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Alkaline Peptone Water-Based Enrichment Method for mcr-3 From Acute Diarrheic Outpatient Gut Samples
Qiaoling Sun1, Yanyan Hu1, Hongwei Zhou1
1Department of Clinical Laboratory, Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, China.
Abstract:
A third plasmid-mediated colistin resistance gene, mcr-3, is increasingly being reported in Enterobacteriaceae and Aeromonas spp. from animals and humans. To investigate the molecular epidemiology of mcr in the gut flora of Chinese outpatients, 152 stool specimens were randomly collected from outpatients in our hospital from May to June, 2017. Stool specimens enriched in alkaline peptone water or Luria-Bertani (LB) broth were screened for mcr-1, mcr-2, and mcr-3 using polymerase chain reaction (PCR)-based assays. Overall, 19.1% (29/152) and 5.3% (8/152) of the stool samples enriched in alkaline peptone water were PCR-positive for mcr-1 and mcr-3, respectively, while 2.7% (4/152) of samples were positive for both mcr-1 and mcr-3. Strains isolated from the samples that were both mcr-1- and mcr-3-positive were subjected to antimicrobial susceptibility testing by broth microdilution. They were also screened for the presence of other resistance genes by PCR, while multilocus sequence typing and whole-genome sequencing were used to investigate the molecular epidemiology and genetic environment, respectively, of the resistance genes. mcr-3-positive Aeromonas veronii strain 126-14, containing a mcr-3.8-mcr-3-like2 segment, and mcr-1-positive Escherichia coli strain 126-1, belonging to sequence type 1485, were isolated from the sample from a diarrheic butcher with no history of colistin treatment. A. veronii 126-14 had a colistin minimum inhibitory concentration (MIC) of 2 µg/mL and was susceptible to antibiotics in common use, while E. coli 126-1 produced TEM-1, CTX-M-55, and CTX-M-14 β-lactamases and was resistant to colistin, ceftazidime, and cefotaxime. Overall, there was a higher detection rate of mcr-3-carrying strains with low colistin MICs from the samples enriched in alkaline peptone water than from samples grown in LB broth.
Insights
The study detected plasmid-mediated colistin resistance genes mcr-1 and mcr-3 in Chinese outpatients
Area of Science:
- Microbiology
- Genetics
- Epidemiology
Background:
- Plasmid-mediated colistin resistance genes, including mcr-3, are increasingly reported in various bacterial species from both animal and human sources.
- Understanding the prevalence and molecular epidemiology of these resistance genes in the human gut microbiome is crucial for public health surveillance.
Purpose of the Study:
- To investigate the molecular epidemiology of mcr genes (mcr-1, mcr-2, mcr-3) in the gut flora of Chinese outpatients.
- To characterize the genetic environment and antimicrobial resistance profiles of bacteria carrying these resistance genes.
Main Methods:
- Collection and screening of 152 stool specimens from outpatients using PCR-based assays for mcr-1, mcr-2, and mcr-3.
- Enrichment of samples using alkaline peptone water and Luria-Bertani (LB) broth.
- Antimicrobial susceptibility testing, PCR for other resistance genes, multilocus sequence typing, and whole-genome sequencing of isolated strains.
Main Results:
- mcr-1 and mcr-3 genes were detected in 19.1% and 5.3% of samples, respectively, with alkaline peptone water enrichment showing higher detection rates.
- mcr-1 and mcr-3 positive strains, including Escherichia coli and Aeromonas veronii, were isolated.
- The study identified specific sequence types and resistance gene profiles, such as E. coli ST1485 carrying mcr-1 and TEM-1, CTX-M-55, CTX-M-14, and A. veronii carrying mcr-3.8-mcr-3-like2.
Conclusions:
- The findings highlight the presence of plasmid-mediated colistin resistance genes mcr-1 and mcr-3 in the gut flora of Chinese outpatients.
- Alkaline peptone water enrichment appears to be a more effective method for detecting mcr-3 carrying strains with low colistin minimum inhibitory concentrations (MICs).
- Further surveillance and characterization of mcr genes in clinical settings are warranted to monitor the spread of colistin resistance.
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