Our study revealed that CRES isolates have unique features compared with CSER isolates, including their AMR genotype [lnu(B)-lsa(E) with aac(6′)-aph(2″)], ST19/CC19, CPS type III, virulence gene profile of rib-lmb-cylE, and in terms of cluster on the phylogenetic tree.
We searched for the presence of CRES S. agalactiae isolates in the Isolates Database on the MLST website (https://pubmlst.org/bigsdb?db=pubmlst_sagalactiae_isolates&page=query). Interestingly, only one CRES isolate was previously recovered from a 61-year-old female patient with bacteremia in Kangwon Province in the north of Korea in 2010 . Another CRES isolate of CPS genotype III was isolated from a patient with bacteremia in Bergen, Norway, in 2010 . Three CRES isolates of serotype III were recovered from clinical specimens in Seoul during 2010–2013 [9, 24]. These findings are in line with our observation that all CRES isolates in Gyeongnam Province in the south of Korea were recovered between March 2010 and August 2011. Thus, the CRES isolates appeared to be epidemic in Korea and other countries during this limited period.
In line with a previous study , we found a significant difference in the distribution of AMR genotype of lnu(B)-lsa(E) between the CRES and CSER isolates. For all CRES isolates, the lnu(B) locus was adjacent to the lsa(E) locus within the same contigs, with a short 53-bp distance between these two loci. Therefore, in our study, the lnu(B)-lsa(E) gene combination seems to mainly contribute to the CRES phenotype. Further observation of the dynamic changes in the CRES phenotype and the corresponding gene transfer is needed.
The erm(B) confers constitutive resistance (cMLSB) through a conformational change in 23S ribosomal RNA methyltransferase . Deletion of a segment in erm(B) sequence in CRES suggests loss of function of the erm(B) protein in this isolate, resulting in an erythromycin-susceptible phenotype. Interestingly, three CRES isolates (NUBL-9601, NUBL-9602, and NUBL-9603) isolated at a hospital in Seoul during 2010–2013 had the identical sequences (accession numbers LC430933, LC430934, and LC430935) (3) [9, 10]. Furthermore, we observed the IS1216E insertion in erm(B) (accession numbers LC512881, LC512882, LC512883, LC512885, and LC512886) in five isolates (GCH16, GCH19, GCH38, GCH55, and GCH58, respectively) of this study. Thus, IS1216E seems to be common among CRES isolates in Korea.
Four CRES isolates (GCH63, GCH64, GCH65, and GCH67) in our study possessed truncated variant sequences of erm(B) (624 and 678 bps) due to insertion of a TAA stop codon into the open reading frame (accession numbers LC512877, LC512878, LC512879, and LC512880), suggesting that an immature erm(B) protein leads to the erythromycin-susceptible phenotype. Three other phenotypes (GCH50, GCH72, and GCH78) also had truncated variant sequences (660, 678, and 510 bps, respectively) (accession numbers LC512884, LC512887, and LC512888, respectively) harboring the erythromycin-susceptible phenotype. Therefore, we may explain the mechanism of the presence of erm(B)-gene with the erythromycin-susceptible phenotype in CRES S. agalactiae.
This study has several limitations. First, clinical data, such as antibiotic treatment, outcome, and complications, does not suffice to demonstrate the relationship with the AMR genotype or virulence gene profile. Second, we cannot explain why the clonal outbreak occurred only during a limited period and is absent nowadays. Third, we did not determine translational activities and enzymatic functions of the IS1216E insertion-erm(B) and the truncated variant sequences.
In conclusion, CRES isolates were obtained during a limited period (2010–2011) and showed a genetic cluster having ST19 and CPS III in Korea as revealed by WGS. This rare AMR phenotype should be meticulously monitored, and the therapeutic efficacy of optimal antibiotics should be further evaluated.