Non-Culture Detection of Antimicrobial Resistance in Neisseria gonorrhoeae

David L. Trees, Ph.D.

Division of STD Prevention

Centers for Disease Control and Prevention

Agar Plate Dilution MIC Determination

Molecular Techniques for the Detection of Antimicrobial Resistance

High level Penicillin and TetracyclineGene based

Ciprofloxacin (flouroquinolones)GyrA/ParC alterations & uptake/efflux

Azithromycin (macrolides)mtrR, 23S rRNA & others?

CephalosporinsExtended spectrum beta-lactamases/others

Mechanisms of Resistance:

Molecular Techniques for the Detection of Antimicrobial Resistance

Probe

PCR

Standard sequencing

Microarray

Transformation (Gonostat)

Tm Analysis

Pyrosequencing

Probe and Standard PCR

Used for the detection of gene based resistances such as plasmid encoded penicillin (PPNG) and tetracycline (TRNG)

Not effective in determining point mutation-based resistance.

Standard Sequencing

Useful in determining point mutations and larger mutations such as those found in some of the azithromycin resistant isolates.

Also good at determining the presence of previously reported mutations that are related to resistance.

Sequencing long stretches of DNA can be time consuming.

TAA

base 1120 base 1606

ATG

153 bp insert

promoters

mtrC mtrR

mtrC mtrR mtrF

AGTGGATTAACAAAAACCAGTACGGCGTTGCCTCGCCTTAGCTCAAAGAGAACGATTCTCTAAGGTGCTGAAGCACCAAGTGAATCGGTTCCGTACTATTTGTACTGTCTGCGGCTTCGTCGCCTTGTCCTGATTTTTGTTAATCCACTATAT

Mtr region of AziR isolates

Ciprofloxacin – Mechanism of Resistance

Fluoroquinolones inhibit the replication of DNA

Mutations in two genes involved in DNA replication results in resistance.

gyrA: encodes for A subunit of DNA gyrase.

parC: encodes for subunit of topoisomease IV.

1 China Samples Second Batch 2 91>Phe, 95>Gly 87>Arg

2 China Samples Second Batch 6 91>Phe, 95>Asn 87>Ile

3 China Samples Second Batch 10 91>Phe, 95>Ala 91>Ala

4 China Samples Second Batch 17 91>Phe, 95>Gly 0

5 China Samples Second Batch 20 91>Phe, 95>Ala 87>Asn

6 China Samples Second Batch 37 91>Phe, 95>Gly 86>Asn

7 China Samples Second Batch 41 91>Phe, 95>Ala 86>Asn

8 China Samples Second Batch 48 91>Phe, 95>Asn 0

9 China Samples Second Batch 49 91>Phe, 95>Ala 0

10 China Samples Second Batch 52 91>Phe 0

11 China Samples Second Batch 62 91>Tyr 0

12 China Samples Second Batch 94 91>Phe, 95>Asn 91>Gly

13 China Samples Second Batch 99 91>Phe, 95>Ala 87>Ile, 91>Ala

14 China Samples Second Batch 100 91>Phe, 95>Gly 91>Gln

15 California Controls 15 0 0

16 California Controls 16 91>Thr, 92>Ala, 93>Val, 94>Tyr 0

17 China Samples Second Batch 6 91>Phe, 95>Asn 87>Ile

18 LA Samples 2001032172-176 2001032175 91>Phe, 95>Ala 87>Asn, 91>Gln

19 Lansing 2001038717 91>Phe, 95>Asn 86>Asn

20 Lansing 2003018721 91>Phe, 92>Ser, 95>Asn 91>Gly

21 NY-NonGISPCipR 2004000379 (86) 91>Phe, 95>Gly 87>Arg

22 NY-NonGISPCipR 2001005845 (39) 91>Phe, 95>Tyr 91>Gly

23 NY-NonGISPCipR 2000032980 (43) 91>Phe, 95>Ala 87>Arg

24 NY-NonGISPCipR 2003000649 (45) 91>Phe, 95>Gly 91>Gly

25 NY-NonGISPCipR 2003007200 (50) 91>Phe 86>Asn

Microarray Assays

Good at detecting known mutations and giving their exact location in the gene sequence.

Will not detect new mutations. Will not confirm resistance.

GyrA Microarrays

ParC Microarrays

.

GyrA (Ser-91)

GyrA (Asp-95)

GyrA (Ser-91, Asp-95)

GyrA (Ser-91), ParC (Asp-86 -> Asn)

GyrA (Ser-91, Asp-95), ParC (Asp-86 -> Asn)

GyrA (Ser-91, Asp-95), ParC (Glu-91-> Gly)

Legend

None

GyrA (Ser-91), ParC (Glu-91 -> Gly)

GyrA (Ser-91, Asp-95), ParC (Ser - 87> Asn)

GyrA (Ser-91, Asp-95), ParC (Asp-86-> Asn, Ser-87-> Asn)

0.125

0.25

0.5

1.0

2.0

4.0

8.0

16.032.0

0 5 10 15 20 0 5 10 15 20 25No. of Isolates No. of Isolates

Cip MIC(µg/ml)

1998 1999

Cip

RC

ipI

Thailand

Tm Analysis

Will detect the presence of a point mutation(s) within a given probe length. Will also detect the presence of non-identified mutations if they occur within the specified DNA sequence.

Will not (at this point) specify the exact location of the mutation and therefore can not identify “silent” mutations.

Melting Curve Analysis

1 China Samples Second Batch 2 91>Phe, 95>Gly 87>Arg

2 China Samples Second Batch 6 91>Phe, 95>Asn 87>Ile

3 China Samples Second Batch 10 91>Phe, 95>Ala 91>Ala

4 China Samples Second Batch 17 91>Phe, 95>Gly 0

5 China Samples Second Batch 20 91>Phe, 95>Ala 87>Asn

6 China Samples Second Batch 37 91>Phe, 95>Gly 86>Asn

7 China Samples Second Batch 41 91>Phe, 95>Ala 86>Asn

8 China Samples Second Batch 48 91>Phe, 95>Asn 0

9 China Samples Second Batch 49 91>Phe, 95>Ala 0

10 China Samples Second Batch 52 91>Phe 0

11 China Samples Second Batch 62 91>Tyr 0

12 China Samples Second Batch 94 91>Phe, 95>Asn 91>Gly

13 China Samples Second Batch 99 91>Phe, 95>Ala 87>Ile, 91>Ala

14 China Samples Second Batch 100 91>Phe, 95>Gly 91>Gln

15 California Controls 15 0 0

16 California Controls 16 91>Thr, 92>Ala, 93>Val, 94>Tyr 0

17 China Samples Second Batch 6 91>Phe, 95>Asn 87>Ile

18 LA Samples 2001032172-176 2001032175 91>Phe, 95>Ala 87>Asn, 91>Gln

19 Lansing 2001038717 91>Phe, 95>Asn 86>Asn

20 Lansing 2003018721 91>Phe, 92>Ser, 95>Asn 91>Gly

21 NY-NonGISPCipR 2004000379 (86) 91>Phe, 95>Gly 87>Arg

22 NY-NonGISPCipR 2001005845 (39) 91>Phe, 95>Tyr 91>Gly

23 NY-NonGISPCipR 2000032980 (43) 91>Phe, 95>Ala 87>Arg

24 NY-NonGISPCipR 2003000649 (45) 91>Phe, 95>Gly 91>Gly

25 NY-NonGISPCipR 2003007200 (50) 91>Phe 86>Asn

GONOSTAT

Detection of gonococcal infection

Non NAAT confirmatory test

Can be used with specimens suspended in BD lysis buffer, Amplicor buffer or M4.

In vitro detection of Cip, Azi, Spc

Tested clinical specimens for Cip

Gonostat is a DNA transformation assay for the detection of N. gonorrhoeae in clinical specimens.

The recipient gonococcus contains a mutation that prevents growth unless it is transformed by a “wild-type” gene provided by gonococcal DNA present in the specimen.

Gonostat does not require a viable isolate and specimen does not require refrigeration or freezing, making the assay extremely useful in resource poor settings.

GONOSTAT

1.  Send swabs to collection site.

2.  Swab can be stored at room temperature for shipment back to testing site.

3.  Place swab in 400 μl of solution A to lyse bacteria.

4.  Add ~400 μl of solution B to neutralize. Will get colorimetric change.

5.  Place 100 μl of solution onto a preplated lawn of Gonostat recipient bacteria.

6.  Incubate for 24-48 hours and look for bacterial growth.

7.  > 10 colonies is positive test.

Standard Gonostat Transformation Detection Assay

•Transformation reaction as described in gonostat protocol.

•Grow up on Choc Agar Plate.

•Harvest all colonies into 2.0 ml Meuller Hinton Broth

•Centrifuge for 5 minutes

•Resuspend in 2.0 ml PBS.

•Place 0.2ml on antibiotic plates in lawn formation.

•Incubate at 37 degrees and read after 24-48 hours. 

Gonostat Transformation Assay for Detection of Antimicrobial Resistance

Sample Retrieval Method Transformant MIC Gyr A Par CDB Diluent - Ethanol Precipitate 8.0 91>Phe, 95>Gly 87>Asn, 91>Lys

DB Diluent - 50 u l 4.0 91>Phe, 95>Gly 87>Asn, 91>LysAmplicor - Ethanol Precipitate 8.0 91>Phe, 95>Gly 87>Asn, 91>Lys

Amplicor - 50 u l 4.0 91>Phe, 95>Gly 87>Asn, 91>Lys

Detection of Ciprofloxacin Resistance from spiked NAAT Buffers;

With and Without Ethanol Precipitation

Positive control transformant gave a MIC of 0.03

GyrA and ParC alterations in the transformant were identical to those in the donor isolate

Detection of decreased susceptibility to ciprofloxacin in clinical specimens: Guangzhou, China

Currently >95% CipR N. gonorrhoeae

Urethral swabs , most with corresponding culture isolate, from 19 patients were tested by Gonostat

The swabs were collected between February and June, 2003 and stored at room temperature and tested at CDC between December 2003 and February 2004.

Of the 19 specimens; Six had no growth on the original transformation plate, two had between 45 to 60 colonies (coded 1+), eight had to many colonies to count (3+), and three had confluent lawn growth (4+) on the original transformation plate.

Acknowledgement to Wei Lai MD, Guangzhou, China

SampleTransformant

GrowthTransformant

MIC Transformant Transformant Original Original

  (Choc Plates) Cip [64.0-0.125] GyrA ParC GyrA ParC

1 3+ 0.125 91>Phe, 95>Gly 0 91>Phe, 95>Gly 87>Arg

2 1+ No Growth NA NA NA NA

3 1+ 0.03 95>Gly 0 91>Phe, 95>Gly 87>Arg

4 3+ 0.25 91>Phe, 95>Ala 0 91>Phe, 95>Ala 87>Arg

5 3+ 0.03 NA NA NA NA

6 4+ 0.06 91>Phe, 95>Ala 0 No Growth No Growth

7 4+ 0.125 91>Phe, 95>Gly 0 No growth No growth

8 4+ 0.125 91>Tyr, 95>Gly 0 No growth No Growth

9 3+ 0.03 NA NA NA NA

10 3+ 0.25 91>Phe, 95>Gly 0 91>Phe, 95>Gly 87>Arg

11 3+ 0.03 95>Gly 0 NA NA

12 3+ 0.125 91>Phe,95>Asn 0 No Growth No Growth

13 3+ 0.06 95>Gly 0 No Growth No Growth

SPH01 4+ 8.0 91>Phe, 95>Gly 87>Asn, 91>Lys 91>Phe, 95>Gly 87>Asn, 91>Lys

Negative Control 0 NA NA NA NA NA

Positive Control 4+ 0.03 95>Gly 0 NA NA

Clinical Studies – Antimicrobial Susceptibilities and Mutation Patterns

CONCLUSIONS

Able to use Gonostat to detect resistance to:

Ciprofloxacin – Both clinical specimens and

spiked specimens

Azithromycin – spiked specimens

Spectinomycin – spiked specimens

Could perform the assay on specimens in NAAT Buffers

Dilution experiments suggest that between 5x103 to 5x105 “chromosomal equivalents” are needed to detect resistance.

Detection did not require viable organisms or refrigeration/freezing equipment

PYROSEQUENCINGPYROSEQUENCING

Real-time sequencing Real-time sequencing for microbial for microbial

identification and identification and resistance typingresistance typing

What is Pyrosequencing?What is Pyrosequencing?

A genetic analysis method based on the A genetic analysis method based on the principle of sequencing by synthesisprinciple of sequencing by synthesisThis system is able to deliver sequence This system is able to deliver sequence information within minutesinformation within minutesThe output data is real sequence data – the The output data is real sequence data – the Gold standardGold standardUses include microbial identification and Uses include microbial identification and resistance detectionresistance detection

Advantages of PyrosequencingAdvantages of Pyrosequencing

Sequencing directly from PCR productSequencing directly from PCR product

Batch analysis of 96 samples in less than 1 hourBatch analysis of 96 samples in less than 1 hour

Only 2 pipetting steps needed to start and Only 2 pipetting steps needed to start and complete the processcomplete the process

Automatic sequence callingAutomatic sequence calling

Direct detection and analysis of SNPs, Direct detection and analysis of SNPs, insertions, and deletionsinsertions, and deletions

Setting up a RunSetting up a Run

Place cooled PSQ plate Place cooled PSQ plate into machineinto machineCan run in SNP or Can run in SNP or SQA modesSQA modes– Enter sequenceEnter sequence

informationinformation– Enter dispensationEnter dispensation

orderorderCan program exact sequenceCan program exact sequenceOr can repeat ACGT for unknownOr can repeat ACGT for unknownor highly variable sequencesor highly variable sequences

ApplicationsApplications

Detecting fluoroquinolone resistance in Detecting fluoroquinolone resistance in Neisseria gonorrhoeaeNeisseria gonorrhoeae conferred by GyrA conferred by GyrA and ParC mutationsand ParC mutations

Sample PyrogramSample PyrogramGyrAGyrA

11.17.2005 - Well A111.17.2005 - Well A1Entry: GYRA2-3SEQx2Entry: GYRA2-3SEQx2Sample: JH1Sample: JH1Result: GATTTCGCAG TTTACGGCAC CATCGTCCGT ATGGCGC Result: GATTTCGCAG TTTACGGCAC CATCGTCCGT ATGGCGC Rev. Comp: GCGCCATACG GACGATGGTG CCGTAAACTG CGAAATC Rev. Comp: GCGCCATACG GACGATGGTG CCGTAAACTG CGAAATC Quality: Quality: PassedPassed (Quality window: 20) (Quality window: 20)

110

120

130

E S A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T5 10 15 20 25 30 35 40 45 50 55 60

Result Result GyrAGyrA

GyrAGyrA WT: GAT TCC GCA GTT TAC GAC ACC ATC WT: GAT TCC GCA GTT TAC GAC ACC ATC

Asp Ser Ala Val Tyr Asp Thr IleAsp Ser Ala Val Tyr Asp Thr Ile

↓ ↓↓ ↓Result: GAT TResult: GAT TTTC GCA GTT TAC GC GCA GTT TAC GGGC ACC ATC C ACC ATC

Asp Asp PhePhe Ala Val Tyr Ala Val Tyr GlyGly Thr Ile Thr Ile

Two Amino Acid changes = Fluoroquinolone ResistanceTwo Amino Acid changes = Fluoroquinolone Resistance

Sample PyrogramSample PyrogramParCParC

12.12.2005 - Well A912.12.2005 - Well A9Entry: PARC1-3SEQEntry: PARC1-3SEQSample: PJH20Sample: PJH20Result: GCCTATGGGG CGATGGTGCG CATGGCTCAGResult: GCCTATGGGG CGATGGTGCG CATGGCTCAGRev. Comp: CTGAGCCATG CGCACCATCG CCCCATAGGC Rev. Comp: CTGAGCCATG CGCACCATCG CCCCATAGGC Quality: Quality: PassedPassed (Quality window: 20) (Quality window: 20)

110

120

130

140

E S A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T A C G T

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Result Result ParCParC

ParCParC WT: WT: GCC TAT GAG GCGGCC TAT GAG GCG

Ala Tyr Glu AlaAla Tyr Glu Ala

↓↓Result:Result: GCC TAT GGCC TAT GGGG GCGG GCG

Ala Tyr Ala Tyr GlyGly Ala Ala

*Amino Acid change = Fluoroquinolone Resistance*Amino Acid change = Fluoroquinolone Resistance

Molecular Techniques

for the Detection of Antimicrobial Resistance

Potential applications?

Used to state that an isolate is resistant?

Used on NAAT specimens to determine resistance?

Used as a surveillance/screening tool to predict prevalence of resistance?

Acknowledgements to “ The Staff ”