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

1. Introduction

Emerging zoonotic diseases are one of the main threats to human and animal health [1,2,3,4,5,6]. According to recent estimations, there are about 1407 human pathogens, of which 58 % are zoonotic and 13% are classified as emerging or reemerging [7]. The infectious agents that involve emerging and reemerging zoonotic diseases include viruses, parasites, fungi, and bacteria among others [6,8,9]. Among these etiological agents of zoonoses, those with a bacterial origin, have great relevance in public health [6,10]. In this context, a report estimated that bacteria make up 538 and 54 (10%) [7], as well as know that the 26 major emerging and reemerging infectious diseases are bacterial origin; most of them originated from an animal, or other sources (i.e., water) and are considered to be zoonoses [3]. Zoonotic bacteria can be transmitted by different animals, rodents being considered major hosts of pathogens [10,11,12], and cause risk to human health or animal species; when they act as a reservoir or amplifying hosts for these microorganisms [13,14,15,16]. 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 [12,13,17,18,19], 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 [18,20]. 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 [21,22,23].

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 [24,25,26], 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 [24]. 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 [14,27,28]. 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 [14,28].

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 close related in the human environment (synanthropic) [14,28]. Therefore, understanding the presence in Panama of rodents 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 [27].

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 [27,29,30], it is important to carry out studies that allow 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 spleen 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.

2. Materials and Methods

2.1. 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°54’24” N, 82°14’19” W, CCG), Comarca Ngäbe-Buglé (8°46’11” N, 81°44’02” W, CNB), Divalá (8°25’12” N, 82°43’12” W), Mercado de Abasto de Curundú (8°59’12” N, 79°32’11” W, MAC), Mercado Público de David (8°26’00” N, 82°26’00” W, MPD), Oajaca Chiguirí Arriba (8°38’12” N, 80°12’22” W, OCA), and Panama Port Balboa (8°57’27” N, 79°33’40” W, PPB), (Table 1, Figure 1). The traps were placed according to Armien’s methodology [27]. 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 [31]. The animals were killed 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 biologic samples were immediately placed into liquid nitrogen. Data collected for all individuals captured according to Armien et al. (2009).

2.2. DNA extraction

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

2.3. DNA amplification

Primers 799 F and 1115R [32,33] were used to amplify a portion of the V5 and V6 region of the 16S rRNA gene. We use these primers because we can reduce the number of chloroplasts in our sequences [32,33], knowing that rodents are consumers of many plants and a wide range of crops [34,35]. 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 10x PCR buffer, 1.5 µl 25mM MgCl₂, 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 ul. Amplifications were conducted as follows: denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 45 seconds, annealing at 50 °C for 60 seconds, and elongation of 72 °C for 90 seconds, and final extension of 72 °C for 10 minutes. We run 2 µl of PCR products on an agarose gel to verify amplifications.

2.4. DNA Library preparation

The three PCR replicates of each sample were pooled and used as a template for a second PCR conducted with primers complementary 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 10X Buffer, 1.5 µl of 25mM MgCl₂, 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 seconds, 50 °C for 60 seconds, 72°C for 1.5 min, and a final extension of 30 seconds 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, Massachusetts) and quality was determined on a BioAnalyzer (Agilent Technologies, Santa Clara, California). Finally, the DNA library was sequenced on an Illumina MiSeq sequencing platform following a 2 x 250 bp Paired-End sequencing (Illumina Inc., San Diego, California).

2.5. Data analysis

Using QIIME 2^TM bioinformatics pipeline [36,37,38], we dereplicated and quality filtered DNA sequences using Divisive Amplicon Denoising Algorithm (DADA2), [39,40]. 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) [41,42] 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 done using QIIME 2.0 as well as the R software for subsequent plotting [43].

2.6. Bacterial diversity, and community composition

For diversity estimation analysis, sequence data from each sample was 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 this was 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 [44,45] and visualized using Principle Coordinates Analysis (PCoA) phyloseq [40], and ggplot2 package [46]. We did not estimate beta diversity between localities due to two sites (Comarca Ngäbe-Buglé and Divalá) were represented by only one rodent specimen (Table 1).

3. Results

We obtained a total of 403,188 sequence reads (per sample Min=6,828; Median=12,830; Maximum=41,263; Mean=15,507) from which 3,352 (ASVs, i.e., putative bacterial species) were detected. Rarefaction curves captured the majority of the bacterial diversity dataset in this study (Figure S1A, Figure S1B).

3.1. The spleen microbiome by rodent species and locality

The spleen microbiome of six species of rodents was composed of 3,352 ASVs. This bacterial community was represented by 25 phyla, 55 classes, 140 orders, 268 families, and 508 genera. The three predominant phyla were Actinobacteria, Firmicutes, and Proteobacteria (Figure 2A, Figure 2B). The five predominant classes were Actinobacteria, Alpha- and Gammaproteobacteria, Bacilli, and Clostridia (Figure 2A, Figure 2B). The most dominant genera were seven: Acinetobacter, Bartonella, Cutibacterium, Enterococcus, Sarcina, Staphylococcus, and Wolbachia. However, there were other bacterial taxa groups in less abundance such as Bradyrhizobium, Chryseobacterium, Clostridium, Corynebacterium, Lactobacillus, Pseudonocardia, Rhodococcus and Sphingomonas. Overall, some of them could be of clinical importance (Table 2, Figure 3A, Figure 3B, Figure 3C). No statistical significance in Alpha diversity was observed by species and locality (Kruskal-Wallis: H= 9.48, p > 0.05; H= 8.36, p > 0.05). Additionally, the spleen microbiome of M. musculus did not show significant differences between sites (Kruskal-Wallis: H= 1.18, p > 0.05) (Figure 4A, Figure 4B). No significant difference was observed in Beta diversity between species (Adonis statistic: R²= 1.78, p =0.057; Anosim statistic: R= 0.09, p > 0.05) (Figure 4C).

4. 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.

4.1. Bacterial composition in the spleen of six species of rodents, role in rodents and implications in zoonotic diseases

Overall, we found 3,352 ASVs associated with the six species of rodents. The most common and abundant bacterial taxa included classes Actinobacteria, Alpha- and Gamma-proteobacteria following Bacilli, Clostridia, and Bacteroidia. Here, genera such 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)[14,47,48,49,50], and some genera or some species belong to these genera are also found in rodents, which are major reservoirs [14,49,50]. For instance, Acinetobacter which we found in all our rodents species and sites, was previously isolated from laboratory mice and rodents [51], and is associated with infections and some species are showed a high drug-resistant [52]. Bartonella, which was only found in high abundance in Z. brevicauda, is a common bacteria found in rodents worldwide [14,50,53,54,55,56], and some species are associated with many clinical manifestations including endocarditis [57,58], neurologic disorders [59], meningitis [60] among others [50]. Additionally, a study showed evidence of the transmission of Bartonella from rodents by fleas [50]. Cutibacterium, which was found in L. adspersus, contains species (i.e., Cutibacterium acnes) that are known as skin infection bacteria [61,62]. Enterococcus was found in high abundance in M. caliginosus, this bacterium is found in different animals including free-living raptors [49], and it is a commensal organism, an opportunistic pathogen associated with the mortality of humans and animals [49,63]. On the other hand, previous studies showed that species such as Enterococcus faecalis were associated with small rodents [48,64], and it has caused inflammatory disease in mice [48]. Another observation is Sarcina that was found in high abundance in M. musculus. Studies have shown some species belonging to Sarcina (i.e., Sarcina ventriculi) is a gram-positive bacterium, able to survive in extremely low pH environments [65], and it is an important pathogen that is associated with a lethal disease in sanctuary chimpanzees [66]. Staphylococcus that 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 [67]. Finally, Wolbachia, interestingly was found in the spleen of rodents, and it was found in high abundance in M. caliginosus. This bacterium is associated with insects [68,69,70,71], and has implications for ecology and reproduction in various insects [68,72,73,74].

This study is a first step in screening bacterial taxa with 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 one have higher potential for zoonosis.

5. Conclusions

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