Molecular Characterization of Invasive Group A Streptococci in Alaska 2000 - 2008 Karen Rudolph, Ph.D. Arctic Investigations Program DEISS, NCPDCID, CCID,

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


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



-291 kb-194-145.5

- 97

- 48.5

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


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


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.


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


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


• 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



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)


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


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

Documents

Molecular Characterization of Invasive Group A Streptococci in Alaska 2000 - 2008 Karen Rudolph, Ph.D. Arctic Investigations Program DEISS, NCPDCID, CCID,