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Molecular Characterization of Invasive Group
A Streptococci in Alaska
2000 - 2008
Karen Rudolph, Ph.D.
Arctic Investigations Program
DEISS, NCPDCID, CCID, CDC
Objectives
1. List two questions molecular strain typing can
address.
2. Describe the two common molecular strain typing
techniques.
3. List three reasons why molecular strain typing
of group A streptococci is important.
Molecular Strain Typing
• Used to address two questions:
 Are isolates recovered from a localized
outbreak of disease the same or different
strains?
 How are strains causing disease in one
geographic area related to those isolated
world wide?
• Method should be highly discriminatory which
refers to the ability to differentiate among unrelated
strains.
Molecular Strain Typing
Pulsed-field Gel Electrophoresis (PFGE)
1. Cell suspension
Bacterial Cell
Intact bacterial cells
embedded in agarose
3. Restriction enzyme
digestion of DNA
Intact bacterial DNA
suspended in agarose
2. Lysis of cells
4. Electrophoresis
Fragments of bacterial DNA
suspended in agarose
Molecular Strain Typing
Pulsed-field Gel Electrophoresis (PFGE)
•
DNA is forced to change
direction
 Large fragments take longer to
realign in each field – move
a shorter distance
 Shorter fragments realign faster and
travel farther
•
Run time of 20 hrs
Molecular Strain Typing
Pulsed-field Gel Electrophoresis (PFGE)
-291 kb
-194
-145.5
- 97
- 48.5
Pulsed-field Gel Electrophoresis (PFGE)
Dendogram
100
90
80
Coefficient of similarity
84.6
96.8
93.6
80.3
96.8
88.5
85.7
100
92.6
74.9
89.7
100
96.3
93.5
100
72.3
86.7
85.7
100
78.6
Molecular Strain Typing
Multilocus Sequence Typing (MLST)
Bacterial chromosomal
DNA
PCR amplify - 450bp fragments
of seven housekeeping genes
aroe
gdh
gki
recP
spi
xpt
ddl
1. Sequence the seven gene fragments on both strands
2. Compare sequences of each gene fragment with the
known alleles at the locus
3. Assign alleles at the seven loci to give the allelic profile
4. Compare the allelic profile with those of isolates
within a central database via the internet
Molecular Strain Typing
Multilocus Sequence Typing (MLST)
1.
>aroe
2.
http://www.mlst.net/databases
GAAGCGAGTGACTTGGCAGAAACAGTGGCCAATATTCGTCGCTACCAGATGTTTGGCA
TCAATCTGTCCATGCCCTATAAGGAGCAGGTGATTCCTTATTTGGATGAGCTGAGCGAT
GAAGCGCGCTTGATTGGTGCGGTTAATACGGTTGTCAATGAGAATGGCAATTTAATTG
GATATAATACAGATGGCAAGGGATTTTTTAAGTGCTTGCCTTCTTTTACAATTTCAGGT
AAAAAGATGACCCTGCTGGGTGCAGGTGGTGCGGCTAAATCAATCTTGGCACAGGCTA
TTTTGGATGGCGTCAGTCAGATTTCGGTCTTTGTTCGTTCCGTTTCTATGGAAAAAACAA
GACCTTACCTAGACAAGTTACAGGAGCAGACAGGCTTTAAAGTGGATTTGTGT
>gdh
3.
AGAACACTTTATCCGTGGACAATACCGCTCTGGTAAGATTGATGGCATGAAATACATCT
CTTATCGTAGCGAACCAAATGTGAATCCAGAATCAACAACTGAAACCTTTACATCTGGTG
CCTTCTTTGTAGACAGCGATCGATTCCGTGGTGTTCCTTTCTTTTTCCGTACAGGTAAAC
GACTGACTGAAAAAGGAACTCATGTCAACATCGTCTTTAAACAAATGGATTCTATCTTTG
GAGAACCACTTGCTCCAAATATTTTGACCATCTATATTCAACCAACAGAAGGCTTCTCT
CTTAGCCTAAATGGGAAGCAAGTAGGAGAAGAATTTAACTTGGCTCCTAACTCACTTGA
TTATCGTACAGACGCGACTGCAACTGGTGCTTCTCCAGAACCATACGAGAAATTGATTT
ATGATGTCCTAAATAACAACTCAACTAACTTTAGCCACTGGGAT
Allelic profile:
aroe_8 gdh_13 gki_13 recP_4 spi_17 xpt_4 ddl_14
4.
Streptococcus pneumoniae - Allelic Profiles query results
Your sequence type is 199
Sequence
Type
aroe
gdh_
gki_
recP
spi_
xpt_
ddl_
Query
8
13
14
4
17
4
14
Molecular Strain Typing
Multilocus Sequence Typing (MLST)
eBURST Analysis:
S. pneumoniae with a central founder
ST199 and 12 linked SLVs;
two of the SLVs have diversified
to produce DLVs. eBURST, unlike cluster
diagrams, trees, or dendograms, uses a simple model
of bacterial evolution in which an ancestral (or founding)
genotype increases in frequency in the population and
while doing so begins to diversify to produce a cluster
of closely related genotypes that are all descended from
the founding genotype.
Multilocus Sequence Typing
Advantages
• Sequencing uncovers all variations at a gene locus.
• Identity of alleles is unambiguous using sequencing
data.
• Electronic portability of DNA sequences - allows
labs to characterize bacterial isolates by submitting
sequence data via the internet to a central MLST
database.
Streptococcus pyogenes
• group A streptococci (GAS), Gram-positive, spherical
or ovoid cells in chains, -hemolytic on blood agar
• Exclusively human pathogen;
transmitted by respiratory droplet or
contact with infected wounds
• Colonize the throat or skin
• Infections range from mild to severe:
- pharyngitis, impetigo, scarlet
fever
- bacteremia, pneumonia, meningitis,
necrotizing fasciitis (NF), streptococcal toxic shock
syndrome (STSS)
Streptococcus pyogenes
Burden of Illness
• Worldwide, GAS is important cause of morbidity and
mortality with an estimated 517,000 deaths each year.
• In the U.S. (2000 – 05), the average annual incidence
rate of invasive GAS disease was 3.5 cases per
100,000 persons with 735 deaths (case fatality rate of 13.7%).
• Highest incidence among persons ≥65 years of age
(9.4 cases per 100,000 persons), and children <1 year
of age (5.3 cases per 100,000 persons).
• Case fatality rate (22.8%) highest among the elderly.
GAS rate per 100,000
persons
Rates of Invasive GAS Disease in
Alaska, 2000 - 06
40
35
30
25
20
15
10
5
0
Native
Non-Native
0-1
2-4
5-17
18-44
45-64
65+
Age Class (Years)
Overall annual incidence rate – 4.7 cases/100,000persons
Streptococcus pyogenes
Control Strategies
• Identification and prevention of risk factors
- young age (<2), elderly (≥65)
• Vaccination
- 26-valent vaccine; in phase 2 clinical trials
• Treatment
- -lactams have been the treatment of
choice
Streptococcus pyogenes
Serotyping
• 1928 – Rebecca Lancefield established method
based on antigenic variation of the M protein
• Considered the gold standard
• >90 M serotypes described
• Problems associated with M serotyping:
- limited availability of M typing antisera
- newly encountered M types (high nontypeability rate)
- difficulty in interpretation
Streptococcus pyogenes
emm sequence typing
• N-terminal hypervariable region of M protein gene.
• Concurs with M serotyping for most serotypes 1:1.
• Validated in the mid-1990s.
• 225 distinct emm types encompassing 450 subtypes.
• Problems associated with M serotyping are avoided.
emm type Distribution of Invasive GAS
Isolates in Alaska, 2000 - 08
12
% of GAS Isolates
10
8
6
4
2
0
1
3
12
41
92
28
87
73
108
114
5
76
82
83
6
49
emm Types
- Top ten emm types account for 66% of isolates.
- Vaccine emm types account for 61% of isolates.
58
emm type Distribution of Invasive GAS
Isolates by Time Period
100%
Percent of Total
80%
60%
40%
20%
0%
1
3
5
6
12
28
41
49
58
73
76
82
83
87
92
emm Types
2000 - 2004
2005 - 2008
- 2000 – 04, N = 99; 2005 – 08, N = 113
- Increase in emm73, emm82, emm108
- Decrease in emm41, emm76, emm83, emm114
- p <0.0001
108
114 Other
emm type Distribution of Invasive GAS Isolates
Alaska and U.S. (lower 48)
25
% of GAS Isolates
20
15
10
5
0
1
2
3
5
6
12
22
28
41
83
emm types
U.S. emm types
AK emm types
87
89
92
103
114
emm type Distribution of Invasive GAS Isolates
Urban vs. Rural
100%
Percent of Total
80%
60%
40%
20%
0%
1
3
12
41
92
28
87
emm Types
Urban
Rural
73
108
114
Other
emm type Distribution of Invasive GAS Isolates
Ethnicity
100%
Percent of Total
80%
60%
40%
20%
0%
1
3
12
41
92
28
87
emm Types
Native
NonNative
Unknown
73
108
114
Other
Antimicrobial Susceptibilities
Antibiotic
Isolates
N=128a (%)
Penicillin
128 (100)
Cefotaxime
Clindamycin
Erythromycin
Tetracycline
128 (100)
127 (99.2)
118 (92.2)
96 (75)
Levofloxacin
Vancomycin
128 (100)
128 (100)
aSusceptibility
testing for GAS isolates began in 2004.
Conclusions
• GAS is an important cause of invasive bacterial
disease particularly among the AK Native population.
• emm types seen in Alaska similar to rest of U.S.
with exception of emm41, emm92, and emm1
• 26-valent GAS vaccine would prevent ~61% of cases
• Continued surveillance is warranted
- to improve understanding of epidemiology
- for notification of possible outbreaks
- to monitor changes in emm types for vaccine development
Acknowledgements
23 Labs participating in the statewide surveillance program
AIP Microbiology Lab
- Alisa Reasonover
- Marcella Harker-Jones
- Julie Morris
AIP Nursing staff
- Debby Hurlburt
- Kim Boyd-Hummel
Tammy Zulz – AIP Surveillance Coordinator
Dana Bruden – Statistician
Debbie Parks – Database Manager
Dr. Mike Bruce – Medical Epidemiologist
The findings and conclusions in this presentation have not been formally disseminated by the
Centers for Disease Control and Prevention (CDC) and should not be construed to represent
any CDC determination or policy