Next-gen sequencing of multi-drug resistant Acinetobacter baumannii to determine antibiotic resistance genotypes

Background

Multi-drug resistant (MDR) Acinetobacter baumannii is an important cause of hospital acquired infection and often increases mortality and length of stay[1–3]. The mechanisms of resistance include: (1) antimicrobial-inactivating enzymes such as □-lactamases, (2) alteration of membrane porin channels, and (3) mutations that change cellular functions [4]. Accurate genotyping and correlation to antimicrobial susceptibility will help prevent and treat outbreaks of Acinetobacter.

The genome of A. baumannii ranges from 3.2 Megabases (Mb) in the drug sensitive SDF strain up to 3.9 Mb in the MDR AYE strain. A surprisingly high proportion of baumannii ORFs, (15%-20%), are located in resistance islands or “alien islands” - long stretches of DNA acquired from a foreign source. The MDR AYE strain has an 86Kb island containing 45-50 drug resistance genes located in an insertion hotspot [5]. Our study aims to sequence several A. baumanni i isolates from Metro Nashville General (NGH) Hospital and conduct a strain-to-reference genomic characterization of clinical virulence factors.

Materials and methods

A retrospective review of the NGH hospital epidemiology data base included 247 isolates of A. baumannii from 164 patients (submitted, BMC Infectious Disease). Cluster Software version 2.11 and TreeView software grouped resistance phenotypes into six categories (see Figure 1) [6].

Figure 1

Clustered baumannii groups. Left Side: full clustergram of all isolate phenotypes. Right Side: zoom in of resistance group 4: meripenem/imipenum and aminoglycoside sensitive.

Legend: red = resistant, green = sensitive, yellow = intermediate, black/gray = no data.

1.Pan resistant

2.Pan sensitive

3.Sensitive to meropenem /imipenem only.

4.Sensitive to meropenem/imipenem and aminoglycoside only.

5.Sensitive to cephalosporins only.

6.Resistant to aminoglycosides only.

We chose a meripenum/imepenum and aminoglycosides sensitive baumanii isolate for strain-to-reference sequencing on an Illumina Genome Analyzer II system at the Vanderbilt University Genome Technology Core (https://gtc.vanderbilt.edu/gtc/tech).

Conclusion

Initial sequencing yielded 5,250,420 reads of 43bp each, yielding 225.76 Mb of total sequence. The reads from our isolate were aligned to MDR baumannii reference strain ACICU (NC_010611.1). Alignment was done with the Bowtie Aligner [7]. Of the 5.2 million total reads, 4,004,012 (76.26%) aligned to AICIU, with a mean coverage depth of 43.96 fold. Roughly 58% of the ACICU genome was covered by at least one read. We will next align the reads further with other baumannii reference strains including MDR AYE (NC_010410) and non-resistant strain SDF (NC_010400) in order to further characterize and annotate our isolate at the genomic level.