Forensic genomics of a novel Klebsiella quasipneumoniae type from an NICU in China reveals patterns of genetic diversity, evolution and epidemiology

Data summary: Genome sequences generated in this study are available in NCBI under BioProject ID PRJNA610124. All bioinformatic protocols used to process the genomic data are available at https://github.com/vjlab/KpIIB_ST2727. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 12, 2020. ; https://doi.org/10.1101/2020.03.07.20032706 doi: medRxiv preprint


INTRODUCTION
Species of Klebsiella are widespread in the environment, found in soil and 21 ground-water, as commensal organisms on plants and are carried widely in animal hosts 22 (1). Klebsiella pneumoniae, is recognized as a leading cause of healthcare-associated 23 urinary tract infections, surgical site infections and pneumonia, increasingly threatening 24 neonates and the immunocompromised (2). However, in recent times, an increasing 25 diversity of Klebsiella species have been identified to cause infection in humans (3,4). 26 Genome sequencing has revealed that bacteria historically classified as K. pneumoniae 27 belonged to the K. pneumoniae species complex containing multiple phylogenetically 28 distinct but closely related bacterial species. These include K. pneumoniae (previously 29 classified as phylogroup KpI), K. quasipneumoniae (subsp. quasipneumoniae (KpIIA) 30 and subsp. similipneumoniae (KPIIB)), K. variicola (KPIII) (5), and two strains forming 31 divergent lineages indicating potentially novel species within this species complex (6). 32 Whole genome sequencing (WGS) has shown that the predominant cause of clinical 33 infection worldwide has been due to KpI (5), although there is an increasing trend, and 34 increasing concern, in the detection of severe cases due to the newly described species 35 (7). Different studies report K. quasipneumoniae isolates causing infections resistant to 36 treatments with ceftazidime and other oxyimino-betalactam antibiotics, and presenting 37 carbapenemase (KPC) genes (3,4,(8)(9)(10)(11). 38 Due to the emergence of a greater diversity of Klebsiella causing severe 39 infections, their ability of colonizing environmental niches such as sinks and ventilators 40 in hospitals (12), and increased detection of hybrid strains with drug resistant and 41 hypervirulent phenotypes, especially in regions such as China where the earliest reports . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted March 12, 2020 Here we report a retrospective study of an incident that occurred in March 2017, 48 in the NICU of a major hospital with >3000 beds in China. A neonate patient suffered 49 severe diarrhea and subsequently developed septicemia and died, with Klebsiella isolated 50 as the causative microorganism from a blood sample. During the same period, severe 51 symptoms of diarrhea were found in three other neonate patients as well as an attending 52 NICU staff member, triggering a response to quell the apparent outbreak. In addition to 53 intensified hygiene procedures, the response included MLST and microbiological AMR 54 screening of stool samples from patients (21 neonates)

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. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 12, 2020 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 12, 2020. ; https://doi.org/10.1101/2020.03.07.20032706 doi: medRxiv preprint fragmented and tagged with adapters using a single transposase enzymatic reaction, 95 followed by amplification using an optimized, limited-cycle PCR protocol and indexing.  (T3SS), type 4 (T4SS) or type 6 (T6SS) (24). The presence of genes encoding a type 2 113 secretion system (T2SS) was detected by BLAST version 2.7.1, using as queries the 114 known T2SS genes (25). Phylogenetic analysis was conducted using 3611 Klebsiella 115 genomes downloaded from the NCBI database (downloaded on 20/06/2018) to find the 116 closest related genomes to the novel strains (Data S1). Core genome alignment of 117 Klebsiella genomes (including ST2727 isolates) was generated using Roary version CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted March 12, 2020. ; https://doi.org/10.1101/2020.03.07.20032706 doi: medRxiv preprint using a ML bootstrap analysis with replicates ranging from 10 to >1000 replicates for the 124 different datasets analysed. Pn/Ps<Dn/Ds, is characteristic of positive selection occurring within strains (29). 136 To confirm that the genome datasets contained sufficient genetic change between CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Clinical scenario and diagnosis 148 This study was initiated when outbreak status was called in a NICU due to a fatal  Genomics-based evaluation of the NICU strains 169 To determine the genomic characteristics of the bacteria recovered in this study,  (Table S2). The GC content of all genomes was 57%. Genomic analysis showed the 174 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Phylogenies generated from a core genome of 3611 globally sampled Klebsiella 180 strains (Fig S1, Data S1), including isolates from the present study made evident that the 181 ST2727 strains belong to K. quasipneumoniae subsp. similipneumoniae (KpIIB) (33) 182 (Fig. 1A, Fig S2). The high similarity of the ST2727 strains suggests a clonal population.  (Table S5), 211 whereas, mutations in the remaining genes were either strongly deleterious or were under 212 neutral selection pressure (29). Many of these genes are fundamental to bacterial cell . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted March 12, 2020. ; https://doi.org/10.1101/2020.03.07.20032706 doi: medRxiv preprint clinical impact (37). Genome sequence data revealed that all isolates were wcaG-( Fig   241   2A). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted March 12, 2020. ; https://doi.org/10.1101/2020.03.07.20032706 doi: medRxiv preprint human tissues (38). Fimbriae are an important feature of Klebsiella, and the gene fimH 249 encodes a well-conserved adhesin subunit that is found in around 90% of Klebsiella 250 strains (39). This gene has high mobility within clones through horizontal transfer (39).

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The gene encoding mrkD is associated with "type III" fimbriae, important for adhesion to 252 promote establishment of bacterial biofilms in harsh environments (40).

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In terms of antimicrobial resistance, two genes encoding beta-lactamases were detected 254 (Fig 2A, Table S3): the ubiquitous ampH, responsible for resistance to penicillin G,   (42), and in Klebsiella the type 2 secretion system (T2SS) is the best 259 characterized of these virulence determinants (43). In silico inspection of the genomes 260 showed that all ST2727 isolates collected have a similar T2SS architecture, typically 261 composed of pulS and pulA-pulM (44) (Fig 2B). HlyD, is a diagnostic component of the 262 type 1 secretion system (T1SS), and was detected in the ST2727 strains (Table S5). Other CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted March 12, 2020 The colonization of the neonate gut is a complex process with contributions from the 283 mother's microbiome and environmental sources, that can not easily be disentangled (46-  It is probable that ST2727 is under antibiotic selective pressure due to their 307 prevalence in the hospital environment for the months prior to the events analysed here.

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This is further confirmed with a MKT indicating positive selective pressure ongoing for 309 genes related with efflux systems that are a key part for the transport of toxic substances 310 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted March 12, 2020. ; https://doi.org/10.1101/2020.03.07.20032706 doi: medRxiv preprint as antibiotics. However, further investigation on evolutionary mechanism selecting for 311 resistant genes and genes related with resistance mechanisms would need to be done in 312 order to assert these findings. Previously, estimates of selection pressure on the genes 313 encoding beta-lactamases in the KpI, KpII and KpIII clades have showed neutral 314 selection of these genes (45). In agreement with this, we did not find either of the genes 315 encoding beta-lactamases in ST2727 to be under natural selective pressure.

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Although made highly unlikely, the data in this study cannot rule out a maternal 317 contribution to the Klebsiella carriage, but we note that (i) 12/20 neonates were delivered 318 via Caesarian section (Table 1) where maternal contribution to microflora should be 319 inconsistent and minimal, and that (ii) ST2727 is probably not a dominant sequence type 320 in the community as it has not been sampled previously, despite the hospital having more 321 than 1500 Klebsiella isolates collected from adult patients over 15 years. We suggest that 322 a substantial contribution to carriage comes from the endemic microflora in the NICU, 323 with this specific built environment colonized by a population corresponding to ST2727.

CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.

ETHICAL APPROVAL
This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (China).

ACKNOWLEDGEMENTS
We thank Dr. Jiangning Song for his input in the early stages of this analysis.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 12, 2020. ; https://doi.org/10.1101/2020.03.07.20032706 doi: medRxiv preprint