molecular characterization of invasive group a streptococci in alaska 2000 - 2008 karen rudolph,...
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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 typingtechniques.
3. List three reasons why molecular strain typingof 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.
Bacterial CellIntact bacterial cells embedded in agarose
Intact bacterial DNAsuspended in agarose
2. Lysis of cells
3. Restriction enzyme digestion of DNA
Fragments of bacterial DNAsuspended in agarose
1. Cell suspension
4. Electrophoresis
Molecular Strain Typing
Pulsed-field Gel Electrophoresis (PFGE)
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
Pulsed-field Gel Electrophoresis (PFGE)Dendogram
100
9080
84.6
96.8
96.8
93.6
88.5
100
92.6
100
96.3
93.5
89.7
85.7
80.3
100
86.7
85.7
74.9
100
78.6
72.3
Coefficient of similarity
Molecular Strain Typing
Multilocus Sequence Typing (MLST)
Bacterial chromosomal DNA
PCR amplify - 450bp fragmentsof seven housekeeping genes
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 isolateswithin a central database via the internet
aroe gdh gki recP spi xpt ddl
Molecular Strain Typing
Multilocus Sequence Typing (MLST)>aroeGAAGCGAGTGACTTGGCAGAAACAGTGGCCAATATTCGTCGCTACCAGATGTTTGGCATCAATCTGTCCATGCCCTATAAGGAGCAGGTGATTCCTTATTTGGATGAGCTGAGCGATGAAGCGCGCTTGATTGGTGCGGTTAATACGGTTGTCAATGAGAATGGCAATTTAATTGGATATAATACAGATGGCAAGGGATTTTTTAAGTGCTTGCCTTCTTTTACAATTTCAGGTAAAAAGATGACCCTGCTGGGTGCAGGTGGTGCGGCTAAATCAATCTTGGCACAGGCTATTTTGGATGGCGTCAGTCAGATTTCGGTCTTTGTTCGTTCCGTTTCTATGGAAAAAACAAGACCTTACCTAGACAAGTTACAGGAGCAGACAGGCTTTAAAGTGGATTTGTGT
>gdhAGAACACTTTATCCGTGGACAATACCGCTCTGGTAAGATTGATGGCATGAAATACATCTCTTATCGTAGCGAACCAAATGTGAATCCAGAATCAACAACTGAAACCTTTACATCTGGTGCCTTCTTTGTAGACAGCGATCGATTCCGTGGTGTTCCTTTCTTTTTCCGTACAGGTAAACGACTGACTGAAAAAGGAACTCATGTCAACATCGTCTTTAAACAAATGGATTCTATCTTTGGAGAACCACTTGCTCCAAATATTTTGACCATCTATATTCAACCAACAGAAGGCTTCTCTCTTAGCCTAAATGGGAAGCAAGTAGGAGAAGAATTTAACTTGGCTCCTAACTCACTTGATTATCGTACAGACGCGACTGCAACTGGTGCTTCTCCAGAACCATACGAGAAATTGATTTATGATGTCCTAAATAACAACTCAACTAACTTTAGCCACTGGGAT
Allelic profile:
aroe_8 gdh_13 gki_13 recP_4 spi_17 xpt_4 ddl_14
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
http://www.mlst.net/databases
1.2.
3.
4.
Molecular Strain Typing
Multilocus Sequence Typing (MLST)
eBURST Analysis:S. pneumoniae with a central founderST199 and 12 linked SLVs;two of the SLVs have diversifiedto produce DLVs. eBURST, unlike clusterdiagrams, trees, or dendograms, uses a simple modelof bacterial evolution in which an ancestral (or founding)genotype increases in frequency in the population andwhile doing so begins to diversify to produce a clusterof closely related genotypes that are all descended fromthe 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.
• 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
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.
Rates of Invasive GAS Disease in Alaska, 2000 - 06
05
101520
25303540
0-1 2-4 5-17 18-44 45-64 65+
Age Class (Years)
GA
S r
ate
pe
r 1
00
,00
0
pe
rso
ns
Native
Non-Native
Overall annual incidence rate – 4.7 cases/100,000persons
• 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
Control Strategies
Streptococcus pyogenes
• 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
Serotyping
Streptococcus pyogenes
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
- Top ten emm types account for 66% of isolates.- Vaccine emm types account for 61% of isolates.
0
2
4
6
8
10
12
1 3 12 41 92 28 87 73 108 114 5 76 82 83 6 49 58
emm Types
% o
f G
AS
Iso
late
s
emm type Distribution of Invasive GAS Isolates by Time Period
- 2000 – 04, N = 99; 2005 – 08, N = 113- Increase in emm73, emm82, emm108- Decrease in emm41, emm76, emm83, emm114- p <0.0001
0%
20%
40%
60%
80%
100%
1 3 5 6 12 28 41 49 58 73 76 82 83 87 92 108 114 Other
emm Types
Per
cen
t o
f T
ota
l
2000 - 2004 2005 - 2008
0
5
10
15
20
25
1 2 3 5 6 12 22 28 41 83 87 89 92 103 114
emm types
% o
f G
AS
Iso
late
s
U.S. emm types AK emm types
emm type Distribution of Invasive GAS Isolates
Alaska and U.S. (lower 48)
emm type Distribution of Invasive GAS Isolates
Urban vs. Rural
0%
20%
40%
60%
80%
100%
1 3 12 41 92 28 87 73 108 114 Other
emm Types
Per
cen
t o
f T
ota
l
Urban Rural
emm type Distribution of Invasive GAS Isolates
Ethnicity
0%
20%
40%
60%
80%
100%
1 3 12 41 92 28 87 73 108 114 Other
emm Types
Per
cen
t o
f T
ota
l
Native NonNative Unknown
Antimicrobial Susceptibilities
Antibiotic Isolates N=128a (%)
Penicillin 128 (100)
Cefotaxime 128 (100)
Clindamycin 127 (99.2)
Erythromycin 118 (92.2)
Tetracycline 96 (75)
Levofloxacin 128 (100)
Vancomycin 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
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
23 Labs participating in the statewide surveillance program
AIP Microbiology Lab AIP Nursing staff - Alisa Reasonover - Debby Hurlburt - Marcella Harker-Jones - Kim Boyd-Hummel - Julie Morris
Tammy Zulz – AIP Surveillance CoordinatorDana Bruden – StatisticianDebbie Parks – Database ManagerDr. Mike Bruce – Medical Epidemiologist
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