A Survey of Zoonotic Bacteria in the Spleen of Six Species of Rodents in Panama

Simple Summary: In this study, we performed microbial community ecology analyses of bacteria present in the spleen of six species of rodents in Panama in order to identify taxa with zoonotic potential in the country. Genera of bacteria containing species with zoonotic potential detected in this study included Acinetobacter , Bartonella , Cutibacterium , Enterococcus , and Staphylococcus . The results obtained are of value for estimating the prevalence and relative abundance of the bacteria found and the potential of different species of rodents as reservoirs of bacterial zoonosis. This study provides information for comparative studies in the Neotropics and other regions of the world and to generate knowledge on the conditions that may drive zoonosis in different rural vs. suburban environmental settings. Abstract: Emerging zoonotic diseases are one of the main threats to human and animal health. Among the agents with the potential for zoonoses, those of bacterial origin have great relevance in Public Health. Rodents are considered one of the main reservoirs of pathogens that represent a risk to human health or animal species. We used massive 16S ribosomal RNA gene amplicon sequencing to survey bacteria present in the spleen of six species of rodents in Panama in order to identify bacterial taxa with zoonotic potential in the country. We found 3352 bacterial Amplicon Sequence Variants (ASVs, i.e., phylogenetic species) in the spleen of


Introduction
Emerging zoonotic diseases are one of the main threats to human and animal health [1][2][3].According to recent estimations, there are about 1407 human pathogens, of which 58% are zoonotic, and 13% are classified as emerging or reemerging [4].The infectious agents that involve emerging and reemerging zoonotic diseases include viruses, parasites, fungi, and bacteria, among others [3,5].Among these etiological agents of zoonoses, those with a bacterial origin have great relevance in public health [3,6].Of the 1407 human pathogens reported, 538 are bacteria, and 54 of them (10%) are considered emerging or reemerging.Most of them originated from an animal or other sources (i.e., water) and are considered to be zoonoses [7].Zoonotic bacteria can be transmitted by different animals, rodents being considered major hosts of pathogens [6,8,9], and cause risk to human health or animal species when they act as reservoirs or amplifying hosts for these microorganisms [10][11][12].This participation of rodents in the epidemiology of human pathogenic bacteria is also favored because they constitute one of the most abundant and diversified groups of mammals [8,9,13,14] and because of their ability to successfully colonize a wide range of habitats, where they often interact with humans, but also with other animal species [14,15].In addition, from the ecological perspective, the transmission of diseases by rodents also involves other factors, including alterations of the ecosystem (anthropogenic or natural) and changes in the number of available hosts and vectors [16,17].
For instance, several authors have also pointed out that the destruction of habitats as a consequence of human expansion and land use across the globe are among the main factors that have led to a defaunation that includes the global reduced abundant of mammals [18,19], which in turn causes an increase in the population of rodents and their pathogens, as observed in the indirect transmission systems of Bartonella spp.from Africa [18].Therefore, an increase in the prevalence of rodent-borne diseases occurs as a result of changes in the abundance of susceptible hosts (rodents) and by closer human-rodent contact [10,20,21].Therefore, taxonomic surveys of microbial communities in different species of rodents can contribute to understanding the natural occurrence and dynamics of pathogenic bacteria in them, and this information is valuable in the development of more precise risk models for these diseases [10,21].
A review of the diversity of rodents that make up the wild mammal fauna of Panama has shown the existence of several species of rodents that in other countries have been reported as reservoirs and hosts of zoonotic agents, which are frequently closely related to the human environment (synanthropic) [10,21].Therefore, understanding the presence of rodents in Panama with the capacity to act as a reservoir for pathogenic bacteria will provide information on the epidemiological links in the country for the circulation and transmission of bacterial zoonoses.On the other hand, the continuous deforestation, land use, and unplanned urbanization in Panama have increased human contact with rodents, which has intensified the number of infections transmitted by rodents in the human population [20].
Based on the above and considering the increase in the cases of zoonoses in various rural and suburban areas with the consequent cases of death [20,22], it is important to carry out studies that allow for the identification of pathogenic bacteria present in different rodents in order to derive the prevalence, co-infection, and interaction of these bacteria and their distribution in natural populations of rodents, and in this way, to know the potential that these animals have to directly or indirectly transmit zoonoses.
In this study, we used massive 16S ribosomal RNA gene amplicon sequencing and microbial community ecology analyses to survey bacteria present in the spleens of six species of rodents in Panama in order to identify and list bacteria with zoonotic potential in the country.This study contributes to generating information on the potential of different species of rodents as reservoirs of bacterial zoonosis in Panama.

The Materials Rodent Surveys and Sample Collection
Six species of rodents (Liomys adspersus, Melanomys caliginosus, Mus musculus, Proechimys semispinosus, Rattus rattus, Zygodontomys brevicauda) were collected from seven sites along Panama: Cañazas Chiriquí Grande (8  1, Figure 1).The traps were placed according to Armien's methodology [20].Trapping grids were separated by a minimum distance of 500 m.All trapping grids were georeferenced with a Global Positioning System (GPS) receiver (Garmin 60 CSx) using the WGS 84 / UTM zone 17 N system, and their central points (centroids) were selected.For the distribution map, we used ArcMap 10.7.1.Mammals were handled according to recommendations by Mills and others [23].The rodent species collected were identified using taxonomic keys, morphometry, and reference books [24][25][26].Information on reproductive stage, age, morphometric measurements, and habitat was recorded for each specimen.The animals were euthanized with inhaled isoflurane.Blood and samples of the spleen, liver, kidneys, heart, and lungs were collected in separate, labeled cryovials, using clean, sterilized instruments for each animal.All biological samples were immediately placed into liquid nitrogen.Data collected for all individuals were captured according to previous work [20].

DNA Extraction
Total DNA was extracted from the spleens using the DNeasy Blood and Tissue Kit (Qiagen, Chatsworth, CA, USA) following the manufacturer's protocol and with final DNA elution in 200 µL of AE buffer.A total of 26 samples were processed.

DNA Extraction
Total DNA was extracted from the spleens using the DNeasy Blood and Tissue Kit (Qiagen, Chatsworth, CA, USA) following the manufacturer's protocol and with final DNA elution in 200 µL of AE buffer.A total of 26 samples were processed.

DNA Amplification
Primers 799 F and 1115R [27,28] were used to amplify a portion of the V5 and V6 regions of the 16S rRNA gene.We use these primers because we can reduce the number of chloroplasts in our sequences [27,28], knowing that rodents are consumers of many plants and a wide range of crops [29,30].These primers contained read adapters for a second PCR needed for DNA library preparation.Each sample was amplified in triplicate PCR using 2.0 µL of DNA, 2.5 µL of 10 X PCR buffer, 1.5 µL 25 mM MgCl 2 , 2.0 µL of 10 mM dNTPs, 0.75 µL of 10 µM of primers (799 F and 1115 R), 0.5 µL of Taq DNA polymerase (Taq DNA polymerase kit of Qiagen (Product catalog 201203 (Qiagen, Valencia, CA, USA), and 15 µL of molecular grade water to obtain a total volume of 25 µL.Amplifications were conducted as follows: denaturation at 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 45 s; annealing at 50 • C for 60 s; elongation of 72 • C for 90 s; and final extension of 72 • C for 10 min.We run 2 µL of PCR products on 1% agarose gel to verify amplification using GelRed stain.

DNA Library Preparation
The three PCR replicates of each sample were pooled and used as templates for a second PCR conducted with complementary primers to read primer adapters and containing indexes and flow cell adapters for Illumina ® DNA sequencing by synthesis technology.Reactions were conducted as follows: 14.75 µL of molecular grade water; 2 µL of 10 X Buffer; 1.5 µL of 25 mM MgCl 2 ; 2 µL of 10 mM dNTPs; 1 µL of 5 µM of each index primer (forward and reverse); 0.25 µL of Taq; and 2 µL of pooled DNA template.PCR reaction started with a denaturation step of 94 • C for 3 min, followed by six cycles of 94 • C for 45 s, 50 • C for 60 s, 72 • C for 1.5 min, and a final extension of 30 s at 72 • C. PCR samples were combined, concentrated, and later purified using Agencourt AMPure XP, following the manufacturer's instructions (Beckman Coulter International, Nyon, Switzerland).The DNA library was quantified using a Qubit fluorometer (Invitrogen, Waltham, MA, USA), and quality was determined on a BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA).Finally, the DNA library was sequenced on an Illumina MiSeq sequencing platform following a 2 × 250 bp Paired-End sequencing (Illumina Inc., San Diego, CA, USA).

Data Analysis
Using the QIIME 2 TM bioinformatics pipeline [31][32][33], we dereplicated and qualityfiltered DNA sequences using the Divisive Amplicon Denoising Algorithm (DADA2) [34,35].Read 1 (R1) was used for subsequent analyses because the sequence quality for Read 2 was low.Continuously, we trained the sequence classifier for our specific region (V5 and V6) using the SILVA database (v.138 for bacteria, www.arb-silva.de,accessed on 17 January 2021) [36,37] that was used to taxonomically annotate amplicon sequence variants (ASVs).DNA sequences of mitochondria, chloroplasts, and unassigned bacterial taxa, as well as ASVs with less than 10 counts, were excluded for further analyses.Community ecology analyses were performed using QIIME 2.0 as well as the R software for subsequent plotting [38].

Bacterial Diversity and Community Composition
For diversity estimation analysis, sequence data from each sample were rarefied to a depth of 3000.Alpha diversity from rodent species and localities was estimated using Faith's phylogenetic diversity (Faith's PD) and analyzed by non-parametric Kruskal-Wallis to determine statistical differences.Faith's PD was used to compare bacterial diversity associated with M. musculus from two sites, as these were the rodent species with a comparable number of samples from two sites.Beta diversity between species was estimated based on weighted UniFrac distance using PERMANOVA and ANOSIM analyses in the vegan package [39,40] and visualized using Principle Coordinates Analysis (PCoA) phyloseq [35] and ggplot2 package [41].We did not estimate beta diversity between localities due to two sites (Comarca Ngäbe-Buglé and Divalá) being represented by only one rodent specimen (Table 1).

Results
We obtained a total of 403,188 sequence reads (per sample Min = 6828; Median = 12,830; Maximum = 41,263; Mean = 15,507), from which 3352 (ASVs, i.e., putative bacterial species) were detected.Rarefaction curves captured the majority of the bacterial diversity dataset in this study (Figure S1A,B).

Discussion
A survey of the spleen microbiome of species of rodents in Panama represents an important opportunity to explore potential zoonosis bacteria in these small mammals, which are considered one of the main hosts of pathogens.Here, we assessed the bacterial community associated with six closely related species of small mammals in Panama using a massive 16S ribosomal RNA gene amplicon sequencing for the first time.
Bacterial Composition in the Spleen of Six Species of Rodents, Role in Rodents, and Implications in Zoonotic Diseases Overall, we found 3352 ASVs associated with the six species of rodents.The most common and abundant bacterial taxa included the classes of Actinobacteria, Alpha-and Gammaproteobacteria, Bacilli, Clostridia, and Bacteroidia.Here, such genera as Acinetobacter, Bartonella, Cutibacterium, Enterococcus, Sarcina, Staphylococcus, and Wolbachia showed high abundance either in species of rodents or sites.Some of these bacterial taxa are pathogens responsible for several zoonotic diseases in humans and animals (i.e., domestic and wild animals) [10,[42][43][44][45], and some genera or some species belonging to these genera are also found in rodents, which are major reservoirs [10,44,45].For instance, Acinetobacter, which we found in all our rodent species and sites, was previously isolated from laboratory mice and rodents [46] and is associated with infections, and some species showed high drug resistance [47].Bartonella, which was only found in high abundance in Z. brevicauda, is a common bacteria found in rodents worldwide [10,45,[48][49][50][51], and some species are associated with many clinical manifestations, including endocarditis [52,53], neurologic disorders [54], and meningitis [55], among others [45].Additionally, a study showed evidence of the transmission of Bartonella from rodents by fleas [45].Cutibacterium, which was found in L. adspersus, contains species (i.e., Cutibacterium acnes) that are known as skin infection bacteria [56,57].Enterococcus was found in high abundance in M. caliginosus; this bacterium is found in different animals, including free-living raptors [44], and it is a commensal organism, an opportunistic pathogen associated with the mortality of humans and animals [44,58].On the other hand, previous studies showed that species such as Enterococcus faecalis were associated with small rodents [43,59], and it has caused inflammatory disease in mice [43].Another observation is Sarcina, which was found in high abundance in M. musculus.Studies have shown that some species belonging to Sarcina (i.e., Sarcina ventriculi) are Gram-positive bacteria, able to survive in extremely low pH environments [60], and it is an important pathogen that is associated with a lethal disease in sanctuary chimpanzees [61].Staphylococcus was found in high abundance in L. adspersus.It contains some species, such as Staphylococcus aureus; this species is a commensal bacteria of the human skin and gastrointestinal tract, which causes infections [62].Finally, Wolbachia was found in the spleen of rodents, and it was also found in high abundance in M. caliginosus.This bacterium is associated with insects [63][64][65][66] and has implications for ecology and reproduction in various insects [63,[67][68][69].
This study is the first step in screening bacterial taxa with the potential for zoonosis in the rodents surveyed in Panama.Although we found several pathogenic bacteria, more studies are needed to accurately estimate their potential for zoonosis in the country.Further research is also needed to assess the core microbiome associated with different species of rodents and which ones have a higher potential for zoonosis.

Conclusions
This study resulted in the identification and relative abundance of important bacterial taxa with the potential for zoonosis in six rodent species in a neotropical country.More studies are needed to determine which of the rodent species studied have a higher potential for bacterial zoonosis and which environmental conditions, for example, rural vs. suburban or urban settings, may drive bacterial zoonosis.

Zoonotic Dis. 2024, 4 167 Figure 2 .
Figure 2. Relative abundance of dominant bacteria taxa associated with rodents from Panama.Abundance was estimated at the level of bacterial class (A) and the level of bacterial genus (B).The x-axis shows the sample, species, and site of rodents.

Figure 2 .
Figure 2. Relative abundance of dominant bacteria taxa associated with rodents from Panama.Abundance was estimated at the level of bacterial class (A) and the level of bacterial genus (B).The x-axis shows the sample, species, and site of rodents.

Figure 3 .
Figure 3. Bacterial diversity associated with rodents from Panama.Graphs represent estimat alpha diversity-based Faith's PD for each species (A) and sites (for M. musculus) (B).Beta dive PCoA based on weighted Unifrac distance among species (C).

Figure 3 .
Figure 3. Bacterial diversity associated rodents from Panama.Graphs represent estimates of alpha diversity-based Faith's PD for each species (A) and sites (for M. musculus) (B).Beta diversity PCoA based on weighted Unifrac distance among species (C).

Table 1 .
Species of rodents collected in this study, sites, and sample size (n).

Table 2 .
Relative abundance (%) of bacteria at the level of genus in the six species of rodents.Sites are shown in abbreviation with exception of Divala.Number of sample sizes are shown in n.Only M. musculus showed relative abundance with same number of n.

Table 2 .
Relative abundance (%) of bacteria at the level of genus in the six species of rodents.Sites are shown in abbreviation with exception of Divala.Number of sample sizes are shown in n.Only M. musculus showed relative abundance with same number of n.
Institutional Review Board Statement: This study was evaluated and approved by the Institutional Animal Care and Use Committee of the Gorgas Memorial Institute for Health Studies (# 001/05 CIUCAL/ICGES, 4 July 2005), using the criteria established in the "International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences (CIOMIS).This study was in accordance with Law No. 23 of 15 January 1997 (Animal Welfare Assurance) of the Republic of Panama.