Download Detecting Emerging and Novel Antimicrobial Resistance Genes

Overview of NARMS Program and Detecting
Emerging/Novel Antimicrobial Resistance
Genes Using WGS
Shaohua Zhao DVM, MPVM, PhD
U.S. Food and Drug Administration
Center for Veterinary Medicine
Office of Research
Laurel, MD
Disclaimer
This communication is consistent with 21 CFR 10.85 (k) and constitutes an informal communication that
represents my best judgment at this time but does not constitute an advisory opinion, does not necessarily
1 views
represent the formal position of FDA, and does not bind or otherwise obligate or commit the agency to the
expressed.
Outline
• Public concern of antimicrobial resistance
• Review US NARMS program
• Detecting Emerging and Novel Antimicrobial Resistance
Genes in Campylobacter Using Whole Genome
Sequencing WGS)
• Using WGS to predict resistant phenotype/application
WGS in NARMS program)
2
The Public Health Action Plan
Four Principal Components
• Surveillance
–
–
Goal 1: Improve the detection, monitoring, and characterization of drug-resistant infections in humans
and animals.
Goal 2: Better define, characterize, and measure the impact of antimicrobial drug use in humans and
animals in the United States.
• Prevention and Control
• Research
–
–
–
–
Goal 1: Facilitate basic research on antimicrobial resistance.
Goal 2: Facilitate the translation of basic research findings into practical applications for the
prevention, diagnosis, and treatment of resistant infections.
Goal 3: Facilitate clinical research to improve the treatment and prevention of antimicrobial drug
resistant infections.
Goal 4: Conduct and support epidemiological studies to identify key drivers of the emergence and
spread of AR in various populations.
• Product Development
NARMS Study Population and Target Organisms
Human Isolates
**
Food-producing animals
1997
•
•
•
•
Campylobacter
Non-typhoidal Salmonella
Generic E. coli
Enterococcus
1996
Retail Food Isolates
2002
•
•
•
•
Campylobacter
Non-typhoidal Salmonella
Generic E. coli
Enterococcus
**also piloted MRSA, C. diff and VRE in foods
•
•
•
•
•
•
Campylobacter
Non-typhoidal Salmonella
E. coli O157:H7
Typhoidal Salmonella
Shigella
Vibrio (2009)
5
Human Salmonella Surveillance Sites*
1996: 14 sites
2002: 28 sites
1999: 17 sites
2003: 53 sites
6
*In 1996, surveillance began in 14 sites. In 2003, participation increased to nationwide: 50 state and three local health
departments, Los Angeles County (joined in 1996), New York City (1996), and Houston, Texas (2003).
Human Campylobacter Surveillance Sites
In 1997, surveillance was initiated in five states. Additional sites joined after 1997. By 2003,
participation included 10 sites: CA, CO, CT, GA, MD, MN, NM, NY, OR, and TN.
7
NARMS Retail Meat Surveillance
Partnership with state FoodNet Sites
•
•
•
•
•
•
CT, GA, MD, MN, TN
CT, GA, MD, MN, TN, OR
CT, GA, MD, MN, TN, OR NY, CA
CT, GA, MD, MN, TN, OR NY, CA, CO, NM
CT, GA, MD, MN, TN, OR NY, CA, CO, NM, PA
CT, GA, MD, MN, TN, OR NY, CA, CO, NM, PA, WA, LA, MO
Sampling scheme
• Each site purchases 10 packages each of
chicken breasts, pork chops, ground turkey,
ground beef per month
• All 14 sites culture for Salmonella and
Campylobacter
• In addition, 3-4 sites (GA, OR, TN, ±MD )
culture for E. coli and Enterococcus
• In 2005, changed from convenience to
randomized sampling
• Sample total = 6,720 per annum
1/2002
9/2002
1/2003
1/2004
1/2008
1/2013
Retail Food Testing Sites
Sampling at Slaughter
HACCP* (1997-Current)
sources: carcass swabs,
rinses, ground product
Swine
Cattle
Campylobacter
Salmonella
Chicken
Turkeys
x
x
x
x
New in-plant cecal
sampling (2013-Current)
x
Swine
(Hogs,
Sows)
Cattle
(Beef,
Dairy)
Young
Chicken
Young
Turkeys
Campylobacter
x
x
x
x
Salmonella
x
x
x
x
E. coli
x
E. coli
x
x
x
x
Enterococcus
x
Enterococcus
x
x
x
x
*HACCP: Hazard Analysis Critical Control Point- samples collected to assess Salmonella
(and now Campylobacter) contamination and i.e. interventions where appropriate.
Sampling became risk based in 2006.
9
Interpretive Criteria Used for Antimicrobial Susceptibility
Testing of Salmonella and E. coli
Breakpoints (µg/ml)
Susceptible
Intermediate
Resistant
Gentamicin
≤4
8
≥ 16
Kanamycin
≤ 16
32
≥ 64
Streptomycin
≤ 32
N/A
≥ 64
≤8/4
16 / 8
≥ 32 / 16
Antimicrobial Class
Antimicrobial Agent
Aminoglycosides
b -Lactam/b -Lactamase
Inhibitor Combinations
Amoxicillin–Clavulanic Acid
Cephems
Cefoxitin
≤8
16
≥ 32
Ceftiofur
≤2
4
≥8
Ceftriaxone
≤1
2
≥4
Sulfisoxazole
≤ 256
N/A
≥ 512
≤ 2 / 38
N/A
≥ 4 / 76
Folate Pathway Inhibitors
Trimethoprim–Sulfamethoxazole
Macrolides
Azithromycin
≤ 16
N/A
≥ 32
Penicillins
Ampicillin
≤8
16
≥ 32
Phenicols
Chloramphenicol
≤8
16
≥ 32
Quinolones
Ciprofloxacin
≤ 0.06
0.12 - 0.5
≥1
≤1
2
≥4
Nalidixic acid
≤ 16
N/A
≥ 32
Tetracycline
≤4
8
≥ 16
Salmonella
E. coli
Tetracyclines
-Breakpoints adopted from CLSI, except for
azithromycin and streptomycin, which have
no CLSI breakpoints. The breakpoints for
azithromycin and streptomycin are NARMSestablished breakpoints developed for
resistance monitoring. They should not be
used to predict clinical efficacy.
-Sulfamethoxazole was tested from 1996
through 2003 and was replaced by
sulfisoxazole in 2004
- The revised ciprofloxacin breakpoint for
invasive Salmonella from the CLSI M100S22 document, published in January 2012, is
used. The revised breakpoints were applied
to all non-typhoidal Salmonella. In previous
NARMS reports, breakpoints from the CLSI
M100-S21 were used.
10
EUCAST Interpretive Criteria Used for Antimicrobial
Susceptibility Testing of Campylobacter
Breakpoints (µg/ml)
jejuni
coli
Antimicrobial Class
Antimicrobial Agent
Aminoglycosides
Gentamicin
0.12 - 32
≤2
≥4
≤2
≥4
Ketolides
Telithromycin
0.015 - 8
≤4
≥8
≤4
≥8
Lincosamides
Clindamycin
0.03 - 16
≤ 0.5
≥1
≤1
≥2
Macrolides
Azithromycin
0.015 - 64
≤ 0.25
≥ 0.5
≤ 0.5
≥1
Erythromycin
0.03 - 64
≤4
≥8
≤8
≥ 16
Phenicols
Florfenicol
0.03 - 64
≤4
≥8
≤4
≥8
Quinolones
Ciprofloxacin
0.015 - 64
≤ 0.5
≥1
≤ 0.5
≥1
Nalidixic acid
4 - 64
≤ 16
≥ 32
≤ 16
≥ 32
Tetracycline
0.06 - 64
≤1
≥2
≤2
≥4
Tetracyclines
Concentration Range (µg/ml) Susceptible
Resistant
Susceptible Resistant
11
Resistance among Salmonella Isolated from Humans
12
Resistance to ≥3 agents increased from <2% in the 1940s to 28% in the 2000s among this collection
MDR among Salmonella from Humans, Poultry and Meats
60%
(R >3 classes )
50%
Percent Resistance
40%
30%
20%
10%
0%
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011
Year
Humans
Retail Chicken
Ground Turkey
Chickens
Turkeys
13
MDR among Salmonella Heidelberg
14
Antibiotic Resistant Gene Database
Antimicrobial Class
Aminoglycoside Resistance
Bacitracin Resistance
Beta-lactam Resistance
Bicyclomycin Resistance
Chloramphenicol Resistance
Fosfomycin Resistance
Fusaric_acid Resistance
Glycopeptide Resistance
Lincosamide Resistance
Macrolide & MLS Resistance
MAR and Efflux Genes
Nucleoside Resistance
Polymyxin Resistance
QAC Resistance
Quinolone Resistance
Rifamyxin Resistance
Streptogramin Resistance
Streptothricin Resistance
Sulfonamide Resistance
Tetracenomycin C Resistance
Tetracycline Resistance
Thiostrepton Resistance
Trimethoprim Resistance
Viomycin Resistance
Grand total
Number of Genes
282
307
1460
58
154
62
4
241
21
207
1372
11
29
45
96
31
47
7
34
10
313
2
150
1
4944
Strategies to detect
genes to the level of:
• Family
• Phylogroup
• Specific variants
Detection of AR genes
• PCR/Sequence
• Microarray
• Whole Genome Sequencing (WGS)
18
Accelerating Technology &
Plummeting Cost
Next
Generation
Sequencing
$795 in 1977
(=$2,800 in
current $)
Lower cost = more innovative and more samples
Cost per bacterial genome
$3,500
$3,000
454
$2,500
$2,000
Illumina
Miseq
$1,500
$1,000
$500
$0
2007
2008
2009
2010
2011
2012
2013
$70/genome
in 2014
$40/genome in 2015 w/
Illumina NextSeq Technology
Lower cost = more innovative and more samples
From WGS to Antibiotic Resistance Genotype
BLAST ®
aac(3)-IIa, aadA1,
aph(3')-Ia
catA1, tetO …
Local Blast AR
Gene Database
Acquired AR genes
DNA from
Single colony
Sequencing
With Illumina Miseq
Assembly
CLC Genomics
Workbench
AR Genotype
Sequences alignment
gyrA gene
23S rRNA gene
Point mutations related
to AR
21
Detecting Novel Gentamycin Resistance
Genes in Campylobacter Isolated from
Human, Retail Chicken and Food Animals
in NARMS Program
22
Aminoglycosides
 They are highly potent, broad-spectrum bactericidal
antibiotics, commonly used in the treatment of
infections caused by aerobic G- bacteria as well as
some selected G+ bacteria
 Common aminoglycoside antibiotics
•
•
•
•
•
•
Gentamicin
Tobramycin
Amikacin
Kanamycin
Neomycin
Streptomycin
 Inhibit protein synthesis
23
Aminoglycosides Resistance
 The primary mechanism of resistance to
aminoglycoside antibiotics is enzymatic inactivation
by three major aminoglycoside-modifying enzymes:
aminoglycoside acetytransferases (AACs)
aminoglycoside nucleotidyltransferases (ANTs)
aminoglycoside phosphotransferases (APHs)
 More than 300 aminoglycoside resistance genes have
been identified
24
Prevalence of GENR Campylobacter coli
from Different Sources
25
PFGE and AST Profiles of GenR Campylobacter
Isolates from Humans and Retail Chicken
GenR and
TetR C.coli
from retail
chicken and
humans
aph(2’’)-Ig
26
Novel Aminoglycosides Resistance Genes
• Seven mono-functional aminoglycoside 2″phosphotransferase genes:
–
–
–
–
–
–
–
aph(2″)-Ib
aph(2″)-Ic
aph(2″)-Ig
aph(2″)-If
aph(2″)-If1
aph(2″)-If3
aph(2″)-Ih
• Two bi-functional aminoglycoside2 ″- phosphotransferase
genes:
– aac(6’)-Ie/aph(2″)-Ia
– aac(6’)-Ie/aph(2″)-If2
27
Percentage of Amino Acid Identity
in the APH(2″) Family
1
*Bifunctional AAC/APH, only the APH part of the enzyme is used to construct the
phylogenetic tree.
28
Timeline of GenR Campylobacter from
Humans (2000-2011) and Retail Chicken (2007-2013)
GenR C.coli
GenR C.jejuni
Humans
Retail
Chicken
2007
2012
2004
2000
GenR C.jejuni
GenR C.coli
Two GenR genes (aph-If and aph-Ig) were shared between human and RT Chicken isolates
Humans
aph-Ig
2012
2008
2009
GenR C.jejuni
Humans
aph-If
2013
2003
2004
29
PFGE and AST Profiles of GenR C. coli
aph-If3
Cluster C:
aph-If by PCR and
WGS
Cluster D:
aac(6’)-Ie/aph(2’’)-Ia
Cluster E:
aph-Ig
aph-Ic
PFGE and AST Profiles of GENR C.jejuni
Cluster A:
aph-If by PCR
aph-Ih by WGS (n=7)
A
aph-If
aph-Ib
aph-If
aph-Ig
B
Cluster B:
aph-If by PCR
aph-Ih by WGS (n=2)
31
Comparison of AR Gene Clusters in MDR
Plasmid pN29710-1 with pCG8245 and SX81
Track 1: AR island in SX81
Track 2: AR gene cluster in
pCG8245
Track 3: AR gene cluster
in pN28710
Track 4: pTet
Track 5: GC content of
pN29710-1
32
Summary
 GenR has increased rapidly in Campylobacter in the U.S.
 9 variants of GenR genes were identified


7 were identified for the first time in Campylobacter
5 were novel aminoglycoside resistance genes
 Human isolates contained more diverse GenR genes than
retail chicken isolates
 PFGE and GenR genotypes indicated that contaminated
retail chicken could serve as a source of GenR C. coli
infections in humans.
 WGS is a powerful tool to detect resistance genotypes.34
Correlation between Antimicrobial Resistance Phenotype and
Genotype in Campylobacter and Salmonella
• Campylobacter spp:
– Sample size: 104 isolates
– Source: Retail meat (n=74), Humans (n=40)
– Representative MDR patterns of human isolates recovered from
2000-2011 and retail meat isolates from 2004-2013
• Salmonella
– Sample size: 285 isolates
– Source: Retail meat (n=181) and Humans (n=104)
– Representative unique combinations of resistance
pattern, source and serotype from 2011 to 2012
35
Resistance genes database at FDA/CVM
Drug class
Aminoglycoside
Beta-lactam
Fosfomycin
Fusaric acid
Glycopeptide
Lincosamide
Macrolide & MLK
Metronidazole
Olaquindox and phenicol
Phenicol
QAC
Quinolone
Rifamyxin
Streptogramin
Streptothricin
Sulfonamide
Tetracenomycin C
Tetracycline
Thiostrepton
Trimethoprim
Viomycin
Total
Gene count
297
1253
14
4
234
19
174
13
2
147
12
80
29
26
7
34
4
271
1
116
1
2738
Cluster count
120
178
10
2
88
8
61
8
2
40
8
13
7
16
3
5
4
55
1
39
1
669
36
Campylobacter Resistance Phenotypes and Genotypes
strain
N13165
N14784
N14840
N15262
N15870
N1630
N1636
N18323
N18725
N20320
N20344
N20402
N23169
N23392
N26070
N26697
N26699
N279
N287
N3506
N3508
N39665
N39671
N39677
N40944
AST
CIP, GEN, NAL, TET,
AZI CIP CLI ERY NAL TEL TET
AZI CLI ERY TEL TET
AZI CLI ERY TEL
AZI CIP CLI ERY NAL TEL TET
CIP NAL TET
AZI CIP CLI ERY NAL TEL TET
AZI CLI ERY TEL TET
AZI CLI ERY TEL TET
AZI CIP CLI ERY NAL TEL TET
GEN, TET,
GEN, TET,
AZI CLI CIP ERY NAL TEL TET
AZI CLI ERY TEL TET
AZI CIP ERY NAL TEL TET
AZI CLI CIP ERY NAL TEL TET
AZI CLI ERY TEL TET
AZI CLI ERY TEL TET
AZI CLI ERY TEL TET
AZI CLI ERY TEL TET
AZI CLI ERY TEL TET
GEN TET
GEN TET
GEN TET
GEN TET
Gene
ant(6), aph(3')-IIIa, blaOXA-61, sat4, tetO,
aph(3')-IIIa blaOXA-61 tetO
aph(3')-IIIa blaOXA-61 tetO
aph(3')-IIIa tetO
blaOXA-61 tetO
aph(3')-Ic tetO
aph(3')-Ic tetO
aph(3')-IIIa blaOXA-61 tetO
blaOXA-61 tetO
aph(2'')-Ic, aph(3')-IIIa, blaOXA-61, tetO,
aph(2'')-Ic, aph(3')-IIIa, blaOXA-61, tetO,
aph(3')-IIIa blaOXA-61 tetO
aph(3')-IIIa tetO
aph(3')-IIIa blaOXA-61 tetO
blaOXA-61 tetO
tetO
tetO
aph(3')-IIIa blaOXA-61 tetO
aph(3')-Ic tetO
aph(3')-Ic tetO
aadE ant(3'') aph(2'')-Ig aph(3')-IIIa sat4 tetO
aadE ant(3'') aph(2'')-Ig aph(3')-IIIa sat4 tetO
aadE ant(3'') aph(2'')-Ig aph(3')-IIIa sat4 tetO
aadE ant(3'') aph(2'')-Ig aph(3')-IIIa blaOXA-61 sat4 tetO
GyrA 86
I
I
T
T
I
I
I
T
T
I
T
T
I
T
I
I
T
T
T
T
T
T
T
T
T
23S 2074
A
A
A
T
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
23S 2075
A
G
G
A
G
A
G
G
G
G
A
A
G
G
G
G
G
G
G
G
G
A
A
A
A
37
Corrections of Resistance Phenotypes and
Genotypes in Campylobacter
68R/6S
24R/50S
Correlation
(%)
98.6
100
100
Macrolide
35R/39S
100
Lincosamides
35R/39S
97.3
Keolides
Total
34R/40S
74
95.9
98.6
Drug class
Gentamicin
Tetracycline
Quinolone
Number of resistance
and susceptible isolates
40R/34S
Correlation: The tested phenotype and the genotype matched bidirectional
38
Salmonella Resistance Phenotypes and Genotypes
(Retail Meat)
CVM_NUMB
ER
N29307
N29309
N29310
N29313
N29315
N29317
N29321
N29323
N29338
N29339
N29343
N29350
N29351
N29355
N29357
N29360
N29362
N29363
N29367
N29369
N29377
N29378
N29379
N32052
AST
Pan susceptible
Pan susceptible
AMP
AMC AMP AXO FIS TET TIO
AMC AMP AXO FOX FIS STR TET TIO
AMP GEN STR TET
AMP GEN KAN FIS STR
AMC AMP AXO KAN FIS TET TIO
AMP GEN STR TET
AMP KAN STR FIS TET
KAN FIS TET
AMP CHL FIS STR TET
AUG AMP FOX TIO AXO GEN
Pan susceptible
GEN FIS STR
TET
AMP GEN TET
STR TET
Pan susceptible
AMP TET
AMP GEN STR TET
FIS STR TET
AMC AMP AXO CHL FOX GEN FIS STR TET TIO
AMP GEN TET
GENE
blaTEM-1
blaCMY-2 sul2 tetA
aph(3'')-Ib aph(6)-Id blaCMY-2 blaTEM-1 sul2 tetA
aac(3)-IIa aadA aph(3'')-Ib aph(6)-Id blaTEM-1 tetA
aadA2 aadB aph(3')-Ia blaTEM-1 sul1
aph(3')-Ia blaCMY-2 sul2 tetA
aac(3)-IIa aadA blaTEM-1 tetA
aph(3')-Ia aph(3'')-Ib aph(6)-Id blaTEM-1 sul2 tetB tetC tetD
aph(3')-Ia sul2 tetA
aadA2 blacarB-2 floR sul1 tetG
blaCMY-61
aac(3)-VI aadA1 sul1
tetB tetC tetD
aac(3)-IIa aadA blaTEM-1 tetA
aph(3'')-Ib aph(6)-Id tetB tetC tetD
blaTEM-1 tetB tetC tetD
aac(3)-IIa aadA blaTEM-1 tetA tetB tetC tetD
aph(3'')-Ib aph(6)-Id sul2 tetA
aac(3)-VI aadA1 aph(3'')-Ib aph(6)-Id blaCMY-2 floR sul1 sul2 tetA
39
aac(3)-IIa aadA blaTEM-1 tetA
Salmonella Resistance Phenotypes and Genotypes
(Clinical Isolates)
CVM
43743
43744
43745
43746
43747
43748
43749
43750
43751
43752
43753
43754
43755
43756
43757
43758
43759
43760
43761
43762
43763
43764
43765
43766
43837
43838
AST Patterns
ASSu
ASSuT
ACSSuTGen
ASSuTKan
CT
Pan susceptible
ASSuTNal
SuGen
Pan susceptible
STKan
Pan susceptible
ASSuSxt
ASSuNal
ACipNal
Sxt
ASuGen
ACSSuT
Pan susceptible
ASSuTNalSxt
CSSuT
SuTSxt
AAuCxCfFox
CSuTKanNalSxt
ACSSuTAuCf
SuSxt
ACSSuTAuCxCfFoxCipNalSxt
gene
aadA12 blaTEM-1 sul1
aph(3'')-Ib aph(6)-Id blaTEM-1 sul2 tetA
aac(3)-VI aadA1 aadA2 blacarB-2 floR sul1 tetG
aph(3')-Ia aph(3'')-Ib aph(6)-Id blaTEM-1 sul2 tetB tetC tetD
catA1 tetA
aph(3'')-Ib aph(6)-Id blaTEM-1 sul2 tetA
aac(3)-VI aadA1 sul1
aph(3'')-Ib aph(3')-II aph(6)-Ic aph(6)-Id ble tetB tetC tetD
aph(3'')-Ib aph(6)-Id blaTEM-1 sul2 dfrA8
aph(3'')-Ib aph(6)-Id blaTEM-1 sul2
blaTEM-1
aac(3)-VI aadA1 dfrA1
aac(3)-VI aadA1 blaHERA-3 sul1
aadA2 blacarB-2 floR sul1 tetG
aph(3'')-Ib aph(6)-Id blaTEM-1 sul2 tetA dfr5
aph(3'')-Ib aph(6)-Id floR sul2 tetA
qnrS sul1 tetA dfrA1
blaCMY-2
aac(3)-IV aadA1 aph(3')-Ia aph(4)-Ia floR sul1 tetA dfrA14
aph(3'')-Ib aph(6)-Id blaCMY-2 floR sul2 tetA
aadA1 sul1 dfrA1
aadA2 aph(3'')-Ib aph(6)-Id blaCMY-2 bleO oqxB oqxA floR qnrB19 sul2 tetA dfrA12
GyrA
83
S
S
S
S
S
S
S
S
S
S
S
S
S
F
S
S
S
S
S
S
S
S
S
S
S
S
GyrA
87
D
D
D
D
D
D
Y
D
D
D
D
D
Y
Y
D
D
D
D
Y
D
D
D
Y
D
40
D
D
ParC
80
S
S
S
S
S
S
S
S
S
S
S
S
S
I
S
S
S
S
S
S
S
S
S
S
S
S
Summary
• Based on current knowledge and technology, WGS predicts
resistance very well
• 98-100% correlation for the drug classes beta-lactam,
tetracycline, chloramphenicol, sulfonamide,
trimethoprim/sulfamethoxazole, macrolides and quinolone
• 92-97% correlation for aminoglycoside, lincosamides and
keolides
• A comprehensive and accurate database of ARG is critical
• Reasons for disconnect




AST interpretation standard
experimental and analytical error
variable gene expression level
unknown mechanisms
41
Benefits of a WGS Strategy in NARMS
WGS has potential to serve as a single assay of NARMS surveillance
and supplant multiple methods
1.
2.
3.
4.
Classical serotyping
PFGE and other molecular typing methods
In vitro antimicrobial susceptibility testing
Multiple PCR assays to detect resistance genes and plasmid typing
And to provide:
1.
2.
3.
4.
Genome surveillance
Virulence profiles
Markers for source attribution
Better understanding of emerging resistance trends, origin, dissemination
and selection pressure
5. Cost saving
42
Acknowledgement
FDA:
NARMS retail meat arm working group
USDA:
NARMS animal arm working group (ARS and FSIS)
CDC:
NARMS human arm working group
CDC PulseNet
FoodNet/State Public Health Laboratories
43