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Applied and Environmental Microbiology, April 2001, p. 1940-1944, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1940-1944.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genotypic Analysis of Escherichia coli Strains from
Poultry Carcasses and Their Susceptibilities to Antimicrobial
Agents
Ifigenia
Geornaras,1
John W.
Hastings,2 and
Alexander
von
Holy1,*
Department of Molecular and Cell Biology,
University of the Witwatersrand, Wits 2050,1 and
School of Molecular and Cellular Biosciences, University of
Natal, Scottsville 3209,2 South Africa
Received 9 August 2000/Accepted 3 January 2001
 |
ABSTRACT |
Plasmid profiling and amplified fragment length polymorphism (AFLP)
analysis were used to genotype 50 Escherichia coli strains from poultry carcasses. Thirty different plasmid profiles were evident,
and clustering of the AFLP data showed that they were a distinctly
heterogeneous group of strains. Susceptibility testing against five
antimicrobial agents used in the South African poultry industry showed
all strains to be susceptible to danofloxacin and colistin, while the
majority (96%) were resistant to two tetracyclines.
| |
TEXT |
Escherichia coli forms
part of the bacterial population of the chicken gastrointestinal tract.
In poultry processing, E. coli is regarded as an indicator
of fecal contamination (19). Levels of E. coli
associated with poultry carcasses can increase or decrease during
processing depending on factors such as levels of fecal contamination
on live birds, length of time and temperature of scalding, efficiency
of evisceration, bacterial load and temperature of the immersion
chiller water, and hygienic practices in the abattoir
(23). E. coli is also regarded as a major
pathogen of worldwide importance in commercially produced poultry and
can result in significant economic losses (20).
Poultry-associated diseases caused by pathogenic E. coli
strains include colibacilliosis and airsacculitis, which can cause high
morbidity and mortality in poultry (20). To control and
prevent poultry diseases, breeders are known to administer
subtherapeutic and therapeutic levels of antimicrobial agents to
chickens via feed and water (7). This practice also
improves feed efficiency and accelerates weight gain (7).
The administration of antimicrobial agents to poultry, however, has
provided a selection pressure for antimicrobial resistance genes, and
as a result, many bacteria associated with chickens and poultry meat
are now resistant to antimicrobial agents (32, 36).
Several molecular typing techniques, including plasmid profiling,
random amplified polymorphic DNA analysis, pulsed-field gel
electrophoresis, and ribotyping have been used to characterize and
determine epidemiological relationships of E. coli
strains (1, 17, 26, 30, 34). Amplified fragment length
polymorphism (AFLP) analysis, based on the principles of restriction
fragment length polymorphism analysis and PCR amplification (25,
37), is a high-resolution typing method which has been used to
differentiate between strains of Campylobacter jejuni
and Campylobacter coli (9); E. coli
O157:H7 (24), Helicobacter pylori
(16), Streptococcus pyogenes (8),
Pseudomonas fluorescens, and Pseudomonas putida (15); and Lactobacillus plantarum and
Leuconostoc mesenteroides (27).
In this study, E. coli strains from poultry carcasses were
analyzed to determine their susceptibilities to antimicrobial agents used in the South African poultry industry, and genetic relationships based on plasmid profiling and AFLP analysis.
Bacterial strains.
The 50 strains examined (Table
1) were obtained from a microbiological
survey of a poultry abattoir where bacterial counts and
populations associated with the neck skins of carcasses at six
processing stages were determined (13, 14). The API 20E system (bioMérieux, Marcy l'Etoile, France) was used to confirm the identity of the E. coli strains. O- and K-antigen
serogrouping of the strains was performed by the Onderstepoort
Veterinary Institute of the Agricultural Research Council
(Onderstepoort, South Africa). Standard E. coli
antisera were used, excluding the antisera against antigens
K21, K64, K65, K77, K92, and K100 through K102 (31). All
50 strains were O rough and K minus.
Strains were stored at
70°C in tryptone soya broth (TSB) (Oxoid,
Basingstoke, United Kingdom) supplemented with 15% (vol/vol)
glycerol.
Antimicrobial susceptibility testing.
MICs for the E. coli strains of the five antimicrobial agents used in the South
African poultry industry were determined by the microdilution method
according to the guidelines of the National Committee for Clinical
Laboratory Standards (NCCLS) (29). Reference powders were
kindly provided by Pfizer (Groton, Conn.) (danofloxacin) and Logos
AgVet (Midrand, South Africa) (colistin sulfate, neomycin sulfate,
chlortetracycline hydrochloride, and oxytetracycline base).
Mueller-Hinton broth (Oxoid) was supplemented with cations, and
concentrations of test strains were standardized to 5 × 105 CFU/ml (29). MICs were read after
18 h of incubation at 37°C. The MIC was interpreted as the
lowest concentration that visibly inhibited growth. E. coli
ATCC 25922 was used as the quality control reference strain
(29).
MIC ranges and MICs at which 50 and 90% of the strains tested are
inhibited (MIC
50s and MIC
90s, respectively) are
shown in
Table
2. MIC breakpoints for
resistance and susceptibility have
not been established by the NCCLS
for any of the antimicrobial
agents tested here. For purposes of this
study, therefore, MIC
breakpoints were assigned to each of the
antimicrobial agents
that were based on breakpoints established by the
NCCLS for related
antibiotics (
29). The strains analyzed
here were thus considered
resistant when MICs were
4 µg/ml for
danofloxacin and
16 µg/ml
for neomycin, chlortetracycline, and
oxytetracycline. An arbitrary
MIC breakpoint for resistance to colistin
of
16 µg/ml was used,
since NCCLS interpretative standards have not
been established
for the polymyxin class of antibiotics, to which
colistin belongs.
Using these breakpoints, all but two of the strains
(strains 217
and 232 [Table
1] were resistant to at least one and at
most
three of the antimicrobial agents. The majority (76%) of the
strains
were resistant to the two tetracyclines only, while 14% were
resistant
to the tetracyclines as well as neomycin. The remaining three
isolates were resistant to neomycin only (Table
1). All the strains
were susceptible to danofloxacin and colistin, with MIC
90s
of
0.125 and 1 µg/ml, respectively (Table
2). Similarly, Watts
et
al. (
38) reported the MIC
90 of danofloxacin
for
E. coli isolates
of veterinary origin to be
0.015
µg/ml. Danofloxacin belongs
to the new fluoroquinolone class of
antimicrobials, which are
highly effective against gram-negative
bacilli (
6,
12). Their
use in the poultry industry,
however, is thought to be inappropriate
due to cross-resistance with
fluoroquinolones used to treat important
human enteric infections
(
10,
11). Fluoroquinolone resistance
has been reported for
Salmonella serotypes (
21,
28),
Campylobacter jejuni (
10), and
E. coli (
11). The susceptibilities of the
strains in
this study to colistin were in agreement with those
reported in a
Spanish study where 468
E. coli strains of avian
origin were
susceptible to this antimicrobial agent (
6). Resistance
to
colistin reportedly does not commonly develop in bacteria originally
susceptible to this antimicrobial agent (
22), which could
possibly
explain the narrow range and low MICs obtained for the
E. coli strains in this study. Neomycin is an aminoglycoside
and is primarily
active against
Escherichia spp., but it is
also effective against
other genera of the
Enterobacteriaceae (
22). In our study, 20%
of
the
E. coli strains tested were resistant to this
antimicrobial
agent. Conversely, 90% of the strains were resistant to
the two
tetracyclines, chlortetracycline, and oxytetracycline
(MIC
90s
of 128 and >512 µg/ml, respectively) (Table
2).
This high level
of resistance is of concern due to possible
cross-resistance with
antibiotics used in human medicine. Recent
studies have suggested
a link between the use of antimicrobial agents
in poultry and
other food-producing animals, and the emergence of human
pathogens
with decreased susceptibilities or complete resistance to
antibiotics
used for treatment of human infections (
4,
5,
28).
Antimicrobial resistance typing was a poor tool for differentiating
between strains in this study since the majority (76%)
shared the same
profile, that is, resistance to the two tetracyclines
(Table
1).
Plasmid profiles.
Plasmid DNA was extracted by the alkaline
lysis method from overnight cultures grown in TSB at 37°C
(18). Plasmids were separated on 0.8% agarose gels,
viewed under UV transillumination, and photographed. Lactococcus
lactis subsp. lactis DSM 4645 plasmids were used as
molecular size markers (3).
All but one of the strains contained between one and six plasmids, with
sizes ranging from 1.5 to 89 kb. One, two, or four
plasmids were
harbored by almost equal proportions of the strains
(24, 28, and 24%,
respectively). Overall, however, plasmid profiles
obtained for all the
strains were diverse, with 30 profiles emanating
from the 50 isolates
(Table
1). Twenty of these profiles were
unique, while the remaining 10 were shared by at least two and
at most nine strains. These nine
strains contained a single 89-kb
plasmid (profile P2 [Table
1]) which
was also present in 86%
of strains containing more than one plasmid.
Profile P19 was shared
by five strains, while profiles P4, P7, P9, P11
through P14, and
P27 were shared by two strains each (Table
1).
Seven of the nine strains isolated from carcasses after the
defeathering stage had different plasmid profiles, while the
profiles
of all the strains originating from carcasses before
evisceration
were different (Table
1). Conversely, 3 and 4 of the 12 strains
from carcasses after evisceration shared profiles P2 and P19,
respectively, while two strains each from carcasses after spray
washing
shared profiles P4, P12, and P13. Furthermore, three and
two strains
from carcasses after the immersion chilling stage
displayed profiles P2
and P11, respectively (Table
1).
No apparent correlation was found between the plasmid profiles of the
strains and their resistance patterns to the antimicrobial
agents
(Table
1).
AFLP analysis.
The NucleoSpin C & T kit (Macherey-Nagel,
Düren, Germany) was used to extract genomic DNA from 1-ml
cultures grown in TSB at 37°C for 18 h. DNA concentrations
were estimated by agarose gel electrophoresis with diluted samples of
DNA (Boehringer Mannheim GmbH, Mannheim, Germany). The AFLP
ligation and preselective amplification kit (Perkin-Elmer, Foster
City, Calif.) was used for AFLP reactions, which were each performed on
250 to 500 ng of DNA as described previously (15).
Amplified fragments were separated on denaturing 4% polyacrylamide
sequencing gels, which were run on a model S2 sequencing gel apparatus
(Gibco, BRL Life Technologies, Gaithersburg, Md.) at 50 W with 1×
Tris-borate-EDTA (TBE) buffer in the upper compartment and 1× TBE
supplemented with 0.5 M sodium acetate in the lower compartment
(2). AFLP fingerprints were detected by the modified
silver staining method described previously (15). Gels
were air dried overnight and then scanned with a Hewlett-Packard
ScanJet IIcx scanner. AFLP patterns were analyzed with the GelCompar
software (version 4.0; Applied Maths, Kortrijk, Belgium). Gels were
normalized by including the 1-kb Plus ladder (Gibco) at four-lane
intervals on every gel as a standard. After conversion, normalization,
and background subtraction, levels of similarity between the AFLP
fingerprints were calculated by using the Pearson product-moment
correlation coefficient (r). Strains were clustered by using
the unweighted pair group method with arithmetic averages (UPGMA)
(33).
AFLP reactions generated between 26 and 44 detectable bands per strain.
The AFLP fingerprints of one
E. coli strain from carcasses
after evisceration and three strains from carcasses after spray
washing
are shown in Fig.
1 as typical examples.
The dendrogram
generated by clustering of the AFLP data by UPGMA is
shown in
Fig.
2. Clearly, the AFLP
fingerprints of the 50 strains analyzed
were highly heterogeneous, with
the highest level of homology
observed between the strains being 96%
and the lowest level of
homology being 27%. Thus, none of the AFLP
fingerprints were shown
by the software to be 100% homologous, even
though they appeared
to be identical by visual inspection. Slight
variations in band
width and mobility as well as background intensities
could explain
this discrepancy. Similarity levels obtained here are
thus not
absolute but were nevertheless useful for determining
relationships
among the
E. coli strains. For purposes of
this study, therefore,
strains whose AFLP patterns were >90% similar
were assumed to
be closely related genetically. At a delineation level
of 90%,
therefore, 41 different AFLP fingerprints were generated for
the
50
E. coli strains (Table
1). Strains with homology
levels of
>90% included strains 212 and 213 from carcasses after
evisceration,
strains 218 and 219, as well as strains 222 and 224 from
carcasses
after spray washing (Fig.
2; Table
1). Strains isolated from
carcasses at different stages of processing were also found to
be
genetically related. These included strains 181 and 239 from
carcasses
after defeathering and immersion chilling, respectively;
strains 184 and 209 from carcasses after defeathering and evisceration,
respectively; and strains 204 and 205 from carcasses before and
after
evisceration, respectively (Fig.
2; Table
1). Finally,
strains 189, 192, 193, and 195 formed the largest cluster of related
strains, with
two of the strains originating from carcasses after
defeathering and
two strains from carcasses before evisceration
(Fig.
2; Table
1). The
heterogeneous nature of the AFLP fingerprints
of the strains possibly
indicates a large number of contamination
sources of carcasses with
E. coli. Sources could include the farm
and processing
environments as well as the processing equipment.
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FIG. 1.
AFLP patterns of one E. coli strain from
carcasses after evisceration (strain 216) and three strains from
carcasses after spray washing (strains 217, 218, and 219). Lane 1, strain 216; lane 2, 1-kb Plus ladder; lane 3, strain 217; lane 4, strain 218; lane 5, strain 219.
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FIG. 2.
Dendrogram based on AFLP fingerprints of 50 E. coli strains from poultry carcasses. The dendrogram was
constructed by using UPGMA. Levels of similarity between AFLP
fingerprints were calculated using the Pearson product-moment
correlation coefficient.
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|
Comparison of the plasmid and AFLP profiles obtained for each of the
strains showed that in almost all cases, strains that
shared plasmid
profiles did not also share the same AFLP profiles
(Table
1). Plasmid
profiling has previously been shown to be
of limited value as a
genotyping method compared to other molecular
typing techniques, mainly
due to the instability of plasmids,
poor reproducibility due to the
variable presence of extra bands
from open and linear forms of
the plasmids, the presence of plasmids
that appear to be
similar, or simply the absence of plasmids in
some of the strains
(
1,
35). In the present study, some of
the strains that
were regarded as genetically closely related
by AFLP analysis also
shared plasmid profiles, that is, strains
204 and 205 (level of
homology, 93%), strains 212 and 213 (level
of homology, 95%), and
strains 218 and 219 (level of homology,
95%) (Fig.
2; Table
1). The
remainder of the related strains,
however, displayed different plasmid
profiles. For instance, strains
189 and 192, whose AFLP fingerprints
were 96% similar, differed
not only in their plasmid profiles but also
in their antimicrobial
resistance profiles (Fig.
2; Table
1). In order
for the AFLP
technique to be sensitive enough to discern these
polymorphisms
in small genomes, such as those in bacteria, it may have
to be
modified to cover more alleles. This could be achieved by using
a
restriction endonuclease that cuts more frequently than the
ones used
in the present
study.
In conclusion, the high-resolution genotyping method of AFLP analysis
showed that the strains isolated from poultry carcasses
during
processing were genetically diverse. This suggests multiple
sources of contamination of carcasses with
E. coli. To pinpoint
these sources, a study including isolates from
the environment
and equipment, as well as intestinal contents of
carcasses and
workers' hands, would have to be
conducted.
| |
ACKNOWLEDGMENTS |
Leigh Morgan is acknowledged for technical assistance with the AFLP analyses.
The National Research Foundation is acknowledged for financial support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, South Africa. Phone: 27 11 717 6374. Fax: 27 11 339 7377. E-mail: alex{at}gecko.biol.wits.ac.za.
| |
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Applied and Environmental Microbiology, April 2001, p. 1940-1944, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1940-1944.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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