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Proceedings of the
55th Annual Convention of
the American Association of
Equine Practitioners
December 59, 2009, Las Vegas, Nevada
Program Chair : Nathaniel A. White

ACKNOWLEDGMENTS
Dr. David D. Frisbie, Educational Programs Committee Chair
Carey M. Ross, Scientific Publications Coordinator

Published by the American Association

of Equine Practitioners
www.aaep.org
ISSN 00657182
American Association of Equine Practitioners, 2009
Published in IVIS with the permission of the AAEP
NON-PREGNANT MARE AND STALLION
How to Diagnose Common Equine Reproductive
Tract Bacterial Pathogens Using
Chromogenic Agar
David P. Beehan, MVB; and
Angus O. McKinnon, BVSc, MSc, Diplomate ACT, ABVP
Authors address: Goulburn Valley Equine Hospital, 905B Goulburn Valley Highway, Congupna,
Victoria 3633, Australia; e-mail: davidpbeehan@eircom.net. 2009 AAEP.
1. Introduction
The most common microbiological pathogens of re-
productive tract disease in the horse are Streptococ-
cus sp., Escherichia coli, Klebsiella pneumoniae,
Staphylococcus aureus, and Pseudomonas aerugi-
nosa. The standard approach to the detection of
these pathogenic bacteria is to culture the reproduc-
tive tracts of the mare or stallion. One or more
general purpose media such as horse blood agar
(HBA) or MacConkey agar are inoculated before aer-
obic incubation occurs at 37C for 24 48 h. The
identification of pathogenic bacteria has been based
on colony size, morphology, pigmentation, hemolytic
patterns, and gram-stain. Such colonial character-
istics rarely permitted more than a presumptive
identification, and biochemical and/or serological
tests are required for definitive diagnosis. This ap-
proach frequently necessitates the testing of com-
mensal bacteria that resemble pathogens,1 which
may be more expensive and labor intensive, espe-
cially in a veterinary practice trying to make accu-
rate and expedient bacterial diagnoses.
The principle of the use of chromogenic agar is that
chromogenic substrates are incorporated into the agar
NOTES
320 2009 Vol. 55 AAEP PROCEEDINGS
Proceedings of the Annual Convention of the AAEP - Las Vegas, NV, USA, 2009
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to reveal the genus- or species-specific enzyme activi-
ties of microorganisms.2 These chromogenic sub-
strates release specific colored dyes when hydrolyzed
by enzymes of pathogenic microorganisms, which re-
sults in readily identifiable colored colonies.3 The in-
clusion of multiple chromogenic substrates into the
culture media facilitates the differentiation of polymi-
crobial cultures by causing the development of multi-
ple colored colonies. Selective chromogenic media
were first used in 1976 for the direct identification of
Escherichia coli in the primary culture of urine.4
Since then, a number of commercially prepared chro-
mogenic agars have been developed for the accurate
and rapid identification of pathogens of the human
urinary tract, because urine cultures in human hospi-
tals contribute greatly to the daily workload of a mi-
crobiological laboratory.5 Brilliance Urinary Tract
Infection (UTI) Agara is one such commercially pre-
pared media (formerly Chromogenic UTI agar). It
allows the identification of Streptococcus sp., Esche-
richia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Staphylococcus sp., Enterobacter aero-
genes, Enterococcus faecalis, Citrobacter sp., Proteus
sp., and Candida sp.2
Published in IVIS with the permission of the AAEP
Fig. 1. A HBA/MacConkey biplate.
The potential of chromogenic agar for use at the
Goulburn Valley Equine Hospital (GVEH) was first
realized after a Google search performed by one of
the authors (AOM) found a practical in-house rapid
and accurate laboratory technique to diagnose
Pseudomonas aeruginosa and Klebsiella pneu-
moniae. The prime directive was a number of
Thoroughbred stud farms, standing breeding stal-
lion farms that were serviced by the GVEH, that
requested that all mares bred by their stallions with
natural service be screened negative for the above-
named pathogens. Additionally, the technique aids
in the rapid identification of bacteria associated with
endometrial infection.
Previously, chromogenic agar was not reported to
be used in this area of veterinary medicine.
2. Materials and Methods
Endometrial and clitoral culture samples were
taken from mares in early estrus. Endometrial
swabs were taken using double-guarded swabs to
minimize vaginal or perineal contamination. The
swabs were transported to the laboratory in trans-
port media. On arrival, the name of the mare,
breeding farm, stallion farm, date, and time were
recorded, and the swabs were then streaked on Bril-
liance UTI Agar, HBA, and MacConkey split agar
plates. (Figs. 1 and 2). Plates were incubated aero-
bically at 37C, and any bacterial growth was regis-
tered at 24 and 48 h. Examination at 24 h was
required to identify heavy mixed cultures that may
have required subculturing so that individual colonies
could be identified at 48 h. Also at 24 h, presumptive
identification of pure uterine cultures permitted anti-
microbial sensitivity testing to begin, and results were
available at 48 h. All plates underwent final exami-
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NON-PREGNANT MARE AND STALLION
Fig. 2. A chromogenic agar plate.
nation again at 48 h. A negative result was only
confirmed at 48 h. Any further subculturing or anti-
microbial sensitivity testing was performed at this
time. When all microorganism identification was
complete and antimicrobial sensitivities were ana-
lyzed, a complete report was made available to both
the stallion farm and broodmare farm. In general, for
reasons of discretion, a positive result was only made
available to the broodmare farm.
Using chromogenic agar, microorganisms are
usually identified by their color and gram-stain
appearance; however, to confirm the presence of
Pseudomonas aeruginosa and Klebsiella pneumoniae,
(Figs. 310) biochemical testing was always per-
formed. (Figs. 310) The three biochemical test
kits used at the practice laboratory (GVEH) are the
RapID NF Plus,b RapID ONE,c and RapID SS/Ud
identification tests. These tests are designed to be
easy to set-up and interpret; they are accurate and
produce same-day (2 4 h) results.6 The RapID NF
Plus test was used for the diagnosis of the medically
important glucose non-fermenting (catalase positive
and oxidase variable) gram-negative rods (e.g.,
Pseudomonas aeruginosa). The RapID ONE test was
used for the identification of medically important En-
terbacteriaceae and other selected, oxidase negative,
gram-negative bacilli (e.g., Klebsiella pneumoniae).
The RapID SS/U test was used for general identifica-
tion of medically important microorganisms commonly
isolated from urine specimens when it was not clear
that either of the other tests were more appropriate.
3. Results
From August 2006 to December 2008, a total of 1374
endometrial swabs and 1158 clitoral swabs were
AAEP PROCEEDINGS Vol. 55 2009 321
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NON-PREGNANT MARE AND STALLION
Fig. 3. Pseudomonas aeruginosa. The cream-colored colonies
are the most common colony types.
collected from Thoroughbred mares on farms ser-
viced by practice veterinarians and other regional
veterinary practices. The color and colony appear-
ance guide provided in Table 1 was used to identify
the isolated bacteria.
The diagnosis of Pseudomonas aeruginosa was
based on the colony appearance, a positive oxidase
test, and the identification by gram stain of medium-
Fig. 4. The unique effects of Pyocyanin and Pyoverdin produced
by Pseudomonas aeruginosa on an antimicrobial sensitivity plate.
322 2009 Vol. 55 AAEP PROCEEDINGS
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Fig. 5. Klebsiella pneumoniae.
sized gram-negative rods. After subculturing to
ensure pure colonies, a RapID NF Plus identification
test was performed as the final confirmation. The
similar appearance of some Staphylococcus sp. and
Pseudomonas aeruginosa colonies required an oxi-
dase test to be performed. Oxidase test stripse
were used to perform this test. (Fig. 11). Also noted
was the unique effect that pyocyanin and pyoverdin,
Fig. 6. The similar appearance of Enterobacter aerogenes (left)
and Klebsiella pneumoniae (right).
Published in IVIS with the permission of the AAEP
Fig. 7. Large colonies of Klebsiella pneumoniae are present with
Streptococcus sp. (small light blue colonies), Staphylococcus sp.
(white colonies), and E. coli (pink colonies).
a blue-green pigment typically produced by Pseudo-
monas aeruginosa, had on sensitivity plates.
The diagnosis of Klebsiella pneumoniae required
the differentiation of these colonies from those of
Enterobacter sp. and Citrobacter sp., because the
color that the Brilliance UTI Agar turns these bac-
teria is similar.3 They were differentiated by sub-
Fig. 8. The pink colonies of Escherichia coli.
Proceedings of the Annual Convention of the AAEP - Las Vegas, NV, USA, 2009
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NON-PREGNANT MARE AND STALLION
Fig. 9. Citrobacter sp. (left) and Pseudomonas aeruginosa
(right).
culturing, when necessary, to isolate single colonies.
A gram stain was used to confirm a gram-negative
rod, and then the RapID One test was performed for
final identification (Figs. 1 8).
The presumptive diagnosis of the other main of-
fending bacteria (i.e., Escherichia coli, Streptococcus
sp., and Staphylococcus sp.) was made by using the
colony identification guide shown in Table 1. Esch-
Fig. 10. Antimicrobial sensitivity testing.
AAEP PROCEEDINGS Vol. 55 2009 323
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NON-PREGNANT MARE AND STALLION

Table 1. Colony Identification Guide

Total No.
Microorganisms
Species Detected Appearance of Colonies on Brilliance UTI Agar

Pseudomonas aeruginosa 29 Variable. Usually cream/pale green or tan/brownish. Oxidase positive.

Good but slow growth. 2.54 mm.
Klebsiella pneumoniae 30 Good growth, Large purple/deep blue/metallic navy colonies. 24 mm.
Escherichia coli 831 Good growth, darkish pink colonies. 23 mm.
Streptococcus sp. 687 Good growth, light blue colonies. 0.51 mm.
Staphylococcus sp. 446 Good growth, white colonies. Oxidase negative, always Catalase
positive. 23 mm.
Enterobacter aerogenes 92 Dark blue/navy colonies. Similar but subjectively smaller than
Klebsiella sp. 24 mm.
Citrobacter sp. 35 Dark blue/navy colonies. Usually smaller than Klebsiella sp. and
Enterobacter sp.
Enterococcus sp. 134 Good Growth, light blue colonies. 0.51 mm.
Proteus sp. 188 Good growth, brown colonies.
Candida sp. 3 Good but slow growth. Typically blue/white colonies.

erichia coli grew rapidly and was the only bacteria
that produced pink colonies (Fig. 9) on Brilliance
UTI Agar. The most significant Streptococcus sp.
associated with endometrial disease is the -hemo-
lytic Streptococcus sp. (e.g., Streptococcus Equi var
zooepidemicus). To diagnose the presence of a
-hemolytic Streptococcus sp., the hemolytic pattern
must be identified from the bacterial growth on
HBA, because chromogenic agar will not show any
hemolytic patterns. This also allows for differenti-
ation between the non-hemolytic and -hemolytic
Streptococcus sp.. The presence of -hemolytic pin-
point colonies that are only present on HBA in con-
junction with the presence of light blue colonies on
Brilliance UTI Agar allows the presumptive diagno-
sis of -hemolytic Streptococcus sp. Enterococcus
sp. is the other main differential for the growth of
Fig. 11. A positive Oxidase test result. A purple color within
20 s is a positive result. Oxidase tests are performed by lightly
rubbing the suspect colony on the test strip. Typically, the color
change occurs in 10 s.
324 2009 Vol. 55 AAEP PROCEEDINGS
light blue colonies on Brilliance UTI Agar; however,
Enterococcus sp. is unique in that it has the ability
to grow on both HBA and MacConkey agar.
Staphylococcus sp. can be differentiated from
Pseudomonas aeruginosa, because it is oxidase neg-
ative and catalase positive. The catalase test is
another simple test that can be used to identify a
species of bacteria. It is especially useful for dis-
tinguishing between Staphylococcus sp. and Strep-
tococcus sp., because both may show hemolysis on
blood agar and are coccoid on gram stain. Typi-
cally, Streptococcus sp. is ovoid, because it divides
only in one plane and is present in chains, whereas
Staphylococcus sp. is perfectly round and present in
clusters as it divides in two planes. The catalyse
test is performed by placing a drop of 3% hydrogen
peroxide on a microscope slide and smearing a bac-
terial colony from chromogenic agar through the
drop. A positive result is bubbling or frothing as-
sociated with the generation of oxygen. Staphylo-
coccus sp. is a strongly catalase positive, and
Streptococcus sp. and Enterococcus sp. are catalyse
negative. The catalase test should not be per-
formed on blood agar, because it contains catalase.
To confirm the presence of Staphyloccus aureus, a
coagulase test must be performed. Coagulase is
an enzyme produced by particular Staphylococcus
sp. and correlates with pathogenicity. The test is
performed by mixing a suspension of staphylococci
with rabbit plasma on a slide. A positive reaction
is indicated by clumping of bacteria within 12
min. Staphyloccus aureus is coagulase positive,
whereas many commensal Staphylococcus sp. are
negative. This test was not routinely performed
at the GVEH.
An endometrial culture was considered positive if
it grew 5 colonies per plate that formed units and
had no more than three different bacterial patho-
gens present. Any positive endometrial swabs that
grew four or more different bacterial colonies were

Proceedings of the Annual Convention of the AAEP - Las Vegas, NV, USA, 2009
Published in IVIS with the permission of the AAEP
Table 2. Common Urinary Pathogens Identified by the RapID SS/U Test
Pseudomonas sp.
Klebsiella sp.
Escherichia coli
Staphylococcus sp.
Enterobacter sp.
Citrobacter sp.
Enterococcus sp.
Proteus sp.
Candida albicans
presumed to be contaminated, and reswabbing of
the affected mare was advised. If an endometrial
swab result showed a positive growth, antimicrobial
sensitivity testing was performed using colonies
taken from the chromogenic agar. The antibiotics
tested at the GVEH were penicillin, ticarcillin, gen-
tamicin, neomycin, ceftiofur, and enrofloxacin.
Quality control measures were used to ensure that
our results were accurate and reliable. These in-
cluded using standard pure cultures of bacterial
specimens, which had been previously validated by
an accredited microbiology laboratory, to compare
color and biochemical tests results. In-house bio-
chemical testing was routinely used on other bacte-
ria in addition to Pseudomonas aeruginosa and
Klebsiella pneumoniae. The RapID SS/U test can
be used to diagnose many of the bacteria that the
GVEH expects to diagnose (Table 2). During our
first year using the new procedure, independent di-
agnosis of bacteria was also confirmed on selected
samples by an accredited microbiological laboratory.
4. Discussion
The use of chromogenic agar is a new and exciting
technique to help identify the pathogens of the
equine reproductive tract. It has revolutionized
microbiological management in our practice because
of the rapid and accurate identification of common
reproductive tract pathogens.
It has a number of advantages over traditional
methods of microbial identification. These include
the reduction in the time required for the laboratory
processing of samples, the ease of colony identifica-
tion, the low level of skill required to interpret re-
sults, the reliability of colonies taken from
chromogenic agar for pathogen identification and
antimicrobial sensitivity testing, and the proven
high level of sensitivity of chromogenic agar in iden-
tifying pathogens.5 The slightly higher cost of
chromogenic agar per plate ($1.15 AUS) compared
with conventional split-plate media ($0.58 AUS) was
offset by a reduced need for complementary reagents
and a reduction in labor hours associated with the
processing of culture plates and suspect pathogens.1
Additionally, the technique removed the often iden-
tified problems associated with samples submitted
immediately before the weekend.
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NON-PREGNANT MARE AND STALLION
The disadvantages of this technique include the
slightly higher cost of chromogenic agar plates, es-
pecially in facilities processing small numbers of
samples, the lack of benefit for the screening and
diagnosis of Taylorella equigenitalis, another poten-
tial venereal pathogen that recently has emerged
again in the United States, and the potential for
misdiagnosis of Candida sp. with Staphylococcus sp.
(both may produce white colonies that are oxidase
negative); however, a gram stain easily differenti-
ates these microorganisms, and Staphylococcus sp.
is invariably catalase positive. The technique does
not differentiate pathogenic from non-pathogenic
bacteria, and any interpretation of the significance
of uterine bacteria isolated must be related to endo-
metrial cytology and clinical examination.
In summary, because of the limited availability of
regional laboratory facilities that offer a rapid, accu-
rate, and continuous weekly screening service in our
area, the introduction of chromogenic agar at the
GVEH has been very useful. It has resulted in a fast
and accurate service that was especially important
when samples were collected from mares close to the
weekend. At that time, traditional services are not
able to provide a report in time to facilitate breeding
when mares are required to be presented with a neg-
ative culture to potential pathogens.
The accuracy and ease of interpretation of bacterial
isolation using chromogenic agar suggests that it is a
technique applicable to many veterinary practices
with similar diagnostic requirements as the GVEH.
The authors would like to acknowledge John F.
Prescott, MA, VetMB, PhD, Natasha Hovanessian,
BVSc, and Padhraic Doran, MVB.
References and Footnotes
1. Perry JD, Freydie`re AM. The application of chromogenic
media in clinical microbiology. J Appl Microbiol 2007;103:
2046 2055.
2. Carricajo A, Boiste S, Thore J, et al. Comparative evalua-
tion of five chromogenic media for detection, enumeration and
identification of urinary tract pathogens. Eur J Clin Micro-
biol Infect Dis 1999;18:796 803.
3. Samra Z, Heifetz M, Talmor J, et al. Evaluation of use of a
new chromogenic agar in detection of urinary tract patho-
gens. J Clin Microbiol 1998;36:990 994.
4. Kilian M, Bulow P. Use of a -glucoridase detecting agar
medium (PGUA agar) for the identification of Escherichia coli
in primary cultures of urine samples. Acta Pathol Microbiol
Scand 1979;8:271276.
5. Fallon D, Ackland G, Andrews N, et al. A comparison of the
performance of commercially available chromogenic agars for
the isolation and presumptive identification of organisms
from urine. J Clin Pathol 2003;56:608 612.
6. Kitch TT, Jacobs MR, Applebaum PC. Evaluation of the
4-hour RapID NF Plus method for identification of 345 gram-
negative non-fermentative rods. J Clin Microbiol 1992;30:
12671270.
a
Brilliance Urinary Tract Infection Agar, Oxoid Ltd., Solaar
House, 19 Mercers Row, Cambridge CB5 8BZ, United Kingdom.
b
c
RapID NF Plus, Remel, Lenexa, Kansas 662153594.
RapID ONE, Remel, Lenexa, Kansas 662153594.
d
RapID SS/, Remel, Lenexa, Kansas 662153594.
e
Oxidase test strips, Oxoid Ltd., Solaar House, 19 Mercers Row,
Cambridge CB5 8BZ, United Kingdom.
AAEP PROCEEDINGS Vol. 55 2009 325