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Current Strategy for Local‑ to Global‑Level Molecular Epidemiological

Characterisation of Global Antimicrobial Resistance Surveillance System
Pathogens

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Special Article

Current Strategy for Local‑ to Global‑Level Molecular

Epidemiological Characterisation of Global Antimicrobial
Resistance Surveillance System Pathogens
Dhiviya Prabaa Muthuirulandi Sethuvel, Naveen Kumar Devanga Ragupathi, Yamuna Devi Bakthavatchalam, Saranya Vijayakumar, Rosemol Varghese,
Chaitra Shankar, Jobin John Jacob, Karthick Vasudevan, Divyaa Elangovan, Veeraraghavan Balaji
Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India

Abstract
The prime goal of molecular epidemiology is to identify the origin and evolution of pathogens, which can potentially influence the public
health worldwide. Traditional methods provide limited information which is not sufficient for outbreak investigation and studying transmission
dynamics. The recent advancement of next‑generation sequencing had a major impact on molecular epidemiological studies. Currently,
whole‑genome sequencing (WGS) has become the gold standard typing method, especially for clinically significant pathogens. Here, we aimed
to describe the application of appropriate molecular typing methods for global antimicrobial resistance surveillance system pathogens based on
the level of discrimination and epidemiological settings. This shows that sequence‑based methods such as multi‑locus sequence typing (MLST)
are widely used due to cost‑effectiveness and database accessibility. However, WGS is the only method of choice for studying Escherichia
coli and Shigella spp. WGS is shown to have higher discrimination than other methods in typing Klebsiella pneumoniae, Acinetobacter
baumannii and Salmonella spp. due to its changing accessory genome content. For Gram positives such as Streptococcus pneumoniae, WGS
would be preferable to understand the evolution of the strains. Similarly, for Staphylococcus aureus, combination of MLST, staphylococcal
protein A or SCCmec typing along with WGS could be the choice for epidemiological typing of hospital‑ and community‑acquired strains.
This review highlights that combinations of different typing methods should be used to get complete information since no one standalone
method is sufficient to study the varying genome diversity.

Keywords: Antimicrobial resistance lineages, global antimicrobial resistance surveillance system, molecular epidemiology, outbreak
investigation, whole genome sequencing

Introduction limited information for outbreak investigation.[2] The recent

Identification of causative agents is a highly important issue in
diagnostic microbiology laboratories, and this has now been
a topic of interest in clinical research. Although conventional
microbiological methods are inexpensive, they have certain
limitations associated with it, especially for the identification
and characterisation of fastidious, slow‑growing, non‑viable
or non‑cultivable organisms which can be easily detected
by molecular methods. Currently, molecular methods are
increasingly being used in the field of epidemiology due to
its rapid turnaround time, sensitivity and specificity although
they are expensive than the conventional methods.[1]
However, the currently available molecular methods are
time‑consuming and lack resolution and provide only
Access this article online
advancement of next‑generation sequencing (NGS) and
whole‑genome sequencing (WGS) methodologies had a major
impact on molecular epidemiology.[3] Previous studies have
shown that WGS had short turnaround time (48–96 h) and
Address for correspondence: Dr. Veeraraghavan Balaji,
Department of Clinical Microbiology, Christian Medical College,
Vellore ‑ 632 004, Tamil Nadu, India.
E‑mail: vbalaji@cmcvellore.ac.in
Received: 18‑10‑2019 Revised: 21‑10‑2019
Accepted: 01-11-2019 Published Online: 19-11-2019
This is an open access journal, and articles are distributed under the terms of the Creative
Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to
remix, tweak, and build upon the work non‑commercially, as long as appropriate credit
is given and the new creations are licensed under the identical terms.

Quick Response Code: For reprints contact: reprints@medknow.com

Website:
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How to cite this article: Muthuirulandi Sethuvel DP, Devanga Ragupathi NK,
Bakthavatchalam YD, Vijayakumar S, Varghese R, Shankar C, et al. Current
DOI: strategy for local- to global-level molecular epidemiological characterisation
10.4103/ijmm.IJMM_19_396 of global antimicrobial resistance surveillance system pathogens. Indian J
Med Microbiol 2019;37:147-62.

© 2019 Indian Journal of Medical Microbiology | Published by Wolters Kluwer - Medknow 147
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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens

unprecedented level of resolution for outbreak investigations
in the clinical setting.[4]
One of the prime goals of epidemiology is to identify the
origin and evolution of a pathogen, which can potentially
influence the public health worldwide. Although several
methods such as multi‑locus sequence typing (MLST),
multiple locus variable number of tandem repeats (MLVA)
and pulse field gel electrophoresis (PFGE) are available,
currently WGS opens up many avenues of approach to analyse
an isolate’s genetic content.[5,6] Generally, variability between
bacterial genomes of the same species occurs for various
reasons that include single nucleotide polymorphism (SNP),
insertion or deletion of one or multiple base pairs (indels) or
by recombination of large blocks of genetic sequences.[7,8]
Among which SNPs are the most common and simplest form
of DNA variations and found to be the important driver of
bacterial evolution and expansion. Therefore, SNPs can be
used as a stable genetic marker for the dissemination of a
particular strain.[9]
WGS has also been used to identify the international spread
of antimicrobial‑resistant pathogens. [2,10] Antimicrobial
resistance (AMR) is a growing problem in both clinical and
socioeconomic setting. In 2015, the World Health Organization
(WHO) launched the global AMR surveillance system
(GLASS) to address this issue, which mainly focus on the
human bacterial pathogens. Further, the accuracy, reliability
and affordability of NGS would permit their use in clinical and
public health laboratory for rapid identification of pathogen,
outbreak investigation and infection control process.[11]
Primarily, all typing methods are eventually useful, but the
challenge is to find what methods are appropriate for specific
settings. Different epidemiological settings require different
typing methods providing different level of discriminatory
power. In this review, we aimed to provide information on
the appropriateness of different molecular typing methods
for GLASS pathogens for different levels of epidemiological
characterisation. The global spread of AMR lineages of these
pathogens was also discussed.
Typing System for Global Antimicrobial
Resistance Surveillance System Pathogens:
Appropriate Molecular Approaches
This could be broadly classified based on the need for local‑,
regional‑  and global‑level discrimination of the strains
[Figure 1]. The best approaches could be as follows.
With WGS, MLST on genome-wide scale such as ribosomal
MLST, core genome MLST and whole‑genome MLST
with increasing discriminatory power is achievable. The
main characteristics of these methods were obtained from
Enterobase database (https://enterobase.warwick.ac.uk/) that
is given in Table 1. Typing methods can be chosen based on
the targets and the level of discrimination required for analysis.
For instance, in VRE outbreak analysis, PFGE is considered

Figure 1: Hierarchical molecular epidemiological typing methods to address outbreak at local, regional and global level. wg: Whole genome,
SNP: Single‑nucleotide polymorphism, MLST: Multi‑locus sequence typing, SLV: Single locus variant, DLV: Double locus variant, Rmlst: Ribosomal
MLST, cg: Core genome

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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens

gold standard though the method is comparably laborious
and time‑consuming and requires experienced personal for
analysis. Alternatively, MLST and MLVA are not reliable for
outbreak analysis due to their less discriminatory power but
can be used when combined with other data such as plasmid
typing, virulence gene profiling, vanA cluster typing. Methods
such as plasmid typing and vanA cluster typing are useful when
used along with the basic methods such as MLST, MLVA and
PFGE but not useful as a standalone method. However, WGS
can provide all the information required for the analysis all at
once.[12]
Genome Features, Resistant Lineages and
Appropriate Typing System for Individual Global
Antimicrobial Resistance Surveillance System
Pathogens – Problems and Solutions
The information on the pathogen specific typing methods for
local‑, regional‑ and global‑level analysis is detailed in Table 2.
Escherichia coli and Shigella
Escherichia coli is the part of normal gut microflora;
however, pathogenic strains also exist and can cause
infection in humans. Though being a commensal, the
genetic flexibility of the species makes it an effective
recipient and donor of AMR determinants through
horizontal gene transfer mechanism.[13] Notably, Shigella
spp., a human restricted pathogen, is considered as a human
adapted E. coli and was viewed as the biotypes/pathotypes
or clones of E. coli. [14] However, recent studies have
indicated that Shigella spp. has emerged at least seven
separate times from E. coli.[15]
Interestingly, multidrug resistance (MDR) E. coli, particularly
strains that produce extended‑spectrum β‑lactamases (ESBLs),
are a rising clinical and public health challenge. Similarly,
Shigella clones with high virulence and MDR have spread
globally. Moreover, differentiation of E. coli from Shigella spp.
is still challenging in many microbiology laboratories because
of their close genetic relatedness.[16] The recent development
in WGS technologies provide accurate differentiation of these
pathogens and enable us to understand the genome features
and the epidemiological characteristics of these pathogens.
In general, E. coli genome comprised only 20% of core
genes and 80% are accessory genes.[17] Similarly, Shigella
spp. has varying accessory genes. Of the predicted E. coli
pan‑genome, the core genome consists of around 5000 genes.
However, previous study on the phylogenomics of Shigella
spp. showed that the predicted pan‑genome has approximately
10,000 genes, which proves the fact that Shigella spp. does not
have the same genomic profile as E. coli. Furthermore, Shigella
contains a smaller genome than E. coli genomes.[18] This could
be due to its distinctive characteristics of losing of genes as a
part of an intracellular pathogenic lifestyle.[19,20]
Further, increasing AMR in these pathogens are a significant
public health concern. Remarkably, mobile genetic elements
(MGEs) plays a major role in dissemination of resistance
mainly through horizontal gene transfer especially among
the enteric pathogens. [13] For instance, ST131 lineage
carrying blaCTX‑M‑15 gene in E. coli has epidemic potential
in spreading the resistance globally. In the same way,
fluoroquinolone‑resistant Shigella sonnei global lineage III
has the ability to spread intercontinentally by acquiring mobile
elements carrying AMR genes. Therefore, it is important to

Table 1: Typing methods based on the targets and the level of discrimination required for analysis (Enterobase)
Features MLST Ribosomal MLST Core genome MLST Whole genome MLST
Loci 7‑8 loci ~50 loci ~1000‑3000 loci ~20,000 loci
Targets Conserved housekeeping genes Ribosomal proteins Core genes Any conserved coding sequence
Variability Highly conserved Highly conserved Variable Highly variable
Resolution Low Medium High High
Typing scheme Different for each species/genus Single scheme Different for each species/genus Different for each species/genus
across tree of life
MLST: Multi‑locus sequence typing, ~: Approximate

Table 2: Pathogen‑specific typing methods for local‑, regional‑ and global‑level analysis
Escherichia coli Shigella Klebsiella Salmonella Acinetobacter Staphylococcus Streptococcus
spp. pneumoniae spp. baumannii aureus pneumoniae
Local wgMLST, PFGE, fimH wgMLST, wgMLST, MLVA, wgMLST, wgMLST, MLST, wgMLST, PGFE, MLST,
typing, fumC typing PFGE, MLVA MLST, PFGE PFGE MLST, PFGE PFGE, spa typing MLVA, PBP typing
Regional SLV/DLV approach, SLV/DLV MLST MLVA MLST MLST, SCCmec MLST, PBP typing
fimH and fumC typing approach typing
Global cgMLST cgMLST MLST, wgMLST MLST, MLST, cgMLST MLST, cgMLST
cgMLST cgMLST
MLST: Multi‑locus sequence typing, wgMLST: Whole‑genome MLST, PFGE: Pulse field gel electrophoresis, MLVA: Multiple‑locus variable number of
tandem repeats, spa: Staphylococcal protein A, PBP: Penicillin‑binding protein, SLV: Single‑locus variant, DLV: Double‑locus variant, SCC: Staphylococcal
cassette chromosome, cgMLST: Core‑genome MLST

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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens
identify the circulating resistant lineage of these pathogens in
the community and hospital settings to manage the spread of
drug‑resistant strains.
Molecular typing methods for Escherichia coli and
Shigella spp.
For E. coli, PFGE is a reliable and highly discriminatory
method and has been considered to be the “gold standard.”
Recently, due to the establishment of Pulse Net, the use of
PFGE has had a major impact on the bacterial sub‑typing and
outbreak investigation.[21,22] However, analysis of multiple
banding pattern leads to inter‑laboratory variability and is
not amenable to create public databases, thus not suitable for
regional‑ or global‑level analysis.[23] Further, fimH and fimC
typing method has been developed and been previously used
for epidemiological surveillance of global pandemic clones
of E. coli. This has shown superior discrimination power than
conventional MLST.[24]
Previous study revealed that the combination of MLST along
with fimH and fumC typing showed good discrimination
of E. coli strains.[24,25] These sub‑typing method has shown
to be mainly applicable among the highly virulent ST131
clonal group as this group is the most predominant human
pathogenic clone being reported.[26] This method is suitable
for screening large collections of E. coli isolates, allowing
for the rapid identification of sequence types (STs) or clonal
complexes (CCs).[23] Similarly, single‑locus or double‑locus
variants (SLV/DLV) approach which is the discrimination
based on difference in one or two MLST housekeeping gene
sequence from the founder genotype is termed SLV/DLV, used
to differentiate closely related species.[16]
Moreover, there is no MLST database accessible currently for
E. coli in pubMLST. However, MLST data can be derived only
from whole‑genome sequences through Enterobase database.
Since WGS has higher discriminatory power than other
typing methods, it can also be used to differentiate sub‑clones
of ST131. For example, the H41 sub‑clone of ST131 is
fluoroquinolone susceptible, while H30 is extended‑spectrum
cephalosporin‑resistant and the prevalence of these sub‑clones
is different. This issue can be resolved with WGS data.[24]
Similarly, epidemiological typing of Shigella spp. has being
done using the E. coli database till today from the evidence
that the Shigella spp. is evolved from E. coli. Alike E. coli,
conventional typing methods such as PFGE and MLVA can be
used for the extent of local outbreak analysis. For local‑ and
regional‑level analysis, SLV/DLV approach can also be used.
However, WGS method is the only method of choice to study
the relatedness of the Shigella strains.
Antimicrobial resistance‑specific lineages of Escherichia
coli
There is a rising evidence that clonal lineages of E. coli ST
131 have more epidemic potential than other lineages within
their species.[27] E. coli ST131 carrying blaCTX‑M‑15 is globally
disseminated in community and healthcare settings. Most of
150 Indian Journal of Medical Microbiology ¦ Volume 37 ¦ Issue 2 ¦ April-June 2019
the MDR E. coli carrying blaCTX‑M‑15 from different countries
in Europe and North America was homogenously grouped
into the E. coli O25:H4‑ST131.[28‑30] This particular lineage
stands out among the other E. coli lineages due to its nature
of resistance to fluoroquinolones and β‑lactams, mediated
by sequential accumulation of chromosomal mutations and
plasmid containing blaCTX‑M‑15 ESBL, respectively.[31]
Particularly, for carbapenem resistance (CR), blaNDM was
found to be carried by ST167 and ST405 in China. In addition,
ST405 carried blaKPC for CR. Recently, ST410 was reported
as a possible international high‑risk clone with B2/H24R,
B3/H24Rx and B4/H24RxC AMR clades. B3/H24Rx was
reported to be evolved by acquisition of the blaCTX‑M‑15 and
an IncFII plasmid. B4/H24RxC emerged by acquiring IncX3
plasmid with blaOXA‑181 known for CR, which further acquired
blaNDM‑5, on a conserved IncFII plasmid. This newly emerging
ST410 has been reported in countries such as Denmark, China,
UK, France and India.[32]
In India, ST167 (18%) and ST405 (20%) were predominant
type followed by ST131 (15%) and ST410 (12%). Among
these STs, ST131 and ST405 clades carried blaCTX‑M‑15 for
cephalosporin resistance, whereas ST167 and ST410 carried
both blaCTX‑M‑15 and blaCMY genes. CR genes, blaNDM was found
in ST405, 167 and 410 as observed elsewhere (unpublished
data). This represents that the global scenario matches with
the Indian data for prevalence of STs and associated AMR
genes. The STs that are widespread and increasingly reported
to be associated with antimicrobial resistance was mapped in
Figure 2a.
Antimicrobial resistance‑specific lineages of Shigella spp.
Studies combining epidemiological and pathogens genome data
are increasingly being reported. This approach has unravelled
the evolutionary history of Shigella species. Specifically,
the phylogenomic studies shows the intercontinental
dissemination of rapidly evolving highly clonal biotype
g S. sonnei lineages.[33] There are four distinct S. sonnei
lineages (lineage I, II, III and IV). The current pandemic
involves globally distributed, MDR clones that belong
to lineage III, which is also referred as global lineage III
and with only low numbers of other lineages. The major
lineage III has three sub‑groups that carry a distinct class II
integron (In2) variant, which is either plasmid‑encoded
(South America III) or integrated into the chromosome (Central
Asia IIIa, Global III).[34]
Further, lineage III was found to carry transposon Tn7 and
class II integron insertion within the chromosome encoding
resistance to streptomycin, trimethoprim‑sulphamethoxazole
and tetracycline and mutations in chromosomal gyrA and
parC, which encodes the target protein for fluoroquinolone.
This ciprofloxacin‑resistant S. sonnei has evolved as a single
clone due to gradual accumulation of the triple mutations
in gyrA and parC, most likely in South Asia, before
spreading internationally to Southeast Asia and Europe.[34,35]
Recent report said that Asia is a primary hub for the recent
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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens

Figure 2: Global distribution of predominant sequence types or lineages associated with antimicrobial resistance identified in Gram‑negative pathogens
is mapped. Sequence types or lineages are differentiated with different shapes and colours. (a) Sequence types associated with antimicrobial resistance
reported in Escherichia coli. (b) Intercontinental dissemination of four distinct Shigella sonnei lineages. (c) Sequence types linked with antimicrobial
resistance identified in Klebsiella pneumoniae. (d) Incidence of typhoidal and non‑typhoidal Salmonella in global context

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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens
international spread of ciprofloxacin‑resistant S. sonnei.
The distribution of antimicrobial resistance genes (ARGs)
and mutations within the chromosomal genes suggests that
selection for MDR played a key role in driving this global
dissemination.
Besides, S. sonnei lineage III has faster mutation rate
than other lineages and appears to have high capability
of acquiring resistance to other antimicrobials including
third‑generation cephalosporins when introduced into new
geographical locations.[34,35] These characteristics make this
particular lineage more predominant worldwide. Based on
the available reports, the global distribution of S. sonnei
lineage III and its subsequent international spread was
mapped [Figure 2b].
Klebsiella pneumoniae
Klebsiella pneumoniae is considered as an opportunistic
pathogen and the most frequent cause of hospital‑acquired
infections for many decades. The WHO has recently recognised
K. pneumoniae as a significant threat to global public health
due to its high rates of hospital outbreaks and deaths associated
with AMR clone.[36] In addition, there is an emergence of
drug resistant hypervirulent K. pneumoniae strains capable
of causing infection in the community.[37,38] However, little
is known about the population structure of K. pneumoniae;
consequently, it is important to understand the emergence of
clinically important resistant clones within this genetically
diverse species. The genome structure, AMR‑specific lineages
and the available tools for epidemiological investigation of
K. pneumoniae are discussed here.
The average genome size of K. pneumoniae is 5.5 Mbp and
encodes ~5500 genes. The whole‑genome comparison of
100 K. pneumoniae isolates indicates that the core genome
comprises of only fewer than 2000 genes and the majority
3500 are accessory genes. This indicates that the species has
access to a vast gene pool and has varied accessory genome
content.[38] Besides, there is evidence that the exchange of
mobile accessory genes within the species through plasmids
and other conjugative elements may contribute to the survival
of the species in different niches.[36,39]
Furthermore, based on the developed molecular epidemiology
and sequencing technologies, studies have demonstrated
that K. pneumoniae can be divided into three distinct
phylogroups (phylogroup KpI represents K. pneumoniae,
KpII and KpIII belong to Klebsiella quasipneumoniae and
Klebsiella variicola, respectively) with variation in their core
and accessory genome.[38] In K. pneumoniae, AMR genes are
commonly carried in diverse plasmid types. In particular, the
pandemic lineage ST258 harbour IncFII plasmid carrying
blaKPC gene, suggestive of successful spread of this clone.
This evidenced that K. pneumoniae can accumulate a large
accessory genome from a larger pool of available genes in
the environment to colonise, infect and resist antibiotics in
humans.[36,39]
152 Indian Journal of Medical Microbiology ¦ Volume 37 ¦ Issue 2 ¦ April-June 2019
Molecular typing methods for Klebsiella pneumoniae
Investigating outbreaks of MDR K. pneumoniae in hospitals
settings can be done by MLST or RAPD to identify the clonal
relatedness of the isolates where WGS is not feasible. Smaller
outbreaks in confined settings can also be identified using
conventional techniques such as PFGE, RAPD and MLST. For
regional‑ or national‑level comparison of isolates, MLST can
be used. Minim‑typing or mini‑MLST has also been described
for K. pneumoniae in which high‑resolution melting analysis
is performed on fragments within MLST loci.[40] However,
minim‑typing in conjugation with AMR screening or WGS
enable quicker surveillance in hospital settings.[40,41]
However, core genome MLST (cgMLST) obtained
through WGS has been used in several hospital outbreak
investigations to decipher information on the relatedness of
the isolates. It helps in identifying the resistance mechanisms,
clone causing outbreak and also the relatedness of the strains
such as single‑nucleotide variants (SNVs).[42] On the other
hand, WGS helps in identifying the transfer of MGEs carrying
AMR genes between various species of Enterobacteriaceae.
Integrons associated with blaIMP among Klebsiella spp.,
Enterobacter cloacae complex, Citrobacter spp. and E. coli
were found to be similar from different regions of the
world.[43]
SNP‑based phylogeny has also aided in the determination
of nosocomial spread of CR K. pneumoniae in about 32
countries in Europe.[44] This enables large‑scale infection
control practices. Average nucleotide identity and genetically
nearest neighbour in the isolate collection were determined to
establish the geographic spread.
Antimicrobial resistance‑specific lineages of Klebsiella
pneumoniae
K. pneumoniae ST258, is the lineage of concern, that carries
plasmid-borne CR gene, blaKPC which confers resistance to all
beta-lactams including carbapenems and cephalosporins.[45]
blaKPC has also been observed in multiple plasmid contexts
within ST258 such as IncFII and IncR. Since multiple
resistance genes are carried on IncF plasmids, this suggests
that this could benefit the successful spreading of epidemic
clones. [31] Figure 2c shows the circulation of AMR K.
pneumoniae clones across the world.
pKpQIL was the first KPC‑encoding plasmid described for
ST258. blaKPC is found on the transposon Tn4401, which
usually spreads through highly conjugative plasmid between
members of Enterobacteriaceae and found K. pneumoniae
ST258 as a highly compatible host.[46] The factors contributing
to the epidemiologic success of this clone still remain unclear;
however, chromosomal or plasmid factors in addition to
AMR may increase the strains fitness. A recent study has
proposed that the dissemination of MDR clones such as ST258
K. pneumoniae and ST131 E. coli could be due to the intergenic
mutations and transcriptional rewiring to overcome fitness cost.
These clones also use anaerobic metabolism to colonise and
disseminate in host.[47]
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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens
Besides, studies showed that antibiotic exposure leads to
the development of resistance strains rather than in‑hospital
transmission.[48] Therefore, it is inevitable to do strain‑typing
for K. pneumoniae to identify the resistant clones that cause
infections in nosocomial settings.
Salmonella spp.
Salmonella spp. is one of the best understood food‑borne
pathogens and one of the leading causes of gastroenteritis in
humans.[49] More than 2500 serotypes have been described
for Salmonella till date and less than 100 serotypes account
for most human infections.[50] Accurate identification and
characterisation of this pathogen is one of the key tasks of
public health laboratory surveillance for outbreak investigation
and long‑term epidemiology.[50]
The pan‑genome of Salmonella enterica is considered
complete since the number of new genes carried by each strain
is not relatively large over the average genome size. S. enterica
and E. coli are close relatives. The recent common ancestor
of S. enterica and E. coli existed about 100 million years
ago, as estimated using the protein clock model.[51] Although
Salmonella is closely related to E. coli, they have an additional
number of virulence genes. Some of which are reported to
be located in the genomic island (GI) of the bacteria, usually
near tRNA genes, which seems to facilitate the integration of
the GIs into the chromosome due to their high conservation.
Many Salmonella‑specific GIs play a major role in virulence
and found to have influence on host specificity as well as on
the degree of invasiveness.[52]
Studies have shown concordant relationship within core
and accessory genome of S. enterica, indicating that the
acquisition of genomic material within accessory genome is
not just a random event, but the selection within specific niches
establishes regulatory elements that enable the survival of the
bacteria. In this regard, the rapid emergence of highly clonal
MDR H58 lineages is found to harbour MDR elements within
the transmissible IncHI1 plasmid. This indicates that certain
lineages may be characterised by the acquisition of specific
accessory genetic markers, which can be used to improve
identification of the source in current epidemics.
Recent studies have shown that combining core genome
analysis with accessory genes, such as pan‑genome‑wide
association studies (pan‑GWASs), has enhanced understanding
on evolutionary and phylogeographic patterns of this
pathogen.[53] Therefore, it is important to study the pan‑genome
of the bacteria to understand why particular clones are more
prevalent or possess particular phenotype.[54]
Molecular typing methods for Salmonella spp.
Classification of Salmonella isolates in outbreak investigation
is generally based on the standard serotyping method, but
now it has been replaced by a range of molecular genotyping
methods. Different typing methods have been employed
for the identification of the source or origin of outbreak
and relatedness of the isolates, includes ribotyping, AFLP,
Indian Journal of Medical Microbiology ¦ Volume 37 ¦ Issue 2 ¦ April-June 2019
MLST, REP‑PCR, ERIC‑PCR, plasmid profiling, MLVA
and PFGE.[55‑57] Among these methods, PFGE is the most
commonly used tool and still remains as a standard method
for Salmonella spp. Sub‑typing for routine surveillance in
microbiology laboratories, worldwide. However, the diversity
of PFGE pattern limits its use in outbreak investigations.[55]
Recently, MLVA has been proposed as a supplementary method
to PFGE for sub‑typing of Salmonella in many countries.
MLVA can able to differentiate the strains that has similar
PFGE pattern. In European countries and Unites States, a
standardised protocol is being used with five or seven VNTR
loci for identification and outbreak investigations.[50] Another
study suggests that MLVA appears a promising alternative to
PFGE sub‑typing Salmonella typhimurium.[58] These suggests
that MLVA can be used for local outbreak analysis due to
its high discrimination and PFGE along with MLVA can be
used for a national‑level data analysis. Banding pattern‑based
molecular sub‑typing methods such as PFGE cannot accurately
sub‑type recently evolved Salmonella serovars. Further,
for long‑term studies of bacterial population structures,
conventional sequence‑based method such as MLST is an
appropriate method but cannot be useful when a species having
high rate of genetic recombination is studied.[58]
A latest study revealed that whole‑genome MLST provides
superior discriminatory power and accurate phylogenetic
inferences with high epidemiological correlation, as well as
provides valuable information on virulence determinants,
drug resistance and genome evolution. WGS has displaced
PFGE as a typing method in both ongoing surveillance and
outbreak investigations.[59,60] However, the method relies on
detailed bioinformatics analysis and not feasible to be a routine
typing method in a low‑resource setting but can be used for
global‑level comparison of the isolates.[50,58]
Antimicrobial resistance‑specific lineages of Salmonella
spp.
The recent rapid emergence of highly clonal multidrug‑resistant
H58 typhoid lineage is a worldwide concern due to its
association with antimicrobial resistance. The H58 isolates
can harbour a complex MDR element residing either on
transmissible IncHI1 plasmids or within multiple chromosomal
integration sites, which may be the primary factor driving
its current expansion. SNP‑based studies suggest that this
clone has followed a specific course of evolution. First,
the strain received an MDR plasmid (IncHI1 plasmid) and
expanded under pressure of first‑line antimicrobials. Second,
chromosomal mutation under fluoroquinolone pressure
extended the clonal expansion. [61] This MDR‑associated
haplotype (H58) has been broadly reported in Asia and locally
in Africa.
Although nontyphoid Salmonella (NTS) predominantly causes
self‑limiting gastrointestinal infections, close to 5% of the
isolates cause invasive bacteraemia.[62] Global monitoring of
Salmonella serovar distribution suggests that S. typhimurium
and S. enteritidis are the most commonly isolated serovars.
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Both S. typhimurium and S. enteritidis have a broad host range
and can cause invasive extra‑intestinal infections, leading
to bacteraemia in humans.[63] Similar to the host adaptation
of Salmonella typhi, recent evolution of S. typhimurium
pathovars to adapt to human hosts found to be an ongoing
process.[64] Phage type DT2, ST ST313 and other monophasic/
non‑motile variants (DT 104) are some of the patho‑variants of
S. typhimurium. Interestingly, patho‑variant ST313 of serovar
typhimurium was considered as the major etiological agent
of fatal bacteraemia in Sub‑Saharan Africa (sSA).[65] Unlike
gastroenteritis causing ST19, ST34 and ST36 genotypes,
ST313 isolates from sSA region are found to have higher
survival rate inside macrophages and cause bacteraemia. MDR
also has contributed to the expansion of S. typhimurium ST313.
Recently, the ST313 clade has acquired resistance to
ceftriaxone, used as a first‑line antibiotic for MDR bacterial
infections. Further, genomic comparison studies showed that
classical ST19 and ST313 shares >4000 genes, and their core
genome differs by ~1000 SNPs.[66] Subsequent genome‑based
studies revealed two distinct phylogenetic lineages of ST313.
Both the lineages are associated with AMR‑mediated by
differing Tn21‑like integrons on the virulence plasmid. The
clonal lineage 1 was replaced by lineage 2 by acquisition of
chloramphenicol resistance.[67] Other than genotype ST313,
S. typhimurium phage types such as DT9, DT204, DT104,
and the current S. 4,[5],12:i: − (monophasic typhimurium
variant) DT193/DT120 are considered as the dominant MDR
pandemic clones.[68] These monophasic phage types harbours
multidrug resistance with antimicrobial susceptibility pattern
of ampicillin, chloramphenicol, streptomycin, sulphonamides
and tetracycline). Phage types such as DT2, DT99 (both variant
copenhagen), DT56, DT56 var, DT40 and DT160 are some
of the other identified S. typhimurium pathovars distributed to
particular zoonotic reservoirs but adapted to human hosts later
on.[68] The global prevalence of typhoidal and non‑typhoidal
clones are illustrated in Figure 2d.
Acinetobacter baumannii
Acinetobacter baumannii is an opportunistic pathogen
responsible for 2%–10% of all Gram‑negative hospital
infections. A. baumannii has reported to acquire resistance to
majority of the antimicrobials in recent decades. The ability
of the pathogen to acquire resistance often through horizontal
gene transfer (HGT) is a contributing factor for the success
of A. baumannii as a nosocomial pathogen.[69] The genome
evolution, resistance acquisition and molecular typing methods
for the identification of MDR clonal lineages in outbreak
settings are described as follows.
The open pan‑genome of A. baumannii is remarkably large
and found to have around 8800 CDS, of which ~1400 are
core genes while ~7300 are acquired genes. This highlights
the significance of gene acquisition and loss events in the
evolution of this human pathogen. The highest contribution
to the species pan‑genome is provided by the accessory genes
that are not shared by all strains, of which 25%–45% of genes
154 Indian Journal of Medical Microbiology ¦ Volume 37 ¦ Issue 2 ¦ April-June 2019
are unique to each strain.[69,70] This suggests that the success
of this species as a human pathogen appears to be associated
with the acquisition of resistance genes through horizontal
gene transfer.
Furthermore, A. baumannii has an ability to adapt to different
habitats; due to a reduction in its genetic diversity along with
the antibiotic pressure, the species led to the evolution and
spread of a highly homogenous clones which are particularly
adapted to the nosocomial environment.[71] The apparent
predominance of a few successful MDR lineages/clones such
as international clone 2 (IC2) worldwide emphasises the
significance of knowledge on the spread and epidemiology
of such A. baumannii strains at national and global level.[72]
Molecular typing methods for Acinetobacter baumannii
Several typing methods have been used to recognise distinct
clonal lineages of A. baumannii during hospital outbreaks such
as PFGE, RFLP, MLST and rep‑PCR.[73] In recent years, MLST
has emerged as the gold standard for classifying A. baumannii
isolates and characterising their continental or global spread
of clonal lineages at national and global level. MLST returns
SNPs in conserved housekeeping genes and therefore more
accurately represents population phylogeny.[74,75]
Currently, two MLST schemes are available for A. baumannii,
the Oxford scheme devised by Bartual et al. and the other is
the Institute Pasteur scheme.[76,77] Each scheme uses seven
housekeeping genes, three of which are shared. Different levels
of resolution have been observed with both the schemes.[78,79]
For example, ST2 under Pasteur scheme covers more than 15
STs in Oxford scheme. Although less resolution with Pasteur
scheme over Oxford scheme has been observed, thus it is
considered significant.[78] Studies have reported that the gpi
gene under Oxford scheme forms a part of the capsule gene
cluster and undergo frequent recombination.[79,80] Another novel
problem with the in silico determination of Oxford profiles is
due to the presence of gdhB paralog, gdhB2.[79] Studies have
suggested that the alleles identified based on the gdhB2 should
be removed from the database and the genomes should be
re‑analysed to identify the correct gdhB allele and the ST.[79]
As a whole, complete agreement has not been achieved with
both the schemes and studies report that to uncover the true
relationships among the isolates, more robust genotyping
methods are required especially with highly dynamic bacterial
pathogen like A. baumannii.[78,79]
Furthermore, studies have reported poor agreement between
PFGE and MLST for A. baumannii and have revealed that
isolates from a single ST type yield markedly different PFGE
patterns. These dissimilar results shows the sensitivity of PFGE
to the changes of accessory genome content and this suggest
that PFGE should not be used to determine relationships
between distantly related bacterial isolates.[74]
Further, the performance of WGS was investigated by various
researchers in outbreak settings and found that the WGS
provided greater resolution than conventional methods in
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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens
typing Acinetobacter at the levels of species identification,
population structure and intra‑hospital outbreak analysis
due to its enhanced discriminatory power.[74] Another study
investigated the epidemiology of CR-A. baumannii outbreaks
have shown that cgMLST can be used for strain sub-typing.
The study also highlights that cgMLST provides additional
information on genetic diversity of the species and can
probably become gold standard for epidemiological typing in
epidemiological investigations.[71]
Antimicrobial resistance‑specific lineages of
Acinetobacter baumannii
Recently, WHO listed Acinetobacter baumannii as the top
priority pathogen in research and for development of new
antibiotics, particularly due to its threat to intensive care
unit setting. [81] There are three main epidemic lineages
of A. baumannii with MDR phenotype that are spread
worldwide and are responsible for the majority of hospital
outbreaks, referred as IC1, IC2 and IC3.[74] The dominant
clone circulating worldwide is CC92 belongs to IC2, which is
associated with class D OXAs along with transposons and/or
insertion sequences. In particular, blaOXA‑23 is the predominant
class D carbapenemase, together with tranposons such as
Tn2006 and Tn2008, and has spread in successful clonal
lineages of A. baumannii.[82] Among the STs of CC92, ST848,
ST451 and ST195 were predominantly reported worldwide
which suggests its global transmission.[83,84] The geographical
distribution of international clones of A. baumannii is shown
in Figure 3.
Staphylococcus aureus
Staphylococcus aureus is one of the major causes of
hospital‑associated infections worldwide. Majority of these
infections are attributed to methicillin‑resistant S. aureus
with the development of AMR. The occurrence of AMR in
S. aureus is well documented and poses significant challenge
to infection control.[85] The genomic features presented here
emphasise the acquisition of resistance driving the emergence
Figure 3: Global spread of international clones of Acinetobacter baumannii with specific to blaOXA‑51 variant. Different international clones are shown
in different shapes and colours
Indian Journal of Medical Microbiology ¦ Volume 37 ¦ Issue 2 ¦ April-June 2019
and spread of MDR MRSA clones that are capable of causing
infection in the community.
S. aureus genome consists of a single circular chromosome
which can range from 2.7 to 3.1 million base pairs.[8,86] The
core genome, which comprises approximately 75% of the
S. aureus genome, is the conserved region across all strains but
not always identical.[8] Genes involved in essential functions,
such as metabolism and survival, comprise the majority of the
genes in the core genome. The accessory genome of S. aureus
contains non‑essential genes that are involved in virulence,
resistance and other metabolic functions.[8,87] The accessory
genome can be highly variable between strains, due to the
HGT of MGEs such as plasmids, transposons, staphylococcal
cassette chromosomes (SCCs) or lysogenic phages.[88] This
exchange is one of the mechanisms by which S. aureus evolves.
There is strong evidence that MGEs contribute to the
emergence of highly virulent and multidrug‑resistant clones;
for example, a clone of the ST772 lineage shows resistance
to six different antibiotics, mostly acquired through HGT
of MGEs.[89] Successful HGT of MGEs is attributed to be
the reason for the evolutionary success and dominance of
certain MRSA clones.[90] Furthermore, recombination plays
an important role in the diversification of MGEs. Large
chromosomal replacements via homologous recombination
are likely to influence the long‑term evolution of MRSA.[91,92]
For example, ST239 is a descendent of ST8 which acquired a
large (635 kb) fragment from ST30[86] and exhibits a mosaic
genome profile.
Molecular typing methods for Staphylococcus aureus
Molecular typing of S. aureus and outbreak investigation is
expansive. Nucleic acid‑based typing approaches including
PFGE, MLST or DNA arrays are commonly used to assess the
epidemiological relatedness of S. aureus.[93] Staphylococcal
protein A (spa) typing of S. aureus is also an important
sequence‑based tool in the study of clonal relatedness and
epidemiology of S. aureus outbreak.[94] Differentiation of
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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens
strains, critically at the genome level, could not achieved using
classical DNA‑based methods.[95]
The investigation of S. aureus are conducted at two levels:
(i) global (extended time frame) and (ii) local (short time
frame).[96] A combination of MLST and SCC mec typing
has been widely used to describe the regional grouping of
MRSA strains.[97] However, these methods do not possess
discriminatory power for the investigation of outbreaks at the
local level. For local investigation, PFGE or MLST along with
spa typing is recommended.[98] A high‑resolution spa typing
was shown to be equally discriminatory to PFGE and can be
used as an alternative method for studying the local MRSA
outbreak.[99] This combinations are appropriate for use in
macro‑epidemiology and evolutionary studies.[100,101]
More recently, WGS provide access to the complete genome,
providing information’s on AMR, virulence, the emergence
and spread of lineages and the population structure of
S. aureus.[102] This approach provides optimal resolution to
infer phylogenetic relatedness, allows accurate characterisation
of transmission events and outbreaks by studying SNVs.[103]
SNVs are considered as the molecular clock to measure the
length of time that takes for the isolates to share a common
ancestor.[103] A study has reported that the global comparison
of ST239 genome revealed the presence of ≤14 SNVs in this
lineage accounts for the population diversity.[104] cgMLST
has reported with higher resolution than MLST and spa
typing for understanding the regional and global S. aureus
epidemiology.[105] Thus, genome analysis would help to link
the genetically related strains having different phenotypes.
Antimicrobial resistance‑specific lineages of
Staphylococcus aureus
I n r e c e n t y e a r s , m e t h i c i l l i n r e s i s t a n t S . a u re u s
(MRSA) epidemiology has changed with the emergence of
community‑associated MRSA (CA‑MRSA) strains. There is
an increasing emergence of CA‑MRSA clones in Africa, Asia
and the Indian sub‑continent.[89] One of such clone, MDR
ST772-MRSA-V which is also called Bengal Bay clone that
was originally isolated from India and Bangladesh has now
become globally disseminated. The clone continued to be
reported in community‑  and healthcare‑associated settings
in India, where it has become one of the dominant epidemic
lineages of CA‑MRSA.[85,106]
There are approximately 11 major S. aureus CCs and most
MRSA isolates in healthcare locations belonging to CC5, CC8,
CC22, CC30 and CC45.[8] In these five CCs, containing both
MRSA and methicillin susceptible S. aureus (MSSA), there
are at least 11 major STs;[107] for example, ST247 is globally
widespread but is commonly known as the Iberian clone,[108]
and the highly transmissible and MDR clone ST239 is known
as the Brazilian clone. There are a number of other major
epidemic (E) MRSA sequence types that are found across
the world; ST5, ST22, ST30 and ST45. Furthermore, MSSA
clones ST8 also contains EMRSA clones that have acquired
SCCmec types II and IV.[107] However, although some strains
156
appear to dominate certain geographic locations, there is still
great diversity seen at one particular location. Figure 4a shows
the global distribution of predominant STs associated with
hospital‑associated MRSA and CA‑MRSA.
Streptococcus pneumoniae
Streptococcus pneumoniae remains a significant risk to
human health and is the leading cause of community‑acquired
pneumonia and meningitis globally. MDR pneumococci
are predominantly related with vaccine serotypes or
serotypes associated with carriage. However, resistant
non‑vaccine‑serotype clones continue to emerge and
expand.[109] S. pneumoniae has received a great attention
from a genomics perspective, with more than 4000 genomes
sequenced till date.[110] Although primary mechanism of
resistance in S. pneumoniae has been identified, genomic
studies continue to provide new insights into evolution and
spread of vaccine and non‑vaccine serotypes of pneumococcal
clones.
S. pneumoniae (the pneumococcus) is a highly recombinogenic
bacterium. The rate of variation through recombination is
much higher than the rate at which the organism acquires
variation through spontaneous mutations.[111] Homologous
recombination, which involves DNA exchange from closely
related loci, occurs in the core genome and can also occur
between MGEs such as plasmids, transposons, bacteriophage,
insertion sequence and integrons. Random mutations and
homologous recombinations are responsible for the variation
in the pneumococcal core genome.[111]
Pneumococci possess a 2.1 megabases (Mb) pair circular
genome that consists of over 2000 predicted protein coding
regions and approximately 5% insertion elements. On average,
the core genome of S. pneumoniae consists of 1647 predicted
coding sequences (including paralogs). The remaining coding
sequences that are not conserved in all members of the species
are collectively referred as the ‘accessory’ genome, which
usually contain dispensable genes that encode proteins that
are not essential to the species; however, it plays a major role
in pathogen evolution.[111] This is largely due to the acquisition
of MGEs that harbour antibiotic resistance determinants
and virulence factors. Being highly recombinant only WGS
can provide high resolution on pneumococcal evolution.
Considering the advantage of pneumococcal WGS, CDC has
initiated Global Pneumococcal Sequencing (GPS) project,
performing WGS free of cost for the S. pneumoniae isolates
from all over the world. Using the global genome dataset,
GPS project has introduced a new scheme for assigning
pneumococcal genomes to Global Pneumococcal Sequence
Clusters (GPSCs) with a reference database https://www.
pneumogen.net/gps/assigningGPSCs. html.[112]
Molecular typing methods for Streptococcus pneumoniae
Recently, it is observed that 74% of the pneumococcal genome
is altered by recombination.[113] High genetic variation demands
continuous genotypic or molecular methods for epidemiologic
typing compared to serotyping. The molecular typing methods
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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens

Figure 4: Global distribution of predominant sequence types or lineages identified in Gram‑positive pathogens is mapped. (a) Sequence types associated
with hospital‑acquired and community‑acquired Staphylococcus aureus infections. (b) Predominance of international pneumococcal lineages, Global
Pneumococcal Sequence Clusters types of Streptococcus pneumoniae and multidrug resistance percentage reported

can be either DNA finger printing or sequence based. The
selection of appropriate method is depending on whether the
interest is short‑term or long‑term epidemiology.[114] Short‑term
studies such as outbreak investigations use molecular
methods based on genes that are highly variable and thereby
provide higher discrimination. Currently, PFGE, MLVA and
penicillin‑binding protein (PBP) typing are the most commonly
used methods for higher discrimination.[115]
Conventional MLST is less discriminatory due to high
recombination events observed in S. pneumoniae but still used
to understand the evolving pneumococcal population over a
long time period. MLVA is based on VNTR loci that are greatly
influenced by environmental factors and highly polymorphic.
However, MLVA can able to further differentiate the isolates
having the same sequence type by MLST.[115] Moreover, MLVA
database consists profiles with unamplified loci making it less
preferable. PFGE is highly discriminative and most widely
used. Since it is gel-based method, data cannot be comparable
with other laboratories. Sequence‑based methods such as
PBP typing have the advantage of reproducible data than
other methods that can be compared between laboratories.
The regional MLST data can be compared globally, with the
availability of large database (www.pubmlst.org). In addition,
PBP typing or PFGE when combined with MLST gives higher
discrimination.[116]
Currently, WGS is the preferred method for understanding
genome evolution of S. pneumoniae. Since the WGS provides
all the whole genome data in a single shot, the sequence can
be analysed in various ways and can be compared with the
global isolates.[117,118] The increase in global whole genome
data raised the need for a genome‑based scheme that can be
used to compare lineages across the countries to assess the
geographical spread of AMR, non‑vaccine types and PCV
impact. The GPS group characterised the global pneumococcal
genome data in to GPSCs, which is the international definition
of pneumococcal lineages.[112] While CCs consist of related

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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens
sequence types grouped by MLST, GPSC consists of cluster
of related CCs which is further divided into sub‑clusters based
on mean pairwise SNP distances. The whole pneumococcal
genome GPSCs were further used to assess lineages
associated with serotype replacement and antibiotic resistance
post‑PCV13 introduction.[119]
Antimicrobial resistance‑specific lineages of
Streptococcus pneumoniae
S. pneumoniae CC320 (ST320) was identified as an MDR
clone responsible for the global emergence of non‑vaccine
serotype 19A in the years following the introduction of
PCV7. The clone is originated from the MDR Taiwan19F‑14
Pneumococcal Molecular Epidemiology Network clone, which
is a double‑locus variant of Taiwan19F‑14– ST236).[120] The
predicted founder of the complex, ST320, was a serotype 19A
clone carrying dual macrolide resistance determinants
(Mega and erm (B)) was the prevalent clone in Asian countries.
The clone represented a 19F‑19A capsule switch and horizontal
acquisition of multiple antibiotic resistance mechanisms,
suggesting that vaccine and antibiotic pressures influenced
its emergence. This highlights the serotype‑specific clonal
evolution of MDR pathogen. Studies suggested that use of
vaccine is not the only factor involved in serotype and ST
changes in a population. It could be due to its highly resistant
nature that contributes to its continued existence in the
post‑vaccine era.[121] In addition, recent findings have suggested
that the ST320 clone was a better coloniser of the nasopharynx
than its progenitor ST236.[110] Based on the new GPSC scheme,
ST320/CC320 belongs to GPSC1 group consists of other CCs,
12229, 2834, 3791 and 420. Considering GPSC association
with resistance, GPSCs and country within GPSCs are found
to be predictors for resistance. Recently, GPSC55 (Israel),
GPSC59 (USA), 138 (USA) and GPSC168 (South Africa)
were found to be associated with 100% penicillin resistance
in the PCV13 era.[119] The predominant GPSCs associated
with resistance and MDR percentages reported in various
countries (SENTRY Surveillance) have been mapped in
Figure 4b.
Figure 5: Schematic representation of metagenomics approach for transmission event analysis of antimicrobial resistant pathogens in a One Health
perspective. PAIs: Pathogenicity islands, AMR: Antimicrobial resistance
158 Indian Journal of Medical Microbiology ¦ Volume 37 ¦ Issue 2 ¦ April-June 2019
Metagenomics: A New Gateway for Transmission
Dynamics and One Health
Resistance has traditionally been viewed as a clinical problem,
but a more holistic approach is needed. The emergence of AMR
is a complex process often involving the interplay between
human, animal and environment. Recently, non‑clinical
environments have been highlighted as an important factor
in the dissemination of ARGs.[122] The emerging field of
metagenomics, the culture‑independent sequencing, has the
potential application in AMR surveillance. Indeed, it has
proven to be useful in investigating novel species, strains and
outbreaks. The success of metagenomics is largely based on
the quality and quantity of the DNA extracted from the sample
due its unique matrix, concentration of the pathogens and
resident microflora.[123]
Metagenomics could also help to track the transmission of
AMR genes and its associated mobile elements.[124] These
sequence data can then be used to predict microbiome,
resistome, virulome, and mobilome.[125] The integration of
genomic data along with epidemiological data could enhance
outbreak investigation and provide potential information on
transmission events [Figure 5].
Challenges of Implementing Next‑Generation
Sequencing in Clinical Laboratories
Although WGS has an excellent discriminatory power, it
has some drawbacks compared to other methods especially
for bioinformatics analysis. Besides, free bioinformatics
tools are widely available, but it is not always easy to find
the appropriate ones. Furthermore, free access software’s
requires bioinformatics expertise and are more laborious and
time‑consuming. The method also requires high computational
power to process and analyse more number of genomes.[58,126]
Furthermore, the ability to translate the sequence data into
relevant information supporting microbiologist, clinicians and
public health epidemiologist to implement control measures in
real‑time is challenging. Further constant update and curation
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Sethuvel, et al.: Molecular epidemiological characterization of GLASS pathogens
of public database are required for the prediction of virulence
and antibiotic resistance from the bacterial genome. In spite of
these above‑mentioned drawbacks, the technology still shows
increased resolution and provides wealth of information which
no other method can accomplish at the same level.
Conclusion
Over the last decade, there are multiple reports of dissemination
of drug‑resistant clones throughout the world. The successful
survival of these bacterial pathogens is probably determined
by a complex of interplay between pathogenicity, epidemicity
and AMR. Earlier studies imply that AMR may be a significant
factor in maintaining existing lineages within specific locales.
In addition, horizontally transferred elements could help
establish the emerging resistant clones. Therefore, more
extensive studies on these lineages can help to understand
the evolution of new clones and spread of the existing clones.
WGS is currently considered as the gold standard for molecular
typing. However, combinations of different typing methods
are widely used for microbial typing and no one method will
be sufficient.
Here, we provide the physicians and microbiologist, an
information on the appropriate use of the different typing
methods to study the molecular epidemiology of outbreaks
caused by GLASS pathogens. Important recommendation are
summarised as follows:
a. WGS is now recommended to be the gold standard method
and prioritised as the method of choice for all outbreak
investigations. The traditional typing methods should be
chosen based on the settings and characteristics of the
outbreak
b. For E. coli and Shigella, WGS is the only appropriate
method for outbreak investigation due to its constantly
changing accessory genome
c. Among the conventional methods, PFGE is still remains
as a standard method for Salmonella spp. Sub‑typing for
routine surveillance. However, currently, MLVA found
to be promising and considered as an alternative method
to PFGE, if WGS is not available
d. PFGE and MLST, in general, are the method of choice
for local outbreak analysis caused by K. pneumoniae and
A. baumannii. MLST can be the early typing method for a
long‑term epidemiological analysis, when a wider view of
a pathogen is needed. Otherwise, WGS provides greater
resolution than conventional typing methods
e. In addition to PFGE or MLST, spa typing is a recognised
methodology for outbreak investigation of MRSA strains.
Combination of MLST and SCCmec typing can be used to
define the regional grouping of MRSA strains. cgMLST
has reported with higher resolution than MLST and spa
typing for phylogenetic relatedness
f. Although MLST is less discriminatory due to high
recombination events observed in S. pneumoniae, it
still can be used for long‑term epidemiological studies.
Targeted sequencing of PBP when combined with MLST
Indian Journal of Medical Microbiology ¦ Volume 37 ¦ Issue 2 ¦ April-June 2019
gives higher discrimination. Recently, WGS is the
preferred method for understanding the evolution of S.
pneumoniae.
Acknowledgement
We gratefully acknowledge the Institutional Review Board of
the Christian Medical College, Vellore, and the Indian Council
of Medical Research, New Delhi.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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