Characterization of Two Escherichia coli Isolates Associated with Poult
Enteritis and Mortality Syndrome1,2
F. W. EDENS,*,3 R. A. QURESHI,* C. R. PARKHURST,* M. A. QURESHI,*
G. B. HAVENSTEIN,* and I. A. CASAS†
*Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7635 and
†BioGaia Biologics, Inc., 6213D Angus Drive, Raleigh, North Carolina 27613
ABSTRACT
Two colonial types (1 and 2) of Escherichia coli are represented predominantly in cultures
isolated from turkey poults with poult enteritis and
mortality syndrome (PEMS). Biotype codes determined
using two systems (BBL: 36570 and 34560 for colony
types 1 and 2, respectively; API-20E: 5144572 and
5144512 for colony types 1 and 2, respectively) clearly
establish these organisms as E. coli. These isolates were
not clearly divergent from the general profile for E. coli,
but colony type 2 differs from colony type 1 with regard
to its negative reactions for ornithine decarboxylase and
the fermentation of dulcitol, rhamnose, sucrose, and
melibiose, suggesting that it is atypical. Colony type 1 is
nonserotypable and nonmotile, whereas colony type 2 is
serotyped as O136: motile because it has H antigens
associated with flagella. Capsular antigens were not
found, but thin capsules were seen on cells from both
colony types in stained preparations. Cultural morphology was different with colony type 1 having a circular,
mucoid, raised morphology and colony type 2 having an
irregular, flat, rough morphology. Colony type 1 has a
doubling time at 37 C of about 20 min, whereas colony
type 2 doubles in 30 min. Furthermore, colony type 1 is
a potent colicin producer, but colony type 2 is not a
colicin producer. Both E. coli isolates have resistance
profiles for multiple antibiotics. Each strain responds to
third generation flouroquinalone antibiotics by changing
their biotypes and become resistant after culturing once
in their presence. These E. coli are proposed as possible
etiological links in the complex series of events that take
place in poults susceptible to PEMS.
(Key words: poult enteritis and mortality syndrome, Escherichia coli, biotype,
colony morphology, plate morphology)
1997 Poultry Science 76:1665–1673
INTRODUCTION
A definitive etiology for Poult Enteritis and Mortality
Syndrome (PEMS) has not been identified at this time
(Barnes et al., 1996; Barnes, 1997; Edens et al., 1997;
Qureshi et al., 1997). However, Edens et al. (1997)
reported that two types of putative Escherichia coli, called
colony type 1 and colony type 2, were isolated
consistently from moribund PEMS-afflicted flocks of
poults brooded in diverse regions of the State of North
Carolina as well as in poults at the North Carolina State
University College of Veterinary Medicine exposed
Received for publication March 3, 1997.
Accepted for publication July 9, 1997.
1Resources supporting this research were provided in part by the
North Carolina Poultry Federation and the Turkey Industry Task Force
on Poult Enteritis and Mortality Syndrome. Salaries and other research
support were provided by state and federal funds appropriated to the
North Carolina Agricultural Research Service at North Carolina State
University, Raleigh, NC 27695.
2The use of trade names in this publication does not imply
endorsement by the North Carolina Agricultural Research Service of
the products named nor criticism of similar products not named.
3To whom correspondence should be addressed.
experimentally to poults that carried the disease.
Because these putative E. coli isolates could not be
isolated from healthy poults on the North Carolina State
University Turkey Research Farm, and because they
were isolated infrequently from healthy poults in
commercial production facilities, it was reasoned that
these putative E. coli isolates were either opportunistic
or pathogenic or both.
Infectivity studies with pure isolates of the two
putative E. coli isolates supported the suspicion that they
were pathogenic and that they caused a severe diarrheal
condition similar to that seen in fulminating field cases
of PEMS (Edens et al., 1997). Both putative E. coli isolates
administered by oral gavage to control and to
cyclophosphamide-treated poults induced severe diarrhea, depressed growth, caused lymphatic tissue involution, induced hard cores in the bursa of Fabricius of
survivors, and ultimately caused high mortalities. Many
of the dead poults exhibited a general wasting of body
musculature (Edens et al., 1997).
Because these two types of putative E. coli appear to
be closely linked to PEMS and its potential etiology, it
was felt that a description of their characteristics would
1665
1666
EDENS ET AL.
be helpful to other PEMS investigators. Thus, the
objective of this report is to describe the colony
morphologies, biochemical, cultural, and structural
characteristics, antibiotic sensitivities, and serotyping of
these two putative E. coli isolates.
MATERIALS AND METHODS
Bacterial Isolation and Culture
The two types of putative E. coli were isolated
separately in pure culture as described previously (Edens
et al., 1997), and approximately 105 cfu of each type were
administered separately by oral gavage to healthy poults 1
or 6 d of age to establish the pathogenicity of each strain.
After 3 wk, the E. coli isolates were reisolated from the
ceca, liver, kidney, heart, spleen, bursa of Fabricius, and
feces of the experimentally infected poults. The cultures
reisolated from the challenged poults were used in studies
designed to determine their biochemical and morphological characteristics.
Twenty colonies of each E. coli type reisolated from the
challenged poults were cultured on MacConkey agar4
with 0.15% bile salts, XLT4 agar,4 tryptose soy agar4 (TSA)
with 5% sheep blood, TSA with 0.02% Congo red dye5
(Spears et al., 1992), and Levine EMB agar.4 All cultures
were grown at 37 C.
The presence of capsules on the two E. coli isolates was
determined with the Hiss method (Lennette et al., 1974). A
loopful of physiological saline suspension of stationary
phase growth from each E. coli strain was mixed with a
loopful of normal turkey serum. This mixture was
smeared on a glass slide and air-dried followed by flame
fixation. The smear was flooded with a 1% aqueous crystal
violet solution, steamed gently for 1 min, and rinsed with
a 20% aqueous solution of copper sulfate. This procedure
yielded preparations in which there were faint blue halos
around dark purple cells.
Growth Curve Analysis
Cultures for determination of growth curves of the two
E. coli isolates were grown overnight in static culture in
dilution bottles in brain heart infusion broth (BHI) at 37 C.
Aliquots (100 mL containing 108 cfu/mL) from each of the
E. coli isolates were used to inoculate 200 mL of fresh BHI,
which was then incubated anaerobically at 37 C. At the
time of inoculation into the fresh BHI and at
15-min intervals for a 6-h period thereafter, 5 mL of the
growth cultures were taken and their optical density at
600 nm were determined immediately.
4Difco Laboratories, Detroit, MI 48232-7058.
5Sigma Chemical Co., St. Louis, MO 63178-9916.
6Becton Dickenson Microbiological Systems, Cockysville, MD 21030.
7bioMerieux Vitek, Inc., Hazelwood, MO 63042.
8American Type Culture Collection, Rockville, MD 20852.
Biochemical Activity and Biotyping
Biochemical profiles of the two E. coli isolates were
obtained using the BBL Enterotube II System6 and the
API 20E System7 according to the manufacturers’ protocols following the procedures outlined by Ewing (1986).
In this procedure, 107 cfu of each strain were added to a
sealed reaction well and allowed to incubate overnight at
37 C. A color change in the medium in the reaction well
indicated a positive biochemical reaction for the E. coli
strain that had been placed in the medium. A total of 26
different biochemical reactions were determined for the
two E. coli colony types with these procedures.
Colicin Production
Following the procedures of Wooley et al. (1992b), 24 h
plate cultures of each strain were placed in a closed
chamber and subjected to chloroform fumes for a period
of 4 h to kill the organisms. These plates were then
overlaid with the colicin-sensitive E. coli K-strain (ATCC
23559).8 The colicin-producing E. coli K-strain (ATCC
23558)8 was used as a positive control for ATCC 23559.
After overnight growth at 37 C, the plates were then
observed for inhibition of growth of colicin-sensitive
organisms.
Electron Microscopy
The two E. coli colony types grown overnight to
stationary phase culture in BHI broth reached a concentration of 108 cfu per mL. A 10-mL aliquot was removed and
the cells were pelleted by centrifugation at 400 × g for 30
min to form a pellet in a conical centrifuge tube. The pellet
was suspended in a 1:1 ice cold glutaraldehyde and 0.1 M
sodium cacodylate buffer, pH 7.4, and was processed for
transmission and scanning electron microscopy as
described previously (Edens et al., 1997). Scanning
electron micrographs at a magnification of 10,000× were
examined and a total of 100 cells from each E. coli strain
were measured with a millimeter ruler. Resultant lengths
and diameters were converted to micron measurements
based upon calibration bars generated by the scanning
electron microscope.
Serotyping, Toxin Production,
and Virulence Factors
The E. coli isolates were submitted for serotyping at the
Pennsylvania State University E. coli Reference Center, at
the University of Missouri E. coli Serotyping Laboratory,
and at the National Institutes of Health, Bethesda, MD.
Toxin production and fimbriae/virulence factors were
determined at each laboratory.
Antibiotic Sensitivity
Using the disc diffusion method, the E. coli isolates
were evaluated for their sensitivity to 10 antibiotics
1667
CHARACTERIZATION OF PEMS E. COLI ISOLATES
TABLE 1. Biochemical comparison of Escherichia coli colony types 1 and 2, compiled with the use of both BBL Enterotube II and
API 20E systems, and compared to biochemical reactions species E. coli strains
Test or substrate
Colony type 1
Colony type 2
BBL Biotype
API 20E Biotype
36570
5144572
34560
5144512
Voges-Proskauer (0)2
Methyl red (99)
Gelatin hydrolysis (0)
Nitrate to nitrite (99)
Oxidase (0)
D-Glucose [acid/gas] (98/92)
Lysine decarboxylase (82)
Ornithine decarboxylase (74)4
H2S (1)
Indole production (92)
Adonitol fermentation (5)
Lactose fermentation (95)
L-Araginose fermentation (90)
D-Sorbitol fermentation (88)
Dulcitol fermentation (60)
Citrate (Simmons) (1)
Urease (1)
Amygdalin (8)
Arginine dihydrolase (3)
Tryptophan deaminase (0)
Mannitol (acid production) (98)
Inositol fermentation (2)
L-Rhamnose fermentation (87)
Sucrose fermentation (42)
Melibiose fermentation (66)
O-nitrophenyl-b-d-galactoside (97)
Negative
Positive
Negative
Positive
Negative
Positive (Gas)
Positive
Positive
Negative
Positive
Negative
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Positive
Positive
Positive
Positive
Negative
Positive
Negative
Positive
Negative
Positive (Gas)
Positive
Negative
Negative
Positive
Negative
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Positive
Species1
Negative
Positive
Negative
Positive
Negative
Positive (Gas)
Different3
Different
Negative
Positive
Negative
Positive
Positive
Different
Different
Negative
Negative
Negative
Different
Negative
Positive
Negative
Different
Positive
Positive
Positive
1Species
Escherichia coli biochemical reactions.
in parentheses indicates percentage of all of the known E. coli genus and species that have tested positive for the biochemical reaction.
3Different indicates that reactions can be positive or negative depending upon the E. coli isolate.
4Italics indicates different biochemical properties between E. coli colony types 1 and 2.
2Number
(gentamicin, ceftiofur, tetracycline, neomycin, streptomycin, lincomycin, apramycin, erythromycin, nalidixic acid,
furazolidone, and sulfisoxazole) that have been or are
widely used in the turkey industry. Additionally, two
fluoroquinolone antibiotics recently made available for
therapeutic use in the turkey industry, sarafloxacin9 and
enrofloxacin,10 were also tested. An overnight BHI broth
culture (108 cfu) was used to inoculate the entire surface of
Mueller-Hinton4 agar plates. After the plates had dried,
four antibiotic-treated diffusion discs were placed on each
agar plate. After 24 h incubation at 37 C, the plates were
evaluated by determining the diameter of inhibitory
zones around the individual Dispens-O-Disc Susceptibility Test Disks9 on the plates. Based on the diameter of the
antibiotic inhibitory zones, a determination of susceptibility, intermediate susceptibility, or resistance was made
using guidelines provided by the vendor of the sensitivity
disks.4
RESULTS
Biotypes determined from the two systems (BBL:
36570 and 34560 for E. coli isolates 1 and 2, respectively;
9Abbott Laboratories, North Chicago, IL 60064.
10Bayer Corp., Animal Health Division, Shawnee Mission,
KS 66201.
API-20E: 5144572 and 5144512 for E. coli isolates 1 and 2,
respectively) clearly establish these organisms as E. coli
(Table 1). Biochemical reactions of the two E. coli colony
types indicate that they were similar but that they
differed in their reactions for ornithine decarboxylase
and the fermentation of dulcitol, rhamnose, sucrose, and
melibiose. Strain 2 is negative for each of these reactions.
Antigenicity serotyping showed that colony type 1 is
nonserotypable as an O serotype and that it has no
flagella as required for H antigen serotyping. Colony
type 2 is classified as an O136 serotype, and it has H
antigens associated with the flagella, suggesting that it is
a variant of the O136 serotype (Table 2). On TSA agar,
colony type 1 has a circular, raised, and mucoid
morphology, whereas colony type 2 has an irregular,
flat, and rough morphology. Both colony types are ahemolytic. Colony type 2 is Congo Red-positive and
colony type 1 is Congo Red-negative. On Levine EMB
agar, both colony types 1 and 2 are metallic blue-green
in color. On MacConkey agar, both colony types have
pink to rose red colonies surrounded by a narrow zone
of precipitated bile. On XLT 4 agar both colony types 1
and 2 produce yellow colonies that have a physical
appearance similar to colonies grown on TSA agar.
Doubling times for colony types 1 and 2 are 20 and 30
min, respectively (Table 2).
1668
EDENS ET AL.
TABLE 2. Morphology and culture characteristics of Escherichia coli colony types 1 and 2 associated
with Poult Enteritis and Mortality Syndrome
Characteristic
Colony type 1
Colony type 2
Morphology
Gram-negative
Nonmotile rods
No flagella
Thin capsule
0.9 m × 0.7 mm
20
Gram-negative
Motile rods
Flagella
Thin capsule
1.1 m × 0.6 mm
30
Nontypable
Nonmotile
O136
Motile (variant)
Doubling time, min
Antigenicity
O (Somatic)
H (Flagellar)
Culture characteristics
TSA (5% Blood)
TSA (0.02% Congo Red)
Levine EMB
MacConkey
XLT-4
Colicin production
Alpha Hemolytic
Alpha Hemolytic
Circular
Irregular
Mucoid
Rough
Raised
Flat
Negative
Positive
Metallic blue-green
Metallic blue-green
Pink to rose red;
Pink to rose red;
Peripheral zone of precipitated bile Peripheral zone of precipitated bile
Yellow
Yellow
Positive
Negative
The colicin-V sensitive E. coli K-12 strain (ATCC
23559) was sensitive to E. coli colony type 1 but not to
colony type 2 (Table 2). The ATCC 23559 Col-V sensitive
E. coli K-12 strain showed no growth in the inhibition
zones around E. coli colony type 1 (> 30 mm in
diameter). Because the E. coli K-12 strain (ATCC 23559),
known to be Col-V-sensitive, was inhibited by Col-V
produced by ATCC 23558, it was concluded that E. coli
colony type 1 produced Col-V and that E. coli colony
type 2 did not.
Other morphological and cultural characterizations of
E. coli colony types 1 and 2 are presented in Table 2.
Colony type 1 has physical dimensions of 0.9 × 0.7 mm,
whereas colony type 2 has physical dimensions of 1.1 ×
0.6 mm (Figure 1). Both colony types had very thin
capsules (Figure 3), but they are not spore formers
(Figures 2 and 3). These two Gram-negative E. coli
isolates differ in physical appearance in that colony type
1 is a nonmotile rod with no flagella whereas colony
type 2 is a motile rod with flagella. In thin sections for
transmission electron microscopy (Figures 2 and 3),
more subtle differences between cells in colony types 1
and 2 can be seen. Examination of the high magnification transmission electron micrographs revealed that
colony type 1 appeared to have a thicker envelope than
colony type 2 cells (Figure 3), but the peptidoglycan of
the cells in colony type 2 appears to be thicker than in
the cells from colony type 1 (Figure 3). Additionally, the
cytoplasmic membrane in the cells from colony type 2
appears to be slightly thicker than the cytoplasmic
membrane in cells from colony type 1.
Both E. coli colony types 1 and 2 were found to be
negative for Shiga-like toxins 1 and 2, heat-labile toxin,
and heat-stable toxins A and B (Table 3). Shiga-like
toxins were not activated in either colony type when
cultured on baboon kidney cells with a nontrypsin
protease. Additionally, both E. coli colony types 1 and 2
were found to be negative for the following virulence
factors: EAE (attaching and effacing), K88, K99, 987P,
F107, CS31A, and F1845.
Commensal E. coli (different from colony types 1 and
2) from the intestinal tracts of the control and infected
poults were variable in sensitivity to antibiotics (data
not included). Within different intestinal regions, some
of the E. coli are susceptible to some antibiotics, and in
another region, the commensal E. coli showed resistance
to antibiotics. However, E. coli colony types 1 and 2 are
generally resistant to antibiotics as determined with
antibiotic-treated diffusion discs (Table 4). Colony type 1
is susceptible initially to sarafloxacin, but within the
zone of inhibition around the diffusion disc, microcolo-
TABLE 3. Toxin and fimbriae/virulence factor characterization of
Escherichia coli colony types 1 and 2
Characterization
Toxins
SLT1 (Shiga-like toxin 1)
SLT2 (Shiga-like toxin 2)
Heat-labile toxin
Heat-stable Toxin A
Heat-stable Toxin B
Fimbriae/virulence factors
EAE (attaching and effacing)
K88
K99
987P
F107
CS31A
F1845
Colony
type 1
Colony
type 2
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
CHARACTERIZATION OF PEMS E. COLI ISOLATES
1669
DISCUSSION
FIGURE 1. Scanning electron micrographs of Escherichia coli colony
type 1 (a) and colony type 2 (b). The small bars = 1 mm.
nies of the organism show inherent resistance. Following
transfer of these microcolonies to another culture plate,
they were resistant to sarafloxacin. Colony type 2 is
sensitive to the initial exposure but also shows intermediate resistance on a subsequent exposure to the
antibiotic. Colony type 1 was highly resistant on first
exposure to enrofloxacin, a third generation fluoroquinolone recently released for use in chicken and
turkey flocks in the U.S., whereas colony type 2 shows
intermediate resistance.
The flouroquinalone (sarafloxacin and enrofloxacin)resistant E. coli from colony types 1 and 2 were cultured
and subjected to biochemical typing. Both colony types 1
and 2 showed a remarkable shift in their biochemical
profiles after exposure to the fluoroquinolone antibiotics
due to the loss of their ability to produce gas from
glucose. This changed the BBL biotypes from 36570 and
34560 to 26570 and 24560 for the E. coli colony types 1
and 2, respectively.
In the search for etiological agent(s) for PEMS, it was
noted that PEMS-infected poults had large numbers of
two colony types of bacteria believed to be E. coli
isolates that could be isolated from cecal and fecal
samples. Additionally, these same isolates could be
found in visceral organs as well and in moribund and
dead poults, these were the predominant E. coli found in
tissues (Edens et al., 1997). These isolates have been
referred to as colony type 1 and colony type 2 (Edens et
al., 1997). It was reasoned that the predominance of
these two isolates implicated their involvement in the
PEMS disease. Subsequent studies (Edens et al., 1997)
have shown that both isolates are capable of causing
severe diarrhea, dehydration, and mortality in poults
kept on warm wet litter. Furthermore, when groups of
poults were treated with cyclophosphamide, an inhibitor
of B cell function due to its alkylation of the B cell DNA,
nearly 70% of the poults died from septicemia and
diarrhea associated with these two isolates of E. coli
(Edens et al., 1997).
If an organism is believed to be associated with a
disease or disease complex, it is important to identify
that organism as precisely as possible. Using selective
media and biochemical characterizations, the two PEMSassociated separate isolates of E. coli were isolated and
characterized. They did not at first appear to be greatly
different or atypical, as previously reported (Edens et al.,
1997), when their biochemical profiles were compared to
the general biochemical profile of E. coli genus and
species (bioMerieux Vitek, 1993). However, they differed
from each other in their biochemical profiles. Colony
type 1 has ornithine decarboxylase and ferments dulcitol, L-rhamnose, sucrose, and melibiose, whereas
colony type 2 lacks these properties. Neither strain is
divergent from the general E. coli profile with regard to
these properties. Colony type 2 differs from the general
profile in that it is negative for sucrose and melibiose
fermentation and could be considered as having some
atypical characteristics.
These two E. coli isolates also differ somewhat in
colony and cellular type. Type 1 produces a circular,
raised, mucoid colonial morphology and is Congo Rednegative, whereas type 2 produces an irregular, rough,
flat colonial morphology and is Congo Red-positive.
Type 1 is a potent colicin producer, possibly Col-V,
whereas type 2 is not. Colony type 1 is nonserotypable
and nonmotile, and colony type 2 is serotyped as O136
and is motile. Colony type 1 cells are apparently shorter
with greater width than colony type 2 cells.
The two organisms are similar with regard to
antibiotic resistance profiles. Both colony type 1 and 2
are resistant to each of the antibiotics tested in this
study, including the third generation fluoroquinolones
sarafloxacin and enrofloxacin, because each type rapidly
developed resistance to them after an initial exposure.
Long-term intensive use of antibiotics in poultry can
1670
EDENS ET AL.
FIGURE 2. Transmission electron micrographs showing Escherichia coli colony type 1 (a) and colony type 2 (b); 1-cm bar = 588 nm.
TABLE 4. Antibiotic sensitivity of 24-h cultures of Escherichia coli colony types 1 and 2 based upon development of inhibitory zone
diameters after application of discs containing specific antimicrobial agents
Escherichia coli
Colony type 1
Colony type 2
Antimicrobial
agents
Sensitive
diameter
Zone
diameter
Status
Zone
diameter
Gentamicin GM10
Sarafloxacin SRF5
(mm)
>15
>20
(mm)
7
22/10
Resistant
Sensitive/Resistant1
(mm)
10
22/15
Enrofloxacin ENO5
Ceftiofur XNL30
Tetracycline TE30
Neomycin N30
Streptomycin S10
Streptomycin S300
Lincomycin L2
Apramycin AP15
Erythromycin E15
Nalidixic Acid NA30
Furazolidone FX100
Sulfisoxazole G300
>15
>18
>19
>17
>15
>19
>2
>18
>18
>19
>17
>16
10
9
0
5
5
7
0
5
0
7
6
0
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
15
13
0
0
0
8
0
7
0
7
8
0
1Resistant
after first exposure.
resistance after first exposure.
2Intermediate
Status
Resistant
Sensitive/
Intermediate2
Intermediate
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
CHARACTERIZATION OF PEMS E. COLI ISOLATES
1671
FIGURE 3. Transmission electron micrographs of Escherichia coli colony type 1 (a) and type 2 (b). OCW = outer cell wall; N = nucleus; P =
peptidoglycan; CM = cell membrane. 1-cm bar = 235 nm.
result in pathogenic E. coli that develop resistance or
express natural resistance to many different antibiotics
(Cloud et al., 1985; Filali et al., 1988; Chulasiri et al., 1989;
Gross, 1991). The observation that these two E. coli
isolates exhibited resistance to multiple antibiotics was
not unusual, but the apparently rapid development of
resistance or inherent resistance to the newest fluoroquinolones used for E. coli and Salmonella treatment is
surprising.
These fluoroquinolones exert their bactericidal activity via the inhibition of the DNA gyrase enzyme
(topoisomerase II), which is responsible for the induction of negative supercoils in circular DNA (Wolfson
and Hooper, 1985). However, the second time that these
E. coli were exposed to the fluoroquinolones, there was
little growth inhibition, indicating a rapid ability to
adapt to the presence of these antibiotics. However,
antibiotic-resistant colonies of these two E. coli isolates
also changed their BBL biotypes from 36570 and 34560
to 26570 and 24560 for the E. coli types 1 and 2,
respectively. The 26570 E. coli biotype had been detected
previously from a PEMS-infected flock that had been
given a 5 d sarafloxacin treatment (data not shown), but
at that time, this biotype was believed to be another E.
coli that was associated with PEMS. In field cases of
PEMS, it has been noted that use of sarafloxacin and
enrofloxacin caused an immediate decrease in PEMSrelated mortality within 24 h after initiation of treatment, but subsequent uses of these antibiotics are often
characterized by little or no effect (Barnes, 1997). This
observation suggests that resistance to the fluoroquinolones is mediated via a chromosomal modification of the
gene(s) encoding DNA gyrase and that there is a
biochemical change, loss of gas production from glucose
fermentation, signaling the development of a biochemical pathway resistant to fluoroquinalones. Antibiotic
resistance to the other antibiotics tested in this study and
commonly used in the turkey industry may also be
mediated by chromosomal changes, but more likely,
most of these resistance factors may be located on R or F
plasmids (Timmis et al., 1985). However, this theory
must be ascertained with additional research.
Colony type 1 is a colicin (possibly Col-V) producer
and colony type 2 is resistant to the colicin produced by
colony type 1, as indicated by their close colonial
development on agar plates. Colicin production has
been associated with virulence in avian E. coli (Vidotto et
al., 1990). Wooley et al. (1992a, 1993, 1994) have shown
1672
EDENS ET AL.
that colicin production provides naturally occurring
intestinal E. coli an ecological advantage in colonization
of the intestinal tract and in the exclusion of pathogenic
invaders that may also have colicin production ability
associated with conjugative R-plasmids. Nevertheless,
there is specificity in the exclusion because Vidotto et al.
(1990) report that virulent colicin-producing avian E. coli
with large molecular weight plasmids do colonize the
intestinal tract. Colicin V plasmids in mammalian
virulent E. coli have been associated with increased
serum survival and resistance to phagocytosis (Waters
and Crosa, 1991), but Bounous et al. (1994) did not find
this association with either virulent or avirulent colicin
V-producing E. coli isolated from chickens. Miller et al.
(1990) showed the significance of virulent Congo Redpositive chicken E. coli isolates because they were
efficiently phagocytized, and Arp and Cheville (1981)
indicated that turkey liver and splenic macrophages
efficiently phagocytized avirulent, weakly piliated, or
nonpiliated isolates of E. coli. The colony type 1 and type
2 E. coli isolates apparently are not efficiently phagocytized by avian macrophages, but of the two colony
types, type 1 is phagocytized more efficiently than
colony type 2 (M. A. Qureshi, unpublished data).
It was of interest that neither colony type 1 nor
colony type 2 produced common toxins and that neither
colony type expressed any of the known virulence
factors tested. Furthermore, these two colony types have
the capability of penetrating the intestinal epithelial
barrier of young turkeys and producing sepsis (Y. M.
Saif, Ohio State University, Wooster, OH 44691, personal
communication). Therefore, it is assumed that these E.
coli are enteroinvasive as suggested by reports from a
North Carolina Regional Diagnostic Laboratory where
these isolates were isolated originally (Edens et al., 1997).
Ewing (1986) indicated that enteroinvasive E. coli in
mammals do not produce heat-labile, heat-stable, or
vero toxins. Earlier work by Edwards and Ewing (1972)
and Ewing (1986) indicated that enteroinvasive E. coli
are less active biochemically than typical E. coli isolates,
that they fail to produce gas from glucose fermentation,
that they do not express many of the common toxins
and virulence factors, and that they often fall into an
O136:nonmotile serotype. This condition is similar to
that of the fluoroquinolone-transformed isolates found
in this study. In this context, colony type 2 should be
more pathogenic than colony type 1, and this was seen
in an earlier study (Edens et al., 1997). However, colony
type 1 is predominant in its appearance in PEMSafflicted poults.
Colibacillosis, characterized by acute septicemia, airsacculitis, pericarditis, or perihepatitis, has long been
known to cause significant economic loss to poultry
producers (Cheville and Arp, 1978; Gross, 1991; Leitner
and Heller, 1992). It has been suggested that in turkeys,
between 6 and 12 wk of age, development of colibacillosis results from interactions among primary infectious
agents (Pierson et al., 1996). However, given certain
conditions, such as extremes in ambient temperature,
resulting in weakened host immunologic reactions or
embryonic exposure to the bacterium, E. coli can cause
primary infections and can be a serious health hazard
for poultry (Gross, 1991). In the PEMS disease, it is
apparent that E. coli colony types 1 and 2 colonize and
damage the intestinal tract and causes an acute septicemia that shares few of the characteristics found in most
cases of colibacillosis (Edens et al., 1997).
There are more than 96,000 isolates of E. coli that have
been biotyped (bioMerieux Vitek, 1993), and many of
these E. coli isolates are considered to be nonserotypable.
If a specific genus and species is determined to be
involved in a disease condition, it is essential that data
be compiled to characterize that organism in order that
patterns of susceptibility to antibiotics and its epidemiological significance can be determined. Furthermore, it is
important to determine whether the E. coli isolates are
classified as being commensal (nonpathogenic), enteropathogenic, enterotoxigenic, enteroinvasive, enterohemorrhagic, or enteroaggregative. In this study with E.
coli associated with the development of PEMS, attempts
were made to characterize only two colony types of E.
coli in terms of physical characteristics, colonial and
cultural morphology, growth rate, biochemical reactions
to certain tests and substrates, serotyping, and characterization of the toxin production and associated virulence factors. The two E. coli isolates described here are
different from each other in several characteristics, but
on comparison with the E. coli general profile they
appear to be normal with only a few exceptions. Based
upon the information presented here, it is difficult to
assign these E. coli isolates to any one of the five classes
listed above, but these isolates exhibit characteristics that
may place them or their antibiotic-transformed derivatives into the enteroinvasive class, because they can
rapidly penetrate the gut epithelial cell barrier be
isolated from the blood and other extraintestinal sites
during the development of PEMS. Additional work is
being conducted with the two characterized PEMSassociated E. coli isolates, and with the completion of
DNA fingerprinting and plasmid isolation and characterization, a clearer understanding of their role in PEMS
should be forthcoming.
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