MINI REVIEW

published: 03 May 2016

doi: 10.3389/fmicb.2016.00644

Advances in Molecular Serotyping

and Subtyping of Escherichia coli†
Pina M. Fratamico 1*, Chitrita DebRoy 2 , Yanhong Liu 1 , David S. Needleman 1 ,
Gian Marco Baranzoni 1 and Peter Feng 3
1
Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA,
USA, 2 Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA, USA,
3
Division of Microbiology, U.S. Food and Drug Administration, College Park, MD, USA

Escherichia coli plays an important role as a member of the gut microbiota; however,
pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and
extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have
traditionally been serotyped using antisera against the ca. 186 O-antigens and 53
H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and
H- serotyping for differentiating and characterizing E. coli have been used for many
years; however, these methods are generally time consuming and not always accurate.
Advances in next generation sequencing technologies have made it possible to develop
genetic-based subtyping and molecular serotyping methods for E. coli, which are more
discriminatory compared to phenotypic typing methods. Furthermore, whole genome
Edited by: sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsed-
Feng Gao, field gel electrophoresis, providing a major advancement in the ability to investigate
Tianjin University, China
food-borne disease outbreaks and for trace-back to sources. A variety of sequence
Reviewed by:
analysis tools and bioinformatic pipelines are being developed to analyze the vast
Andrea Isabel Moreno Switt,
Universidad Andrés Bello, Chile amount of data generated by WGS and to obtain specific information such as O- and
Séamus Fanning, H-group determination and the presence of virulence genes and other genetic markers.
University College Dublin, Ireland
*Correspondence: Keywords: Escherichia coli, molecular serotyping, subtyping, detection, identification, whole genome
sequencing, O-group, H-type
Pina M. Fratamico
[email protected]

Specialty section: INTRODUCTION

This article was submitted to
Evolutionary and Genomic Escherichia coli strains are commensal organisms that are part of the normal intestinal microflora
Microbiology, of humans and other mammals. The traditional method for identifying E. coli uses antibodies
a section of the journal to test for surface antigens: the O- polysaccharide antigens, flagellar H-antigens, and capsular
Frontiers in Microbiology
K-antigens (described below). There are currently ∼186 different E. coli O-groups and 53 H-types,
Received: 26 February 2016 so serotyping is highly complex. There are also many pathogenic groups of E. coli that cause disease
Accepted: 18 April 2016 in humans and animals, including diarrheagenic E. coli and the extra-intestinal pathogenic E. coli
Published: 03 May 2016
(ExPEC) that cause illness outside of the GI-tract. Diarrheagenic E. coli that cause human illness
Citation: have been classified based on specific sets of virulence genes they carry and the characteristics
Fratamico PM, DebRoy C, Liu Y, of the disease they cause (Kaper et al., 2004). These pathotypes include the enteropathogenic
Needleman DS, Baranzoni GM
E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative
and Feng P (2016) Advances
in Molecular Serotyping
E. coli (EAEC), Shiga toxin-producing E. coli (STEC), diffusely adherent E. coli (DEAC), and
and Subtyping of Escherichia coli.
Front. Microbiol. 7:644. † Mention of trade names or commercial products is solely for the purpose of providing specific information and does not

doi: 10.3389/fmicb.2016.00644 imply recommendation or endorsement by the U.S. Department of Agriculture.

Frontiers in Microbiology | www.frontiersin.org 1 May 2016 | Volume 7 | Article 644

Fratamico et al. E. coli Subtyping and Molecular Serotyping

adherent invasive E. coli (AIEC) that have been associated with Rapid and accurate molecular methods are critically needed
Crohn’s disease. There are also hybrid pathotypes, including the to detect and trace pathogenic E. coli in food and animals
enteroaggregative hemorrhagic E. coli (EAHEC) that carry STEC- and for epidemiological investigations to enhance food safety
and EAEC-associated virulence genes. As an example, EAHEC and animal and human health, as well as to minimize the size
serotype O104:H4, an EAEC that acquired the phage that carried and geographical extent of outbreaks. As opposed to traditional
the Shiga toxin gene of STEC, caused a large outbreak in 2011 serotyping using antisera raised against the different E. coli O-
associated with illness in over 3800 individuals and 54 deaths and H-types, molecular serotyping generally refers to genetic-
(Frank et al., 2011). Certain E. coli serotypes are often associated based assays targeting O-group-specific genes found within the
with specific pathotypes, such as STEC O157:H7 and O103:H21 E. coli O-antigen gene clusters and the H-antigen genes that
(Kaper et al., 2004) that are important STEC, often referred to encode for the different flagellar types. Although determining the
as enterohemorrhagic E. coli (EHEC). Therefore, pathogenic E. E. coli serotype could be considered a component of subtyping
coli constitutes a genetically heterogeneous family of bacteria, and (differentiation beyond the species level), methods used for
they continue to evolve. molecular subtyping such as pulsed-field gel electrophoresis
Extra-intestinal pathogenic E. coli cause illness outside of (PFGE), multilocus sequence typing (MLST), and whole genome
the gastrointestinal tract, including urinary tract infections, sequencing (WGS) generate a unique “fingerprint” of the
meningitis, pneumonia, septicemia, and other types of infections bacterium that can be used in outbreak investigations and to
(Russo and Johnson, 2003; Smith et al., 2007). ExPEC that determine the source of illnesses. There are many problems
cause illness in poultry are known as avian pathogenic E. coli associated with traditional serotyping for determining the
(APEC). Avian colibacillosis caused by APEC is a major cause E. coli O- and H-groups. It is costly, labor-intensive and
of morbidity and mortality associated with economic losses in time consuming, cross reactivity of the antisera with different
the poultry industry throughout the world. The human gut is serogroups occurs, antisera are available only in specialized
a reservoir for ExPEC that cause human illness. When ExPEC laboratories, batch-to-batch variations in antibodies can occur,
leave the GI tract and infect other parts of the body such and many E. coli strains isolated from various sources are non-
as the urinary tract, the blood, or the lungs, illness results typeable (Lacher et al., 2014). Thus, molecular serotyping offers
(Smith et al., 2007). Animals, particularly, poultry and poultry alternative methods for E. coli serotyping, and furthermore, they
products (eggs), pork/pigs, and beef/cattle, and also companion can be coupled with assays for specific virulence gene enabling
animals may carry ExPEC, and thus, these pathogens may be the determination of O- and H-group, pathotype, and the strain’s
acquired through the food supply, and zoonotic pathogens may pathogenic potential simultaneously.
also be acquired via contact with animals (Vincent et al., 2010;
Nordstrom et al., 2013; Mitchell et al., 2015; Singer, 2015).
Investigations of community-acquired UTI and outbreaks of E. coli O-, K-, AND H-ANTIGENS
UTI suggested common point sources, such as contaminated
food products (Nordstrom et al., 2013). Indeed, high genetic The outer membrane of E. coli is composed of
similarity, including antibiotic resistance and virulence gene lipopolysaccharides (LPS) that includes lipid A, core
patterns, between APEC and ExPEC strains causing disease in oligosaccharides, and a unique polysaccharide, referred to
poultry and humans, respectively, has been observed (Smith et al., as the O-antigen. Loss of the O-antigens results in attenuated
2007; Manges and Johnson, 2012). The ability to differentiate virulence suggesting their importance in host–pathogen
commensal E. coli from ExPEC and other pathotypes is important interactions (Sarkar et al., 2014). Based on the antigenic
for risk assessment and epidemiological and ecological studies. diversity among the different O-antigens, they have been
However, a rapid and reliable typing/identification system or targeted as biomarkers for classification of E. coli since the
criteria that allows this type of discrimination and that also 1940s (Kaufmann, 1943, 1944, 1947). Later, Ørskov et al.
provides information on the organism’s evolutionary history, (1977) presented a comprehensive serotyping system for 164
fitness, and pathogenic potential has not yet been established. E. coli O-groups and developed a typing scheme based on the
Determining whether an E. coli strain is an ExPEC and whether it presence of three principal surface antigens, O-antigens, flagellar
is pathogenic is based on its source, O:K:H serotype, phylogenetic H-antigens, and capsular K-antigens. Since few laboratories
background, virulence factor profile, and experimental virulence had capabilities to type the K antigen, serotyping based on O-
in an animal model. ExPEC belong to specific phylogenetic and H-antigens became the gold standard for E. coli typing.
groups (A, B1, B2, and D) determined based on multilocus Currently, O-groups numbered O1-O188 have been defined,
enzyme electrophoresis, ribotyping, or by triplex PCR targeting except for O31, O47, O67, O72, O94, and O122 that have not
the genes chuA and yjaA and a particular DNA fragment known been designated (Ørskov and Ørskov, 1984; Scheutz et al., 2004),
as TSPE4.C2. ExPEC strains belonging to phylogenetic groups B2 and four groups have been divided into subtypes O18ab/ac,
and D show higher virulence in humans (Clermont et al., 2000; O28ab/ac, O112ab/ac, and O125ab/ac, giving a total of 186
Smith et al., 2007). It has become evident that certain ExPEC O-groups.
lineages or clonal groups are responsible for a large fraction of The conventional serotyping method is based on agglutination
human extraintestinal E. coli infections, and these lineages are reactions of the O-antigen with antisera that are generated
becoming increasingly multi-drug resistant (Smith et al., 2007; in rabbits against each of the O-groups (Ørskov and Ørskov,
Manges and Johnson, 2012). 1984). The method is easy to carry out; however, it is

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Fratamico et al. E. coli Subtyping and Molecular Serotyping

laborious and error-prone, and thus, molecular methods are acid differences within the central region, which is the surface-
better alternatives for O-typing (Ballmer et al., 2007; Lacher exposed antigenic part of the flagellar filament (Namba et al.,
et al., 2014). The genes that encode for O-antigens are 1989). Thus, PCR methods developed to distinguish H-types
located on the chromosome in a cluster designated as the target the variable region of the fliC gene (Machado et al.,
O-antigen gene cluster (O-AGC). These are flanked by two 2000); however, these regions of some H-types such as H1
conserved sequences called JUMPstart, a 39 bp-element at the and H12 and H25 and H28 are very similar, making them
50 end (Hobbs and Reeves, 1994), which is downstream of difficult to distinguish. However, a two-step PCR method was
galF (UTP-glucose-1-phosphate uridylyltransferase) and gnd (6- developed that can distinguish between fliCH1 and fliCH12
phosphogluconate dehydrogenase) at the 30 end. Analysis of (Beutin et al., 2015, 2016). Other methods such as Matrix-
the O-AGCs of all E. coli O-groups (Iguchi et al., 2015a; Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-
DebRoy et al., 2016) showed that the sizes of the O-AGCs TOF)-based peptide mass fingerprinting in conjunction with a
and their gene content vary considerably, which results in custom E. coli H-antigen data base (Cheng et al., 2014) has been
the variability of O-antigens. O-antigens are composed of 10– also utilized to distinguish H-types (Chui et al., 2015).
25 repeating units of two to seven sugar residues and are
processed by three mechanism of which the most common
is Wzy (O antigen polymerase) dependent, followed by an METHODS USED FOR SUBTYPING AND
ABC transporter dependent system, and the third mechanism, MOLECULAR SEROTYPING OF E. coli
which involves a synthase dependent pathway (Greenfield
and Whitfield, 2012) by which the O-antigens are flipped Subtyping methods that allow for differentiation of E. coli beyond
across the outer membrane. The pathways for biosynthesis of the species and subspecies level are critical for determining
the O-AGCs and assembly of O-antigens have been studied the source of outbreaks and establishing transmission pathways
extensively (Samuel and Reeves, 2003). Each of the O-antigens (Eppinger et al., 2011; Frank et al., 2011). Several phenotype-
that utilize Wzy-dependent pathway carries two unique genes based and genotype-based methods for subtyping E. coli are listed
wzx (O-antigen flippase) and wzy (O-antigen polymerase). Wzx in Table 1. Phenotypic culture methods, in conjunction with
proteins translocates the O-units across the inner membrane, biochemical-based testing, serotyping, phage typing, multilocus
and Wzy polymerizes the O-antigen (Samuel and Reeves, 2003). enzyme electrophoresis have been used for many years
For the ABC transporter-dependent pathway, wzm (O-antigen and could be considered gold standard methods; however,
ABC transporter permease gene) and wzt (ABC transporter they are time and labor intensive and may not be very
ATP-binding gene) are involved in O-AGC synthesis. The discriminatory.
O-AGCs are composed of nucleotide sugar biosynthesis genes Compared to phenotypic methods, genetic subtyping methods
that are involved in the synthesis of O-antigen nucleotide sugar that are based on bacterial DNA, generally have better
precursors, the glycosyl transferases that transfer the various discriminatory ability. Of the various methods used for E. coli
sugar precursors to form the oligosaccharide, and the O-antigen subtyping, PFGE is a reliable and highly discriminating method
processing genes described above. and has been considered to be the “gold standard” of typing
All of the O-AGC clusters have been sequenced, and sequence methods. Through the establishment of PulseNet (Ribot et al.,
analyses revealed that some O-AGCs are 98–100% identical 2006), use of PFGE has had a major impact on pathogen
(Iguchi et al., 2015a; DebRoy et al., 2016) while others have subtyping and outbreak investigation.
point mutations or insertion sequences which causes these to In contrast to traditional serotyping, Luminex -based R

type as different serogroups (Liu et al., 2008, 2015) . Therefore, suspension assays allow for simultaneous testing for multiple
there is a need to resolve these discrepancies, merge or eliminate serogroups in a single assay. Lin et al. (2011) performed
serogroups and to revise the E. coli serotype nomenclature PCR assays targeting the wzx and wzy genes of ten Shiga
(DebRoy et al., 2016). Furthermore, many of the E. coli toxin-producing E. coli (STEC) serogroups, and then used the
O-AGCs have been found to be identical to those of other Luminex system to identify the 10 serogroups through binding
R

Enterobacteriaceae members such as Shigella and Salmonella of the PCR products to fluorescent microspheres conjugated to
(Wang et al., 2007). Out of 34 distinct Shigella O-antigens, 13 specific DNA probes for each of the ten serogroups. Clotilde
were unique to Shigella; however, the other 21 were also found et al. (2015) used the Luminex technology, both antibody- and
R

in E. coli (Liu et al., 2008). Similarly, out of 46 O-AGCs of multiplex PCR-based, and compared them to traditional E. coli
Salmonella, 24 of were found to be identical or closely related to serotyping. The results of the two Luminex assays were mostly
R

E. coli O-antigens (Liu et al., 2014). consistent, and 11 STEC isolates that were previously untypeable
Serology has defined 53 H-flagellar antigens (Ørskov and by traditional serotyping were able to be typed.
Ørskov, 1984; Ewing, 1986) that are numbered from H1 to Multiplex PCR-based assays targeting unique regions within
H56, but H-types 13, 22, and 50 are not in use (Ørskov et al., the E. coli O-AGCs have been used to determine the O-groups.
1975; Centers for Disease Control and Prevention [CDC], 1999). A review by DebRoy et al. (2011) describes many of these
Molecular H-typing methods are based on the sequences of fliC assays, most of which target the E. coli wzx and wzy genes.
gene that encode for the FliC, the flagellar filament structural Based on O-AGC sequence data for all O-groups, Iguchi et al.
protein (Wang et al., 2003). The N- and C-terminals of FliC (2015b) designed 162 PCR primer pairs for identification and
are highly conserved, so different H-types are due to amino classification of E. coli O-serogroups. The primer pairs were

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Fratamico et al. E. coli Subtyping and Molecular Serotyping

TABLE 1 | Phenotype- and genotype-based methods for subtyping and molecular serotyping of E. coli.

Phenotype-based methods O-antigena H-antigena Virulence- SNPs and Reference

related and other markers
other genes

Immunological O/H typing (serotyping) X X Ørskov et al., 1977

Bacteriophage typing X Ahmed et al., 1987;
Krause et al., 1996
Multilocus enzyme electrophoresis X Ochman and Selander, 1984;
(MLEE) Selander et al., 1986;
Campos et al., 1994
MALDI-TOF X X Chui et al., 2015
Genotype-based methods
Restriction length polymorphism (RFLP) X X X Coimbra et al., 2000;
Beutin et al., 2005;
Moreno et al., 2006;
Abbadi and Strockbine, 2007
Luminex-based suspension assay X Lin et al., 2011;
Clotilde et al., 2015
Amplified fragment length X Hahm et al., 2003;
polymorphisms (AFLP) Leung et al., 2004
Optical mapping X X Kotewicz et al., 2007, 2008;
Miller, 2013
Ribotyping X Martin et al., 1996;
Carson et al., 2001
Multilocus variable number tandem X Hyytiä-Trees et al., 2006;
repeat analysis (MLVA) Byrne et al., 2014
Pulsed-field gel electrophoresis X X Krause et al., 1996;
Ribot et al., 2006
Multilocus sequence typing (MLST) X Eichhorn et al., 2015;
Manges et al., 2015
High throughput real-time PCR X X X Bugarel et al., 2010a,b, 2011a,b;
Delannoy et al., 2013;
Tseng et al., 2014
Multiplex PCR X X X Fratamico and DebRoy, 2010;
Botkin et al., 2012; Doumith
et al., 2012; Fratamico et al.,
2014; Iguchi et al., 2015b
Whole genome sequencing and SNP X X X X Zhang et al., 2006; Eppinger
analysis et al., 2011; Norman et al., 2012,
2015; Joensen et al., 2014, 2015;
Griffing et al., 2015; DebRoy
et al., 2016; Ison et al., 2016
Virulence gene profiles X Nandanwar et al., 2014;
Manges et al., 2015
CRISPRs X Shariat and Dudley, 2014;
Delannoy et al., 2015
Microarray X X X X Liu and Fratamico, 2006; Hegde
et al., 2013; Lacher et al., 2014
NeoSEEKTM (PCR-mass spectroscopy) X X X X Stromberg et al., 2015
a The O-somatic and H-flagellar antigens define the E. coli serotype. Agglutination assays using antibodies that react with the specific O- and H-antigens are the basis for
traditional serotyping. Molecular serotyping methods are generally based on genetic targets specific to the O- and H-antigens.

used in 20 separate multiplex PCR assays with each assay EHEC-like O26 pathogenic strains, and avirulent O26 strains
containing 6–9 primer pairs that amplified products of different (Bugarel et al., 2011a).
sizes so that they could be distinguished. A high-throughput Clustered regularly interspaced short palindromic repeats
PCR method based on the GeneDisc array targeted virulence
R
(CRISPR) are short, highly conserved DNA repeats separated
genes and O- and H-type-specific genes for identification of by unique sequences of similar length, and they have been used
STEC associated with severe illness (Bugarel et al., 2010b). for subtyping, identification, and detection of bacteria (Shariat
Another high-throughput method, known as the BioMarkTM and Dudley, 2014). Based on spacer content or sequencing
real-time PCR system (Fluidigm), used a panel of virulence genes of CRISPR loci, CRISPR-based typing analyses can be used
as discriminative markers to differentiate EHEC O26 strains, to differentiate strains for epidemiological investigations or

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Fratamico et al. E. coli Subtyping and Molecular Serotyping

for detection. Delannoy et al. (2012) utilized CRISPR loci of some challenges need to be addressed before standardization
seven important EHEC serotypes to develop real-time PCR and full implementation of this technology. The bioinformatic
assays, generating results based on CRISPR polymorphisms that analyses required to analyze enormous amounts of sequence
correlated with specific EHEC O:H serotypes and the presence of data generated by WGS are necessitating the development of
EHEC virulence genes. analysis pipelines to enhance the assembly, annotation, and
DNA microarrays have also been developed for molecular interpretation of the data, which will require a coordinated
serotyping of E. coli (Liu and Fratamico, 2006; Ballmer et al., international approach (Franz et al., 2014; Oulas et al., 2015).
2007; Geue et al., 2014; Lacher et al., 2014). One microarray Currently, the following databases for WGS and advanced
method to identify E. coli serogroups involved spotting O-group- detection are available: the 100K Genome Project1 , GenomeTrakr
specific wzx or wzy gene oligonucleotides or PCR products onto Network2 , Global Microbial Identifier3 , and Advanced Molecular
the chip and hybridized with labeled PCR products of the entire Detection4 . These databases are creating a vast resource of
O-AGCs (Liu and Fratamico, 2006). Lacher et al. (2014) reported microbial genome information for WGS-based surveillance of
on the use of an FDA-ECID (E. coli identification) microarray for microbial pathogens. Furthermore, detailed analysis of WGS
O- and H-typing of E. coli. The ECID chip was designed based on data can determine the E. coli O- and H-type and provide
>250 E. coli genomes and incorporates over 40,000 E. coli genes, information on the resistome (antibiotic resistance gene profile)
including O- and H-group-specific genes, and approximately of the isolate, and the presence of specific virulence genes,
9800 single nucleotide polymorphisms (SNPs). Antibody-based prophages, and plasmids, as well as other genetic information
microarrays have also been developed to detect important non- important to identify E. coli pathotypes as well as utility in
O157 STEC serogroups (Gehring et al., 2013; Hegde et al., 2013). evolutionary studies. The advantages of WGS approaches are
Although this method is rapid and has the potential to be used being recognized by academic, government, industry, and the
for high throughput screening, the utilization of this method is private sector for addressing regulatory and public health needs.
dependent on the availability of antibodies with good specificity. However, as we move toward the use of these genetic approaches
The commercial introduction of next-generation sequencing for non-culture-based detection, characterization, subtyping,
technologies has made it possible to perform routine WGS of trace backs, and outbreak investigations, it will be critical to
E. coli and other bacteria relatively rapidly and at affordable establish bioinformatics pipelines that are capable of analyzing
costs (Franz et al., 2014). Since WGS typing has discriminatory and handling the large amounts of data that are generated.
power superior to other typing methods, it has the potential
to revolutionize bacterial subtyping. A MLST webserver was
designed to determine sequence types (STs) of bacteria using AUTHOR CONTRIBUTIONS
WGS data. STs were determined from uploaded preassembled
complete or partial genome sequences or short sequence reads PF, CD, YL, DN, GB, and PF have made a substantial, direct,
obtained from different sequencing platforms (Larsen et al., and intellectual contribution to the work and approved it for
2012). Based on SNPs observed from WGS data, Norman publication.
et al. (2015) identified unique STEC O26 genotypes in human
and cattle strains. These isolates had similar virulence gene
profiles and did not cluster in separate polymorphism-derived
genotypes, and thus human and cattle strains could not be
FUNDING
distinguished within the phylogenetic clusters. An approach
This work was supported in part by an appointment to the
based on targeted amplicon sequencing for SNP genotyping
Agricultural Research services (ARS) Research Participation
was used to determine the relationship of stx-positive and stx-
Program which is administered by the Oak Ridge Institute
negative E. coli O26:H11 strains from cattle compared to the
for Science and Education (ORISE) through an interagency
genomes of human clinical isolates (Ison et al., 2016). Joensen
agreement between the U.S. Department of Energy (DOE) and
et al. (2015) described SerotypeFinder, a publicly available
the USDA. ORISE is managed by ORAU under DOE contract
web tool hosted by the Center for Genomic Epidemiology,
number DE-AC05-06OR23100. All opinions expressed in this
Denmark, which enables WGS-based serotyping of E. coli. Typing
manuscript are the author’s and do not necessarily reflect the
is based on wzx, wzy, wzm, and wzt, as well as flagellin-
policies and views of USDA, ARS, DOE, or ORAU/ORISE.
associated genes. Similar to SerotypeFinder, the VirulenceFinder
tool can be used to determine virulence genes in E. coli 1
http://100kgenome.vetmed.ucdavis.edu/
to determine different pathogenic groups (Joensen et al., 2
http://www.fda.gov/Food/FoodScienceResearch/WholeGenomeSequencing
2014). ProgramWGS/
3
Whole genome sequencing typing has the potential to be http://www.cdc.gov/amd/project-summaries/index.html
4
the new “gold-standard” for pathogen subtyping. However, http://www.globalmicrobialidentifier.org/

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Fratamico et al. E. coli Subtyping and Molecular Serotyping

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bacterial population genetics and systematics. Appl. Environ. Microbiol. 51, Attribution License (CC BY). The use, distribution or reproduction in other forums
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