Inverse PCR for subtyping of Acinetobacter baumannii carrying ISAba1

Abstract

Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla(OXA-51-like), bla(OXA-23-like), and bla(ampC), which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes--bla(ampC), bla(OXA-66-like), and csuD--were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.

DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences

“…The analysis of anti‐microbial resistance genes in bacterial genomes, particularly those representing a small fraction of the microbiome and hence, potentially difficult to detect by shotgun sequencing, represents another clinical application. Similarly, SIP could also be applied to assist with the assembly and annotation of divergent viruses, many of which have genomes within the range of PacBio sequencing read lengths (Geldenhuys et al., 2018), as well as strain level detection across bacteria and parasites (Kim et al., 2016).…”

“…Long reads might enable strain level detection. Targeting antibiotic resistance genes coupled with long-read sequencing is of relevance as well Kim et al (2016) Non-invasive disease dignostics Upstream and downstream flanking regions of a target gene could be studied to identify or detect promotors and enhancers of that gene. Additionally, genes with unknown function within a flanking region could be investigated.…”

Protocol to retrieve unknown flanking DNA sequences using semi-site-specific PCR-based genome walking

“…Genome-walking is extensively used to probe unknown flanking DNA sequences. 2 , 3 , 4 , 5 , 6 , 7 , 8 Recently, we have devised Wristwatch PCR for genome-walking. 9 The Wristwatch PCR involves a set of three random walking primers that form a wristwatch-like structure with each other.…”