Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Animal Microbiome https://doi.org/10.1186/s42523-023-00229-9 RESEARCH Open Access The fecal bacterial microbiome of the Kuhl’s pipistrelle bat (Pipistrellus kuhlii) reflects landscape anthropogenic pressure Lourdes Lobato‑Bailón1*, Manuel García‑Ulloa2, Andrés Santos2, David Guixé3, Jordi Camprodon3,4, Xavier Florensa‑Rius3, Raúl Molleda3,5, Robert Manzano3, Maria P. Ribas1, Johan Espunyes1, Andrea Dias‑Alves1, Ignasi Marco1, Lourdes Migura‑Garcia6,7, Jaime Martínez‑Urtaza2† and Oscar Cabezón1,6† Abstract Background Anthropogenic disturbance has the potential to negatively affect wildlife health by altering food avail‑ ability and diet composition, increasing the exposure to agrochemicals, and intensifying the contact with humans, domestic animals, and their pathogens. However, the impact of these factors on the fecal microbiome composition of wildlife hosts and its link to host health modulation remains barely explored. Here we investigated the composition of the fecal bacterial microbiome of the insectivorous bat Kuhl’s pipistrelle (Pipistrellus kuhlii) dwelling in four environ‑ ture and diversity through full-length 16S rRNA metabarcoding using the nanopore long-read sequencer MinION™. mental contexts with different levels of anthropogenic pressure. We analyzed their microbiome composition, struc‑ We hypothesized that the bacterial community structure of fecal samples would vary across the different scenarios, showing a decreased diversity and richness in samples from disturbed ecosystems. Results The fecal microbiomes of 31 bats from 4 scenarios were sequenced. A total of 4,829,302 reads were obtained with a taxonomic assignment percentage of 99.9% at genus level. Most abundant genera across all scenarios were Enterococcus, Escherichia/Shigella, Bacillus and Enterobacter. Alpha diversity varied significantly between the four scenarios (p < 0.05), showing the lowest Shannon index in bats from urban and intensive agriculture landscapes, while the highest alpha diversity value was found in near pristine landscapes. Beta diversity obtained by Bray–Curtis distance showed weak statistical differentiation of bacterial taxonomic profiles among scenarios. Furthermore, core community analysis showed that 1,293 genera were shared among localities. Differential abundance analyses showed that the highest differentially abundant taxa were found in near pristine landscapes, with the exception of the family Alcaligenaceae, which was also overrepresented in urban and intensive agriculture landscapes. Conclusions This study suggests that near pristine and undisturbed landscapes could promote a more resilient gut microbiome in wild populations of P. kuhlii. These results highlight the potential of the fecal microbiome as a non- invasive bioindicator to assess insectivorous bats’ health and as a key element of landscape conservation strategies. Keywords Anthropogenic disturbance, Chiroptera, Wildlife, Conservation, Health indicator, Nanopore sequencing, MinION, 16S rRNA † Jaime Martínez-Urtaza and Oscar Cabezón contributed equally to this work. *Correspondence: Lourdes Lobato‑Bailón l.lobatobailon@gmail.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 2 of 14 Background conditions often entailing inaccessible sites or technical The majority of Earth’s ecosystems are dominated by training [9, 11, 12]. human activity and suffer significant and continuously The bacterial communities that inhabit the gut of all growing disturbances. Consequently, we are witness- animal species constitute the intestinal bacterial micro- ing a global biodiversity crisis in which current species biota. The genetic and structural elements (e.g., lipids, extinction rates far exceed background estimates [1, 2]. proteins) and metabolites (e.g., signaling molecules, inor- In turn, biodiversity sustains many ecosystem services ganic and organic molecules, toxins) produced by these needed by humans, and its loss is entailing clear eco- organisms in a specific environment, and their theater logical, public health and economic costs at a global of activity, are referred to as the bacterial microbiome scale [3]. [13]. Most of the knowledge about the normal gastro- The Order Chiroptera contributes to worldwide bio- intestinal microbial community of bats comes from tra- diversity with more than 1400 different species, making ditional microbiological studies [14, 15] and they are up one-fourth of terrestrial mammals [4]. This diverse particularly focused on the presence of infectious agents group of mammals inhabits all continents except Antarc- with zoonotic potential [16, 17]. Current studies on the tica and is responsible to promote and support ecosystem metagenomic profiling for fecal bacterial communities of health by means of pollination, seed dispersal and insect bat species have contributed to broadening the knowl- and vector-borne diseases regulations [5]. Nonetheless, it edge of species-specific microbial diversity, zoonotic is among the most endangered group of mammals with pathogens [18–25], diet and niche adaptation, and evolu- more than 200 bat species around the world considered tion [26–28]. threatened by the International Union for the Conserva- Two decades of microbiome studies in a wide range of tion of Nature [4]. species suggest that intestinal microbiota may contrib- Most of the current research efforts relevant to bat ute not only to the gut’s health but to the overall host’s conservation worldwide are linked to trends in species immunity [29, 30]. Among other characteristics, species- diversity and abundance, and distribution patterns [6–8]. rich communities in a given microbial system appear to Furthermore, investigation into the threats affecting bat be more resilient and to prevent establishment of exog- species has focused mainly on climate change, habitat enous microbes -including pathogens- than species-poor degradation and other related human disturbances such communities [31]. This bacterial richness further pro- as over-hunting, pollution, or collisions with wind energy motes a better functioning of the community by resource turbines [9]. Except for the white nose syndrome, which specializing and, in turn, using limited resources more has given rise to extensive research, very little investi- efficiently [32]. As it happens in macroecosystems [33], gation has been done on bat health in contrast to other the inherent properties of resource specializing and resil- endangered species [4, 6]. In Europe, bat populations ience from bacterial species-rich communities may be have declined considerably over the last decades presum- reflected in the overall health status of the host. Given ably due to multiple factors of anthropogenic origin (e.g., that the microbiome is composed of a dynamic commu- pesticides use in agriculture, wind turbines or habitat loss nity of bacteria, it is constantly susceptible to change due and fragmentation) [9]. Understanding the factors that to age, diet, environment, and diseases among other fac- contribute to such declines and the differential responses tors [34]. Hence, substantial changes in species propor- of bat species to habitat disturbance is critical for world- tions or richness within the gastrointestinal microbial wide bat conservation. community of a host may lead to dysbiosis, which has Health can be broadly defined as a state of physical been associated to digestive, neurologic, metabolic, and and psychological well-being and the subsequent abil- respiratory affections in mammals [35]. ity to adapt and cope with changing environment [10]. Land-use changes for agricultural use and urbaniza- Measuring the health of a group of individuals or pop- tion can negatively affect wildlife health, especially by ulations, particularly in free-ranging species, can be altering food availability and diet composition, increas- challenging. Health indices, such as body condition or ing the exposure to agrochemicals, and increasing the hematological values, are quantifiable parameters used to contact with humans, domestic animals, and their refer to the health state of a group of animals or species pathogens. These factors may disrupt the normal gut [10]. A specific framework to assess this particular issue microbiota and, in consequence, increase the incidence in populations of insectivorous bats around the globe of pathogens which may contribute to the emergence of has not yet been developed. Difficulties inherent to this diseases [36, 37]. Particularly, agriculture development broad taxonomic group are not only the requirement of has been shown to create selective pressure on inver- species-specific knowledge of ecology, anatomy, disease tebrates [38] and soil microbes [39] through intensifi- susceptibility and pathology, but also arduous sampling cation practices and pesticide use. Their impact on the Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 3 of 14 nutrient cycling of soil and its microbes further affects Animals and samples the food webs of ecosystems [40, 41], which eventu- We selected the Kuhl’s pipistrelle (Pipistrellus kuhlii), a ally may alter the intestinal microbiome of animals. sedentary and synanthropic bat species in NE-Spain, as However, evidence for an association between habitat a study model. This species indistinctively inhabits open degradation and gut microbiota changes in bat spe- forests and anthropogenic landscapes from the Mediter- cies has barely been explored [42, 43]. Furthermore, ranean basin and extends throughout Europe. From what information on bat microbial communities -especially is known so far, the home range of this species is less these detected in feces- and their role in bat’s health than 2 ­km2, and foraging sites can beas far as 4.5 km [46]. remain scarce. Understanding the complex relation- Its ability to dwell in pristine and altered ecosystems ship between host habitat and fecal bacterial composi- makes this species an interesting model for assessing the tion could contribute to developing a new operational impact of anthropogenic disturbance on fecal bacterial framework for the assessment of bats’ health which can composition. be applied to guide future conservation decisions. Thirty-one Kuhl’s pipistrelles were captured across the Here, we contribute to the information gathered world- four studied landscapes from mid-July to early Septem- wide about the fecal microbiome of insectivorous bats ber 2021 (Additional file 1: Table S2). Bats were captured by exploring the hypothesis that land-use changes and using harp traps and mist nets and placed in individual anthropogenic disturbances could shape differences in and clean cloth holding bags until sampling. Fecal sam- the composition of the fecal bacterial microbiome of ples were collected directly from bats or from cloth hold- P. kuhlii. Because bats dwelling in intensive agriculture ing bags using sterile forceps. Samples were individually and urban landscapes may have access to a less diverse placed in sterile Phosphate Buffered Saline (PBS) (Lonza, diet and be more exposed to pesticides and pollutants, Basel, Switzerland) and immediately deposited in dry we hypothesized that bats residing in near pristine eco- ice. Once in the laboratory facilities, they were stored at systems would show an increased alpha diversity of fecal -80ºC until DNA extraction. samples in comparison with bats dwelling in human-dis- Bats included in this study were individually marked turbed ecosystems. We also expected to find significant with a circular wing biopsy (3 mm punch) which was differences in the structure of the fecal bacterial commu- used for other research purposes but also allowed us to nity between the different scenarios. avoid resampling. All sampling procedures followed the EUROBATS best practices [47] and at least one wild- life veterinarian was present in all the captures in order Methods to guarantee the welfare of the captured individuals. No Study areas bats resulted harmed or died during the performance of Our study areas were located in Catalonia, in the north- the study. east of the Iberian Peninsula. We selected four environ- mental contexts with different levels of anthropogenic environmental degradation and separated by a mini- DNA extraction, generation of 16S rRNA gene amplicon mum of 10 km from each other (Additional file 1: Figure sequences and library preparation for next‑generation S1 and Table S1): mature and old-growth forest (D0), sequencing extensive farming and agriculture (D1), immature and Fecal DNA was extracted using QIAamp PowerFe- secondary forest (D2), and urban and intensive agricul- cal Pro DNA Kit (Qiagen, Hilden, Germany) following ture landscape (D3). Mature forests from this study were manufacturer’s instructions. Initial DNA of the samples composed of large trees with abundant suitable roosts was quantified by Qubit Fluorometric Quantification for bats and were assumed to be free of human distur- High Sensitivity Assay (Invitrogen, California, USA). bance. The selected youth forest had been under log- 16S rRNA was selectively amplified from genomic DNA ging pressure and habitat fragmentation for decades and by the polymerase chain reaction (PCR) according the was represented by a less complex vegetation structure SQK-RAB201 Nanopore Kit using universal bacterial than mature forests and scarce suitable roosts for bats. primers 27F (5′-AGA​GTT​TGA​TCC​TGG​C TC​AG-3′) In scenario D1, the main use of the soil was pastureland and 1492R (5′-GGT​TAC​C TT​GTT​ACG​ACT​T-3′), ena- for bovine grazing and crop fields with traditional pest bling the amplification of approximately 1500 bp of the management and moderate pesticide use. The urban and 16S rRNA gene. PCR amplification was performed in intensive agriculture landscape selected (D3) was located 50 µl of PCR mix comprising 25 µl mix reaction buffer in the Segrià County, which comprises one of the largest 2 × (LongAmp Taq 2X master mix, New England Bio- intensive pig industry and intensive agriculture area of labs); 14 µl of ultra-pure water; 1 µl of each primer Spain, and Europe [44, 45]. 10 µM; and 10 µl of DNA. The temperature and cycling Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 4 of 14 conditions were as follows: first, preheating at 95 °C for Results 1 min; then 25 cycles at 95 °C for 20 secs; 55 °C for 30 The entire 16S gene (≈ 1.5 kb) from the fecal micro- secs; 75 °C for 2 min; and a final incubation at 65 °C biomes of 31 bats was sequenced, for which a total of for 5 min. Library construction was performed using 4,829,302 reads (7.2 Gb) were obtained with an average the Rapid 16S Amplicon Barcoding Kit (SQK-RAB201) of 114,988 ± 43,577 reads per sample. The percentage of from Oxford Nanopore Technologies (ONT, Oxford, taxonomic assignment at the genus level was 99.9% of the United Kingdom). Two sequencing runs of 20 and 11 total sequences obtained and rarefaction curves of rich- multiplexed samples were carried out on a MinION ness against sequence sample size reached asymptotic sequencer (ONT) using a brand new R9.6 flow cell. growth (Additional file 1: Figure S2). Taxonomic composition Data analysis Firmicutes and Proteobacteria were the most abun- All 16S rRNA sequences were obtained by the Min- dant phyla in all scenarios (Fig. 1). Firmicutes showed KNOW suite [48] and basecalled with Guppy 3.0. a relative abundance of 58.6% ± 31.1 in scenario D0, (ONT). Reads were filtered by length (> 1500 bp) and 70.63% ± 33.7 in scenario D1, 45.3% ± 28.8 in scenario quality (> 10) using NanoFilt 1.1.0 [49]; adapters and D2 and 58.1% ± 22 in scenario D3. On the other hand, barcodes were trimmed with qcat-1.1.0 (ONT). Taxo- Proteobacteria showed an abundance of 39.88% ± 31.9 in nomic assignment at genus level was carried out with scenario D0, 28.32% ± 33.8 in scenario D1, 54.3% ± 29 in Centrifuge 10.3-beta [50], using Silva 132 database [51] scenario D2 and 41.4% ± 22 in scenario D3. Actinobac- based on a 95% of identity threshold. Afterwards, taxa teria was the third most abundant phyla in all the sam- with single read counts were removed. In addition, low ples. Nevertheless, its relative abundance was < 1% for all count filter was set to a minimum read count of 4 with scenarios, ranging from 0.09% in scenario D2 to 0.40% in a 20% prevalence in the samples. Finally, filtered data scenario D0. was normalized using total sum scaling (TSS). Plots Although there was not a strict pattern of relative and analysis of microbiomes structure and diversity abundance across all scenarios, Enterococcus, Escheri- were made with Pavian-0.3 [52] and MicrobiomeAna- chia/Shigella, Bacillus and Enterobacter were consistently lyst [53]. found as the most abundant genera, showing relative abundances (among scenarios) of 15.4% ± 15.1 for Ente- rococcus, 10.5% ± 10 for Escherichia/Shigella, 6.7% ± 7 Statistical analysis for Bacillus and 5.5% ± 6 for Enterobacter (Fig. 2). Alpha diversity of each sample was estimated by the Enterobacter displayed both high and low dominance Shannon index. Differences between alpha diversity indi- across samples with a range of 0.02% to 20%. The most ces were assessed using the Kruskal–Wallis test [54] with abundant genera in particular scenarios included Ser- a significance threshold set at p < 0.05. Additionally, beta- ratia (4.56% ± 0.61), Lachnoclostridium (4.13% ± 0.25), diversity was determined using Bray–Curtis distances Candidatus Soleaferrea (4.12% ± 0.29), Pseudomonas and localities were compared using the nonparametric (3.7% ± 0.59), and Carnobacterium (3.38% ± 0.56) in analysis of similarities (ANOSIM) test [55]. Regarding D0; Staphylococcus (11.7% ± 3), and Vespertiliibac- the study of differential taxa among scenarios, a Linear ter (2.59% ± 3) in D1; Serratia (4.23% ± 0.32), Lons- Discriminant Analysis Effect Size (LEfSe) [55] algorithm dalea (3.58% ± 0.6), Hafnia (3.38% ± 0.7), Ricketsiella with a LDA effect size threshold of 2 (on a l­og10 scale) (3.1% ± 0.8) and Klebsiella (2.8% ± 0.56) in D2; and Lacto- was applied at phylum, family, and genus levels. Moreo- coccus (8.9% ± 0.12), Carnobacterium (3.51% ± 0.85) and ver, core (taxa shared by 100% of samples), accessory Klebsiella (3% ± 0.6) in D3. (taxa shared by samples from 2 or 3 scenarios) and exclu- sive microbiomes (taxa found exclusively in one scenario) Diversity analyses were identified using the R package vegan [56]; and the Alpha diversity analyses (Fig. 3A) revealed differential Venn diagram was obtained using the VennDiagram diversity patterns for the studied scenarios, showing Shan- package [57]. Abundance threshold for the core micro- non index values varying between 2.8 (Scenario D3) and biome analysis was set to 0.01. Random effects of sex 3.4 (Scenario D0). Scenario D0 showed the highest mean and locality on alpha and beta diversity were tested with alpha diversity (Shannon’s Index = 3.45 ± 0.38), followed Kruskal–Wallis and a Permutational Mantel test (9999 by D1 (3.14 ± 0.57), D2 (3.1 ± 0.38) and D3 (2.7 ± 0.38). permutations) based on Spearman’s rank correlation rho, Furthermore, Shannon index varied significantly between respectively. All statistical analyses were performed using the four scenarios (H: 7.935; p < 0.05), while no significant R version 4.1.3 [58]. variance was found between localities (H: 10.45, p > 0.05) Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 5 of 14 Fig. 1 Stacked bar plot of relative abundances of phyla from scenarios D0 (mature and old growth forest), D1 (extensive farming and agriculture), D2 (immature and secondary forest), and D3 (urban and intensive agriculture). Fecal samples are displayed on the bottom and scenarios displayed at the top and the sex of the bats (H: 105, p > 0.05). Pair-wise com- Scenarios D0 and D3 displayed the greatest differenti- parisons of Shannon values showed significant differen- ation between each other, both in terms of composition tiation between scenarios D0 and D3 (Additional file 1: regardless of taxonomic hierarchy (Figs. 1 and 2), and Table S3). On the other hand, beta diversity obtained by alpha and beta diversities (Fig. 4). Because only sce- Bray–Curtis distance showed weak statistical differen- nario D0 included lactating females, we tested whether tiation of community structure across scenarios (Fig. 3B; these samples were influencing the overall results. ANOSIM’s R: 0.12; p < 0.02), and pair-wise comparison No significant differentiation in alpha (Kruskal–Wal- showed significant differentiation between D0-D3 and lis statistic: 7; p = 1) and beta diversity (ANOSIM’s R: D1-D3 (Additional file 1: Table S3). No significant correla- -0.16923; p = 0.783) were found between males and tion between beta diversity and geographic distance was females (all lactating) within scenario D0 (Additional found (Mantel’s r: 0.013, p > 0.05). file 1: Figure S3). Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 6 of 14 Fig. 2 Stacked bar plot of relative abundances of the top 30 most abundant genera from scenarios D0, D1, D2 and D3. Fecal samples are displayed on the bottom and scenarios displayed at the top Differential abundance analyses Gemmatimonadaceae and Rhodocyclaceae were sig- Linear discriminant analyses effect size (LEfSe) of all nificantly more abundant in scenario D0. Phylum Spi- scenarios showed a distinct pattern of differentially rochaetota; orders Spirochaetales and Coxiellales; and abundant taxa, which were found to be significantly families Spirochaetaceae and Coxiellaceae were sig- more abundant in scenarios D0 and D1 than D2 and nificantly more abundant in scenario D1. With the D3 (Fig. 5). Phylum Gemmatimonadota; orders Bur- exception of Alcaligenaceae, which appeared in high-to- kholderiales, Xanthomonadales, Bdellovibrionales moderate abundance in scenario D3 and low-to-mod- and Gemmatimonadales; and families Xanthomona- erate abundance in D1, all aforementioned taxa were daceae, Oxalobacteraceae, Bdellovibrionaceae, Nitros- found in low abundance in scenarios D2 and D3. No sig- omonadaceae, Alcaligenaceae, Nocardioidaceae, nificant taxa were found at the class and genus levels. Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 7 of 14 Fig. 3 Alpha (A) and beta (B) diversity measures of the four scenarios: D0, D1, D2 and D3. Boxplot of Shannon’s Diversity Index (Kruskal–Wallis statistic: 7.935; p = 0.047) is depicted in panel (A). A PcoA of Bray–Curtis distance matrix (ANOSIM’s R: 0.12; p < 0.02) is shown in panel (B). Different color intensities in dots from scenarios D0 and D3 are used to indicate the different localities sampled within each scenario (Additional file 1: Figure S1 and Table S1 and S2) Fig. 4 Alpha (A) and beta (B) diversity measures of scenarios D0 and D3. Shannon’s Diversity Index for D0 and D3 is shown in panel A (Kruskal– Wallis statistic: 64; p = 0.005). Bray–Curtis distance matrix (ANOSIM’s R: 0.28; p < 0.002) for scenarios D0 and D3 is illustrated in panel (B). Alpha diversity is also shown in the ordination plot through a color gradient from green (low value) to red (high value) Core microbiome distance matrix calculated from binary data (presence/ Core community analysis indicated that 1,293 genera absence of genera) roughly depicted two partially overlap- were shared by all sites (Fig. 6A). Scenario D0 contained ping clusters composed of Scenarios D0-D1 and D2-D3 the largest number of exclusive genera (n = 239), while (Fig. 6). scenario D2 comprised the least (n = 50). A plot of the Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 8 of 14 Fig. 5 Linear discriminant analysis effect size (LEfSe) of all scenarios. Phylum, order and family taxonomic levels are respectively shown in panels (A, B and C). LEfSe threshold of 2 (on a ­log10 scale) and a significance threshold of 0.05 were set. p values were adjusted for false discovery rate (FDR) method Fig. 6 Core community analysis of the four scenarios studied. A Venn Diagram of genera shared across and exclusive to localities is shown in panel (A). A Non-metric Multidimensional Scaling plot from a Jaccard distance matrix of presence/absence data is depicted in panel (B) Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 9 of 14 Discussion microbiota capable of nutrient assimilation [66]. In this Similar fecal microbial communities are shared sense, significant differences in microbiome composition among P. kuhlii and other insectivorous bats, regardless between frugivorous, insectivorous and piscivorous bats of the quality of the environment have been reported by several authors [62, 67–69]. More According to the literature and supported by our results, recently, different bat dietary habits have also been linked chiropteran’s gut microbiota is mainly represented by to differences in metagenome functions which may be Firmicutes and Proteobacteria, while the anaerobic phy- linked to specific metabolic pathways [28]. Several most lum Bacteroidetes is underrepresented in bats, but highly abundant genera from our study (Carnobacterium, Serra- dominant in other terrestrial mammals [59]. The fast gas- tia, Hafnia, Enterococcus and Lonsdalea) were also found trointestinal transit of bats and their adaptation to flight to be significantly abundant in some insectivorous bat is believed to be responsible for this Proteobacteria-dom- species across Europe, China, Israel, Mexico and Costa inated gut microbiota, as also observed in birds [60]. Rica, compared to piscivorous bat species [68]. Notably, Although the fecal microbiome is strongly influenced we found that Serratia, Hafnia and Lonsdalea genera by gut microbial communities, guano samples may also were differentially abundant in P. kuhlii from scenario be strongly influenced by environmental factors [61]. D2. Insectivorous bat species from the former study Nonetheless, the most abundant genera found in fresh included Pipistrellus kuhlii, other vespertilionid and rhi- feces of P. kuhlii from the four scenarios of our study are nolophid bats, but specific characteristics of the sampling consistent with the most common taxa identified by NGS localities were not specified. Moreover, none of the most in guano samples from different bat species around the differentially abundant genera present in piscivorous bats globe, particularly Enterococcus and Bacillus genera [20, were represented in our study [68], further supporting 22, 62]. the current scientific data about the significant impact of Bacteriological analyses showed the presence of Escher- trophic guilds on fecal bacterial communities [66]. ichia adecarboxylata, Citrobacter freundii, Klebsiella Additionally, the predominant bacterial genera in oxytoca, Enterobacter agglomerans, E. aerogenes, E. gergo- fecal samples from scenario D0 were found to particu- viae, Proteus vulgaris and Streptococcus faecalis in feces larly overlap with fecal samples of P. pipistrellus and of P. kuhlii from Italy [15]. While Enterobacter, Klebsiella, other insectivorous bats from the Netherlands, shar- Streptococcus, Escherichia, and Citrobacter genera were ing Carnobacterium, Serratia and Pseudomonas as the also present in our analyses, different isolation and detec- most abundant genera [17]. Interestingly, we would have tion techniques could have contributed to different find- anticipated greater similarities between either scenario ings such as the high abundance of Enterococcus genera D1, D2 or D3 since the bats from the latter study were across all our studied scenarios. Since most microbes captured in lime-stone mines near villages rather than in remain unculturable, using culture-independent analyses near pristine habitats. These counterintuitive results may in microbiome studies is essential to better understand be explained by the fact that mines are usually located in and characterize bacterial communities and their func- rural areas and, once mine activities cease, sites tend to tions [63]. As demonstrated by Newman et al. in 2018, be partially restored to pre-mining conditions, with mini- the observation of a high abundance of the genus Vesper- mal human presence [70]. However, persistent environ- tiliibacter in scenario D1 from our study would have been mental pollutants of different mining activities can also neglected by culture-dependent analyses [64]. This Pas- alter the fecal microbiome of bats and their impacts still teurellaceae was isolated for the first time in Germany in need to be clarified. the upper respiratory tract of three different bat species of the family Vespertilionidae, including one P. pipistrel- Intensive agriculture and urban landscapes lus [65]. More recently, it has also been isolated in guano may be enhancing intestinal bacterial lineages samples from a maternal colony of Tadarida brasilien- with pollutant‑degrading properties sis (family Molossidae) in New Mexico (US), showing a By linear discriminant analyses effect size (LEfSe), dif- greater presence on fresh guano samples over surface or ferential abundance taxa, including the order Burkholde- subsurface guano samples [63]. However, the role of the riales, appeared in high abundance in scenario D0. The genus Vespertiliibacter as a commensal or opportunistic order Burkholderiales constitutes a metabolic and eco- pathogen for bats is yet to be elucidated. As a dominant logically diverse bacterial linage, which includes human genus in samples from scenario D1, it may be responsible opportunistic bacteria (mostly nosocomial), animal and of meaningful functions in the microbiome of these bats. plant pathogens, bacteria present in wastewater and Despite partial overlapping of fecal microbial commu- sludge, and bacteria naturally present in soil, freshwater nities exists between bats with different trophic strategies and sediment [71, 72]. This order comprises the families [27], diet specialization is generally linked with specific Alcaligenaceae, Burkholderiaceae, Comamonadaceae, Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 10 of 14 Oxalobacteraceae, Sutterellaceae, Sphaerotilaceae and comparison to those foraging in organic or conventional Burkholderiales genera incertae sedis [73]. Our results banana monocultures where pesticides were used [43]. showed that, while Oxalobacteraceae and Alcaligenaceae As a highly diverse bacterial linage, further research is were highly abundant in scenario D0, Alcaligenaceae was needed to understand Alcaligenaceae’s functional signifi- also the only family overrepresented in scenario D3. cance in the gut microbiome of bats. Species belonging to the family Oxalobacteraceae have shown the ability to invade and persist in different niches, Multiple variables may explain why healthier ecosystems such as plant tissues, Antarctic soil, rivers and lakes, support more resilient fecal bacterial microbiomes groundwater and contaminated soils among others [72]. We demonstrated that the richness and diversity of the Moreover, species belonging to this family have been fecal bacterial microbiome of P. kuhlii bats were affected recovered from patients with clinical disease and are con- by the level of anthropogenic disturbance (Shanon Index; sidered mild and opportunistic human pathogens [72]. p < 0.05), while gender and geographical distance between On the other hand, the family Alcaligenaceae includes sampling sites had no significant impacts on the micro- some well-recognized primary animal and human patho- biome composition (Mantel test; p > 0.05). Variation in gens such as the genus Bordetella [72] and Taylorella [74]. gut microbial diversity of bats has been linked to shifts Bacteria from the family Alcaligenaceae have also been in season (mainly pre- and post-hibernation) and specific isolated from biogas slurry (genus Advenella) [75], acti- reproductive states such as lactation or pregnancy [89, vated sludge (genera Caenimicrobium and Pusillimonas) 90]. Three females from scenario D0 were captured dur- [76, 77] and other samples from wastewater treatment ing the lactation period, when microbial diversity often plants (genera Parapusullimonas and Pigmentiphaga) increases [88]. However, no significant differences in [78, 79]. Other bacteria from this family have been iso- alpha and beta diversity were found between these lactat- lated from freshwater sources (genus Parvibium) [80] or ing females and the rest of the bats from the same sce- have shown the ability to thrive in different environments nario, thereby ruling out any potential effect of lactation such as the genus Pusillimonas, isolated from wastewater on the overall microbial diversity. treatment plants, farm soil and poultry manure among In our study, P. kuhlii inhabiting pristine forests dis- others [77, 81, 82]. Although the ecological function of played richer fecal bacterial microbiomes than those bacteria shouldn’t be defined solely by their source of iso- dwelling in degraded environments such as farmlands lation, many genera from the family Alcaligenaceae have and urban landscapes. Defining what represents a healthy shown the ability to degrade Polychlorobiphenyl (PCB) gut microbiome has proven difficult, particularly due to [83], neonicotinoids [84] and aromatic hydrocarbons [71, the high interindividual variability [91]. However, resist- 85], pointing out their potential in removing environ- ance and resilience to external perturbations are among mental pollutants. The high abundance of Alcaligenaceae the crucial characteristics of a healthy microbiome [92, in bats from scenario D3 may be explained by the gut 93] and both are positively affected by high microbial bacterial community’s response to pesticides and other diversity [32]. Accordingly, the higher alpha diversity dis- pollutants exposure, promoting the growth of detoxifying played by feces of Kuhl’s pipistrelles from undisturbed bacteria. The differentially high abundance of this family forests may be indicative of a more resilient gut micro- in scenario D0 in contrast to D1 and D2 may not be so biome compared to that of bats dwelling in disturbed straightforward. Evidence of atmospheric transport and environments. deposition of pollutants into mountain ecosystems (cold- The gut microbiota at the population level is supposed trapping effect) have been reported for several pollutants, to be evolutionarily selected and to remain relatively including microplastics [86] and semi-volatile organic stable across individuals of the same species [94]. Nev- chemical pollutants [87]. Lles de Cerdanya, one of the ertheless, environmental factors can mediate bacterial sampled localities in scenario D0, is located in a montane community structure as demonstrated in human and area of the southeastern Pyrenees. Therefore, despite other animal studies. Such is the case of industrialization, being far from major populations or industrial centers, which has been related to shifts in the relative abundance the high elevation of this village may aid the deposition of of bacterial lineages and reduced microbiota diversity in air chemical pollutants through rain, snow, fog or wind. human populations [95, 96]. Human activities have also The family Alcaligenaceae has been described in the modified how wild animals move through the landscape fecal microbiome of different bat species [24, 25, 88], and interact with its biological components [97]. In this regardless of their dietary habits or geographic distri- sense, shifts in intestinal microbiota composition and bution. Furthermore, this family of proteobacteria was diversity from wild animals have been reported as conse- found to be characteristic of Palla’s Long-tongued bats quences of habitat fragmentation and encroachment [98, (Glossophaga soricine) foraging in natural forests in 99]. Human-disturbed and fragmented landscapes were Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 11 of 14 also associated with significantly lower alpha diversity bats foraging intensively managed plantations and those in the gut microbiome of Tome’s spiny rats (Proechimys foraging organic plantations, suggesting that reduction in semispinosus) [100]. However, when considering habitat gut microbiota diversity may be caused by other factors fragmentation without additional anthropogenic distur- such as the use of pesticides [43]. The use of pesticides bances such as contact with humans and domesticated in the studied agricultural, logged, and urban landscapes animals, no impacts were detected [100]. Similar to our from Catalonia is a common practice (personal observa- study, Barelli et al. [101] showed that colobus monkeys tion). Therefore, the lower alpha diversity of Kuhl’s pipis- (Procolobus gordonorum) inhabiting undisturbed forests trelles from these scenarios may be further explained by from Tanzania had significantly higher alpha diversity similar factors to those suggested in G. soricine [43]. than those from disturbed forests. Through functional analyses they concluded that this variation was associ- ated with dietary changes and diversity derived from Fecal bacterial microbiome analysis as a potential health anthropogenic habitat degradation [101]. indicator for free‑ranging insectivorous bat populations The Kuhl’s pipistrelle is considered a selective-oppor- Significant differences in community structure found tunist feeder, targeting larger preys over the smaller ones. between scenarios D0 and D3, which were not explained The most important orders of insects in the diet of P. by geographic distance or different reproductive status, kuhlii are Coleoptera, Diptera, Hemiptera, Heteroptera, highlight the potential impact of human activities on the Hymenoptera and Lepidoptera. In the Iberian Peninsula, fecal microbiome composition of insectivorous bats. Fur- Diptera and Lepidoptera account for the main represent- thermore, we showed that the fecal bacterial richness of ative orders of their diet, and these include several agri- wild populations of Kuhl’s pipistrelles decreased along a cultural pests such as Pectinophora gossypiella (i.e. cotton gradient of increasing urbanization and other anthropo- pest) [46]. However, as an opportunistic feeder, variation genic disturbances such as the presence of domesticated in insect diversity and volumetric representation may be animals. Due to the fact that loss of microbiota diversity encountered in the diet of Kuhl’s pipistrelles across differ- opens up niches for opportunistic pathogens [35], our ent types of landscape. Further explorations of P. kuhlii’s results suggest that anthropogenic disturbance may mod- diet across the different scenarios from this study could ify the gut composition and functionality, and pose a risk provide a better overview of how insects respond to to the health of insectivorous bats. these specific anthropogenic changes and their potential Owing to its non-invasive nature and simplicity of sam- effect on the fecal microbiome of this bat species. Land- pling, we propose the fecal bacterial microbiome as a use changes, particularly urbanization and agriculture, health indicator for free-ranging insectivorous bats. Nev- are probably responsible for the recent decline in insect ertheless, larger sample sizes, and experimental and longi- abundance and biotic homogenization [102–104]. Future tudinal studies are needed to explore the dynamics of bat’s research assessing the potential effects of diet homogeni- gastrointestinal microbiome through life and its adaptation zation on the health of insectivorous bats may highlight processes to environmental disturbances. Future studies the relevance of specific habitat conservation strategies. should also explore other factors that may modulate differ- Another consequence of human activities is the release ences in microbiome composition such as bat species, sea- of toxic substances into air, water and land at harm- sonality, diet, and reproductive and physiologic status. ful rates for living beings [105]. Therefore, wildlife that manages to thrive and adapt to urban and agricultural Conclusion landscapes faces constant or temporary exposure to Our study demonstrates a correlation, rather than pollutants and pesticides. A growing body of evidence causal relationship, between anthropogenic pres- indicates that pesticides may induce changes in the gut’s sure and the fecal bacterial microbiome of Kuhl’s pip- microbiota composition and lead to dysbiosis or altera- istrelles. Nonetheless, the inverse correlation found tion of the homeostasis in several host species [106]. between the level of anthropogenic disturbance and Insectivorous and frugivorous bats forage equally in plan- the fecal bacterial microbiome richness and diversity tations -feeding on pests or crops respectively- as well of P. kuhlii indicates that near pristine and undisturbed as in forests or natural areas. Alpízar et al. [43] revealed landscapes could promote more resilient gut microbi- that nectar-feeding bats Glossophaga soricine foraging omes. Studies on physiological parameters for different intensively managed banana plantations had a signifi- bat species during their variable and complex life cycle cantly lower alpha diversity than those foraging organic are needed to establish reference values and, in turn, to banana plantations, and far lower than bats foraging be able to link microbiome changes to health and dis- natural forests. Although a less diverse diet may explain ease status. this association, no differences in diet were seen among Lobato‑Bailón et al. Animal Microbiome (2023) 5:7 Page 12 of 14 The results of this study generate new questions about Author details 1 Wildlife Conservation Medicine Research Group (WildCoM), Departament de the impact of degraded habitats on the fitness of bats via Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, 08193 Bella‑ the microbiome and highlight its potential as a non-inva- terra, Catalonia, Spain. 2 Departament de Genètica i Microbiologia, Universitat sive bioindicator to assess insectivorous bats’ health. We Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain. 3 Centre de Ciència i Tecnologia Forestal de Catalunya, 25280 Solsona, Catalonia, Spain. 4 BETA, believe that such studies will, additionally, serve as a key Universitat de Vic-Universitat Central de Catalunya, 08500 Vic, Catalonia, Spain. element of landscape conservation strategies. 5 Asociación Naturalista MUR, 39300 Torrelavega, Cantabria, Spain. 6 Unitat Mixta d’Investigació IRTA‑UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Supplementary Information 08193 Bellaterra, Catalonia, Spain. 7 IRTA, Programa de Sanitat Animal, Centre The online version contains supplementary material available at https://​doi.​ de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de org/​10.​1186/​s42523-​023-​00229-9. Barcelona (UAB), 08193 Bellaterra, Catalonia, Spain. Additional file 1. Figure S1. Map of sampling locations. Localities and Received: 3 May 2022 Accepted: 1 February 2023 their associated scenarios are depicted in the map with colored dots covering the foraging range of Pipistrellus kuhlii (4.5km): D0 (mature and old-growth forest), D1 (extensive farming and agriculture), D2 (imma‑ ture and secondary forest), and D3 (urban and intensive agriculture landscape). Figure S2. Rarefaction curves of each sample, separated References by scenario. Sequence sample size (number of reads generated) and 1. Powers RP, Jetz W. Global habitat loss and extinction risk of terrestrial genus richness are depicted on the X and Y-axis, respectively. Figure vertebrates under future land-use-change scenarios. Nat Clim Change. S3. Comparison of diversity between males and lactating females from 2019;9:323–9. scenario D0. No significant differentiation in alpha diversity given by 2. Cardillo M, Mace GM, Jones KE, Bielby J, Bininda-Emonds OR, Sechrest Shannon’s Index (Panel A; Kruskal-Wallis statistic: 7; p = 1) and beta W, et al. Multiple causes of high extinction risk in large mammal spe‑ diversity given by Bray-Curtis distance (Panel B; ANOSIM’s R: -0.16923; p = cies. Philos Trans R Soc London Ser B. 2005;2:379. 0.783) were found. Table S1. Environmental information of the scenarios 3. Hosseini PR, Roche B, Mills JN, Ne Prieur-Richard A-H, Ezenwa VO, Bailly selected. Table S2. Biological data of sampled bats, time and location of X, et al. Does the impact of biodiversity differ between emerging and sampling. Table S3. Pair-wise comparison of alpha and beta diversities endemic pathogens? The need to separatethe concepts of hazard and between scenarios. Significant results are signaled with *. Table S4. Biosa‑ risk. Philos Trans B. 2016;372:20160129. mple accession numbers for datasets obtained in this study. 4. Hutson AM, Mickleburgh SP, Racey PA. Global status survey and conser‑ vation action plan: microchiropteran bats. IUCN, Gland, Switzerland and Cambridge, UK.: IUCN/SSC Chiroptera Specialist Group; 2001. Acknowledgements 5. Ramirez-Francel LA, Garcia-Herrera LV, Losada-prado S, Reinoso-flórez The authors thank the veterinary and biological sciences students who col‑ G, Sánchez-hernández A, Estrada-villegas S, et al. Bats and their vital laborated in the field work. ecosystem services: a global review. Integr Zool. 2022;17:2–23. 6. Feijó A, Wang Y, Sun J, Li F, Wen Z, Ge D, et al. Research trends on bats in Author contributions China: a twenty-first century review. Mamm Biol. 2019;98:163–72. Capture and identification of bats was carried out by DG, JC, XFR, R Molleda 7. Preble JH, Ohte N, Vincenot C. In the shadow of the rising sun: a sys‑ and R Manzano. The sampling was carried out by ADA, JE, MPR, OC and tematic review of Japanese bat research and conservation. Mamm Rev. LLB. 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