Genomic Characteristics Behind the Spread of Bacteremic Group A Streptococcus Type emm89 in Finland, 2004–2014
Correspondence: J. Vuopio, Department of Medical Microbiology and Immunology, University
of Turku, Kiinamyllynkatu
Genomic Characteristics Behind the Spread of Bacteremic Group A Streptococcus Type emm89 in Finland, 2004-2014
Francesca Latronico 0 2
Waleed Nasser 3
Kai Puhakainen 1 2
Jukka Ollgren 2
Hanne-Leena Hyyryläinen 2
Stephen B. Beres 3
Outi Lyytikäinen 2
Jari Jalava 2
James M. Musser 3
Jaana Vuopio 1 2
0 European Programme for Public Health Microbiology Training, European Centre for Disease Prevention and Control , Stockholm , Sweden
1 Department of Medical Microbiology and Immunology, University of Turku , Finland
2 Department of Infectious Diseases, National Institute for Health and Welfare , Helsinki
3 Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute , Texas
Background. Many countries worldwide have reported increasing numbers of emm89 group A Streptococcus (GAS) infections during last decade. Pathogen genetic factors linked to this increase need assessment. Methods. We investigated epidemiological characteristics of emm89 GAS bacteremic infections, including 7-day and 30-day case-fatality rates, in Finland during 2004-2014 and linked them to whole-genome sequencing data obtained from corresponding strains. The Fisher exact test and exact logistic regression were used to compare differences between bacteremic infections due to emm89 GAS belonging to different genetic clades and subclades. Results. Out of 1928 cases of GAS bacteremic infection, 278 were caused by emm89 GAS. We identified 2 genetically distinct clades, arbitrarily designated clade 2 and clade 3. Both clades were present during 2004-2008, but clade 3 increased rapidly from 2009 onward. Six subclades (designated subclades A-F) were identified within clade 3, based on phylogenetic core genome analysis. The case-fatality rate differed significantly between subclades (P < .05), with subclade D having the highest 30-day estimated case-fatality rate (19% vs 3%-14%). Conclusions. A new emm89 clone, clade 3, emerged in 2009 and spread rapidly in Finland. Patients infected with certain subclades of clade 3 were significantly more likely to die. A specific polymerase chain reaction assay was developed to follow the spread of subclade D in 2015.
Group A Streptococcus (GAS) is a gram-positive
human-adapted pathogen with the ability to cause diseases with a wide
clinical range, from mild infections such as pharyngitis to severe
infections such as bacteremia and necrotizing fasciitis. When
the isolation of GAS from a normally sterile body site is
associated with disease, it is referred as an invasive GAS (iGAS)
infection. Worldwide, GAS infections are responsible for >600
million cases of mild infections and for 663 000 new cases of
iGAS per year, with incidence rates ranging from 2.45 to
46.00 cases per 100 000 population and case-fatality rates
(CFRs) of up to 25% [1, 2].
The emm gene, which encodes M protein, one of the most
important GAS virulence factors, is a target for genotyping.
To date, >240 emm types have been reported, and genetic
diversity can exist among strains of the same emm type [3, 4].
Dynamic changes and fluctuations in emm type frequency in
iGAS infection occur over time and space , including
emergence and replacement of new clones or subtypes [5–7]. Some
emm types, such as emm1, have been linked to more severe
disease manifestations and higher CFRs [4, 8–10].
Our recent study on iGAS infection in Finland showed that
although the overall incidence of iGAS infections remained
relatively stable during last few years, the incidence of cases caused
by emm89 strains increased . Similar findings have been
reported from other countries in Europe [4, 6, 12–15], North
America [7, 9, 16], and elsewhere [17–19].
The emergence of emm89 GAS strains has led investigators to
perform bacterial genomic studies to understand reasons
behind it. Thus far, the study of the largest sample of 1125
emm89 isolates, covering 2003–2013 from 3 countries,
including Finland, revealed that emm89 strains underwent
recombinational replacement events previously reported for the successful
intercontinental epidemic emm1 clone [5, 7].
The goal of this study was to analyze, in detail, temporal,
spatial, genomic, and clinical aspects of emm89 bacteremic infections
in Finland during 2004–2014. We linked the epidemiological
information with whole-genome sequencing (WGS) data for
the corresponding 272 strains. We found that progeny of a
new genetic clone (designated clade 3) emerged in 2009,
spread rapidly in Finland, and caused a significantly increased
number of iGAS infections throughout the country. Patients
infected with certain subclades of this new clone were
significantly more likely to die from the infection. A specific
polymerase chain reaction (PCR) assay was developed to permit
rapid identification of a distinct subclade for epidemiologic
MATERIALS AND METHODS
Surveillance of Bacteremic Infections Due to GAS
The Finnish healthcare system consists of 20 healthcare
districts that are organized in 5 tertiary healthcare districts
( population range, 740 000–1 900 000), for this study
designated as southern, eastern, northern, central, and western
Finland. Since 1995, all clinical microbiological laboratories
electronically report each GAS isolate cultured from blood
and/or cerebrospinal fluid to the National Infectious Disease
Register (NIDR) maintained by the National Institute for
Health and Welfare (THL). Each notification includes patient
demographic data (date of birth, sex, and age), information
regarding the specimen (type and date of sampling), name of the
laboratory, date of notification, place of treatment, and, since
2004, the national personal identity code. Through the
personal identity code, NIDR data are linked to the National
Population Register (NPR) to obtain place of residence and date of
death. In case of multiple notifications from the same patient
within 90-day interval, notifications are merged and reported
as a single case. In addition, clinical microbiological
laboratories submit the corresponding GAS isolate to the National
Reference Laboratory (NRL) for further analysis such as emm
Case Definition and Outcome
A case of bacteremic GAS disease was defined as isolation of
Streptococcus pyogenes (GAS) from a blood culture in Finland
during 2004–2014 and as an emm89 GAS bacteremic infection
when emm89 was specifically detected. All bacteremic GAS
cases reported to the NIDR (n = 1977) and all available
corresponding isolates (n = 1928) submitted to NRL from January
2004 to December 2014 were included in this study. Data
about deaths of bacteremic GAS cases with appropriate
personal identity codes (n = 1915) within 7-day and 30-day follow-up
periods after GAS blood isolation were obtained from the
National Population Register and used to assess 7-day and 30-day
CFRs, respectively. In case of suspected epidemiological link
among emm89 bacteremic GAS cases, medical records were
reviewed by infectious diseases physicians at healthcare districts
to assess common exposure or contacts between the patients
and to identify underlying conditions known as risk factors
All isolates sent to the NRL were analyzed for emm type and
subtype according to the guidelines of the Centers for Disease
Prevention and Control (Atlanta, GA) .
Methods used for genome sequencing and analysis were
recently described [5, 7, 22]. Briefly, after chromosomal DNA
extraction from overnight cultures using the DNeasy 96 Blood and
Tissue Kit (Qiagen), sequencing libraries were prepared using
Nextera XT DNA Sample and Nextera XT Index V2 Prep kits
(Illumina) and sequenced using Illumina instruments
(HiSeq2500, NextSeq, and MiSeq). Raw reads were quality
filtered using Trimmomatic , and errors were corrected
using Musket . Reads were aligned to the emm89 clade 3
reference genome MGAS27601 , using SMALT software
(Wellcome Trust Sanger Institute), while genetic variant calls
compared to the reference genome were detected using FreeBayes
software. Single-nucleotide polymorphism (SNP) multiple
sequence alignments were generated using Prephix and Phrecon
scripts (available at: https://github.com/codinghedgehog).
Genetic distances among strains and branches were calculated using
MEGA6  and R statistical packages (available at: https://
www.r-project.org/). De novo assemblies of large contigs
containing the ndrI to fabG chromosomal RB-15 region were
generated using SPAdes software . The nature of each SNP
and INDEL category (coding/noncoding and synonymous/
nonsynonymous) was interrogated using the in-house–developed
script SNPfx.pl. All genome sequence data were deposited under
accession number SRP059971 and SUB1086963 in the Sequence
Read Archive (National Center for Biotechnology Information).
PCR to Identify an Emerging emm89 Subclade
A specific PCR targeting the nrdl to fabG chromosomal region
was developed to rapidly identify emm89 bacteremic GAS strains
belonging to clade 3 subclade D. Primer 1 (5′-GACTAAAA
CAGCTAAGAAGAAGGAC -3′) was designed to detect all
subclades, primer 2 (5′-CATACAAGGTACTATCTTCCTCAAG-3′)
to be specific for A-B-C-E-F subclades, and primer 3
(5′GTCTCTTTTTGATATCACCTCC-3′) to be specific for subclade
D. The expected size of amplified products was 626 bp for
subclade D and ≥1034 bp for all other subclades. Strains were
cultivated overnight on blood agar at 35°C with 5% CO2, and DNA
was extracted from bacterial colonies after suspension in 150 µL
of Milli-Q water, incubation at 99°C for 10 minutes, and
centrifugation at high speed for 5 minutes. Each reaction contained
15.5 µL of nuclease-free water, 25 µL of GoTaq Master Mix
(G2 Hot Start polymerase, Promega), 2.5 µL of each primer
(10 µM), and 2 µL (50–100 ng) of DNA template. PCR was
performed using T100 Thermal Cycler (Biorad, Singapore) with
initial denaturation at 95°C for 2 minutes; 30 cycles at 95°C for 30
seconds, 55°C for 30 seconds, and 72°C for 1 minute and 10
seconds; and final extension for 2 minutes at 72°C. ATCC 700294
was used as positive control.
Data Analysis and Statistics
NIDR data for notified cases and emm typing results for
corresponding isolates were linked using personal identity code and
date of specimen. Annual incidence rates for all bacteremic GAS
and emm89 bacteremic GAS in the entire country and in each of
the 5 tertiary healthcare districts were calculated using
population data for the corresponding year as reported by Statistics
Finland. CFRs within 7-day and 30-day follow-up periods
after emm89 bacteremic GAS isolation was calculated for clades
and subclades within emm89 bacteremic GAS cases. Fisher
exact test and exact logistic regression were used to compare
differences in 7-day and 30-day CFRs. Yearly overall and district
relative increases in emm89 proportion were estimated by
binary regression with log-link. Differences were considered
significant when P values were < .05. Data were analyzed with Stata 13
(Statacorp, College Station, Texas).
Ethical Study Approval
Ethical committee clearance was not required as all aspects of
this study fall within THL legal mandate defined by the Finnish
Communicable Diseases Act . No personal information
concerning cases was shared with non-THL investigators.
During 2004–2014, 1928 bacteremic GAS cases were identified
(range by year, 112–218 cases). The median age of cases was 54
years (range, 0–100 years), and 54% were males. The average
annual incidence rate during 2004–2014 was 3.3 cases per
100 000 population (range by year, 2.1–4.1 cases per 100 000
population), with a peak of 4.1 cases per 100 000 population
in 2008 (Figure 1). The average incidence rate was highest in
the age groups of 55–65 years (4.5 cases per 100 000 population)
and >65 years (5.7 cases per 100 000 population). The 7-day
CFR was 7% (127 of 1915), and the 30-day CFR was 9% (176
emm89 GAS Cases
During 2004–2014, 278 bacteremic GAS cases with available
corresponding strains were caused by emm89 (range by year,
5–58 cases). The median age of emm89 bacteremic GAS cases
was 55 years (range, 0–97 years), equally distributed between
males and females. The 7-day and 30-day CFRs of emm89
bacteremic GAS were 4% (12 of 278) and 7% (20 of 278),
respectively, which were not significantly different from those caused
by all other emm types combined (7-day CFR, P = .12; 30-day
CFR, P = .26).
The proportion of emm89 cases among bacteremic GAS
cases varied from 4% (5 of 112) in 2005 to 28% (58 of 207)
in 2012, corresponding to incidence rates of 0.1 and 1.1 cases
per 100 000 population, respectively (Figure 1). In 2004,
emm89 bacteremic cases were only notified in southern, central,
and western Finland, but within 3 years it caused bacteremic
cases throughout all country (Figure 2). The proportion of
emm89 increased significantly in all tertiary healthcare districts
(mean relative increase, 19%; range, 17.8%–27.9%; P < .005).
The majority of emm89 bacteremic GAS cases (276 of 278)
were caused by subtype emm89.0. One strain of emm89.0b
and one of emm89.1 subtypes were also identified.
A total of 272 emm89 bacteremic GAS strains isolated in
Finland during 2004–2014 were analyzed by WGS, and 2
genetically distinct clades, designated clade 2 and clade 3, as recently
described , were identified. Clade 2 included 6% (16 of
272), while clade 3 accounted for the remaining 94% of emm89
isolates (256 of 272). A total of 56% of clade 2 cases were in
males, whereas clade 3 cases were equally distributed between
males and females. Cases with clade 3 strains were significantly
older than those with clade 2 (median age, 56 vs 44 years;
P < .05). Both clades caused bacteremic GAS cases during
2004–2008, but thereafter clade 3 rapidly increased in
frequency, causing virtually all cases from 2009 onward. By 2012, all
emm89 bacteremic GAS strains were of clade 3 (Figure 3).
Inasmuch as clade 3 strains increased rapidly in frequency
and have showed enhanced virulence in animal models of
invasive infection , we tested the hypothesis that patients
infected with strains of the 2 different clades differed significantly in
outcome. Within the 7-day or 30-day follow-up periods, none
of the clade 2 cases died, while among clade 3 cases, the 7-day
CFR was 5% (12 of 255), and the 30-day CFR was 8% (20 of
255). However, these observations were not statistically
significant. On the basis of SNP variation among the core genomes of
clade 3 emm89 bacteremic GAS strains, 6 distinct subclades
were identified, arbitrarily designated as subclades A through
F (Figure 4A and 4B). Subclade A included 74, subclade B 41,
subclade C 14, subclade D 32, subclade E 46, and subclade F 49
of the 256 isolates. We identified variations in the temporal
distribution of the 6 subclades, and a noteworthy increase of cases
caused by subclade D strains occurred during 2013–2014
(Figure 4A and 4B).
CFRs differed significantly between subclades, both with
respect to the 7-day CFR (P = .001) and the 30-day CFR (P = .04;
Table 1). Subclade C had the highest 7-day CFR (14%), followed
by subclade D, B, E, and both A and F. For the 30-day CFR,
subclade D showed the highest rate (19%), followed by subclades C,
B, E, F, and A. Subclade A was assigned as a reference when we
calculated the estimates of age-adjusted 7-day and 30-day CFR
odds (Table 1). Subclade C had the highest age-adjusted 7-day
odds of death due to GAS, followed by subclade D, B, E, and
F. Subclade D had the highest age-adjusted 30-day odds of
death due to GAS, followed by subclade B, C, E, and F.
Subclade D strains differed from other clade 3 strains in the
ndrI to fabG chromosomal region RB-15 (Figure 5A). In
addition, 6 subclade D strains had a nonsynonymous SNP
(Lys214Arg) in the 3-component regulatory system liaS gene. Of
note, 11 subclade D strains had also 2 unique SNPs ,
resulting in amino acid replacements in CovR (Ser130Asn) and ParC
(Asp83Gly) genes, which are part of regulatory systems involved
Subclade A B C
Abbreviations: CFR, case-fatality rate; CI, confidence interval; OR, odds ratio.
in virulence and DNA topoisomerase IV chromosome
partitioning, respectively. Five of these 11 subclade D strains were
isolated from cases in eastern Finland, and 4 were isolated
from cases in southern Finland, at the border with eastern
Finland. Nine cases were identified within a 5-month period in
2014, and 2 cases died within 7 days after the detection of
bacteremia. Based on medical records, no epidemiological link
could be observed among cases, but they all possessed ≥1
known risk factor for bacteremic GAS infection, such as old
age, delivery, alcoholism, liver chronic disease, and malignancy.
Rapid PCR Identification of Recently Emerged Subclade D Strains
The PCR specific for subclade D was validated using 69 emm89
bacteremic GAS strains representing the different subclades
isolated during 2009–2014. A band of 600 bp was obtained from
all 33 subclade D strains as expected, whereas bands of
≥1000 bp were obtained from 36 A-B-C-E-F subclade strains
(Figure 5). After validation, this PCR was used to screen all
33 emm89 bacteremic GAS strains isolated in 2015, and 9
subclade D strains (27%) were identified.
GAS has been shown to spread in epidemic waves through the
emergence and dissemination of certain strains [5, 7, 29].
Genomic alterations and molecular events leading to events such as
upregulated expression of virulence factors contributed to the
spread of certain successful GAS lineages, including emm1
. Recently, reports of similar events taking place in emm89
strains have been published [7, 30]. In our present study, we
investigated the emergence and spread of emm89 GAS among
bacteremic cases in Finland during 2004–2014, using
nationwide comprehensive, population-based infectious disease
surveillance data and the cognate strain collection. We show how
emm89 increase is particularly linked to the recent emergence of
clade 3 strains, a genetically distinct clade recently described [7,
22, 28]. The current emergence of 1 subclade among the 6 in
clade 3, namely subclade D, was studied in more depth. As
we discovered that cases caused by this subclade rapidly
increased in frequency among all emm89 cases and had a
significantly higher 30-day CFR, targeted surveillance by PCR specific
for subclade D was performed during 2015, which revealed its
sustained circulation during this period.
The increase of emm89 GAS strains in prevalence and
incidence has been reported in many countries [4, 6, 7, 9, 12–17, 19,
31, 32]. The first 2 emm89 bacteremic GAS strains in Finland
were reported in 1996 , and since then this emm type has
progressively increased in number . emm89 GAS strains
among bacteremic cases were already reported by all Finnish
tertiary healthcare districts in 2007, but its proportion increased
nationally, becoming the second most prevalent emm type from
2009 onward. In addition, already during 2008–2009, emm89
isolates were common among a various different infections in
Finland, including pharyngitis and deep-tissue infections .
Interestingly, the highest number and corresponding
proportion of emm89 strains (58 of 207 [28%]) among all bacteremic
GAS strains occurred in 2012, a year when the annual incidence
of bacteremic GAS cases also peaked at 3.9 cases per 100 000
population. The proportion of bacteremic GAS cases due to
emm89 remained constant after 2012, representing at least
one fourth of all bacteremic GAS cases annually, although
yearly fluctuations were detected on tertiary healthcare district level.
Previous studies of annual incidence and emm type
distribution among iGAS strains showed fluctuations over years with
emergence and reemergence of specific emm types [13, 14]. In
Finland, during past decades, in addition to emm89 strains,
these periodic fluctuations were associated with peaks of disease
activity caused by emm1 [8, 33, 34], emm28 [8, 11], emm84 ,
and emm33 [11, 36] strains. For decades, the reasons behind
fluctuations in emm type distribution were considered to be
primarily associated with the spread of distinct emm types in
immunological naive populations. Recent studies based on WGS
have shown that genetic changes in the organism due to
acquisition or upregulation of virulence factors [5, 6, 28, 29] also
participate. We show that the change in emm type distribution
overlapped changes in emm89 population genomics, and the
increasing number of emm89 bacteremic GAS cases during 2009–
2012 coincided with the clade 2 to clade 3 shift. Turner et al [6,
37] described a similar event with the emergence of new ST101
clade in England and Wales during 2005–2009, and Friães et al
 suggested, although without the support of WGS data, that
this also happened in Portugal. Based on available information,
it thus seems that emm89 ST101 emerged in many countries in
a closely similar time frame during the first decade of the 2000s,
accompanied by a clade swift from 1 to 2 and subsequent
emergence of clade 3. However, clade 1 strains have not been
identified in Finland [7, 22].
So far, few studies have been conducted to address molecular
mechanisms responsible for the increasing number of emm89
GAS cases [5–7, 28, 37]. Zhu et al  analyzed a set of emm89
isogenic mutant strains for biochemical, pathogenesis, and ex
vivo studies and demonstrated that the molecular trigger
underlying the current emm89 epidemic is associated with the
upregulation in the expression of 2 secreted cytotoxins, known as
S. pyogenes NADase and streptolysin O. Recently, we showed
that a distinct genetic subpopulation of Finnish emm89
bacteremic GAS isolates had an enhanced ability to survive in human
saliva ex vivo , and this could promote person-to-person
spread. In addition, GAS can persist inside host cells  and
thus evade action of antibiotics and perhaps even reach distant
body sites from the point of entry though a Trojan horse
emm89 bacteremic GAS cases did not differ from bacteremic
GAS cases caused by all other emm types when compared by
age. The sex distribution and mean age of these 2 groups were
similar. However, when emm89 cases were analyzed at the clade level,
clade 3 cases were significantly older than clade 2 cases. Tamayo
et al  also described an increased number of emm89 cases,
especially in the older age group, in Spain during 2009–2011.
The 7-day (4%) and 30-day (7%) CFRs of emm89 bacteremic
GAS were lower, compared with values from a previous report
. No statistically significant differences were found when
comparing 7-day and 30-day CFRs between overall bacteremic
GAS cases and emm89 bacteremic GAS cases or between clades
2 and 3, as also previously described . On the contrary,
statistically significant differences were found within clade 3
subclades, using exact logistic regression adjusted by age.
However, the small numbers among subclades represents a
limitation, and, thus, the interpretation needs precaution. The
eventual concomitant role of underlying conditions and risk
factors could not be assessed because this information is not
collected by the surveillance system. Subclade C and subclade D
had the highest 7-day and 30-day CFRs, with values of 14% (2 of
14) and 19% (6 of 32), respectively. Although subclade C had
the highest 7-day CFR, we focused particularly on subclade D
because of its increase in frequency among all emm89 cases
during 2013–2014 and additional deaths reported after the 7-day
and within the 30-day follow-up periods among cases of this
subclade, while no additional deaths after 7-day follow-up
and no increase in subclade C cases were observed during our
study period. Finnish emm89 bacteremic GAS clade 3 subclade
D strains differed from other clade 3 strains in ndrI-to-fabG
chromosomal region RB-15, as recently described . A
specific PCR based on WGS data analysis was developed to detect
subclade D among emm89 bacteremic GAS strains during 2015,
and, not surprisingly, this subclade was still circulating in the
country. However, no obvious clustering of cases could be
observed as the 9 subclade D cases occurred in all 5 tertiary
healthcare districts (data not shown). The circulation of subclade D
strains still warrant further surveillance of this subclade.
Our study describes the characteristics of the spread of a
newly emerged emm89 GAS clone causing bacteremia in
Finland during 2004–2014. WGS identified potentially
more-virulent clones, such as clade 3 and its subclade D. The spread of
subclade D strains was further followed by the use of a specific
PCR during 2015. To our best knowledge, this is the first study
that systematically combines comprehensive, population-based
surveillance data and full-genome analyses of corresponding
isolates to highlight the importance of their combined use to
better understand the evolution and spread of emm89 GAS.
The need for targeted public health strategies to control these
life-threatening infections necessitates the establishment of
comprehensive iGAS infection surveillance systems, including
those for collecting clinical data, and research to develop an
effective GAS vaccine should continue.
Acknowledgments. We thank Teemu Möttönen, for technical expertise
in retrieving and combining data from several database sources; Aino
Makkonen, Tuula Rantasalo, and Toni Huovinen, for excellent technical
assistance; Tiina Sorvari, Pekka Suomalainen, Jukka Heikkinen, and Katariina
Kainulainen, for assistance in collecting patient background data; Aftab
Jasir, for critical comments on the manuscript; and the clinical microbiology
laboratories that submitted notifications and isolates.
Financial support. This work was partially supported by the Academy
of Finland (grant 255636), the Hospital District of Southwest Finland, and
the Fondren Foundation.
Potential conflicts of interest. All authors: No reported conflicts. All
authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the content
of the manuscript have been disclosed.
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