key: cord-0305765-cig2fjvj
authors: Martin, Melissa J.; Corey, Brendan W.; Sannio, Filomena; Hall, Lindsey R.; MacDonald, Ulrike; Jones, Brendan T.; Mills, Emma G.; Stam, Jason; Maybank, Rosslyn; Kwak, Yoon; Schaufler, Katharina; Becker, Karsten; Hübner, Nils-Olaf; Cresti, Stefania; Tordini, Giacinta; Valassina, Marcello; Cusi, Maria Grazia; Bennett, Jason W.; Russo, Thomas A.; McGann, Patrick T.; Lebreton, Francois; Docquier, Jean-Denis
title: Anatomy of an Extensively Drug Resistant Klebsiella pneumoniae Outbreak in Tuscany, Italy
date: 2021-06-02
journal: bioRxiv
DOI: 10.1101/2021.06.02.446696
sha: 37978a3438df7ecb88a91702dbd8b741079e8b59
doc_id: 305765
cord_uid: cig2fjvj

A protracted outbreak of New Delhi metallo-beta-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae, started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-month period from 76 patients at several health care facilities in South-East Tuscany. All isolates belonged to high-risk clone ST-147 and were typically non-susceptible to all first line antibiotics. Albeit sporadic, resistances to colistin, tigecycline and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic, highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates) and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM–1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncH-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats. Significance Statement Carbapenem-resistant Klebsiella pneumoniae belong to the “critical priority” tier of bacterial pathogens as identified by the World Health Organization. Emerging “high-risk” lineages are responsible for difficult-to-treat, hospital-acquired infections and outbreaks around the globe. By integrating genomic and epidemiological data for isolates collected over 20 months, this study revealed both the high, regional prevalence and the rapid spread, within a single hospital, of K. pneumoniae ST-147 in Italy. Besides resistance to nearly all antibiotics, we showed that this lineage carried a hybrid plasmid harboring a set of biomarker genes previously linked to hypervirulence. Convergence of multidrug resistance and hypervirulence is a major concern and these findings highlight the need for robust, global surveillance to monitor the emergence of high-risk K. pneumoniae.

hybrid plasmid resulted in increased siderophore production but did not confer virulence to the Carbapenem-resistant Klebsiella pneumoniae belong to the "critical priority" tier of bacterial 53 pathogens as identified by the World Health Organization. Emerging "high-risk" lineages are 54 responsible for difficult-to-treat, hospital-acquired infections and outbreaks around the globe. By 55 integrating genomic and epidemiological data for isolates collected over 20 months, this study 56 revealed both the high, regional prevalence and the rapid spread, within a single hospital, of K. 57 pneumoniae ST-147 in Italy. Besides resistance to nearly all antibiotics, we showed that this 58 lineage carried a hybrid plasmid harboring a set of biomarker genes previously linked to 59 hypervirulence. Convergence of multidrug resistance and hypervirulence is a major concern and 60 these findings highlight the need for robust, global surveillance to monitor the emergence of 61 high-risk K. pneumoniae. In recent years, a distinct hypervirulent pathotype (hvKp) has been identified, and is recognized 77 clinically by invasive and disseminated infections, in otherwise healthy individuals, that include 78 meningitis, liver abscesses, and endophthalmitis (2). However, the defining features of Compounding the problem, convergent K. pneumoniae lineages with both virulence and 87 resistance genes have been observed, albeit infrequently (3, 9, 10). Genotypic convergence most 88 frequently occurs when MDR-cKp lineages acquire mobile genetic elements that carry the 89 aforementioned virulence biomarker genes (11). Alarmingly, large hybrid plasmids that harbor In this report, we investigated the emergence, genotypic convergence, phenotypic virulence, as 96 well as the regional and nosocomial spread of a NDM-producing ST-147 clone causing an 97 outbreak in Tuscany, Italy (17). This outbreak caused by an extensively drug-resistant (XDR) K. 98 pneumoniae was first identified in November 2018 in the Tuscany region where a significant 99 increase in reported cases from seven hospitals led to expanded surveillance practices (18, 19 (19)) were monophyletic (node 1) and showed high genetic relatedness (average pairwise 119 distance from nearest neighbor was 5.5 SNPs) (Fig. 1) . 120 When compared to international ST-147 genomes (from both public databases and the MRSN 121 collection) the outbreak clone from Italy most closely resembled (shortest distance was 45 SNPs) 122 isolates collected from the Middle East (ME) between 2005 and 2015 ( Fig. 1 and S1 ). All 123 isolates were predicted to be K-antigen capsular biosynthesis loci K64 and O-antigen type O2 124 variant 1 (O2v1) ( Table S1 ). Further, the isolates from Italy carried a blaNDM-1 metallo-β-125 lactamase gene on an IncFIB-type plasmid (54,064 bp; hereby named pSI0739-NDM) (Fig S2a) 126 and a comparable plasmid (99% identity and 87% coverage) was found in 9 of 14 isolates from 127 the ME (Fig 1) .

Convergence of virulence and resistance genes. 130 Genomic comparisons further identified that, since divergence from the ancestor shared with the 131 ME lineage, the ST-147 clone from Italy acquired a large hybrid IncH-type plasmid (335,489 132 bp), subsequently referred to as pSI0739-ARMA-Vir (Fig S2b) . When compared to public 133 databases, pSI0739-ARMA-Vir was highly related to hybrid plasmids previously characterized loci (rmpADC/rmpA2), and the metabolite transporter, peg-344 ( Fig. 1 and S2b) . 92% of isolates 140 also carry genes encoding the yersiniabactin siderophore system (ybt9 lineage harbored by a 141 chromosomal ICEKp3).

Within the outbreak isolates from Tuscany, the presence or absence of plasmid-bound resistance 143 and virulence genes was used to identify excision events or plasmid loss (labelled A-K) ( Fig. 1   144 and S2b). In four independent instances (A, B, I, and J) a ~ 60 to 90 Kb segment containing the 145 virulence genes was excised from pSI0739-ARMA-Vir ( Fig. 1 and S2b) . Insertion sequences 146 (IS) were often identified at the boundaries of the virulence island, suggesting transposition 147 could play a role in the mosaic structure of these plasmids, as previously proposed (14). Notably, 148 excision event B occurred within the host where (2 out of 5) serial isolates from patient 19 149 eventually lost all virulence genes ( Fig. 1 and S2b) . The armA gene cassette, flanked by IS26 150 elements, was also excised on three separate occasions (D, E and F; the latter as the likely result 151 of in-host evolution within serial isolates from patient 4). By contrast, the complete pSI0739-152 ARMA-Vir was lost in three of the later isolates (July and August 2020; event K); all highly 153 genetically related but from distinct patients ( Fig. 1 and S2b) . Plasmid loss was also observed 154 for pSI0739-NDM (three distinct events D, G, and H) (Fig. S2b) . As expected, susceptibility to 155 the aminoglycosides and/or carbapenems was restored in these isolates (Table S2) . 

Phylogenetic analysis of the isolates from Tuscany revealed the existence of two clades 159 (differing by 32.9 SNPs on average), A and B (Fig. 1) . Clade A was relatively heterogenous 160 (average pairwise distance from nearest neighbor was 11.1 SNPs) and comprised 31 isolates 161 from 23 patients at 3 distinct healthcare facilities in SE Tuscany ( Fig. 1 and Table S1 ). 162 Additionally, clade A includes 9 genomes from a hospital in NW Tuscany for which patient 163 metadata is unknown (19). In contrast, clade B was highly homogeneous (average distance of 2.4 164 SNPs from nearest neighbor) and grouped 86 isolates collected from 57 inpatients from the Siena 165 University Teaching hospital. 166 Temporally, detection of the first clade A isolate from each patient (i.e. removing serial isolates) 167 was relatively stable (1.2 new cases per month on average) across the 20-month sampling period 168 (Fig. 2a) . By contrast, a large increase of new cases due to clade B isolates (7.1 cases per month) 169 was observed over a 6-month period (July to December 2019). Further, the majority (90%) of the cardiology clinic in July and August of 2019, shared genetically identical isolates (Fig. 2bc) . 176 While the last case detected in the cardiology clinic was in September 2019, this clone had 177 seemingly spread throughout the hospital. Indeed, between September 2019 and October 2020, 178 the internal medicine and emergency wards accounted for 75% of primary cases due to a clade B 179 isolate ( Fig. 2bc) . Of note, the apparent reduction in isolates collected from patients between Positive selection on resistance and virulence determinants. 186 In addition to some isolates independently excising regions within or the whole pSI0739-187 ARMA-Vir and pSI0739-NDM plasmids, mutational convergence was also observed within 188 specific genes and functional clusters. The most striking example was gene glpT that encodes the isolates, mostly from clade A (Fig. 3) . This is in agreement with a previous report of glpT 193 mutations found in clinical, epidemic ST-147 isolates that was likely responsible for reduced 194 fosfomycin susceptibility (21). Four distinct NSY mutations in the cusSRA cluster, previously 195 associated with copper and silver, and tigecycline resistance (22), were found in 6 isolates from 196 both clades (Fig. 3) . In addition, the ramR-ramA gene cluster, consisting of the romA-ramA 197 operon and the divergently transcribed ramR (resistance antibiotic multiple protein A), was 198 mutated at 7 distinct occurrences (including 2 predicted LOF in ramR and one in romA) in 20 199 strains (Fig. 3) . Two serial isolates from patient 4 with the same frameshift mutation in ramR 200 demonstrated tigecycline resistance (MIC ≥ 8 mg/L), consistent with previous reports in K. 201 pneumoniae (23) (Fig. S4b and Table S2 ). Notably, isolate 752623 also evolved tigecycline 202 resistance, but the genetic determinants remain unclear ( Table S2) Here, isolate 849820 carried a truncated mgrB (Table S1) but remained susceptible to colistin 210 (MIC < 0.25 mg/L). Positive selection was also observed for genes involved in cell wall 211 synthesis (pbp2, pbp3, rlpA) and glycosylation (pglA), adhesion to mucosal surfaces and 212 virulence (fimH), nitrate respiration (narLXG), and iron binding and transport (sufBDS) (Fig. 3) . 213 The latter was recently reported as a potential factor underlying the persistence of an outbreak 214 involving high-risk ST-258 K. pneumoniae (24).

Fixation of variants through patient transmission and within host evolution. 217 To further identify traits that possibly contributed to the success and spread of the ST-147 clone 218 in hospitals in Tuscany, we identified variants that became fixed in the population at nodes of 219 interest (excluding SYN and intergenic variants) (Fig. S3a) . Using parsimony, we identified 8 220 variants found in all isolates from Italy that were missing in all ME genomes (node 1). Of Fig. S2b ) resulted in siderophore levels comparable to the cKP1 control (Fig. S4a) . 249 However, despite carrying the virulence genes rmpADC and rmpA2, isolates 752019 and 752165 250 showed mucoviscosity levels comparable to the cKP1 control ( Fig. 4b and S4b ). Moreover, a 251 subcutaneous (SQ) model of infection with immunocompetent CD1 mice determined that 752019 252 and 752165 were non-lethal at a challenge inoculum of 10 3 CFU, unlike canonical hypervirulent 253 isolate hvKP2 (Fig. 4c) . Further, no lethality was observed with a 10 5 CFU inoculum of the ST- (Table S3) , resulted in mortality rate of 20% at the highest inoculum (not reaching statistical 260 significance) compared to the cKP1 control (Fig. S5) . East (32), by acquiring a hybrid resistance/virulence plasmid. Furthermore, we also confirm the 270 early concern about cross-border spread (17), as highly genetically related isolates (Fig. S1) , 271 with near identical plasmids (Table S3) 

Bacterial Isolates and Antibiotic Susceptibility Testing. The complete set of XDR K. 337 pneumoniae isolates (Table S1) were collected from the University Teaching Hospital of Siena 338 (Azienda Ospedaliera Universitaria Senese AOUS), Italy, a 700-bed regional health care facility 339 providing microbiological analyses in the frame of a regional surveillance network for several 340 other local healthcare facilities part of the South-East district and long-term healthcare structures. to identify resistance alleles from draft assemblies and processed reads, respectively, followed by 368 deduplication of redundant alleles calls. cgMLST allele assignment and minimum spanning tree 369 generation were performed with SeqSphere (47). 370 We created a SNP phylogeny for ST-147 including the 117 outbreak isolates, 9 previously 371 published genomes (19), and 14 MRSN genomes from ME and Germany (Table S1) (Table S4 ). The core SNP alignment was filtered for recombination using Gubbins To quantify siderophore production, the isolates of interest were grown overnight at 37°C in with 10 3 , 10 5 , 10 7 , or 10 8 CFU of outbreak isolate 752019 or reference isolates hvKP2 and cKP1.

Total n=10 (n=5 in each of 2 independent experiments) for each titer for each strain. 593   Table S1 . Characteristics of all isolates used in this study. 594 Table S2 . Antibiotic susceptibility profiles for a selection of ST-147 isolates. 595   Table S3 . Overview of plasmid relatedness with pSI0739-ARMA-Vir. . 

Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy

Typing Methods, and Pathogenicity Factors

Hypervirulent Klebsiella pneumoniae

Finding Order in the Chaos: Outstanding Questions in Klebsiella 436 pneumoniae Pathogenesis

Risk Klebsiella pneumoniae Clones ST307 and ST147

Population genomics of Klebsiella pneumoniae

A Klebsiella pneumoniae ST307 outbreak clone from Germany demonstrates 443 features of extensive drug resistance, hypermucoviscosity, and enhanced iron acquisition

The Small Protein RmpD Drives 446 Hypermucoviscosity in

Metabolite Transporter PEG344 Is 448 Required for Full Virulence of Hypervirulent Klebsiella pneumoniae Strain hvKP1 after 449 Pulmonary but Not Subcutaneous Challenge

A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae 451 in a Chinese hospital: a molecular epidemiological study. The Lancet Infectious Diseases

Genomic surveillance for hypervirulence and multi-drug resistance in invasive 454 Klebsiella pneumoniae from South and Southeast Asia

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant 456 and virulent clones of Klebsiella pneumoniae

Genomic surveillance framework and global population structure for 458 Klebsiella pneumoniae

Virulence genes in isolates of Klebsiella pneumoniae from the UK during 2016, 461 including among carbapenemase gene-positive hypervirulent K1-ST23 and 'non-hypervirulent' 462 types ST147, ST15 and ST383

Hybrid Resistance and Virulence Plasmids in "High-Risk" Clones of Klebsiella 464 pneumoniae, Including Those Carrying blaNDM-5. Microorganisms

Novel Subclone of Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 466 11 with Enhanced Virulence and Transmissibility

Emergence of Hypervirulent Carbapenem-Resistant Klebsiella 468 pneumoniae Coharboring a bla NDM-1 -Carrying Virulent Plasmid and a bla KPC-2 -Carrying Plasmid 469 in an Egyptian Hospital

European Centre for Disease Prevention and Control, Regional outbreak of New Delhi metallo-471 beta-lactamase-producing carbapenem-resistant Enterobacteriaceae

Prolonged outbreak of New Delhi metallo-beta-lactamase-producing 473 carbapenem-resistant Enterobacterales (NDM-CRE)

Extremely drug-resistant NDM-9-producing ST147 Klebsiella pneumoniae 476 causing infections in Italy

Genetic Plurality of OXA/NDM-Encoding Features Characterized From 478

Characterization of Extensively Drug-Resistant or Pandrug-Resistant 480

Sequence Type 147 and 101 OXA-48-Producing Klebsiella pneumoniae Causing Bloodstream 481 Infections in Patients in an Intensive Care Unit

CusS-CusR Two-Component System Mediates Tigecycline Resistance in 484 Carbapenem-Resistant Klebsiella pneumoniae

Effect of ramR loss-of-function insertion on 486 tigecycline resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae

Evolution of Outbreak-Causing Carbapenem-Resistant Klebsiella pneumoniae 489 ST258 at a Tertiary Care Hospital over 8 Years

Induction kinetics of the 491 Staphylococcus aureus cell wall stress stimulon in response to different cell wall active antibiotics

Identifying virulence determinants of 494 multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

A systematic analysis of hypermucoviscosity and capsule reveals distinct and 497 overlapping genes that impact Klebsiella pneumoniae fitness

The Effects of Methionine Acquisition and Synthesis on Streptococcus 499 Pneumoniae Growth and Virulence

Lipoprotein Contribute to Serum Resistance, Antiphagocytosis, and Proinflammatory Cytokine 502 Stimulation

Identification of Biomarkers for Differentiation of Hypervirulent Klebsiella 504 pneumoniae from Classical K. pneumoniae

An Assessment of Siderophore Production, Mucoviscosity, and Mouse 506

Infection Models for Defining the Virulence Spectrum of Hypervirulent Klebsiella pneumoniae

Antibiotic Resistance and Mobile Genetic Elements in Extensively Drug-509

Resistant Klebsiella pneumoniae Sequence Type 147 Recovered from Germany

Treatment of carbapenem-resistant Klebsiella 512 pneumoniae : the state of the art

Enables the Growth/Survival of Hypervirulent 515 (Hypermucoviscous) Klebsiella pneumoniae Ex Vivo and In Vivo

Aerobactin Mediates Virulence and Accounts for Increased Siderophore 518 Production under Iron-Limiting Conditions by Hypervirulent (Hypermucoviscous) Klebsiella 519 pneumoniae

Increased Relative Abundance of Klebsiella pneumoniae Carbapenemase-524 producing Klebsiella pneumoniae Within the Gut Microbiota Is Associated With Risk of 525

Bloodstream Infection in Long-term Acute Care Hospital Patients

Animal Model To Study Klebsiella pneumoniae Gastrointestinal Colonization 528 and Host-to-Host Transmission

The Cytokine IL-22 Promotes Pathogen Colonization by Suppressing Related 530

Biosynthetic Gene Distribution and Production across Environmental and Faecal Populations of 533 Escherichia coli

A plasmid locus associated with Klebsiella clinical infections encodes a 535 microbiome-dependent gut fitness factor

A Diverse Panel of Clinical Acinetobacter baumannii for Research and 537 Development

Unicycler: Resolving bacterial genome 539 assemblies from short and long sequencing reads

Genomic surveillance framework and global 541 population structure for Klebsiella pneumoniae

Validating the AMRFinder Tool and Resistance Gene Database by Using 544

Antimicrobial Resistance Genotype-Phenotype Correlations in a Collection of Isolates

ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads

Updating benchtop sequencing performance comparison

Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection

Prokka: rapid prokaryotic genome annotation

Rapid phylogenetic analysis of large samples of recombinant bacterial whole 554 genome sequences using Gubbins

RAxML-NG: a fast, scalable and 556 user-friendly tool for maximum likelihood phylogenetic inference

Interactive Tree Of Life (iTOL) v4: recent updates and new developments

A) Fixation of shared variants within the K. pneumoniae ST-147 outbreak strains 616 (nodes 1-4 corresponding to Fig. 1). (B) Stepwise, fixation of variants found in 6 serial isolates 617 from patient 4, clade B. For each gene