Analysis of a Streptococcus pyogenes puerperal sepsis cluster by use of whole-genome sequencing

Abstract

Between June and November 2010, a concerning rise in the number of cases of puerperal sepsis, a postpartum pelvic bacterial infection contracted by women after childbirth, was observed in the New South Wales, Australia, hospital system. Group A streptococcus (GAS; Streptococcus pyogenes) isolates PS001 to PS011 were recovered from nine patients. Pulsed-field gel electrophoresis and emm sequence typing revealed that GAS of emm1.40, emm75.0, emm77.0, emm89.0, and emm89.9 were each recovered from a single patient, ruling out a single source of infection. However, emm28.8 GAS were recovered from four different patients. To investigate the relatedness of these emm28 isolates, whole-genome sequencing was undertaken and the genome sequences were compared to the genome sequence of the emm28.4 reference strain, MGAS6180. A total of 186 single nucleotide polymorphisms were identified, for which the phylogenetic reconstruction indicated an outbreak of a polyclonal nature. While two isolates collected from different hospitals were not closely related, isolates from two puerperal sepsis patients from the same hospital were indistinguishable, suggesting patient-to-patient transmission or infection from a common source. The results of this study indicate that traditional typing protocols, such as pulsed-field gel electrophoresis, may not be sensitive enough to allow fine epidemiological discrimination of closely related bacterial isolates. Whole-genome sequencing presents a valid alternative that allows accurate fine-scale epidemiological investigation of bacterial infectious disease.

Fig 1

PFGE profiles of the five emm28.8 isolates examined in this study, using SmaI on the left of the marker lane (Mk) and AscI on the right.

Fig 2

Visualization of the reads selected for each strain mapped onto the S. pyogenes MGAS6180 reference genome. The innermost circles represent the GC content (black), GC skew (purple/green), and rRNA operons of MGAS6180 (pink boxes). BRIG (1) shows the distribution of the number of reads for each individual strain mapped onto the central reference using a window size of 500, arranged from inner to outer colored circles as follows: resequenced reference MGAS6180 (pink), PS001 (yellow), PS006 (orange), PS005 (red), PS007 (maroon), and PS008 (purple). Additional strain-specific regions of difference (RODs) (ϕPS008 and ICESpPS008) are represented as insertions. The outermost circle represents previously reported regions of difference in MGAS6180, namely, prophage elements 6180.1 and 6180.2, prophage remnants 6180.3 and 6180.4, and regions of difference 6180.RD1 and 6180.RD2 (15) (black).

Fig 3

Phylogeny of the emm28 strains based on SNPs. An unrooted maximum-likelihood tree including the five emm28.8 strains and the reference strain MGAS6180 was constructed using the 172 core SNPs out of the 186 total SNPs identified compared to the sequence of MGAS6180, which were obtained by excluding 14 SNPs associated with recombination events, as described in the supplemental material. Numbers on internal nodes represent the percentage of replicated trees (>50%) in which the associated samples clustered together in the bootstrap test (based on 1,000 replicates). The scale bar represents the number of base substitutions per site.