Polymorphisms of Gene Cassette Promoters of the Class 1 Integron in Clinical Proteus Isolates.

Linlin Xiao,Xiaotong Wang,Long Zhang,Quhao Wei,Mei Cao,Nana Kong,Weiwei Liu

doi:10.3389/fmicb.2019.00790

Abstract

ObjectiveTo describe the polymorphisms of gene cassette promoters of the class 1 integron in clinical Proteus isolates and their relationship with antibiotic resistance.MethodsPolymorphisms of the gene cassette promoter in 153 strains of Proteus were analyzed by PCR and nucleotide sequencing. Variable regions of atypical class 1 integrons were detected by inverse PCR and nucleotide sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyze the phylogenetic relationships of class 1 integron-positive clinical Proteus isolates. Representative beta-lactamase genes (bla), including blaTEM,blaSHV,blaCTX-M-1,blaCTX-M-2,blaCTX-M-8,blaCTX-M-9,blaCTX-M-25 and blaOXA-1, and plasmid-mediated quinolone resistance (PMQR) genes including qnrA, qnrB, qnrC, qnrD, qnrS, oqxA, oqxB, qepA, and aac(6′)-Ib were also screened using PCR and sequence analysis.ResultsFifteen different gene cassette arrays and 20 different gene cassettes were detected in integron-positive strains. Of them, aadB-aadA2 (37/96) was the most common gene cassette array. Two of these gene cassette arrays (estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3) have not previously been reported. Three different Pc-P2 variants (PcS, PcWTGN-10, PcH1) were detected among the 96 Proteus strains, with PcH1 being the most common (49/96). Strains carrying the promoters PcS or PcWTGN-10 were more resistant to sulfamethoxazole, gentamicin and tobramycin than those carrying PcH1. Strains with weak promoter (PcH1) harbored significantly more intra- and extra-integron antibiotic resistance genes than isolates with strong promoter (PcWTGN-10). Further, among 153 isolates, representative beta-lactamase genes were detected in 70 isolates (blaTEM-1, 54; blaOXA-1, 40; blaCTX-M-3, 12; blaCTX-M-14, 12; blaCTX-M-65, 5; blaCTX-M-15, 2) and representative PMQR genes were detected in 87 isolates (qnrA, 6; qnrB, 3; qnrC, 5; qnrD, 46; qnrS, 5; oqxA, 7; aac(6′)-Ib, 13; aac(6′)-Ib-cr, 32).ConclusionTo the best of our knowledge, this study provides the first evidence for polymorphisms of the class 1 integron variable promoter in clinical Proteus isolates, which generally contain relatively strong promoters. Resistance genotypes showed a higher coincidence rate with the drug-resistant phenotype in strong-promoter-containing strains, resulting in an ability to confer strong resistance to antibiotics among host bacteria and a relatively limited ability to capture gene cassettes. Moreover, strains with relatively weak integron promoters can “afford” a heavier “extra-integron antibiotic resistance gene load”. Furthermore, the gene cassettes estX, psp and the gene cassette arrays estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 have been confirmed for the first time in clinical Proteus isolates. Beta-lactamase genes and PMQR were investigated, and blaTEM-1 and blaOXA-1 were the most common, with qnrD and aac (6′)-Ib-cr also being dominant.

Highlights

P. mirabilis is an important causative pathogen of various community and healthcare-associated infections, such as wound infections, primary bacteremia, pneumonia and urinary tract infections, among patients with anatomical or functional urinary tract abnormalities or indwelling urinary catheters (Ahn et al, 2017)
We found that the relatively weak promoter (PcH1) strains carried 6.88 resistance genes on average, of which 5.35 resistance genes were located in the integrons, and there were 1.53 resistance genes not located on the integrons
Multiple resistance genes were detected in isolates, and we compared the antibiotic resistance “gene load” of strains with different promoters. It further explains the fitness of the clinical bacteria. These results demonstrate that strains with relatively weak integron promoters can “afford” a heavier intra- and extra-integron antibiotic resistance gene load

Summary

Introduction

P. mirabilis is an important causative pathogen of various community and healthcare-associated infections, such as wound infections, primary bacteremia, pneumonia and urinary tract infections, among patients with anatomical or functional urinary tract abnormalities or indwelling urinary catheters (Ahn et al, 2017). Integration of exogenous antibiotic resistance genes (Guerin et al, 2009; Grieb et al, 2017) via site-specific recombination is an important pathway in the development of clinical antibiotic-resistant strains. Class 1 integrons are highly mobile and repetitive bacterial elements that integrate foreign gene cassettes and promote the expression of genes in the gene cassettes (Frumerie et al, 2010; Loot et al, 2012; Nivina et al, 2016). Class 1 integrons can be integrated into chromosomes, plasmids, or transposons, carrying resistance genes with them, play an important role in the formation and dissemination of drug-resistant bacterial strains (Collis et al, 2002; Ghazi et al, 2015; Makena et al, 2015; Moyo et al, 2015). The classical structure of class 1 integrons includes an integrase gene intI1, a recombination site attI1, an integrase gene transcription promoter, a lexA-binding site that regulates integrase gene expression, and a variable region gene cassette promoter (Collis et al, 1998, 2002; Collis and Hall, 2004; Demarre et al, 2007)

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