Escherichia coli strains possessing a four amino acid YRIN insertion in PBP3 identified as part of the SIDERO-WT-2014 surveillance study

Abstract Background In addition to carbapenemases, dissemination of recently reported Escherichia coli lineages possessing a four amino acid insertion in PBP3 (encoded by ftsI) that confers reduced susceptibility to PBP3-targeted β-lactams, such as ceftazidime, can pose a threat of antimicrobial resistance. Objectives To evaluate genotypic and phenotypic characteristics of E. coli possessing the mutated PBP3 collected during SIDERO-WT-2014 surveillance. Methods A subset of 65 E. coli clinical isolates with MICs ≥2 mg/L for ceftazidime/avibactam, ceftolozane/tazobactam or cefiderocol, among a total of 1529 isolates from the multinational surveillance study, were subjected to gene analysis and antimicrobial susceptibility testing. Isogenic PBP3 mutants were constructed to confirm experimentally an impact on antimicrobial susceptibility. Results Eleven strains possessing a YRIN-inserted PBP3 were identified, consisting of nine strains collected from the same hospital in Turkey (ST1284) and one each from the USA and Italy (ST361). Strains associated with each ST lineage possessed similar genetic backgrounds including β-lactamase genotypes; all nine strains from Turkey carried CMY-42, OXA-1 and the OXA-181 carbapenemase (five strains additionally carried CTX-M-15 ESBL), whereas the two other strains carried CMY-42 and TEM-1, indicating dissemination driven by selective pressure. The presence of the YRIN insertion contributed to reduced susceptibility to aztreonam, ceftazidime, cefepime and ceftolozane/tazobactam, although the strains remained susceptible to ceftazidime/avibactam despite relatively high MICs. Conclusions E. coli strains of both ST1284 and ST361 lineages, possessing YRIN-inserted PBP3, are disseminating in several regions. The YRIN insertion in PBP3 occurred with multiple β-lactamases, which indicates frequent cross-resistance to other β-lactams.


Introduction
Unusual PBP3 substitutions, consisting of a four amino acid insertion of YRIN or YRIK after position P333 or TIPY after position Y334, have been reported in Escherichia coli clinical isolates. 1,2 These altered PBP3 proteins conferred reduced susceptibility to a broad range of b-lactams, such as ceftazidime, cefepime and aztreonam, due to the decreased accessibility of the b-lactams to the transpeptidase pocket of PBP3. The YRIN(K) insertion in PBP3 was first identified in nine MLST lineages of E. coli, including ST101, ST405 and ST410, isolated in Asian and Middle Eastern countries, especially India, and was enriched among NDM-type carbapenemasecarrying strains. 1 In a recent report, the four amino acid insertion in PBP3 was observed in several E. coli lineages according to NCBI database analysis, and the presence of this mutation as well as mutations in ompC and ompF was associated with acquisition of carbapenemase genes. It is still unknown which lineages carrying YRIN(K)-inserted PBP3 are widespread or limited in geographic distribution and which have clinical importance. 3 Here, we evaluated the genotypic and phenotypic characteristics of E. coli strains possessing four amino acid insertions in PBP3 by re-analysing isolates collected as part of the SIDERO-WT-2014 surveillance study. 4

Screening for amino acid insertion in PBP3
The susceptibility data from 1529 E. coli isolates reported for SIDERO-WT-2014, the first multinational surveillance programme (North America and V C The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Analyses of ftsI, MLST, b-lactamases and SNPs
Sanger sequencing and/or WGS by the Illumina MiSeq system (San Diego, CA, USA) were conducted as described in the Supplementary data (available as Supplementary data at JAC Online). The sequences of ftsI and the translated PBP3 protein from WT E. coli K-12 (GenBank accession number NC_000913) were used as references for analysis. MLST profiles were determined by comparison of seven allele sequences in the public database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). 5 b-Lactamase sequences were detected using the WU-blast 2.0 algorithm (http://genetics.bwh.harvard. edu/msblast/) and the ResFinder database (https://cge.cbs.dtu.dk/services/ ResFinder/) using Genedata Selector 5.2.3 (Genedata, Switzerland). For SNP analysis, the trimmed reads were mapped against the E. coli K-12 genome and variant calls were performed using Microbial Genomics Module 3.5.1 in CLC Gx.

Substitutions and amino acid insertions in PBP3
A total of 65 isolates with MIC 2 mg/L for one or more of three PBP3-targeted cephalosporins were selected for analysis of ftsI gene sequences. Eleven strains (9 strains from Turkey and 1 each from Italy and the USA) encoded a PBP3 protein possessing the YRIN insertion after amino acid P333 and the amino acid substitution I532L; two or three additional substitutions were found in two strains ( Table 1 and Table S1; additional substitutions in other strains). The SNPs E349K and I532L were previously reported in association with the YRIN insertion in PBP3, and these ftsI alleles are suggested to be disseminated by horizontal gene transfer. 3 The nine strains were collected from only one hospital in Turkey, although five medical institutions in that country participated in the SIDERO-WT-2014 surveillance programme (Table S2). Isolates with PBP3 possessing the YRIK or TIPY insertion were not found in this study.

Genetic backgrounds of the strains possessing the YRIN insertion
All nine strains from Turkey belonged to ST1284, whereas two strains from Italy and the USA belonged to ST361 (Table 1). Strains belonging to each sequence type displayed similar b-lactamase genotypes, with all nine strains from Turkey carrying CMY-42,   (Table 1). SNP analysis revealed that quite similar genetic backgrounds were shared among the nine strains collected in Turkey, with eight strains differing by only zero to four SNPs (Table S3). The two strains collected in Italy and the USA were less closely related (268 differences), but there were approximately 32 600 differences in SNPs between strains collected in Turkey and Italy/USA.

Antimicrobial susceptibility of the strains possessing the YRIN insertion and isogenic mutants
All 11 sequenced strains showed reduced susceptibility to cefepime (MIC 4 to .64 mg/L) and ceftolozane/tazobactam (MIC 64 to .64 mg/L); the presence of a CTX-M-15 ESBL in 4 strains undoubtedly contributed to the high cefepime MIC of .64 mg/L that was observed (Table 1). 9 In comparison, the strains remained susceptible (by CLSI breakpoints) to ceftazidime/avibactam and cefiderocol, with MICs ranging from 1 to 4 and 0.25 to 4 mg/L, respectively. 7 The strains were susceptible to meropenem and colistin, with the exception of one strain, SR202171, that was meropenem resistant (MIC 4 mg/L), but all were resistant to ciprofloxacin (MIC .8 mg/L). Isogenic mutants possessing YIRN(K)inserted PBP3 displayed 8-fold reduced susceptibility to several tested cephalosporins, including aztreonam and ceftazidime, and 4-fold reduced susceptibility to ceftazidime/avibactam ( Table 2). No fold change was observed for meropenem.

Discussion
All the identified strains of ST1284 lineage possessing the YRIN insertion from Turkey had a genetically clonal background and were collected from the same hospital, suggesting the occurrence of an outbreak limited to this hospital (Table S2). In addition, the two ST361 lineage strains also had a genetically clonal background, but these strains were isolated from distant countries, Italy and the USA. Although it is only possible to speculate how E. coli strains with YRIN-inserted PBP3 emerged and disseminated in each region, antibiotic pressure caused by inadequate treatment with cephalosporins or other b-lactam antibacterials may have contributed to the dissemination of these particular strains, which also carry several acquired b-lactamases. Strains of ST1284 and ST361 lineages possessing YRIN-inserted PBP3 were previously reported (including as part of NCBI database analysis), but the b-lactamases carried by these strains were consistent with the strains in the current study in only some cases. 1 The YRIN insertion in PBP3 is predicted to have a negative impact on the antibacterial activity of several b-lactam antibacterials, such as cephalosporins and monobactams, that mainly target E. coli PBP3. 1 In addition, since the identified E. coli strains possessing YRIN-inserted PBP3 also acquired multiple b-lactamases, including carbapenemases, the resulting cross-resistance and lack of treatment options would be problematic in the clinical setting. Indeed, in a study of isogenic mutants, the presence of the YRIN insertion appeared to impact susceptibility to several well-used antibacterials. Regarding ceftazidime/avibactam, the MICs of this agent against clinical strains possessing YRIN-inserted PBP3 remained susceptible but were relatively higher (1-4 mg/L) than the MICs obtained against all 1529 E. coli isolates in the SIDERO-WT-2014 study (MIC 50 and MIC 90 0.12 and 0.25 mg/L, respectively). 4 There were only 21 strains among 1529 E. coli isolates that showed a ceftazidime/avibactam MIC of 1 mg/L, including 11 strains possessing YRIN-inserted PBP3 and one VIM-type metallob-lactamase producer (Y. Yamano, Shionogi, unpublished data). Therefore, a four amino acid insertion in PBP3 might be suspected in clinical E. coli isolates with ceftazidime/avibactam MIC of 1 mg/L that do not carry metallo-b-lactamases.
In summary, we analysed the characteristics of E. coli possessing PBP3 substitutions that affect cephalosporin susceptibility using the clinical isolates collected as part of the SIDERO-WT-2014 surveillance study. The similarities in the genetic backgrounds of the strains identified in our study and strains reported by others JAR suggest that some clonal strains of the ST1284 and ST361 lineages have disseminated among multiple regions. Although they remained susceptible to several antibacterials, these strains need to be carefully monitored because of cross-resistance to b-lactam antibacterials caused by the YRIN insertion as well as acquired blactamases.

Limitations
Because the ftsI gene was only sequenced in a subset of E. coli isolates with MICs 2 mg/L for one of three PBP3-targeted cephalosporins, the true prevalence of isolates possessing four amino acid insertions in PBP3 cannot be determined for the SIDERO-WT-2014 surveillance collection.