Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Bacteria richness and antibiotic-resistance in bats from a protected area in the Atlantic Forest of Southeastern Brazil

  • Vinícius C. Cláudio ,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Writing – original draft, Writing – review & editing

    vcclaud@gmail.com

    Affiliations Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, SP, Brazil, Fundação Parque Zoológico de São Paulo, São Paulo, SP, Brazil, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

  • Irys Gonzalez,

    Roles Conceptualization, Formal analysis, Methodology, Supervision, Writing – review & editing

    Affiliation Fundação Parque Zoológico de São Paulo, São Paulo, SP, Brazil

  • Gedimar Barbosa,

    Roles Methodology, Project administration, Writing – review & editing

    Affiliations Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, SP, Brazil, Fundação Parque Zoológico de São Paulo, São Paulo, SP, Brazil

  • Vlamir Rocha,

    Roles Methodology, Project administration, Writing – review & editing

    Affiliation Centro de Ciências Agrárias, Universidade Federal de São Carlos, Araras, SP, Brazil

  • Ricardo Moratelli,

    Roles Conceptualization, Project administration, Writing – review & editing

    Affiliation Fiocruz Mata Atlântica, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil

  • Fabrício Rassy

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Writing – review & editing

    Affiliation Fundação Parque Zoológico de São Paulo, São Paulo, SP, Brazil

Bacteria richness and antibiotic-resistance in bats from a protected area in the Atlantic Forest of Southeastern Brazil

  • Vinícius C. Cláudio, 
  • Irys Gonzalez, 
  • Gedimar Barbosa, 
  • Vlamir Rocha, 
  • Ricardo Moratelli, 
  • Fabrício Rassy
PLOS
x

Abstract

Bats play key ecological roles, also hosting many zoonotic pathogens. Neotropical bat microbiota is still poorly known. We speculate that their dietary habits strongly influence their microbiota richness and antibiotic-resistance patterns, which represent growing and serious public health and environmental issue. Here we describe the aerobic microbiota richness of bats from an Atlantic Forest remnant in Southeastern Brazil, and the antibiotic-resistance patterns of bacteria of clinical importance. Oral and rectal cavities of 113 bats from Carlos Botelho State Park were swabbed. Samples were plated on 5% sheep blood and MacConkey agar and identified by the MALDI-TOF technique. Antibiotic susceptibility tests were performed using Kirby-Bauer’s antibiotic disc diffusion technique.We identified 596 isolates at the genus level and tentatively to the species level. Proteobacteria was the most abundant phylum in all the dietary guilds, representing 87% of the total identified samples. The most common bacteria within bat individuals were Escherichia coli, Klebsiella oxytoca and Serratia marcescens, and within bat species were Serratia marcescens, Pseudomonas sp. and Staphylococcus sp. Frugivores presented the most diverse microbiota. In general, the antibiogram results indicated a low occurrence of resistance on eigth potentially pathogenic bacteria species. The resistance to antibiotics found on our samples was related mostly to the intrinsic resistance of the tested species.The low occurrence of resistant bacteria in our samples could be related to the well preserved environment where bats were caught. Once the major causes of resistance-acquiring are related to anthropic activites, the controlled access of tourists on certain regions of the Park seems to be effectively protecting the environment.

Introduction

Bats as a group are distributed worldwide, with more than 1300 species, representing ca. 20% of the world mammals [1]. They are highly diversified ecologically, bringing together the most diversified feeding strategies among terrestrial vertebrates. Dietary strategies include frugivory, hematophagy, insectivory, nectarivory, carnivory, piscivory and omnivory [1, 2]. Some species allocated in one of these categories include different food sources in their diet [1, 2]. Due to this diversified diet they provide important ecosystem services such as seed dispersal, pollination and pest control, but also carry many pathogens, some of them of zoonotic potential [3, 4]. Little is known about Neotropical bat microbiota, which is in great part studied for Old World species and mostly related to the gastrointestinal diversity [511];. Also, studies focused on the interaction, influence and ecologic role of bats oral and rectal microbiota are scarce, despite their importance on the digestion, vitamin synthesis, protection against harmful microorganisms and also public health [1217].

Previous studies of bat gut microbiota showed that the bacteria diversity is in part related to the host diet, with a partial overlap between species in different dietary guilds, once these species can compensate the lack of some requirements with different food sources during resources shortages [9, 18]. Besides the microbiota diversity, the bacteria antibiotic-resistance patterns could be also modulated by dietary habits [1921]. Among the major causes of resistance acquiring is the contact with anthropic environments [21, 22]. Antimicrobial resistant bacteria are a growing and serious problem to the public health and environment, and are reported to be present even on remote habitats [17, 23]. The presence of antimicrobial resistance in wildlife brings implications, as it can drive animals to become potential reservoirs of resistant bacteria, and also impose limits to the efficiency of antibiotics used on the control of human and wildlife diseases [17, 21].

Against that background, we aimed (1) to describe the oral and rectal aerobic microbiota richness of bats in five dietary guilds from the Carlos Botelho State Park (CBSP), a protected area on the Atlantic Forest of Southeastern Brazil, focusing bacteria of clinical importance; (2) to identify the antibiotic-resistance profile of eight potentially pathogenic bacteria for those bats; and (3) to evaluate whether the protected area is preserving the wildlife from antibiotic resistant bacteria.

Material and methods

Sampling

Fieldwork was conducted monthly from October 2016 to September 2017 on the Carlos Botelho State Park (CBSP; 24°12'–24°4'S, 47°47'–48°7'W), which is a protected area in the Brazilian Southeastern Atlantic Forest, created in 1982. The phytophysiognomy is mostly represented by the ombrophilous forest, with ca. 23,300 ha composed by pristine forests [24]. Bats were captured using with mist-nets and during searches for roosts, under the permits SISBIO/ICMBIO 54.381-1/2016 and COTEC/SMA-IF 260108006.479/2016. Monthly, oral and rectal cavities of one bat of each species captured were swabbed with sterile cotton swabs, which were then separately transported in Stuart’s transport medium and refrigerated. Samples used in this study were collected from 113 bats of 33 species, divided into five dietary guilds (frugivores [FRU]; insectivores [INS]; nectarivores [NEC]; sanguivores [SAN]; and carnivores [CAR]).

Isolation and identification of the microbiota

Samples collected in fieldwork were plated on 5% sheep blood agar and MacConkey agar, and incubated aerobically at 36°C for 24h. Colonies were further isolated by morphotype and preserved in Tryptic Soy Broth and 20% glycerol at -80°C; all the isolates are stocked at the Culture Collection of the Fundação Parque Zoológico de São Paulo. The isolates were later identified by the matrix-assisted laser desorption/ionization (MALDI) technique, using MALDI Biotyper System in collaboration with the Proteomics Laboratory at Universidade Federal de São Paulo [25]. The database of this technique is mostly composed by pathogenic species, therefore a great part of the identifications tend to result on pathogenic bacteria species. Isolates were analyzed using a formic acid-based direct, on-plate preparation method. Small amounts of a single colony were smeared directly onto a spot of the MALDI-TOF MS steel anchor plate. Each spot was then overlaid with one microliter of 70% formic acid and allowed to dry. The dried mixture was overlain with 1 μl of matrix solution (α-cyano-4-hydroxycinnamic acid [HCCA]) dissolved in 50% acetonitrile, 47.5% water, and 2.5% trifluoroacetic acid and allowed to dry prior to analysis using the MALDI Biotyper. An Escherichia coli (ATCC 25922) isolate was used for instrument calibration. Two positive controls (Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923) were included with each run [26].

Antibiotic sensitivity

Antibiotic susceptibility tests were performed on Mueller Hinton agar using Kirby-Bauer’s antibiotic disc diffusion technique [27]. The tests were performed for the most potentially pathogenic bacteria species Acinetobacter baumannii, Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella sp., Serratia marcescens, Stenotrophomonas maltophilia, and Stenotrophomonas sp. The antibiotics used on the tests were selected according to the bacteria characteristics [28], and the discs were firmly placed on the seeded plates, which were incubated at 36°C for 24h. The susceptibility of each isolate for different antibiotics was evaluated by the zones of inhibition, which were measured and compared with the susceptibility pattern of each antibiotic defined by the Clinical and Laboratory Standards Institute [28].

The antibiotics tested for Acinetobacter baumannii were: amikacin (AMI, 30 μg), ceftazidime (CAZ, 30 μg), ceftriaxone (CRO, 30 μg), ciprofloxacin (CIP, 5 μg), chloramphenicol (CLO, 30 μg), gentamicin (GEN, 10 μg), imipenem (IPM, 10 μg) and norfloxacin (NOR, 10 μg). The antibiotics tested for Pseudomonas aeruginosa were: ceftazidime (CAZ, 30 μg), ceftriaxone (CRO, 30 μg), ciprofloxacin (CIP, 5 μg), gentamicin (GEN, 10 μg), imipenem (IPM, 10 μg) and norfloxacin (NOR, 10 μg). The antibiotics tested for Stenotrophomonas maltophilia and Stenotrophomonas sp. were: ceftazidime (CAZ, 30 μg), ceftriaxone (CRO, 30 μg), ciprofloxacin (CIP, 5 μg), gentamicin (GEN, 10 μg), imipenem (IPM, 10 μg), norfloxacin (NOR, 10 μg) and trimethoprim-sulphamethoxazole (SUT, 1.25/23.75 μg). The antibiotics tested for Escherichia coli, Klebsiella oxytoca, Salmonella sp. and Serratia marcescens were: amikacin (AMI, 30 μg), ceftazidime (CAZ, 30 μg), ceftriaxone (CRO, 30 μg), ciprofloxacin (CIP, 5 μg), chloramphenicol (CLO, 30 μg), gentamicin (GEN, 10 μg), imipenem (IPM, 10 μg), doxycycline (DOX, 30 μg), ampicillin (AMP, 10 μg), amoxicillin-clavulanate (AMC, 20/10 μg) and cephalexin (CFL, 30 μg).

Results

Oral and rectal microbiota

We isolated 830 morphotypes of bacteria from bats in five different dietary guilds (carnivores, frugivores, insectivores, nectarivores and sanguivores). A total of 596 morphotypes were identified at the genus level and tentatively to the species level by the MALDI-TOF methodology, including 243 from the oral cavity and 353 from the rectal cavity. Successfully identified isolates from the oral cavity are represented by: 14 isolates from two species of carnivores; 15 isolates from two species of sanguivores; 25 isolates from three species of nectarivores; 75 isolates from 14 species of insectivores; and 113 isolates from 10 species of frugivores (Table 1). Successfully identified isolates from the rectal cavity are represented by: 11 isolates from two species of carnivores; 27 isolates from two species of sanguivores; 60 isolates from two species of nectarivores; 90 isolates from 15 species of insectivores; and 165 isolates from 11 species of frugivores (Table 2).

thumbnail
Table 1. Successfully identified oral microbiota from bats of Carlos Botelho State Park, São Paulo State.

https://doi.org/10.1371/journal.pone.0203411.t001

thumbnail
Table 2. Successfully identified rectal microbiota from bats of Carlos Botelho State Park, São Paulo State.

https://doi.org/10.1371/journal.pone.0203411.t002

Isolates belong to four bacteria phyla, divided into 15 families. Proteobacteria was the most abundant phylum in all the dietary guilds, representing 87% of the total samples, followed by Firmicutes with 12%, and Actinobacteria and Bacteriodetes counting together 1% of the total identified samples. The family Enterobacteriaceae represented 73% of the samples, followed by Pseudomonadaceae, with 7%, and the other 20% are composed by small sums of the families Aeromonadaceae, Bacillaceae, Brucellaceae, Burkholderiaceae, Caulobacteraceae, Enterococcaceae, Lysobacteraceae, Microbacteriaceae, Micrococcaceae, Moraxellaceae, Neisseriaceae, Sphingobacteriaceae, Staphylococcaceae and Streptococcaceae. The phylum Actinobacteria, represented by Arthrobacter sp. and Microbacterium sp. was found only in the oral cavity, while the phylum Bacteroidetes is represented only by Sphingobacterium sp. in the rectal cavity.

Sixty-two taxa of bacteria were identified in the oral cavity and 72 in rectal cavity of the bats. The Venn diagram analysis (Figs 1 and 2) indicates that the major proportion of the bacteria within the dietary guilds is shared between two or more guilds. The oral richness shared between guilds varies from 49% to 75%, whereas the rectal richness varies from 59% to 87% of bacteria taxa shared with at least one other guild. However, only the species S. marcescens is shared between all five guilds when the oral richness is analyzed alone, and only the species K. oxytoca and S. marcescens are shared between all the guilds when considered the rectal richness. Comparing the dietary guilds, higher richness was found on frugivores (58 taxa), followed by insectivores (50 taxa), nectarivores (37 taxa), sanguivores (21 taxa) and carnivores (11 taxa).

thumbnail
Fig 1. Venn-diagram showing the distribution of bacterial taxa from oral swabs of five dietary guilds of bats on Carlos Botelho State Park, São Paulo State.

The number of taxa within each guild is represented in parenthesis. The abundance of each taxa on bat species is presented in the graph, and separated by dietary guilds.

https://doi.org/10.1371/journal.pone.0203411.g001

thumbnail
Fig 2. Venn-diagram showing the distribution of bacterial taxa from rectal swabs of five dietary guilds of bats on Carlos Botelho State Park, São Paulo State.

The number of taxa within each guild is represented in parenthesis. The abundance of each taxa on bat species is presented in the graph, and separated by dietary guilds.

https://doi.org/10.1371/journal.pone.0203411.g002

Antibiotic sensitivity

Strains of one A. baumannii, 20 E. coli, 13 K. oxytoca, two P. aeruginosa, two Salmonella sp., 36 S. marcescens, two S. maltophilia and five Stenotrophomonas sp. were selected as the most potentially pathogenic isolates and tested for their susceptibility for antibiotics. The A. baumannii isolate was resistant only to ciprofloxacin, intermediate to ceftriaxone and sensible to all the other tested antibiotics. The two P. aeruginosa isolates were sensible to all the antibiotics tested. The two Salmonella sp. isolates exhibited different sensitivity, with one sensible to all the antibiotics tested, and the other resistant to the antibiotics ampicillin and cephalexin. Two S. maltophilia and five Stenotrophomonas sp. isolates also exhibited differences in sensitivity, with all the isolates resistant to the antibiotics ceftriaxone and imipenem, only one isolate sensible to the antibiotic gentamicin, and with variable sensitivity to the antibiotic ceftazidime (Table 3).

thumbnail
Table 3. Antibiotic-resistance patterns of Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella sp., Stenotrophomonas maltophilia and Stenotrophomonas sp. from swabs of bats on Carlos Botelho State Park, Brazil.

The resistance patterns are classified as Sensitive (S), Intermediate (I) and Resistant (R). See Materials and Methods section for description of diet and antibiotics.

https://doi.org/10.1371/journal.pone.0203411.t003

The 20 E. coli isolates responses to the antibiotics tested were variable. Resistance to the antibiotics was absent for 16 of the isolates (80% of E. coli isolates), one isolate (5% of E. coli isolates) was resistant to ampicillin, one isolate (5% of E. coli isolates) was resistant to ampicillin and cephalexin, and two (10% of E. coli isolates) were resistant to amoxicillin-clavulanate, ampicillin and cephalexin (Table 4). From the 13 K. oxytoca isolates, seven (54% of K. oxytoca isolates) showed resistance to ampicillin, five (38% of K. oxytoca isolates) were intermediate to ampicillin, and one (8% of K. oxytoca isolates) was resistant to amoxicillin-clavulanate, ampicillin and cephalexin (Table 5). From the 36 S. marcescens isolates, 34 (95% of S. marcescens isolates) presented resistance to the antibiotics amoxicillin-clavulanate, ampicillin and cephalexin, and only two isolates (5% of S. marcescens isolates) were not resistant to amoxicillin-clavulanate and ampicillin (Table 6).

thumbnail
Table 4. Antibiotic-resistance patterns of Escherichia coli from swabs of bats on Carlos Botelho State Park, Brazil.

The resistance patterns are classified as Sensitive (S), Intermediate (I) and Resistant (R). See Materials and Methods section for description of diet and antibiotics.

https://doi.org/10.1371/journal.pone.0203411.t004

thumbnail
Table 5. Antibiotic-resistance patterns of Klebsiella oxytoca from swabs of bats on Carlos Botelho State Park, Brazil.

The resistance patterns are classified as Sensitive (S), Intermediate (I) and Resistant (R). See Materials and Methods section for description of diet and antibiotics.

https://doi.org/10.1371/journal.pone.0203411.t005

thumbnail
Table 6. Antibiotic-resistance patterns of Serratia marcescens from swabs of bats on Carlos Botelho State Park, Brazil.

The resistance patterns are classified as Sensitive (S), Intermediate (I) and Resistant (R). See Materials and Methods section for description of diet and antibiotics.

https://doi.org/10.1371/journal.pone.0203411.t006

Discussion

Bacteria richness

Gram-negative bacteria in the phylum Proteobacteria seems to be common in bat microbiota on studies based both on culture protocols and DNA sequencing, being isolated from oral and rectal cavities [8], intestine [9, 18] and saliva [10]. The phyla Actinobacteria, Bacteriodetes and Firmicutes were also previously reported as common on bats [10, 11, 18].

The mammalian gut microbiota diversity is related to the host diet, and should increase from animal-based diets to omnivorous to herbivore diets [29]. In our results, the frugivores microbiota was the most diverse among the five analyzed dietary guilds, and agrees to the mammalian gut microbiota theory. The less diverse microbiota in our survey was found in carnivores, which is also in agreement to the mammal microbiota theory. However, insectivores also showed high microbiota richness, and diverge from the expected, which could be explained by the inclusion of different alimentary items, rather than insects, on the diet of many species classified as insectivores. Species such as Glyphonycteris sylvestris, Lampronycteris brachyotis, Micronycteris microtis and Myotis nigricans analyzed in this study are reported to complement their diet with fruits and/or pollen [3032], which could increase the general microbiota richness of the insectivore bats guild analyzed here.

Another possible explanation for the richness observed in the different bat guilds lies within the number of bats sampled for each guild, whereas the most diverse guilds are also the ones with more bat captures.Though most of the results are in agreement to other studies based on DNA sequencing, the general bacteria richness of bats from CBSP may be biased by the identification technique and the culture step. On the other hand, some bacteria genera, including pathogenic ones, are hard to speciate using DNA sequencing techniques [11], making comparisons even harder.

Some bacteria genera, such as Arthrobacter, Burkholderia, Microbacterium, Neisseria and Rahnella were found only in the oral cavity. Arthrobacter is composed by soil bacteria, and was also found on bats’ wing sacs, chin and axillae by other authors [3335]; strains of Arthrobacter and Rahnella were identified as effective inhibitory antagonists of the growth of Pseudogymnoascus destructans, the fungus that causes white-nose syndrome, a letal bat disease [36]. Burkholderia and Microbacterium were previously found on bats’ saliva, urine, faeces, and intestine [9, 10]. Neisseria was previously found on bat saliva samples [10], and is closely related to mucosal and dental surfaces, being a consistent component of human oral microbiota and also found in different mammals [37]. The rectal cavity exclusive genus Enterococcus was also isolated from bats’ wings [35]. Brevundimonas, found only on the rectal cavity, was originally isolated from water and hospital-related material. This bacterium has been previously reported for marine mammals and is not common in bats [38, 39].

Bacteria genera observed within different dietary guilds were also divergent, with some exclusive occurrences. Edwardsiella was found only in sanguivores. This bacterium was previously isolated from bovine faeces and latter from cattle meat, wild mammals and birds [40, 41]. Thus, the occurrence of this bacterium only in this guild appears to be related to the feeding habit, which is based on blood from domestic and wild mammals and birds [42]. Plesiomonas, Proteus and Yokenella were identified only in insectivores. The genus Proteus, however, was also found in sanguivores and frugivores in other studies [8, 43]. The genus Plesiomonas is reported to be isolated from freshwater and surface water samples [44], and many species of insectivores are associated to these environments [4547], where they forage and could be exposed to bacteria. Yokenella was previously isolated from the intestinal tracts of insects and faeces of insect-feeding animals, including bats [48, 49]; therefore it is probably related to this kind of diet. Vagococcus, here observed only in carnivores, has been recovered from animals, water, soil and human sources [50]. S.marcescens and K. oxytoca were found on all five dietary guilds. S.marcescens was reported in other studies and various dietary guilds, including frugivores [7], sanguivores [8, 43], and insectivores[11, 35]. K. oxytoca was previously reported in frugivores [7, 51] and insectivores[14, 52]; however, K. oxytoca was highly related to vespertilionid (insectivores) bats rather than to any other Australian mammal on previous studies [53].

Antibiotic sensitivity

Generally, the resistance to antibiotics found on our samples was related to the intrinsic resistance of the tested species [54] and independent of dietary guilds of the bats. The species P. aeruginosa and S. maltophilia did not show any resistance besides their expected intrinsic resistance patterns. The species A. baumannii and Stenotrophomonas sp. showed resistance to the antibiotics ciprofloxacin and ceftazidime, respectively; those resistances are not intrinsic and could be acquired from both clinical or environmental antibiotic resistance genes sources, disseminated on the environment. A. baumannii is one of the most important pathogens in hospitals, and the development of multidrug-resistant strains has become of great concern for antibiotic therapies. Ciprofloxacin is a very potent antibiotic used as first line agaist A. baumannii infections [5557] and previous studies have isolated high rates of ciprofloxacin resistant strains of A. baumannii [5860]. The development of resistance on A. baumannii strains has been previously related to mutations in the quinolone resistance determining regions and efflux pump mechanisms [58, 59]. The only strain of A. baumannii was isolated from an insectivore bat that was found during the day on the floor of a Visitors Center on CBSP, and the possible contact of the bat with human leavings could have influenced on the acquiring of resistant strains. The Stenotrophomonas sp. resistant strains were found on frugivorous and nectarivorous and could outcome from the contact with water or fruits and even casual ingestion of insects [1921].

Additionally, once the contact with anthropic and agricultural environments is one of the major sources of acquired resistance, the activity pattern and diet of carnivores, insectivores and sanguivores bats would make them more susceptible to exposure to antimicrobials [21, 61, 62]. Therefore, it could be expected that carnivore, insectivore and sanguivore bats would present a higher rate of antibiotic resistant strains, when compared to frugivores and nectarivores. However, this pattern is not clear when we analyse the results obtained for the antibiograms of the abundant bacteria species E. coli, K. oxytoca and S. marcescens to compare the dietary guilds. A larger number of samples and complementary analysis could help to better evaluate this question.

The tested K. oxytoca isolates presented only one resistant strain (5%) and none of the S. marcescens isolates presented any resistance besides the intrinsic ones. The small rates of resistant bacteria observed on CBSP in consistently different from those observed in other studies conducted on areas influenced by anthropic activities [17, 20, 63, 64]. A study conducted on Krakatau Islands found a great number of resistant bacteria on local bats and rats, which they correlated, in part, to anthropic influence on the local islands [20]. The antibiotic-resistance pattern found for E. coli isolates from Nigerian bats also showed a great number of resistant isolates; the resistance was attributed to the use of antibiotics on poultry feed or on poultry itself [17]. Analyzing all of our tested isolates, 71 out of 81 (87%) did not present any resistance besides the expected from the intrinsic pattern, which could be related to the effectiveness of CBSP on the conservation of the wildlife and environment present on the preserved area of the Park. Once some of the sampling sites were close to the Park limits and some Brazilian bats are know to forage on distances of 0.5 to 15 km [6567], it seens that bats from CBSP prefer to forage on the pristine environments rather than anthropized surroundings. Moreover, the restriction of the contact to antibiotics would not lead to the decline of acquired resistances; therefore, it is reasonable to expect that resistance patterns on CBSP were always similar to the results presented here and no previous chronic exposures existed [68]. This result is in agreement to previous studies [22, 69], which reported a lack of human-acquired antibiotic resistance on environments with minimal anthropic influence and no chronic exposure to antibiotics.

Besides direct exposure to antibiotics, bacterial resistance can be originated through horizontally mobile elements such as conjugative plasmids, integrons and transposons [21]. Therefore, the low rate of resistance found on the Enterobacteriaceae from CBSP also suggests a small probability of the diffusion of acquired resistance on the Park. Many authors reported that bacteria from remote areas could work as sentinels and help to evaluate the impact of anthropic pressure on wildlife and the role of wild-species and natural environments on the process of resistance acquiring, which includes not only the exposure to antibiotics but also horizontal transference [2123]. Our findings reinforce the need of monitoring antimicrobial resistance in wildlife from remote areas, appearing to be an effective tool to evaluate the environment responses to anthropic pressures. On this way, more efforts should be carried out on the Park to better evaluate local resistance patterns, the impact that the human activites of the surroundings on the Park environment and the role of wildlife as reservoirs of resistant bacteria.

Acknowledgments

We are thankful to the staff of Parque Estadual Carlos Botelho for the logistical support, and to the Proteomics Laboratory from the Universidade Federal de São Paulo (UNIFESP) to provide the MALDI Biotyper for this research.

References

  1. 1. Fenton MB, Simmons N. Bats: A World of Science and Mystery. Chicago: The University of Chicago Press; 2014.
  2. 2. Herrera GL, Gutierrez E, Hobson KA, Altube B, Díaz WG, Sánchez-Cordero V. Sources of assimilated protein in five species of New World frugivorous bats. Oecologia. 2002;133: 280–287. pmid:28466224
  3. 3. Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006;19: 531–545. pmid:16847084
  4. 4. Peracchi AL, Lima IP, Reis NR, Nogueira MR, Ortencio Filho H. Ordem Chiroptera. In: Reis NR, Peracchi AL, Pedro WA, Lima IP, editors. Mamíferos do Brasil. Londrina: Editora Universidade Estadual de Londrina; 2006. pp. 153–230.
  5. 5. Souza V, Rocha M, Valera A, Eguiarte LE. Genetic structure of natural populations of Escherichia coli in wild hosts on different continents. Appl Environ Microbiol. 1999;65: 3373–3385. pmid:10427022
  6. 6. Costa LP, Leite YLR, Mendes SL, Ditchfield AD. Mammal Conservation in Brazil. Conserv Biol. 2005;19: 672–679.
  7. 7. Daniel DS, Ng YK, Chua EL, Arumugam Y, Wong WL, Kumaran JV. Isolation and identification of gastrointestinal microbiota from the short-nosed fruit bat Cynopterus brachyotis brachyotis. Microbiol Res. 2013;168: 485–496. pmid:23706760
  8. 8. Galicia J, Marcela M, Buenorostro SA, García GJ. Diversidad específica bacteriana en murciélagos de distintos gremios alimenticios en la sierra sur de Oaxaca, México. Rev Biol Trop. 2014;62: 1673–1681.
  9. 9. Banskar S, Mourya DT, Shouche YS. Bacterial diversity indicates dietary overlap amoung bats of different feeding habits. Microbiol Res. 2016;182: 99–108. pmid:26686618
  10. 10. Dietrich M, Kearney T, Seamark EC, Markotter W. The excreted microbiota of bats: evidence of niche specialisation based on multiple body habitats. FEMS Microbiol Lett. 2017;364: 1–7. pmid:27986822
  11. 11. Vengust M, Knapic T, Weese JS. The fecal bacterial microbiota of bats; Slovenia. PLoS ONE 2018;13: e0196728. pmid:29791473
  12. 12. Klite PD. Intestinal bacteria flora and transit time of three neotropical bat species. J Bacteriol. 1965;90: 375–379. pmid:14329450
  13. 13. Pinus M, Müller HE. Enterobacteria of bats (Chiroptera). Zentralbl Bakteriol. 1980;247: 315–322.
  14. 14. Di Bella C, Piraino C, Caracappa S, Fornasari L, Violani C, Zava B. Enteric microflora in Italian Chiroptera. J Mt Ecol. 2003;7: 221–224.
  15. 15. Whitaker JO, Dannelly HK Jr., Prentice DA. Chitinase in insectivorous bats. J Mammal. 2004;85: 15–18.
  16. 16. Mühldorfer K, Wibbelt G, Haensel J, Riehm J, Speck SY. Species isolated from bats, Germany. Emerg Infect Dis J. 2010;16: 578–580.
  17. 17. Oluduro AO. Antibiotic-resistant commensal Escherichia coli in faecal droplets from bats and poultry in Nigeria. Vet Ital. 2012;48: 297–308. pmid:23038076
  18. 18. Carrillo-Araujo M, Taş N, Alcántara-Hernández RJ, Gaona O, Schondube JE, Medellín RA, et al. Phyllostomid bat microbiome composition is associated to host phylogeny and feeding strategies. Front Microbiol. 2015;6: 1–9.
  19. 19. Remington JS, Schimpff SC. Please don’t eat the salads. N Engl J Med. 1981;304: 433–435. pmid:7453770
  20. 20. Graves SR, Kennelly-Merrit SA, Tidermann CR, Rawlinson PA, Harvey KJ, Thornton IWB. Antibiotic-resistance patterns of enteric bacteria of wild mammals on the Krakatau Islands and West Java, Indonesia. Phil Trans R Soc Lond B. 1988;322: 339–353.
  21. 21. Radhouani H, Silva N, Poeta P, Torres C, Correia S, Igrejas G. Potential impact of antimicrobial resistance in wildlife, environment and human health. Front Microbiol. 2014;5: 1–12.
  22. 22. Thaller MC, Migliore L, Marquez C, Tapia W, Cedeno V, Rossolini GM, et al. Tracking acquired antibiotic resistance in commensal bacteria of Galapagos land iguanas: no man, no resistance. PLoS ONE 2010;5: e8989. pmid:20126545
  23. 23. Smith S, Wang J, Fanning S, McMahon BJ. Antimicrobial resistant bacteria in wild mammals and birds: a coincidence or cause for concern? Ir Vet J. 2014;67: 1–3.
  24. 24. Paulo São (State). Plano de Manejo do Parque Estadual Carlos Botelho. São Paulo: Secretaria do Meio Ambiente/Instituto Florestal; 2008.
  25. 25. Veen SQ, Claas ECJ, Kuijper EJ. High-Through put Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories. J Clin Microbiol. 2010;48: 900–907. pmid:20053859
  26. 26. Schmitt BH, Cunningham SA, Dailey AL, Gustafson DR, Patel R. Identification of anaerobic bacteria by Bruker Biotyper matrix assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation. J Clin Microbiol. 2013;51: 782–786. pmid:23254126
  27. 27. Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1966;45: 493–496. pmid:5325707
  28. 28. NCCLS. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard, 2nd Ed. NCCLS document M31-A2. Pennsylvania: NCCLS; 2002.
  29. 29. Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JG. Worlds within worlds: evolution of the vertebrate gut microbiota. Nat Rev Microbiol. 2008;6: 776–788. pmid:18794915
  30. 30. Giannini NP, Kalko EKV. The guild structure of animalivorous leaf–nosed bat of Barro Colorado Island, Panama, revisited. Acta Chiropt. 2005;7: 131–146.
  31. 31. Williams SL, Genoways HH. Subfamily Phyllostominae. In: Gardner AL, editor. Mammals of South America, Volume I. Marsupials, Xenarthrans, Shrews, and Bats. Chicago: University of Chicago Press; 2008. pp. 255–300.
  32. 32. Novaes RLM, Souza RF, Ribeiro EA, Siqueira AC, Greco AV, Moratelli R. First evidence of frugivory in Myotis (Chiroptera, Vespertilionidae, Myotinae). Biodivers Data Journal. 2015;3: 1–5. pmid:26696768
  33. 33. Conn HJ, Dimmick I. Soil bacteria similar in morphology to Mycobacterium and Corynebacterium. J Bacteriol. 1947;54: 291–303. pmid:16561362
  34. 34. Studier EH, Lavoie KH. Microbial involvement in scent production in noctilionid bats. J Mammal. 1984;65: 711–714.
  35. 35. Voigt CC, Caspers B, Speck S. Bats, bacteria, and bat smell: sex-specific diversity of microbes in a sexually selected scent organ. J Mammal. 2005;86: 745–749.
  36. 36. Micalizzi EW, Mack JN, White GP, Avis TJ, Smith ML. Microbial inhibitors of the fungus Pseudogymnoascus destructans, the causal agent of white-nose syndrome in bats. PloS ONE. 2017;12: e0179770. pmid:28632782
  37. 37. Bennett JS, Bratcher HB, Brehony C, Harrison OB, Maiden MC. The Genus Neisseria. In: Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F, editors. The Prokaryotes. Berlin: Springer; 2014. pp. 881–900.
  38. 38. Segers P, Vancanneyt M, Pot B, Torck U, Hoste B, Dewettinck D, et al. Classification of Pseudomonas diminuta Leifson and Hugh 1954 and Pseudomonas vesicularis Büsing, Döll, and Freytag 1953 in Brevundimonas gen. nov. as Brevundimonas diminuta comb. nov. and Brevundimonas vesicularis comb. nov., respectively. Int J Syst Evol Microbiol. 1994;44: 499–510. pmid:8068543
  39. 39. Wallace CC, Yund PO, Ford TE, Matassa KA, Bass AL. Increase in antimicrobial resistance in bacteria isolated from stranded marine mammals of the Northwest Atlantic. Ecohealth. 2013;10: 201–210. pmid:23636484
  40. 40. Ewing WH, McWhorter AC, Escobar MR, Lubin AH. Edwardsiella, a new genus of Enterobacteriaceae based on a new species, E. tarda. Int J Syst EvolMicrobioly. 1965;15:33–38.
  41. 41. Van Damme LR, Vandepitte J. Isolation of Edwardsiella tarda and Plesiomonas shigelloides from mammals and birds in Zaire. Rev Elev Med Vet Pays Trop. 1984;37: 145–151. pmid:6531491
  42. 42. Oliveira GR, Porto GS, Lima IP. Subfamília Desmodotinae Wagner 1840. In: Reis NR, Peracchi AP, Batista CB, Lima IP, Pereira AD, editors. História Natural dos Morcegos Brasileiros: Chave de Identificação de Espécies. Rio de Janeiro: Technical Books Editora; 2017. pp. 109–115.
  43. 43. Chaverri G. Flora bacteriana aeróbica del tracto digestivo del vampiro común, Desmodus rotundus (Chiroptera: Phyllostomidae). Rev Biol Trop. 2006;54: 717–724. pmid:18491612
  44. 44. Niedziela T, Lukasiewicz J, Jachymek W, Dzieciatkowska M, Lugowski C, Kenne L. Core Oligosaccharides of Plesiomonas shigelloides O54: H2 (Strain CNCTC 113/92) Structural and serological analysis of the lipopolysaccharide core region, the o-antigen biological repeating unit, and the linkage between them. J Biol Chem. 2002;277: 11653–11663. pmid:11796731
  45. 45. Handley CO Jr.. Mammals of the Smithsonian Venezuelan Project. Brigham Young Univ Sci Bull. 1976;20: 1–89.
  46. 46. López-González C, Presley SJ, Owen RD, Willig MR.Taxonomic status of Myotis (Chiroptera: Vespertilionidae) in Paraguay. J Mammal. 2001;82: 138–160.
  47. 47. Meyer CFJ, Weinbeer M, Kalko EKV. Home–range size and spacing patterns of Macrophyllum macrophyllum (Phyllostomidae) foraging over water. J Mammal. 2005;86: 587–598.
  48. 48. Cassel-Beraud AM, Richard C. The aerobic intestinal flora of the microchiropteran bat Chaerephon pumila in Madagascar. Bull Soc Pathol Exot Filiales. 1988;81: 806–810. pmid:3240566
  49. 49. Oliveira SMP, Morais BA, Gonçalves CA, Giordano-Dias CM, Vilela ML, Brazil RP, et al. Digestive tract microbiota in female Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) feeding on blood meal and sucrose plus blood meal. Cad Saude Publica. 2001;17: 229–232. pmid:11241946
  50. 50. Lawson PA. The genus Vagococcus. In: Holzapfel WH, Wood BJB, editors. Lactic Acid Bacteria: Biodiversity and Taxonomy. West Sussex: John Wiley & Sons, Ltd; 2014. pp. 229–237.
  51. 51. Anand AAP, Sripathi K. Digestion of cellulose and xylan by symbiotic bacteria in the intestine of the Indian flying fox (Pteropus giganteus). Comp Biochem Physiol AMol Integr Physiol. 2004;139: 65–69. pmid:15471682
  52. 52. Gordon DM, Lee J. The genetic structure of enteric bacteria from Australian mammals. Microbiol. 1999;145: 2673–2682. pmid:10537189
  53. 53. Gordon DM. Geographical structure and host specificity in bacteria and the implications for tracing the source of coliform contamination. Microbiol. 2001;147: 1079–1085. pmid:11320111
  54. 54. Leclercq R, Cantón R, Brown DF, Giske CG, Heisig P, MacGowan AP, et al. EUCAST expert rules in antimicrobial susceptibility testing. Clin Microbiol Infect. 2011;19: 141–160. pmid:22117544
  55. 55. Higgins PG, Wisplinghoff H, Stefanik D, Seifert H. Selection of topoisomerase mutations and overexpression of adeB mRNA transcripts during an outbreak of Acinetobacter baumannii. J Antimicrob Chemother 2004;54:821–3. pmid:15355942
  56. 56. Meric M, Kasap M, Gacar G, Budak F, Dundar D, Kolayli F, et al. Emergence and spread of carbapenem-resistant Acinetobacter baumannii in a tertiary care hospital in Turkey. FEMS Microbiol Lett. 2008;282: 214–218. pmid:18371065
  57. 57. Benjamin AE, Ahmed H, Sebastian GBA. The Rise of Carbapenem-Resistant Acinetobacter baumannii. Curr Pharm Des. 2013;19: 223–238. pmid:22894617
  58. 58. Ardebili A, Lari AR, Talebi M. Correlation of ciprofloxacin resistance with the AdeABC efflux system in Acinetobacter baumannii clinical isolates. AnnLab Med. 2014;34: 433–438. pmid:25368818
  59. 59. Maleki MH, Jalilian FA, Khayat H, Mohammadi M, Pourahmad F, Asadollahi K, et al. Detection of highly ciprofloxacin resistance Acinetobacter baumannii isolated from patients with burn wound infections in presence and absence of efflux pump inhibitor. Mædica (Burchar), 2014;9: 162–167.
  60. 60. Chen LK, Kuo SC, Chang KC, Cheng CC, Yu PY, Chang CH, et al. Clinical Antibiotic-resistant Acinetobacter baumannii strains with higher susceptibility to environmental phages than antibiotic-sensitive strains. Sci Rep. 2017;7: 6319. pmid:28740225
  61. 61. Alonso CA, González-Barrio D, Tenorio C, Ruiz-Fons F, Torres C. Antimicrobial resistance in faecal Escherichia coli isolates from farmed red deer and wild small mammals. Detection of a multiresistant E. coli producing extended-spectrum beta-lactamase. Comp Immunol Microbiol Infect Dis. 2016;45: 34–39. pmid:27012919
  62. 62. Reis NR, Peracchi AL, Batista CB, Lima IP, Pereira AD. História Natural dos Morcegos Brasileiros: Chave de Identificação de Espécies,1st ed. Rio de Janeiro: Technical Books; 2017.
  63. 63. Sherley M, Gordon DM, Collignon PJ. Variations in antibiotic resistance profile in Enterobacteriaceae isolated from wild Australian mammals. Environ Microbiol. 2000;2: 620–631. pmid:11214795
  64. 64. Costa D, Poeta P, Sáenz Y, Vinué L, Coelho AC, Matos M, et al. Mechanisms of antibiotic resistance in Escherichia coli isolates recovered from wild animals. Microb Drug Resist. 2008;14: 71–77. pmid:18321208
  65. 65. Mello MAR, Kalko EKV, Silva WR. Movements of the bat Sturnira lilium and its role as a seed disperser of Solanaceae in the Brazilian Atlantic forest. J Trop Ecol. 2008;24: 225–228.
  66. 66. Peracchi AL, Lima IP, Reis NR, Nogueira MR, Ortencio Filho H. Ordem Chiroptera. In: Reis NR, Peracchi AL, Pedro WA, Lima IP, editors. Mamíferos do Brasil, 2nd ed. Londrina: Editora Universidade Estadual de Londrina; 2011. pp. 155–234.
  67. 67. Sekiama ML, Rocha VJ, Peracchi AL. Subfamília Carolliinae. In: Reis NR, Fregonezi MN, Peracchi AL, Shibatta OA. Morcegos do Brasil: guia de campo. Rio de Janeiro: Technical Books Editora; 2013. pp. 109–133.
  68. 68. Gilliver MA, Bennett M, Begon M, Hazel SM, Hart CA. Enterobacteria: antibiotic resistance found in wild rodents. Nature. 1999;401: 233. pmid:10499578
  69. 69. Österblad M, Norrdahl K, Korpimaki E, Huovinen P. How wild are wild mammals? Nature. 2001;409: 37–38.