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Geographical distribution, disease association and diversity of Klebsiella pneumoniae

KL and O antigens in India: roadmap for vaccine development

Varun Shamanna1,2,*, Srikanth Srinivas1, Natacha Couto3, Geetha Nagaraj1, Shyama Prasad
Sajankila2, Harshitha Gangaiah Krishnappa1, Kavitha Arakalgud Kumar1, David Aanensen3,
Kadahalli Lingegowda Ravikumar1, GHRU India Consortium

1
Central Research Laboratory, KIMS, Bengaluru, India
2
Department of Biotechnology, NMAM Institute of Technology, Nitte, Udupi, India
3
Centre for Genomic Pathogen Surveillance, Big Data Institute, University of Oxford,
Oxford, United Kingdom

*
Corresponding Author: Varun Shamanna (varunshamanna4@gmail.com)

Keywords: K-Locus types, O antigen, Serotypes, K. pneumoniae, India, WGS, vaccine

Repositories: All the WGS data has been submitted to ENA under the Bioproject numbers
PRJEB29740 and PRJEB50614. All the R scripts used in the study have been deposited in
Figshare (https://doi.org/10.6084/m9.figshare.25414807.v1)
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

Abstract

Klebsiella pneumoniae poses a significant healthcare challenge due to its multidrug resistance
and diverse serotype landscape. This study aimed to explore the serotype diversity of 1072 K.
pneumoniae and its association with geographical distribution, disease severity and
antimicrobial/virulence patterns in India. Whole-genome sequencing was performed on the
Illumina platform, and genomic analysis was carried out using the Kleborate tool. KL64
(n=264/1072, 26%), KL51 (249/1072, 24%), KL2 (n=88/1072, 8%), O1/O2v1 (n=471/1072,
44%), O1/O2v2 (n=353/1072, 33%), and OL101 (n=66/1072, 6%) were the most prevalent
serotypes. The study identified 119 different sequence types (STs) with varying serotypes,
with KL64 being the most predominant in ST231 (26%). O serotypes were strongly linked
with STs, with O1/O2v1 predominantly associated with ST231 (44%). Simpson's diversity
index and Fisher’s exact test revealed higher serotype diversity in the north and east regions,
along with intriguing associations between specific serotypes and resistance profiles. No
significant association between KL or O types and disease severity was observed.
Furthermore, we found no specific association of virulence factors with KL types or O
antigen types (P>0.05). Conventionally described hypervirulent clones (i.e., KL1 and KL2) in
India lacked typical virulent markers (i.e., aerobactin), contrasting with other regional
serotypes. The cumulative distribution of KL and O serotypes suggests that future vaccines
may have to include either ~20 KL types or 4 O types to cover >85% of the
carbapenemase-producing Indian K. pneumoniae population. The findings underscore the
need for a vaccine with broad coverage to address the diverse landscape of K. pneumoniae
strains in different regions of India. Understanding regional serotype dynamics is pivotal for
targeted surveillance, interventions, and tailored vaccine strategies to tackle the diverse
landscape of K. pneumoniae infections across India.
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

Data Summary

● All the sequenced data has been submitted to the European Nucleotide Archive
(ENA) under the Bioproject numbers PRJEB29740 and PRJEB50614. Run
Accessions and Biosample numbers are provided in Supplementary Table 1 with
corresponding metadata for each sample used in the study.
● The Microreact link for the genomic analysis is provided
(https://microreact.org/project/oqKM84GBszEPW9Emt2FKnP-klebsiella-pneumonia
e-indian-serotypes).
● The pipelines used in the study are published in gitlab
(https://gitlab.com/cgps/ghru/pipelines).
● The tools’ details and the implementation of the pipelines are described in
protocols.io
(https://www.protocols.io/view/ghru-genomic-surveillance-of-antimicrobial-resista-bp
2l6b11kgqe/v4).
● The R scripts used with all the input files used for each script have been published in
Fishare (https://doi.org/10.6084/m9.figshare.25414807.v1)

Impact Statement

Klebsiella pneumoniae produces polysaccharide capsules, which serve as both

epidemiological markers and significant virulence factors. The increasing accessibility of
whole genome sequencing has made it easier than ever to investigate this capsule diversity.
This study is the first of its kind in India to comprehensively investigate the serotype
diversity of K. pneumoniae strains and their association with disease severity, antimicrobial
resistance/virulence patterns, and geographical distribution across various regions of the
subcontinent. This multi-dimensional analysis not only provides valuable insights into the
molecular epidemiology of K. pneumoniae in India but also offers crucial data for the
development of targeted interventions, including vaccine formulations tailored to address the
prevailing serotypes. These findings serve as a foundation for informed decision-making in
the management and prevention of K. pneumoniae infections, ultimately contributing to
improved public health outcomes in the region.
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

Introduction

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative bacterium that is a major cause

of nosocomial infections [1], including pneumonia, sepsis, and urinary tract infections [2];
[3]. Its increasing resistance to antimicrobials poses a serious public health threat [4].
Genome-based surveillance is urgently needed to control the emerging threat of K.
pneumoniae [5]. Recent advances in understanding the population structure of K.
pneumoniae have revealed an immense genomic diversity, providing a framework for
pathogen tracking [6]; [7]. The emergence of multidrug-resistant (MDR) K. pneumoniae
strains, resistant to multiple antimicrobials, is a significant concern, especially in countries
like India, where treatment becomes challenging. [8]; [9]; [10]; [11]. MDR hypervirulent K.
pneumoniae (hvKp) strains have been emerging, spawning a new generation of hypervirulent
"superbugs" [12]. Newer therapeutics, such as vaccines or monoclonal antibodies, are needed
to combat MDR K. pneumoniae. Vaccines could help to prevent infections from occurring in
the first place and/or they could help to reduce the severity of infections in affected
individuals. Human monoclonal antibodies, on the other hand, can rapidly progress to
innovative prophylactic and therapeutic solutions (Roscioli et al., 2024).

Capsular polysaccharides (CPS, encoded by the K locus) and lipopolysaccharides (LPS,

encoded by the O locus) are major virulence factors of K. pneumoniae, and they are
responsible for protecting the bacterium from the host's immune system. Bacterial
capsule-targeted vaccines, such as those designed for Streptococcus pneumoniae, Neisseria
meningitidis, and Haemophilus influenzae, have demonstrated significant effectiveness in
preventing illnesses caused by these encapsulated pathogens [13]. Currently, efforts are in
place to develop vaccines or monoclonal antibodies against K. pneumoniae. The increased
immunogenicity and enhanced surface exposure of CPS and LPS in K. pneumoniae render
them appealing candidates [13–15]. A bioconjugation approach based on glycoengineered
Escherichia coli expressing K. pneumoniae KL1 and KL2 antigens led to the production of
IgG against both glycans in mice conferring protection against lethal challenges with KL1
and KL2 strains [16]. Affinivax is working on an LPS-based formulation that combines
antigens O1, O2, O3, and O5 with the type III fimbriae adhesion MrkA to generate a
multiple-antigen presenting system (MAPS) [17]. However, the wide range of CPS and LPS
types makes it challenging to provide comprehensive coverage [18]. The structural
variability, defective capsule or O-antigen production, and variations in the geographic
distribution of serotypes limit the potential coverage of CPS/LPS-based vaccines or
monoclonal antibodies [19].

There are over 80 capsular serotypes (encoded by the K-loci) of K. pneumoniae [20]; [21],
and the prevalence of these serotypes varies from region to region. Understanding the
distribution of K. pneumoniae serotypes is important for vaccine and monoclonal antibodies
development, as a vaccine targeting the most prevalent serotypes in a given region is more
likely to be effective. Among the various capsular (KL) types, KL1 and KL2 are often linked
to high virulence, and more concerningly, isolates with KL47 and KL64 are often linked to
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
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made available under aCC-BY 4.0 International license.

both hypervirulence and carbapenem resistance [22], which present significant challenges for
antimicrobial therapy [23,24]. These isolates are referred to as hypervirulent
carbapenem-resistant K. pneumoniae (hv-CRKP) emphasizing the urgent need to design and
develop broad-spectrum therapeutic drugs or vaccines against K. pneumoniae isolates of
serotypes KL1, KL2, KL47, and KL64 [25]. In India, there are few studies with a limited
number of samples. These have reported KL51 and KL64 as the most common K.
pneumoniae serotypes, which are associated with specific sequence types (STs), ST231-KL51
and ST147-KL64 [26]; [27]. The frequency of other KL types varies in different studies based
on the sample sizes. The globally prevalent, hypervirulent serotypes KL1, KL2, and KL20
were rarely found in these Indian studies.

The O-antigen is a significant virulence factor for K. pneumoniae. It aids the bacterium in
evading the host's immune system and attaching to host cells. The various O-antigen
serogroups have different antigenic properties, which can affect the bacterium's ability to
cause disease. The O-antigen of K. pneumoniae can be divided into different groups based on
their unique structures and antigenic properties. K. pneumoniae has eleven characterised LPS
serogroups. Just four serogroups: O1, O2a, O3, and O5 are expressed by over 80% of all
isolates [13]; [15]. In India, O1 and O2 are the most prevalent types, collectively constituting
over 70% of the isolates, as indicated in prior studies [28]; [10]; [26]. However, it is worth
mentioning that the number of studies conducted in the Indian setting is limited and these
studies represent smaller-scale investigations with relatively modest sample sizes, underlining
the need for more comprehensive research in this context.

Understanding the prevalence of K. pneumoniae serotypes in India will help the design of an
effective vaccine that covers the main circulating lineages in the country. For that reason, in
this study, we aim to identify the K and O loci of a large collection of country-wide K.
pneumoniae strains causing infection in India. Furthermore, our study explores the
relationship between these serotypes and disease severity, geographic distribution, and
virulence and antimicrobial resistance (AMR) characteristics, which adds to our
understanding of their intricate interactions in the Indian setting. This multi-pronged
approach will help India to develop effective and specialised vaccines against K. pneumoniae.
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

Materials and Methods

Bacterial Isolates and Phenotypic Characterization

The bacterial isolates used for this study comprised of 1072 putative K. pneumoniae isolates
primarily sourced from hospital infections and obtained from the years 2014 to 2022 across
India. This includes 307 isolates from our previous study [26]. The phenotypic
characterization was done at the Central Research Laboratory, Kempegowda Institute of
Medical Sciences (KIMS) using the VITEK 2 (bioMérieux, Marcy-l'Étoile, France) compact
system. The Ethical approval for the study was obtained from the KIMS ethical committee
with the study number KIMS/IEC/27/2017. The strain details are provided in Supplementary
Table 1.

Sequencing and Genomic Analyses

● Whole-genome sequencing, assembly, and annotation

Genomic DNA was extracted and isolated from the bacterial isolates using the QIAamp DNA
mini kit (Qiagen, Hilden, Germany) and quantified using the Qubit double-stranded DNA kit
(ThermoScientific, Massachusetts, United States) as instructed by the manufacturer.
Double-stranded DNA libraries with 450 bp insert size were prepared using the ultraFS-II kit
(New England Biolabs, London, United Kingdom). The QC check for the prepared libraries
was done on an Agilent Tapestation (Santa Clara, California, USA) and libraries were
sequenced on the Illumina MiSeq platform (Illumina, San Diego, California, United States of
America) with paired-end reads of 250 bp length. All the WGS data generated as a part of
this study were submitted to the European Nucleotide Archive (ENA) under the Bioproject
numbers PRJEB29740 and PRJEB50614 with accession IDs provided in Supplementary
Table 1.

The bioinformatic analysis was conducted using Nextflow pipelines created as part of the
Genomic Surveillance of Antimicrobial Resistance-AMR project available at protocols.io
[29]. The pipeline performs assembly using SPApades assembler v3.14 [30]. Quality control
of sequence data was evaluated for the following parameters: (i) the basic statistics of raw
reads, (ii) the assembly statistics, (iii) contamination due to single nucleotide variants (SNV)
and sequences from different species, (iv) Species prediction using Bactinspector and (v)
Overall QC as Pass, Warning or Fail for each isolate based on these different parameters as
described in the pipeline. All the quality metrics were combined using Multiqc and Qualifyr
to generate web-based reports [31]. The passed assemblies were annotated with Prokka v1.5
[32].

● In silico genomic characterization

Kleborate v2.3.2 (https://github.com/katholt/Kleborate) is a designated genotyping tool
developed for Klebsiella spp. It integrates multiple analysis steps, including multi locus
sequence typing (MLST), and identification of virulence and acquired resistance genes, to
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
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provide a comprehensive genotypic profile of the isolates [33]. KL and O antigens were
identified using Kaptive [34].

● Variant detection and phylogenetic analysis

Genome mapping of the 1072 isolates to the reference genome of K. pneumoniae (strain
NTUH-K2044, GCF_009497695.1) was done using the GHRU-SNP phylogeny pipeline
v1.2.2 (https://gitlab.com/cgps/ghru/pipelines/snp_phylogeny). The mobile genetic elements
(MGEs) were masked in the pseudo genome alignment using MGEmasker [35], and the
recombinant regions of the genome were removed using the Gubbins algorithm v2.0.0 [36].
A maximum-likelihood tree was built utilising the non-recombinant SNPs using IQ-tree [37]
with 100 bootstrap replicates and parameters -czb to collapse near-zero branches, and a
general time-reversible (GTR) model. Phylogeographic analysis and visualisation were
performed on Microreact [38].

● Statistical Analysis and Plots

We used the Rstudio server v2023.03.0 Build 386 for descriptive and statistical analysis.
Plots were generated using the ggplot package [39] and for data interpretation, the dplyr [40]
and tidyr [41] packages were used. All the code used in the study is published on Zenodo and
the link is provided.

Results

Overview of the collection

The collection included 1072 isolates from 38 different hospitals located in 19 different states
across India from 60% (646/1072) male and 40% (426/1072) female population. Of the 1072
isolates, 65 (1%) isolates were obtained from children <24 months of age, and overall patient
age ranged from 1 to 96 years with a median age of 48 years. The geographical representation
and the timeline of the samples are shown in Supplementary Figure 1 and also provided in
Supplementary Table 1.

The isolates were classified into two categories: invasive and non-invasive based on the site
of collection and specimen type. Among the invasive specimen types, blood samples emerged
as the predominant source, yielding 217 K. pneumoniae isolates followed by endotracheal
aspirates (ETA) with 147 isolates and body fluids with 50 isolates. In the non-invasive
specimen types, urine samples were predominant, with 257 isolates. Pus specimens were
followed by 154 isolates and sputum samples with 140 isolates. It is noteworthy that 90% of
the K. pneumoniae isolates were limited to 6 sample types and were not commonly found
from other sources. The complete distribution of different specimens is represented in Figure
1.
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
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Figure 1. Distribution of specimen types, classified as invasive and non-invasive, from which
Klebsiella pneumoniae was recovered.

K and O loci Diversity

In our collection of non-redundant genomes, we identified 79 distinct KL. The most prevalent
K-loci were KL64 (n = 274), KL51 (n = 249), KL2 (n = 88), and KL10 (n = 50). The top
three K-loci contributed to 59% of the isolates. The top 10 major KL types are provided in
Table 1 and the complete distribution of each K-loci is provided in Supplementary Table 2.
The major KL types in both invasive and non-invasive specimen sources were similar, i.e.
KL64 was a major KL in invasive sources (n=122) and also in non-invasive sources (n= 152)
(Figure 2) (Suppl. Table 2). Hence, we assessed the association between KL sample types
(invasive and non-invasive) using Fisher's exact test [42]. The most prevalent K-loci, namely
KL64 (P-value: 0.441), KL51 (P-value: 0.469), and KL2 (P-value: 0.266) were not
statistically associated either with invasive or non-invasive disease. Only KL10 was
significantly associated with non-invasive disease (P-value: 0.041), indicating a potential
link.

The theoretical coverage provided by multi-valent vaccines targeting increasing numbers of

KL (ordered by KL frequency in the population) is shown in Fig. 3a. The diversity of KL
types within our sample collection was assessed using Simpson’s Diversity Index (D). This
index gives a numerical representation of the diversity of KL in the population, ranging from
0 (no diversity) to 1 (highest diversity). The KL were very diverse in age groups <2 years
(D-value: 0.915), indicating that there is no specific KL associated with paediatric infections.
The diversity among other age groups was also very high and the diversity calculation for
each age group is provided in Suppl. Tables 3 and 4.
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
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Figure 2. Distribution of KL and O types among invasive and noninvasive specimens. The
bars are stratified by the number of samples in each KL and O type. The bars are stacked to
100%.

Table 1. KL distribution among invasive and non-invasive specimen types. The top 10 KL of
frequency >15 are shown individually and the lesser predominant KL <15 counts are
grouped. The top 10 STs are shown individually and others are grouped as ST_other.

Non- STs
K-locus Invasive Total
Invasive (n=count)
ST231 (n=9), ST147 (n=102), ST395 (n=90),
KL64 122 152 274
ST14 (n=6), ST_other (n=13), ST2096 (n=54)
ST231 (n=212), ST147 (n=20), ST_other (n=4),
KL51 121 128 249
ST16 (n=13)
ST14 (n=55), ST_other (n=17), ST101 (n=2),
KL2 46 42 88
ST11 (n=5), ST15 (n=9)
KL10 16 34 ST147 (n=46), ST_other (n=4) 50
KL36 22 20 ST_other (n=1), ST437 (n=41) 42
ST147 (n=2), ST_other (n=5), ST11 (n=1), ST16
KL81 17 17 34
(n=26)
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KL17 8 14 ST_other (n=2), ST101-1LV (n=11), ST101 (n=9) 22

KL112 8 13 ST15 (n=21) 21
KL52 8 9 ST_other (n=1), ST38 (n=11), ST437 (n=5) 17
KL62 10 7 ST_other (n=6), ST48 (n=11) 17
KL102 8 7 ST_other (n=3), ST307 (n=12) 15
ST231 (n=4), ST147 (n=9), ST395 (n=4), ST14
KL Other (n=1), ST_other (n=155), ST101 (n=1), ST1710
113 130 243
(count <15) (n=12), ST23 (n=12), ST469 (n=12), ST307
(n=1), ST11 (n=13), ST15 (n=8), ST16 (n=11)
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
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made available under aCC-BY 4.0 International license.

Figure 3. The cumulative coverage of the KL and the O types represents the percentage of
isolates covered. The KL and O types are sorted with the highest to lowest frequency. The
orange dotted line shows cumulative carbapenemase-positive strains.

Our study assessed the prevalence and associations of different O types with disease. Our
collection had a diverse distribution of O antigens with 14 different O types. The most
prevalent O-loci were O1/O2v1 (44%, 471/1072) and O1/O2v2 (33%, 353/1072), O10 (7%,
66/1072), which were collectively the top 3 O-loci constituting 83% (890/1072) of the
collection. Other O-loci, such as OL101 (66/1072), O3b (54/1072), O4 (52/1072), and
O3/O3a (50/1072), were also identified in Table 2). In the assessment of associations with
sample sources, a Fisher’s exact test [42]) for O1/O2v1 (P-value: 0.388), O1/O2v2 (P-value:
0.696), and O101 (P-value: 0.445) revealed no significant associations, implying a similar
distribution between invasive and non-invasive isolates. Serotype distributions in KL and O
types showed no significant differences by age group, as confirmed by Fisher's exact test. The
cumulative coverage of O types shows that if 5 types are incorporated in a vaccine
formulation, it will cover 90% of the isolates from different specimen types and disease
conditions (Figure 3).

Table 2. O-loci distribution among the invasive and non-invasive isolates and their associated
STs. The top 10 STs are shown individually and others are grouped as ST_other.
Non Grand
O antigen Invasive Invasive STs Total
ST231 (n=9), ST147 (n=104), ST14 (n=62), ST395
(n=90), ST_other (n=69), ST101 (n=9), ST101-1LV
(n=11), ST11 (n=9), ST15 (n=37), ST16 (n=6),
O1/O2v1 212 259 ST2096 (n=54), ST48 (n=11) 471
ST231 (n=216), ST147 (n=21), ST395 (n=4),
ST_other (n=81), ST101 (n=3), ST11 (n=4), ST23
O1/O2v2 161 192 (n=12), ST307 (n=12) 353
ST147 (n=4), ST_other (n=11), ST11 (n=4), ST16
OL101 34 32 (n=30), ST307 (n=1), ST38 (n=11), ST437 (n=5) 66
ST147 (n=3), ST_other (n=24), ST16 (n=13),
O3b 29 25 ST1710 (n=3), ST469 (n=11) 54
O4 27 25 ST_other (n=9), ST11 (n=2), ST437 (n=41) 52
O3/O3a 17 33 ST147 (n=46), ST_other (n=4) 50
OL104 7 3 ST1710 (n=9), ST469 (n=1) 10
O5 4 2 ST_other (n=6) 6
O12 4 ST_other (n=4) 4
OL103 1 1 ST_other (n=1), ST15 (n=1) 2
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Novel
(O1/O2v1) 1 ST_other (n=1) 1
Novel
(O3/O3a) 1 ST147 (n=1) 1
Novel
(OL101) 1 ST16 (n=1) 1
O1/O2v3 1 ST_other (n=1) 1

Sequence types and their serotypes association

The 1072 genomes of K. pneumoniae encompass 119 different STs, some persisting for
several years. Most of them have been reported globally while others are mainly restricted to
Southeast Asia, such as ST231 (n=228), ST147 (n= 180), ST395 (n=94) and ST14 (n=62)
(Supplementary Table 1). Analysis of the KL types revealed a distinct pattern of variation,
where some STs were associated with single K-locus types and others had multiple K-locus
types (Figure 4). For example, within ST231, KL51 is the dominant allele while KL64 is
present in a small subset. The second most prevalent ST, ST147, also exhibited remarkable
K-locus heterogeneity, hosting KL64 (n=110), KL51 (n=21), and KL10 (n=49) alleles and
three additional K-loci, highlighting the intricate diversity within this clonal group. On the
other hand, ST395 exclusively harboured KL64, while ST14 exhibited a strong association
with KL2. This suggests lineage-specific selective pressures or functional constraints shaping
KL diversification within the bacterial population.

O serotypes were strongly linked with STs. O1/O2v2 was seen in 220 isolates of ST231 and
21 of the ST147 isolates but ST147 also carried clade-specific associations with O1/O2v1
and O3/O3a. ST395, ST14, ST15, ST2096 and ST101 only carried O1/O2V1. ST16 was
associated with 3 different O-types namely O101, O1/O2v1 and O3b. Other minor STs had
varied O-types but some were associated with single O-types. This pattern of O-types
suggests ancient independent acquisition of O-types with subsequent clonal spread. The
distribution of K- and O-loci among different STs is shown in Figure 4 and also in the
microreact views (microreact.org/klocusvsst and microreact.org/olocusvsst)
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
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made available under aCC-BY 4.0 International license.

Figure 4. The circular view of the SNP tree was constructed using 100 bootstrap replicates.
The tree is midpoint rooted and the scale bar represents the SNPs per variable site. The inner
ring represents the K-loci types and the outer ring represents the O antigen types. The nodes
are coloured by ST.

Geographical distribution of KL and O antigens across India

We also examined the geographical distribution of KL and O antigens across India, revealing
the diversity of serotype prevalence. The distribution of KL and O antigens was
heterogeneous, with some loci being more prevalent in particular parts of the nation than
others. The ST147 clone with KL64 and O1/O2V1 locus and the ST395 clone with KL64 and
O1/O2V1 locus were the two most prevalent types in the Northern part of the subcontinent.
Twelve isolates of ST1710 having KL169 and O104 or O3B were seen in only one sentinel
site from Gujarat.
From the Simpson’s diversity index, the calculated overall D value of 0.8663956 indicates a
relatively high level of diversity among KL types. On the other hand, the regional variation in
the K loci diversity is quite remarkable. The Suppl. Table 5 reveals the Simpson’s Diversity
Index for six major political zones across India. We observed that specific zones exhibited a
higher degree of diversity in KL types compared to the others. Notably, the Southern and
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
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made available under aCC-BY 4.0 International license.

Central zones displayed the highest diversity in K loci, with Simpson's Diversity Index values
of 0.914 and 0.894, respectively. The diversity was relatively lower in the Northern and
Western zones (0.759 and 0.794, respectively) (Suppl. Table 5). The major KL types KL64
and KL51 and O types O1/O2v1 and O1/2v2 were distributed across all the zones and the
sentinel sites.

Virulence factors associated with capsular (KL) and O antigen serotypes

We screened for all 6 known virulence factors in K. pneumoniae, i.e. yersiniabactin (ybt),
aerobactin (iuc), salmochelin (iro), the genotoxin colibactin (clb), and the hyper mucoid locus
rmpADC and rmpA2. From the Kleborate virulence scoring, 314 out of 1072 (26%) isolates
in our collection had a virulence score of 4. The most common KL found in these was KL51
(179/314, 57%) including 132 ST231, followed by KL64 (91/314, 29%) including 38
ST2096. Only 9/14 isolates of KL1, a globally known hypervirulent serotype, had a virulence
score of 5 with all the virulence factors. 69/88 isolates of KL2 had a virulence score of 1 and
only 9/88 of them carried the hypervirulence marker aerobactin with a virulence score of 4.
There was no specific association of virulence factors with the KL or O antigen types
(P>0.05). These results show no particular association between serotypes and virulence
factors. These findings underscore the complex and varied nature of virulence among K.
pneumoniae isolates, highlighting the need for further research to elucidate the factors
influencing pathogenicity in this bacterium. Interestingly, it was observed that conventionally
described hypervirulent clones did not carry virulence markers, while other regional
serotypes exhibited the presence of hypervirulence markers, adding a layer of complexity to
the understanding of virulence in K. pneumoniae.

Resistance profiles associated with capsular (KL) and O antigen serotypes

In this study, Kleborate identified 64 distinct genes associated with antimicrobial resistance.
These genes are linked to resistance across 10 antimicrobial classes. All isolates carried at
least one genetic resistance determinant and 73% (786/1072) were MDR having resistance to
more than 3 classes of drugs. Notably, a significant majority, comprising 78% (841/1072),
exhibited specifically carbapenem-resistance markers. The majority of the isolates in our
collection (70%, 748/1072) had a resistance score of 2, followed by 14% (158/1072) of the
isolates having a resistance score of 1, notably 4% (43/1072) had the highest resistance score
of 3 and only 11% (123/1072) had a resistance score of 0.

Forty (12%, n=129/1072) of the KL types did not carry any carbapenemase genes. The other
39 types carried at least one carbapenamase gene. The predominant KL types, KL51 and
KL64, exhibited varied associations with different carbapenemase genes, with KL64
primarily linked to blaOXA-48-like variants (particularly blaOXA-181 (63/274) and blaOXA-232
(124/274)), while KL51 strains carried predominantly blaOXA-232 (183/249), followed by
co-carriage of blaNDM and blaOXA-232 (38/249). The carbapenemase gene distribution among
different KL types is provided in Suppl. Tables 6 and 7. There was no significant association
between O antigen types and carbapenemase genes (P>0.05). The predominant O types,
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

O1/O2v1 and O1/O2v2 carried both blaOXA-48-like and blaNDM genes. Four different O types
comprising 1.4% (16/1072) of the isolates did not carry any carbapenemase genes
(Supplementary table 4). The acquisition of the carbapenemase genes was independent of
serotypes and largely driven by the STs. The cumulative distribution of the KL and O types in
Figure 3 showed that the vaccine formulas against K. pneumoniae in India need to
incorporate either ~20 KL types to cover >85% of the carbapenemase-producing population
or 4 OL types to cover the same population.

Discussion

K. pneumoniae is a human commensal and opportunistic pathogen that can cause severe
hospital-acquired infections, especially among patients with compromised immune systems.
K. pneumoniae infections are tough to cure due to the organism's thick capsule [43] and the
emergence of MDR strains has made the majority of current antimicrobials ineffective [44].
Effective measures for preventing K. pneumoniae infections are desperately needed and
vaccines are one of the proposed alternatives [18]. Capsular polysaccharides in K.
pneumoniae have emerged as promising targets for vaccine development due to their role in
virulence and the bacteria's resistance mechanisms [19]. This study aimed to
comprehensively investigate serotype prevalence and epidemiology in India, a region
grappling with antimicrobial resistance and healthcare-associated infections.
The analysis of 1072 K. pneumoniae isolates from 2014 to 2022 in India revealed significant
genomic diversity within the KL and O types. This highlights a varied genetic landscape
among K. pneumoniae strains circulating in Indian hospitals. The extensive diversity
observed within the KL of K. pneumoniae poses a considerable challenge for vaccine
development. With the identification of 79 distinct KL types and the top three KL (KL51,
KL64, KL2) collectively representing only 59% of the isolates, it is evident that creating a
vaccine or monoclonal antibodies targeting a wide spectrum of prevalent KL types will be
complex. The recently described development of anti-KL64 antibodies would only cover
about 26% of the Indian K. pneumoniae isolates [45]. Several studies have consistently
reported a high diversity of KL types in K. pneumoniae strains across different regions [46];
[47]; [48]; [28]; [49]. In Southeast (SE) Asia particularly, studies have highlighted the
regional disparity of KL types in K. pneumoniae strains causing infection. For instance, a
study by Holt and colleagues [7] found that KL2 and KL47 were predominant in Thailand,
while KL107 was more prevalent in Cambodia. Similarly, a study by Wyres and colleagues
[28] reported variations in KL types among K. pneumoniae isolates in Malaysia, with KL1,
KL2, and KL102 being the most common. The development of the Inventprise 25-valent
pneumococcal conjugate vaccine candidate, utilising the patented Hz-PEG-Hz linker
technology platform, represents a significant advancement in vaccine technology [50]. It is
anticipated that this vaccine candidate will offer the broadest coverage against pathogenic
pneumococcal serotypes encountered by populations worldwide, irrespective of geographical
location. Similarly, if a K. pneumoniae vaccine targeting 25 KL types were to be developed, it
could potentially provide coverage for up to 90% of the Indian K. pneumoniae population
along with 85% of CRKP clones, illustrating the profound impact such a vaccine could have
on public health and disease prevention efforts.
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

Research has highlighted the variability in disease associations linked to specific KL types.
Some studies have identified certain KL types, such as KL1 and KL2, as being more
prevalent and associated with poorer disease outcomes [51]; [52]; [53], particularly in
hypervirulent strains causing community-acquired invasive infections. Conversely, others
have noted the rarity of these KL types associated with severe infections in certain
geographic regions [54]; [27]; [49]. Our study also highlights that certain KL types within K.
pneumoniae strains were found in both invasive and non-invasive diseases, indicating their
versatile pathogenic potential. Additionally, we did not observe an association between
hypervirulence and certain KL types or disease severity, as noted before. This finding further
complicates our understanding of the complex relationships between K. pneumoniae
virulence factors and disease manifestation, emphasising the need for further research to
elucidate the mechanisms underlying KL-associated pathogenicity.

We discovered a considerable diversity of 14 different O types among K. pneumoniae isolates

compared to previous studies [28]; [55]. However, a few strong O-loci, especially O1/O2v1,
O1/O2v2, and O10 stood out, accounting for a significant proportion (83%) of the isolates
analysed. The cumulative coverage analysis revealed that incorporating only five types of O
antigens (O1, O2v1, O2v2, O10 and O3b ) would cover approximately 90% of the isolates
across different specimen types and disease conditions in India. Previous studies have also
explored the inclusion of a narrower selection of O types in vaccine formulations. For
instance, Wyres and colleagues [28] suggested that incorporating a subset of prevalent O
antigens could provide significant coverage against K. pneumoniae infections. Studies have
highlighted the importance of targeting specific O types, such as O1, O2, and O3b, in vaccine
development strategies due to their high prevalence and association with disease
manifestation [13]. A recent study reported a heptavalent O-antigen bioconjugate vaccine
[56] which exhibits promising efficacy against some, but not all, K. pneumoniae isolates.
While some studies [57] say that O antigen is accessible by antibodies irrespective of the
capsule type other studies are highlighting that hyperproduction of CPS may inhibit the
vaccine-induced O-antigen antibody binding [57]; [58]. A more recent study evaluating
monoclonal antibodies against K. pneumoniae ST147NDM-1, concluded that highly bactericidal
anti O-antigen antibodies are not protective against this hypervirulent and pan drug-resistant
strain ([45]). This suggests more studies are needed to clarify the effectiveness of anti
O-antigen vaccine or monoclonal antibody formulations against the broader K. pneumoniae
population.

Conclusion

The findings provide crucial insights into the genetic diversity, evolution, and potential
adaptation mechanisms of K and O loci within the K. pneumoniae Indian population, with
implications for understanding its epidemiology. Additionally, the insights regarding its
diversity underscore the challenges in formulating vaccines or monoclonal antibodies that
adequately cover the diverse array of K. pneumoniae strains including the
carbapenemase-producing ones. The lack of a straightforward correlation between specific
serotypes and virulence factors challenges conventional assumptions about hypervirulence
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

clones and necessitates further exploration into the multifaceted nature of virulence
determinants in K. pneumoniae. Understanding the epidemiology of K. pneumoniae can help
tailor effective prophylactic and therapeutic solutions against KL and O antigens in India.

Author contributions

This study was conceptualised by V.S. and D.A., V.S. performed the genomic analysis of the
samples collected in this study. S.S. was involved in statistical analysis, table generation, and
figure generation. N.C. guided the manuscript preparation and reviewed the manuscript.
Funding for the study was provided through grants to K.L.R and D.A., G.N. and H.G.K. were
involved in the sample collection. H.G.K. performed the microbiology part of the analysis &
G.N. did the sequencing. All authors reviewed the manuscript and suggested improvements.

Conflicts of interest
The authors report no conflicts of interest.

Funding Statement
The sample collection and whole genome sequencing work was supported by the Official
Development Assistance (ODA) funding from the National Institute for Health Research
[grant number 16_136_111] and the Wellcome Trust grant number 206194.

The views expressed in this publication are those of the authors and not necessarily those of
the NHS, the National Institute for Health Research, or the Department of Health.

Acknowledgements

Members of the NIHR Global Health Research Unit on Genomic Surveillance of

Antimicrobial Resistance: Sophia David, Monica Abrudan, Julio Diaz Caballero,
Emmanuelle Kumaran, Georgina Lewis-Woodhouse, Khalil Abudahab and Ben Pascoe of the
Centre for Genomic Pathogen Surveillance, Big Data Institute, University of Oxford, Old
Road Campus, Oxford, UK, and Wellcome Genome Campus, Hinxton, UK; Pilar
Donado-Godoy of the Colombian Integrated Program for Antimicrobial Resistance
Surveillance, Coipars, CI Tibaitatá, Corporación Colombiana de Investigación Agropecuaria
(AGROSAVIA), Tibaitatá, Mosquera, Cundinamarca, Colombia; Shreedhanya D M, Shincy
M. R., Sravani D., K. N. Ravishankar of the Central Research Laboratory, Kempegowda
Institute of Medical Sciences, Bengaluru, India; Iruka N. Okeke, Anderson O. Oaikhena of
the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ibadan,
Oyo State, Nigeria; Sonia Sia, Celia Carlos, Marietta L. Lagrada and June M. Gayeta of the
Antimicrobial Resistance Surveillance Reference Laboratory, Research Institute for Tropical
Medicine, Muntinlupa, the Philippines; John Stelling, The Brigham and Women’s Hospital,
Boston, MA, USA; and Carolin Vegvari, Imperial College London, London, UK. NMAMIT,
Dept. of Biotechnology: Ujwal P, Vidya S M and Chethan D M. GHRU India Consortium:
Dr. Anuradha Sharma, AIIMS, Jodhpur, Rajasthan, India; Dr. Ujjwayini Ray, Apollo Multi
Scpeciality Hospital, Kankurgachi, Kolkata, West Bengal, India; Dr. Manick Das, Apollo
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

Medical College, Hyderabad, India; Dr. Maneesha Sahu, BALCO Medical Center, Raipur,
Chhattisgarh, India; Dr. Aruna Poojary, Breach Candy Hospitals, Mumbai, Maharashtra,
India; Dr. Lakshmi, Biocare Research Lab, Gandhinagar, Gujarat, India; Dr. Shwetha,
Bangalore Medical College, Bangalore, Karnataka, India; Dr.malini sahroff, BPS womens
Medical college, Sonipat, Haryana, India; Dr. Shubranshu mandal, Calcutta Medical
Research Institute, Kolkata, West Bengal, India; Dr. Frincy, Excel Health care, Guwahati,
Assam, India; Dr. Anitha, Government medical college, Trichy, Tamil Nadu, India; Dr.
Varsha Guptha, GMC, Chandigarh, India; Dr. Namrata Rai, Indira Gandhi Institute of
Medical Sciences, Patna, Bihar, India; Dr. Bhattacharyya, All India Institute of Hygiene &
Public Health, Kolkata, West Bengal, India; Dr. Naveena, Sri Jayadeva Institute of
Cardiovascular Sciences and Research, Bangalore, Karnataka, India; Dr. Sujatha, Jawaharlal
Institute of Postgraduate Medical Education and Research, Pondicherry, Pondicherry, India;
Dr. Sheetal Verma, King George's Medical University, Lucknow, Uttar Pradesh, India; Dr.
Shrikala baliga, Kasturba Medical College, Mangalore, Karnataka, India; Dr. Lakshmi,
Kamineni Hospital, Hyderabad, Telangana, India; Dr. Shalab Malik, Dr Lal PathLabs, Delhi,
India; Dr. Vishwanath, Mediquest Diagnostics, Hyderabad, Telangana, India; Dr. Sohan Lal,
Malabar Institute of Medical Sciences, Kozhikode, Kerala, India; Dr. veenakumari,
NIMHANS, Bangalore, Karnataka, India; Dr. Milan, Neugen Laboratories, Rajkot, Gujarat,
India; Dr. Venkatraman Kandi, Prathima Institute of Medical sciences, Karimnagar,
Telangana, India; Dr. Smita Sood, Rukmani Birla Hospital, Jaipur, Rajasthan, India; Dr.
Keerthi Lakshmi, RajaRajeswari Medical College, Bangalore, Karnataka, India; Dr. Jyothi
EK, SCTIMST, Thiruvananthapuram, Kerala, India; Purna chandra, Kolar medical college,
Kolar, Karnataka, India; Dr. Vaidehi, Sundaram Medical Foundation, Chennai, Tamil Nadu,
India; Dr. Jagatheeswary, Saveetha Medical College, Kuthambakkam, Tamil Nadu, India; Dr.
B G Vishwanath, Sreekar Lab, Hyderabad, Telangana, India; Dr.Shruthi Uppoor, Siddiah
Referral Hospital, Bangalore, Karnataka, India; Dr. chitra rajalakshmi, Trichy SRM Medical
College Hospital and Research Centre, Trichy, Tamil Nadu, India; Dr. Mohit Bhatia, AIIMS,
Rishikesh, Uttarakhand, India; Dr. Sowmusharee, VIMSAR, Burla, Odisha, India; Dr. Malini
Sahriff, Vallabhbhai Patel Chest Institute, Delhi, India
bioRxiv preprint doi: https://doi.org/10.1101/2024.03.15.585133; this version posted March 19, 2024. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.

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