Genomic analysis reveals the presence of emerging pathogenic Klebsiella lineages aboard the International Space Station

ABSTRACT Klebsiella species, such as Klebsiella pneumoniae, Klebsiella aerogenes, and Klebsiella quasipneumoniae, are opportunistic pathogens that commonly cause infections in humans. Hypervirulent Klebsiella pneumoniae (hvKP) is a subgroup of K. pneumoniae, which has gained attention due to its global dissemination, its capacity to cause invasive infections in community settings among immunocompetent individuals, and its escalating antibiotic resistance levels. Our study presents the first comprehensive phenotypic and genotypic analysis including mobile genetic elements (MGEs) of Klebsiella isolates from the International Space Station (ISS). The genomes of K. pneumoniae, K. aerogenes, and K. quasipneumoniae offered insights into their antimicrobial resistance, virulence, thermotolerance, disinfectant resistance, and MGEs. All isolates were part of emerging pathogenic lineages with K. quasipneumoniae ST138 presenting spatial and temporal persistence aboard the ISS, possibly due to its genotypic profile encoding for numerous resistance genes to disinfectants and heavy metals. We also report on the isolation of a yersiniabactin-encoding K. pneumoniae, belonging to the emerging high-risk ST101 clone, aboard the ISS. A potential dissemination of hvKp strains on ISS might pose a risk to the immunocompromised crew members. Moreover, MGEs containing virulent loci could enable horizontal gene transfer to other benign microorganisms on the ISS, possibly enhancing their virulence potential. Additionally, some Klebsiella genomes exhibited genetic divergence from their respective lineages, which we hypothesize results from the unique spaceflight associated environmental pressures. These findings underscore the need to monitor microbial communities in space to comprehend their survival mechanisms and implications for human health. IMPORTANCE The International Space Station (ISS) is a unique, hermetically sealed environment, subject to environmental pressures not encountered on Earth, including microgravity and radiation (cosmic ionising/UV). While bacteria's adaptability during spaceflight remains elusive, recent research suggests that it may be species and even clone-specific. Considering the documented spaceflight-induced suppression of the human immune system, a deper understanding of the genomics of potential human pathogens in space could shed light on species and lineages of medical astromicrobiological significance. In this study, we used hybrid assembly methods and comparative genomics to deliver a comprehensive genomic characterization of 10 Klebsiella isolates retrieved from the ISS. Our analysis unveiled that Klebsiella quasipneumoniae ST138 demonstrates both spatial and temporal persistence aboard the ISS, showing evidence of genomic divergence from its Earth-based ST138 lineage. Moreover, we characterized plasmids from Klebsiella species of ISS origin, which harbored genes for disinfectant resistance and enhanced thermotolerance, suggestin possible adaptive advantages. Furthermore, we identified a mobile genetic element containing a hypervirulence-associated locus belonging to a Klebsiella pneumoniae isolate of the “high-risk” ST101 clone. Our work provides insights into the adaptability and persistence of Klebsiella species during spaceflight, highlighting the importance of understanding the dynamics of potential pathogenic bacteria in such environments.

Resistive Exercise Device (ARED), and Lab 3 of the ISS.Using culture-independent methods, it has been reported that K. pneumoniae can be identified in multiple locations on the ISS, with documented succession over time (22,23).K. pneumoniae exhibited noticeable persistence between different flights among propidium monoazide-treated surface samples.Additionally, metabolic modeling indicated that Klebsiella species play a key role in the ISS microbiome, benefiting co-existing microorganisms (e.g., Pantoea species).It was also found that K. pneumoniae displays parasitic interactions with Aspergillus species and amensalistic interactions with Penicillium species.The overall importance of understanding the genomic background, evolution, and persis tence of potentially pathogenic microorganisms during spaceflight, in terms of medical astromicrobiology, has also been recently discussed (24).
The aim of this research communication is twofold: first, to generate complete genomes and circular chromosomal maps of the 10 Klebsiella strains isolated from the ISS, investigating their antimicrobial resistance, virulence, thermotolerance, heavy metal resistance, and MGEs and would provide the first complete Klebsiella plasmids from the ISS.Secondly, to employ large-scale population genomics to understand the evolution ary paths and potential divergences of the ISS Klebsiella isolates from the Earth-based lineages.

Antimicrobial susceptibility testing
As expected, the Klebsiella isolates (n=10) exhibited resistance to ampicillin and rifampicin.Among the strains, three displayed resistance to a second-generation cephamycin (cefoxitin), five exhibited intermediate susceptibility to nalidixic acid, two to aminoglycosides (kanamycin), and one to sulphonamides (Table S1).

Assembly statistics
Using hybrid assembly methods with data from both Illumina and ONT sequencing platforms, circular plasmid and closed chromosomal genomes were possible for all isolates.The assembly statistics are provided in Table S2.A direct genomic comparison of the chromosomes of all 10 ISS Klebsiella strains is shown in Fig. 1A.

Comparative genomics of K. pneumoniae ST101
The K. pneumoniae ST101 is a globally distributed clone with over 696 isolates reported in over 33 countries on the Pathogenwatch genomic surveillance database.K. pneumo niae ST101 is widely associated with hypervirulent pathotypes.Overall, the ST101 lineage genomes exhibited high genetic similarity (ANI > 99.4%; Fig. 2A) and are predominantly associated with Europe and South-East Asia (Fig. 2B).F3-2P (2), the strain from the ISS, is most closely related to ERR985162, a genome retrieved from sewage wastewater in the UK in 2014, with an ANI of 99.86%.Phylogenetic analysis revealed that F3-2P (2) belongs to a distinct sub-clade within the ST101 lineage, along with 41 other genomes (Fig. 2C).Out of the 696 K. pneumoniae ST101 isolates reported worldwide, 87.07%(606/696) were found to carry the ybt gene cluster, which is a determinant of virulence associated with the presence of yersiniabactin, suggesting that yersiniabactin plays a key role in the pathogenicity of this clone.Among these isolates, only nine encoded for additional siderophore virulence factors such as colibactin or aerobactin.Moreover, 98.2% (595/606) of the ybt-positive genomes encoded for ybt9, as part of a horizontally acquired ICEKp3.The integrative conjugative element ICEKp5, which was identified in the ISS isolate F3-2P (2) and carries the ybt14 locus, is a rarity, identified in only four other ST101 genomes.

Comparative genomics of K. aerogenes ST103
The genomes of the K. aerogenes ST103 isolates (n = 7), including the ISS strain IIIF7SW-P1, were found to be highly related with an ANI > 99.8% (Fig. 3A).The associated ST103 isolates were geographically confined, with isolates obtained from human hosts in the USA (n = 4) and Japan (n = 1) (Fig. 3B).The genetically most akin to strain IIIF7SW-P1 was SRR5666484, which was isolated from a human with a urinary tract infection in the USA in 2015 (ANI = 99.97%).Based on core-genome phylogeny, IIIF7SW-P1 formed a distinct node within the ST103 clone along with uropathogenic strain SRR5666484.

Comparative genomics of K. quasipneumoniae ST3234
K. quasipneumoniae ST3234 is a seldom encountered clone with only two genomes listed in the Pathogenwatch database.The genomes of K. quasipneumoniae IIIF3SW-P1 and those of two strains from Oxford, England were found to be highly similar, with an ANI > 99.8% (Fig. 4A).The two UK strains were isolated from cases of invasive bloodstream infections (Fig. 4B).When analyzed using core-genome phylogenetics, IIIF3SW-P1 was part of a separate node from the two strains from Oxford (Fig. 4C).

Comparative genomics of K. quasipneumoniae ST138
The K. quasipneumoniae ST138 lineage exhibits a broader spatial distribution and higher genetic diversity compared to ST3234, with ANI > 99.3% (Fig. 5A and B).The genome most closely related to the seven ISS K. quasipneumoniae ST138 isolates was that of ERR4635453, isolated from a human in Tacloban city, Philippines in 2017, with ANI = 99.77%.The ISS strains (n = 7) formed a distinct clade within the ST138 lineage based on core genome phylogeny, indicating potential evolutionary divergence from the main ST138 lineage.

Antimicrobial resistance genes
The ISS strains (n = 10) were found to each carry an average of 22.70 ± 0.64 antimicro bial-resistance genes (ARGs) (Fig. 6A).Among the genomes, K. pneumoniae F3-2P(2) (ST101) was identified to carry the β-lactamase gene blaSHV-1, which is known to confer resistance to certain penicillin and first-generation cephalosporin antibiotics (Fig. 6B).In the K. quasipneumoniae genomes of ST138, the chromosomal blaOKP-B-8 was identified.The genome of K. quasipneumoniae strain IIIF3SW-P1 (ST3234) was found to carry a blaOKP-B-3 allelic variant.The OKP-B chromosomal β-lactamase group is known to confer resistance mainly to aminopenicillins and carboxypenicillins and remains susceptible to the presence of inhibitors such as clavulanic acid (25).K. aerogenes strain IIIF7SW-P1 (ST103) had three missense mutations in the outer membrane protein encoding ompK gene, which are linked with increased resistance to cephalosporins and cephamycins explain the observed phenotypic resistance to cefoxitin (Table S5).
Furthermore, all 10 ISS Klebsiella genomes encoded for the virulence-associated enterotoxin (entA/B/C/E/F).K. quasipneumoniae strain IIIF3SW-P1 (ST3234) encoded for the enterotoxin entD and wcaG/H/I/J, a gene cluster involved in the biosynthesis of the outer core lipopolysaccharide.K. aerogenes strain IIIF7SW-P1 was the only strain identified to contain the flgH/G/M/N genes, which encode for regions of the bacterial flagellar switch protein (Fig. 6D).

DISCUSSION
This study highlights the presence of Klebsiella isolates associated with human patho genic lineages.It also reports on the first identification of a yersiniabactin-encoding K. pneumoniae and associated Klebsiella plasmids aboard the ISS.Of note, isolate F3-2P (2) belongs to the emerging nosocomial high-risk clone ST101, which is linked with carbapenem and colistin resistance and exhibits a hvKp pathotype (26,27).The ST101 clone has been implicated in numerous outbreaks of bloodstream infections worldwide, including in southern Italy and India (28,29).With isolates reported from over 33 countries, ST101 poses a persistent public health concern and ST101 induced infections have been associated with 11% increased mortality compared to non-ST101 infections genomes.(C) Core-genome phylogenetic relatedness of the K. quasipneumoniae ST3234 genomes.For more details, an interactive dashboard is available here.(30).Widely disseminated, high-risk clones like ST101 are known to be able to hori zontally acquire hypervirulence gene clusters, potentially precipitating the emergence of hypervirulent clonal lineages (31).In line with this, our analysis suggests that the predominant virulence factor associated with ST101 is yersiniabactin, as part of a horizontally acquired and self-mobilizable ICEKp.
K. pneumoniae strain F3-2P (2) was identified to encode for 75 VF genetic determi nants, including the siderophore genes (irp1/2), the enterobactin gene cluster, and the yersiniabactin (ybt-14) gene cluster as part of a self-mobilizable ICEKp5.K. pneumoniae clinical isolates harboring yersiniabactin on ICEKp5 are known to prevail in intensive care units and tertiary hospital settings and are linked with invasive infections (32)(33)(34).Among the 696 ST101 genomes available on the Pathogenwatch surveillance database, only four esxhibited the same ICEKp5 harboring the ybt-14 gene cluster.These strains were distributed widely in different countries such as the USA, China, and the Philippines.The mode of transportation of a potential pathogen such as K. pneumoniae F3-2P (2) to the ISS could be through cargo or crew, although relevant crew data were not available as NASA does not mandate the monitoring of microbial diversity.Similar to screening and surveillance protocols in clinical settings, targeted screening of cargo and crew for potential opportunistic pathogens could mitigate any putative pathogen-associated risks.
The genome of K. pneumoniae strain F3-2P (2) also revealed the presence of an IncFIIK conjugative plasmid containing several heavy-metal resistance and thermoresistance genes.Although ESBL CTX-M-15-encoding plasmids are commonly associated with the FIIK-9 replicon, the plasmid of F3-2P (2) lacked ESBLs likely due to the absence of antibiotic-induced selective pressure (35).This strain was isolated from the WHC of the ISS.The role of the WHC is the removal and containment of human solid waste and urine.While it is unclear if the strain originated from the ISS crew or cargo, K. pneumoniae strains have been reported to survive on surfaces for up to 6 weeks, with the longest survival rates observed for stainless steel (36,37).When a uman infection is established with hvKp isolates, while both enterobactin and yersiniabactin virulence loci are present, it has been demonstrated that could result in multi-site infection due to metastatic spread (38).Furthermore, the hypervirulent, multimetal resistance, and thermotolerant genomic profile of the K. pneumoniae F3-2P (2) strain could possibly allow it to evade the existing disinfecting protocols aboard the ISS.Further work is required to clarify the phenotypic translation of these genotypic traits.The presence of the resistance and hypervirulence determinants in MGEs further poses a consideration for their dissemina tion across the ISS microbiome.
The persistence of K. quasipneumoniae ST138 in the ISS environment was evidenced by the isolation of seven strains over 18 months from three different locations (Per manent Multipurpose Module, Cupola, and WHC).Despite the spatial and temporal distribution of their collection, the ST138 strains showed remarkable genetic similar ity, suggesting the possible propagation of a single clone in the closed system for a prolonged period.The genomes of the ST138 isolates shared several chromosomal genetic determinants of resistace, including these encoding for resistance to quater nary ammonium compounds (QACs), peroxide, fosmidomycin, methyl viologen, tellurite, nickel, cobalt, zinc, and the multiple stress-resistance protein BhsA encoded by bhsA.These determinants may confer adaptability advantages to the ST138 lineage during spaceflight, given most cleaning agents and disinfectants contain several of these heavy metals and QACs.On Earth, K. quasipneumoniae ST138 strains have been isolated from untreated and treated water in a wastewater treatment plant in Slovenia (38).ESBL cefotaximase (CTXM-9)-producing strains of K. quasipneumoniae ST138 have also been reported to cause community-onset infections in China (38).Overall, K. quasipneumoniae ST138 shows temporal and spatial persistence within the ISS environment, more than any other Klebsiella lineage.The ISS K. quasipneumoniae ST138 strains lacked any MGEs and, therefore, their adaptability advantage is likely ntrinsic.Further research is required to elucidate the properties of this lineage, which allow it to adapt and propagate to the high-pressure environment of the ISS.
K. quasipneumoniae subsp.similipneumoniae strain IIIF3SW-P1 was the only ST3234 strain identified on ISS, and it was isolated from the crew exercise platform (ARED).
There is not much known about the dissemination and persistence of K. quasipneumo niae ST3234 as only two isolates have been reported on surveillance databases.Both Earthly strains were associated with invasive bloodstream infections in humans and were isolated at John Radcliffe Hospital in Oxford, UK between 2009 and 2012.
K. aerogenes strain IIIF7SW-P1 (ST103) was isolated from the ISS Lab3 in 2015.This strain belongs to the globally rare K. aerogenes ST103 lineage, with only five strains deposited in the Pathogenwatch database, all of which are associated with UTIs and reported in the USA.K. aerogenes IIIF7SW-P1 presented phenotypic resistance to ampicillin and the second generation cephamycin cefoxitin as expected, given the intrinsic resistance of K. aerogenes due to the presence of a chromosomal ampC.The isolate was susceptible to the third generation cephalosporin cefpodoxime and to the cefpodoxime/clavulanic acid combination tested.This is in liaison with the litera ture supporting that only overproduction of AmpC is linked with resistance to third generation cephalosporins (39).Interestingly, IIIF7SW-P1 harbored two uncharacterized plasmids, one of which (pIIF7SW-P1_1) was predicted to be mobilizable and contained the pilL gene responsible for thin pilus biogenesis (40).PilL is known to promote bacterial adhesion and colonization, providing a selective advantage in the high-selective-pres sure environment of the ISS.The second plasmid (pIIF7SW-P1_2) was a high copy plasmid, harboring the heat shock gene htrC, which has been linked with increased thermoresistance in E. coli (41).The genetic makeup of IIIF7SW-P1 suggests that it may have acquired plasmids enabling it to survive and persist in the challenging conditions of the ISS, which warrants further investigation.
In sumary, this study provides a first complete genomic characterization of Klebsiella strains isolated from the ISS .This study reports on the genomic makeup of 10 Klebsiella isolates, revealing an abundance of heavy metal and QAC-resistance genes amongst the genomes, similar to the ones seen in clinical settings where the use of disinfectants is intense (42).However, clinically relevant ESBLs and carbapenemase-producing genes were notably absent.This study reports on the isolation of a yersiniabactin-encoding K. pneumoniae from a human-associated pathogenic lineage aboard the ISS as well as on the persistence and possible genomic divergence of K. quasipneumoniae ST138 isolates.The prevalence and seemingly adaptability of K. quasipneumoniae ST138 in spaceflight conditions should be considered for further investigation.Additional epidemiological studies are needed to map and understand the prevalent Klebsiella lineages that could persist during prolonged spaceflight.Ongoing surveillance of potentially problematic STs is feasible on the ISS due to its small crew size and could help mitigate associated risks.These efforts will aid in developing suitable countermeasures for eradicating potentially problematic pathogens in closed habitats for future human missions to the Moon, Mars, and beyond.

Isolation of the strains from the ISS
Various locations were sampled on the ISS using polyester wipes, and the metadata associated with the samples and their collections were published elsewhere (43).Briefly, each wipe was aseptically removed from the zip lock bag and transferred to a 500 mL bottle containing 200 mL of sterile phosphate-buffered saline (pH 7.4) and concentrated with a concentrating pipette (Innova Prep, Drexel, MO, USA) using a 0.22 µm Hollow Fiber Polysulfone tips (Cat #: CC08022).Suitable aliquots (100 µL) of each sample were plated on Reasoner's 2A agar in duplicate and incubated at 25°C for 7 days, and well-matured colonies were picked, archived in semisolid R2A (agar media diluted 1:10), and stored at room temperature.A loopful of purified microbial culture was directly subjected to PCR.The targeted fragment was amplified (colony PCR) to amplify the 1.5 kb 16S rRNA gene to identify the bacterial strains.The following primers were used for the 16S rRNA gene amplification: the forward primer, 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′), and the reverse primer, 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) (44,45).The PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 35 cycles consisting of denaturation at 95°C for 50 s, annealing at 55°C for 50 s, and extension at 72°C for 1 min 30 s and finalized by extension at 72°C for 10 min.The sequencing was performed using 27F and 1492R primers, and the sequences were assembled using SeqMan Pro from DNAStar Lasergene Package (DNASTAR Inc., Madison, WI, USA).The bacterial sequen ces were searched against EzTaxon-e database (46) and the initial identification was based on the closest percentage similarity (>97%) to previously identified microbial type strains.

DNA extraction and sequencing
Cultures of the 10 Klebsiella strains were grown overnight on MacConkey agar plates at 37°C.DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions, with no pre-enrichment steps.The extracted DNA was quantified using a Qubit fluorometer (Thermo-Fisher Scientific, USA) and the dsDNA High Sensitivity kit (Thermo-Fisher Scientific, USA).DNA purity was evaluated by measuring the absorbance ratio at 260 and 280 nm (A260/280) on a Jenway Genova Nano Micro-volume Spectrophotometer (ThermoFisher Scientific, USA).For sequencing, DNA samples with a suitable quantity (>12.5 ng/µL) and purity (A260/280 of 1.80-2.00)were selected for downstream processing.Illumina NovaSeq 6000 sequencing was performed on these samples at Oxford's Genomics Centre (PE150).In addition, long-read sequencing for the DNA of all 10 isolates was carried out using an Oxford MinION Mk1C platform with a R9.4.1 flow cell (Oxford, UK).

Antimicrobial susceptibility testing
Antimicrobial susceptibilities for the 10 Klebsiella spp.isolates were determined by the Kirby-Bauer disc diffusion method for a range of 8 β-lactam and 10 non-β-lactam antibiotics as previously described (47) (Table S6).The diffusion test was performed on Mueller Hinton agar as a culture medium.Classification into susceptibility categories was determined using the clinical breakpoints provided for Enterobacterales defined by the European Committee on Antimicrobial Susceptibility Testing (v.13.0, 2023) (48).For nalidixic acid and kanamycin, breakpoints provided by the Clinical and Labora tory Standards Institute (30th edition) were applied (49).K. pneumoniae ATCC 700603, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were utilized as quality controls.

Large scale comparative genomics
To compare the ISS Klebsiella genomes with Earthly analogs of the same ST, all available genomes from the STs identified for the ISS strains were retrieved from the Pathogen watch database (Table S6).Their assemblies were annotated using PROKKA (v1.14.5) (60).A pan-genome analysis for each ST, which included the corresponding ISS strains and the species typing strain, was conducted using roary (v3.11.2) (61).Core genome align ment was determined using mafft (v7.47.1) (62).Phylogenetic analysis was performed to infer the evolutionary relationships among the core gene sequences with RAxML (v.8.2.12) using the General Time Reversible-gamma model with rapid bootstrapping of 200 bootstrapping replicates (63).The presence-absence results from the pan-genome analysis and the phylogenetic tree generated were visualized using microreact (64).Data were also visualized using the R programming language.
Phylogenetic trees displaying branch distances and bootstrap support values are presented in Fig. S2.

Hypervirulence multiplex PCR assay
An in-house developed and validated multiplex PCR assay, designed to detect hyperviru lence in clinical K. pneumoniae isoalates, was used to verify the presence of yersiniabactin identified in strain F3-2P (2).The assay targets the ybtQ gene of the yersiniabactin gene cluster, the iucA gene of the aerobactin gene cluster, and the 16SrRNA gene as a positive control (Table S7).The multiplex PCR was conducted on a SensQuest Thermocycler with the following conditions: 2 min at 50°C, 10 min at 95°C (15 s at 95°C, 1 min at 60°C) × 35.

ACKNOWLEDGMENTS
Part of the research described in this manuscript was performed at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with NASA.
We would like to thank Aleksandra Checinska-Sielaff for isolating the strains.We thank astronaut Captain Terry Virts for collecting samples aboard the ISS and the Implementation Team at NASA Ames Research Center (Fathi Karouia) for coordinating this effort.We also acknowledge the Jet Propulsion Laboratory supercomputing facility staff, notably Narendra J. (Jimmy) Patel and Edward Villanueva, for their continuous support in providing the best possible infrastructure for BIG-DATA analyses.Government sponsorship acknowledged.
The research described in this manuscript was funded by a 2012 Space Biology NNH12ZTT001N grant no.19-12829-26 under Task Order NNN13D111T award to K.V. Financial support for this research was also provided by a research grant from the University of Galway's School of Medicine: 2021_ECRAward, awarded to G.M. K.V., G.M., and N.K.S. conceived and designed the experiments.A.T. designed and conducted the multiplex PCR assay.L.O'C. and G.M. generated genomic libraries and conducted the long-read sequencing.G.M. and F.MD. conducted the microbiological phenotypic characterization.G.M., K.V., N.K.S., and F.MD. conducted the genomic analysis.G.M. generated the figures, and the manuscript was compiled by all authors.The authors have read and agreed to the published version of the manuscript.
This manuscript was prepared as an account of work sponsored by NASA, an agency of the US Government.The US Government, NASA, California Institute of Technology, Jet Propulsion Laboratory, and their employees make no warranty, expressed or implied, or assume any liability or responsibility for the accuracy, completeness, or usefulness of information, apparatus, product, or process disclosed in this manuscript, or represent that its use would not infringe upon privately held rights.The use of and references to any commercial product, process, or service does not necessarily constitute or imply endorsement, recommendation, or favoring by the US Government, NASA, California Institute of Technology, or Jet Propulsion Laboratory.Views and opinions presented herein by the authors of this manuscript do not necessarily reflect those of the US Government, NASA, California Institute of Technology, or Jet Propulsion Laboratory, and shall not be used for advertisements or product endorsements.

AUTHOR AFFILIATIONS
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FIG 5 FIG 6
FIG 5 Phylogenetic relationships within K. quasipneumoniae ST138.(A) ANI between the genome of K. quasipneumoniae ST138 ISS strains (n = 7), the genome of K. quasipneumoniae typing strain 07A044, and all K. quasipneumoniae genomes of ST138 (n = 20).(B) Temporal dissemination of the K. quasipneumoniae ST138 genomes.(C) Core-genome phylogenetic relatedness of the K. quasipneumoniae ST138 genomes.The clade of the ISS isolates is highlighted.For more details, an interactive dashboard is available here.