Transcript resentation - Grain Science and Industry

Antibiotic resistant and virulent enterococci in
animal feed
Lakshmikantha H.C1, Bh. Subramanyam1,
and L. Zurek2
1Department
of Grain Science and Industry
2Department of Entomology
Kansas State University
Introduction
animal feed: antibiotics: enterococci
Antimicrobials in animal feed

In the US, 3,000 feed mills produce 121 million tons of feed for
various animal species (Feedstuffs 2003)

Antibiotic use in feed as a :

>70% of antibiotics (>22 million pounds) produced in USA are
used by the feed industry (Walker et al. 2005)

Selection pressure
¤ Growth promotion
¤ Prophylactic
¤ Therapeutic
Antibiotic resistant bacteria
Importance of enterococci

Enterococci are normal fauna of the
gastrointestinal tract of humans and
animals

Enterococci are not typical foodborne pathogens

Reservoirs for drug-resistant genes
and mobile genetic elements
Enterococcus faecalis
Antimicrobial resistance and public health

76 million cases of food borne illness reported
annually in the United States (CDC)

>70% bacterial infections in the world are resistant
to at least one antimicrobial (CDC)

Vancomycin-resistant enterococci infections, MRSA
have become common in U.S. hospitals (National Institute
of Allergy and Infectious Diseases)
Research questions

Does animal feed carry enterococci?

Do enterococci harbor drug resistant & virulence genes?

Are these virulence genes expressed?

Is there any gene transfer from Enterococcus spp. to
other species via horizontal gene transfer (HGT)
Horizontal transmission of resistance genes
among species
Levy and Miller, (1989). Gene transfer in the environment. Science. 247: 350-351
Hypothesis
Animal feed carry enterococci with
antibiotic resistant and virulent genes
Research objectives

Isolation, quantification, and species level identification
of enterococci isolated from animal feed using 16S rRNA
gene

Molecular characterization of antibiotic resistance and
virulence genes assessed by phenotypic and genotypic
assays

Conjugation ability and transfer of virulent genes from
Enterococcus spp. to other species via horizontal gene
transfer
Materials & Methods
Collection of samples
● ●
●


Feed samples: 89
No. enterococcal isolates: 208
●
Materials and Methods
Feed
Isolation
Quantification
Antibiotic resistance
Specific primers; 16S rRNA gene sequences
Multiplex PCR
Species level identification
Antibiotics used in this study
• Tetracycline 30µg (TET)
• Ampicillin 10µg (AMP)
TET
• Erythromycin 15µg (ERY)
VAN
CIP
• Vancomycin 30µg (VAN)
ERY
CHL
• Chloramphenicol 30µg (CHL)
AMP
• Ciprofloxacin 5µg (CIP)
• Gentamicin 120 µg (GEN)
• Kanamycin 20µ g (KAN)
Diffusion disk assay
• Streptomycin 20µg (STR)
Followed the guidelines of the Clinical and Laboratory Standards Institute
(formerly NCCLS)
Results
Prevalence and distribution of enterococci
in animal feed
Prevalence Total
(%)
isolates
Mean ± SEM
CFU/g
Source
Feed
samples
Swine farms
41
24 (58)
160
8.1 ± 6.3 x 103
Feed mills
48
16 (33)
48
1 ± .15 x 101
Total
89
40 (43)
208
4 ± 3 x 103
Enterococcal species distribution in animal
feeds
Type of
sample
Total
E.
E.
E.
E.
E.
isolates faecalis faecium gallinarum casseliflavus hirae
Feed on
farm
160
6
29
29
85
11
Unprocessed
feed
33
2
4
6
20
1
Processed
feed
15
0
4
2
9
0
Total
208
8
37
37
114
12
Antibiotic resistance profiles of enterococci
isolated from feed environments
% prevalence
Feed
(n=208)
(208)
50
45
40
35
30
25
20
15
10
5
0
VAN
TET
AMP
CHL
CIP
Antibiotics
ERY
KAN
STR
GEN
Tetracycline resistance profiles of enterococci
isolated from animal feeds
% prevalence
60
Feed (n=208)
(208)
50
40
30
20
10
0
tetM
tetS
tetK
tetO
tetA
tetC
Tetracycline resistance genes
tetQ
tetW
Virulence determinants of enterococci isolated
from animal feed
Genotypic assay
Phenotypic assay
Feed (n=208)
50
% prevalence
% prevalence
60
40
30
20
10
0
Hemolysis Gelatinase
Virulence factor
asa
20
18
16
14
12
10
8
6
4
2
0
cylA
gelE
asa1
Virulence factor
esp
Distribution of virulence determinants in Enterococcus
species isolated from animal feed
% isolates
positive genotypically
% isolates
positive phenotypically
Feed
Feed
asa1
3.8
50
esp
24
ND
cylA
2.5
40
gelE
18.2
79
Virulence
determinants
Feed ( n = 208)
Highlights and outcomes of research-to-date

Majority of the isolates showed resistance to tetracycline
(48 %), erythromycin (14.4 %), and streptomycin (13 %)

58 % of isolates were resistant to at least one antimicrobial
agent

26 % of isolates were resistant to two or more antimicrobial
agents

20.6 % of isolates harbors at least one virulence gene and
resistance to at least one antimicrobial agent
Conclusions

Animal feed collected from feed mills and livestock
facilities harbored antibiotic-resistant and potentially
virulent enterococci

Source of enterococcal contamination in animal feed
is yet to be determined

Can these drug-resistant genes be transferred to other
enteric pathogens via horizontal gene transfer (HGT)?
Acknowledgements
Dr Leland McKinney
Dr Aqeel Ahmad
Research reported here was funded by USDA/CSREES
under agreement number 2002-3438-112187
Thank you
Questions?