Bats (Mammalia, Chiroptera) play an important ecological role in many different ecosystems, especially as pollinators and seed dispersers (Ramírez-Fráncel et al., 2022), as well for their importance as potential vectors and natural reservoirs for several pathogens (e.g., virus, bacteria, fungi) (Han et al., 2015; Allocati et al., 2016) and hosts for ectoparasites (e.g., flies, ticks, mites) (Stuckey et al., 2017). In addition to their large number of species (1,460 worldwide; 181 in Brazil) (Garbino et al., 2022; Upham et al., 2023) and broad geographic distribution, bats can also live in various habitats (wild, rural and urban) and have different dietary strategies (insectivore, carnivore, omnivore, frugivore, nectarivore, piscivore and hematophagy), which favor the exposure to a wide variety of environments and microorganisms (Avena et al., 2016; Alcantara et al., 2022).

Just like other mammals, many biological functions, such as immune system efficiency (Zhang et al., 2013; Thaiss et al., 2016), digestion, and individual development and growth, are influenced by the coevolution of these animals with microorganisms, especially bacteria (Lutz et al., 2022). For example, bats have been implicated as the likely ancestral hosts of all Bartonella bacteria associated with mammals and seem to have played a significant role in the initial geographic spread of this genus. Their coevolution with Bartonella might have had a profound impact on the bacteria evolutionary diversification and their transmission to other orders of mammals, including humans (McKee et al., 2021).

Since bats live in association with many different bacteria, the composition of their microbiomes is directly influenced by environmental (e.g., geographic location, climatic conditions) (Lemieux-Labonté et al., 2016; Lutz et al., 2022), behavioral (e.g., diet, frequented habitats) (Carrillo-Araujo et al., 2015; Corduneanu et al., 2023) and individual (e.g., species, age) features (Hughes et al., 2018; Jones et al., 2022).

While the microbiome’s significance in bat adaptation, survival and evolution is undeniable, many bat-associated microorganisms can be pathogenic to other animals, including humans (Dimkić et al., 2021). Among pathogenic agents, the Phylum Proteobacteria stands out and comprises Gram-negative bacteria with several different genera of phototrophic, plant- and arthropod-associated symbionts, as well as pathogens. This phylum is sub-divided into six different classes and comprises important pathogenic/zoonotic genera commonly detected in bats, such as Anaplasma, Ehrlichia, Neorickettsia (Alphaproteobacteria, Anaplasmataceae), Bartonella (Alphaproteobacteria, Bartonellaceae) and Coxiella (Gammaproteobacteria, Coxiellaceae) (Cicuttin et al., 2013; Rizzatti et al., 2017; Ferreira et al., 2018; Ikeda et al., 2020, 2021).

The microbiome composition of many biological samples (feces, skin, urine, organs, fluids) collected from over 200 bat species (mostly insectivorous bats) from 32 different countries have already been assessed (Jones et al., 2022). Many authors report a high abundance of Proteobacteria in bat gut flora. In South Africa, Mehl et al. (2021) analyzed the composition of intestinal bacteria of insectivorous bats (Neronomica nana) sampled on wastewater treatment work and reference sites. The authors reported Proteobacteria abundance (range of 19.9 to 46.6%) comprising four different classes (Alphaproteobacteria, Betaproteobacteria, Epsilonproteobacteria and Gammaproteobacteria). Additionally, the authors reported that the bat sampling site was the most influential parameter on microbiome constitution and diversity (Mehl et al., 2021).

The gut microbiome composition of two insectivorous bat species (Rhinolophus sinicus and Myotis altarium) was described and compared according to different gut sections (i.e., small and large intestine) and feces. Proteobacteria was the dominant phylum in both bat species (average of 43.5% in R. sinicus and 42.5% in M. altarium), comprising over half of the microbiome composition in the large intestine of M. altarium and feces of R. sinicus (Wu et al., 2019). Indeed, Hughes et al. (2018) demonstrated the high presence of Proteobacteria (42.8–57.4%) in bat guts in comparison to other mammals, when analyzing rectal swab samples from Myotis myotis insectivore bats from western France.

Although the majority of studies on bat-associated microbiomes have been performed on gut-associated samples, such as guano or small and large intestines, it is crucial to elucidate the microorganism composition of other body sites aiming at gaining a better understanding of coevolution, ecology and pathogen transmission (Lutz et al., 2019; Dimkić et al., 2021). For instance, potentially zoonotic bacteria were discovered in saliva, urine and feces of insectivorous bats from South Africa (Dietrich et al., 2017). Proteobacteria were detected in all examined body sites with different sequence abundances (>90% in saliva; 32.6% in urine; 31.2% in feces), including some in clades with established zoonotic arthropod-borne bacteria, such as Bartonella, Rickettsia, and Coxiella (Dietrich et al., 2017).

The bat skin microbiome is also inhabited by a high abundance of Proteobacteria. Winter et al. (2017) used whole body cotton swabs from cave bats in the southwestern United States to evaluate skin and fur microbiome. Proteobacteria comprised 24.6% of the microbiota, ranging from 8.15 to 39.98%, with Alphaproteobacteria representing the most abundant class. A higher abundance of Proteobacteria in the skin microbiome of bats (> 60%), using whole body cotton swabs, was also reported in the Northeastern region of the United States (Avena et al., 2016).

Proteobacteria showed abundance ranging from 1.31 to 99.7% among 10 heart samples (8 individual and 2 pooled) of insectivorous bats from Central and South Eastern Europe (Corduneanu et al., 2021). Furthermore, specific PCR assays based on the gltA and rpoB encoding genes and ITS region proved the presence of Bartonella sp. in one pooled heart sample; additionally, PCR assays targeting the gltA and ompA genes confirmed the presence of Rickettsia sp. in one individual sample.

In Brazil, studies on the composition of bat-associated microbiomes are scarce. Cláudio et al. (2018) described the oral and rectal cavity-associated microbiota from bats sampled in southeastern Brazil, which were divided into five feeding guilds (insectivores, frugivores, nectarivores, carnivores, and hematophagous). After isolating bacteria, taxon identification was performed by matrix-assisted laser desorption/ionization (MALDI-TOF) technique. Proteobacteria represented over 87% of the microbiome from bats presenting all dietary strategies, without further identification of the bacterial classes/genera. Additionally, previous studies in Brazil have already detected, by using PCR assays followed by sequencing and phylogenetic inferences, potential zoonotic Proteobacteria in Brazilian bats, namely Bartonella spp. (Ikeda et al., 2017, 2020; André et al., 2019), Ehrlichia sp. (Ikeda et al., 2021), Neorickettsia sp. (Ikeda et al., 2021), Anaplasma spp. phylogenetically associated to A. phagocytophilum and A. bovis (Ikeda et al., 2021), and Coxiella burnetti (Ferreira et al., 2018).

Moreover, bats are hosts for many hematophagous ectoparasites, including flies (Diptera: Streblidae and Nycteribiidae), soft ticks (Ixodida: Argasidae) and mites (Mesostigmata: Macronyssidae and Spinturnicidae), whose microbiome composition is also of great significance (Szentiványi et al., 2019). Although the diversity of bacteria that comprise those microbiomes is mainly associated with the evolution and ecology of ectoparasite species, recent studies suggest that this composition is also influenced by environmental factors and host ecology (Speer et al., 2022). The investigation of the microbiome composition of seven different Nycteribiidae bat fly species in the Malagasy Region revealed that Alphaproteobacteria represented 17% of the identified species, while Betaproteobacteria and Gammaproteobacteria constituted 3 and 78%, respectively, with 55% of the sequences corresponding to Wolbachia, 26% to Bartonella, and 17% to Rickettsia. Phylogenetic analyses, based on the concatenation of fbpA, ftsZ and hcpA genes, positioned Wolbachia sequences into the F subgroup, which comprises strains associated with many different arthropod orders (Diptera, Scorpiones, Hemiptera, Coleoptera, Phthiraptera). The gltA-based phylogeny clustered the Bartonella genotypes into five different groups along with similar bat and bat-fly associated sequences (Wilkinson et al., 2016).

In Brazil, Speer et al. (2022) reported a significant difference in the bacterial microbiome composition of Streblidae and Nycteribiidae flies collected from bats in the Atlantic Forest. Based on next generation sequencing (NGS) of the V4 region of the 16S rRNA gene, Alphaproteobacteria were highly abundant in nycteribiid bat flies, mainly Wolbachia and Bartonella (> 75%). Both genera were also detected in streblid bat flies, but at a much lower prevalence (> 10%). Arsenophonus (Gammaproteobacteria) showed a higher abundance in streblids (> 70%) when compared to nycteribiid flies (> 1%).

Although the microbiome composition of bat mites has not yet been studied, these arthropods are known to act as hosts for many pathogenic agents. Two studies carried out in China demonstrated that Spinturnicidae bat mites (Spinturnix sp. and Eyndhovenia sp.) collected from insectivorous bats harbored Bartonella genotypes associated with Bartonella mayotimonensis (Han et al., 2022) as well as hemoplasma genotypes, including one with high similarity to the human-pathogenic ‘Candidatus Mycoplasma haemohominis’ (Wang et al., 2023). In midwestern Brazil, Ikeda et al. (2020) demonstrated the presence of Ehrlichia sp., closely related to E. ruminantum and E. minasensis, in Macronyssidae (Steatonyssus sp.) and Spinturnicidae (Periglischrus sp., Periglischrus torrealbai and Periglischrus acustisternus) mites collected from non-hematophagous bats and analyzed using PCR assays based on the dsb gene.

Considering the role of bats as carriers of pathogens with medical and veterinary importance (Federici et al., 2002; Van Brussels and Holmes, 2002), the assessment of the bacterial microbiome composition of bats and associated ectoparasites can contribute to the understanding of the mechanisms involved in the adaptability and coevolution of this group of mammals with symbionts and pathogenic agents. Therefore, the present work aimed to investigate the bacterial microbiome composition of non-hematophagous bats and associated ectoparasites sampled in midwestern Brazil.

The microbiome composition of both oral (ranging from 13 to 86%) and rectal (ranging from 13 to 99%) swab samples showed that Gammaproteobacteria was the most abundant bacterial class, accounting for 42 and 70% of bacterial taxa, respectively. In addition to Gammaproteobacteria, other bacterial classes were also found in high relative abundance in both sort of samples, including (Firmicutes) Bacilli (26% in oral and 6% in rectal swabs), (Proteobacteria) Betaproteobacteria (8% in oral and 2.3% in rectal swabs) and (Actinomycetota) Actinomycetia (4% in oral and rectal swabs).

Lutz et al. (2019) also found a high relative abundance of Gammaproteobacteria in the oral and distal gut microbiome of insectivore and frugivore bats from Uganda and Kenya. Our findings were also similar to those found in previous studies performed in Mexico (Carrillo-Araujo et al., 2015), France (Hughes et al., 2018), southeastern Brazil (Cláudio et al., 2018), China (Xiao et al., 2019), and South Africa (Mehl et al., 2021), which have also found a high relative abundance of Proteobacteria in the gut microbiome of bats. Indeed, chiropterans have a higher proportion of Proteobacteria in their gut microbiome than other mammals, harboring a common core gut flora, irrespective of their geographic location or dietary habits. The reason for this difference is likely to be linked to the bat adaptation to flight and their rapid intestinal transit (Hughes et al., 2018; Nishida and Ochman, 2018; Jones et al., 2022).

Among the bacterial genera that were found in high abundance in rectal swab samples are Enterobacter, Klebsiella, Escherichia, and Serratia (Gammaproteobacteria: Enterobacteriaceae) as well as Enterococcus and Streptococcus (Bacilli: Streptococcaceae), which have also been detected in the rectal microbiome of Myotis myotis bats from Poland (Rózalska et al., 1998) and France (Hughes et al., 2018) and in the distal gut microbiome of Rhinolophus monoceros bats from India (Selvin et al., 2019). Although these bacteria play an essential role in the digestive system functioning, being present in the gut flora of most mammals, many may act as opportunistic pathogens to humans and animals (e.g., Escherichia coli, Salmonella sp., Vibrio cholerae, Coxiella burnetii, Pseudomonas aeruginosa, Yersinia pestis) (Garrity, 2007). Indeed, multidrug-resistant Enterococcus faecalis and Escherichia coli strains, which have been associated with human diseases, have already been found in frugivorous bats rectal and guano samples from Poland (Nowakiewicz et al., 2020, 2021). Similarly, novel strains of Klebsiella africana and Klebsiella pneumoniae were described in frugivorous Pteropus poliocephalus bats from Australia (McDougall et al., 2021), and septic-encephalitis causing Streptococcus dysgalactiae were detected in Desmodus rotundus from Brazil (Mioni et al., 2018). While the exact role of bats in transmitting pathogenic agents to humans is not yet fully understood, it is crucial that researchers conduct extensive studies on bat microbiomes and possible bat-bacteria associations. These studies will help predict the potential for bacterial antibiotic resistance and the transmission of pathogens to other animals and humans.

Similar to other mammals, bats have a diverse oral microbiome that is usually dominated by Firmicutes and Proteobacteria. Some studies have suggested that the oral microbiome of bats may be shaped by their diet, host phylogeny and environmental conditions (Carrillo-Araujo et al., 2015). Previous studies involving bats from Brazil (Cláudio et al., 2018) and South Africa (Dietrich et al., 2017) support our findings, indicating Proteobacteria as the most abundant phylum in oral samples. In contrast to our study, Presley et al. (2021) reported a predominance of Actinobacteria (30.6%) and Firmicutes (29.2%) in the oral microbiome of frugivorous and herbivorous bats from Puerto Rico, with only 0.4% of the sequences corresponding to Proteobacteria. Such a discrepancy could be attributed to environmental fluctuations, including factors such as diet, temperature, and humidity. These fluctuations can impact the diversity of microorganisms present in the bat oral microbiome (Klausenstock, 2016).

Out of the genera detected in oral swab samples, Streptococcus, Neisseria, Actinomyces and Anaplasma stand out among the most abundant taxa. Indeed, Streptococcus and Neisseria species are usually found in the oral cavity associated with dental and mucosal surfaces and saliva in mammals (Bennett et al., 2014; Abranches et al., 2018). Although previous studies have not reported the presence of Anaplasma species in the oral microbiome of bats, Gerbáčová et al. (2020) detected Anaplasma in the guano microbiome of Myotis myotis and Rhinolophus hipposideros bats from Slovakia. Anaplasma phagocytophilum DNA has already been detected in guano from horseshoe bat (R. hipposideros) maternity roosts in France (Afonso and Goydadin, 2018). In agreement with the abovementioned authors, a possible explanation for such a finding could be a persistent infection of bats with Anaplasma, with release of Anaplasma DNA in bat feces or, alternatively, caused by the consumption of arthropods containing the bacteria.

Although in our study both types of samples had comparable predominant taxa in common, it was possible to determine a higher bacterial diversity in rectal swab samples (21 classes and 189 genera) in comparison to oral swab samples (18 classes and 157 genera). There can be a few reasons why the rectal microbiome of bats is more diverse than the oral microbiome. One conceivable explanation is that the digestive system of bats is designed to extract as many nutrients as possible from their food, and this may lead to a higher bacterial richness in their gut (Nishida and Ochman, 2018). The anatomical structure of the digestive system may also play a role in shaping the microbial communities in different parts of the intestine (Hughes et al., 2018). Additionally, as already mentioned, the oral microbiome can be subjected to more environmental fluctuations, directly interfering with the bacterial richness. However, further investigation is required to fully understand the factors contributing to the differences in bacterial diversity between the oral and rectal microbiomes of bats.

The microbiome composition of the analyzed Spinturnicidae and Macronyssidae mites exhibited differences. Actinomycetia showed high relative abundance in both families (24.2% in spinturnicids and 15.8% in macronyssids), while Alphaproteobacteria dominated in spinturnicids (67.2%) and Gammaproteobacteria in macronissids (78.9%). Although there is limited research on the microbiome composition of these mites, especially those parasitizing bats, their microbial community composition is directly influenced by factors such as living environment, host bloodmeal source, and host species (Guo et al., 2020).

While Wolbachia (Alphaproteobacteria) was the most abundant genus found in Spinturnicidae (65.3%), Arsenophonus (Gammaproteobacteria) was found in high abundance in Macronyssidae (77.3%) mites. These two genera encompass symbiotic bacterial species found in many arthropods, such as flies, beetles, butterflies, ticks, bees, and wasps. They can establish either a commensal or mutualistic relationship with their hosts, providing protection against other infectious agents, such as parasitic nematodes, parasitoid wasps, fungi, and viruses (Kautz et al., 2013) However, in certain species, they can also act as pathogens, eliminating male progeny, inducing male feminization, and causing cytoplasmic incompatibility (Kautz et al., 2013). Cutibacterium (Actinomycetia) was found to be highly abundant in the microbiome composition of both mite families. The Cutibacterium genus, along with other closely related genera, such as Acidipropionibacterium, Propionibacterium, and Pseudopropionibacterium, are commensal microorganisms found on the skin of humans and animals and have been associated with the development of acne and various other skin conditions in humans (Patrick and Mcdowell, 2015; Mayslich et al., 2021). Although there are no reports on the presence of these bacteria in the skin microbiome composition of bats, Díaz-Sánchez et al. (2019) observed that Cutibacterium was the most prevalent genus in the microbiome of wild-caught Ixodes ventalloi ticks from Italy. The authors highlighted that Cutibacterium is commonly found in the microbiome of blood-feeding arthropods. These bacteria release volatile molecules from the sebaceous glands of their vertebrate hosts, which attract these ectoparasites and may be associated with their host-seeking behavior. It is worth noting that Microbacterium (Actinomycetia), which was also relatively abundant in the analyzed mite microbiome composition, has been reported in laboratory reagent contamination and can be overrepresented in microbiome analyses as a result (Salter et al., 2014).

Herein, the presence of Bartonella and Anaplasma was observed, in much lower abundances, in both Spinturnicidae and Macronyssidae families, whereas Ehrlichia was found only in Macronyssidae mites. The presence of Bartonella spp. in Spinturnicidae mites and associated bats have already been reported in China (Han et al., 2022). Some of the genotypes identified were shared between mites and bats, which could result from bloodsucking behavior. Furthermore, our research group has already detected by PCR followed by Sanger sequencing Ehrlichia sp. closely related to Ehrlichia ruminantium and Ehrlichia minasensis in the same Spinturnicidade and Macronyssidae mites used in the present work (Ikeda et al., 2021). Such difference in the results achieved by NGS and PCR followed by Sanger sequencing might have been due to different target genes used for each technique (dsb in PCR versus 16S rRNA in NGS). Although there is no current evidence supporting mites as competent to vector the abovementioned agents to bats, understanding the microbiome composition of these arthropods is crucial for advancing research on their microbial community compositions, vector competence and the epidemiology of arthropod-borne diseases.

A high abundance of Mollicutes sequences (59.9%), followed by Alpha (36.6%) and Gammaproteobacteria (1.2%), was found in the microbiome composition of the analyzed Streblidae bat flies. In contrast to our research findings, Speer et al. (2022) documented that Gammaproteobacteria (mostly Arsenophonus) constituted the most abundant class in the microbiome composition of Streblidae flies from southeastern Brazil. This observation aligns with the findings of Morse et al. (2012), who also reported Gammaproteobacteria (Arsenophonus) as the dominant class in flies from various regions including Puerto Rico, Panama, Peru, Costa Rica, Dominican Republic, Mexico, and the United States. The two most abundant genera observed in the present study were Spiroplasma (Mollicutes) and Wolbachia (Alphaproteobacteria), both of which are commonly found symbionts in various arthropod species (Cisak et al., 2015).

Following those genera, Bartonella was the third most abundant genus present in the microbiome composition. Out of the 48 flies analyzed, Bartonella sequences were detected in 37 (77%) specimens. Streblidae flies have already been identified as important hosts for many Bartonella genotypes and are possibly capable of transmitting the bacteria vertically (between themselves) or horizontally (between bats) (Morse et al., 2012; Do Amaral et al., 2018; Braga et al., 2020; Ikeda et al., 2020). Despite the high abundance of Bartonella sequences detected in Streblidae flies, we were unable to establish a connection between the microbiome of bat flies and bat oral and fecal swabs. Our findings revealed that only two bat swabs, one oral and one rectal, showed the presence of Bartonella. The observed lack of connection between infected flies and bats in terms of Bartonella sequences could potentially be attributed to the type of sample analyzed. It is worth noting that the prevalence of Bartonella in bats varies considerably, ranging from 7.3 to 54.4%, depending on the bat species, location and dietary niches and has already been documented in Africa, Americas, Asia and Europe (Stuckey et al., 2017). Furthermore, Bartonella species are known to be intraerythrocytic agents, with higher prevalence in blood-related samples. Since our study focused on oral and rectal swabs, which may not directly reflect the presence of Bartonella circulating in a bat bloodstream, it is possible that the detection of Bartonella in oral and rectal samples was limited. To obtain a more comprehensive understanding of the relationship between infected flies and bats, further investigations utilizing blood-related samples are much needed.

In summary, investigating the microbiome composition of bats and their ectoparasites provides valuable descriptive insights to guide future studies regarding the ecological dynamics of these organisms, as well as the potential for pathogen transmission and anti-microbial resistance. Future research should focus on understanding the interactions among bats, ectoparasites, and their associated microbiomes to better comprehend impacts on public health and wildlife conservation efforts.

The present study reports the oral and rectal microbiome composition of non-hematophagous bats and associated ectoparasites (Streblidae flies and Macronyssidae and Spinturnicidae mites) from Midwestern Brazil. In addition to the presence of symbionts, it was possible to observe the occurrence of bacterial classes and genera that include some species of animal and human medical importance. The microbiome composition of both oral and rectal bat swab samples showed that Gammaproteobacteria was the most abundant bacterial class. Spiroplasma, Wolbachia and Bartonella represented the most abundant genera in Streblidae flies. While Wolbachia (Alphaproteobacteria) was the most abundant genus found in Spinturnicidae, Arsenophonus (Gammaproteobacteria) was found in high abundance in Macronyssidae mites. In addition to characterizing the microbiome of each sample at the class and genus taxonomic levels, our results reveal the potential presence of medically significant bacteria able to infect both animals and humans. This includes those identified in oral (Streptococcus and Anaplasma) and rectal swabs (Enterobacter, Klebsiella, Escherichia, Enterococcus, Streptococcus), Macronyssidae (Anaplasma, Bartonella, Ehrlichia) and Spinturnicidae (Anaplasma, Bartonella) mites as well as Streblidae flies (Spiroplasma, Bartonella). Besides expanding the knowledge of the bacterial microbiome of non-hematophagous bats and Streblidae flies from Brazil, the present work describes the bacterial community of bat-associated Macronyssidae and Spinturnicidae mites.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at this link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1011525?reviewer=i6m1ps7ghm3i13r476ov2270q0 repository, accession number PRJNA1011525.

The animal study was approved by ICMBio – SISBIO number 57450-1 and Ethics Committee on the Use of Animals (CEUA – FCAV/UNESP), registered under number 010050. The study was conducted in accordance with the local legislation and institutional requirements.

MA: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Writing – original draft, Writing – review & editing. PI: Conceptualization, Investigation, Methodology, Writing – review & editing. DL: Investigation, Visualization, Writing – original draft, Writing – review & editing. RA: Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. LC: Data curation, Formal analysis, Investigation, Methodology, Software, Writing – review & editing. DP: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – review & editing. JT: Methodology, Writing – review & editing. VM: Methodology, Writing – review & editing. GR: Writing – review & editing, Methodology. RC: Writing – review & editing, Methodology. EL: Methodology, Writing – review & editing. CO: Methodology, Writing – review & editing. HH: Methodology, Writing – review & editing. DB-B: Methodology, Writing – review & editing. RM: Investigation, Writing – review & editing. KB-L: Writing – review & editing, Methodology, Funding acquisition, Resources. CD: Methodology, Resources, Validation, Writing – review & editing. JD: Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing.