Transcript Slide 1

Animal Feed Components and
The Impacts of Land-applying Manure
on Soil Bacterial Biodiversity and
Antibiotic Resistance
Amy R. Sapkota, PhD, MPH
University of Maryland College Park
School of Public Health
Background
Animal-based food products constitute a large proportion
of the U.S. diet
– U.S. per capita consumption of total meats is 90.5 kg/year (199
lbs/year) (USDA, 2005)
Quality of food products is directly related to animal
feeding practices
Farms and feeding practices have undergone changes
– Small family farms  Concentrated animal feeding operations
– Corn or grass diet  Complex mixtures
U.S. feed industry has grown to be
the largest feed producer in the world
– >120 million tons of feed
– $25 billion industry
Source: USDA, 2005. http://www.ers.usda.gov/data/
What is in Animal Feed and Does
it Affect Human Health?
Previously, no comprehensive, peerreviewed source existed regarding
specific feed ingredients and their
potential impacts on human health
Existing sources focused on one feed
contaminant and one health effect
Methods
We reviewed the literature and FDA, USDA, and
CDC databases:
1. To summarize existing data on current feed
production practices and feed ingredients
2. To describe biological, chemical and other etiologic
agents that have been detected in feed
3. To evaluate evidence whether current feeding
practices may lead to adverse human health effects
4. To identify data gaps that prevent comprehensive
assessments of human health risks associated with
feed
Compiled a review article targeted to public
health researchers
What is in animal feed?
Source: Sapkota et al. 2007. EHP 115;5:663-670.
Etiologic Agents Detected in Feed
and Potential Human Health Effects
Source: Sapkota et al. 2007. EHP 115;5:663-670.
From Animal Feed Ingredients to Human Health
Animal Feed Ingredients
Contaminated plant- and
animal-based ingredients
Additional Routes
of Contamination
Insects, birds, feral
animals, humans,
fomites
Environmental
Factors
Affecting
Bacterial Growth
Feed Production
Milling, mixing, storage
and transport
Animal Feeding
Operation
Crops
Flies
Air
Live Animals
Manure
Groundwater
Human Biosolids
Surface
water
Processing and
Packaging Plant
Farmers,
Workers and
Neighbors
Meats
Pets
General
Human
Population
Susceptible subpopulations, other
secondary contacts
Take-home Messages
No comprehensive nationwide animal feed
surveillance system to monitor:
– The amounts and types of specific feed ingredients
– The levels of contaminants in feeds and food
products
Human health effects data are not appropriately
linked to the minimal amount of surveillance
data that do exist
Difficult to pinpoint the extent to which human
health risks are associated with animal feed
We need increased federal funding
and surveillance from “farm to fork”
The Impacts of Land-applying Manure
on Soil Bacterial Biodiversity and
Antibiotic Resistance
Background
500 million to 1 billion tons of animal manure is landapplied each year
Manure contains nutrients and contaminants, including
antibiotic residues and resistant bacteria
When swine manure is land-applied, resistance genes
can be transferred between swine-associated bacteria
and soil bacteria, perpetuating the spread of resistance
(Agerso 2005; Jensen 2002)
We hypothesized that antibiotic selective pressures
resulting from the land-application of swine manure
could modify soil bacterial biodiversity as well
Sources: Agerso et al. 2005. Appl Env Microbiol 71:7941-7947; Jensen et al. 2002. Env Int 28:487-491.
Study Objectives
To use traditional culture techniques and
metagenomic methods to explore
antibiotic resistance and bacterial diversity
in agricultural soils amended with swine
manure compared to control soils
Methods
Figure 1: Soil and manure sampling locations at a swine farm*
No pigs or manure
8a,b,c
Old pig grazing field,
manure applied
Upgradient
7a,b,c
Winter wheat field,
manure applied
Pig Barns
Downgradient
10
6a,b,c
9
5a,b,c
4a,b,c
3a,b,c
Spectinomycin
2
1
Pig grazing field
Manure sample
Soil sample
*Not to scale
Sapkota et al. [In preparation].
Methods: Sample Analysis
Culture techniques:
– Culturable bacteria (susceptible and resistant)
were isolated from each sample and identified
by PCR and sequencing
– Isolates were tested for the aad(A) resistance
gene and the resulting genes were sequenced
Metagenomic methods:
– Total DNA was extracted from each sample
– 16S rRNA genes in each sample were amplified,
purified, and labeled using an in vitro
transcription method
– RNA was hybridized to a 16S rRNA-based
taxonomic microarray
– Microarray results were analyzed by principal
components analysis
– The aad(A) resistance gene was quantified
using real-time PCR, cloned and sequenced
Bacterial Biodiversity in
Culturable Isolates
Table 1: Predominant bacterial species cultured from manure and soil samples collected at a swine
farm
Sample Type
Bacteria
Frozen manure
Planococcus spp., Acinetobacter spp., Arthrobacter spp.,
Enterobacter ludwigii, Pseudomonas spp.
Soil from pig grazing field
Stenotrophomonas spp., Bacillus spp., Acinetobacter spp.,
Pseudomonas spp.
Soil from downgradient field
Stenotrophomonas spp., Pseudomonas spp., Bacillus spp.
Soil from upgradient field, no pigs or manure
Serratia spp., Pseudomonas spp., Bacillus spp.,
Streptomyces spp., Bacteroidetes spp.
Fresh solid manure sample
Enterococcus inusitatus
Sapkota et al. [In preparation].
Bacterial Biodiversity in Metagenomic
DNA Samples
Figure 3
d = 0.02
PC1 20%
Metagenomic microarray approach revealed more soil biodiversity, in general,
and more differences between manure-amended and control soils.
Figure 4
Flav o2
PF8c(38.0)A01
PF8c(38.0)A05
PF8c(38.0)A09
PF7a(37.5)A05
PF7b(39.0)A05
PF7a(37.5)A09
PF7b(39.0)A01
PF7a(37.5)A01
PF7b(39.0)A09
PF4b(37.5)A09
PF4b(37.5)A01
PF7cR(38.0)A05
PF7cR(38.0)A09
PF7cR(38.0)A01
PF6aR(38.5)A09
PF6aR(38.5)A01
PF6aR(38.5)A05
PF5bR(40.0)A01
PF5a(37.0)A09
PF5a(37.0)A05
PF6b(38.0)A09
PF5cR(38.0)A09
PF6b(38.0)A05
PF5cR(38.0)A01
PF5a(37.0)A01PF6b(38.0)A01
PF6c(38.5)A05
PF4a(38.0)A09
PF5bR(40.0)A09PF6c(38.5)A09
PF6c(38.5)A01
PF4a(38.0)A01
PF4a(38.0)A05
PF4c(39.0)A09
PF3c(37.0)A09
PF4c(39.0)A05
PF4c(39.0)A01
PF3c(37.0)A01
PF3c(37.0)A05
PF4b(37.5)A05
PF3b(42.0)A01
PF3b(42.0)A09
PF3a(42.5)A05
PF3a(42.5)A01
PF3a(42.5)A09
PF3b(42.0)A05
PC1 23%
d = 0.005
Burcep
CF319.m
Verru1
CF319
PseuPsJ
CFB562A
PF5bR(40.0)A05
PF5cR(38.0)A05
PC1 23%
PF8b(38.5)A05
PC1 20%
PF8b(38.5)A09
PF8b(38.5)A01
PF8a(39.5)A01
PF8a(39.5)A05
PF8a(39.5)A09
HGC664.IN16
Acido.c
Plancto4.mB
Plancto12
HGC664.IN14
Alpha.683.IN21
CFB562C
CFB562B Alpha.683.IN23
PseuD
Alpha.683.IN20
Plancto4.mA
AcidoUnc3
ARC915
PseubC2.9
Mogi12 HGC236.m
HGC664.IN17
Pseu1 BET940
Ly ng1
Pola1
HGC664.m
CFX109
EUB338II
BONE23Am
HGC664.IN18
ClostIII.m
Ace1
Nso190
Nitbri2
Raltaiw2
Epsi5
Psdjess1
AcidUnc
Psdenit1b
Herb1
PseubC3.6
PseubC1.6
PseubC1.5
Acine1
Pseu32 HGC664.IN33
LGC354B
Blatta1
PLA46
PseubC2.10
LGC354A
Rhodopseud
Acido.a
EUK309
Nit1A
PseuChlo
Pseu9
PseuCicho2
Nit1B
Aci2
PseubC2BC3.2
PseuNZ17.4
Pseu27
Nit1C
Ser2
Ornit3
PseuPseu2
Entero1
Kleb3
PseubC2BC3
Plancto1
Burkho4A
Alpha.683.IN19
Sphingo5B
Poly
Spiro5
Sele1
Act1
cell Clost
Kluy
TDRNO1030
3.2.1
HGC664.IN53
Rzbc1247
CFB562D
Nitocea1
Achro1
Mann1
Cau2
Geli3
Pseu3
HGC664.IN30
Ep4
HGC664.IN48
Pho1
Azorhizobi5
Methy
Mega4
UNIV1389b
HGC664.IN58
HGC664.IN40
Hy lo2
me3
Alpha.683.IN12
NSR1156
Entero2
Rgal157
Verru3
Rhodobact1A
Diaz1
HGC664.IN13
Brady
6APseuDhA.91
Acine3
Legiob
GAM.mrc2
PseuNZ17.1
HGC664.IN5
Kluy
3.3.1
HGC664.IN37
Nostoc1
Rhi
Plancto9
Alpha.683.IN9Alpha.683.IN18
Ehrli2 Xan
Alpha.683.IN22
Chloro2 Rhizo157
Psdcorr1
Plancto5
Alpha.683.IN6
Alpha.683.IN16
DELTA495a
Alpha.683.IN4
Rhodobact1B
Brady 4
Alpha.683.IN13
HGC664.IN32
HGC664.IN24
Ecoli2
Plancto8
TM7.2
HGC664.IN47
Alpha.683.IN11
Alpha.683.IN10
TM7.1
Alpha.683.IN2
Alpha.683.IN3
Alpha.683.IN14
Campy
Rhodov o1B
Alpha.683.IN15
Acidocella1
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HGC664.IN38
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Alpha685
HGC664.IN31
Sapkota et al. [In preparation].
Spectinomycin Resistance in
Culturable Isolates
Spectinomycin Resistance in Culturable Soil and Manure Bacterial Isolates
Recovered from a Swine Farm
Percent resistant to spectinomycin
25
Spectinomycin Resistance
20
15
10
5
0
Fresh Manure
Frozen
Manure
Soil/
Soil/
Soil/
Soil/ Wheat
Downgradient Downgradient Downgradient field manure1
2
3
applied
Soil/ Old pig
grazing
Soil/
Upgradient
The aad(A) resistance gene was only detected in isolates from
manure samples and soils impacted by manure.
aad(A) Resistance Gene in Metagenomic
DNA Samples
Similar to results observed in culturable isolates
Higher copy numbers of the aad(A) resistance
gene were in soils impacted by manure
Samples
Standards
NC
Currently, we are analyzing all aad(A) resistance
gene sequences from culturable bacteria and
metagenomic samples to understand diversity in
the gene
Conclusions
Microarray results indicate that some differences
exist in bacterial biodiversity between soils
amended with swine manure and control soils
Spectinomycin-resistant bacteria were isolated
only from manure samples and soils recovered
from pig grazing fields
The aad(A) resistance gene was only detected
in manure samples and soils impacted by
manure
The application of swine manure to soils impacts
both antibiotic resistance and bacterial
biodiversity in soil
Acknowledgements
Funding: Center for a Livable Future, Johns Hopkins
Bloomberg School of Public Health
École Centrale de Lyon Colleagues: Timothy Vogel,
Pascal Simonet, Elizabeth Navarro, Maude David, JeanMichel Monier, Audra Nemir, Benoit Remenant, Saliou
Fall, Sandrine Demaneche
JHU Colleagues: Kellogg Schwab, Ellen Silbergeld,
Robert Lawrence, Shawn McKenzie, Polly Walker, Lisa
Lefferts