Descriptive and Molecular
Epidemiology of Antibiotic
Resistant Gram-Negative Enteric
Bacteria from Dairy Cattle
Ashish Sawant, MVSc
Department of Veterinary Science
The Pennsylvania State University
University Park, PA 16802
Overview of Presentation
‰ Introduction to antibiotics in animal agriculture
‰ Assessment of management practices associated with
antibiotic usage on dairy farms
‰ Prevalence and distribution of antibiotic resistance
bacteria in dairy animals
‰ Phenotypic and genotypic analysis of tetracycline and
beta-lactam resistant E. coli
March 2005
Ashish Sawant-PhD Thesis Defense
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Introduction
‰Antimicrobial:
A substance of natural, semisynthetic, or synthetic origin
that kills or inhibit the growth
of microorganisms
S.B. Levy, Scientific American
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Antimicrobials in Animal
Agriculture
‰ Prophylactic: Preventive measure
‰ Transport of animals
‰ Weaning of animals
‰ Growth promotion:
‰ Increase weight gain
‰ Therapy: Treatment of clinical cases
‰ Some countries report that more than 50% of
their total output of antimicrobial compounds is
used in food animal agriculture.
March 2005
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Beef, Dairy, Sheep, Swine, & Poultry Contribute
More than $87 Billion to the Nation’s Economy
‰ 18 million lbs. of antibiotics were used with
90% used for subtherapeutic use.
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Introduction of antibiotics, reduced
mortality in food animals, added 10 years
to the average life expectancy of humans
but ……………..What is the concern about
antibiotics?
Bacterial resistance to antibiotics
Drug Residues in animal origin food
Antibiotic Resistance
‰ WHO
‰ CDC
‰ CARC
‰ AMA
‰ PAHO
‰ OIE
‰ FAO
‰ USDA-FSIS
‰ FDA-CVM
‰ AVMA
Resistance to antimicrobials
‰ 1960s: Resistance in Salmonella from calves lead to ban in
penicillin and tetracycline as feed additive
‰ 1970s: reports of multidrug resistant bacteria
‰ 1978: WHO defines rules for monitoring bacterial resistance in
veterinary and human origin organisms
‰ 1980-90s: Emergence of antimicrobial resistance
‰ Vancomycin resistance Enterococci
‰ Multidrug resistance S. Typhimurium DT104
‰ Floroquinolone resistant Campylobacter
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The US perspective
‰ United States National Antimicrobial Resistance Monitoring System
(NARMS) established in 1996
‰ Campylobacter, Salmonella, E. coli O157, and Shigella
‰ FDA proposed framework (1999)
‰ 1) Categorization of antibiotics based on their importance in human
medicine
‰ 2) Revision of the pre-approval safety assessments for new animals
drug applications to access microbial safety
‰ 3) Post-approval monitoring for resistance development
‰ 4) Collection of food animal antibiotics use data
‰ 5) Establishment of regulatory thresholds
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Assessment of
Antibiotic Usage in
Dairy Herds in
Pennsylvania
Chapter 1
Survey for Antibiotic Usage
‰ 248 dairy farms (36 counties) in Pennsylvania were
solicited for survey
‰ 173 dairy farms indicated interest to participate
‰ 126 dairy producers met the criteria for the survey study
‰ A total of 113 dairy producers participated in study
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Survey for Antibiotic Usage
‰ 113 dairy producers (n=13 counties)
‰ Categorized on basis of herd size
‰<100 lactating cows (n=42 herds, 3-4 herds/county)
‰100-199 lactating cows (n=45 herds, 3-5 herds/county)
‰>200 lactating cows (n=26 herds, 2 herds/county)
‰ 33 of 113 maintained complete records on individual
health and antibiotic usage
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Findings
‰Positive attributes
‰Detrimental attributes
‰ Consulted a veterinarian
before extra-label use (79%)
‰ No records on antibiotic usage
(50%)
‰ Milked treated animals last (85%)
‰ Written protocols for treating sick
animals (21%)
‰ Milk free of antibiotic residues
(6 months) (97%)
‰ Consulted a veterinarian before
antibiotic use (32%)
‰ Screened fresh cows for antibiotic
residues (58%)
‰ Completed the antibiotic
treatment course (24%)
‰ Antibiotics administered by other
than a veterinarian (93%)
Reported conditions on 33 farms
Enteritis
Pneumonia
Calves (88%)
Lactating cows (33%)
Calves (100%)
Lactating cows (27%)
Metritis
Lactating cows (79%)
Heifers (27%)
Mastitis
Foot rot
Lactating cows (100%)
Dry cows (21%)
Lactating cows (100%)
Heifers (30%)
Percent animals treated with
antibiotics
Enteritis
Pneumonia
Calves (25%)
Lactating cows (3%)
Calves (36%)
Lactating cows (2%)
Metritis
Lactating cows (11%)
Heifers (4%)
Mastitis
Foot rot
Lactating cows (16%)
Dry cows (5%)
Lactating cows (14%)
Heifers (5%)
Antibiotics approved in dairy cattle
Pneumonia
Amoxicillin
Ampicillin
Ceftiofur
Tetracyclines
Florfenicol
Penicillin G
Spectinomycin
Sulfadimethoxine
Enteritis
Tetracyclines
Neomycin
Metritis
Ceftiofur
Penicillin G
Medicated milk replacer
Mastitis
Prophylactic
Tetracycline-Neomycin
Foot rot
Amoxicillin
Ceftiofur
Tetracyclines
Florfenicol
Neomycin
Sulfadimethoxine
Amoxicillin
Cephapirin
Cloxacillin
Erythromycin
Hetacillin
Novobiocin
Penicillin G
Pirlimycin
Antibiotics used on 33 farms
Pneumonia
Amoxicillin
Danofloxacin
Ampicillin
Tilmicosin
Ceftiofur
Tetracyclines
Florfenicol
Penicillin G
Spectinomycin
Sulfadimethoxine
Enteritis
Tetracyclines
Neomycin
Lincomycin
Spectinomycin
Metritis
Ceftiofur
Penicillin G
Medicated milk replacer
Mastitis
Prophylactic
Tetracycline-Neomycin
Bacitracin
Bambermycin
Carbadox
Foot rot
Amoxicillin
Ceftiofur
Tetracyclines
Florfenicol
Neomycin
Sulfadimethoxine
Amoxicillin
Cephapirin
Cloxacillin
Erythromycin
Hetacillin
Novobiocin
Penicillin G
Pirlimycin
Ceftiofur
Commonly used antibiotics on 33 farms
Pneumonia
Ampicillin (45%)
Ceftiofur (48%)
Florfenicol (30%)
Spectinomycin (42%)
Enteritis
Spectinomycin (30%)
Metritis
Ceftiofur (15%)
Medicated milk replacer
Prophylactic
Mastitis
Tetracycline-Neomycin
(70%)
Cephapirin
52% dry cow
49% mastitis
Novobiocin (27%)
Penicillin G (46%)
Ceftiofur (18%)
Foot rot
Sulfadimethoxine (27%)
Conclusions
‰Current practices on most of the dairy farms
are not structured for prudent use of
antibiotics
‰Current practices related to antimicrobial
usage could contribute to the emergence of
antibiotic resistant bacteria
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RESEARCH QUESTION
What is the influence of antibiotic
usage on the prevalence and
distribution of antibiotic resistant
gram-negative commensal bacteria
from healthy lactating cattle?
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Prevalence of Antimicrobial
Resistant Gram Negative
Commensal Bacteria in
Lactating Dairy Cattle
Chapter 2
Study Design
‰ Fecal samples from
‰ 313 lactating cattle from 33 Dairy farms
‰ 93 calves from 13 farms
‰ Ten fold dilutions of fecal sample plated on
MacConkey’s agar supplemented with
antibiotics
‰
‰
‰
‰
‰
‰
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Ampicillin (64μg/ml)
Enrofloxacin (8μg/ml)
Florfenicol (16μg/ml)
Neomycin (512μg/ml)
Oxytetracycline (32μg/ml)
Spectinomycin (256μg/ml)
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Prevalence* of antimicrobial resistant gramnegative enteric bacteria (GN-EB)
Number and percent cows shedding antimicrobial resistant GN-EB
Antimicrobial agent
Farm (n=33)
Cows (n=313)
No.
%
No.
%
20/33
61
98/313
31
Enrofloxacin (8μg/ml)
0/33
0
0/313
0
Florfenicol (16μg/ml)
11/33
33
23/313
7
Neomycin (512μg/ml)
2/33
6
4/313
1
Oxytetracycline (32μg/ml)
21/33
64
97/313
31
Spectinomycin (256μg/ml)
7/33
21
14/313
5
Ampicillin (64μg/ml)
*One concentration higher than recommended by NCCLS
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Prevalence of antibiotic resistant GN-EB
MAC plate
with
antibiotics
Control MAC
plate with no
antibiotics
Table 3. Relationship between resistant bacteria and total GNB flora
Antimicrobial
Cows
(N)
Mean Total
GNB (cfu/g)
Mean Resistant
GNB (cfu/g)
Ratio: Mean (R-GNB/T-GNB)%
Mean %
Range %
Ampicillin
72
3.8x106
3.0x105
9.26
0.01 - 96.5
Florfenicol
18
1.2x106
2.9x103
4.86
0.00 - 63.0
Neomycin
4
4.2x105
350
1.2
0.01 - 3
Oxytetracycline
89
6.9x106
3.9x105
13.94
0.01 - 100.0
Spectinomycin
10
1.9x106
6.2x104
9.83
0.004 - 89
Data from 23 farms
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Using medicated milk replacers in feed
(218 lactating cattle from 13 dairy herds)
Fed medicated milk
replacers with
tetracycline to
calves
Tetracycline resistant GNEB in lactating cattle
Detected
Not detected
Total
Yes
89
23
112
No
56
50
106
Total
145
73
218
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Test of significance:
χ2 (P < 0.05) = 17.27
(0.00003)
Odds ratio (confidence
interval)
3.45 (1.83-6.56)
25
Resistant GNB species (n=264)
Species
1. Citrobacter koseri
2. Enterobacter aerogenes
3. Escherichia coli
4. Morganella morganii
5. Klebsiella oxytoca
6. Klebsiella pneumonia
7. Kluyuera spp.
8. Providentia alcaligenes
9. Providentia stuartii
10. Pasturella spp.
n=14
n=4
n=229
n=1
n=3
n=1
n=1
n=1
n=1
n=2
11. Pseudomonas aeruginosa
12. Pseudomonas fluorescens
13. Pseudomonas spp.
n=1
n=5
n=1
March 2005
ƒ Group 5: Facultative anaerobic gramnegative rods
ƒ Intestinal inhabitants of human and
animals
ƒ Family Enterobacteriaceae
ƒ Family Pasteurellaceae
ƒ Group 4: Gram-negative
aerobic/microaerophillic rods and cocci
ƒ Widely distributed in nature
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Disk diffusion
assay
‰ Isolates: E. coli (n=229)
‰ Antimicrobials: Antimicrobial agents approved
for use in animal species by the FDA-CVM
‰ Assay performed according to NCCLS
‰
NCCLS document M31-A2 [ISBN 1-56238-461-9], 2002
Measuring
zone of
inhibition
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Antimicrobial agents
ampicillin
chloramphenicol
gentamicin
enrofloxacin
spectinomycin
tetracycline
ticarcillin
ticarcillin/CA
ceftiofur
27
Antimicrobial
resistance:
E. coli isolates
ƒPredominant resistance to
ampicillin and tetracycline
ƒ26 resistance profiles
ƒLargest groups
ƒMultidrug resistance (≥3 to <9)
observed in 29% isolates
Resistant isolates (%)
Ampicillin
137 (59.8)
Ceftiofur
25 (10.9)
Chloramphenicol
43 (18.8)
Enrofloxacin
0 (0.0)
Gentamicin
5 (2.2)
Spectinomycin
50 (21.8)
Tetracycline
215 (93.9)
Ticarcillin
47 (20.5)
Ticarcillin/Clavulanic acid
25 (10.9)
Frequently observed
resistance profiles
ƒTetracycline resistant (36.7%)
ƒAmpicillin-tetracycline resistant
(25.3%)
March 2005
Antimicrobial agent
No. of isolates
(%)
TET
84 (36.7)
AMP
10 (4.4)
AMP-TET
58 (25.3)
AMP-SPT-TET
10 (4.4)
AMP-SPT-TET-TIC
9 (3.9)
AMP-CHL-SPT-TET
4 (1.7)
AMP-CHL-SPT-TET-TIC-TIM-XNL
15 (6.6)
AMP-CHL-GEN-SPT-TET-TIC-TIM-XNL
3 (1.3)
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Survival of multidrug resistant bacteria in
water at refrigeration temperature
Average weekly cfu/ml
30000
cfu/ml
25000
20000
15000
10000
5000
0
0
7
14
21
28
35
42
49
56
63
Day of Experim ent
‰ Multidrug resistant E. coli isolate A92 was used in the experiment
‰ Resistance phenotype (AMP-CHL-SPT-TET-TIC-TIM-XNL)
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Survival of multidrug resistant bacteria in
water at refrigeration temperature
Mean=0.0255
X <=0.0038
5%
0.35
X <=0.047
95%
0.3
0.25
0.2
0.15
0.1
0.05
0
-3
-2
-1
0
1
2
3
4
5
6
7
8
Survival of resistant bacteria in water at
refrigeration temperature
‰ K=0.025 (mean) was used
to calculate the die-off rate
Resistant bacteria die off K= 0.025
6
‰ A 3 log reduction in the
counts of the E. coli isolate
could occur in 126 days
Log cfu/g
5
4
3
2
1
12
6
11
2
98
84
70
56
42
28
14
0
0
days
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Degree of relatedness among
commensal E. coli
‰ Pulse field gel electrophoresis (PFGE)
‰ Determine clonal subtypes in tetracycline
and ampicillin resistant E. coli isolates
using PFGE typing system
20
‰ PFGE pattern relatedness
by dendogram analysis
40
60
80
100
‰ 66 PFGE patterns from
99 tetracycline resistant
E. coli
(
Farm A
T-19 (1)
‰ 44 PFGE patterns from 69
ampicillin resistant E. coli
)
Farm B Farm C
T-20 (1) T-9 (2)
T-21 (1)
T-22 (1)
T-04 (7)
T-6 (3)
Farm D Farm E
ND T-23 (1)
T-24 (1)
T-7 (5)
Farm K Farm L Farm M Farm N Farm O
T-14 (2) T-10 (2) T-16 (2) T-12 (1) T-11 (3)
T-34 (1) T-41 (1) T-42 (1) T-17 (2) T-50 (1)
T-35 (1)
T-43 (1) T-18 (1)
T-36 (1)
T-44 (1) T-46 (1)
T-37 (1)
T-45 (1) T-47 (1)
T-38 (1)
T-5 (4) T-48 (1)
T-39 (1)
T-49 (1)
T-40 (1)
Farm F Farm G Farm H
T-25 (1) T-26 (1) T-30 (1)
T-27 (1)
T-28 (1)
T-29 (1)
T-3 (2)
Farm P
T-1 (8)
T-18 (1)
T-51 (1)
T-52 (1)
T-53 (1)
T-54 (1)
Farm Q
T-2 (3)
T-55 (1)
T-56 (1)
T-57 (1)
T-58 (1)
Farm I
T-1 (1)
T-12 (2)
T-13 (1)
T-31 (1)
T-32 (1)
T-33 (1)
Farm J
T-8 (2)
Farm R Farm S
T-15 (2) T-13 (1)
T-59 (1)
T-60 (1)
Farm A
A-53 (1)
Farm B Farm C Farm D Farm E Farm F Farm G Farm H Farm I
A-4 (2) A-6 (2) A-5 (1) A-48 (1) A-5 (9) A-38 (1) A-35 (1) A-25 (1)
A-44 (1) A-39 (1)
A-57 (1) A-42 (1) A-47 (1)
A-46 (1) A-7 (2)
A-3 (2) A-43 (1)
A-55 (1)
A-10 (3)
A-56 (1)
A-51 (1)
A-9 (1)
A-45 (1)
A-36 (1)
A-37 (1)
Farm K Farm L Farm M Farm N
A-19 (1) A-28 (1) A-16 (1) A-1 (4)
A-2 (3)
A-13 (1)
A-17 (1)
A-29 (1)
Farm O Farm P Farm Q Farm R Farm S
A-27 (1) A-9 (3) A-41 (1) A-52 (1) A-49 (1)
A-61 (1)
A-8 (3)
A-54 (1)
A-40 (1)
No. of isolates
T-7 (5)
PFGE type
(5): isolates from the same cow
(5): isolates from different cows
Farm J
A-23 (1)
A-26 (1)
A-22 (1)
Research Questions
‰What are the genetic markers associated with
tetracycline resistant E. coli?
‰What are the phenotypic and genotypic
characteristics of beta-lactam resistant E. coli?
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Molecular Characterization of
Tetracycline Resistant
Determinants in Commensal E.
coli from Lactating Dairy Cattle
Chapter 3
Efflux mediated resistance in gramnegative bacteria
No Tetracycline inside the bacterial cell
R
tet(R)
PR
P
tet(B)
Outer
membrane
Porin Ompf
Gram-negative bacterial cell
+
Tetracycline-Mg+ complex
enters the bacterial cell
Mg
R
Tet-Mg+
Repressor
protein
+
+
+
Periplasm
Tet
R
tet(R)
R
P
Efflux pump
P
tetB
Efflux gene expressed
R: Tetracycline Repressor Protein
P: Promotor
Membrane based
Efflux pump
+
+
+
+ Protons
+
Cytoplasmic
membrane
Tetracycline Mg+ complex
Tetracycline resistance determinants
in 113 E. coli isolates
ƒ tet(B) gene was predominant: n=105 (93%)
ƒ tet(A) gene was observed in remaining 8 (7%) isolates
1000bp
1078bp
500bp
603bp
tet(B)
(650bp)
100bp
ФX174 Hae III
DNA ladder
March 2005
300bp
310bp
tet(A)
(210bp)
Ashish Sawant-PhD Thesis Defense
100bp
DNA ladder
37
Small and large plasmids
Small size plasmid on 0.8% gel
1 2 3 4 5 6 7 8 9 10 11 12 Sa
Large plasmids on PFGE gel
1 2 3 4 5 6 7 8 9 10 11 12 13
16210 bp
14174 bp
12138 bp
10102 bp
8066 bp
7045 bp
6030 bp
5012 bp
3990 bp
2972 bp
2067 bp
Small size plasmid blotting results
Large size plasmid blotting results
None of the plasmids carried tet gene
Sa: Supercoiled DNA ladder
Genomic DNA
1
PFGE Genomic DNA
2 3 4 5 6 7 8 9 10 Sb
917 kb
614.9 kb
487.4 kb
321.6 kb
307.6 kb
251.53 kb
230.6 kb
185.1 kb
174.0 kb
123.8 kb
102.7 kb
78.5 kb
65.7 kb
45.1 kb
35.8 kb
1 2 3
Blotting results
4 5 6 7 8 9 10 Sb
Sb: Salmonella ser. Newport Standard strain am01144 (XbaI digested)
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Genetic elements associated with tet(B)
tet(B) Genomic Library*
2.5-3.5 Kb region
selected for cloning
Electroporation
E. coli (strain T8)
No extrachromosomal DNA
(phenotype- Tet-R)
DNA extracted with
CTAB-NaCl
Digested with Sau3A1
pTrc plasmid vector
Electrocompetent
DH5α cells
Primary selection on LB
medium with oxytetracycline
(12µg/ml)
*Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhal K. 1998. Current protocols
in molecular biology. John Wiley and sons, Inc.
Isolation of tet(B) gene
‰ Plasmids were extracted from clones that showed
resistance to tetracycline
‰ Plasmids were isolated and send for sequencing
pTrc Forward 5’ GAGGTATATATTAATGTATCG 3’
pTrc Reverse 5’ GATTTAATCTGTATCAGG 3’
‰ Sequence was compared with NCBI genebank using
the BLAST program
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Total sequence of tetracycline
resistant genes and their location*
Tn10
5kb
(Accession no AF162223)
Tn10
6kb
tetA
tetR (membrane bound efflux
(repressor)
Pump)
7kb
tetC
8kb
tetD
9kb
IS10-right
(transposase)
Open reading frame map of Tn10
‰ Functions of tetC and tetD are not yet known
*
Lawley TD, Burland V, Taylor DE. 2000. Plasmid. 43:235-9.
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Gene sequence deposited in NCBI genebank
Conclusions
‰ This is perhaps the first report of sequence data of Tn10 based
class B tetracycline resistance from the chromosome of commensal
E. coli from lactating cattle
Commensal E. coli could serve as a reservoir for
tetracycline resistance determinants in dairy environment
even in the absence of tetracycline selective pressure
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Phenotypic and Genotypic Analysis
of Beta-Lactam and Extended
Spectrum Cephalosporin
Resistance in
E. coli from Lactating Cattle
Chapter 4
Susceptibility profiles of 94 ampicillin
resistant E. coli
‰ Susceptibility to different antimicrobial agents
‰ Pasco MIC gram-negative panel
‰ Evaluating susceptibility to penicillins, 1st, 2nd, 3rd, and 4th
generation of cephalosporins
‰ NCCLS document M100-S10
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Resistance profiles of
94 ampicillin resistant
E. coli isolates on disk
diffusion assay
AMP Ampicillin
CHL Chloramphenicol
GEN Gentamicin
SPT Spectinomycin
TET Tetracycline
TIC Ticarcillin
TIM Ticarcillin/CA
XNL Ceftiofur
No.
Antimicrobial resistance patterns
No. of isolates
1
AMP
10
2
AMP-TET
39
3
AMP-TIC
1
4
AMP-CHL-TET
1
5
AMP-SPT-TET
9
6
AMP-TET-TIC
3
7
AMP-CHL-SPT-TET
1
8
AMP-CHL-TET-TIC
3
9
AMP-GEN-SPT-TET
1
10
AMP-TET-TIC-XNL
1
11
AMP-CHL-SPT-TET-XNL
1
12
AMP-TET-TIC-TIM-XNL
1
13
AMP-GEN-SPT-TET-TIC
1
14
AMP-CHL-SPT-TET-TIC-XNL
1
15
AMP-CHL-SPT-TET-TIM-XNL
1
16
AMP-CHL-TET-TIC-TIM-XNL
2
17
AMP-CHL-SPT-TET-TIC-TIM-XNL
15
18
AMP-CHL-GEN-SPT-TET-TIC-TIM-XNL
3
No.
Resistance patterns
Pasco gram-negative
panel
Penicillins
1st gen
2nd gen
3rd gen
Antimicrobial agents:
AMP Ampicillin
SAM Amp/Sulbactam
PIP Piperacillin
TZP Pipera/tazobactam
ATM Aztreonam
CFZ Cefazolin
CEF Cephalothin
CXM Cefuroxime
CTT Cefotetan
FOX Cefoxitin
CRO Ceftriaxone
CPD Cefpodoxime
CFP Cefoperazone
CAZ Ceftazidime
CTX Cefotaxime
ZOX Ceftizoxime
GEN Gentamicin
TOB Tobramycin
CHL Chloramphenicol
NIT Nitrofurantoin.
Antimicrobial resistance patterns
No. of isolates
6
1
AMP
2
AMP-PIP
42
3
AMP-SAM
1
4
AMP-SAM-PIP
9
5
AMP-PIP-GEN
1
6
AMP-PIP-CHL
2
7
AMP-PIP-CEF
3
8
AMP-PIP-CEF-CHL
2
9
AMP-SAM-PIP-CEF
1
10
AMP-SAM-PIP-CHL
1
11
AMP-SAM-PIP-TOB-GEN
1
12
AMP-SAM-CEF-FOX-CPD-CHL
1
13
AMP-CFZ-CEF- FOX-CPD-CHL
3
14
AMP-SAM-CFZ-CEF- FOX-CPD-CHL
1
15
AMP-SAM-PIP-CFZ-CEF-CXM-FOX-CPD
1
16
AMP-SAM-CFZ-CEF-CXM-FOX-CPD-CHL
4
17
AMP-SAM-CFZ-CEF-CXM-FOX-CPD-CAZ-CHL
1
18
AMP-SAM-PIP-CFZ-CEF-CXM-FOX-CPD-CHL
1
19
AMP-SAM-CFZ-CEF-CXM-FOX-CPD-CAZ-CHL
4
20
AMP-SAM-CFZ-CEF-CXM-FOX-CRO-CPD-CAZ-CHL
1
21
AMP-SAM-CFZ-CEF-CXM-FOX-CPD-CAZ-ZOX-CHL
1
22
AMP-SAM-PIP-CFZ-CEF-CXM-CTT-FOX-CPD-CAZ
1
23
AMP-SAM-PIP-CFZ-CEF-CXM-CTT-FOX-CPD-CAZ-CHL
2
24
AMP-SAM-PIP-TZP-ATM-CFZ-CEF-CTT-FOX-CRO-CPD-CFP-CAZ-ZOX-CHL
1
25
AMP-SAM-PIP-ATM-CFZ-CEF-CXM-CTT-FOX-CRO-CPD-CFP-CAZ-ZOX-CHL
1
26
AMP-SAM-PIP-TZP-ATM-CFZ-CEF-CXM-FOX-CRO-CPD-CFP-CAZ-CTX-ZOX-CHL
1
27
AMP-SAM-PIP-TZP-ATM-CFZ-CEF-CXM-CTT-FOX-CRO-CPD-CFP-CAZ-ZOX-CHL
1
Beta-lactamases that confer
resistance to ampicillin
ƒ73 of 94 Amp-R E. coli
isolates encoded for blaTEM-1
gene
bla TEM-1 gene
(861bp)
ƒOnly 4 ceftiofur resistant
isolates encoded the blaTEM-1
gene
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Integrons class 1
Sample A73: Integron size: 1165 bp
5’
3’
5’ conserved region
dfrA gene
Hypothetical protein 3’ conserved region
1.6 kb
Sample A107: Integron size: 1598 bp
Sequenced 534 bp
Not-sequenced 536 bp
Sequenced 528 bp
3’
5’
dfr17 gene
5’ conserved region
aadA5 gene
3’ conserved region
1.2 kb
1 kb
Sample A92: Integron size: 942 bp
5’
5’ conserved region
3’
aadA23 gene
3’ conserved region
dfr gene : Confers resistance to Trimethoprim
aad gene: Confers resistance to streptomycin and spectinomycin
ƒ15 out of 75 E. coli isolates showed presence of an integron
ƒSequence analysis revealed that all of them were Integron-1
Screening for extended spectrum beta-lactamases
ESBL positive control
ESBL negative culture
Ceftazidime
Cefotaxime
Ceftazidime + CA
> 5mm zone
Cefotaxime + CA
> 5mm zone
Clavulanic acid (CA)
effect
No Increase in zones when in
combination with clavulanic acid
Results of ESBL analysis of 25 Cef-R
and extended spectrum cephalosporins
No zone of inhibition
No CA effect
None of the isolates
showed CA effect, and
were not phenotypically
ESBL producers
Screening for cephamycinases
ampC : Salmonella enterica subsp. enterica
serovar Choleraesuis plasmid (AY253913)
1774
I:1851
II:1851
III:1851
IV:1851
V:1851
1924
I:2001
II:2001
III:2001
IV:2001
V:2001
ampC gene sequence region: 1774-2919
bla CMY-2: K. pneumoniae plasmid (X91840)
ampC gene sequence region: 1924-3069
ampC: Salmonella senftenberg extended
spectrum beta-lactamase bla LAT-3 (U77414)
1
I:78
II:78
III:78
IV:78
V:78
ampC gene sequence region: 1-1143
PCR product of blaCMY gene sequencing data
I:
Isolate A92
945bp (99% match)
II:
Isolate A113
940bp (100% match)
III:
Isolate C1
946bp (99% match)
IV:
Isolate F10
939bp (100% match)
V:
Isolate S16
937bp (100% match)
I:2785
II:2789
III:2785
IV:2786
V:2784
I:2935
II:2939
III:2935
IV:2936
V:2934
I:1012
II:1016
III:1012
IV:1013
V:1011
2
937
2
940
6
941
4
939
4
937
2919
3069
1143
blaCMY (1kb)
All the 25 multi-drug and
ceftiofur resistant E. coli
encoded for the
plasmid based blaCMY gene
Conclusion
‰ The prevalence of multidrug resistance and
extended spectrum cephalosporin resistance in
commensal E. coli is an important public health
concern
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Recommendations
‰ Educate veterinarians and dairy producers on prudent
use of antimicrobials
‰ Antibiotic usage: an integral part of quality assurance
programs
‰ Further study influence of antibiotic selective pressure
‰ Monitoring of antibiotic resistance should include
commensal flora
March 2005
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Recommendations
‰ Understand the role of Tn10 as the reservoir of tet(B) determinants
‰ Useful for developing risk assessment modeling
‰ Analyze treatment protocols on their influence on treatment
outcomes
‰ Continuing monitoring for multidrug and cephalosporin resistance in
dairy environment
‰ Cephalosporin like ceftiofur is the latest and most valuable
antimicrobial agent available to the dairy industry. Attempts to
judiciously use this antibiotic needs to be widely promoted
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