Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris.

Candida auris (C. auris) was first discovered in Japan in 2009 and has since spread worldwide. It exhibits strong transmission ability, high multidrug resistance, blood infectivity, and mortality rates. Traditional diagnostic techniques for C. auris have shortcomings, leading to difficulty in its timely diagnosis and identification. Therefore, timely and accurate diagnostic assays for clinical samples are crucial. We developed a novel, rapid recombinase-aided amplification (RAA) assay targeting the 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 28S rRNA genes for C. auris identification. This assay can rapidly amplify DNA at 39 °C in 20 min. The analytical sensitivity and specificity were evaluated. From 241 clinical samples collected from pediatric inpatients, none were detected as C. auris-positive. We then prepared simulated clinical samples by adding 10-fold serial dilutions of C. auris into the samples to test the RAA assay's efficacy and compared it with that of real-time PCR. The assay demonstrated an analytical sensitivity of 10 copies/µL and an analytical specificity of 100%. The lower detection limit of the RAA assay for simulated clinical samples was 101 CFU/mL, which was better than that of real-time PCR (102-103 CFU/mL), demonstrating that the RAA assay may have a better detection efficacy for clinical samples. In summary, the RAA assay has high sensitivity, specificity, and detection efficacy. This assay is a potential new method for detecting C. auris, with simple reaction condition requirements, thus helping to manage C. auris epidemics.

Synthetic consortia of four strains promote Schisandra chinensis growth by regulating soil microbial community and improving soil fertility.

MAIN CONCLUSION: Synthetic consortia performed better in promoting Schisandra chinensis growth than individual strains, and this result provides valuable information for the development of synthetic microbial fertilizers. Schisandra chinensis is an herbal medicine that can treat numerous diseases. However, the excessive reliance on chemical fertilizers during the plantation of S. chinensis has severely restricted the development of the S. chinensis planting industry. Plant growth-promoting rhizobacteria (PGPR) can promote the growth of a wide range of crops, and synthetic consortia of them are frequently superior to those of a single strain. In this study, we compared the effects of four PGPR and their synthetic consortia on S. chinensis growth. The pot experiment showed that compared with the control, synthetic consortia significantly increased the plant height, biomass, and total chlorophyll contents of S. chinensis, and their combined effects were better than those of individual strains. In addition, they improved the rhizosphere soil fertility (e.g., TC and TN contents) and enzyme activities (e.g., soil urease activity) and affected the composition and structure of soil microbial community significantly, including promoting the enrichment of beneficial microorganisms (e.g., Actinobacteria and Verrucomicrobiota) and increasing the relative abundance of Proteobacteria, a dominant bacterial phylum. They also enhanced the synergistic effect between the soil microorganisms. The correlation analysis between soil physicochemical properties and microbiome revealed that soil microorganisms participated in regulating soil fertility and promoting S. chinensis growth. This study may provide a theoretical basis for the development of synthetic microbial fertilizers for S. chinensis.

Detecting and quantifying Veillonella by real-time quantitative PCR and droplet digital PCR.

Veillonella spp. are Gram-negative opportunistic pathogens present in the respiratory, digestive, and reproductive tracts of mammals. An abnormal increase in Veillonella relative abundance in the body is closely associated with periodontitis, inflammatory bowel disease, urinary tract infections, and many other diseases. We designed a pair of primers and a probe based on the 16S rRNA gene sequences of Veillonella and conducted real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) to quantify the abundance of Veillonella in fecal samples. These two methods were tested for specificity and sensitivity using simulated clinical samples. The sensitivity of qPCR was 100 copies/µL, allowing for the accurate detection of a wide range of Veillonella concentrations from 103 to 108 CFU/mL. The sensitivity of ddPCR was 11.3 copies/µL, only allowing for the accurate detection of Veillonella concentrations from 101 to 104 CFU/mL because of the limited number of droplets generated by ddPCR. ddPCR is therefore more suitable for the detection of low-abundance Veillonella samples. To characterize the validity of the assay system, clinical samples from children with inflammatory bowel disease were collected and analyzed, and the results were verified using isolation methods. We conclude that molecular assays targeting the 16S rRNA gene provides an important tool for the rapid diagnosis of chronic and infectious diseases caused by Veillonella and also supports the isolation and identification of Veillonella for research purposes. KEY POINTS: • With suitable primer sets, the qPCR has a wider detection range than ddPCR. • ddPCR is suitable for the detection of low-abundance samples. • Methods successfully guided the isolation of Veillonella in clinical sample.

The role of integration host factor in biofilm and virulence of high-alcohol-producing Klebsiella pneumoniae.

Klebsiella pneumoniae is a well-known human nosocomial pathogen with an arsenal of virulence factors, including capsular polysaccharides (CPS), fimbriae, flagella, and lipopolysaccharides (LPS). Our previous study found that alcohol acted as an essential virulence factor for high-alcohol-producing K. pneumoniae (HiAlc Kpn). Integration host factor (IHF) is a nucleoid-associated protein that functions as a global virulence regulator in Escherichia coli. However, the regulatory role of IHF in K. pneumoniae remains unknown. In the present study, we found that deletion of ihfA or ihfB resulted in a slight defect in bacterial growth, a severe absence of biofilm formation and cytotoxicity, and a significant reduction in alcohol production. RNA sequencing differential gene expression analysis showed that compared with the wild-type control, the expression of many virulence factor genes was downregulated in ΔihfA and ΔihfB strains, such as those related to CPS (rcsA, galF, wzi, and iscR), LPS (rfbABCD), type I and type III fimbriae (fim and mrk operon), cellulose (bcs operon), iron transporter (feoABC, fhuA, fhuF, tonB, exbB, and exbD), quorum sensing (lsr operon and sdiA), type II secretion system (T2SS) and type VI secretion system (T6SS) (tssG, hcp, and gspE). Of these virulence factors, CPS, LPS, fimbriae, and cellulose are involved in biofilm formation. In addition, IHF could affect the alcohol production by regulating genes related to glucose intake (ptsG), pyruvate formate-lyase, alcohol dehydrogenase, and the tricarboxylic acid (TCA) cycle. Our data provided new insights into the importance of IHF in regulating the virulence of HiAlc Kpn. IMPORTANCE Klebsiella pneumoniae is a well-known human nosocomial pathogen that causes various infectious diseases, including urinary tract infections, hospital-acquired pneumonia, bacteremia, and liver abscesses. Our previous studies demonstrated that HiAlc Kpn mediated the development of nonalcoholic fatty liver disease by producing excess endogenous alcohol in vivo. However, the regulators regulating the expression of genes related to metabolism, biofilm formation, and virulence of HiAlc Kpn remain unclear. In this study, the regulator IHF was found to positively regulate biofilm formation and many virulence factors including CPS, LPS, type I and type III fimbriae, cellulose, iron transporter, AI-2 quorum sensing, T2SS, and T6SS in HiAlc Kpn. Furthermore, IHF positively regulated alcohol production in HiAlc Kpn. Our results suggested that IHF could be a potential drug target for treating various infectious diseases caused by K. pneumoniae. Hence, the regulation of different virulence factors by IHF in K. pneumoniae requires further investigation.

A novel phage carrying capsule depolymerase effectively relieves pneumonia caused by multidrug-resistant Klebsiella aerogenes.

BACKGROUND: Klebsiella aerogenes can cause ventilator-associated pneumonia by forming biofilms, and it is frequently associated with multidrug resistance. Phages are good antibiotic alternatives with unique advantages. There has been a lack of phage therapeutic explorations, kinetic studies, and interaction mechanism research targeting K. aerogenes. METHODS: Plaque assay, transmission electron microscopy and whole-genome sequencing were used to determine the biology, morphology, and genomic characteristics of the phage. A mouse pneumonia model was constructed by intratracheal/endobronchial delivery of K. aerogenes to assess the therapeutic effect of phage in vivo. Bioinformatics analysis and a prokaryotic protein expression system were used to predict and identify a novel capsule depolymerase. Confocal laser scanning microscopy, Galleria mellonella larvae infection models and other experiments were performed to clarify the function of the capsule depolymerase. RESULTS: A novel lytic phage (pK4-26) was isolated from hospital sewage. It was typical of the Podoviridae family and exhibited serotype specificity, high lytic activity, and high environmental adaptability. The whole genome is 40,234 bp in length and contains 49 coding domain sequences. Genomic data show that the phage does not carry antibiotic resistance, virulence, or lysogenic genes. The phage effectively lysed K. aerogenes in vivo, reducing mortality and alleviating pneumonia without promoting obvious side effects. A novel phage-derived depolymerase was predicted and proven to be able to digest the capsule, remove biofilms, reduce bacterial virulence, and sensitize the bacteria to serum killing. CONCLUSIONS: The phage pK4-26 is a good antibiotic alternative and can effectively relieve pneumonia caused by multidrug-resistant K. aerogenes. It carries a depolymerase that removes biofilms, reduces virulence, and improves intrinsic immune sensitivity.

Glucose Induces Resistance to Polymyxins in High-Alcohol-Producing Klebsiella pneumoniae via Increasing Capsular Polysaccharide and Maintaining Intracellular ATP.

High-alcohol-producing K. pneumoniae (HiAlc Kpn) causes nonalcoholic fatty liver disease (NAFLD) by producing excess endogenous alcohol in the gut of patients with NAFLD, using glucose as the main carbon source. The role of glucose in the response of HiAlc Kpn to environmental stresses such as antibiotics remains unclear. In this study, we found that glucose could enhance the resistance of HiAlc Kpn to polymyxins. First, glucose inhibited the expression of crp in HiAlc Kpn and promoted the increase of capsular polysaccharide (CPS), which promoted the drug resistance of HiAlc Kpn. Second, glucose maintained high ATP levels in HiAlc Kpn cells under the pressure of polymyxins, enhancing the resistance of the cells to the killing effect of antibiotics. Notably, the inhibition of CPS formation and the decrease of intracellular ATP levels could both effectively reverse glucose-induced polymyxins resistance. Our work demonstrated the mechanism by which glucose induces polymyxins resistance in HiAlc Kpn, thereby laying the foundation for developing effective treatments for NAFLD caused by HiAlc Kpn. IMPORTANCE HiAlc Kpn can use glucose to produce excess endogenous alcohol for promoting the development of NAFLD. Polymyxins are the last line of antibiotics and are commonly used to treat infections caused by carbapenem-resistant K. pneumoniae. In this study, we found that glucose increased bacterial resistance to polymyxins via increasing CPS and maintaining intracellular ATP; this increases the risk of failure to treat NAFLD caused by multidrug-resistant HiAlc Kpn infection. Further research revealed the important roles of glucose and the global regulator, CRP, in bacterial resistance and found that inhibiting CPS formation and decreasing intracellular ATP levels could effectively reverse glucose-induced polymyxins resistance. Our work reveals that glucose and the regulatory factor CRP can affect the resistance of bacteria to polymyxins, laying a foundation for the treatment of infections caused by multidrug-resistant bacteria.

ArcR contributes to tolerance to fluoroquinolone antibiotics by regulating katA in Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that shows a unique ability to quickly respond to a variety of antibiotics. The Crp/Fnr family transcriptional regulator ArcR controls expression of arginine deiminase pathway genes arcABDC, which enable the utilization of arginine as an energy source for cell growth under anaerobic conditions. However, ArcR shares low overall similarity with other Crp/Fnr family proteins, suggesting that they differ in the response to environmental stress. In this study, MIC and survival assays were performed to determine the role of ArcR in antibiotic resistance and tolerance. The results showed that deletion of arcR reduced tolerance of S.aureus to fluoroquinolone antibiotics, mainly through a defect in the response to oxidative stress. In ΔarcR mutant, the expression of the major catalase gene katA was downregulated, and katA overexpression restored bacterial resistance to oxidative stress and antibiotics. We showed that ArcR directly regulated katA transcription by binding to the promoter region of katA. Therefore, our results revealed the contribution of ArcR in bacterial tolerance to oxidative stress and subsequently to fluoroquinolones antibiotics. This study added our understanding on the role of Crp/Fnr family in bacterial susceptibility to antibiotics.

Microbial community roles and chemical mechanisms in the parasitic development of Orobanche cumana.

Orobanche cumana Wallr. is a holoparasite weed that extracts water and nutrients from its host the sunflower, thereby causing yield reductions and quality losses. However, the number of O. cumana parasites in the same farmland is distinctly different. The roots of some hosts have been heavily parasitized, while others have not been parasitized. What are the factors contributing to this phenomenon? Is it possible that sunflower interroot microorganisms are playing a regulatory role in this phenomenon? The role of the microbial community in this remains unclear. In this study, we investigated the rhizosphere soil microbiome for sunflowers with different degrees of O. cumana parasitism, that is, healthy, light infection, moderate infection, and severe infection on the sunflower roots. The microbial structures differed significantly according to the degree of parasitism, where Xanthomonadaceae was enriched in severe infections. Metagenomic analyses revealed that amino acid, carbohydrate, energy, and lipid metabolism were increased in the rhizosphere soils of severely infected sunflowers, which were attributed to the proliferation of Lysobacter. Lysobacter antibioticus (HX79) was isolated and its capacity to promote O. cumana seed germination and increase the germ tube length was confirmed by germination and pot experiments. Cyclo(Pro-Val), an active metabolite of strain HX79, was identified and metabolomic and molecular docking approaches confirmed it was responsible for promoting O. cumana seed germination and growth. And we found that Pseudomonas mandelii HX1 inhibited the growth of O. cumana in the host rhizosphere soil. Our findings clarify the role of rhizosphere microbiota in regulating the parasite O. cumana to possibly facilitate the development of a new weed suppression strategy.