Multiple factors trigger the formation and resuscitation of the VBNC state in alcohol-producing Klebsiella pneumoniae.

Klebsiella pneumoniae can enter a viable but nonculturable (VBNC) state to survive in unfavorable environments. Our research found that high-, medium-, and low-alcohol-producing K. pneumoniae strains are associated with nonalcoholic fatty liver disease. However, the presence of the three Kpn strains has not been reported in the VBNC state or during resuscitation. In this study, the effects of different strains, salt concentrations, oxygen concentrations, temperatures, and nutrients in K. pneumoniae VBNC state were evaluated. The results showed that high-alcohol-producing K. pneumoniae induced a slower VBNC state than medium-alcohol-producing K. pneumoniae, and low-alcohol-producing K. pneumoniae. A high-salt concentration and micro-oxygen environment accelerated the loss of culturability. Simultaneously, both real-time quantitative PCR and droplet digital PCR were developed to compare the quantitative comparison of three Kpn strain VBNC states by counting single-copy gene numbers. At 22°C or 37°C, the number of culturable cells decreased significantly from about 108 to 105-106 CFU/mL. In addition, imipenem, ciprofloxacin, polymyxin, and phiW14 inhibited cell resuscitation but could not kill VBNC-state cells. These results revealed that the different environments evaluated play different roles in the VBNC induction process, and new effective strategies for eliminating VBNC-state cells need to be further studied. These findings provide a better understanding of VBNC-state occurrence, maintenance, detection, and absolute quantification, as well as metabolic studies of resuscitation resistance and ethanol production.IMPORTANCEBacteria may enter VBNC state under different harsh environments. Pathogenic VBNC bacteria cells in clinical and environmental samples pose a potential threat to public health because cells cannot be found by routine culture. The alcohol-producing Kpn VBNC state was not reported, and the influencing factors were unknown. The formation and recovery of VBNC state is a complete bacterial escape process. We evaluated the influence of multiple induction conditions on the formation of VBNC state and recovery from antibiotic and bacteriophage inhibition, and established a sensitive molecular method to enumerate the VBNC cells single-copy gene. The method can improve the sensitivity of pathogen detection in clinical, food, and environmental contamination monitoring, and outbreak warning. The study of the formation and recovery of VBNC-state cells under different stress environments will also promote the microbiological research on the development, adaptation, and resuscitation in VBNC-state ecology.

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.

Inhibition of the ATP synthase increases sensitivity of Escherichia coli carrying mcr-1 to polymyxin B.

Bacterial infections caused by multidrug-resistant (MDR) gram-negative strains carrying the mobile colistin resistance gene mcr-1 are serious threats to world public health due to the lack of effective treatments. Inhibition of the ATP synthase makes bacteria such as Staphylococcus aureus and Klebsiella pneumoniae more sensitive to polymyxin. This provides new strategies for treating infections caused by polymyxins-resistant bacteria carrying mcr-1. Six mcr-1-positive strains were isolated from clinical samples, and all were identified as Escherichia coli. Here we investigated several ATP synthase inhibitors, N,N'-dicyclohexylcarbodiimide (DCCD), resveratrol, and piceatannol, for their antibacterial effects against the mcr-1-positive strains combined with polymyxin B (POL). Checkerboard assay, time-kill assay, biofilm inhibition and eradication assay indicated the significant synergistic effect of ATP synthase inhibitors/POL combination in vitro. Meanwhile, mouse infection model experiment was also performed, showing a 5 log10 reduction of the pathogen after treatment with the resveratrol/POL combination. Moreover, adding adenosine disodium triphosphate (Na2ATP) could inhibit the antibacterial effect of the ATP synthase inhibitors/POL combination. In conclusion, our study confirmed that inhibition of ATP production could increase the susceptibility of bacteria carrying mcr-1 to polymyxins. This provides a new strategy against polymyxins-resistant bacteria infection.

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.

Prophylactic vitamin C supplementation regulates DNA demethylation to protect against cisplatin-induced acute kidney injury in mice.

Cisplatin-induced acute kidney injury (AKI) restricts the use of cisplatin as a first-line chemotherapeutic agent. Our previous study showed that prophylactic vitamin C supplementation may act as an epigenetic modulator in alleviating cisplatin-induced AKI in mice. However, the targets of vitamin C and the mechanisms underlying the epigenetics changes remain largely unknown. Herein, whole-genome bisulfite sequencing and bulk RNA sequencing were performed on the kidney tissues of mice treated with cisplatin with prophylactic vitamin C supplementation (treatment mice) or phosphate-buffered saline (control mice) at 24 h after cisplatin treatment. Ascorbyl phosphate magnesium (APM), an oxidation-resistant vitamin C derivative, was found that led to global hypomethylation in the kidney tissue and regulated different functional genes in the promoter region and gene body region. Integrated evidence suggested that APM enhanced renal ion transport and metabolism, and reduced apoptosis and inflammation in the kidney tissues. Strikingly, Mapk15, Slc22a6, Cxcl5, and Cd44 were the potential targets of APM that conferred protection against cisplatin-induced AKI. Moreover, APM was found to be difficult to rescue cell proliferation and apoptosis caused by cisplatin in the Slc22a6 knockdown cell line. These results elucidate the mechanism by which vitamin C as an epigenetic regulator to protects against cisplatin-induced AKI and provides a new perspective and evidence support for controlling the disease process through regulating DNA methylation.

Alterations in gut microbiota and metabolite profiles in patients with infantile cholestasis.

BACKGROUND: Infantile cholestasis (IC) is the most common hepatobiliary disease in infants, resulting in elevated direct bilirubin levels. Indeed, hepatointestinal circulation impacts bile acid and bilirubin metabolism. This study evaluates changes in the gut microbiota composition in children with IC and identifies abnormal metabolite profiles associated with microbial alterations. RESULTS: The gut microbiota in the IC group exhibits the higher abundance of Veillonella, Streptococcus and Clostridium spp. (P < 0.05), compared to healthy infants (CON) group. Moreover, the abundance of Ruminococcus, Vibrio butyricum, Eubacterium coprostanogenes group, Intestinibacter, and Faecalibacterium were lower (P < 0.05). In terms of microbiota-derived metabolites, the levels of fatty acids (palmitoleic, α-linolenic, arachidonic, and linoleic) (P < 0.05) increased and the levels of amino acids decreased in IC group. Furthermore, the abundances of Ruminococcus, Eubacterium coprostanoligenes group, Intestinibacter and Butyrivibrio are positively correlated with proline, asparagine and aspartic acid, but negatively correlated with the α-linolenic acid, linoleic acid, palmitoleic acid and arachidonic acid. For analysis of the relationship between the microbiota and clinical index, it was found that the abundance of Veillonella and Streptococcus was positively correlated with serum bile acid content (P < 0.05), while APTT, PT and INR were negatively correlated with Faecalibalum and Ruminococcus (P < 0.05). CONCLUSION: Microbiota dysbiosis happened in IC children, which also can lead to the abnormal metabolism, thus obstructing the absorption of enteral nutrition and aggravating liver cell damage. Veillonella, Ruminococcus and Butyrivibrio may be important microbiome related with IC and need further research.

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.

Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy.

Mycoplasma pneumoniae is a common causative pathogen of community-acquired pneumonia. An accurate and sensitive detection method is important for evaluating disease severity and treatment efficacy. Digital droplet PCR (ddPCR) is a competent method enabling the absolute quantification of DNA copy number with high precision and sensitivity. We established ddPCR for M. pneumoniae detection, using clinical specimens for validation, and this showed excellent specificity for M. pneumoniae. The limit of detection of ddPCR was 2.9 copies/reaction, while that for real-time PCR was 10.8 copies/reaction. In total, 178 clinical samples were used to evaluate the ddPCR assay, which correctly identified and differentiated 80 positive samples, whereas the real-time PCR tested 79 samples as positive. One sample that tested negative in real-time PCR was positive in ddPCR, with a bacterial load of three copies/test. For samples that tested positive in both methods, the cycle threshold of real-time PCR was highly correlated with the copy number of ddPCR. Bacterial loads in patients with severe M. pneumoniae pneumonia were significantly higher than those in patients with general M. pneumoniae pneumonia. The ddPCR showed that bacterial loads were significantly decreased after macrolide treatment, which could have reflected the treatment efficacy. The proposed ddPCR assay was sensitive and specific for the detection of M. pneumoniae. Quantitative monitoring of bacterial load in clinical samples could help clinicians to evaluate treatment efficacy.

Roles of the Crp/Fnr Family Regulator ArcR in the Hemolysis and Biofilm of Staphylococcus aureus.

Staphylococcus aureus is an opportunistic human pathogen that is often involved in severe infections such as pneumonia and sepsis in which bacterial virulence factors play a key role. Infections caused by S. aureus are often difficult to eradicate, particularly when they are associated with biofilm. The physiological roles of the Crp/Fnr family regulator ArcR are elusive in S. aureus. In this study, it was found that the deletion of arcR increased the hemolytic ability and biofilm formation in S. aureus. Differential gene expression analysis by RNA-seq and real-time quantitative reverse transcription PCR showed that genes associated with hemolytic ability (hla and hlb) and biofilm formation (icaA, icaB, icaC and icaD) were significantly upregulated compared with those in the wild-type strain. The results revealed that ArcR regulated the expression of the hla and ica operon by binding to their promoter regions, respectively. This study provided new insights into the functional importance of ArcR in regulating the virulence and biofilm of S. aureus.

Detection and Quantification of Klebsiella pneumoniae in Fecal Samples Using Digital Droplet PCR in Comparison with Real-Time PCR.

This study aimed to develop a rapid and sensitive droplet digital PCR (ddPCR) assay for the specific detection of Klebsiella pneumoniae in fecal samples, and to evaluate its application in the clinic by comparison with real-time PCR assay and conventional microbial culture. Specific primers and a probe targeting the K. pneumoniae hemolysin (khe) gene were designed. Thirteen other pathogens were used to evaluate the specificity of the primers and probe. A recombinant plasmid containing the khe gene was constructed and used to assess the sensitivity, repeatability, and reproducibility of the ddPCR. Clinical fecal samples (n = 103) were collected and tested by the ddPCR, real-time PCR, and conventional microbial culture methods. The detection limit of ddPCR for K. pneumoniae was 1.1 copies/µL, about a 10-fold increase in sensitivity compared with real-time PCR. The ddPCR was negative for the 13 pathogens other than K. pneumoniae, confirming its high specificity. Clinical fecal samples gave a higher rate of positivity in the K. pneumoniae ddPCR assay than in analysis by real-time PCR or conventional culture. ddPCR also showed less inhibition by the inhibitor in fecal sample than real-time PCR. Thus, we established a sensitive and effective ddPCR-based assay method for K. pneumoniae. It could be a useful tool for K. pneumoniae detection in feces and may serve as a reliable method to identify causal pathogens and help guide treatment decisions. IMPORTANCE Klebsiella pneumoniae can cause a range of illnesses and has a high colonization rate in the human gut, making it crucial to develop an efficient method for detecting K. pneumoniae in fecal samples.

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.

Bacteriophage targeting microbiota alleviates non-alcoholic fatty liver disease induced by high alcohol-producing Klebsiella pneumoniae.

Our previous studies have shown that high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) in the intestinal microbiome could be one of the causes of non-alcoholic fatty liver disease (NAFLD). Considering antimicrobial resistance of K. pneumoniae and dysbacteriosis caused by antibiotics, phage therapy might have potential in treatment of HiAlc Kpn-induced NAFLD, because of the specificity targeting the bacteria. Here, we clarified the effectiveness of phage therapy in male mice with HiAlc Kpn-induced steatohepatitis. Comprehensive investigations including transcriptomes and metabolomes revealed that treatment with HiAlc Kpn-specific phage was able to alleviate steatohepatitis caused by HiAlc Kpn, including hepatic dysfunction and expression of cytokines and lipogenic genes. In contrast, such treatment did not cause significantly pathological changes, either in functions of liver and kidney, or in components of gut microbiota. In addition to reducing alcohol attack, phage therapy also regulated inflammation, and lipid and carbohydrate metabolism. Our data suggest that phage therapy targeting gut microbiota is an alternative to antibiotics, with potential efficacy and safety, at least in HiAlc Kpn-caused NAFLD.

Combined Methylation and Transcriptome Analysis of Liver Injury of Nonalcoholic Fatty Liver Disease Induced by High Alcohol-Producing Klebsiella pneumoniae.

It has been known that high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) is one of causative agents of nonalcoholic fatty liver disease (NAFLD). However, how HiAlc Kpn promotes liver injury remains unclear. Recent findings suggest that DNA methylation might associate with the pathogenesis of NAFLD. Herein, the role of DNA methylation in HiAlc Kpn-induced liver injury was investigated. Murine models of NAFLD were established in C57BL/6N wild-type mice by gavaging HiAlc Kpn for 8 weeks. The liver injury was assessed based on the liver histopathology and biochemical indicators. In addition, DNA methylation in hepatic tissue was assessed by using dot bolt of 5-mC. RNA sequencing analysis and whole-genome bisulfite sequencing (WGBS) analysis were also performed. HiAlc Kpn significantly increased the activity of aspartate transaminase (AST), alanine transaminase (ALT), triglycerides (TGs), and glutathione (GSH), while hypomethylation was associated with liver injury in the experimental mice induced by HiAlc Kpn. The GO and KEGG pathway enrichment analysis of the transcriptome revealed that HiAlc Kpn induced fat metabolic disorders and DNA damage. The conjoint analysis of methylome and transcriptome showed that hypomethylation regulated related gene expression in signal pathways of lipid formation and circadian rhythm, including Rorα and Arntl1genes, which may be the dominant cause of NAFLD induced by HiAlc Kpn. Data suggest that DNA hypomethylation might play an important role in liver injury of NAFLD induced by HiAlc Kpn. Which possibly provides a new sight for understanding the mechanisms of NAFLD and selecting the potential therapeutic targets. IMPORTANCE High alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) is one of causative agents of nonalcoholic fatty liver disease (NAFLD) and could induce liver damage. DNA methylation, as a common epigenetic form following contact with an etiologic agent and pathogenesis, can affect chromosome stability and transcription. We conjointly analyzed DNA methylation and transcriptome levels in the established murine models to explore the potential mechanisms for further understanding the role of DNA methylation in the liver damage of HiAlc Kpn-induced NAFLD. The analysis of the DNA methylation landscape contributes to our understanding of the entire disease process, which might be crucial in developing treatment strategies.

Three Klebsiella species as potential pathobionts generating endogenous ethanol in a clinical cohort of patients with auto-brewery syndrome: a case control study.

BACKGROUND: Patients with auto-brewery syndrome (ABS) become inebriated after the ingestion of an alcohol-free, high-carbohydrate diet. Our previous work has shown that high-alcohol-producing (HiAlc) Klebsiella pneumoniae can generate excessive endogenous ethanol and cause non-alcoholic fatty liver disease (NAFLD). Therefore, it is reasonable to speculate that such bacteria might play an important role in the pathogenesis of ABS. METHODS: The characteristics and metabolites of the intestinal flora from a clinical cohort of patients with ABS were analysed during different stages of disease and compared to a group of healthy controls. An in vitro culture system of relevant samples was used for screening drug sensitivity and ABS-inducing factors. Rabbit intestinal and murine models were established to verify if the isolated strains could induce ABS in vivo. FINDINGS: We observed intestinal dysbiosis with decreased abundance of Firmicutes and increased of Proteobacteria in patients with ABS compared with healthy controls. The abundance of the genus Klebsiella in Enterobacteriaceae was strongly associated with fluctuations of patient's blood alcohol concentration. We isolated three species of HiAlc Klebsiella from ABS patients, which were able to induce ABS in mice. Monosaccharide content was identified as a potential food-related inducing factor for alcohol production. Treatments with antibiotics, a complex probiotic preparation and a low-carbohydrate diet not only alleviated ABS, but also erased ABS relapse during the follow-up observation of one of the patients. INTERPRETATION: Excessive endogenous alcohol produced by HiAlc Klebsiella species was an underlying cause of bacterial ABS. Combined prescription of appropriate antibiotics, complex probiotic preparation and a controlled diet could be sufficient for treatment of bacteria-caused ABS. FUNDING: The funders are listed in the acknowledgement.

Rapid detection of mpox virus using recombinase aided amplification assay.

A recent, unprecedented outbreak of human mpox virus infection has led to cases in non-African nations, and the number of confirmed or suspected cases outside of Africa has exceeded 1,000 within 5 weeks. Mpox may pose a double threat to public health in the context of the ongoing COVID-19 pandemic. It is difficult to distinguish mpox virus infection from other diseases in the early stages, and patients are contagious from the onset of nonspecific symptoms; therefore, it is crucial to develop rapid and specific diagnostic methods. The diagnosis of mpox relies on real-time polymerase chain reaction, a time-consuming method that requires a highly sophisticated thermal cycler, which makes it unsuitable for widespread use in underdeveloped areas, where the outbreak is still severe. In this study, we developed a recombinase-aided amplification (RAA) assay that can detect mpox virus within 5-10 minutes. The conserved regions of the A27L gene and F3L gene were selected as targets, as they amplify well from different mpox virus clades with no cross-reaction from other pathogens. The sensitivity of this RAA assay is 10 copies/reaction for the A27L gene and 102 copies/reaction for the F3L gene. When applied to simulated clinical samples, both targets showed 100% specificity, and the detection limits were consistent with the sensitivity results. Moreover, through clinical blinded sample detection, RAA exhibits the same detection power as RT-PCR. In summary, the RAA mpox assay described here exhibits rapid detection, high sensitivity and specificity, and low operational difficulty, making it suitable for mpox virus detection in less developed countries and regions.

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.

Recombinase-Aided Amplification Assay for Rapid Detection of Hypervirulent Klebsiella pneumoniae (hvKp) and Characterization of the hvKp Pathotype.

Hypervirulent Klebsiella pneumoniae (hvKp) is a major human pathogen associated with liver abscess, pneumonia, meningitis, and endophthalmitis. It is challenging to differentiate hvKp from classical Klebsiella pneumoniae (cKp) using conventional methods, necessitating the development of a rapid, sensitive, and convenient assay for hvKp detection. In this study, we constructed a recombinase-aided amplification (RAA) method targeting hvKp genes peg344 and rmpA, and also analyzed the pathogenic characteristics of hvKp. We optimized the reaction temperature and system, and evaluated its sensitivity, specificity, and clinical application. The primer and probe sets peg344-set1 and rmpA-set2 delivered significant fluorescent signals at 39°C with the shortest gene amplification times (sensitivity: 20 copies/reaction). This RAA assay showed no cross-reactivity with 15 other common pathogenic bacteria. Its applicability was confirmed by the evaluation of 208 clinical specimens, of which 45 were confirmed to be hvKp. The sensitivity and specificity of the RAA assay were both 100% compared with real-time PCR as the reference standard. To verify the assay, we also assessed the diversity of molecular characteristics among the hvKp isolates and identified serotype K1 and sequence type ST23 as the dominant clone. Virulence factors iroN and iutA were highly associated with virulence level. In conclusion, our novel RAA assay is a powerful tool for early diagnosis and epidemiological surveillance of hvKp. IMPORTANCEKlebsiella pneumoniae is the most common opportunistic bacterial species and a major threat to public health. Since the 1990s, hvKp has received increasing attention from public health officials and infectious disease specialists. Hypervirulent strains differ from classical strains in terms of phenotypic features and clinical outcomes. It is hard to identify hvKp from cKp using the conventional methods including colony morphology analysis, serum killing assays, mouse lethality assays, string tests, and real-time PCR. In this study, we established a rapid, sensitive and convenient recombinase-aided amplification assay for hvKp detection targeting virulence genes peg344 and rmpA. Our RAA assay provides an important tool for the rapid diagnosis of infectious diseases caused by hvKp, particularly in primary laboratories.

Inhibitory Effect of Verapamil on the Growth of Human Airway Granulation Fibroblasts.

OBJECTIVES: To explore the inhibitory effect of verapamil, a calcium channel blocker, on the growth of human airway granulation fibroblasts to provide an experimental basis for the clinical use of calcium channel blockers in preventing and treating benign airway stenosis. METHODS: Primary human airway normal fibroblasts and human airway granulation fibroblasts were cultured by tissue block attachment culture method, and the experimental studies were carried out using 3-8 generation cells. Cell Counting Kit-8 (CCK-8) was used to test the proliferation of human normal airway fibroblasts and human airway granulation fibroblasts and the semi-inhibitory concentration of verapamil on normal airway fibroblasts and airway granulation fibroblasts. A scratch test detected the migration effect of verapamil on human airway granulation fibroblasts. The mRNA relative expression levels of related factors were detected by PCR to compare the differences between normal airway fibroblasts and airway granulation fibroblasts. Western blot was used to detect the relative amount of related proteins and compare the differences between normal airway fibroblasts and granulation airway fibroblasts. After 48 hours of treatment with half of the inhibitory concentration of Vera Pammy for granulation airway fibroblasts, the relative expression levels of related factors on mRNA and protein were observed. RESULTS: Human normal airway fibroblasts and human airway granulation fibroblasts with a purity of more than 95% could be obtained from primary culture by tissue block adherence method. CCK8 results showed that the proliferation rate of human airway granulation fibroblasts was faster than that of the normal human airway fibroblasts. The semi-inhibitory concentration of verapamil on human normal airway fibroblasts was 92.81 ug/ml, while the semi-inhibitory concentration on human airway granulation fibroblasts was 69.57 ug/ml. The scratch test indicated that the cell migration rate of human airway granulation fibroblasts treated with verapamil decreased significantly (P < 0.05). PCR results showed that the mRNA relative expression levels of TGFß1, COL1A1, Smad2/3, VEGFA, IL6, and IL8 in human airway granulation fibroblasts were significantly higher than those in normal human airway fibroblasts (P < 0.05). The mRNA relative expressions of TGFß1, smad2/3, and COL1A1 in human airway granulation fibroblasts treated with semi-inhibited verapamil for 48h were down-regulated (P < 0.05), while the mRNA relative expressions of VEGFA, IL6 and IL8 had no significant changes (P > 0.05). WB test showed that the relative protein expressions of TGFß1, Smad2, and VEGFC in human airway granulation fibroblasts were upregulated (P < 0.05) but downregulated after verapamil treatment compared with before treatment (P < 0.05). CONCLUSION: Calcium channel blockers can inhibit the proliferation of human airway granulation fibroblasts through TGFß1/ Smad pathway, which may be a method to prevent and treat benign airway stenosis.