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Antibiotic resistance in Staphylococci isolated from the vaginas of captive female Leontopithecus (CallitrichidaeЦPrimates).

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American Journal of Primatology 68:825–831 (2006)
RESEARCH ARTICLE
Antibiotic Resistance in Staphylococci Isolated
From the Vaginas of Captive Female Leontopithecus
(Callitrichidae–Primates)
WALTER LILENBAUM1, ISMAR A. MORAES2, VERÔNICA S. CARDOSO1,
RENATO G. VARGES1, ANA MARIA R. FERREIRA3, AND ALCIDES PISSINATTI4
1
Department of Microbiology and Parasitology, Universidade Federal Fluminense,
Niterói/RJ, Brazil
2
Department of Physiology and Pharmacology, Universidade Federal Fluminense,
Niterói/RJ, Brazil
3
Department of Pathology, Universidade Federal Fluminense, Niterói/RJ, Brazil
4
Centro de Primatologia do Rio de Janeiro-FEEMA, Centro Universitário Plı´nio Leite,
Fundac- ão Educacional Serra dos Órgãos, Teresópolis/RJ, Brazil
The purpose of this study was to provide current data on Staphylococcus
species from the vaginas of clinically normal captive lion tamarins and to
determine the antimicrobial susceptibility of these isolates. Samples were
collected from 25 adult lion tamarins, processed to isolate Staphylococcus
species, and tested for susceptibility to penicillin G, gentamicin, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, streptomycin,
ampicillin, and rifampicin. Isolates with the typical characteristics of the
genus Staphylococcus were recovered from all 25 samples. Coagulasenegative species were the most common (68% of the isolates), and the most
frequently isolated species (10 samples) was S. simulans. Other coagulasenegative species, including S. saprophyticus (n 5 5), S. epidermidis (n 5 1),
and S. arlettae (n 5 1), were also recovered. Coagulase-positive Staphylococci were obtained from eight animals (six of from the S. aureus species
and two from S. intermedius). Resistance to antibiotics was frequently
observed, and 88% of the isolates (23 samples) showed resistance to at least
one drug. Resistance to penicillin G was a common finding, and the most
active antimicrobial agents were chloramphenicol and gentamicin. Coagulase-positive strains were more frequently resistant to antibiotics (79.7%,
average 5 6.4 drugs) than coagulase-negative strains (38.2%, average 5 3.0
drugs). The high frequency of resistance observed in those isolates is
surprising and very alarming. A detailed history of the use of antimicrobial
drugs in these subjects did not reveal any previous exposure to any of
the tested antibiotics that could justify the observed resistance rate. Am. J.
Primatol. 68:825–831, 2006. c 2006 Wiley-Liss, Inc.
Contract grant sponsor: Fundac- ão de Amparo a Pesquisa no Estado do Rio de Janeiro.
Correspondence to: Dr. Walter Lilenbaum, Rua Hernani Mello, 101 Niterói/RJ, Brazil.
E-mail: mipwalt@vm.uff.br
Received 9 February 2004; revised 19 March 2004; revision accepted 5 March 2004
DOI 10.1002/ajp.20282
Published online in Wiley InterScience (www.interscience.wiley.com).
r 2006 Wiley-Liss, Inc.
826 / Lilenbaum et al.
Key words: Staphylococcus; tamarins; primates; monkey; Leontopithecus sp.
INTRODUCTION
The Callitrichidae family is composed of the genera Leontopithecus,
Saguinus, Cebuella, and Callithrix [Hershkovitz, 1977]. Genus Leontopithecus
includes four recognized species: the golden lion tamarin (L. rosalia), goldenheaded lion tamarin (L. chrysomelas), black lion tamarin (L. chrysopygus), and
black-faced lion tamarin (L. caissara) [Coimbra-Filho, 1990]. These species have
been considered as critically endangered by the International Union for
Conservation of Nature (IUCN) Species Survival Commission [Anonymous,
2002]. Concern regarding the survival of these tamarins has given rise to major
research, management (including reintroduction of captive-born animals), and
environmental education programs over the last two decades [Beck et al., 1991;
Coimbra-Filho, 1990]. Therefore, captive breeding of these animals is an
important option for increasing their population and reintroducing more animals
into their natural habitats.
Other than a comprehensive study about the microbiology of laboratory-kept
marmosets [Deinhardt et al., 1967], little research has focused on Leontopithecus
in this regard. Thus, our knowledge about the physiology and pathology of
reproduction in these animals is still limited [De Vleeschouwer et al., 2001].
Knowledge about normal bacterial flora is considered to be of paramount
importance for the proper diagnosis and treatment of pathologic abnormalities
of the reproductive tract [Doyle et al., 1991]. Therefore, a better comprehension of
the Staphylococci species that occur in the vaginal flora and their susceptibility to
antimicrobial agents may contribute to the correct management of vaginitis and
other infections of the reproductive tract. Isolates of the Staphylococcus genus
from pyometra [Lang & Benjamin, 1969] and toxigenic vaginal infections [Pollack
et al., 1983], as well as ascending intrauterine infections [Swindle et al., 1982],
have been reported in rhesus monkeys.
One of the most common alterations of the genital tract in all primates is
vaginitis. It is caused by a variety of organisms that can act at times as
opportunistic secondary invaders. They may also contribute to clinically overt
disease, and even to complications from septic abortion [Isenberg & D’Amato,
1995]. Aerobic vaginitis is characterized by the presence of clinical signs such as
red inflammation of the vagina, yellowish discharge, and abundant vaginal
leucocytes. It is correlated with the recovery of S. aureus, since the more severe
the microscopic findings, the more likely these organisms will be cultured
[Donders et al., 2002].
Bacteria from the genus Staphylococcus have been described as members of
the normal vaginal flora of nonhuman primates, such as chimpanzees [Boncyk &
Kalter, 1972], baboons [Skangalis et al., 1979], howler monkeys [Claver et al.,
1984], rhesus monkeys [Doyle et al., 1991], and pig-tailed monkeys [Lichtenwalner
et al., 2000]. Nevertheless, to our knowledge, no studies of lion tamarins
(Leontopithecus sp.) in this regard have been conducted.
The purpose of this study was to provide current data on the aerobic resident
bacterial flora (particularly Staphylococcus species) of the vagina of clinically
normal captive lion tamarins, and to determine the antimicrobial susceptibility of
these isolates.
Am. J. Primatol. DOI 10.1002/ajp
Staphylococci From Vaginas of Captive Lion Tamarins / 827
MATERIALS AND METHODS
Animals
Samples were obtained from 25 unmedicated adult females (two golden lion
tamarins (Leontopithecus rosalia), 14 golden-headed lion tamarins (L. chrysomelas),
and nine black lion tamarins (L. chrysopygus) from the Centro de Primatologia do
Rio de Janeiro, Brazil. The animals were judged to be free of reproductive disease
following a detailed history and physical examination. Animals with a recent
history of disease or administration of local or systemic drugs or antimicrobial
agents in the last 6 months were excluded from the study. Each animal was
cultured once.
Sample Collection and Processing
A sterile cotton swab was used to collect samples from the vaginal area. The
samples were immediately inoculated in Brain Heart Infusion broth (Merck,
Darmstadt, Germany) and incubated at 371C. No methodology was employed to
isolate anaerobic bacteria. If growth occurred after 24 or 48 hr, smears were
made, Gram-stained, and examined microscopically for organisms. Samples with
morphology consistent with that of Staphylococcus sp. were transferred to tryptic
soy agar, 5% sheep blood agar, and mannitol salt agar (Merck).
Identification of Isolates
After growth, Staphylococci were identified on the basis of colony characteristics, Gram stain, pigment production, hemolysis on 5% bovine blood agar, and
biochemical reactions, including catalase activity, tube coagulase test (rabbit
plasma), nitrate reduction, phosphatase, acetoin and arginine dihydrolase
production, mannitol fermentation, urease, mannose and maltose fermentation,
novobiocin resistance, and a deoxyribonuclease test [Kloos & Bannerman, 1995].
Staphylococcus species were classified as described in previous studies [Lilenbaum
et al., 1999] and according to Bergey’s Manual of Determinative Bacteriology [Holt
et al., 1994]. Isolates of other bacterial genera (e.g., Gram-negative rods) were
not considered in this study. One colony from each isolate was transferred to
stock agar.
Disk Diffusion Test
Susceptibility to a panel of eight antimicrobial agents was determined by
the disk diffusion method in Mueller-Hinton agar (Merck) according to the guidelines of the National Committee for Clinical Laboratory Standards [Anonymous,
2003]. Discs of penicillin G (10 U), gentamicin (10 mg), chloramphenicol (30 mg),
tetracycline (30 mg), trimethoprim-sulfamethoxazole (25 mg), streptomycin (10 mg),
ampicillin (10 mg), and rifampicin (30 mg) were tested. After the antimicrobial zone
diameters were measured, the strains were categorized according to the
standards as being susceptible or resistant to the drugs.
Statistics
The correlation between the occurrence of Staphylococcus species and
the tamarin species, as well as the age of the animals, was evaluated by means
of the chi-square (w2) test and Fischer’s test using a required confidence level of
95% (Po0.05).
Am. J. Primatol. DOI 10.1002/ajp
828 / Lilenbaum et al.
RESULTS
Isolates with the typical characteristics of the genus Staphylococcus
were recovered from all 25 samples, which confirms the frequent occurrence
of Staphylococcal strains in the vaginal microflora of lion tamarins. The
coagulase-negative Staphylococcus species (CoNS) were the most common,
representing 68% of the isolates. The most frequently isolated Staphylococcus
species (10 samples) was S. simulans. Other CoNS, such as S. saprophyticus
(five isolates), S. epidermidis (one isolate), and S. arlettae (one isolate), were also
obtained.
Coagulase-positive species of Staphylococci (CoPS) were isolated from
eight animals (six from the species S. aureus and two from S. intermedius).
Even though we recovered all of the isolates of S. intermedius species from
L. chrysopygus and none from L. chrysomelas, the percentage of total CoPS was
similar in both species (33.3% and 35.7%, respectively). A possible association
between S. intermedius and L. chrysopygus species is suggested, but since two
isolates of these species were recovered, we were unable to perform a statistical
analysis to confirm that hypothesis. Therefore, this phenomenon remains to be
elucidated in further studies. The frequency of isolation of the different
Staphylococci species is shown in Table I.
No correlation was observed between the age of the animal and
the occurrence of CoPS. We considered 24 months as the time when the
females reached complete sexual maturity. Of the 25 tamarins in this study, 15
were considered mature, seven were young, and no age information was available
for three. The occurrence of CoPS was 33.3% (5/15) in older animals and 28.5%
(2/7) in younger ones. This slight difference was not statistically significant
(P40.05).
Resistance to antibiotics was frequently observed, and 88% of the isolates
(23 samples) showed resistance to at least one drug (Table II). Resistance to
penicillin G was a common finding and was observed in 21 (84%) of the 25
isolates. Twelve isolates were resistant to tetracycline (48%), 16 to rifampin
(64%), 13 to ampicillin (52%), and 10 to streptomycin or trimethoprimsulfamethoxazole (40%). The most active antimicrobial agents against Staphylococci isolated from the vagina of healthy tamarins were chloramphenicol and
gentamicin, with only four samples showing resistance to those drugs (16% of the
isolates).
Coagulase-positive strains were more frequently resistant to antibiotics
(79.7%, average 5 6.4 drugs) than coagulase-negative strains (38.2%, average 5 3.0 drugs). This finding was more evident for trimethoprim-sulfamethoxazole (with 100% of coagulase-positive and 11.8% of coagulase-negative resistant
TABLE I. Frequency of Different Staphylococcal Species Isolated from Vagina
of 25 Clinically Healthy Lion Tamarins
Species
Coagulase
Number of isolates
%
S. aureus
S. intermedius
S. simulans
S. saprophyticus
S. epidermidis
S. arlettae
TOTAL
Positive
Positive
Negative
Negative
Negative
Negative
6
2
10
5
1
1
25
24
8
40
20
4
4
100
Am. J. Primatol. DOI 10.1002/ajp
Staphylococci From Vaginas of Captive Lion Tamarins / 829
TABLE II. Antibiotic Resistance of Different Staphylococcal Species Isolated
from Captive Lion Tamarins
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Species
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
rosalia
rosalia
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysomelas
chrysopygus
chrysopygus
chrysopygus
chrysopygus
chrysopygus
chrysopygus
chrysopygus
chrysopygus
chrysopygus
CLO GEN PEN TET SUT EST RIF AMP
S
S
S
S
R
S
S
S
S
S
S
S
R
R
R
S
S
S
S
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
R
S
R
S
S
S
S
S
S
S
S
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
S
S
R
R
S
R
R
R
R
R
R
S
R
R
R
S
S
S
S
S
R
R
R
R
S
S
R
S
S
S
S
R
R
S
S
S
R
R
R
S
S
R
R
S
S
S
S
R
R
S
S
S
S
R
S
R
R
S
S
S
S
S
R
R
R
S
S
S
R
S
S
S
R
R
S
S
S
R
R
S
S
R
R
S
R
R
R
R
R
S
S
S
R
R
R
R
R
S
S
S
S
R
R
R
S
R
R
S
R
R
R
R
R
R
R
S
S
R
S
S
R
S
R
R
S
S
S
S
R
S
R
R
S
S
Isolate
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
S.
simulans
epidermidis
simulans
aureus
aureus
simulans
simulans
saprophyticus
aureus
simulans
saprophyticus
arlettae
simulans
aureus
aureus
saprophyticus
saprophyticus
simulans
simulans
intermedius
saprophyticus
intermedius
aureus
simulans
simulans
CLO, cloramphenicol; GEN, gentamicin; PEN, penicillin; TET, tetracycline; SUT, trimethoprim-sulfamethoxazole; EST, streptomycin; RIF, rifampicin; AMP, ampicillin; S, susceptible; R, resistant.
samples) and penicillin G (with 100% of coagulase-positive and 76.4% of
coagulase-negative samples showing resistance).
DISCUSSION
Knowledge about normal microbial populations has been recognized as
an important factor in elucidating the pathophysiology of disease in human beings
and animals. It is believed that in addition to the presence of bacteriocins,
antagonism, occupation of adhesins, and other competitive systems, a normal
microbial population inhibits the proliferation of pathogenic organisms by depriving them of nutrients. It is normally harmless, but with the presence of predisposing factors (e.g., trauma or concurrent infection), some of these organisms
may become potential pathogens, multiplying and causing bacterial disease.
The present study confirms the presence of Staphylococcal strains in
the vaginal microflora of lion tamarins, as evidenced by the recovery of
Staphylococcus strains from 100% of the vaginal swabs. The predominance of
CoNS was an expected finding, since previous reports on other nonhuman
primates revealed that flora to be very similar to that found in human vaginas.
S. epidermidis has been recovered from the vaginas of baboons [Skangalis et al.,
1979], captive howler monkeys [Claver et al., 1984], rhesus monkeys [Doyle et al.,
1991], and both live and postmortem marmosets [Hill et al., 1978].
Am. J. Primatol. DOI 10.1002/ajp
830 / Lilenbaum et al.
The isolation of eight strains of CoPS is a very important finding, since they
are often related to acute purulent infections in humans and other animals,
including nonhuman primates. CoPS have been described in cases of clinical
vaginitis and metritis in rhesus and cynomolgus monkeys [Doyle et al., 1991].
S. aureus is the most commonly reported species, and has been described in acute
external otitis in a marmoset [Hill et al., 1978], in suppurative arthritis in an
infant orangutan [Hoopes et al., 1978], and in postoperative wound infections
[Blouse et al., 1976], acute pyometra [Lang & Benjamin, 1969], and intrauterine
infections [Swindle et al., 1982] in rhesus monkeys. S. saprophyticus is known to
be a common cause of urinary tract infection in women and has been reported as a
cause of acute infection of both the lower and upper urinary tracts in grivet
monkeys, leading to cystitis, ureteritis, and pyelonephritis [Mardh et al., 1980].
CoPS have also been described as members of the normal vaginal flora
of chimpanzees [Boncyk & Kalter, 1972] and rhesus monkeys [Doyle et al., 1991].
With regard to the susceptibility to antimicrobial agents of the strains
isolated in this study, some unexpected results were observed. In the present
study the strains tested were from normal microflora of animals with no recent
history of disease. Therefore, the high frequency of resistance observed in these
isolates is surprising and very alarming. Isolates from normal microflora usually
present less resistance to antimicrobial drugs. Infectious diseases that require
antimicrobial administration are uncommon in those animals. Moreover, a
detailed history of the use of antimicrobial drugs did not show previous exposure
to any of the tested antibiotics that could justify the observed resistance rate.
CONCLUSIONS
Staphylococcus strains were recovered from 100% of the animals, and
therefore constitute members of the normal microflora of the vagina. During
changes in the microbial balance of the microflora, these species may play an
important role in vaginitis and other infections of the urinary and reproductive
tracts. Therefore, for better comprehension, diagnosis, and treatment of those
diseases, knowledge about the different species of this genus, the resistance
pattern of those strains, and their role in the normal microflora of the tamarin
vagina is fundamental.
ACKNOWLEDGMENTS
We thank the CPRJ-FEEMA team, Fundac- ão Estadual de Engenharia do
Meio Ambiente-Rio de Janeiro, Durrell Wildlife Conservation Trust (Jersey),
American Zoo and Aquarium Association, Ministério Público Federal, and IBAMA
for their support and cooperation.
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