Characterization of Tigecycline Resistance Among Tigecycline Non-susceptible Klebsiella pneumoniae Isolates From Humans, Food-Producing Animals, and in vitro Selection Assay

Frontiers in Microbiology ◽

10.3389/fmicb.2021.702006 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mohaddeseh Moghimi ◽

Mehri Haeili ◽

Hanieh Mohajjel Shoja

Keyword(s):

Klebsiella Pneumoniae ◽

In Vitro Selection ◽

Nonsense Mutations ◽

Frameshift Mutations ◽

Distinct Sequence ◽

Broth Macrodilution ◽

Sequence Types ◽

Animal Isolates

Emergence of extensively drug-resistant isolates of Klebsiella pneumoniae has prompted increased reliance on the last-resort antibiotics such as tigecycline (TGC) for treating infections caused by these pathogens. Consumption of human antibiotics in the food production industry has been found to contribute to the current antibiotic resistance crisis. In the current study, we aimed to investigate the mechanisms of TGC resistance among 18 TGC-non-susceptible (resistant or intermediate) K. pneumoniae (TGC-NSKP) isolates obtained from human (n = 5), food animals (n = 7), and in vitro selection experiment (n = 6). Isolates were genotyped by multilocus sequence typing (MLST). ramR, acrR, rpsJ, tetA, and mgrB (for colistin resistance) genes were sequenced. The presence of tetX, tetX1, and carbapenemase genes was examined by PCR. Susceptibility to different classes of antibiotics was evaluated by disc diffusion and broth macrodilution methods. The expression level of acrB was quantified by RT-qPCR assay. The 12 TGC-NSKP isolates [minimum inhibitory concentrations (MICs) = 4–32 mg/l] belonged to 10 distinct sequence types including ST37 (n = 2), ST11, ST15, ST45, ST1326 (animal isolates); ST147 (n = 2, human and animal isolates); and ST16, ST377, ST893, and ST2935 (human isolates). Co-resistance to TGC and colistin was identified among 57 and 40% of animal and human isolates, respectively. All human TGC-NSKP isolates carried carbapenemase genes (blaOXA–48, blaNDM–1, and blaNDM–5). tetX/X1 genes were not detected in any isolates. About 83% of TGC-NSKP isolates (n = 15) carried ramR and/or acrR alterations including missense/nonsense mutations (A19V, L44Q, I141T, G180D, A28T, R114L, T119S, Y59stop, and Q122stop), insertions (positions +205 and +343), or deletions (position +205) for ramR, and R90G substitution or frameshift mutations for acrR. In one isolate ramR amplicon was not detected using all primers used in this study. Among seven colistin-resistant isolates, five harbored inactivated/mutated MgrB due to premature termination by nonsense mutations, insertion of IS elements, and frameshift mutations. All isolates revealed wild-type RpsJ and TetA (if present). Increased expression of acrB gene was detected among all resistant isolates, with the in vitro selected mutants showing the highest values. A combination of RamR and AcrR alterations was involved in TGC non-susceptibility in the majority of studied isolates.

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PENGEMBANGAN TEKNOLOGI SELEKSI IN VITRO BERULANG (REPEAT CYCLING–IN VITRO SELECTION) TOLERAN STRES KEKERINGAN UNTUK PERBAIKAN MUTU GENETIK DAN PRODUKSI TANAMAN KEDELAI BERMIKORIZA

Jurnal Ilmiah Ilmu Terapan Universitas Jambi|JIITUJ| ◽

10.22437/jiituj.v1i1.3753 ◽

2017 ◽

Vol 1 (1) ◽

pp. 74-84

Author(s):

Ahmad Riduan ◽

Rainiyati Rainiyati ◽

Yulia Alia

Keyword(s):

Arbuscular Mycorrhizae ◽

In Vitro Selection ◽

Soil Samples ◽

Single Spore ◽

Isolation And Characterization ◽

Single Spore Culture ◽

Spore Culture ◽

Glomus Sp

Every plant rhizospheres in any ecosystem there are various living microorganisms including Arbuscular Mycorrhizae Fungi (AMF). An isolation and characterization is required to investigate the species or type of the AMF. This research was aimed at studying the isolation and characterization of AMF sporulation in soybean rhizospheres in Jambi Province. The results of evaluation on soil samples before trapping showed that there are spores from three genus of AMF twelve types Glomus , two types Acaulospora and one type of Enthrophospora. Following single spore culture in soybean rhizosphere, 5 spore types were obtained: Glomus sp-1, Glomus sp-4, Glomus sp-7, Glomus sp-8 Glomus sp-10.

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Enterobacteriaceae isolates carrying the New Delhi metallo-β-lactamase gene in Yemen

Journal of Medical Microbiology ◽

10.1099/jmm.0.073767-0 ◽

2014 ◽

Vol 63 (10) ◽

pp. 1316-1323 ◽

Cited By ~ 24

Author(s):

Alima Gharout-Sait ◽

Samer-Ahmed Alsharapy ◽

Lucien Brasme ◽

Abdelaziz Touati ◽

Rachida Kermas ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Susceptibility Testing ◽

Enterobacter Cloacae ◽

New Delhi ◽

Carbapenem Resistant ◽

Genes Encoding ◽

Phenotypic Tests ◽

Lactamase Gene ◽

Sequence Types

Ten carbapenem-resistant Enterobacteriaceae (eight Klebsiella pneumoniae isolates and two Enterobacter cloacae) isolates from Yemen were investigated using in vitro antimicrobial susceptibility testing, phenotypic carbapenemase detection, multilocus sequence typing (MLST) and replicon typing. Carbapenemase, extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance determinant genes were identified using PCR and sequencing. All of the 10 carbapenem-resistant Enterobacteriaceae were resistant to β-lactams, tobramycin, ciprofloxacin and cotrimoxazole. Imipenem, doripenem and meropenem MICs ranged from 2 to >32 mg l−1 and ertapenem MICs ranged from 6 to >32 mg l−1. All of the K. pneumoniae isolates showed ESBL activity in phenotypic tests. Genes encoding bla NDM were detected in all strains. All K. pneumoniae strains produced CTX-M-15 ESBL and SHV β-lactamases. TEM-1 β-lactamase was detected in seven isolates. Nine isolates were qnr positive including QnrB1, QnrA1 and QnrS1, and six isolates produced AAC-6′-Ib-cr. MLST identified five different sequence types (STs): ST1399, ST147, ST29, ST405 and ST340. Replicon typing showed the presence of IncFII1K plasmids in four transformants. To the best of our knowledge, this is the first report of NDM-1-producing Enterobacteriaceae isolates in Yemen.

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In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Journal of Virology ◽

10.1128/jvi.77.6.3669-3679.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3669-3679 ◽

Cited By ~ 85

Author(s):

Caterina Trozzi ◽

Linda Bartholomew ◽

Alessandra Ceccacci ◽

Gabriella Biasiol ◽

Laura Pacini ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Serine Protease ◽

In Vitro Selection ◽

Three Dimensional ◽

Host Cells ◽

Dimensional Structure ◽

In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

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