Heptaplex PCR melting curve analysis for rapid detection of plasmid-mediated AmpC β-lactamase genes

Liu, Zanzan; Zhang, Jiaqin; Rao, Shitao; Sun, Lei; Zhang, Jie; Liu, Ran; Zheng, Gangsen; Ma, Xiaoxuan; Hou, Shuyi; Zhuang, Xinguo; Song, Xiaomin; Li, Qingge

doi:10.1016/j.mimet.2014.12.019

Cited by 12 publications

(11 citation statements)

References 29 publications

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“…A panel of various, relevant antimicrobial resistance genes were amplified by PCR, and the positive amplicons were further confirmed by DNA sequencing. These resistance genes included those encoding the extended spectrum β-lactamase genes ( bla CTX-M , bla PER , bla SHV , bla TEM and bla VEB ) [ 24 ], carbapenemase genes ( bla GES , bla IMI/NMC-A , bla SME , bla KPC , bla VIM , bla IMP , bla NDM and bla OXA-48 ) [ 25 , 26 ], AmpC β-lactamase genes ( bla CMY , bla FOX , bla MOX , and bla DHA ) [ 27 ], plasmid-mediated quinolone resistance (PMQR) genes ( qnrA , qnrB , qnrC , qnrD , qnrS , aac (6′)-Ib-cr ) [ 28 ], oqxAB multidrug efflux pump genes [ 29 ] and the aminoglycoside resistant genes ( aac(6′)–Ib , aac(3′)–Ib , rmtB , armA , APH , ANT , rmtA , rmtB rmtC and rmtD ) [ 30 – 32 ]. DNA sequences were characterized by sequence comparisons using BLAST ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) [ 33 ].…”

Section: Methodsmentioning

confidence: 99%

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling

et al. 2020

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Background: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a looming threat to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC β-lactamase, a systematic study on the treatment-emergence of porins alteration in antibiotic resistance does not yet exist. The aim of this study was to investigate the underlying mechanism of resistance of K. pneumoniae during carbapenem treatment. Results: Here, we report three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that that the first isolate, FK-2624, was susceptible to almost all tested antimicrobials, being resistant only to fosfomycin. The subsequent isolates FK-2723 and FK-2820 were multidrug resistant (MDR). After imipenem therapy, FK-2820 was found to be carbapenem-resistant. PCR and Genome Sequencing analysis indicated that oqxA, and fosA5, were identified in all three strains. In addition, FK-2624 also harbored bla SHV-187 and bla TEM-116. The bla SHV-187 and bla TEM-116 genes were not detected in FK-2723 and FK-2820. bla DHA-1 , qnrB4, aac (6′)-IIc, and bla SHV-12 , EreA2, CatA2, SulI, and tetD, were identified in both FK-2723 and FK-2820. Moreover, the genes bla DHA-1, qnrB4, aac (6′)-IIc were co-harbored on a plasmid. Of the virulence factors found in this study, ybtA, ICEKp6, mrkD, entB, iroN, rmpA2-6, wzi16 and capsular serotype K57 were found in the three isolates. The results of pairwise comparisons, multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK36, with genome sequence data validating that there was a premature stop codon in the ompK36 gene and real-time RT-PCR suggesting high turnover of the ompK36 nonsense transcript in FK-2820, with the steady-state mRNA level 0.007 relative to the initial isolate.

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Section: Methodsmentioning

confidence: 99%

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling

et al. 2020

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show abstract

“…A panel of various, relevant antimicrobial resistance genes were amplified by PCR, and the positive amplicons were further confirmed by DNA sequencing. These resistance genes included those encoding the extended spectrum β-lactamase genes (bla CTX-M , bla PER , bla SHV , bla TEM and bla VEB ) [10], carbapenemase genes (bla GES , bla IMI/NMC-A , bla SME , bla KPC , bla VIM , bla IMP , bla NDM and bla OXA-48 ) [11,12], AmpC β-lactamase genes (bla CMY , bla FOX , bla MOX , and bla DHA ) [13], plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr) [14], oqxAB multidrug efflux pump genes [15] and the aminoglycoside resistant genes (aac(6')-Ib, aac(3')-Ib, rmtB, armA, APH, ANT, rmtA, rmtB rmtC and rmtD) [16][17][18]. DNA sequences were characterized by sequence comparisons using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) [19].…”

Section: Antimicrobial Susceptibility Testing Detection Of Resistancmentioning

confidence: 99%

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling

Tian

Wang

New

et al. 2020

Preprint

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Background:The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a looming threat to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC β-lactamase, a systematic study on the treatment-emergence of porins alteration in antibiotic resistance does not yet exist. The aim of this study was to investigate the underlying mechanism and evolution of resistance of K. pneumoniae during carbapenem treatment. Results:Here, we report three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that that the first isolate, FK-2624, was susceptible to almost all tested antimicrobials, being resistant only to fosfomycin. The subsequent isolates FK-2723 and FK-2820 were multidrug resistant (MDR). After imipenem therapy, FK-2820 had evolved to be carbapenem-resistant. PCR and Genome Sequencing analysis indicated that oqxA, and fosA5, were identified in all three strains. In addition, FK-2624 also harbored bla SHV-187 and blaTEM-116. The blaSHV-187 and blaTEM-116 genes were not detected in FK-2723 and FK-2820. blaDHA-1, qnrB4, aac (6’)-IIc, and blaSHV-12, EreA2, CatA2, SulI, and tetD , were identified in both FK-2723 and FK-2820. Moreover, the genes bla DHA -1, qnrB4, aac(6’)-IIc were co-harbored on a plasmid. Of the virulence factors found in this study, ybtA, ICEKp6, mrkD, entB, iroN, rmpA2-6, wzi16 and capsular serotype K57 were found in the three isolates. The results of pairwise comparisons, multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK36, with genome sequence data validating that there was a premature stop codon in the ompK36 gene and real-time RT-PCR suggesting high turnover of the ompK36 non-sense transcript in FK-2820, with the steady-state mRNA level 0.007 relative to the initial isolate. Conclusion:This study in China highlight that the alteration of outer membrane porins due to the 14-day use of imipenem play a potential role in leading to clinical presentation of carbapenem-resistance. This is the first description of increased resistance developing from a carbapenem-susceptible K. pneumoniae with imipenem treatment driven by outer membrane remodeling.

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“…2020). The genes encoding plasmid-mediated AmpC β-lactamase are harbored by mobile elements that could confer transmissible resistance to environmental bacteria and pathogens, which may accelerate cephalosporin resistance dissemination in the environment (Liu et al 2015;Pazda et al 2019). Therefore, the detection rates of MOX and EBC were very high (85.71%).…”

Section: Comparison Of Arg Elimination Efficiency By Processes Among mentioning

confidence: 99%

Comparison of the elimination effectiveness of tetracycline and AmpC β-lactamase resistance genes in a municipal wastewater treatment plant using four parallel processes

et al. 2020

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Municipal wastewater treatment plants (mWWTPs), considered reservoirs of antibiotic resistance genes (ARGs), are selected to compare the contributions of technology and process to ARG removal. Fifteen ARGs (tetA, tetB, tetC, tetE, tetG, tetL, tetM, tetO, tetQ, tetS, tetX, MOX, CIT, EBC, and FOX) and two integron genes (intI1, intI2) were tracked and detected in wastewater samples from a large-scale mWWTP with four parallel processes, including three biological technologies of AAO (anaerobic-anoxic-oxic), AB (adsorption-biodegradation), and UNITANK, two different disinfection technologies, and two primary sedimentation steps. The results showed that ARGs were widely detected, among which tetA and tetM had the highest detection rate at 100%. AAO was the most effective process in removing ARGs, followed by the AB and UNITANK processes, where the separation step was critical: 37.5% AmpC β-lactamase genes were reduced by the secondary clarifier. UV disinfection was more efficient than chlorination disinfection by 47.0% in ARG removal. Both disinfection and primary sedimentation processes could effectively remove integrons, and the swirling flow grit chamber was a more effective primary settling facility in total ARG removal than the aerated grit chamber. The tet genes and AmpC β-lactamase genes were significantly correlated with the water quality indexes of BOD 5 , COD Cr , SS, TP, TOC, pH and NH 4 +-N (p < 0.05). In addition, the correlation between efflux pump genes and AmpC β-lactamase genes was strongly significant (r 2 = 0.717, p < 0.01). This study provides a more powerful guide for selecting and designing treatment processes in mWWTPs with additional consideration of ARG removal.

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Heptaplex PCR melting curve analysis for rapid detection of plasmid-mediated AmpC β-lactamase genes

Cited by 12 publications

References 29 publications

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling

First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling

Comparison of the elimination effectiveness of tetracycline and AmpC β-lactamase resistance genes in a municipal wastewater treatment plant using four parallel processes

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