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  • Zhao, Hanqing  (17)
  • 1
    Online Resource
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    Rapid Detection of Multi-Resistance Strains Carrying mcr-1 Gene Using Recombinase-Aided Amplification Directly on Clinical Samples
    Frontiers Media SA ; 2022
    In:  Frontiers in Microbiology Vol. 13 ( 2022-3-31)
    Details
    In: Frontiers in Microbiology, Frontiers Media SA, Vol. 13 ( 2022-3-31)
    Abstract: With the increasingly severe problem of bacterial resistance, colistin, as the last line of defense, has attracted attention again. Mobile colistin resistance ( mcr -1) gene is involved in the horizontal transmission of colistin resistance in Gram-negative bacteria (GNB), which is a serious threat to human health. Therefore, rapid detection of mcr -1 gene presence in clinical samples is crucial. In this study, a Recombinase-aided amplification(RAA) method for mcr -1 was successfully constructed, with sensitivity of 20 copies/reaction. In addition, amplification signal could only be detected in the strain containing mcr -1 gene among 14 different bacterial species. The method was then used to test a total of 672 clinical samples from a pediatric hospital in Beijing. Five strains harbored mcr -1 genes were isolated from mcr -1-positive clinical samples and identified as Escherichia coli . Multi-locus sequence typing (MLST) analysis showed that the five E. coli belonged to different ST types. Notably, the mcr -1 gene from the isolates could be transferred conjugately to the recipient strain E. coli J53, with highest transfer efficiency up to 57–58%, suggesting that the mcr -1 gene was located on the plasmid. These findings showed that the RAA assay has potential to be a rapid and sensitive mcr -1 gene screening test for clinical samples, and mcr -1 could be transmitted vertically and horizontally between and within bacterial species in a plasmid-mediated manner.
    Type of Medium: Online Resource
    ISSN: 1664-302X
    URL: Article
    DOI: 10.3389/fmicb.2022.852488
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2587354-4
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  • 2
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    A novel phage carrying capsule depolymerase effectively relieves pneumonia caused by multidrug-resistant Klebsiella aerogenes
    Springer Science and Business Media LLC ; 2023
    In:  Journal of Biomedical Science Vol. 30, No. 1 ( 2023-08-31)
    Details
    In: Journal of Biomedical Science, Springer Science and Business Media LLC, Vol. 30, No. 1 ( 2023-08-31)
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 1423-0127
    URL: Article
    DOI: 10.1186/s12929-023-00946-y
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 1482918-6
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  • 3
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    Prophylactic vitamin C supplementation regulates DNA demethylation to protect against cisplatin-induced acute kidney injury in mice
    Elsevier BV ; 2023
    In:  Biochemical and Biophysical Research Communications ( 2023-12), p. 149463-
    Details
    In: Biochemical and Biophysical Research Communications, Elsevier BV, ( 2023-12), p. 149463-
    Type of Medium: Online Resource
    ISSN: 0006-291X
    URL: Article
    DOI: 10.1016/j.bbrc.2023.149463
    RVK:
    WA 15000
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2023
    detail.hit.zdb_id: 1461396-7
    SSG: 12
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  • 4
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    Table_1.DOCX
    Frontiers Media SA ; 2023
    In:  Frontiers in Microbiology Vol. 14 ( 2023-6-22)
    Details
    In: Frontiers in Microbiology, Frontiers Media SA, Vol. 14 ( 2023-6-22)
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 1664-302X
    URL: Article
    URL: Article
    DOI: 10.3389/fmicb.2023.1177273
    DOI: 10.3389/fmicb.2023.1177273.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2023
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  • 5
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    Glucose Induces Resistance to Polymyxins in High-Alcohol-Producing Klebsiella pneumoniae via Increasing Capsular Polysaccharide and Maintaining Intracellular ATP
    American Society for Microbiology ; 2023
    In:  Microbiology Spectrum Vol. 11, No. 4 ( 2023-08-17)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 11, No. 4 ( 2023-08-17)
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/spectrum.00031-23
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 2807133-5
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  • 6
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    Table_2.pdf
    Frontiers Media SA ; 2022
    In:  Frontiers in Microbiology Vol. 12 ( 2022-1-17)
    Details
    In: Frontiers in Microbiology, Frontiers Media SA, Vol. 12 ( 2022-1-17)
    Abstract: Streptococcus pneumoniae ( S. pneumoniae ) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63°C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies/μL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4–100%), specificity 99.0% (95% CI 97.8–99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae . This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy.
    Type of Medium: Online Resource
    ISSN: 1664-302X
    URL: Article
    URL: Article
    URL: Article
    URL: Article
    DOI: 10.3389/fmicb.2021.816997
    DOI: 10.3389/fmicb.2021.816997.s001
    DOI: 10.3389/fmicb.2021.816997.s002
    DOI: 10.3389/fmicb.2021.816997.s003
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2587354-4
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  • 7
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    Data_Sheet_1.doc
    Frontiers Media SA ; 2023
    In:  Frontiers in Microbiology Vol. 14 ( 2023-2-24)
    Details
    In: Frontiers in Microbiology, Frontiers Media SA, Vol. 14 ( 2023-2-24)
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 1664-302X
    URL: Article
    URL: Article
    DOI: 10.3389/fmicb.2023.1106340
    DOI: 10.3389/fmicb.2023.1106340.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2023
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  • 8
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    Recombinase-Aided Amplification Assay for Rapid Detection of Hypervirulent Klebsiella pneumoniae (hvKp) and Characterization of the hvKp Pathotype
    American Society for Microbiology ; 2023
    In:  Microbiology Spectrum Vol. 11, No. 2 ( 2023-04-13)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 11, No. 2 ( 2023-04-13)
    Abstract: 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. IMPORTANCE Klebsiella 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.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/spectrum.03984-22
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
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  • 9
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    Genetic Diversity and Pathogenic Features in Klebsiella pneumoniae Isolates from Patients with Pyogenic Liver Abscess and Pneumonia
    American Society for Microbiology ; 2022
    In:  Microbiology Spectrum Vol. 10, No. 2 ( 2022-04-27)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 10, No. 2 ( 2022-04-27)
    Abstract: While Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, including pneumonia and pyogenic liver abscess, little is known about the population structure of this bacterium. In this study, we investigated the prevalence and molecular characteristics of K. pneumoniae isolates from carriers, pyogenic liver abscess patients, and pneumonia patients, and genomic and phenotypic assays were used to determine the differences among the isolates. A total of 232 K. pneumoniae isolates were subtyped into 74 sequence types (STs). The isolates from different sources had their own STs, and the predominant subtypes in liver abscess and pneumonia patients were ST23 and ST11, respectively. Pangenome analysis also distinguished three phylogroups that were consistent with the isolate sources. The isolates collected from liver abscess patients carried significantly more virulence factors, and those from pneumonia patients harbored significantly more resistance genes and replicons. Almost all isolate STs (93/97 [95.88%]) from liver abscesses strongly correlated with the virulence factor salmochelin, while most pneumonia isolate STs (52/53 [98.11%] ) from pneumonia did not correlate with salmochelin. The isolates collected from liver abscesses showed higher virulence in the cytotoxicity and mouse models. These data provide genomic support for the proposal that isolates collected from carriers, liver abscess patients, and pneumonia patients have distinct genomic features. Isolates from the different sources are largely nonoverlapping, suggesting that different patients may be infected via different sources. Further studies on the pathogenic mechanisms of salmochelin and other virulence factors will be required. IMPORTANCE While Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, including pneumonia and pyogenic liver abscess, little is known about the population structure of this bacterium. We collected 232 isolates from carriers, pyogenic liver abscess patients, and pneumonia patients, and the isolates from different sources had their own sequence types. Pangenome analysis also distinguished three phylogroups that were consistent with the isolate sources. The isolates collected from liver abscess patients carried significantly more virulence factors, and those from pneumonia patients harbored significantly more resistance genes and replicons. Besides, there was a strong link between salmochelin and liver abscess. The isolates collected from liver abscesses also showed higher virulence in the cytotoxicity and mouse models. Isolates collected from different sources have distinct genomic features, suggesting that different patients may be infected via different sources.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/spectrum.02646-21
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
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  • 10
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    Development of a Loop-Mediated Isothermal Amplification Method for Rapid and Visual Detection of Monkeypox Virus
    American Society for Microbiology ; 2022
    In:  Microbiology Spectrum Vol. 10, No. 5 ( 2022-10-26)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 10, No. 5 ( 2022-10-26)
    Abstract: Monkeypox virus (MPXV) is a human pathogenic virus that belongs to the genus Orthopoxvirus . In 2022, MPXV caused an unprecedented number of infections in many countries. As it is difficult to distinguish MPXV from other pathogens by its symptoms in the early stage of infection, a rapid and reliable assay for MPXV detection is needed. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the specific detection of MPXV and evaluated its application in simulated clinical samples. The A27L-1 and F3L-1 primer sets were identified as the optimal primers, and 63°C was the most appropriate reaction temperature for sequence amplification. The detection limits of the LAMP assay using primer sets A27L-1 and F3L-1 were both 20 copies/reaction mixture, which were 〉 100-fold higher in terms of sensitivity, compared with conventional PCR. The LAMP assay findings were negative for all 21 non-MPXV pathogens, confirming the high specificity of our assay. All three types of simulated clinical samples were clearly identified by our LAMP assay, and the detection limits were consistent with the sensitivity results, indicating efficient clinical sample identification. Our rapid and reliable MPXV LAMP assay could be useful for MPXV detection and on-site diagnosis, especially in primary hospitals and rural areas. IMPORTANCE MPXV outbreaks rapidly grew in the first half of 2022, and this virus has been recognized as an increasing public health threat, particularly in the context of the COVID-19 pandemic. Thus, developing reliable and fast detection methods for MPXV is necessary.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/spectrum.02714-22
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
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