The sample of municipal sewage was obtained from the influx of wastewater-related plant in Luzhou City (Sichuan Province, China) in March 2019. The sewage was 1:10 diluted and an aliquot (10 μL) was streaked onto a CHROM Agar Orientation (CHROMAgar, Paris, France) agar plate containing 2 mg/L meropenem (Solarbio, China) and then incubated at 37 °C overnight. Bacterial species identification was carried out by the Vitek2 system (BioMérieux, France), 16srRNA sequencing and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The minimal inhibitory concentrations (MICs) of 15 antimicrobial agents (Solarbio, China) including meropenem, imipenem, cefepime, cefotaxime, ceftazidime, piperacillin-tazobactam, amoxicillin-clavulanic acid, gentamicin, amikacin, aztreonam, erythromycin, chloramphenicol, sulfadiazine, colistin and ciprofloxacin were determined by broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2013, M100-S23). Escherichia coli strain ATCC 25,922 was used as quality control. Polymerase chain reaction (PCR) amplification and DNA sequencing were performed to identify the key carbapenemase-encoding genes (blaNDM and blaKPC) as previously reference.7 (link)
Meropenem
Manufactured by Solarbio
Sourced in China
Meropenem is a broad-spectrum antibiotic that belongs to the carbapenem class of antibiotics. It is used to treat a variety of bacterial infections.
Automatically generated - may contain errors
Lab products found in correlation
Imipenem, by Solarbio (2 mentions)
Tigecycline, by Maclin Biochemical Technology (1 mentions)
Colistin, by Solarbio (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Mueller hinton agar (mha), by Solarbio (1 mentions)
Ampicillin, by Sangon (1 mentions)
Meropenem, by Meilun (1 mentions)
Imipenem, by Fresenius (1 mentions)
Ceftriaxone, by Solarbio (1 mentions)
Penicillin g, by Solarbio (1 mentions)
Hepes, by Sangon (1 mentions)
Piperacillin, by Solarbio (1 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
Orientation, by CHROMagar (1 mentions)
5 protocols using meropenem
1
Carbapenem-Resistant Bacterial Isolation
2
CRKP Antimicrobial Susceptibility Testing
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The antimicrobial susceptibility testing of the CRKP from HWW, recipient and transconjugants was performed by Vitek-2 (Vitek-AST-GN16) (BioMerieux, France) according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2022). The antibiotics included ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, cefazolin, cefoxitin, ceftriaxone, cefepime, aztreonam, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, nitrofurantoin, and trimethoprim/sulfamethoxazole. Among them, MICs of ertapenem (Macklin, China), imipenem (Solarbio, China), meropenem (Solarbio, China), tigecycline (Macklin, China) and colistin (Solarbio, China) were determined using broth dilution method. E. coli ATCC 25,922 was used as quality control.
3
Antibacterial Assay of Meropenem and Energy Modulators
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The antibacterial assay
was conducted following previously established protocols. A single
colony was inoculated into 100 mL of LB medium in 250 mL flasks and
grown for 16 h at 37 °C. The bacterial culture was harvested
and centrifuged at 8000 rpm for 5 min. The resulting pellets were
washed three times with saline, followed by resuspension in M9 buffer
containing NaAc (10 mM), MgSO4 (2 mM), and CaCl2 (0.1 mM) and adjusted to an OD600 of 0.2. The samples
were then supplemented with Meropenem (Solarbio) and AMP or ATP (Sigma-Aldrich),
followed by incubation at 37 °C for 6 h. To determine bacterial
counts, 100 μL of the samples underwent a 10-fold serial dilution,
and subsequently, 5 μL of each dilution was applied onto 2%
LB agar plates for cultivation at 37 °C for 18 h. The viability
was assessed by calculating the ratio of clone numbers observed in
the treated group compared with the control group.
was conducted following previously established protocols. A single
colony was inoculated into 100 mL of LB medium in 250 mL flasks and
grown for 16 h at 37 °C. The bacterial culture was harvested
and centrifuged at 8000 rpm for 5 min. The resulting pellets were
washed three times with saline, followed by resuspension in M9 buffer
containing NaAc (10 mM), MgSO4 (2 mM), and CaCl2 (0.1 mM) and adjusted to an OD600 of 0.2. The samples
were then supplemented with Meropenem (Solarbio) and AMP or ATP (Sigma-Aldrich),
followed by incubation at 37 °C for 6 h. To determine bacterial
counts, 100 μL of the samples underwent a 10-fold serial dilution,
and subsequently, 5 μL of each dilution was applied onto 2%
LB agar plates for cultivation at 37 °C for 18 h. The viability
was assessed by calculating the ratio of clone numbers observed in
the treated group compared with the control group.
4
Restoring Meropenem Efficacy Against VIM-2 E. coli
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The pUC57-ISAba125-pelB-VIM-2 plasmid was constructed by multi-step gene cloning and transformed into E. coli DH5α for susceptibility test. Meropenem was purchased from Dalian Meilun Biotechnology Co., Ltd. Ampicillin was obtained from Sangon Biotech (Shanghai) Co., Ltd. MHB and MHA were purchased from Solarbio (Beijing, China).
Strains of E. coli DH5α containing plasmids pUC57-ISAba125-pelB-VIM-2 (E. coli-VIM-2) and ATCC 25922 (as control) were used to assess the ability of inhibitors in restoring the antimicrobial activity of β-lactam antibiotic Meropenem. Meropenem were tested alone or in combination with inhibitors at 100 or 10 μg/mL. Minimal inhibitory concentration (MIC) values were determined by the standard broth micro-dilution method according to the Clinical and Laboratory Standards Institute (CLSI, M07-A9, 2012) guideline.
Strains of E. coli DH5α containing plasmids pUC57-ISAba125-pelB-VIM-2 (E. coli-VIM-2) and ATCC 25922 (as control) were used to assess the ability of inhibitors in restoring the antimicrobial activity of β-lactam antibiotic Meropenem. Meropenem were tested alone or in combination with inhibitors at 100 or 10 μg/mL. Minimal inhibitory concentration (MIC) values were determined by the standard broth micro-dilution method according to the Clinical and Laboratory Standards Institute (CLSI, M07-A9, 2012) guideline.
5
Purification and Characterization of NDM-1
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Penicillin G, Piperacillin, Ceftriaxone, Cefepime, Cefazolin, Meropenem were purchased from Solarbio Co., Ltd. (Beijing, China). Imipenem was bought from Fresenius Kabi (Bad Homburg, Germany). HEPES was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). All other reagents were purchased from Sigma-Aldrich Co (St. Louis, USA). The controlled pore glass (CPG) beads (125–140 μm particle diameter and 50 nm pore size) were originally purchased from VEB Trisola, Steinach, Germany. The construction of plasmid encoding NDM-1, expression and purification of recombinant NDM-1(rNDM-1) were performed as previously described (Meng et al., 2021a (link); Meng et al., 2021b (link)). The purity of the purified rNDM-1 is > 95%, The unit activity of rNDM-1 to hydrolyze Meropenem is 108 IU/mg.
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