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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.
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Klebsiella pneumoniae , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Antibacterianos/farmacología , Temperatura , Alcoholes/metabolismo , Alcoholes/farmacologíaRESUMEN
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.
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Deoxynivalenol (DON) is one of the most common mycotoxins distributed in food and feed, which causes severe liver injury in humans and animals. Cold atmospheric plasma (CAP) has received much attention in mycotoxin degradation due to the advantages of easy operation, high efficiency, and low temperature. So far, the majority of studies have focused on the degradation efficiency and mechanism of CAP on DON, while there is still little information available on the hepatotoxicity of DON after CAP treatment. Herein, this study aimed to investigate the effect of CAP on DON-induced hepatotoxicity both in vitro and in vivo and its underlying mechanisms. The results showed that 120-s CAP treatment achieved 97â¯% degradation of DON. The vitro hepatotoxicity of DON in L02 cells was significantly reduced with CAP treatment time. Meanwhile, CAP markedly alleviated DON-induced liver injury in mice including the balloon-like degeneration of liver tissues and elevation of AST and ALP level. The underlying mechanism for CAP detoxification of DON-induced hepatotoxicity was further elucidated. The results showed that DON caused severe oxidative stress in cells by suppressing the antioxidant signaling pathway of Nrf2/HO-1/NQO-1, consequently leading to mitochondrial dysfunction and cell apoptosis, accompanied by cellular senescence and inflammation. CAP blocked DON inhibition on the Nrf2/HO-1/NQO-1 signaling pathway through the efficient degradation of DON, accordingly alleviating the oxidative stress and liver injury induced by DON. Therefore, CAP is an effective method to eliminate DON hepatotoxicity, which can be applied in the detoxification of mycotoxin-contaminated food and feed to ensure human and animal health.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Gases em Plasma , Tricotecenos , Animales , Ratones , Tricotecenos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Estrés Oxidativo/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Hígado/metabolismo , Apoptosis/efectos de los fármacos , Masculino , Humanos , Inactivación Metabólica , Línea CelularRESUMEN
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.
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Candida auris , Técnicas de Amplificación de Ácido Nucleico , Recombinasas , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , Recombinasas/metabolismo , Candida auris/genética , Candidiasis/diagnóstico , Candidiasis/microbiología , Límite de Detección , ADN de Hongos/genética , ADN de Hongos/análisisRESUMEN
INTRODUCTION: Zearalenone (ZEN) is one of the most widely contaminated mycotoxins in world, posing a severe threat to human and animal health. Atmospheric cold plasma (ACP) holds great penitential in mycotoxin degradation. OBJECTIVES: This study aimed to investigate the degradation efficiency and mechanisms of ACP on ZEN as well as the cytotoxicity of ZEN degradation products by ACP. Additionally, this study also investigated the degradation efficiency of ACP on ZEN in cereals and its effect on cereal quality. METHODS: The degradation efficiency and products of ZEN by ACP was analyzed by HPLC and LC-MS/MS. The human normal liver cells and mice were employed to assess the cytotoxicity of ZEN degradation products. The ZEN artificially contaminated cereals were used to evaluate the feasibility of ACP detoxification in cereals. RESULTS: The results showed that the degradation rate of ZEN was 96.18 % after 30-W ACP treatment for 180 s. The degradation rate was dependent on the discharge power, and treatment time and distance. Four major ZEN degradation products were produced after ACP treatment due to the oxidative destruction of CC double bond, namely C18H22O7 (m/z = 351.19), C18H22O8 (m/z = 367.14), C18H22O6 (m/z = 335.14), and C17H20O6 (m/z = 321.19). L02 cell viability was increased from 52.4 % to 99.76 % with ACP treatment time ranging from 0 to 180 s. Mice results showed significant recovery of body weight and depth of colonic crypts as well as mitigation of glomerular and liver damage. Additionally, ACP removed up to 50.55 % and 58.07 % of ZEN from wheat and corn. CONCLUSIONS: This study demonstrates that ACP could efficiently degrade ZEN in cereals and its cytotoxicity was significantly reduced. Therefore, ACP is a promising effective method for ZEN detoxification in cereals to ensure human and animal health. Future study needs to develop large-scale ACP device with high degradation efficiency.
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Wnt/ß-catenin signaling plays a pivotal role in the pathogenesis of chronic kidney diseases (CKD), which is associated with macrophage activation and polarization. However, the relative contribution of macrophage-derived Wnts in the evolution of CKD is poorly understood. Here we demonstrate a critical role of Wnts secreted by macrophages in regulating renal inflammation and fibrosis after various injuries. In mouse model of kidney fibrosis induced by unilateral ureteral obstruction (UUO), macrophages were activated and polarized to M1 and M2 subtypes, which coincided with the activation of Wnt/ß-catenin signaling. In vitro, multiple Wnts were induced in primary cultured bone marrow-derived macrophages (BMDMs) after polarization. Conversely, Wnt proteins also stimulated the activation and polarization of BMDMs to M1 and M2 subtype. Blockade of Wnt secretion from macrophages in mice with myeloid-specific ablation of Wntless (Wls), a cargo receptor that is obligatory for Wnt trafficking and secretion, blunted macrophage infiltration and activation and inhibited the expression of inflammatory cytokines. Inhibition of Wnt secretion by macrophages also abolished ß-catenin activation in tubular epithelium, repressed myofibroblast activation and reduced kidney fibrosis after either obstructive or ischemic injury. Furthermore, conditioned medium from Wls-deficient BMDMs exhibited less potency to stimulate fibroblast proliferation and activation, compared to the controls. These results underscore an indispensable role of macrophage-derived Wnts in promoting renal inflammation, fibroblasts activation and kidney fibrosis.
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Insuficiencia Renal Crónica , beta Catenina , Animales , Ratones , Macrófagos , Miofibroblastos , Inflamación , RiñónRESUMEN
Food contaminated by mycotoxins has become a worldwide public problem with political and economic implications. Although a variety of traditional methods have been used to eliminate mycotoxins from agri-foods, the results have been somewhat less than satisfactory. As an emerging non-thermal processing technology, atmospheric cold plasma (ACP) has great potential for food decontamination. Herein, this review mainly presents the degradation efficiency of ACP on mycotoxins in vitro and agri-foods as well as its possible degradation mechanisms. Meanwhile, ACP effects on food quality, factors affecting the degradation efficiency and the toxicity of degradation products are also discussed. According to the literatures, ACP could efficiently degrade many mycotoxins (e.g., aflatoxin, deoxynivalenol, zearalenone, ochratoxin A, fumonisin, and T-2 toxin) both in vitro and various foods (e.g., hazelnut, peanut, maize, rice, wheat, barley, oat flour, and date palm fruit) with little effects on the nutritional and sensory properties of food. The degradation efficacy was dependent on many factors including ACP treatment parameter, working gas, mycotoxin property, and food substrate. The mycotoxin degradation by ACP was mainly attributed to the reactive oxygen and nitrogen species in ACP, which can damage the chemical bonds of mycotoxins, consequently reducing the toxicity of mycotoxins.
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Fumonisinas , Micotoxinas , Gases em Plasma , Zearalenona , Micotoxinas/toxicidad , Gases em Plasma/química , Contaminación de Alimentos/análisis , Fumonisinas/análisisRESUMEN
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.
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Enfermedades Inflamatorias del Intestino , Veillonella , Niño , Humanos , Bioensayo , Enfermedades Inflamatorias del Intestino/diagnóstico , Mamíferos , Reacción en Cadena en Tiempo Real de la Polimerasa , ARN Ribosómico 16S/genéticaRESUMEN
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.
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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.
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Bacteriófagos , Enterobacter aerogenes , Neumonía , Animales , Ratones , Bacteriófagos/genética , Cinética , Antibacterianos , Modelos Animales de EnfermedadRESUMEN
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.
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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.
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BACKGROUND: Intercellular communication between macrophages and peritoneal mesothelial cells (PMCs) has been suggested as a key factor regulating peritonitis development. Here, we explored whether PPARγ (peroxisome proliferator-activated receptor gamma) can be packaged into macrophage exosomes to mediate intercellular communication and regulate peritonitis. METHODS: Macrophage exosomes were isolated by ultracentrifugation and identified by nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of macrophage-derived exosomes was performed using mass spectrometry. Co-culture models of supernatants or exosomes with PMCs, as well as a mouse peritonitis model induced by lipopolysaccharide (LPS), were employed. RESULTS: In this study, using stable Raw264.7 cells overexpressing GFP-FLAG-PPARγ (OE-PPARγ), we found that PPARγ inhibited LPS-induced inflammatory responses in Raw264.7 cells and that PPARγ was incorporated into macrophage exosomes during this process. Overexpression of PPARγ mainly regulated the secretion of differentially expressed exosomal proteins involved in the biological processes of protein transport, lipid metabolic process, cell cycle, apoptotic process, DNA damage stimulus, as well as the KEGG pathway of salmonella infection. Using co-culture models and mouse peritonitis model, we showed that exosomes from Raw264.7 cells overexpressing PPARγ inhibited LPS-induced inflammation in co-cultured human PMCs and in mice through downregulating CD14 and TLR4, two key regulators of the salmonella infection pathway. Pretreatment of the PPARγ inhibitor GW9662 abolished the anti-inflammatory effect of exosomes from Raw264.7 OE-PPARγ cells on human PMCs. CONCLUSIONS: These results suggested that overexpression of PPARγ largely altered the proteomic profile of macrophage exosomes and that exosomal PPARγ from macrophages acted as a regulator of intercellular communication to suppress LPS-induced inflammatory responses in vitro and in vivo via negatively regulating the CD14/TLR4 axis.
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Fenómenos Biológicos , Peritonitis , Ratones , Humanos , Animales , PPAR gamma/metabolismo , Lipopolisacáridos/farmacología , Receptor Toll-Like 4/metabolismo , Proteómica , Macrófagos/metabolismo , Peritonitis/inducido químicamenteRESUMEN
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.
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Klebsiella pneumoniae , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Klebsiella pneumoniae/genética , Reproducibilidad de los Resultados , HecesRESUMEN
This work aimed to compare the effects of helium and air surface micro-discharge (SMD) plasma on the microbial safety and quality of beef tissues. For the beef tissue model, the concentration and diffusion depth of hydroxyl radical and ozone have different change patterns over plasma treatment time and distance in helium and air SMD plasma. The inactivation efficiency of helium plasma depended on the plasma treatment time and distance, while the inactivation efficiency of air plasma only depended on the treatment time. For the fresh beef slices, air SMD plasma treatment exhibited a higher antimicrobial activity against S. aureus and E. coli than helium SMD plasma treatment (1.5 versus 0.9; 0.9 versus 0.28 log CFU/g at 10 min). However, air SMD plasma treatment caused more adverse effects on beef quality, leading to a smooth surface, extensive lipid oxidation, protein structure damage, low pH and discoloration compared to helium SMD plasma treatment. This work provides valuable guidelines for the working gas choice in the practical application of plasma to meat decontamination.
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Escherichia coli , Microbiología de Alimentos , Animales , Bovinos , Recuento de Colonia Microbiana , Helio/farmacología , Staphylococcus aureusRESUMEN
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.
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Infecciones por Klebsiella , Enfermedad del Hígado Graso no Alcohólico , Humanos , Polimixinas/farmacología , Polimixinas/metabolismo , Klebsiella pneumoniae , Glucosa/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Etanol/metabolismo , Polisacáridos/metabolismo , Adenosina Trifosfato/metabolismo , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiologíaRESUMEN
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.
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Bacteriófagos , Microbiota , Enfermedad del Hígado Graso no Alcohólico , Masculino , Animales , Ratones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Klebsiella pneumoniae/genética , Etanol/metabolismo , Hígado/metabolismo , Antibacterianos/uso terapéutico , Antibacterianos/metabolismoRESUMEN
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.
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Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Klebsiella pneumoniae/genética , Ratones Endogámicos C57BL , Hígado/metabolismo , Etanol/toxicidad , Etanol/metabolismo , Perfilación de la Expresión Génica , Metilación de ADNRESUMEN
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.
Asunto(s)
Etanol , Enfermedad del Hígado Graso no Alcohólico , Animales , Ratones , Conejos , Etanol/efectos adversos , Etanol/metabolismo , Klebsiella , Nivel de Alcohol en Sangre , Estudios de Casos y Controles , BacteriasRESUMEN
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.