Annex 5: Patterns of Multiple Antimicrobial Resistant Escherichia coli on Broiler
Farms
Eve Pleydell, Meenaxi Sharma, Lourdes Migura, Rob Davies
Aims
 To compare the occurrence of multiple antimicrobial resistant E. coli on a conventional
and an organic broiler farm.
 To assess whether the carriage of ampicillin- or chloramphenicol-resistance is associated
with a multiple antimicrobial resistant phenotype.
 To assess sources of multiple antimicrobial resistant E. coli on broiler farms.
 To assess changes in patterns of multiple antimicrobial resistant E. coli shed in the
faeces of broiler chickens over time.
Summary
This study found that E. coli isolated from the faecal droppings of chickens on a conventional
broiler farm expressed a significantly higher number of resistances per isolate than those E.
coli isolated from organic-broiler droppings. However low numbers of multiple-resistant E.
coli with up to 10 resistances were found on the organic farm, thus indicating that these
highly multiple-resistant E. coli were not able to become a predominant part of the faecal
flora on this farm. Conversely, the level of multiple-resistance seen for E. coli that had been
grown on media containing chloramphenicol was equally high on both farms, with a median
number of resistances per isolate of 6. The reasons for the persistence of these multipleresistant organisms in an environment where antimicrobials are not used should be
investigated further.
On both farms it was seen that day-old chicks brought largely sensitive organisms onto the
farm with them, whereas the level of resistances expressed by E. coli remaining in the
houses after cleaning was more similar to that expressed by the faecal E. coli isolated from
the birds as they grew. Thus, whilst it is likely that the E. coli remaining in the houses
reflected that excreted by the previous birds, it is also feasible that the next flock of birds
could be colonised by the bacteria persisting in the houses. Therefore one could envisage
that over a period of time this could develop into a stable self-sustaining system, with the
actual level of multiple-resistance reflecting the individual farm policy regarding the use,
non-use, or historical use of antimicrobials.
When the pattern of multiple-resistance in E. coli isolated at different times throughout the
growth cycle of the birds was investigated, it was seen that the E. coli on the conventional
farm went from largely sensitive to multiple-resistant within the first few days of life. This level
of multiple-resistance then persisted until the birds went to slaughter. This farm was
administering a 3-day course of lincomycin-spectinomycin to the day-old chicks upon arrival.
Therefore it appears that dosing chickens at an age before their gut flora has become
established may act to eliminate the less antimicrobial-resistant E. coli, that arrived with the
chicks, from the flock. Furthermore in a closed system, such as a biosecure conventional
broiler house, by definition there should be limited opportunities for further bacteria to enter
the houses and therefore there are no sensitive E. coli to re-adjust the balance of resistance
within the birds intestines.
Further characterisation of these isolates would help ascertain whether these multipleresistant E. coli are also carrying fitness traits that may be enhancing their persistence. This
information is important for the design of farm management policies with a view to
decreasing reservoirs of resistant organisms and genes on livestock farms. The results of
this work also implies that studies should be undertaken to investigate the effect of
medicating birds of different ages with regularly-used products with respect to the
persistence of multiple-resistant E. coli within a flock. Furthermore, a role for the potential
use of post-medication competitive exclusion agents has been elucidated which should be
further investigated using controlled intervention studies.
Methods
A panel of 857 E. coli was tested for phenotypic antimicrobial resistance against 17
antimicrobials using custom-made 90-well micro-dilution Sensititre® plates (for further details
of the Sensititre® plates see Annex 2). The E. coli were selected from isolates that had been
stored from studies on two broiler farms, one conventionally managed and the other an
organic farm (for more details regarding the study farms see Annex 1). The criteria used for
selecting the E. coli panel are shown in Table 1.
Table 1. The sources of the panel of 857 E. coli isolates that were selected for phenotypicresistance characterisation.
Conventional Broiler Farm
Sample Type Number Selection Criteria
Isolates
Faeces
240
 17 birdsa per week
 1 per type of E. coli per
week
Post95c
When present
in a
Cleaning
sample:
 2 non-specific E. coli
per area of house
 1 ampicillin-E. coli per
area of house
 1 chloramphenicol-E.
coli per area of house
Chicks
& 87
Chicks
Chick Box 1 isolate per growthLiners
positive plate (58)
Liners
 3 E. coli per E. colitype per house (29)
Organic Broiler Farm
Number Selection Criteria
Isolates
212
 16 birdsa per fortnightb
 1 per type E. coli per
fortnight
206c
When present
in a
sample:
 2 non-specific E. coli
per area of house
 1 ampicillin-E. coli per
area of house
 1 chloramphenicol-E.
coli per area of house
17
Liners
 3 E. coli per E. colitype
per
incoming
group of birds
Statistical methods
The box-and-whiskers plots were produced using R 2.1.11. Linear mixed effects models
were also fitted in R 2.1.1.
a
Wherever possible the isolates were selected from the same birds each week to reduce potential variation in
resistance-phenotypes due to between-bird differences.
b As there was only a finite number of Sensititre® plates it was not possible to select isolates from every week
of the longer organic growth cycle.
c The lower numbers of samples from the conventional farm represent the lower level of E. coli-positive
samples in the cleaned conventional houses compared with the organic farm (see Annex 4)
Results and Discussion
Broad comparisons of multiple-resistance
Figure 1 shows the median number of resistances expressed per E. coli isolated from the
faeces of the broiler chickens on each of the two farms. The group entitled “All Faecal E.
coli” are those isolates that grew on plain CHROMagar ECC isolation agar within which no
antimicrobials had been incorporated. The ampicillin- and chloramphenicol-resistant E. coli
were isolated from the same samples as the non-specific E. coli by incorporating the
antimicrobials within the CHROMagar. Therefore the AR and CR E. coli groups represent
subsets of the total E. coli population that is in turn represented by the non-specific E. coli
isolates.
From Figure 1, it is apparent that the general E. coli population on the conventional broiler
farm express a greater degree of multiple-resistance than the E. coli on the organic farm
(median number of resistances per isolate of 5 and 1 respectively). Nonetheless, E. coli with
up to nine resistances were also present on the organic farm, however they were not the
predominant part of the faecal E. coli flora. Furthermore the ampicillin-resistant E. coli subpopulation on the organic farm also expressed a lower number of resistances than their
conventional-derived counterparts (median number of resistances of 2 and 6 respectively). In
contrast, the chloramphenicol-resistant sub-populations on both farms were of equal
multiple-resistance with 6 resistances being expressed per isolate.
Linear mixed-effects models were fitted to the data in order to assess the significance of
these observations (see Table 1). The response variable of number of resistances
expressed per E. coli was modelled against type of farm and type of E. coli. Due to the
clustered nature of the data, a random effects hierarchy was incorporated to describe the
isolation of faecal E. coli from individual chickens that in turn were clustered within houses.
Table 1 shows that most of the random variance in this model occurs at the level of the
residual error. In this model the residual variance will include the within-chicken variance;
therefore this result implies that repeat-isolates from the same chicken expressed markedly
different numbers of resistances.
From the fixed effects detailed in Table 1 it can be seen that there is indeed a significant
negative association between the organic broiler isolates and the level of multiple-resistance.
Furthermore the increases in resistance expressed by the ampicillin- and chloramphenicolresistant sub-populations of E. coli, compared with the full E. coli populations on the two
farms, are also statistically significant. These results suggest that on both farms there is a
minority population of chloramphenicol-resistant E. coli (CR E. coli) that are highly multipleresistant. Furthermore these CR E. coli are persisting on the organic farm in the absence of
antimicrobial administration. This may imply that whilst they are not the fittest E. coli within
the broiler gut, they nonetheless remain fit enough to persist at low levels despite the large
number of resistances that they carry. In fact, on the organic farm, the faecal concentration
of CR E. coli was seen to increase during the last two weeks of the broilers lives. (Further
details have been provided about the dynamics and characteristics of these CR E. coli in
Annex 4).
Figure 1. Box-and-whiskers plots illustrating the number of resistances concurrently
expressed by isolates of Escherichia coli that had been obtained from faecal samples on two
broiler-farms. The total faecal E. coli populations are displayed concurrently with the
ampicillin- and chloramphenicol-resistant subsets of these populations.
Organic Broilers
Conventional Broilers
All Faecal E.coli
Amp-R E.coli
Chlor-R E.coli
0
2
4
6
8
10
0
2
4
6
8
10
Number of resistances expressed per E.coli
Table 1. Linear mixed-effects model describing the number of resistances expressed per
Escherichia coli isolate from faecal samples on two broiler-farms.
Random Effects
House
Chicken
Residual
Variance
0.21
0.09
3.08
Fixed Effects
Coefficient
Farm
Conventional broiler
Organic broiler
Strata of E. coli Population
Total population
95% Confidence P-Value
Interval
Ref
-2.25
-3.01 - -1.49
0.0001
Ref
Ampicillin-resistant subset
1.44
1.06 – 1.82
<0.0001
Chloramphenicol-resistant subset
2.74
2.31 – 3.18
<0.0001
Patterns of multiple-resistance
The following comparisons were made between groups of E. coli that had been isolated on
plain CHROMagar media without the incorporation of antimicrobials.
1. Conventional Farm
Although E. coli expressing up to four resistances were isolated from samples taken from
day-old chicks and chick-box liners, the majority of the E. coli brought on to the farm with the
chicks appear to be fully-sensitive (median number resistances of 0) or resistant to a single
antimicrobial (see Figure 2). In contrast, the E. coli that persisted in the houses after cleaning
expressed a median number of resistances of 4.5-6 within a range stretching from 0-10. The
phenotypes of the E. coli isolated from the growing birds more closely resembled the
phenotypes found in the cleaned houses rather than those brought onto the farm by the
chicks.
2. Organic Farm
The median levels of multiple-resistance for all the isolates from the organic poultry houses
and growing birds were lower than those seen on the conventional farm (see Figure 3), and
once again the chicks were not found to be bringing high levels of multiple-resistant E. coli
onto the farm.
Using linear mixed effects models, and controlling for differences between the farms, the
number of resistances expressed by E. coli isolated from chicks coming onto the farms was
seen to be significantly lower than E. coli from the other sample groups (see Table 2).
Table 2. Linear mixed-effects model describing the level of expression of multipleresistances for Escherichia coli isolates that had been derived from different sources on two
broiler farms.
Random Effects
Flocka
Residual
Variance
0.02
3.77
Fixed Effects
Coefficient
Farm
Conventional broiler
Organic broiler
95% Confidence P-Value
Interval
Ref
-2.94
(-3.55 - -2.34)
<0.0001
Derivation of Samples
Growing birds
Ref
Incoming day-old chicks
-3.54
-4.14 - -2.95
<0.0001
Cleaned houses
-0.06
-0.51 – 0.40
0.80
A single random effect of “Flock” was used in this model, this denotes both the group of birds and the house
that they are growing in.
a
Figure 2. A comparison of the level of multiple-resistance expressed by E. coli isolates
collected from the environment of the conventional poultry houses after cleaning and
disinfection; from chicks brought onto the farm and from further faecal samples collected
from those birds as they grew.
Isolates from Conventional Broilers
Day-Old Chicks
Post C&D Before Flock
Growing Birds
Post C&D After Flock
0
2
4
6
8
Number of resistances expressed per E.coli isolate
10
Figure 3. A comparison of the level of multiple-resistance expressed by E. coli isolates
collected from the environment of the organic poultry houses after cleaning and disinfecting,
from chicks brought onto the farm and from further faecal samples collected from the birds
as they grew.
Isolates from Organic Broilers
Chick-Box Liners
Post-C&D (brooders)
Brooding Birds
Post-C&D (mobiles)
Growing Birds
0
2
4
6
8
Number of resistances expressed per E.coli
10
Patterns of multiple-resistant E. coli isolated from growing birds
Further analyses were undertaken by stratifying the resistance-phenotypes of E. coli isolated
from the faeces of marked individual chickens by the age of bird at the time of sampling.
These analyses were carried out separately for each farm.
1. Conventional Farm
Figure 4 illustrates that by the second week of age the level of multiple-resistance per E. coli
has risen from 0-1 to 4-6 expressed resistances. This level of multiple-resistance then
persists until the birds are processed. The y-axis in Figure 4 has been construed so as to
highlight the changes occurring in the level multiple-resistance expressed by E. coli over the
first few days of the birds lives on the farm. This highlights the facts that, the few E. coli that
were isolated from the day-old chicks were mainly sensitive to antimicrobials, but by day two
the range of resistances expressed had increased from 0 to 5 and by the second half of that
first week this had stabilised out to the persistent median of 5 resistances per E. coli.
Figure 4. Patterns of multiple-resistance expressed by Escherichia coli isolated from the
faeces of conventionally-reared broiler chickens throughout a single growth cycle.
Isolates from Conventional Broilers
Week 6
Week 5
Age of Birds
Week 4
Week 3
Week 2
Days 3-7
Day-2
Day-Old
0
2
4
6
8
Number of resistances expressed per E.coli isolate
10
A linear mixed-effects model was fitted to the dataset (see Table 4). Because the isolates
had originated from known birds a random effect term of “Chicken” was incorporated to
account for the clustering of isolates within birds. The model confirms that the increases in
resistances expressed from day-old chicks to two-days to 3-days or more are significant. The
birds on this farm were dosed with lincomycin-spectinomycin, as a prophylactic therapeutic
agent, for the first 3-days after arrival on the farm. It is over the course of these 3-days that
this change in multiple-resistance occurs in the faecal E. coli population. Thus it would seem
that the use of this broad-spectrum antimicrobial could be selecting for the survival of
multiple-resistant E. coli. Furthermore this level of multiple-resistance remains high
throughout the growing cycle.
Table 4. Linear mixed effects model describing the expression of multiple-resistances by E.
coli isolated from conventionally-reared broiler chickens throughout the duration of the
growing period.
Random Effects
Chicken
Residual
Variance
0.06
1.63
Fixed Effects
Coefficient
Age of Birds
Incoming day-old chicks
95% Confidence P-Value
Interval
Ref
2-day chicks
3.74
(2.56 – 4.93)
<0.0001
3-7 day chicks
5.67
(4.67 – 6.68)
<0.0001
Birds >7 days of age
5.43
(4.57 – 6.30)
<0.0001
A possible explanation of this finding is that the intestines of newly hatched chicks are
microbiologically naïve and are rapidly colonised by the bacteria the chicks ingest in the first
few days of life. If a strong selective pressure is placed upon those early colonisers, such as
a 3-day course of antimicrobial therapy, then the resultant colonisers will be those bacteria
that are resistant to the actions of the antimicrobial. Thus the susceptible flora brought onto
the farm by the chicks are likely to be excluded from the flock. Furthermore, we have already
ascertained that the bacteria in the house that survive the cleaning process are also multipleresistant (see Figure 2). Therefore it is plausible that the persistence of these multipleresistant E. coli throughout the entire growth cycle of the birds reflects the closed nature of a
bio-secure conventional broiler house into which the entry of susceptible E. coli is limited.
Thus, in essence, there is little or no susceptible E. coli remaining in the system and the
faecal flora cannot therefore be repopulated with susceptible E. coli. At the risk of stretching
this hypothesis still further, referring to the Week 6 group in Figure 4 suggests that although
the median number of expressed resistances has remained fairly stable at 4.5, the lower
range has extended down to 1 resistance. The group of birds had decreased in Week 6
because some of the cock-birds had already gone for slaughter (a process known as
thinning of the flock) during Week 5. Therefore could the increased numbers of E. coli
expressing fewer than 4 resistances in Week 6 reflect an increase in the diversity of E. coli
present in the house & flock after the thinning-team had been walking around removing the
large cock-birds?
It would be extremely interesting to explore these bacterial dynamics more closely. This farm
had withdrawn the use of the antimicrobial growth-promoter avilamycin three crops before
the flock that was studied. During this study two of the flocks on-site showed signs of the
clinical syndrome dysbacteriosis and were treated with amoxicillin. Dysbacteriosis is thought
to be caused by an inappropriate balance of intestinal bacteria. It would be extremely
interesting to further investigate whether the administration of antimicrobials to young chicks
is playing a role in the emergence of dysbacteriosis as a clinical problem in the UK broiler
industry by restricting the long-term diversity of the enteric bacterial flora within a flock.
2. Organic Farm
Unlike the conventional farm, the median number of resistances expressed per E. coli
remained stable throughout the life of the organic birds (see Figure 5).
Figure 5. Patterns of multiple-resistance expressed by Escherichia coli isolated from the
faeces of organically-reared broiler chickens throughout a single growth cycle.
Isolates from Organic Broilers
Week 9
Age of Birds
Week 6
Week 4
Week3
Week 1
0
2
4
6
8
Number of resistances expressed per E.coli isolate
10
However by Week 3, the last in the brooder houses, higher numbers of more multipleresistant E. coli were isolated. Thus the mean, or average, number of resistances had
increased compared with the E. coli isolated from birds during their first week of life. This is
reflected by the results of the linear mixed effects model for this data, where a significant
increase in number if resistances expressed per E. coli was seen when comparing these two
time-points (see Table 5). This increase in mean number of resistances was then maintained
until the end of the growing period, with the possible exception of 3-weeks after transfer to
the fields, although this latter result was not significant at the 95% confidence level (i.e. P is
not < 0.05).
The model reported in Table 5 also incorporated flock of birds as an explanatory variable.
Whilst the number of resistances per E. coli between the four flocks was not significantly
different at the 95% confidence level, there is a suggestion that the flock from Brooder
House 2, and subsequently Mobile House 1, may have harboured E. coli expressing a higher
number of resistances. As the sample size within each group was low the standard errors
were high and therefore this may have masked potential differences between the different
flocks.
Table 5. Linear mixed effects model describing the expression of multiple-resistances by E.
coli isolated from organically-reared broiler chickens throughout the duration of the growing
period.
Random Effects
Chicken
Residual
Variance
0.05
6.37
Fixed Effects
Coefficient
Age of Birds
<7 days
95% Confidence P-Value
Interval
Ref
End of brooding period
1.40
(0.16 – 2.64)
0.03
1-week after transfer to field
1 .2 6
(0.02 – 2.50)
0 .0 5
3-weeks after transfer to field
0.58
(-0.60 – 1.77)
0.33
End of growing period
1.31
(0.21 – 2.41)
0.02
Flock of Birds
Brooder 7 / Mobile 16
Ref
Brooder 8 / Mobile 15
0.34
(-0.74 – 1.43)
0.53
Brooder 1 / Mobile 2
-0.55
(-1.71 – 0.62)
0.33
Brooder 2 / Mobile 1
1 .0 7
(-0.07 – 2.21)
0 .0 6
Reference List
1. R Development Core Team. R: A Language and Environment for Statistical Computing.
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