LETTER
GES-type extended-spectrum β-lactamases (ESBLs), such as GES-1, mutate by Gly170Asn/Ser substitutions into carbapenem-hydrolyzing variants, of which GES-5 is seen relatively more frequently (1, 2). In some countries GES-5-producing Enterobacterales have occurred repeatedly in clinical cases and/or outbreaks (1, 3–7) and in hospital or aquatic environmental samples (7–10). blaGES-Like genes reside mainly in class 1 integrons found on diverse plasmids or chromosomes in various species and clones (3, 4, 6–8, 10–12). We report a cluster of infant cases with carbapenem-nonsusceptible GES-positive Klebsiella pneumoniae isolates.
(The results were presented in part at the 28th European Congress of Clinical Microbiology and Infectious Diseases, 21 to 24 April 2018, Madrid, Spain [13].)
Between June 2017 and May 2019, a children’s hospital in Krakow, Poland, recorded 11 unique K. pneumoniae clinical/surveillance isolates, flagged as carbapenemase producers by BD Phoenix (BD, Franklin Lakes, NJ) (Table 1). PCR sequencing identified GES-5 in the first 3 isolates and the GES-1 ESBL in 8 others. In March 2019, a GES-5-producing K. pneumoniae isolate colonized a newborn in Bytom, a city ∼100 km from Krakow. Susceptibility testing by EUCAST guidelines (http://www.eucast.org/) showed resistance of GES-5-positive isolates to all carbapenems, whereas GES-1 isolates had varied carbapenem MICs (see Table S1 in the supplemental material). Failure of a mating-out assay but positive electroporation results indicated that GES-5 and GES-1 were encoded by non–self-transmissible plasmids.
Basic clinical epidemiological data for GES-5- and GES-1-producing K. pneumoniae infection/colonization cases
All 12 isolates were sequenced by MiSeq (Illumina, San Diego, CA); contigs were assembled with SPAdes 3.13.2 (14). The first isolate, 4661/17, was also sequenced by MinION (Oxford Nanopore Technologies, Oxford, UK), with hybrid reads assembly by MaSuRCA v.3.2.6 (https://github.com/alekseyzimin/masurca). MLST and core genome MLST (cgMLST), done with BIGSdb (15), revealed that all isolates were ST45 and had 689 identical cgMLST loci. The SNP analysis, utilizing BioNumerics v.7.6.3 (Applied Maths, Sint-Martens-Latem, Belgium) and 4661/17 as a reference, confirmed high genetic homogeneity of the isolates, with 0 to 7 SNPs between the closest relatives (see Fig. S1 in the supplemental material). All K. pneumoniae genomes available at GenBank (n = 8,130 as of 3 December 2019) were screened for ST45 using MLST (https://github.com/tseemann/mlst), which yielded 150 entries. These were aligned against the 4661/17 isolate, inferring SNP-based phylogeny with Parsnp v.1.2 (16). Isolate 4661/17 formed a clade with 31 GES-negative ST45 isolates, recorded mainly and widely in Europe (Fig. 1). The K-type antigen of all Polish isolates was KL24 (https://github.com/katholt/Kaptive).
SNP-based phylogenetic tree of the GES-5-producing isolate 4661/17 (NMI4661_17) and 31 related K. pneumoniae ST45 isolates, constructed using Parsnp software and visualized with the iTOL tool (https://itol.embl.de). The numbers on the right correspond to assembly numbers available in GenBank.
PlasmidFinder 2.0 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) identified four plasmid replicons in all of the study isolates, i.e., IncFIBK, IncP-6, Col(RNAI), and IncQ2, assigned to circular MinION contigs for 4661/17 (see Table S2 in the supplemental material). blaGES-5/-1 Genes within new class 1 In1525 integrons (dfrB1-blaGES-5/-1-blaOXA-10-aacA4) resided on IncP-6 pKRA-GES-5 plasmids (25,010 bp). Compared with the IncP-6 archetype Rms149 (17), pKRA-GES-5 had an ∼100% identical ∼12.7-kb backbone with partitioning, replication, and mobilization loci (see Fig. S2 in the supplemental material). Similar segments were found in a few more recent plasmids, such as pJF-786 with blaGES-5, but in another integron (GenBank accession no. KX912255). ResFinder 3.2 showed more acquired resistance genes, located on other plasmids (Table S2) (18), and resistomes fit well to resistance patterns. However, carbapenem nonsusceptibility of GES-1 isolates was unclear, despite its regular emergence in ESBL producers by porin deficiency (19). Genes of OmpK36 and OmpK35 porins in the isolates were neither disrupted nor had premature stop codons, but their functionality has not been assessed.
This is one of few reports on carbapenem-resistant Enterobacterales isolates in children in Poland, which surprisingly were GES-5/-1 producers and have still not been recorded in adults. All isolates were of the same genotype, a European-spread K. pneumoniae ST45 sublineage, but with unique blaGES-5/-1 genetic platforms. The temporal replacement of GES-5 isolates by GES-1-positive isolates in Krakow is confusing, considering that reverse GES mutation is unlikely. It might be assumed that a carbapenem-susceptible K. pneumoniae ST45 GES-1 variant has spread in regional pediatric centers, repeatedly segregating resistant variants by GES-1 to GES-5 convergent evolution or unknown auxiliary mechanisms. The epidemiology of GES producers is not completely known, and their prevalence or transmission seems to be underestimated. Broader implementation of genomics in high-quality surveillance may unveil complex and still hidden phenomena (7).
Accession number(s).The nucleotide sequences of the pKRA-GES-5 plasmid, K. pneumoniae 4661_17 chromosome, and pKRA_Col_RNAI, pKRA_IncFIB, and pKRA_IncQ2-like plasmids have been submitted to the GenBank nucleotide sequence database and assigned accession numbers MN436715 and CP050455 to CP050458, respectively.
ACKNOWLEDGMENTS
We thank Anna Baraniak for helpful discussion, Małgorzata Herda and Katarzyna Malinowska for excellent assistance, and Thomas Jové for consultation on the integron analysis.
The study was supported by grants SPUB MIKROBANK from the Polish Ministry of Science and Higher Education, Narodowy Program Ochrony Antybiotyków from the Polish Ministry of Health, and DS-4.47/2018-19 from the National Medicines Institute.
FOOTNOTES
- Accepted manuscript posted online 6 July 2020.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
