Hindawi

BioMed Research International

Volume 2019, Article ID 9530732, 9 pages
https://doi.org/10.1155/2019/9530732

Research Article
Identification and Characterization of Escherichia coli,
Salmonella Spp., Clostridium perfringens, and C. difficile
Isolates from Reptiles in Brazil

Carolina Pantuzza Ramos,1 Jordana Almeida Santana,1

Fernanda Morcatti Coura,1,2 Rafael Gariglio Clark Xavier,1 Carlos Augusto Gomes Leal,1
Carlos Augusto Oliveira Junior,1 Marcos Bryan Heinemann ,3 Andrey Pereira Lage ,1
Francisco Carlos Faria Lobato,1 and Rodrigo Otávio Silveira Silva 1
1
Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais,
31270-901 Belo Horizonte, Brazil
2
Departamento de Ciências Agrárias, Instituto Federal de Minas Gerais, 38900-000 Bambuı́, Brazil
3
Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia,
Universidade de São Paulo, 05508-270 São Paulo, Brazil

Correspondence should be addressed to Rodrigo Otávio Silveira Silva; rodrigo.otaviosilva@gmail.com

Received 7 February 2019; Revised 9 April 2019; Accepted 2 May 2019; Published 27 May 2019

Academic Editor: Gabriele Gentile

Copyright © 2019 Carolina Pantuzza Ramos et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Considering the increasing popularity of reptiles as pets and their possible role as reservoirs of pathogenic microorganisms, the aim
of this study was to isolate Escherichia coli, Salmonella spp., Clostridium perfringens, and C. difficile strains from reptiles in Brazil
and to characterize the isolated strains. The characterization was based on phylogenetic typing of E. coli, identification of virulence
genes of E. coli, C. perfringens, and C. difficile, serotyping of Salmonella spp., ribotyping and MLST of C. difficile and antimicrobial
susceptibility test of pathogenic strains. Cloacal swabs were collected from 76 reptiles, of which 15 were lizards, 16 chelonians,
and 45 snakes, either living in captivity, in the wild, or as companion animals. E. coli was isolated from 52 (68.4%) reptiles, of
which 46 (88.4%) were characterized as phylogroup B1. The virulence factor CNF1 of E. coli was found in seven (9.2%) sampled
animals, whereas the gene of EAST1 was found in isolates from two (2.6%) reptiles. Three isolates positive for CNF1 were resistant
to cephalothin, one of which was also resistant to ciprofloxacin, trimethoprim/sulfamethoxazole, and chloramphenicol, being then
classified as multidrug resistant strain (MDR). Salmonella enterica was identified in 26 (34.2%) reptiles, of which 13 belonged to
the subspecies enterica. Serotypes such as S. Mbandaka, S. Panama, S. Infantis, S. Heidelberg, and S. Anatum were identified. One
isolate of S. enterica subsp. houtenae was resistant to cephalothin and ciprofloxacin. C. perfringens type A was isolated from six (7.8%)
animals. C. difficile was isolated from three (3.9%) reptiles. Two of these isolates were toxigenic and classified into ribotypes/MLST
081/ST9 and 106/ST42, which have been previously reported to infect humans. In conclusion, reptiles in Brazil can harbor toxigenic
C. difficile and potentially pathogenic E. coli and Salmonella enterica subsp. enterica, thus representing a risk to human and animal
health.

1. Introduction homes have reptiles as pets [1, 2]. This same trend can be
Several countries have shown an increase in reptiles as also seen in European countries, with a growing population
companion animals over the years. From 2001 to 2016, the of pet reptiles [3, 4]. Based on data from the latest census of
number of households in the United States (US) with reptiles, companion animal population in Brazil, it was ranked as the
such as turtles, snakes, and lizards, as pets increased from country with the 9th largest number of domesticated reptiles,
1.7 to approximately 4.7 million [1]. Today, almost 4% of US with approximately 2.2 million animals [5].
2 BioMed Research International

The close contact between reptiles and humans is a public Mendes de Conservação da Biodiversidade (ICMBio) under
health concern since these animals have been character- the protocol 49195-1.
ized as carriers of zoonotic agents, mainly Salmonella spp.,
which is associated with human salmonellosis [2]. In fact, 2.2. Escherichia coli. For E. coli isolation, samples were plated
reptiles are responsible for approximately 6% of sporadic onto MacConkey agar (Difco, USA) and incubated for 24 h
cases of human salmonellosis [6]. In addition to Salmonella at 37∘ C and characteristic lactose-fermenting colonies were
spp., other zoonotic bacteria of the genera Mycobacterium, identified using the EPM-MILI-Simmons Citrate Enterobac-
Chlamydia, and Leptospira have been associated with reptiles teriaceae identification test [15]. To identify the phylogenetic
[7]. Some reports have also shown Escherichia coli carriage groups of E. coli (A, B1, B2, C, D, E, or F), a quadruplex
by reptiles and one study described the phylogenetic group PCR commonly used to characterize E. coli was performed
B1 as more common in these animals [8, 9]. Curiously, [16]. Characterization of pathovars such as enterotoxigenic E.
studies have shown B1 phylogroup commonly associated with coli (ETEC), Enteropathogenic E. coli (EPEC), Enterohemor-
diarrheogenic pathovar, although there is no study focusing rhagic E. coli (EHEC), Shiga-toxin producing E. coli (STEC),
on detection of pathogenic E. coli in reptiles’ isolates [10]. C. Necrotoxigenic E. coli (NTEC), Enteroinvasive E. coli (EIEC),
difficile is an emerging pathogen responsible for the majority Diffusely Adherent E. coli (DAEC), and Enteroaggregative E.
of nosocomial diarrhea cases in humans [11]. Interestingly, a coli (EAEC) was also performed according to the presence
strain of C. difficile was recently isolated from a lizard (Pogona of specific virulence genes. Genes encoding virulence factors
vitticeps), being the first report of this agent in reptiles, associated with each E. coli pathovars was identified by
although no toxigenic potential was found in this isolate [12]. PCR. The reference strains used were EDL933 (eae+ , stx1+ ,
Also, enterotoxigenic C. perfringens, responsible for human stx2+ , hlyA+ , iha+ , toxB+ , efa1+ ), EAEC O42 (ast1+ , aggR+ ,
disease, was already found in a tortoise with diarrhea, but aaf + , pet + ), B41 (f41+ , f5+ , sta+ ), S5 (f17 + , cnf2+ ), NTEC-1
potential risk of reptiles is cloudy by the absence of studies (cnf1), STECLBA05 (saa+ ), E2348/69 (bfpA+ ), PA58 (aidaI +),
with C. perfringens strains isolated from these animals [13]. EIEC (ipaH +), 2568 (stb+ , f18+ , stx2e+ ), 2569 (lt + , k88+ ),
Despite the increasing popularity of reptiles as pets, in 2570 (987p+ ), ECSTh (sth+ ), 4833 (cs2+ , cs3+ ), PB176 (cs1+ ),
Brazil, little is known about their carriage of potentially E17018A (cs5+ , cs6+ ), H10407 (cfa1+ ), E8775 (cs4+ ) and 29
pathogenic microorganisms, including the main subspecies (cs12+ ). The primers used to detect virulence genes associated
and serotypes of Salmonella spp. Thus far, there have been with diarrheagenic E. coli were described in Table 1S of
few studies focusing on detecting important human and Supplementary Material.
animal enteropathogens in reptiles. Therefore, the present
study aimed to realize an investigation into the carriage of E. 2.3. Salmonella Spp. For isolation of Salmonella spp., samples
coli, Salmonella spp., C. perfringens, and C. difficile in fecal were plated onto Hektoen enteric agar (Oxoid, USA) and
material of reptiles living in different habitats in Brazil and to XLT4 agar (Prodimol Biotechnology, Brazil) and incubated at
characterize the isolates. 37∘ C for 18 - 24 h. Additionally, samples were also preenriched
in tetrathionate broth (Oxoid, USA) at 37∘ C for 24 h prior
2. Material and Methods to plating on Hektoen enteric agar (Difco, USA). Sulfite-
reducing colonies of Salmonella spp. were identified by genus-
2.1. Samples. Fecal material was obtained from swabs of specific PCR using the reference strain ATCC 14028 [17].
cloacae from reptiles between July 2016 and September 2017. Strains confirmed as Salmonella spp. were differentiated into
A convenience sampling of 76 apparently healthy reptiles species and subspecies [18] and afterwards serotyped by anti-
after clinical examination was performed, consisting of sam- genic characterization based on the White-Kauffmann-Le
ples obtained from 60 scaled reptiles (order Squamata), Minor scheme at the Brazilian National Reference Laboratory
comprising 15 lizards (suborder Sauria) and 45 snakes (sub- of Enterobacteria of Oswaldo Cruz Foundation (FIOCRUZ),
order Serpentes), and 16 chelonians (order Chelonii)—ten Brazilian Ministry of Health, being serotyping the most com-
from the suborder Pleurodira and six from the suborder mon method to differentiate strains of Salmonella [19]. The
Cryptodira. The reptiles were selected from the following antigenic characterization was performed by slide agglutina-
habitats: private owners volunteers (n = 23), free-living from tion with somatic (O), flagellar (H), and occasionally capsular
metropolitan area of Belo Horizonte captured for monitoring (Vi) poly- and monovalent antisera. The identification of
(n = 37), and captivity (n = 16) randomly selected from specific serovars was performed and represented according
the Wildlife Screening Center (CETAS) and Ezequiel Dias to the criteria reported by Grimont and Weill [20]. After
Foundation (FUNED). For sampling procedures, a sterile identification, the antibiotic resistance patterns of pathogenic
swab (BactiSwab; Remel, Lenexa, KA, USA) was introduced E. coli and Salmonella subspecies or serotypes were evaluated
5–6 cm into the cloaca and rotated five times, as described by the disc diffusion method according to the Clinical and
by Ives et al. [14]. The swab was vigorously agitated in 500 𝜇L Laboratory Standards Institute (CLSI) manual VET01-A4
of phosphate-buffered saline (PBS), stored in a transport box [21]. Briefly, four to five isolated bacterial colonies were
with ice packs and transported to the Bacterial and Research incubated in Mueller-Hinton broth (Difco, USA) at 35∘ C until
Laboratory of Veterinary School of Federal University of obtaining turbidity comparable to that of the 0.5 McFarland
Minas Gerais for immediate processing. The study was standard. The suspension was inoculated over the surface
approved by the Ethical Committee on Animal Use of UFMG of a Mueller-Hinton agar plate, followed by the application
(CEUA) under the protocol 238/2015 and by Instituto Chico of the drug-impregnated disks. The plates were incubated
BioMed Research International 3

at 35∘ C ± 2∘ C for 16 to 18 hours and the size of inhibition on sterile 0.9% saline to McFarland standard 1 from a pure
zones was interpreted as recommended by the VET01-A4 culture after 24 hours’ growth in Brucella agar (Oxoid, USA).
[21]. The antibiotic disks used were ciprofloxacin (5 𝜇g), The test was performed on Brucella agar with 5% lysed
trimethoprim/sulfamethoxazole (25 𝜇g), chloramphenicol blood supplemented with hemin (Difco, USA) and vitamin K
(30 𝜇g), ceftriaxone (30 𝜇g), and cephalothin (30 𝜇g) (DME, (Sigma, USA). The plates were incubated at 37∘ C in anaerobic
Brazil), and the control used for antimicrobial susceptibility atmosphere and the MIC end point were measured and
was E. coli ATCC 25922. interpreted according to cut-off values from M100-S25 [28]
and EUCAST guidelines [29] after 48 h of incubation.
2.4. Clostridium perfringens. The isolation of C. perfringens
was performed according to Silva et al. [22]. Briefly, each 2.6. Statistical Analysis. Associations between the categorical
sample was inoculated into 10 mL of brain heart infusion variables (order or suborder of reptile, diet, and habitat
broth (Difco, USA) for enrichment. After incubation at 37∘ C of reptile) and the frequency of microorganisms identified
for 24 h in an anaerobic atmosphere, 10 𝜇L of the culture were studied using univariate analysis with the Chi-square
was plated onto SPS agar (Difco, USA) and anaerobically test or Fisher’s exact test within Stata 12 software (Stat-
incubated at 35 ± 2∘ C for 24 - 48 h. After isolation, at aCorp, USA), with a p value of ≤0.05 being considered
least three rounded sulfite-reducing colonies were subjected significant.
to PCR for the detection of genes encoding C. perfringens
major toxins alpha (cpa), beta (cpb), epsilon (etx), iota (ia), 3. Results
additionally to beta-2 toxin (cpb-2), and enterotoxin (cpe),
associated with human and animals’ disease [23]. Also, PCR E. coli was isolated from 52 (68.4%) out of 76 sampled
protocols for detection of additional virulence factors, such animals, of which 38 (50%) isolates were from snakes, 7
as NetB- and NetE-, NetF- and NetG-encoding genes were (9.2%) were from lizards, 4 (5.2%) from the suborder Pleu-
used, as described in Table 1S of Supplementary Material. rodira, and 3 (3.9%) from the Cryptodira. When compared
The following C. perfringens strains were used as controls: to lizards (p = 0.003), suborder Pleurodira (p = 0.002) and
BAA1481 (cpa+ , ia+ ), ATCC 3626 (cpa+ , cpb+ , etx+ , cpb2+ , and Cryptodira (p = 0.04), the frequency of isolation of E. coli
pfoA+ ), D7 (cpe+ , netE+ , netF + , netG+ ), and CP149 (netB+ ). was higher in snakes (Table 1). Of the free-living and pet
reptiles, 62.1% and 65.2% were positive for E. coli, respectively.
2.5. Clostridium difficile. A previously described protocol The frequency of isolation in captivity reptiles was 87.5%, and
was used for isolation of C. difficile on cycloserine-cefoxitin no statistic difference was found among the positivity of E.
fructose agar supplemented with 7% horse blood and 0.1% coli according to the reptile habitats (Table 2). The isolation
sodium taurocholate (Sigma, USA) [24]. After anaerobic frequency of E. coli in carnivores was significantly higher than
incubation for 72 h at 37∘ C, all colonies with characteristic for omnivorous and herbivorous reptiles (p = 0.01) (Table 2).
C. difficile morphology (flat, irregular, and with ground-glass Considering phylogenetic groups, almost 89% (46/52) of the
appearance) were subjected to multiplex-PCR for detection E. coli strains belonged to the B1 group (Table 1). It was
of housekeeping gene (tpi), toxin A gene (tcdA), toxin B found a greater propensity of isolation of B1 E. coli from
gene (tcdB), and binary toxin gene (cdtB) (Table 1S of snakes and lizards (p = 0.00004) while the groups A and B2
Supplementary Material). The reference strain C. difficile were more frequent in chelonians (p = 0.0001). Also, there
ATCC 9689 were used as control. C. difficile strains were is a positive association between phylogroup B1 and E. coli
submitted to PCR ribotyping, a molecular tool largely used strains isolated from carnivorous reptiles (p = 0.0001). Of
for C. difficile typing since 1990s [25]. Intergenic spacer the sampled reptiles, approximately 9.2% were positive for
regions were amplified using Bidet primers, as previously the virulence gene cnf1 of NTEC pathovar, corresponding to
described [26]. Amplification products were separated by seven positive strains (13.4%) from B1, B2, and F groups. The
electrophoresis in a 3% agarose gel (Bio-Rad, USA) for 5 h virulence factor gene encoding EAST1 (enteroaggregative E.
at 2.5 V/cm, and the gel was photo-documented and then coli heat-stable enterotoxin 1) was identified in two (3.8%)
analyzed using BioNumerics 7.6 (Applied Maths, Belgium). strains, classified as F and B1, respectively (Table 3). Three
The PCR ribotypes were designated by the international positive strains for CNF1 were resistant to cephalothin, one
Cardiff nomenclature. Multilocus sequence typing (MLST) shows intermediate resistance to cephalothin and the last
was conducted as previously described by Griffiths et al. [27], one strain, isolated from a wildlife testudine, was resistant to
using a typing scheme with discriminatory power similar cephalothin, ciprofloxacin, trimethoprim/sulfamethoxazole,
to PCR ribotyping [25], being the following housekeeping and chloramphenicol. On the other hand, the EAST1 strains
genes: adk, atpA, dxr, glyA, recA, sodA, and tpi. Ampli- were sensitive to all antimicrobial agents tested. Two E. coli
con sequences were compared with the MLST database cnf1+ strains were isolated from a lizard and a snake that were
(https://pubmlst.org/cdifficile/) to identify the allelic profiles also positive for S. Johannesburg and S. Ndolo, respectively.
and the corresponding Sts. The minimal inhibitory concen- Additionally, one toxigenic C. difficile strain was isolated from
tration (MIC) for metronidazole, vancomycin, clindamycin, a pet testudine that carried E. coli cnf1+ with intermediate
and sulfamethoxazole/trimethoprim, commonly associated resistance to cephalothin. One reptile was simultaneously
with CDI was performed by gradient test with the M.I.C. positive for E. coli, S. enterica houtenae, and nontoxigenic C.
Evaluator (M.I.C.E.) strips (Oxoid, USA). Briefly, a sus- difficile, while other two were positive for S. enterica subsp
pension of the toxigenic strains of C. difficile was prepared enterica, E. coli, and C. perfringens type A.
4 BioMed Research International

Table 1: Frequency of isolation and phylogenetic groups of E. coli from reptiles in Brazil.

No. E. coli groups∗ (%)

Host group No. (%) E. coli
A B1 B2 D F
Serpentes (n=45) 38/45 (84.4) 0 38/38 (100) 0 0 0
Sauria (n=15) 7/15 (46.6) 1/7 (14.2) 5/7 (71.4) 0 0 1/7 (14.2)
Cryptodira (n=6) 3/6 (50) 1/3 (33.3) 2/3 (66.6) 0 0 0
Pleurodira (n=10) 4/10 (40) 0 1/4 (25) 2/4 (50) 1/4 (25) 0
Total 52/76 (68.4) 2/52 (3.8) 46/52 (88.4) 2/52 (3.8) 1/52 (1.9) 1/52 (1.9)
∗ None E. coli strains belonged to phylogenetic groups C and E.

Table 2: Frequency of isolation of E. coli and Salmonella spp. from reptiles based on dietary habits and animal habitats.

Dietary habit No. Samples No. E. coli-positive (%) No. Salmonella-positive (%)
Carnivore 52 40/52 (76.9) 22/52 (42.3)
Herbivore 5 4/5 (80) 3/5 (60)
Omnivore 19 8/19 (42.1) 1/19 (5.2)
Animal habitat No. Samples No. E. coli-positive(%) No. Salmonella-positive (%)
Pet 23 15/23 (65.2) 7/23 (30.4)
Captivity 16 14/16 (87.5) 8/16 (50)
Wild 37 23/37 (62.1) 11/37 (29.7)

Table 3: Frequency and characteristics of E. coli, Salmonella spp., C. difficile and C. perfringens isolated from reptiles in Brazil.

E. coli (%) Salmonella spp. (%) C. difficile (%) C. perfringens Type A

Host group
Isolates EAST1 CNF1 Isolates A+B+
Sauria (n=15) 7 (46.6) 2 (28.5) 3 (42.8) 7 (46.6) 0 0 2 (13.3)
Serpentes (n=45) 38 (84.4) 0 1 (2.6) 18 (40) 2 (4.4) 1 (50) 3 (6.6)
Pleurodira (n=10) 4 (40) 0 3 (75) 1 (10) 1 (10) 1 (100) 1 (10)
Cryptodira (n=6) 3 (50) 0 0 0 0 0 0
Total (n=76) 52 (68.4) 2 (3.8) 7 (13.4) 26 (34.2) 3 (3.9) 2 (66.6) 6 (7.8)

Salmonella spp. was isolated from 26 (34.2%) reptiles, was identified in each of these strains, being classified as C.
with higher rates of carriage in lizards and snakes than perfringens type A. Not additional virulence factors tested
chelonians (p = 0.007) (Table 4), and in carnivores (p = were identified.
0.003) and herbivores (p = 0.003) than omnivores (Table 2). C. difficile was isolated from three (3.9%) reptiles, being
Of the captivity reptiles 50% were positive for Salmonella two toxigenic strains (A+B+CDT-) and one nontoxigenic (A-
spp., while the frequency of isolation in companion animals B-CDT-) (Table 3). One of the toxigenic strains, identified
and free-living reptiles were respectively 30.4% and 29.7% as ribotype (RT) 081 and strain type (ST) 09, was recov-
(Table 2) and no statistic difference was found between the ered from a captive snake (Bothrops alternatus). The other
different habitats. All 26 Salmonella isolates were classified toxigenic strain was isolated from a domesticated chelonian
as S. enterica with 13 strains belonging to the subspecies from the suborder Pleurodira (Phrynops geoffroanus) and
enterica (Table 4). The other S. enterica isolates comprised the classified as RT106 and ST042. The two toxigenic strains
subspecies houtenae, arizonae, and diarizonae. Ten different were susceptible to metronidazole, vancomycin and sul-
serovars of S. enterica subspecies were identified in the famethoxazole/trimethoprim, but resistant to clindamycin.
present study, including the important zoonotic serovars The nontoxigenic C. difficile strain was isolated from a pet
S. Infantis, S. Mbandaka, S. Heidelberg, and S. Panama corn snake (Pantherophis guttatus) and was classified as
(Table 5). All but one of the Salmonella isolates were sensitive RT009 and ST457.
to the four classes of antimicrobial agents tested. The excep-
tion was a strain of S. enterica subsp. houtenae, obtained from 4. Discussion
a domesticated lizard (Iguana iguana), which was resistant to
cephalothin and ciprofloxacin. This domiciled lizard was also Being a common inhabitant of the intestinal tract of warm-
positive for EAST1 E. coli. blooded vertebrates, E. coli can also be isolated in cold-
C. perfringens was isolated from six animals (7.8%) and blooded animals, such as reptiles, which frequency is highly
no difference in isolation between captivity, free-living, or pet dependent on their diet and contact with other animals [8].
reptiles was identified (Table 3). The alpha toxin gene (cpa) The frequency of isolation of E. coli found in the present
BioMed Research International 5

Table 4: Frequency of isolation of Salmonella enterica subspecies from reptiles in Brazil.

No (%) Salmonella enterica subspecies

Host group No. samples No (%) Salmonella
enterica houtenae arizonae diarizonae
Sauria 15 7/15 (46.6) 3/7 (42.8) 4/7 (57.1) 0 0
Serpentes 45 18/45 (40) 10/18 (55.5) 4/18 (22.2) 2/18 (11.1) 2/18 (11.1)
Pleurodira 10 1/10 (10) 0 1/1 (100) 0 0
Cryptodira 6 0 0 0 0 0
Total 76 26/76 (34.2) 13/26 (50) 9/26 (34.6) 2/26 (7.6) 2/26 (7.6)

Table 5: Salmonella enterica serotypes isolated from reptiles.

Serotype No. of isolates Host Origin

Anatum 1 Serpentes Captivity
Heidelberg 1 Serpentes Captivity
Infantis 1 Serpentes Captivity
Johannesburg 1 Sauria Wild
Mbandaka 2 Serpentes and Sauria Pet and Captivity
Ndolo 3 Serpentes Captivity
Panama 1 Serpentes Wild
16: - : - 1 Serpentes Wild
6,7: - : - 1 Sauria Pet
Rough: - : - 1 Serpentes Wild

study was higher than reported in reptiles by previous studies complexity of their gastrointestinal tract [8, 30]. Although the
[8, 9] although these reports have stated different method- present study analyzed isolates from carnivorous reptiles, the
ologies and objectives. The higher frequency in snakes when results suggested that, as demonstrated for mammals, the diet
compared to other reptiles might be due to the diet of these may also influence in the phylogenetic distribution of E. coli
animals, since all snakes are carnivores. Interestingly, these isolated from these animals.
results contrast previous studies that indicate that E. coli is Pathogenic strains of E. coli have been reported as the
more likely to be isolated from omnivorous mammals [8]; causative agent of intestinal and extra intestinal diseases in
however, the present results could be influenced by the low humans and animals, although there have been no studies
number of omnivores in the sample population. focusing on the detection of pathogenic E. coli in reptiles.
Each of the E. coli isolates from reptiles was classified Therefore, to the best of our knowledge, this is the first
into one of the seven phylogroups (A, B1, B2, C, D, E, and report of the CNF1 and EAST1 factors in reptilian E. coli
F) according to the quadruplex PCR developed by Clermont strains. E. coli cnf1+ are responsible for diarrhea and extra
et al. [16]. The high frequency of E. coli isolates belonging to intestinal diseases such as cystitis and meningitis in humans
group B1 was similar to the only previous study evaluating and domestic animals [31–33], while EAST1 positive strains
phylogroups of E. coli isolates from reptiles [8]. This finding have been associated with several outbreaks of diarrhea in
also corroborated previous reports that show that most E. humans [34]. Thus, the present study suggests that reptiles
coli from animals belong to group B1, whereas, in humans, positive for EAST1 and CNF1 strains may represent a risk for
groups A and B2 are predominant [10]. Previous studies have human and animals health. As described, five cnf1+ strains
already suggested that phylogenetic groups are associated were not susceptible to cephalothin, an antimicrobial agent
with different hosts [30], which could justify the greater that could be used for treatment of urinary tract infections
propensity for isolation of B1 E. coli from snakes and lizards, (UTI) in humans [35, 36]. Considering their resistance to
already suggested by Gordon and Cowling [8], as well as the cephalothin, ciprofloxacin, trimethoprim/sulfamethoxazole,
higher frequency of groups A and B2, commonly described and chloramphenicol, the cnf1+ strain isolated from a tes-
in humans, from chelonians. Curiously, all samples that tudine was classified as multidrug resistant (MDR) [37].
were identified as A or B2 strains were isolated from pet The occurrence of MDR strains is of high public health
reptiles, raising the question of whether close contact of concern, since it could contribute to therapeutic failure and
reptiles and humans may have resulted in colonization of increased patient morbidity and mortality [38]. Curiously,
these animals with phylogroups commonly associated with ciprofloxacin is a fluoroquinolone recommended for compli-
humans. cated infections and urinary tract infections (UTI) caused by
In mammals, E. coli genotype distribution seems to MDR Gram-negative bacteria, such as E. coli [36]. Addition-
depend on several factors, including the climate, host diet, ally, chloramphenicol and trimethoprim/sulfamethoxazole
and host body mass. In addition, phylogroup B1 is more are also common choices for treatment of UTI and diarrhea
common in carnivorous mammals, probably due to the lower associated with E. coli in humans and animals [36, 39, 40].
6 BioMed Research International

Salmonella enterica has been described as an animal and [43, 48, 51, 53]. In fact, half of the serotypes identified here
human enteropathogen. There are six known subspecies of have been reported to infect humans (Table 5). Some of them
S. enterica (enterica, salamae, arizonae, diarizonae, houtenae, (including S. Mbandaka, S. Panama, and S. Infantis) are listed
and indica) and more than 2500 serovars that have been as the most common isolates associated with human infection
associated with different types of infection [20]. Previous in Brazil [55, 56], and in the United States and European
reports in other countries, including a single study in Brazil, countries, including S. Heidelberg, S. Panama, and S. Infantis
have showed that Salmonella shedding by reptiles is frequent, [57, 58]. It should also be highlighted that almost 70% of
suggesting that they are natural hosts that eventually become the serotypes identified in the present study, including S.
ill [41–43]. It is important to highlight that only one cloacal Heidelberg, S. Infantis, and S. Mbandaka, were isolated from
swab was collected from each animal in the present work. pet reptiles that were kept in close contact with humans.
Since it is known that Salmonella shedding by reptiles is Also, this study seems to be the first to report isolation of S.
intermittent [44], the number of asymptomatic colonized Johannesburg and S. Ndolo from reptiles, serotypes already
reptiles might be much higher than the 34% reported here described to infect humans in Brazil [56, 59].
thus may represent some risk to the carrier as well as to people Resistance to cephalothin and ciprofloxacin, found in
in close contact with these animals [2]. one strain of S. enterica subsp. houtenae, may make it
Previous reports have shown a highest shedding of difficult to treat salmonellosis in humans [28]. As described,
Salmonella spp. by carnivores’ reptiles, a result also found ciprofloxacin is a fluoroquinolone critically important in
in the present study when compared to omnivorous reptiles human medicine and largely used to treat serious infections.
[45]. This result was also found for E. coli shedding, which Additionally, Salmonella fluoroquinolone-resistant is one of
may have influenced the higher rate of cocolonization of these 12 bacterial agents for which new antibiotics are urgently
animals in contrast with omnivore and herbivore reptiles, needed [60]. It is important to note that this resistant strain
most of them from Chelonii order. Curiously, previous of Salmonella was isolated from a pet reptile also positive for
reports have shown that shedding of Salmonella spp. is greater EAST1 E. coli, thus reinforcing the possible risk of these ani-
in carnivore reptiles fed by contaminated reptile feeder mals for human health. Altogether, our results demonstrate
mice [46]. In fact, feeder rodents are potential carriers of the importance of reptiles as reservoirs of Salmonella spp. and
Salmonella spp. [47]. On the other hand, the higher carriage E. coli and highlight the need to study these agents in view of
rate of Salmonella spp. by herbivores compared to omnivores One Health.
may not represent what actually occurs in nature, considering C. perfringens is a widespread gram-positive anaerobic
the small sampling of herbivores reptiles in the present study bacillus, commonly found as part of the microbiota of
(five samples, 6.5% of the total), of which three were positive animals and humans [61]. However, there is little information
for Salmonella spp. regarding the occurrence of C. perfringens in reptiles. Most
The higher Salmonella carriage rate among lizards and studies are restricted to its isolation from the oral microbiota
snakes compared with chelonians is consistent with findings and venom of some snake species [62, 63] or on the effect
of previous studies [41]. Chelonians have been recognized as of diet on the occurrence of clostridia species [64]. In
sources of human salmonellosis since the mid-1960s, mainly the present study, the frequency of reptiles positive for C.
due to their popularity as pets [48]. On the other hand, special perfringens was much lower than previously reported for
attention has been recently given to lizards and snakes, since other animal species, which is commonly above 75% [65, 66].
the domestication of these reptiles has considerably increased These results suggest that C. perfringens is less frequently
[49], even in spite of the indication by several studies that isolated from the microbiota of reptiles than from that of most
these animals seem to have a high rate of Salmonella shedding warm-blooded animals.
[4, 42, 50]. This concern seems legitimate, since a recent C. perfringens may also be classified into five types (A to
report has shown that lizards became an important source E) according to the production of four major toxins: alpha,
of Salmonella spp. in human reptile-associated salmonellosis beta, epsilon, and iota [23]. In addition to the major toxins,
[51]. the bacterium produces additional virulence factors that are
The subspecies houtenae, arizonae, and diarizonae, which associated with the pathogenesis of some diseases in humans
corresponded to 50% of the isolates of S. enterica identified and animals, such as enterotoxin, beta-2 toxin, NetB, NetE,
in the present study, are common in cold-blooded animals NetF, and NetG [67, 68]. Interestingly, no additional virulence
[4] and are occasionally associated with human salmonellosis factors tested were identified, including the enterotoxin-
[52, 53]. On the other hand, Salmonella enterica subsp. encoding gene (cpe), which is commonly associated with
enterica is the most common subspecies associated with disease in humans [61] and is already suggested as a cause
human disease [45]. Notably, half of the Salmonella strains of diarrhea in red-footed tortoise (Geochelone carbonaria)
isolated from reptiles in the present study belonged to the [13]. Thus, considering the small frequency of isolation of C.
subspecies enterica, a proportion higher than described in perfringens and the absence of additional virulence factors in
other studies [43, 54]. This difference might be due to several these isolates beyond the alpha toxin encoding gene, present
factors, including the geographical origin of the animals, in all C. perfringens strains, the present work suggests that the
sampling, and living conditions (captive, as pets, or free- fecal shedding of this agent by reptiles may not represent a
living) that these reptiles were subjected to (Table 2) [41, 45]. public health concern.
Several serotypes of Salmonella are recognized as etio- C. difficile is an anaerobic gram-positive bacterium con-
logical agents of reptile-associated salmonellosis in humans sidered an emerging pathogen, being responsible for the
BioMed Research International 7

majority of nosocomial diarrhea cases in humans [11]. In Acknowledgments

animals, C. difficile infection (CDI) is common in piglets and
horses, but also occurs in several other species, including We thank CETAS-BH and FUNED/MG for the support and
some wild animals [12, 69]. There are few studies on C. difficile animal samples, IBAMA for the permission to work with wild
shedding by wild animals and, to the best of our knowledge, species, and Danielle F. M. Soares and Rômulo A. R. Toledo
this is the first report on toxigenic C. difficile isolated from for their invaluable help with wild animal sample collection.
reptilian species. Some other free-living or captive species Acknowledgments also are due to Maja Rupnik and Dr.
are reported as possible reservoirs of C. difficile strains, but Dominique S. Blanc, for contribution to PCR ribotyping
the role of these animals in the epidemiology of CDI in and MLST performed. This work was supported by the
humans and domestic species, in addition to the risk for Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Supe-
reptiles’ health, is still poorly understood [12, 65, 70, 71]. It rior (CAPES), the Conselho Nacional de Desenvolvimento
is important to note that none of the positive animals were Cientı́fico (CNPq), the Fundação de Amparo à Pesquisa do
undergoing antibiotic therapy which is known to increasing Estado de Minas Gerais (FAPEMIG), and the Pró-Reitoria de
the shedding of C. difficile in humans and animals [11, 69], Pesquisa da UFMG (PRPq/UFMG).
including wild species [65].
The nontoxigenic C. difficile strain found in the present Supplementary Materials
study, classified as RT009, was described with a high capacity
to colonize different host species, being one of the most com- The primers used to detect genes encoding virulence factors
mon ribotypes isolated from humans and domestic animals associated with diarrheagenic E. coli in humans and animals
[72]. Interestingly, this isolate was classified as ST457, a novel and their references are described in Table S1 in the Supple-
strain type of C. difficile. The only study of C. difficile in rep- mentary Material. (Supplementary Materials)
tiles was recently published, describing another nontoxigenic
C. difficile strain isolated from a lizard (Pogona vitticeps) that References
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