Abstract
In this work, we characterized a novel chromosome-encoded AmpC β-lactamase gene, blaPRC–1, in an isolate of a newly classified Pseudomonas species designated Pseudomonas wenzhouensis A20, which was isolated from sewage discharged from an animal farm in Wenzhou, China. Susceptibility testing, molecular cloning, and enzyme kinetic parameter analysis were performed to determine the function and enzymatic properties of the β-lactamase. Sequencing and comparative genomic analysis were conducted to clarify the phylogenetic relationship and genetic context of the blaPRC–1 gene. PRC-1 is a 379-amino acid AmpC β-lactamase with a molecular weight of 41.48 kDa and a predicted pI of 6.44, sharing the highest amino acid identity (57.7%) with the functionally characterized AmpC β-lactamase PDC-211 (ARX71249). blaPRC–1 confers resistance to many β-lactam antibiotics, including penicillins (penicillin G, amoxicillin, and amoxicillin-clavulanic acid) and cephalosporins (cefazolin, ceftriaxone, and cefotaxime). The kinetic properties of PRC-1 were compatible with those of a typical class C β-lactamase showing hydrolytic activities against β-lactam antibiotics, and the hydrolytic activity was strongly inhibited by avibactam. The genetic context of blaPRC–1 was relatively conserved, and no mobile genetic element was predicted in its surrounding region. Identification of a novel β-lactamase gene in an unusual environmental bacterium reveals that there might be numerous unknown resistance mechanisms in bacterial populations, which may pose potential risks to human health due to universal horizontal gene transfer between microorganisms. It is therefore of great value to carry out extensive research on the mechanism of antibiotic resistance.
Highlights
MATERIALS AND METHODSThe genus Pseudomonas, first described by Professor Mikula in 1894, is one of the most common bacteria in the world and has been found in various natural environments, human clinical specimens, and infected plants (Peix et al, 2009)
The strain P. wenzhouensis A20 carrying the novel β-lactamase gene blaPRC-1 was isolated from sewage discharged from an animal farm in Wenzhou, China
The strain P. wenzhouensis A20 was isolated from sewage discharged from an animal farm in Wenzhou, China. 16S ribosomal RNA (rRNA) homologous gene analysis demonstrated that P. wenzhouensis A20 showed the closest relationship with the strain Pseudomonas hydrolytica DSWY01 (NR_170428.1), at 98.50% identity and 100% coverage
Summary
The genus Pseudomonas, first described by Professor Mikula in 1894, is one of the most common bacteria in the world and has been found in various natural environments, human clinical specimens, and infected plants (Peix et al, 2009). AmpC β-lactamases are usually not expressed or are underexpressed in E. coli because of a weak promoter and a transcriptional attenuator preceding the ampC gene; due to mutations in β-lactamases or induction by specific βlactams, these enzymes can be expressed at high levels and increase the minimum inhibitory concentrations (MICs) of β-lactams (Fisher and Mobashery, 2014; Juan et al, 2017). In this study, based on the complete genome sequencing of an isolate of a newly classified Pseudomonas species, Pseudomonas wenzhouensis A20, we characterized a novel chromosomeencoded AmpC β-lactamase, designated PRC-1, and analyzed the kinetic parameters of the β-lactamase. The strain P. wenzhouensis A20 carrying the novel β-lactamase gene blaPRC-1 was isolated from sewage discharged from an animal farm in Wenzhou, China. The nucleotide sequence of the blaPRC-1 gene with the upstream promoter region was amplified using PrimeSTAR HS DNA Polymerase (Takara Bio Inc., Dalian, China), and P. wenzhouensis A20 genomic DNA was used as the template. According to previous studies on the phylogeny of Pseudomonas based on 16S ribosomal DNA (rDNA) sequences, there was not enough discrimination at the species level (Mulet et al, 2010),
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