Azithromycin Resistance in Shigella spp. in Southeast Asia

Abstract

Infection by Shigella spp. is a common cause of dysentery in Southeast Asia. Antimicrobials are thought to be beneficial for treatment; however, antimicrobial resistance in Shigella spp. is becoming widespread. We aimed to assess the frequency and mechanisms associated with decreased susceptibility to azithromycin in Southeast Asian Shigella isolates and use these data to assess appropriate susceptibility breakpoints. Shigella isolates recovered in Vietnam and Laos were screened for susceptibility to azithromycin (15 μg) by disc diffusion and MIC. Phenotypic resistance was confirmed by PCR amplification of macrolide resistance loci. We compared the genetic relationships and plasmid contents of azithromycin-resistant Shigella sonnei isolates using whole-genome sequences. From 475 available Shigella spp. isolated in Vietnam and Laos between 1994 and 2012, 6/181 S. flexneri isolates (3.3%, MIC ≥ 16 g/liter) and 16/294 S. sonnei isolates (5.4%, MIC ≥ 32 g/liter) were phenotypically resistant to azithromycin. PCR amplification confirmed a resistance mechanism in 22/475 (4.6%) isolates (mphA in 19 isolates and ermB in 3 isolates). The susceptibility data demonstrated the acceptability of the S. flexneri (MIC ≥ 16 g/liter, zone diameter ≤ 15 mm) and S. sonnei (MIC ≥ 32 g/liter, zone diameter ≤ 11 mm) breakpoints with a <3% discrepancy. Phylogenetic analysis demonstrated that decreased susceptibility has arisen sporadically in Vietnamese S. sonnei isolates on at least seven occasions between 2000 and 2009 but failed to become established. While the proposed susceptibility breakpoints may allow better recognition of resistant isolates, additional studies are required to assess the impact on the clinical outcome. The potential emergence of azithromycin resistance highlights the need for alternative options for management of Shigella infections in countries where Shigella is endemic.

Keywords: Shigella; Southeast Asia; azithromycin; breakpoints; genome analysis; resistance.

FIG 1

Distribution of azithromycin MICs for S. flexneri and S. sonnei in Southeast Asia. The histograms show the number of S. sonnei and S. flexneri isolates collected in 7 studies performed in Southeast Asia between 1994 and 2012 exhibiting different MICs against azithromycin.

FIG 2

Relationship between azithromycin MIC and inhibition zone size in Southeast Asian Shigella spp. The plots show the relationship between inhibition zone size (x axis) and the MIC (y axis) for azithromycin in S. flexneri (left) and S. sonnei (right). The squares are colored with respect to the number of isolates in each group, and the number of isolate in each group is additionally provided.

FIG 3

Phylogenetic tree of 261 S. sonnei genomes (global lineage III) and an additional 54 genomes of isolates collected during the same period (1995 to 2011) in Southeast Asia. The tree was constructed through the use of 2,812 chromosomal SNPs. Phylogenetic reconstruction was performed using multiple-sequence alignments of the SNPs by maximum likelihood-based phylogenetic inference; the tree was displayed and annotated using the iTOL tool. The year/period of isolation is highlighted in the outer ring, and the organisms with reduced susceptibility to azithromycin are identified; mphA-positive isolates are highlighted in red, and ermB-positive isolates are highlighted in blue.

FIG 4

Maps of azithromycin resistance plasmids pDE105 (A) and pEG430_2 (B) from Vietnamese S. sonnei isolates. The coding sequences are numbered consecutively, and notable genes/regions, including DNA transfer, replication, and antimicrobial resistance regions and the azithromycin resistance genes (ermB and mphA), are highlighted. The size of each plasmid is shown in the center.