Research in Veterinary Science 78 (2005) 117–121

www.elsevier.com/locate/rvsc

Salmonella enterica isolates from faeces of domestic reptiles

and a study of their antimicrobial in vitro sensitivity
a,*
V.V. Ebani , D. Cerri a, F. Fratini a, N. Meille, P. Valentini b, E. Andreani a

a
Department of Animal Pathology, Prophylaxis and Food Hygiene, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy
b
Center for Pathogen Enterobacteria for Central Italy, via S. Zeno 35-39, 56127 Pisa, Italy

Accepted 13 August 2004

Abstract

From October 2001 to February 2002, the faecal samples of 305 reptiles (165 saurians, 99 ophidians and 41 chelonians) were
bacteriologically examined to detect Salmonella enterica. S. enterica was isolated from 73 (23.93%) faecal samples including 44
(60.27%) samples collected from saurians, 15 (20.55%) from chelonians and 14 (19.18%) from ophidians; considering the number
of samples taken for each reptile group, S. enterica was isolated from the 36.58% of chelonians, 26.66% of saurians and 14.14%
of ophidians. The isolates were distributed among 38 serotypes. Sixty-nine (94.52%) isolates were resistant to erythromycin. About
one-third of the isolates was resistant to sulfisoxazole (35.61%), gentamycin (32.88%), amoxycillin (31.51%) and ampicillin (27.40%).
All but one of the isolates were sensitive to chloramphenicol. A high percentages of isolates were sensitive to enrofloxacin (84.93%),
nitrofurantoin (80.82%), trimethoprim (76.71%) and tetracycline (75.34%).
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: Salmonella; Reptiles; Faeces; Antimicrobial sensitivity

1. Introduction lae mainly through direct contact with other infected

Until a few years ago, reptiles were not considered as
pet animals and their presence was limited to specialized
centers. However, during the last 10 years, we observed
an ever-increasing importation of exotic animals des-
tined to live under domestic conditions. Terrestrial and
aquatic turtles are the most common exotic animals,
but other reptiles such as snakes and iguanas are fre-
quently kept as pets. Domesticated as well as free-living
reptiles have been frequently shown to be important res-
ervoirs of zoonotic microorganisms.
Salmonellosis, one of the most important infectious
disease affecting human and animals, is widespread over
the world. Reptiles may become infected with salmonel-
*
Corresponding author. Tel.: +39 50 570310; fax: +39 50 540644.
E-mail address: vebani@vet.unipi.it (V.V. Ebani).
reptiles. Turtles kept in large breeding ponds are often
overcrowded, exposed to offal and refuse feeding that
may be contaminated with enteric pathogens. Reptile
eggs buried in moist soil or sand may be contaminated
with organisms in that substrate or become exposed
during the passage through the cloaca. Salmonellae were
reported to readily penetrate turtle eggs with contamina-
tion of the internal contents within 1 h of exposure (Fee-
ley and Treger, 1969). Infection can also occur through
transovarial transmission (Austin and Wilkins, 1998).
Human cases of salmonellosis are often associated
with food, but frequently people contract the infection
from infected material, mainly faeces, eliminated by
warm-blooded domestic animals. It is now recognized
that human salmonellosis associated with exotic pets
represents a rapidly emerging public health problem
(Aleksic et al., 1996; Woodward et al., 1997; Austin

0034-5288/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rvsc.2004.08.002
118 V.V. Ebani et al. / Research in Veterinary Science 78 (2005) 117–121
and Wilkins, 1998; Nowinski and Albert, 2000; Jafari
et al., 2002).
Reptiles usually do not show signs of disease from
infection with Salmonella enterica and the relationship
may indeed be a saprophytic one. However, S. enterica
has been found to cause septicemia, pneumonia, coe-
lomitis, abscess, granuloma, hypovolemic shock and
death (Onderka and Finlayson, 1985; Frye, 1991). In
all cases, salmonellae are carried in the gastrointestinal
tract and may be shed intermittently during times of
stress (Chiodini and Sundberg, 1981; Bradley et al.,
2001).
Antimicrobial resistance in S. enterica isolated from
samples of animal origin is an emerging problem. These
strains may be a source of resistance genes for other bac-
teria and cause therapeutic failures in veterinary and
human diseases for which antimicrobial treatment is
essential.
In the present study the occurrence of S. enterica in
faeces of reptiles (chelonians, ophidians and saurians),
kept in a specialized sale centre in central Italy, was
investigated and the antimicrobial sensitivity of the
isolates were tested.
2. Material and methods
2.1. Animals
From October 2001 to February 2002, the faecal sam-
ples of 305 reptiles (165 saurians, 99 ophidians and 41
chelonians) were examined for S. enterica at the Bacteri-
ology Laboratory of the Department of Animal Pathol-
ogy, Prophylaxis and Food Hygiene, Faculty of
Veterinary Medicine in Pisa. Samples were collected
from animals in a sale centre in central Italy. Faecal
samples were collected into aseptic tubes from individ-
ual cages and pools.
2.2. Bacteriology
Faecal samples (about 3 g) were incubated in 10 ml of
buffered peptone water at 37 °C for 24 h. One ml of this
culture was then transferred to 10 ml of Selenite and to
10 ml of Rappaport-Vassiliadis broths (Difco, Becton
Dickinson, Sparks, MD, USA) and the tubes incubated
for 24 h at 43 and 42 °C, respectively. One loopful from
each broth culture was streaked onto duplicate plates of
Brillant Green Agar – BGA (Difco) and Salmonella
Shigella Agar – SSA (Difco). The plates were incubated
at 37 °C for 24 h. Suspected colonies were then picked
and inoculated into tubes containing Triple Sugar Iron
Agar – TSIA (Oxoid Ltd., Basingstoke, UK) which were
incubated at 37 °C for 24 h. Colonies showing typical
reactions on TSIA were purified and biochemically char-
acterized using urease, oxidase and o-nitrophenyl-b-D -
galactopyranoside (ONPG) tests. The isolates were
investigated further to determine somatic (O) antigens
with anti-OMA, OMB, OMC, OMD, OME, OMF,
OMG salmonella polyvalent sera (Bio-Rad, Marnes la
Coquette, France) and the serotypes identified according
to the Kaufmann–White Salmonella serotyping scheme
(Popoff and Le Minor, 1992) at the Center for Pathogen
Enterobacteria for Central Italy, Pisa.
2.3. Antimicrobial resistance test
Seventy-three isolates were tested for their sensitivity
to 12 antimicrobial agents by the disk diffusion method
(Bauer et al., 1966). Briefly, 5 ml of tryptic soy broth
were inoculated with one loop of culture. The suspen-
sion obtained was uniformly spread onto the surface
of dry Mueller–Hinton (Oxoid) agar plates using an
impregnant swab. The following antibiotics (Oxoid)
were tested: ampicillin (AMP), 10 lg; amoxycillin
(AMX), 25 lg; amoxycillin–clavulanic acid (AMC),
20 + 10 lg; cefotaxime (CTX), 30 lg; chloramphenicol
(CHL), 30 lg; gentamycin (GEN), 10 lg; nitrofurantoin
(NIT), 300 lg; tetracycline (TET), 30 lg; enrofloxacin
(ENR), 5 lg; erythromycin (ERY), 15 lg; sulfisoxazole
(SUL), 300 lg; trimethoprim (TMP), 5 lg.
For each isolate, the zone of inhibition around each
disk was measured, after incubation at 37 °C for 24 h.
The isolates were classified as susceptible (S), intermedi-
ate (I) or resistant (R), using the zone diameter stand-
ards published by Oxoid.
3. Results
Salmonella enterica was isolated from 73 of 305
(23.93%) faecal samples. Among the positive samples,
44 (60.27%) were collected from saurians, 15 (20.55%)
from chelonians and 14 (19.18%) from ophidians. Con-
sidering the number of samples taken for each reptile
group, S. enterica was isolated from the 36.58% of
chelonians, 26.66% of saurians and 14.14% of
ophidians.
Seventy-one isolates were typed, two isolates
were untypable because they were in R-phase
(Tables 1 and 2). The 71 isolates were distributed among
38 serovars. The serovars most frequently encountered
were: S. Berta (nine isolates from turtles Chrysemys scri-
pta), Salmonella IV 44:z4z23:- (five isolates from iguanas
Iguana iguana and two from snakes Python regius), S.
Fluntern (four isolates from geckos Eublepharis macu-
laris), S. Trimdon (two isolates from tortoises Testudo
hermanni, one isolate from a lizard Tiliqua scincoides
and one from a water dragon Physignathus concincinus),
S. Apapa (three isolates from iguanas Iguana iguana), S.
Ebrie (one isolate from a chamaleon Chamaeleo verruco-
sus, one from a gecko Eublepharis macularis, one from a
 V.V. Ebani et al. / Research in Veterinary Science 78 (2005) 117–121 119

Table 1
Salmonella enterica serotypes isolated from faeces samples of reptiles
Salmonella serotype No. of organisms isolated Animal source
S. Agona 1 Chamaleon (Chamaeleo verrucosus)
S. Anatum 1 Gecko (Eublepharis macularis)
S. Apapa 3 Iguanas (Iguana iguana)
S. Berta 9 Turtles (Chrysemys scripta)
S. Blukwa 1 Snake (Python regius)
S. Caracas 2 Gecko (Eublepharis macularis)
Lizard (Physignathus concincinus)
S. Ceyco 1 Lizard (Physignathus concincinus)
S. Chichiri 1 Tortoise (Testudo hermanni)
S. Durban 1 Chamaleon (Chamaeleo verrucosus)
S. Duval 1 Turtle (Chrysemys scripta)
S. Ebrie 3 Gecko (Eublepharis macularis)
Lizard (Eumeces schneideri)
Chamaleon (Chamaeleo verrucosus)
S. Fluntern 4 Geckos (Eublepharis macularis)
S. Friedrichsfelde 1 Dragon lizard (Pogona vitticeps)
S. Israel 1 Lizard (Tiliqua scincoides)
S. Kapemba 1 Corn snake (Elaphy guttata)
S. Kottobus 1 Tortoise (Testudo hermanni)
S. Kuntair 1 Gecko (Eublepharis macularis)
S. Memphis 1 Snake (Boa constrictor)
S. Midway 2 Snake (Lampropeltis getula)
Lizard (Uromastyx acanthinurus)
S. Montevideo 2 Gecko (Hemitheconyx caudicinctus)
Tortoise (Testudo hermanni)
S. Muenchen 1 Snake (Python regius)
S. Othmarschen 3 Water dragons (Physignathus lesueurii)
S. Reading 1 Corn snake (Elaphy guttata)
S. Trimdon 4 Tortoises (Testudo hermanni) (two isolates)
Lizard (Tiliqua scincoides)
Water dragon (Physignathus concincinus)
S. Senftemberg 2 Snake (Python regius)
Iguana (Iguana iguana)
S. Veneziana 1 Gecko (Eublepharis macularis)

Table 2
Salmonella enterica serotypes isolated from faeces samples of reptiles
Salmonella serotype No. of organisms isolated Animal source
Salmonella subsp. II 9,46:z39:1,7 1 Water dragon (Physignathus concincinus)
Salmonella subsp. II 16:g,m,s,t:- 1 Snake (Python regius)
Salmonella subsp. II 48:d:z6 1 Iguana (Iguana iguana)
Salmonella subsp. II 50:b:z6 3 Iguanas (Iguana iguana)
Salmonella subsp. IIIa 48:z4Z23:- 1 Snake (Python regius)
Salmonella subsp. IIIb 48:z4Z23:- 1 Snake (Python regius)
Salmonella subsp. IIIb 50:k:z 1 Iguana (Iguana iguana)
Salmonella subsp. IIIb 50:z:z52 1 Iguana (Iguana iguana)
Salmonella subsp. IIIb 50:r:e,n,x,z15 1 Iguana (Iguana iguana)
Salmonella subsp. IIIb 65:k:z53 2 Geckos (Hemitheconyx caudicinctus)
Salmonella subsp. IV 18:z36z38:- 1 Snake (Boa constrictor)
Salmonella subsp. IV 44:z4z23:- 7 Iguanas (Iguana iguana) (five isolates)
Snakes (Python regius) (two isolates)
R-phase Salmonella 2 Gecko (Eublapharis macularis)
Snake (Python regius)
120 V.V. Ebani et al. / Research in Veterinary Science 78 (2005) 117–121

Table 3
Number of Salmonella enterica strains sensitive, intermediate and resistant to the antimicrobial agents tested
Antimicrobial agenta Sensitive isolates Intermediate isolates Resistant isolates
AMP 48 (65.75%) 5 (6.85%) 20 (27.40%)
AMC 52 (71.23%) 11 (15.07%) 10 (13.70%)
AMX 48 (65.75%) 2 (2.74%) 23 (31.51%)
CHL 72 (98.63%) 0 1 (1.37%)
GEN 47 (64.38%) 2 (2.74%) 24 (32.88%)
CTX 52 (71.23%) 15 (20.55%) 6 (8.22%)
ERY 0 4 (5.48%) 69 (94.52%)
ENR 62 (84.93%) 8 (10.96%) 3 (4.11%)
NIT 59 (80.82%) 9 (12.33%) 5 (6.85%)
SUL 42 (57.54%) 5 (6.85%) 26 (35.61%)
TET 55 (75.34%) 16 (21.92%) 2 (2.74%)
TMP 56 (76.71%) 10 (13.70%) 7 (9.59%)
a
AMP: ampicillin, AMC: amoxycillin–clavulanic acid, AMX: amoxycillin, CHL: chloramphenicol, GEN: gentamycin, CTX: cefotaxime, ERY:
erythromycin, ENR: enrofloxacin, NIT: nitrofurantoin, SUL: sulfisoxazole, TET: tetracyclin, TMP: trimethoprim.

lizard Eumeces schneideri), S. Othmarschen (three iso-
lates from water dragons Physignathus concincinus),
and Salmonella II 50:b:z6 (three isolates from iguanas
Iguana iguana).
The results of antimicrobial resistance tests are shown
in Table 3. All the isolates tested were resistant to one or
more antibiotics, and 19 isolates were multiresistant.
Sixty-nine (94.52%) isolates were resistant to erythromy-
cin and the remaining four were intermediately suscepti-
ble to the same antibiotic. A large percentage of isolates
was resistant to sulfisoxazole (35.61%), gentamycin
(32.88%), amoxycillin (31.51%) and ampicillin
(27.40%). All but one isolates were sensitive to chloram-
phenicol. A high percentage of isolates was sensitive to
enrofloxacin (84.93%), nitrofurantoin (80.82%), trimeth-
oprim (76.71%) and tetracycline (75.34%).
4. Discussion
The presence of S. enterica in exotic pets constitutes a
serious public health problem. During the last few years,
a high percentages of human salmonellosis have been
associated with reptiles kept as domestic pets (Ackman
et al., 1995; Dalton, 1995; Mermin et al., 1997; Wood-
ward et al., 1997; Olsen et al., 2001).
During the present investigation, S. enterica was iso-
lated from saurians, particularly iguanas. Uncommon
serovars were isolated from iguanas: three isolates
belonged to S. enterica serotype Apapa; five isolates
were serotype IV 44:z4z23:- and three isolates were II
50:b:z6. Serovars Senftenberg, II 48:d:z6, IIIb 50:k:z,
IIIb 50:z:z52, and IIIb 50:r:e,n,x,z15 were each detected
in one isolate.
The biggest number of isolates was obtained from the
chelonian group. From 33 aquatic turtles (Chrysemys
scripta) examined, 10 serovars, nine isolates belonging
to Berta and one to Duval, were identified. Two isolates
belonging to Trimdon, one to Kottobus and one to
Montevideo were isolated from four of eight terrestrial
turtles (Testudo hermanni, Sternotherus odoratus and
Sternotherus carinatum).
In previous bacteriological investigations, which were
carried out in Tuscany (central Italy), 106 (79%) S. ent-
erica was isolated from 135 cloaca samples examined
(Pasmans et al., 2000). The serovars found during this
survey were different from those isolated in the present
study.
Fifteen serotypes belonging to Salmonella subsp. IIIa,
IIIb, and IV, which are traditionally observed in cold-
blooded vertebrates, were isolated from five snakes (four
Python regius and one Boa constrictor), eight iguanas
(Iguana iguana) and two geckos (Hemitheconyx
caudicinctus).
Our results and those obtained by other authors, con-
firm that reptiles housed as pet animals, particularly ter-
restrial and aquatic turtles, which are the most
commonly present in domestic houses, may be a serious
source of infection for humans. For these reasons, it is
important that veterinarians inform new reptile owners
about the importance of hygiene and dangers of infec-
tion in these animals. Owners need to be award about
handling reptiles to avoid injuries by venomous or
aggressive species and to protect themselves from zoo-
notic infections. Good care of household reptiles is
important for animal health and to reduce stress, which
may cause the excretion of pathogenic organisms such
as salmonellae (Chiodini and Sundberg, 1981).
It is also important that, other household pets, such
as dogs and cats, are not in-contact with reptiles, their
cages, water pool, faeces and feed to reduce spread of
transmission of salmonellae and other pathogens.
Children, the elderly, pregnant women and patients
on immunosoppressive therapy are at high risk of infec-
tion, and all reptile owners should follow some simple
rules to prevent salmonellosis and other bacterial zoo-
noses. Particularly, they should wash the hands after
handling reptiles, cages and accessories, frequently
 V.V. Ebani et al. / Research in Veterinary Science 78 (2005) 117–121
disinfect cages and accessories, keep reptiles away from
food preparation and eating areas.
A relevant number of S. enterica isolated in the
present study belongs to exotic serotypes, rarely
encountered in humans. Salmonella Agona, Anatum,
Berta, Midway, Montevideo, Muenchen, Reading,
Senftemberg and Veneziana are serotypes which had
been often isolated in Italy, as reported by on-line
data of Italian Superior Health Institute. All Salmo-
nella serovars should be considered potential human
pathogens.
Human salmonellosis associated with exotic pets usu-
ally results in diarrhea, abdominal cramp, vomiting and
fever. However, the infection can spread to the blood-
stream, bone marrow and nervous system, leading to se-
vere illness (Aleksic et al., 1996; Woodward et al., 1997;
Austin and Wilkins, 1998; Nowinski and Albert, 2000;
Jafari et al., 2002).
In the present study, most isolates were sensitive to
enrofloxacin, which is the antibiotic of choice against
salmonellae and other bacterial infections (Mader,
1998; Mitchell et al., 2001). Only three isolates belonging
to S. Berta were resistant to enrofloxacin, with six other
Berta classified as intermediate.
All but one isolates were sensitive to chloramphenicol
in vitro, but this antimicrobial agent is not recom-
mended for in vivo use, because it can cause bone
marrow suppression (Aiello, 1998).
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