Comparative Immunology, Microbiology and Infectious Diseases 104 (2024) 102094

Contents lists available at ScienceDirect

Comparative Immunology, Microbiology and

Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid

Impact of human created environments in the pathogenic potential and

antimicrobial resistance of staphylococci from wild neotropical primates
in Brazil
Indiara Sales a, b, Olney Vieira-da-Motta b, Ana Tavares c, Carlos Ramón Ruiz-Miranda d,
Hermínia de Lencastre c, e, Maria Miragaia a, *
a
Laboratory of Bacterial Evolution and Molecular Epidemiology, Instituto de Tecnologia Química e Biológica António Xavier, NOVA University (ITQB-NOVA), Oeiras,
Portugal
b
Universidade Estadual do Norte Fluminense Darcy Ribeiro-UENF, Centro de Ciências e Tecnologias Agropecuárias, Laboratório de Sanidade Animal-Setor Doenças
Infecto-contagiosas, Rio de Janeiro, Brazil
c
Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica António Xavier, NOVA University (ITQB-NOVA), Oeiras, Portugal
d
Universidade Estadual do Norte Fluminense Darcy Ribeiro-UENF, Centro de Biociências e Biotecnologia, Laboratório de Ciências Ambientais, Campos dos Goytacazes,
Rio de Janeiro, Brazil
e
Laboratory of Microbiology & Infectious Diseases, The Rockefeller University, New York, USA

A R T I C L E I N F O A B S T R A C T

Keywords: The non-human primate (NHP) Leontopithecus rosalia is an endangered species native of Brazil and lives in forest
Staphylococcus fragments with different levels of contact with humans (natural, private and urban). Other NHPs – Callithrix spp. -
Clonal types were introduced by humans and co-exist and interact with the native species in these forests.
Antimicrobial resistance
To evaluate if living in or close to human-modified environments could constitute a risk for L. rosalia, we
Dissemination
compared the prevalence, genetic background, antibiotic susceptibility and virulence gene content of staphy­
lococci collected from the native and the introduced species from different forest fragments.
We found that presence in human-dominated environments increased the colonization rate of L. rosalia with
Mammaliicoccus sciuri (former Staphylococcus sciuri) from 18 % to 85 % (p = 0.0001) and of Callithrix spp with
Staphylococcus aureus from 6 % to 100 % (p = 0.0001). According to molecular typing data obtained differences
probably resulted from dissemination of these bacterial species from the invader NHP species and from humans.
Changes in microbiota were paralleled by an increase in the prevalence of Panton-Valentine Leukocidin gene and
in resistance to beta-lactams, macrolides and/or lincosamides as exposure to human environment increased. In
particular, erythromycin resistance in S. aureus from Callithrix spp. increased from 0 % to 50 % and resistance rate
to at least one antibiotic in coagulase-negative staphylococci species from L. rosalia increased from 13 % to 56 %
(p = 0.0003).
Our results showed that contact of native animal species with human-created environments increased the
content of antimicrobial resistant and pathogenic bacteria on their commensal microbiota, which ultimately can
impact on their health.
Importance: Endangered animal species are vulnerable to environmental alterations and human activities have
been repeatedly identified as factors driving drastic changes in the natural landscape. It is extremely important to
monitor changes in the environment surrounding protected species, because this could lead to early detection of
any potential threats. In this study, we found that the contact of L. rosalia - a protected non-human primate from
Brazil - with human environments is related to changes in their commensal microbiota. These included an in­
crease in the number of pathogenic and antibiotic resistant bacteria, which have a higher potential to cause
infections that are more difficult to treat. We provided evidence for the harmful impact human contact has on

* Correspondence to: Laboratory of Bacterial Evolution and Molecular Epidemiology, Instituto de Tecnologia Química e Biológica António Xavier, NOVA University
(ITQB-NOVA), Avenida da República, Estação Agronómica Nacional, 2780-157 Oeiras, Portugal.
E-mail address: [email protected] (M. Miragaia).

https://doi.org/10.1016/j.cimid.2023.102094
Received 3 October 2023; Received in revised form 5 November 2023; Accepted 13 November 2023
Available online 17 November 2023
0147-9571/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
I. Sales et al. Comparative Immunology, Microbiology and Infectious Diseases 104 (2024) 102094

L. rosalia. Also, our results suggest that monitoring of commensal microbiota of protected animal species might
be a useful way of sensing the risks of protected species to human exposure.

1. Introduction extensive contact with humans (Water Island, Ilha d’Água, Guanabara
Bay); a rural region wherein marmosets co-exist with tamarins in forest
Antibiotic resistance development in bacteria is associated to the fragments (Private Reserves of National Heritage - RPPNs); and two
continuous use and misuse of antibiotics in humans and human derived protected forests in which tamarins do not have contact with marmosets
activities [1]. Antibiotic resistant bacteria have become wide spread in (Biologic Reserve (ReBio) Poço das Antas, Silva Jardim; ReBio União,
hospitals, production and companion animals and in human modified Rocha Leão) (see Fig. 1).
environments [2–7]. It has been hypothesized that contact with human Tamarins (L. rosalia) and marmosets (Callithrix spp.) were captured
and human modified environments and the consequent transmission of using Tomahawk model traps (18 × 18 × 60 cm) baited with bananas,
potentially pathogenic antibiotic resistant bacteria could constitute a set on platforms 1.5 m above ground. Traps with captured animals were
risk to the survival of wild animals, such as the free-ranging neotropical covered with cloth or paper in order to minimize stress. Tamarins and
non-human primate (NHP) Leontopithecus rosalia (golden lion tamarins). marmosets from the forest areas were transported by car to the field
Staphylococcus aureus, Mammaliicoccus sciuri (former Staphylococcus laboratory at the Mico-leão dourado (L. rosalia) Association (AMLD).
sciuri) and other coagulase-negative staphylococci (CoNS) [8,9] are Marmosets from Water Island were taken to the SERCAS, a captive fa­
common colonizers of human and non-human primates (NHP) and have cility at the Universidade Estadual do Norte Fluminense, where they
been recognized as important reservoirs of antibiotic resistance genes. remained captive. The animals were anesthetized with ketamine hy­
Moreover, some staphylococcal species, like S. aureus have the ability to drochloride (10 mg kg-1) to allow for a clinical examination and the
cause serious infections in NHP, namely septicemia, abscess, stomatitis, collection of oral and rectal swabs (Cary-Blair, Plast-Labor, Brazil). After
arthritis, myocarditis, meningoencephalitis, aerosaculitis, and pneu­ recovering from anesthesia, animals were released in the same location
monia [10–14]. Thus, colonization by antibiotic resistant S. aureus they were captured. Although NHP can be colonized in the nose with
might constitute an increased risk to wild NHP as treatment of infections staphylococci, nasal swabs were not collected. This was due to the fact
would be more difficult. that besides collecting staphylococci, the project aimed to screen also for
Several lines of evidence suggest that transmission of antibiotic other bacteria inhabiting in mouth and rectum/gut. Additionally,
resistant bacteria occurs between human and NHP. Examples of this staphylococci are known to be also isolated from the mouth and gut.
include the finding that old world NHP kept in captivity were colonized The tamarins were from groups monitored by the AMLD. For most
by human-related MRSA and MSSA clonal types [14,15]. More scarce, is animals, date of birth and familial relations are known. Among the
the information on the colonization of MR-staphylococci in free-ranging golden lion tamarins captured, 23 were classified as adults, 17 as young
neotropical NHP. individuals, and seven as infants, with 55.3 % males and 44.7 % females.
Although Brazil is recognized as a country with the largest NHP The marmosets groups had not been part of a long-term monitoring
population worldwide [16], data about their microbiota are scarce [17]. program. The captured marmosets consisted of 19 adults and four young
In particular, the genetic characteristics of antibiotic-resistant and animals, 56.6 % females and 43.4 % males. All animals were apparently
pathogenic staphylococci isolated from these animals are poorly char­ healthy when sampled.
acterized, and published information about pathogenicity of S. aureus
clones in NHP is restricted to captive animals [14]. 2.3. Bacterial isolates
Golden lion tamarins (L. rosalia) are endangered primates endemic to
the Atlantic forest of the state of Rio de Janeiro, Brazil. Their population Swabs were enriched in 2.0 mL of BHI broth (HiMedia, India) con­
of 2600 animals is distributed over a mosaic of forest fragments with taining 7.5 % NaCl and aerobically incubated at 37 ◦ C during 24 h. After
different degrees of protection (biological reserves and private-land centrifugation (13.000 rpm, 5 min), pellets were cultured in mannitol
forests) that vary in the level of human presence and activity [18,19]. salt agar (HiMedia, India) aerobically at 37 ◦ C during 24–48 h and
One threat to the conservation of the tamarins is the presence of pop­ isolates were conserved at − 70 ◦ C. Staphylococci were presumptively
ulations of hybrids of two other Brazilian NHP, the marmosets Callithrix identified based on colony morphology, Gram staining, catalase, oxi­
jacchus and C. penicillate [20]. The introduction of Callithrix spp in these dase, coagulase production (Staphaurex Plus kit, Remel, United
forests is a consequence of the illegal wildlife trade [21], therefore this Kingdom), and hemolytic pattern (blood agar base supplemented with 5
NHP species are managed as invasive species. When in the same forest % (v/v) defibrinated sheep blood aerobically, HiMedia, India).
fragments, marmosets and tamarins interact frequently over food sour­
ces [22]. 2.4. Species identification
In this study, we aimed to assess if the co-habitation of NHP with
humans and human modified environments could constitute a risk for S. aureus were confirmed by amplification of nuc gene by PCR [1] and
colonization of the native neotropical NHP L.rosalia with antibiotic coagulase-negative staphylococci (CoNS) species were identified by
resistant and pathogenic staphylococci. For this purpose, we isolated sequencing of an internal fragment of tuf gene (elongation factor Tu)
and compared staphylococci from free-ranging Callithrix sp. – the [23].
invasive species - and L.rosalia – the native species, from areas with
different levels of contact with humans: an industrial park, and natural 2.5. Antimicrobial susceptibility testing
forests with different degrees of human presence.
Antimicrobial susceptibility was conducted by the agar disk diffusion
2. Material and methods method (Oxoid, United Kingdom) according to Clinical and Laboratory
Standards Institute guidelines [24] for the following 13 antibiotics:
2.2. Study design penicillin, oxacillin, vancomycin, linezolid, gentamycin, ciprofloxacin,
erythromycin, clindamycin, quinupristin/dalfopristin, tetracycline,
Ninety-three non-human primates (NHP) were captured from three rifampicin, fusidic acid, and sulfamethoxazole-trimethoprim. For iso­
regions in Rio de Janeiro, Brazil: an industrial area with a small forest lates showing resistance or intermediary resistance to oxacillin, vanco­
wherein marmosets did not have contact with tamarins, but had mycin or fusidic acid, the minimum inhibitory concentration [19] was

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I. Sales et al. Comparative Immunology, Microbiology and Infectious Diseases 104 (2024) 102094

determined by Etests (bioMérieux, France), according to CLSI and [hlb]) were detected by multiplex PCR, as previously described [32–34].
EUCAST (fusidic acid) [24,25]. The breakpoint for oxacillin in M. sciuri
was considered to be 3 µg/mL as previously established [26]. 2.8. Detection of antibiotic resistance genes

The presence of mecA and mecC genes was determined by uniplex

2.6. Molecular characterization of S. aureus.
PCR for all S. aureus and CoNS isolates [35,36]. Isolates that presented
resistance to penicillin but that did not carry mecA and mecC were
Pulsed-field gele electrophoresis (PFGE) was performed after SmaI
additionally screened for blaZ by PCR [37] and for blaZ and mecA by
digestion of total DNA, as described [27]. The resulting band patterns
Southern blotting. The SmaI DNA macrorestriction fragments were
were analyzed with BioNumerics software (version 4.61; Applied Maths,
transferred by vacuum blotting [38] to a nylon membrane (GE Health­
Saint-Martens-Latem, Belgium) using an optimization of 0.5 %, and a
care, Amersham, UK) and hybridized with purified PCR amplicons of
tolerance of 1.3 %. Strains were considered to belong to the same PFGE
mecA and blaZ (1039 bp and 533 bp, respectively) using ECL direct
type if the similarity of their macrorestriction was above 80 % and were
Prime Labeling and detection systems (Amersham Biosciences, Buck­
considered to belong to the same PFGE subtype if their similarity was
inghamshire, UK), according to manufacturer instructions.
higher than 95 % [28]. The spa type was determined by sequencing as
Isolates showing resistance to fusidic acid were additionally screened
described [29] using the Ridom-Staph software and database
for the presence fusB and fusC by multiplex PCR, as previously described
(http://spaserver.ridom.de). MLST was conducted [30] for 10 selected
[39].
S. aureus strains, including at least one representative of each spa type
and PFGE subtype. Gene allele numbers and sequence types (STs) were
2.9. Statistical analysis
attributed by using MLST database (http://www.mlst.net/). For the
remaining 31 S. aureus isolates, the ST was inferred based on the com­
The significance of difference between proportions was tested by the
bination of spa and PFGE type.
Qui2 test using the GraphPad software and considering a 95 % confi­
dence level.
2.7. Detection of virulence factors in S. aureus

ACME allotype (type I to III) [31], and genes codifying the virulence
factors Panton-Valentine leukocidin (PVL), LukE LukD leukocidin
(lukED), class F leukocidin (lukM), staphylococcal enterotoxins (sea-e,
seg-j, sep, sel), toxic shock syndrome toxin (tsst), exfoliative toxins (eta,
etb, etd), and hemolysins (gamma [hlg], gamma variant [hlgv], and beta

Fig. 1. Map showing the location of the Biological and Private Reservations where Callithrix spp. and L. rosalia were sampled in this study. Biological reservations
(PDA and U) are delimited by a black line; Callithrix spp. are represented by a white triangle; L. rosalia are represented by white circles; regions of interaction between
L. rosalia and Callithrix spp. are depicted by black stars and constitute the private reservations.

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I. Sales et al. Comparative Immunology, Microbiology and Infectious Diseases 104 (2024) 102094

3. Results (18/21, 85 %) than those collected from biological reserves (4/22, 18 %)

(p = 0.0001), where the contact with humans is rare or inexistent (see
3.1. The colonization rate and relative distribution of staphylococcal Fig. 3A).
species among Lion tamarins and marmosets is different
3.3. Staphylococcus aureus strains collected from lion tamarins and
A total of 93 staphylococci (41 S. aureus and 52 CoNS) strains were
marmosets are genetically different
obtained from the 70 NHP analyzed (n = 47 tamarins; n = 23 marmo­
sets); 43 were collected from the rectum and 50 from the oral cavity.
S. aureus isolates recovered from L. rosalia were different from
The amount of S. aureus colonization in L. rosalia (68 %; 32/47) was
S. aureus collected from Callithrix spp. In L.rosalia seven clonal types
significantly higher than that of Callithrix spp. (39 %; 9/23) (p = 0.03).
were found: ST6-t701 (n = 17), ST188-t189 (n = 6), ST6-t5271 (n = 5),
The overall CoNS colonization rate did not differ between the two spe­
ST6-t7396 (n = 1), ST2985-t189 (n = 2) and ST133-NT (n = 1). In
cies (L. rosalia: 76 %; Callithrix spp.: 69 %) (p = 0.56). However, they
Callithrix spp., only three clonal types were found: ST398-t1451 (n = 7),
differed in the rate of colonization of individual species: S. xylosus
ST1-t13736 (n = 1), and ST2984-t13737 (n = 1) (see Table 1 and
colonization rate was higher in Callithrix spp. when compared to
Fig. 4). From the clonal types identified, the most widespread were the
L. rosalia (17 % and 4 %, respectively (p = 0.08) and S. simiae colonized
ST6-t701 (n = 30 countries), the ST188-t189 (n = 30) and the ST398-
L. rosalia (21 %) only.
t1451 (n = 15) (SpaRidom database), which were all previously
Among all staphylococci, the most frequently isolated species among
described in human and animal [40–45]. The remaining six clonal types
L. rosalia was S. aureus (47 %) followed by M. sciuri (32 %), S. simiae (16
identified in this study were not reported in the literature, but were
%), S. xylosus (3 %) and S. saprophyticus (1.5 %). The relative distribu­
listed in the SpaRidom database as being isolated in only one or two
tion of the staphylococcal species among Callithrix spp. was different:
countries. Noteworthy, only the clonal type ST398-t1451 was previously
M. sciuri was the most common species (46 %), followed by S. aureus,
reported in Brazil [46].
S. xylosus and S. saprophyticus (33 %, 17 % and 4 %, respectively) (see
Additionally, the S. aureus clonal types identified depended on the
Fig. 2) and S. simiae was not found.
geographic origin of the NHP L. rosalia analyzed. S. aureus collected from
L. rosalia were colonized by a higher number of staphylococcal
L. rosalia inhabiting the Biological Reserves belonged mainly to ST6 (81
species and were frequently co-colonized with different species, a phe­
%; 22/27); and those inhabiting Private Forest Reserves areas were
nomenon that was not observed in Callithrix spp. A total of 21 % (10/47)
mostly from ST188 (67 %;4/6). For Callithrix spp., the S. aureus found in
of L. rosalia were co-colonized with different pairs of staphylococcal
the animals from Water Island belonged mainly to ST398 (87.5 %; 7/8)
species, including S. aureus/M. sciuri; M. sciuri/S. simiae, and S. aureus/
(Table 1). Interestingly, this was the only staphylococcal species isolated
S. xylosus.
from this specific group of primates. The results suggest that not only
There was no difference in distribution of staphylococci between
each mammalian species has a specific microbiota but also that
young and adult animals. The data showed a higher prevalence of
dissemination of S. aureus between the two species of non-human pri­
staphylococci in the oral cavity (71 %, 50/70), compared to rectal cavity
mates is rare. Furthermore contact with human created environment
(61 %, 43/70) (p = 0.2829), although distribution of individual species
lead to changes in microbiota of both L. rosalia and Callithrix spp.
among the sites of collection was similarly proportional. The only
exception was S. simiae that was more frequently isolated from the rectal
cavity. 3.4. S. aureus collected from non-human primates in anthopogenic
environments have decreased susceptibility to erythromycin and carry PVL

3.2. Contact of non-human primates with anthropogenic environments Overall, S. aureus isolated from non-human primates were highly
was associated to a change in the relative prevalence of staphylococcal susceptible to almost all classes of antibiotics tested (see Table 1), which
species was expected given the low use of antibiotics in this type of natural
environments. All isolates were susceptible to penicillin and oxacillin,
We observed that NHPs living in the urban/industrial environment and carried no mecA or mecC genes, as determined by PCR and Southern
were colonized by specific staphylococcal species. In privately owned blotting. The only antibiotic for which a decreased susceptibility was
forest 6 % (1/17) of the marmosets were colonized with S. aureus, but in observed was erythromycin. A total of 10 % of the strains (4/41), all
the industrial environment (Water Island), all marmosets were colo­ isolated from Callithrix spp. from Water island and belonging to ST398-
nized with S. aureus (p = 0.0001). We also found that L. rosalia from t1451, were resistant to erythromycin, suggesting that contact with
private forest fragments were significantly more colonized with M. sciuri humans might constitute a risk factor for the acquisition of antibiotic

Fig. 2. Distribution of Staphylococcus species among L. rosalia and Callithrix spp.

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I. Sales et al. Comparative Immunology, Microbiology and Infectious Diseases 104 (2024) 102094

A B
100%

Frequency of R staphylococci in L. rosalia

100% 90%
100%
S, auerus
S. aureus in in marmosets
marmosets 80%
90% 85%

80% M. sciuri
S. sciuri in lion
in Lion tamarins
tamarins 70%
n

70% 60% 56%

60% 50%

50%
40%
Frequency of c

40%
30%
30%
18% 20%
20% 13%
10%
10% 6%

0% 0%
Biological Reserve Private Reserves Water Island Biological Reserve Private Reserves

Fig. 3. Frequency of colonization with staphylococci of non-human primates from environments with different levels of contact with humans. A) Frequency of
colonization of Callithrix spp. with S. aureus and of L. rosalia with M. sciuri; B) Frequency of colonization of L. rosalia with staphylococci resistant to at least one
antibiotic tested.

Table 1
Genotypic and phenotypic characteristics of S. aureus collected from non-human primates in environments with different levels of contact with humans.
Environment Level of human Host (no CClonal type by MLST-spa type (MLST- Antibiotic resistance (no Virulence genes (no isolates)
contact isolates) SCCmec) (no isolates) isolates)

Water Island High Callithrix sp. ST398-t1451 (7) EryR (4), FusR (3) hlg (3)
(8) ST2984-t13737 (1) – hlgv, lukE/D (1)
Private Low L. rosalia (6) ST188-t189 (4) – hlb (4), hlgv (4), lukED (4), PVL (4)
Reservations ST133-NT (1) – hlb (1)
ST6-t701 (1) – hlb (1), hlgv (1), lukED (1)
Callithrix sp. ST1-t13736 (1) – hlb (1), hlgv (1), lukED (1), PVL (1)
(1)
Biological None L. rosalia (26) ST6-t701 (16) – hlb, (16), hlgv (16), lukED (16) hlb
Reservations hlgv lukED
I
ST6-t5271 (5) Ery (1) hlb, (5), hlgv (5), lukED (5)
ST188-t189 (2) – hlb, (2), hlgv (2), lukED (2)
ST2985-t189 (2) – hlb, (2), hlgv (2), lukED (2)
ST6-t7396 (1) – hlb, (1), hlgv (1), lukED (1)

resistant bacteria. Strains belonging to this clonal type with resistance to dissemination of isolates between animals that are in close contact.
erythromycin were previously described as a cause of infections in Examples of this are isolates SJ21, SJ22 and SJ23, all belonging to PFGE
humans [42] and an isolate belonging to a related clone (ST398-t034), type F and collected from different animals inhabiting RPPN FC (PT2).
also resistant to macrolides, was found as a cause of a fatal pneumonia in However, we also observed the occurrence of the same PFGE in M. sciuri
an oncology patient in São Paulo, Brazil [47]. isolates collected from L. rosalia inhabiting different geographic loca­
To understand the pathogenic potential of S. aureus isolated from tions (see Table 2). In addition, we identified one case in which M. sciuri
non-human primates S. aureus isolates were screened for a set of viru­ isolated from one L. rosalia has a SmaI macrorestriction pattern (PFGE
lence factors by PCR. The great majority of strains carried LukE-LukD type E) similar to a M. sciuri isolated collected from a Callithrix spp. from
leukocidin (80 %) and hemolysins beta and gamma variant (76 %, the same private forest, suggesting that, although rare, dissemination of
each), including representatives of almost all the clonal types (ST1, ST6, M. sciuri between the two non-human primates might occur. For the
ST188, ST2984 and ST2984). The only exceptions were the isolate remaining CoNS species, we observed a similar situation, wherein
belonging to ST133 that carried beta-hemolysin only and isolates of related SmaI macrorestriction PFGE patterns were found both in isolates
ST398 that carried the gamma-hemolysin only (3/7). A total of 12.2 % sharing the same host and geographic region and in isolates from
(5/41) of the isolates carried PVL, including isolates of ST188 collected different geographic places.
from L. rosalia (n = 4) and isolates of ST1 (n = 1) from Callithrix spp., all
collected from private forests. Strains belonging to these genetic back­
grounds and carrying PVL have been found to cause infections in 3.6. CoNS collected from marmosets in contact with humans are
humans in different regions of the world, including in Brazil [48]. reservoirs of antibiotic resistance

Although in general CoNS were susceptible to most of the antibiotics

3.5. CoNS staphylococci can disseminate between non-human primate tested, their frequencies of resistance were higher than those observed
species for S. aureus. M. sciuri and S. xylosus. were the CoNS species that showed
the highest resistance rates and the species that accumulated resistance
M. sciuri isolates showed a high genetic diversity, comprising as to more antibiotics. M. sciuri isolates showed resistance to penicillin
many as 19 different PFGE types (Table 2). Overall, isolates belonging to (n = 6/33), oxacillin (n = 2/33) and fusidic acid (n = 33/33) and
the same PFGE type were collected from the same species of primates intermediary resistance to clindamycin (n = 2/33) and S. xylosus
and the same geographic region, suggesting the occurrence of showed resistance to penicillin (2/6), erythromycin (3/6) and a

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I. Sales et al. Comparative Immunology, Microbiology and Infectious Diseases 104 (2024) 102094

Fig. 4. SmaI PFGE profiles of 33 S. aureus isolated from wild marmoset (Callithrix spp.) and golden lion tamarins (L. rosalia) in private and Biological Reserves in
Brazil. A cut-off of 80 % similarity was used to define the PFGE types. Multilocus sequence type and spa type of each strain are indicated as well as the origin and host
of the isolate. aControl strains; bEach distinct code corresponds to a different location in Biological (R) or private (RP) reserves.

Table 2
Genotypic and phenotypic characteristics of coagulase-negative staphylococci (CoNS) collected from non-human primates in environments with different levels of
contact with humans.
Environment Level of human Host (no Bacterial species (no PFGE type (no isolates) Antibiotic resistance (no isolates)
contact isolates) isolates)

Private Reserve Low L rosalia M. sciuri (18) X (2), C (1), E (1), H (1), I (3), J (5), L(1), M PenR (3), OxaR (1), ClinR (4), FusR
(26) (1), Q (1) (18)
S. simiae (7) AA (3), DD (3), GG (1) –
S. saprophyticus (1) b (1) FusR
Callithrix spp. M. sciuri (11) B (2), U (1), E (2), G (2), F (1), A (2), V (1) PenR (4), FusR (11)
(16) S. xylosus (4) AAA (2), BBB (1), CCC (1) PenR (2), EryR (3)
S. saprophyticus (1) a (1) FusR (1)
Biological None L. rosalia (10) S. simiae (4) FF (1), EE (1), BB (1), CC (1) –
reserve M. sciuri (4) P (1), ND (1), A (1), R (1) OxaR (1), ClinR (3), FusR (4)
S. xylosus (2) DDD (1), EEE (1) ClinR (1)

decreased susceptibility to clindamycin (1/6). Resistance to penicillin forests (18/32, 56 %) than those from the biological reserves (5/37, 13
was observed in isolates collected from both L. rosalia and Callithrix spp; %) (p = 0.0003) (see Fig. 3B).
all found in private forest. Resistance to oxacillin was observed both in
Private forests and Biological reserves in L. rosalia. In all CoNS strains 4. Discussion
presenting resistance to oxacillin and penicillin there was no detection
of mecA/mecC and blaZ genes, respectively, either by PCR or Southern We evaluated the colonization prevalence of virulent and antibiotic
blotting. The great majority of the isolates showing phenotypic resis­ resistant staphylococci in an endangered protected NHP species –
tance to beta-lactams, but lacking mecA, belonged to the M. sciuri species L. rosalia - and in introduced populations of hybrids of other NHPs -
(n = 8/10); the remaining isolates were S. xylosus (n = 2/10). Callithrix jacchus and penicillate [20,49]. L. rosalia is a native species of
Regarding the remaining staphylococci isolated, the only two the state of Rio de Janeiro, Brazil with populations occurring in gov­
S. saprophyticus isolates were resistant to fusidic acid and susceptible to ernment conservation units (biological reserves) and in neighboring
the remaining antibiotics and S. simiae were susceptible to all antibiotics forests in private lands, wherein the contact with humans, their animals
tested. Overall, the frequency of staphylococcal isolates showing resis­ or environments is higher. One of the results of human activities has
tance to at least one antibiotic was higher among L. rosalia from private been the introduction of Callithrix spp, into urban and industrial regions

6
I. Sales et al. Comparative Immunology, Microbiology and Infectious Diseases 104 (2024) 102094

(like Water Island) as well as into private forests, where they co-exist study. The low number of NHP sampled could have impacted on the
with L. rosalia. diversity of staphylococcal species and clonal types identified and on the
Our results showed that the contact of L. rosalia and Callithrix spp. number of epidemiological links found between staphylococci of the two
with human environments has led to changes in their microbiota and in species of primates. Sampling of wild animals is difficult and dependent
their virulence genes content, and antibiotic susceptibility. L. rosalia on a number of uncontrollable factors, like the number of wild animals
from private forests were enriched in M. sciuri when compared to bio­ that occur in the specific sampling region in the specific sampling day.
logical reserves and Callithrix spp. from Water Island were enriched in Even so, epidemiological links were established, and the conclusions
S. aureus, when compared to private forests. In particular, the frequency taken from our study have statistical support. In this study transmission
of S. aureus resistant to erythromycin and carrying PVL was higher in was assessed through PFGE, a high-resolution methodology used for
samples collected from Callithrix spp in the human environment, when many years as the state of the art method to assess bacterial trans­
compared to environments with less contact with humans. Furthermore, mission. Currently, with the advent of whole genome sequencing
the frequency of resistance to at least one antibiotic (beta-lactams, transmission events are usually detected by genomics approaches based
macrolides and lincosamides) in the human-influenced environments on the analysis of single-nucleotide polymorphisms or core genome
(the island and the private forests) was higher for CoNS collected both multilocus sequence typing that have higher resolution than PFGE.
from L. rosalia and Callithrix spp. The origin of such alterations is not However, these are more appropriate to detect recent transmission
clear but could have resulted from the contact of bacteria with envi­ events in closed environments like hospitals that presuppose a high
ronmental pressure associated to human activities, such as the similarity between strains, which is not the case of our study.
contamination of water and/or soils with antibiotics, dissemination of These results support the hypothesis that human activities and
antibiotic resistant bacteria from farms, the contact between the native human contact influence the bacterial microbiota of wild and protected
and invader NHP species or the direct interaction of Callithrix spp. with animals. We found evidence that L. rosalia in locations with increased
humans in Water Island. human contact have a higher prevalence of colonization with antibiotic
Penicillin, macrolides and lincosamides are extremely important resistant and pathogenic bacteria than wild populations with reduced
antimicrobials for the treatment of infections in production animals contact with humans.
(cattle and pigs) [50] and have been described to be important con­
taminants of farm waters and ground soil in farms [51]. Farming ac­ Ethical statement
tivities occur in the land surrounding the private forests in this region in
Brazil and might be the source of antimicrobial resistant bacteria or of This study was authorized by the Instituto Chico Mendes de Con­
contamination of water and soil that could in its turn be the environ­ servação da Biodiversidade (ICMBio), certified by Authorization and
mental pressure inducing the NHP microbiota change and the increased Information Biodiversity System (SisBio) (protocol # 10596-2), and was
antimicrobial resistance observed. approved by the Ethical Committee from Universidade Estadual do
Introduced Callithrix spp represent a risk for the transmission of po­ Norte Fluminense Darcy Ribeiro (protocol # 148). CEUA was approved
tential pathogenic and antibiotic resistant S. aureus to L. rosalia, however in November, 2011, with four years validation.
our data did not support the existence of transmission of S. aureus be­
tween the two NHP species. On the other hand, the most important
Declaration of Competing Interest
S. aureus clonal types found in NHP inhabiting the regions with the
highest contact with humans, namely the private forests and Water is­
No conflicts of interests to declare.
land (ST398, ST188, ST133, ST1, ST6), were previously identified
colonizing humans in Brazil [52]. It is possible that dissemination of
Acknowledgements
S. aureus between humans and NHP, and not the dissemination between
the two species of NHP, might have caused the observed increase in
We would like to thank the Associação Mico Leão Dourado (AMLD)
S. aureus in Callithrix spp. Other studies have documented the dissemi­
staff for trapping monkeys and logistic support. The capture of monekys
nation of antimicrobial resistant S. aureus between humans and NHP and
at Water Island was funded by a contract from TRANSPETRO to C.Ruiz-
PVL was previously found in S. aureus strains from NHP in captivity
Miranda to erradicate the animals from the island. We thank the staff
[15].
and students from SERCAS (Setor de Etologia aplicada a Reintrodução e
A somewhat different pattern was found for CoNS. The M. sciuri
Conservação de Animais Silvestres - Brazil) for captures and assistance
isolates collected from Callithrix spp and L. rosalia from the same
in sample collection. This work was partially supported by ONEIDA
geographic regions had closely related PFGE profiles. This suggests the
project (LISBOA-01–0145-FEDER- 016417) co-funded by FEEI - "Fundos
dissemination of CoNS between the two primate hosts. Contrarily to
Europeus Estruturais e de Investimento - Portugal" from "Programa
what was observed for S. aureus, the results suggest that the contact
Operacional Regional Lisboa 2020" and by national funds from FCT -
between the native and invader NHP species could have caused the
"Fundação para a Ciência e a Tecnologia - Portugal.” and by Projects
observed increase in M. sciuri prevalence and the occurrence of peni­
LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e
cillin resistance among L. rosalia in private forests, where Callithrix also
Celular) and UID/Multi/04378/2019) funded by FEDER - Portugal
lives. A high resistance to penicillin was also previously found in
funds through COMPETE2020 - Programa Operacional Competitividade
staphylococci from vaginal mucosa of captive Azara’s night monkey
e Internacionalização (POCI). This study was supported by projects
(Aotus azarai infulatus) and L. rosalia in Brazil [53,54].
PTDC/BIA-EVF/117507/2010 and PTDC/FIS-NAN/0117/2014, from
In Staphylococcus the most widely spread mechanism of beta-lactams
FCT and European Society of Clinical Microbiology and Infectious Dis­
resistance is associated to mecA – the gene that encodes a penicillin
eases (ESCMID) Research Grants 2010, awarded to M. Miragaia. Addi­
binding protein with low affinity for beta-lactams (Pbp2A). In our study
tionally, it was funded by TRANSPETRO - Brazil (No 4600007468) and
most of the M. sciuri isolates were resistant to beta-lactam antibiotics,
SERCAS - FAPERJ - APQ1 (No E26/171.530/2004) and by Coordenação
but did not carry the mecA as detected by PCR and Southern blotting.
de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) -
This apparent discrepancy was previously described, and was proved to
Finance Code 001.
be due to the overexpression of mecA1, a mecA homolog, which is
ubiquitous in M. sciuri and that is believed to be the mecA ancestral [26].
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