Transcript Association of Enterococci with Stored Products and Stored

Association of Enterococci with Stored
Products and Stored-Product Insects:
Public Health Importance and Implications
H. C. Lakshmikantha, Bh. Subramanyam,
Z. A. Larson, and L. Zurek†
Department of Grain Science and Industry
†Department of Entomology
Kansas State University, Manhattan 66506, USA
Presented at the 9th International Working Conference on
Stored-Product Protection, 15-18 October 2006, Campinas, Brazil
Introduction
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Enterococci are ubiquitous, grampositive, catalase negative, cocci
Enterococci are normal flora of
the gastro-intestinal tract of
humans and animals
Enterococci are not food-borne
pathogens
Reservoirs for drug-resistant
genes
Enterococcus spp.
Medical importance of enterococci

Readily develop resistance to antibiotics and can transfer
resistance genes to other bacteria

Second most common cause of hospital-acquired infections
in the United States
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Documented resistance to numerous antibiotics, including
vancomycin
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Vancomycin resistant enterococci (VRE) and VRE infections
have become a worldwide public health threat

In the United States, the prevalence of antibiotic-resistant
enterococci (ARE) may be, in part, due to the use of
antibiotics in feed as growth promoters for farm animals
Antibiotics and the US feed industry
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In the United States, about 3,000 feed mills produce 121
million tons of feeds annually for various domestic animals
An estimated 70% (nearly 2.5 million tons) of the antibiotics
used in the United States each year are used as feed
additives for chickens, hogs, and beef cattle
11 antimicrobials are commonly used in animal feeds
 Examples: bacitracin, chlortetracycline, dynafac,
mycostatin, oxytetracycline, oleandomycin, penicillin,
streptomycin, bambermycins, tilmicosin, and tylosin
Antibiotic use in feed may lead to development of resistance
in bacterial strains of clinical importance
Association of stored-product insects
with bacteria
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Stored-product insects are cosmopolitan in distribution
and occur in diverse habitats from the farm to the
consumer’s home
The digestive tract of these insects provides a
microclimate for bacterial multiplication
Stored-product insects have been reported to harbor
potentially pathogenic bacteria (Apuya et al. 1994; Larson
2004)
Stored-product insects may be of medical and veterinary
interest, because they could serve as vectors for
antibiotic resistant bacteria (Larson 2004)
Importance of stored products and storedproduct insects in the food web
http://res2.agr.ca/winnipeg/storage/images/lo-res/control/ecosys-l.jpg
Materials and methods
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Insects: 392
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Mar-Nov, 2003 [298]
Apr-Jun, 2006 [94]
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Stored products: 28
Stored products were collected from a feed mill and two livestock
facilities in Kansas.
Materials and methods
Insect/feed
Isolation
Quantification
Antibiotic resistance
Specific primers; 16S rRNA gene sequences
PCR
Species level identification
Specific media used for bacterial
isolation
• Trypticase soy broth agar:
• MacConkey sorbitol agar:
• mEnterococcus agar:
for general bacteria
for Enterobacteriaceae bacteria
for Enterococcus spp.
Antibiotic resistance assays conducted
using the diffusion disk assay
• Tetracycline (30 µg)
• Ampicillin (10 µg)
• Erythromycin (15 µg)
• Vancomycin (30 µg)
• Chloramphenicol (30 µg)
• Ciprofloxacin (5 µg)
Measuring antibiotic resistance
Resistance is determined by
the bacterial carpet growing
up to the diffusion disk
No resistance and intermittence
is determined by measuring the
zone of inhibition (dotted line)
created by the antibiotic around
the disk
Primers and multiplex PCR conditions used for species
identification of Enterococcus
a
Sequence (5'–3')
Primer
conc.
(pmol)
Temp
(°C)
Size
(bp)
Reference
F
GGTATCAAGGAAACCTC
2.5
54
822
Kariyama et al.,
2000
ATCC 49579
R
CTTCCGCCATCATAGCT
2.5
54
822
Kariyama et al.,
2000
E. casseliflavus
ATCC 25788
F
CGGGGAAGATGGCAGTAT
2.5
54
484
Kariyama et al.,
2000
E. casseliflavus
ATCC 25788
R
CGCAGGGACGGTGATTTT
2.5
54
484
E. faecalis
ATCC 19433
F
TCAAGTACAGTTAGTCTTTATTAG
5.0
54
941
E. faecalis
ATCC 19433
R
ACGATTCAAAGCTAACTGAATCAGT
5.0
54
941
E. faecium
ATCC 19434
F
TTGAGGCAGACCAGATTGACG
1.25
54
658
E. faecium
ATCC 19434
R
TATGACAGCGACTCCGATTCC
1.25
54
658
16S rRNA gene
F
GGATTAGATACCCTGGTAGTCC
2.5
54
320
Kariyama et al.,
2000
16S rRNA gene
R
TCGTTGCGGGACTTAACCCAAC
2.5
54
320
Kariyama et al.,
2000
Primer
type
Positive
control
Directiona
E. gallinarum
ATCC 49579
E. gallinarum
F, forward; R, reverse.
Kariyama et al.,
2000
Dutka-Malen et
al., 1995
Dutka-Malen et
al., 1995
Dutka-Malen et
al., 1995
Dutka-Malen et
al., 1995
Results
Percentage distribution of stored-product insects
collected from various facilities for this study
(n= 392)
ID
no.
Insect species
Number of insects (%)
1
Alphitobius diaperinus
2
Cryptolestes spp.
17 (4.33)
3
Palorus ratzeburgi
1 (0.25)
4
Rhyzopertha dominica
5
Sitophilus zeamais
6
Stegobium paniceum
12 (3.06)
7
Tribolium castaneum
156 (39.79)
8
Tribolium confusum
145 (36.98)
9
Trogoderma variabile
10
Ahasverus advena
Total
4 (1.02)
15 (3.82)
2 (0.51)
36 (9.18)
4 (1.02)
392
Number of stored product insects sampled from each mill and
number of colony forming units (CFU) per insect on three
media
Sitea
mEnterococcus agar
N
b
Trypticase soy broth
agar
MacConkey agar
n (%)c
mean CFU
n (%)c
mean CFU
n (%)c
mean CFU
1
57
5 (8.7)
1.7 x 105
12 (21.1)
3.0 x 104
1 (1.8)
3.5 x 103
2
22
0
0
11 (50.0)
5.9 x 102
1 (4.5)
2.4 x 103
3
65
1 (1.5)
4.8 x 103
42 (64.6)
3.1 x 103
4 (6.2)
6.0 x 103
4
45
1 (2.2)
1.2 x 101
9 (20.0)
4.1 x 102
1 (2.2)
3.6 x 102
5
53
45 (84.9)
6.5 x 103
43 (81.1)
1.6 x 104
10 (18.9)
2.4 x 102
6
56
1 (1.8)
8.4 x 103
23 (41.1)
2.4 x 104
2 (3.6)
3.5 x 103
7
10
3 (30)
4 x 101
7 (70)
4.2 x 102
6 (60)
2.5 x 102
8
08
6 (75)
4 x 101
7 (87.5)
4.3 x 102
6 (75)
4 x 101
9
09
7 (77.7)
3.8 x 101
8 (88.8)
2.4 x 103
7 (77.7)
4.3 x 102
10
47
47 (100)
3.7 x 101
47 (100)
4.1 x 102
10 (100)
2.2 x 102
11
12
8 (66.6)
3.7 x 101
11 (91.6)
3.7 x 102
9 (75)
7.5 x 101
12
08
8 (100)
4.1 x 101
8 (100)
1.4 x 103
8 (100)
3.4 x 102
aSamples
in mills 1-6 were collected during March to November, 2003 (Larson, 2004); samples in mills 7-12 were
collected by H. C. Lakshmikantha during April to June, 2006.
bN = Total number of insects sampled from each mill. cn = Number of positive samples (% of positive samples).
Number of stored products sampled from each
mill/facility and number of colony forming units (CFU)
per g sample on three media
Sitea
mEnterococcus agar
N
b
n (%)c
Trypticase soy broth
agar
MacConkey agar
mean CFU
n (%)c
mean CFU
n (%)c
mean CFU
1
4
4 (100)
9.3 x 102
4 (100)
6.4 x 103
4 (100)
2.2 x 103
2
15
3 (60)
2.7 x 102
5 (100)
2.7 x 103
3 (60)
8.3 x 102
3
12
8 (66.6)
3.1 x 102
12 (100)
5 x 103
10 (83.3)
1.4 x 103
4
7
5 (71.4)
3.12 x 102
7 (100)
5.5 x 103
6 (85.7)
2.8 x 103
aSites
1 and 3 are swine farms in Salina, Kansas; site 2 is the Kansas State University pilot feed mill and site
4 is the swine farm at Kansas State University
bN = Total number of stored products sampled from each site
cn = Number of positive samples (% of positive samples)
Fig 1. Percentage of enterococcal isolates (n = 67) from storedproduct insects with antibiotic resistance
Percentage of isolates
Resistant
Intermediate
Susceptible
100
90
80
70
60
50
40
30
20
10
0
Vancomycin
Tetracycline
Ampicillin
Chloramphenicol
Antibiotics tested
Ciprofloxacin
Erythromycin
Fig 2. Percentage of enterococcal isolates (n = 125) from stored
products with antibiotic resistance
Percentage of isolates
Resistant
Intermediate
Susceptible
100
90
80
70
60
50
40
30
20
10
0
Vancomycin
Tetracycline
Ampicillin
Chloramphenicol
Antibiotics tested
Ciprofloxacin
Erythromycin
Prevalence and identification of enterococci isolated from stored
products and stored-product insects collected during April to
June, 2006
No. species identified (%)
Source
No.
samples
Total no.
enterococci
isolates
No.
isolates
identified
(%)
Storedproduct
insects
94
67
43 (64.1)
0
10 (23.2)
7 (16.2)
26 (60.4)
Stored
products
28
125
45 (36)
2 (4.4)
8 (17.7)
2 (4.4)
33 (73.3)
E.
faecalis
E.
faecium
E.
gallinarum
E.
casseliflavus
Prevalence, identification, and distribution of enterococci isolated
from stored products and stored-product insects collected during
April to June, 2006
Insect Species
Total isolates
E. faecalis
E. faecium
E. gallinarum
E. casseliflavus
Red flour beetle
18
-
4
4
10
Confused flour beetle
11
-
4
7
Warehouse beetle
8
-
1
7
Lesser grain borer
3
-
1
Foreign grain beetle
3
-
Total
43
-
Type of sample
Total isolates
E. faecalis
Stored products (farm)
37
Stored products
(feed mill)
Before processing
2
2
1
E. faecium
E. gallinarum
E. casseliflavus
1
8
2
26
6
1
-
-
5
Stored products
(feed mill)
After processing
2
-
-
-
2
Total
45
Conclusions
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Our findings provide evidence that stored
products and stored-product insects harbor
antibiotic resistant and potentially virulent
enterococci
Our data reinforces the need for pest
management to reduce the availability of
insect vectors, particularly stored-product
insects, in the feed mill and livestock
environments
Acknowledgements
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Dr. Aqeel Ahmad
Dr. Keith Behnke
Consortium for Integrated Management of Stored
Product Insect Pests (CIMSPIP)
Research reported here was funded by the United
States Department of Agriculture, Cooperative State
Research and Education, and Extension Service’s Risk
Assessment and Mitigation Program under Agreement
Number 2002-3438-112187.
References
Apuya, L. C., S. M. Stringham, J. J. Arends, and W. M. Brooks. 1994 Prevalence of
protozoan infections in darkling beetles from poultry houses in North Carolina. Journal of
Invertebrate Pathology. 63: 255-9
Larson, A.Z. 2004. Diversity and Medical importance of stored- product insects in feed mills.
M.S Thesis, Kansas state university, USA.
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Thank you