNGS and conventional microbiological tests (CMTs). RESULTS: The detection rate of mNGS was 96.15%, higher than CMTs (45.05%) with a significant difference (χ 2 = 114.46, P < .01). The pathogen spectrum identified by mNGS was significantly wider than CMTs. Interestingly, the detection rate of mNGS was substantially higher than that of CMTs (70.33% vs 23.08%, P < .01) in patients with but not without antibiotic exposure. There was a significant positive correlation between mapped reads and pro-inflammatory cytokines (interleukin-6 and interleukin-8). However, mNGS failed to predict antimicrobial resistance in 5 of 12 patients compared to phenotype antimicrobial susceptibility testing results. CONCLUSIONS: Metagenomic next-generation sequencing has a higher detection rate, a wider pathogen spectrum, and is less affected by prior antibiotic exposure than CMTs in identifying Gram-negative pathogens. The mapped reads may reflect a pro-inflammatory state in GNB-infected patients. Inferring actual resistance phenotypes from metagenomic data remains a great challenge.

Ciprofloxacin enhances the biofilm formation of Staphylococcus aureus via an agrC-dependent mechanism.

Staphylococcus aureus readily forms biofilms on host tissues and medical devices, enabling its persistence in chronic infections and resistance to antibiotic therapy. The accessory gene regulator (Agr) quorum sensing system plays a key role in regulating S. aureus biofilm formation. This study reveals the widely used fluoroquinolone antibiotic, ciprofloxacin, strongly stimulates biofilm formation in methicillin-resistant S. aureus, methicillin-sensitive S. aureus, and clinical isolates with diverse genetic backgrounds. Crystal violet staining indicated that ciprofloxacin induced a remarkable 12.46- to 15.19-fold increase in biofilm biomass. Confocal laser scanning microscopy revealed that ciprofloxacin induced denser biofilms. Phenotypic assays suggest that ciprofloxacin may enhance polysaccharide intercellular adhesin production, inhibit autolysis, and reduce proteolysis during the biofilm development, thus promoting initial adhesion and enhancing biofilm stability. Mechanistically, ciprofloxacin significantly alters the expression of various biofilm-related genes (icaA, icaD, fnbA, fnbB, eap, emp) and regulators (agrA, saeR). Gene knockout experiments revealed that deletion of agrC, rather than saeRS, abolishes the ciprofloxacin-induced enhancement of biofilm formation, underscoring the key role of agrC. Thermal shift assays showed ciprofloxacin binds purified AgrC protein, thereby inhibiting the Agr system. Molecular docking results further support the potential interaction between ciprofloxacin and AgrC. In summary, subinhibitory concentrations of ciprofloxacin stimulate S. aureus biofilm formation via an agrC-dependent pathway. This inductive effect may facilitate local infection establishment and bacterial persistence, ultimately leading to therapeutic failure.

[Soil Olsen-P content changing trend and its relationship with phosphorus surplus and crop yield under long-term fertilization in loessial soil region on the Loess Plateau, China]. / é•¿æœŸæ–½è‚¥é»„ç»µåœŸæœ‰æ•ˆç£·å«é‡æ¼”å˜åŠå ¶ä¸Žç£·ç´ å¹³è¡¡å’Œä½œç‰©äº§é‡çš„å ³ç³».

The changing trend of soil available phosphorus (Olsen-P) content in soil and its relationship with soil phosphorus surplus and crop yield are fundamental when making appropriate phosphate fertilizer recommendations. In this paper, the influences of long-term fertilization on crops phosphorus uptake, soil phosphorus surplus, changing trend of soil available phosphorus content and relationships of soil available phosphorus content with soil phosphorus surplus and crop yield were investigated through 34 years (1981-2015) long-term trial in loessial soil region on the Loess Plateau. The experiment had a completely-randomized-block split-plot design in triplicate. Two main-plot treatments were no farmyard manure and farmyard manure (M), and four subplot treatments were CK (no fertilizer), N (application of chemical fertilizer N), NP (application of chemical fertilizer NP) and NPK (balanced application of chemical fertilizer NPK), respectively. The results showed that fertilization treatments and crop types significantly influenced uptake amount of phosphorus and soil phosphorus surplus. Averaged over time from 1981 to 2015, wheat mean phosphorus uptake amounts of CK, N, NP, NPK, M, MN, MNP and MNPK were 8.63, 10.64, 16.22, 16.21, 16.25, 17.83, 20.39 and 20.27 kg·hm-2, while rape phosphorus uptakeamounts of eight treatments were 4.40, 8.38, 15.08, 15.71, 10.52, 11.23, 17.96 and 17.66 kg·hm-2, respectively. The surplus amount of soil phosphorus significantly correlated with the amount of phosphorus applied to soil. When soil phosphorus surplus amount equal zero, wheat and rape phosphorus input amounts were 10.47 kg·hm-2 and 6.97 kg·hm-2, respectively. Soil phosphorus surplus amount significantly influenced the changing trend of available phosphorus content in soil. CK and N treatments had no phosphorus input, and soil available phosphorus content exhibited a declining trend, annually decreased by 0.16 mg·kg-1 and 0.15 mg·kg-1, respectively. In contrast, NP, NPK, M, MN, MNP and MNPK six treatments were applied with phosphate fertilizer every years, and available phosphorus content gradually increased along with the duration of trial, with annual increase by 0.02-0.33 mg·kg-1. Soil available phosphorus content significantly correlated with phosphorus accumulative surplus amount, and the linear models were y=0.012x+9.33 and y=0.009x+11.72 in manure and no manure treatments, respectively. In no manure treatments, wheat yields significantly positively correlated with soil available phosphorus content, however, in manure treatments, their relationships did not reach a significant level. The relationship of wheat grain yield with available phosphorus content could be significantly fitted by piecewise linear model, and available phosphorus agronomy threshold of wheat was 14.99 mg·kg-1. Rape grain yield also increased with increasing soil available phosphorus content, but the relationship was not significant. This indicated when soil available P content is higher than 14.99 mg·kg -1, application of phosphate fertili-zer should be reduced or even avoided for planting wheat in loessial soil region on the Loess Plateau.

[Effects of long-term fertilization on nitrogen fertilizer use efficiency in loessial soil region on the Loess Plateau, Northwest China]. / 长期施肥对黄土高原黄绵土氮肥利用率的影响.

Nitrogen fertilizer use efficiency is a key parameter to determine recommended nitrogen fertilizer amount and evaluate the effects of nitrogen fertilizer application. The effects of long-term fertilization on annual and accumulated nitrogen fertilizer use efficiency and the relationship between them were studied using a 34-year fertilization experiment (1981-2015) in loessial soil region. The results showed that fertilization had significant influence on annual and accumulated nitrogen ferti-lizer use efficiency of wheat, rape and flax in the period of 1983-2015. The highest mean annual nitrogen fertilizer use efficiency of wheat was achieved in N, P and K combination (NPK), thesecond was N combined with P (NP), 77.7% and 62.0% higher than N application alone respectively. The highest mean annual nitrogen fertilizer use efficiency of rape was achieved in NPK, the second was manure (M), N, P and K combination (MNPK), 93.7% and 65.6% higher than N application alone respectively. The annual nitrogenfertilizer use efficiency of M combined with N (MN) increased significantly compared with N application alone. The annual nitrogen fertilizer use efficiency of M, N and P combination (MNP) decreased significantly compared with NP. The annual nitrogen fertilizer use efficiency of MNPK decreased significantly compared with NPK.There was significant positive linear relationship between annual nitrogen fertilizer use efficiency and crop yield. The correlation between accumulated nitrogen fertilizer use efficiency and crop yield was not significant. It indicated that the annual nitrogen fertilizer use efficiency could be more effective in representing fertilizer use efficiency characteristics under given soil fertility levels, crop varieties and environmental conditions compared with accumulated nitrogen fertilizer use efficiency.

Host genetic background determines whether IL-18 deficiency results in increased susceptibility or resistance to murine Leishmania major infection.

Interleukin-18 (IL-18) plays an important role in innate and acquired immunity. IL-18 gene deficient (IL-18-/-) mice of the 129 x CD1 strain were reported to be more susceptible to Leishmania major infection than the wild-type mice. In contrast IL-18-/- mice of the C57BL/6 background were found to be as resistant as the wild-type (WT) mice. To resolve this discrepancy, IL-18 gene deficiency was introduced by backcrossing on to the highly susceptible BALB/c, or the moderately resistant DBA/1 backgrounds. Here we have demonstrated that BALB/c IL-18-/- mice were more resistant to L. major infection than WT BALB/c mice, whereas DBA/1 IL-18-/- mice were markedly more susceptible than their WT littermates. BALB/c IL-18-/- mice produced less IFNgamma and IL-4, whereas DBA/1 IL-18ko mice produced more IFNgamma and IL-4 than their respective WT controls. These result clearly demonstrate that the role of IL-18 in resistance or susceptibility to L. major is determined by host genetic background.

Clinicopathological study on TTV infection in hepatitis of unknown etiology.

AIM: To investigate the state of infection, replication site, pathogenicity and clinical significance of transfusion transmitted virus (TTV) in patients with hepatitis, especially in patients of unknown etiology. METHODS: Liver tissues taken from 136 cases of non-A non-G hepatitis were tested for TT virus antigen and nucleic acid by in situ hybridization (ISH) and nested-polymerase chain reaction (PCR). Among them, TT virus genome and its complemental strand were also detected in 24 cases of autopsy liver and extrahepatic tissues with ISH. Meanwhile, TTV DNA was detected in the sera of 187 hepatitis patients by nested-PCR. The pathological and clinical data of the cases infected with TTV only were analyzed. RESULTS: In liver, the total positive rate of TTV DNA was 32.4% and the positive signals were located in the nuclei of hepatocytes. In serus, TTV DNA was detected in 21.4% cases of hepatitis A-G, 34.4% of non-A non-G hepatitis and 15% of healthy donors. The correspondence rate of TTV DNA detection between liver tissue with ISH and sera with PCR was 63.2% and 89.3% in the same liver tissues by ISH and by PCR, respectively. Using double-strand probes and single-strand probes designed to detect TTV genome, the correspondence rate of TTV DNA detected in liver and extrahepatic tissues was 85.7%. Using single-strand probes, TTV genome could be detected in liver and extrahepatic tissues by PCR, but its complemental strands (replication strands) could be observed only in livers. The liver function of most cases infected with TTV alone was abnormal and the liver tissues had different pathological damage such as ballooning, acidophilia degeneration, formation of apoptosis bodies and focus of necrosis, but the inflammation in the lobule and portal area was mild. CONCLUSION: The positive rate of TTV DNA among cases of hepatitis was higher than that of donors, especially in patients with non-A non-G hepatitis, but most of them were coinfected with other hepatitis viruses. TTV can infect not only hepatocytes, but also extrahepatic tissues. However, the chief replication place may be liver. The infection of TTV may have some pathogenicity. Although the pathogenicity is comparatively weak, it can still damage the liver tissues. The lesions in acute hepatitis (AH) and chronic hepatitis (CH) are mild, but in severe hepatitis (SH), it can be very serious and cause liver function failure, therefore, we should pay more attention to TTV when studying the possible pathogens of so-called "liver hepatitis of unknown etiology".