Development and clinical application of a rapid and visual loop-mediated isothermal amplification test for tetM gene in Clostridioides difficile strains cultured from feces

Lin, Minyi; Li, Zitong; Lin, Qiuxia; Wang, Pu; Liu, Wei; Yuan, Jing; Hong, Zhongsi; Chen, Ye

doi:10.1016/j.ijid.2022.07.032

Cited by 7 publications

(3 citation statements)

References 59 publications

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“…TetM gene is the most common tetracycline resistance determinant that encodes a ribosomal protection protein which prevents the binding of tetracycline to 16S rRNA (Brown and Roberts, 1987; LaPlante et al, 2022). Many studies report that TetM gene is acquired within mobile genetics mobile elements or transposons by horizontal transfer (Dönhöfer et al, 2012; Dong et al, 2014; Imwattana et al, 2021; Lin et al, 2022) In CDC20121308, TetM was located in a 6,579 bp insertion together with others six genes, one of which corresponds to a macrolide-efflux protein transporter (mefH (Supplementary Table 4)). Tn5397, Tn916 and Tn6190 transposons often carry the TetM gene in C. difficile (Spigaglia et al, 2011; Imwattana et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

Genome sequence and characterization of a hypervirulent BI/NAP1/027Clostridioides difficile(CDC20121308)

Español,

Palumbo,

Barbero

et al. 2024

Preprint

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Clostridioides difficile is a gram-positive bacterium implicated in antibiotic-associated diarrhea. The use of antibiotics alters the gut microbiota, rendering the host susceptible to infection by C. difficile. This pathogen colonizes the large intestine of humans and animals leading to asymptomatic carriage or clinical manifestations such as toxic megacolon and fulminant colitis depending on a wide range of pathogen and host factors. The emergence of BI/NAP1/027 strains in North America and the spread of these hypervirulent ribotypes worldwide have been linked to the increase in incidence and severity of C. difficile infections (CDI) over the last decade. In this work, we aimed to characterize the BI/NAP1/027 C. difficile commercial strain CDC20121308 widely employed in the study of host-pathogen interactions. The genome sequence was obtained using a whole-genome shotgun strategy. A total of 3,717 coding sequences (CDS) and 45 tRNAs were predicted. The annotation of the CDC20121308 strain identified 26% of CDS into RAST subsystems. We also detected the presence of RT 027 lineage markers such as thyA, cdtA, cdtB and tcdC 18bp-deletion. Moreover, the genome of CDC20121308 had 11 genes devoted to resistance to toxic compounds, antibiotics (e.g. Tetracycline (Tet) and Vancomycin (Van)) and disinfecting agents as predicted using CARD. C. difficile CDC20121308 resistance to Van and Tet was confirmed by broth microdilution assay. Crystal violet staining demonstrated biofilm formation, which could be associated with antibiotic resistance and pathogenicity. Additionally, we observed a spreading diffuse growth in soft agar tubes, suggesting a motile phenotype. Lastly, a genomic region containing Type 4 Secretory System components such as virD4, virB4, and virB6 was identified. In conclusion, our results allowed a genomic and functional characterization of the BI/NAP1/027 C. difficile CDC20121308 strain. We demonstrated the presence of several genes associated with pathogenesis that were validated by experimental assays. This study provides additional data for the use of this highly virulence commercial strain of epidemiological relevance in research works involving in vitro and in vivo approaches.

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Section: Resultsmentioning

confidence: 99%

Genome sequence and characterization of a hypervirulent BI/NAP1/027Clostridioides difficile(CDC20121308)

Español,

Palumbo,

Barbero

et al. 2024

Preprint

View full text Add to dashboard Cite

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“…Recently, Lin et al developed a rapid and visual calcein-LAMP assay targeting the tetM gene in Clostridioides difficile strains. The detection limit of LAMP was 36.1 pg/µL, which was 100-fold more sensitive than that of PCR [ 66 ].…”

Section: Monitoring Methods For Lampmentioning

confidence: 99%

Principles and Applications of Loop-Mediated Isothermal Amplification to Point-of-Care Tests

Park

2022

Biosensors

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For the identification of nucleic acids, which are important biomarkers of pathogen-mediated diseases and viruses, the gold standard for NA-based diagnostic applications is polymerase chain reaction (PCR). However, the requirements of PCR limit its application as a rapid point-of-care diagnostic technique. To address the challenges associated with regular PCR, many isothermal amplification methods have been developed to accurately detect NAs. Isothermal amplification methods enable NA amplification without changes in temperature with simple devices, as well as faster amplification times compared with regular PCR. Of the isothermal amplifications, loop-mediated isothermal amplification (LAMP) is the most studied because it amplifies NAs rapidly and specifically. This review describes the principles of LAMP, the methods used to monitor the process of LAMP, and examples of biosensors that detect the amplicons of LAMP. In addition, current trends in the application of LAMP to smartphones and self-diagnosis systems for point-of-care tests are also discussed.

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“…Similar patterns were found in the present study, where the relative abundances of tetM and adeF increased after the anaerobic digestion of livestock wastewater samples (Table 2); the relative abundances of QnrB74, lnuG, and vanTC increased significantly after the high-temperature fermentation of manure (Table 3). There are several possible reasons for this phenomenon: the first factor may be due to the chemical stability of the antibiotics themselves [33]; the second factor may be that the enrichment of these genes is due to the aggregation of resistance genes on mobile genetic elements [34,35]; and the third factor may be that the host bacteria containing these ARGs are thermophilic microbes or facultative anaerobic fermentation bacteria [36,37]. In summary, the waste treatment process in this study was able to remove 29.3-33.6% of ARGs and ARB; hence, there could be an potential risk of spreading ARGs and ARB to the farmland if this treated wastewater is used for irrigation.…”

Section: Args Removal Efficiency Of Current Livestock Wastewater and ...mentioning

confidence: 99%

Identification and Distribution of Antibiotic Resistance Genes and Antibiotic Resistance Bacteria in the Feces Treatment Process: A Case Study in a Dairy Farm, China

Wang,

Gao,

Zheng

et al. 2024

Water

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The overuse of antibiotics has resulted in the prevalence of antibiotic resistance genes (ARGs) and antibiotic resistance bacteria (ARB) in the environment. High-density livestock farming is one of the major industries for antibiotic overuse. In this study, we sampled wastewater and manure at different stages of the feces treatment process from a dairy farm, as well as the soil in the farmland where the treated wastewater was being used for irrigation purpose. High-throughput Illumina sequencing was used to analyze the profiles of bacteria communities and ARGs. The results showed that the main ARG types were multidrug, aminoglycoside, glycopeptide, and tetracycline resistance genes, and Actinobacteria, Proteobacteria and Firmicutes were the main host bacteria phyla of these ARGs. The genus Nocardioides sp. and Ornithinimicrobium sp. were closely associated with the ARGs in the investigated samples. The relative abundances of ARGs in wastewater and manure were reduced by 68.5% and 62.1%, respectively, by the existing feces treatment process. Anaerobic fermentation and high-temperature fermentation were the most efficient treatment steps; the relative abundances of ARGs were reduced by 29.3% and 33.6% in the treated wastewater and manure, respectively. Irrigation with the treated wastewater significantly increased the abundance and diversity of ARGs and ARB in the surface soil of the farmland. The residual ARGs were found to transit through vertical gene transfer (VGT) and horizontal gene transfer (HGT) in soil. Therefore, the direct application of this inadequately treated wastewater and/or manure could risk spreading ARGs into the environment, and potentially impact human health. In order to effectively restrain the spread of ARGs, it is necessary to modify the wastewater and manure treatment processes and improve the regulations and guidelines of applying treated wastewater for irrigation.

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Development and clinical application of a rapid and visual loop-mediated isothermal amplification test for tetM gene in Clostridioides difficile strains cultured from feces

Cited by 7 publications

References 59 publications

Genome sequence and characterization of a hypervirulent BI/NAP1/027Clostridioides difficile(CDC20121308)

Genome sequence and characterization of a hypervirulent BI/NAP1/027Clostridioides difficile(CDC20121308)

Principles and Applications of Loop-Mediated Isothermal Amplification to Point-of-Care Tests

Identification and Distribution of Antibiotic Resistance Genes and Antibiotic Resistance Bacteria in the Feces Treatment Process: A Case Study in a Dairy Farm, China

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