Bacterial Infections in Humans and Nonhuman Primates from Africa: Expanding the Knowledge.

The close phylogenetic relationship between humans and other primates creates exceptionally high potential for pathogen exchange. The surveillance of pathogens in primates plays an important role in anticipating possible outbreaks. In this study, we conducted a molecular investigation of pathogenic bacteria in feces from African nonhuman primates (NHPs). We also investigated the pathogens shared by the human population and gorillas living in the same territory in the Republic of Congo. In total, 93% of NHPs (n=176) and 95% (n=38) of humans were found to carry at least one bacterium. Non-pallidum Treponema spp. (including T. succinifaciens, T. berlinense, and several potential new species) were recovered from stools of 70% of great apes, 88% of monkeys, and 79% of humans. Non-tuberculosis Mycobacterium spp. were also common in almost all NHP species as well as in humans. In addition, Acinetobacter spp., members of the primate gut microbiota, were mainly prevalent in human and gorilla. Pathogenic Leptospira spp. were highly present in humans (82%) and gorillas (66%) stool samples in Congo, but were absent in the other NHPs, therefore suggesting a possible gorillas-humans exchange. Particular attention will be necessary for enteropathogenic bacteria detected in humans such as Helicobacter pylori, Salmonella spp. (including S. typhi/paratyphi), Staphyloccocus aureus, and Tropheryma whipplei, some of which were also present in gorillas in the same territory (S. aureus and T. whipplei). This study enhances our knowledge of pathogenic bacteria that threaten African NHPs and humans by using a non-invasive sampling technique. Contact between humans and NHPs results in an exchange of pathogens. Ongoing surveillance, prevention, and treatment strategies alone will limit the spread of these infectious agents.

Introduction

More than 60% of known infectious diseases have a zoonotic origin, the majority being caused by pathogens of wild origin [1]. The close phylogenetic relationship between humans and other primates creates high potential for pathogen exchange [2]. As a result, diseases emerge in humans as an unintended consequence of the hunting and butchering of the African great apes and other contacts. They were responsible for human Ebola outbreaks and the global AIDS pandemic [3,4], as well as the high mortality observed in wild chimpanzee populations (Pan troglodytes) associated with the anthropozoonotic transmission of human respiratory viruses [5].

In the tropics, in many rural areas, human population growth and changes in land use are leading to a growing overlap between humans and wild primates [6]. This is mainly the result of large-scale activities, such as extractive industries (ie, logging, mining); as well as small-scale interfaces, like subsistence use of natural resources, ecotourism, and research. These changes increase contact between people and non-human primates (NHPs), and they result in more intimate contact with wild primates [7-9]. By contrast, the increasing fragmentation of habitats is forcing primates to seek resources more widely, including through the active use of human-dominated systems (eg, crop raiding in agricultural fields and urban occupation) [10,11]. All these scenarios are likely to increase the risk of transmission of zoonoses [12,13]. Some NHPs that persist in anthropogenically modified landscapes, such as monkeys, are susceptible to many of the same pathogens as humans. As a result, these resilient species have the capacity to act as sentinels for ecosystem health and provide early warning of potential risks to human health [1].

Several bacteria have been reported to be transmitted by direct as well as indirect contacts from NHPs to humans [9]. Further, it had been demonstrated that proximity between wild primates and people can promote transmission of the common gastrointestinal bacterium Escherichia coli, as well as other pathogenic microorganisms, such as Cryptosporidium [7] and Shigella. Other studies stressed that direct contact between species is not mandatory for interspecific disease transmission (ie, Shigella, Salmonella, E. coli, etc.) [9]. Demonstration of human pathogens negatively impacting wild primates has sparked considerable debate regarding the costs and benefits to endangered primate populations of scientific research, ecotourism, and current conservation and management paradigms. Despite the disease-related risks, the consensus is that both research and tourism have contributed in overwhelmingly positive ways to primate conservation, enhancing their long-term survival by increasing their scientific and economic value. Nevertheless, such activities as well as overlap of humans and NHPs may have unintended consequences for the health and survival of wild primate populations.

The central hypothesis of this work is that key NHPs could carry pathogen agents for both humans and animals. In addition, human behaviors, wildlife behaviors, ecological conditions and landscape features increase the risks of interspecific disease transmission.

Thus, given the deadly epidemics and pandemics that have already occurred related to NHPs (HIV, malaria, Ebola, etc.), it is of utmost importance to study microorganisms common to both NHPs and humans. It is difficult to carry out such studies because, in most cases, we cannot capture them and collect the necessary samples. Using stool samples, it is possible to find not only enteric pathogens, but also blood and urinary pathogens (like filaria, Plasmodia, and Leptospira). Its collection is absolutely non-invasive. Here, we performed an extensive epidemiological survey for bacteria on NHP feces, using polymerase chain reaction (PCR) systems known for their specificities and sensitivities. Moreover, in a One Health context in Congo, we studied a human population sharing the same living area as gorillas in order to study the transmission of pathogens between species.

Material and Methods

Ethical Statement, Animals, and Study Area

In Senegal, in August 2016, 48 western chimpanzees (Pan troglodytes verus) feces were collected. The study was approved by the Senegalese Ministry of the Environment (Direction of the National Parks, No. 1302, 16 October 2015). The Direction des Eaux, Forêts, Chasses et Conservation des Sols of the Republic of Senegal gave authorization to collect and export fecal samples (No. 1914/DEF/DGF of 5 June 2016) in Collaboration with Jane Goodall Foundation (https://www.janegoodall.org/). Between 2017 and 2019, in the Republic of Congo, 38 gorilla (Gorilla gorilla) and 38 human feces were collected as part of a collaborative project carried out by the Government of the Republic of Congo and the Aspinall Foundation, which manages a protected area of 170,000 ha located about 140 km north of Brazzaville. In addition, the project was authorized by the Ministry of Health (No 208/MSP/CAB.15 of 20 August 2015) and the Forest Economy and Sustainable Development (No 94/MEFDD/CAB/DGACFAP-DTS of 24 August 2015) of the Republic of Congo. In this country, wildlife protected areas are not fenced and there is close interaction between apes and the communities. In some instances, they even share open-well water sources and/or gardens. This can lead to microorganism transmission between humans and NHPs. In the Republic of Djibouti, six fecal samples of hamadryas baboons (Papio hamadryas) were collected in 2017. This collection was carried out in partnership with the Center for Studies and Research of Djibouti. Finally, in Algeria, fecal samples were collected from 69 Barbary macaques (Macaca sylvanus), with the authorization of the management of the Chréa National Park (CNP). These primates were synanthropic and lived in close contact with the people who provided them with food (Figure 1).

Figure 1

Map indicating the countries where fecal samples were collected. In Senegal: 48 fecal samples of western chimpanzees (Pan troglodytes verus) were collected from three sites located within the Dindefelo Community Natural Reserve in the Kédougou region. Site 1: three samples (12°22′57.1404″ N, 12°17′16.7172″ W), Site 2: seven samples (12°22′53.1732″N, 12°17′26.7936″W), and Site 3: 38 samples (12°22′47.7084″ N, 12°17′48.588″ W). Four green monkey (Chlorocebus sabaeus) and seven Guinea baboon (Papio papio) fecal samples were collected near the Niokolo forestry guardhouse of the NKNP (13°04′28.6″ N 12°43′18.2″ W). In the Republic of Congo: 28 gorilla (Gorilla gorilla) feces were collected from the Lésio-Louna (LLR) and South-West Léfini gorilla reserves (2°58′33.1″S 15°28′33.4″E), 10 from Odzala-Kokoua National Park (OKNP) (1.3206°”N, 14.8455°”E), 35 human feces from (1.3206°”N, 14.8455°”E) village of Mbomo, located within the OKNP, and three from eco-guards in the LLR. In Djibouti: six hamadryas (Papio hamadryas) feces. These baboons lived outside the village of Oueah, 38 km from the city of Djibouti (11°29′56.1″N 42°51′14.8″E). In Algeria: 69 Barbary macaques (Macaca sylvanus), including 30 samples collected from two sites, the Stream of Monkeys and the Gorges of la Chiffa in Blida Province, 50 km north of Algiers (36°23′42.9″N 2°45′53.6″E), and 39 samples from Cap Carbon (36°46′31.6″ N 5°06′11.2″ E) in the suburbs of Béjaïa, 250 km east of Algiers.

The sampling was non-invasive and did not disturb any wild animal. For NHPs, feces were collected at sleeping sites, feeding sites, and places where the primates had been observed. Human stool samples were collected after obtaining the verbal consent of all the participants because of their low level of literacy.

All the humans in our study were apparently healthy. In addition, the fact that the NHP stools were not diarrheal may indicate that they were also in relatively good health. All collected samples were transported to the IHU Méditérranée Infection Laboratory, 13005 Marseille, France, for analysis. They had been identified and stored at either -20°C or -80°C.

DNA Extraction

Initially, 40 mg of stool were mixed with 360 µL of G2 lysis buffer from EZ1®DNA Tissue Kit (Qiagen, Hiden, Germany). This was mechanically lysed with tungsten beads (Qiagen, Hiden, Germany) using FastPrep-24TM 5G Grinder for 40 sec. After 10 min of incubation at 100°C to allow for complete lysis, tubes were centrifuged at 10,000g for 1 min. Subsequently, 200μL of supernatant was enzymatically digested using 20μL of proteinase K (20mg/mL, Qiagen) and incubated overnight at 56°C. DNA was extracted from 200µL of sample using the EZ1®DNA Tissue Kit on BIOROBOT EZ1 (Qiagen, Hiden, Germany), according to the manufacturer’s instructions. Elution was performed in 200µL volume, then aliquoted in individual tubes of pure extracted DNA, dilutions to 1:10 and to 1:100.

The extraction quality and the absence of PCR inhibitors were controlled using the universal eubacterial qPCR targeting the 16S rRNA bacterial genes [14] on pure DNA, dilutions to 1:10 and to 1:100. By comparison of the Ct values obtained, the dilution to 1:10 was chosen for the analysis. DNA tubes were stored at -20°C until use.

Molecular Screening for Bacteria by Real-time PCR Assays (qPCR)

The approach consists of screening for bacteria that may be pathogenic for humans and NHPs. We used single target real-time genus or species-specific qPCR assays. We targeted pathogens of medical interest, previously reported in NHPs. At least 12 qPCR assays targeting bacterial genera and 16 species-specific qPCRs, all known for their specificity and sensitivity, were used in multiparallel assays, as shown in Table S1 (Appendix A). For Rickettsia spp., a new qPCR was designed and validated targeting the 16S rRNA gene. qPCR was tested for its specificity using several laboratory-maintained colonies as well as DNA from arthropods, humans, monkeys, donkeys, horses, cattle, mice, and dogs as described previously [15].

Assays were carried out in 20 µl final volume containing 10µl of Master Mix Roche (Eurogentec), 0.5µl each primer per reaction at the concentration of 20µM, 0.5 µl UDG, 0.5µl of each probe at the concentration of 5µM and 5µl of the DNA template. The qPCR amplifications were performed in a CFX96 Real-Time system (Biorad Laboratories, Foster City, CA, USA). The thermal conditions included two hold steps at 50°C for 2 minutes, followed by 95°C for 15 minutes and 40 cycles of two steps each (95°C for 30 sec and 60°C for 30 sec). Each PCR plate contains 96 wells. Known microorganisms’ DNAs or plasmids were used as positive controls and master mixtures as a negative control in each reaction.

Genetic Amplification by Standard PCR, Sequencing, and Phylogeny

In addition to species-specific qPCR screening, we continued identification by PCR/sequencing for Treponema spp., Acinetobacter spp., Rickettsia spp., Mycobacterium spp., and Leptospira spp. Positive samples in qPCR assays were subjected to standard PCRs targeting genes and using primer pairs summarized in Table S1 (Appendix A).

Genetic amplifications were carried out in 50µl volume consisting of 5µl of DNA template, 25µl of AmpliTaq Gold master mix, 18µl of ultra-purified water DNAse-RNAse free and 1µl of primers at 20µM of concentration. This was performed in a thermocycler (Applied Biosystem, Paris, France). Protocols of amplification were as follows: incubation step for 15 minutes at 95°C, 40 cycles (one minute at 95°C, 30 sec at the annealing temperature, an elongation step at 72°C), and a final extension step for 5 minutes at 72°C. Amplicons were visualized on 2% agarose gel and were then purified using NucleoFast 96 PCR plates (Macherey Nagel EURL, Hoerdt, France) as per the manufacturer’s instructions. Amplicons were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems). Generated electropherograms were assembled and edited using ChromasPro software (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia) and compared with those available in the GenBank database by NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The fragments obtained were compared with each other and with the related fragments available in the GenBank database. The phylogenetic analyses were inferred using neighbor joining methods and tree reconstructions were performed using MEGA software version 7 (https://www.megasoftware.net/). Bootstrap analyses were conducted using 1000 replicates.

Results

In this study, we were able to collect NHP stool samples from different African countries, including 59 samples from Senegal, 6 from Djibouti, and 69 from Algeria. In addition, in Republic of Congo, we collected 38 samples from humans and 38 from gorillas sharing the same territory. In total, 93% (160/172) of NHPs tested positive for at least one (min: 1; max: 6) of the bacterial species tested in their feces while 94.7% (36/38) of humans were positive for at least one (min: 1; max: 7) bacterium. Prevalence in NHPs and humans are summarized in Figure 2; Table 1.

Figure 2

Infections detected in African NHPs and humans.

Table 1

Prevalence of bacterial infections detected on feces from different African NHP species as well as humans.

Species	Origin	n=	Leptospira spp.		Treponema spp.		Mycobacterium spp.		Acinetobacter spp.		Rickettsia spp.		Wolbachia spp.*		Mycoplasma spp.		S. aureus		H. pylori		Salmonella spp.		T. whipplei
			Pos	%	Pos	%	Pos	%	Pos	%	Pos	%	Pos	%	Pos	%	Pos	%	Pos	%	Pos	%	Pos	%
Gorilla	Congo	38	25	65,8	38	100,0	20	52,6	13	34,2	5	13,2	3	7,9	3	7,9	3	7,9	1	2,6	0	0	1	2,6
Chimpanzee	Senegal	48	1	2,1	22	45,8	38	79,2	1	2,1	2	4,2	0	0,0	0	0,0	0	0,0	0	0,0	0	0	0	0,0
Guinea baboon		7	0	0,0	6	85,7	2	28,6	0	0,0	0	0,0	0	0,0	0	0,0	0	0,0	0	0,0	0	0	0	0,0
Hamadryas baboon	Djibouti	6	0	0,0	6	100,0	0	0,0	0	0,0	0	0,0	0	0,0	0	0,0	0	0,0	0	0,0	0	0	0	0,0
Green Monkey	Senegal	4	0	0,0	3	75,0	0	0,0	0	0,0	0	0,0	0	0,0	1	25,0	0	0,0	0	0,0	0	0	0	0,0
Macaque	Algeria	69	20	29,0	61	88,4	6	8,7	3	4,3	1	1,4	0	0,0	0	0,0	0	0,0	0	0,0	0	0	0	0,0
Great Apes	-	86	26	30,2	60	69,8	58	67,4	14	16,3	7	8,1	3	3,5	3	3,5	3	3,5	1	1,2	0	0	1	1,2
Monkeys	-	86	20	23,3	76	88,4	8	9,3	3	3,5	1	1,2	0	0,0	1	1,2	0	0,0	0	0,0	0	0	0	0,0
Total NHP	-	172	46	26,7	136	79,1	66	38,4	17	9,9	8	4,7	3	1,7	4	2,3	3	1,7	1	0,6	0	0	1	0,6
Human	Congo	38	31	81,6	30	78,9	12	31,6	28	73,7	0	0,0	0	0,0	0	0,0	4	10,5	9	23,7	4	10,5	4	10,5

In bolt: Number include 2 samples positive by S. typhi/paratyphi qPCR. *: where also positive by Anaplasmataceae qPCR

Positive rates for all of Treponema (non-pallidum) (79.1%), Mycobacterium (non-tuberculosis) (38.4%), pathogenic Leptospira spp. (26.7%), and Acinetobacter spp. (9.9%) were high in NHPs as well as in humans, 78.9%, 31.6%, 81.6%, and 73.7% in humans respectively. Bacteria such as Rickettsia (4.7%), Mycoplasma (2.3%), and Wolbachia spp. (1.7%) were found only in NHPs. By contrast, H. pylori (23.7%), S. aureus (10.5%), and T. whipplei (10.5%) were more prevalent in humans than in NHPs, for which the prevalence rates were 1.7%, 0.6%, 0.6% respectively. Salmonella spp. was detected only in humans, 10.5% (4/38), including two samples (5.2%) positive for S. typhi/paratyphi.

No significant differences were observed between great apes (n=86) and monkeys (n=86) for pathogens (great apes %, monkeys %; Z test p-value) such as Leptospira spp. (30.2%, 23.3%; 0.388), Rickettsia spp. (8.1%, 1.2%; 0.066), Wolbachia spp. (3.5%, 0.0%; 0.240), Mycoplasma spp. (3.5%, 1.2%; 0.612), S. aureus (3.5%, 0.0%; 0.240), H. pylori (1.2%, 0.0%; 1.000), T. whipplei (1.2%, 0.0%; 1.000). By contrast, significant differences were observed for Treponema spp. (69.8%, 88.4%; 0.004), Mycobacterium spp. (67.4%, 9.3%; <0.0001) and Acinetobacter spp. (16.3%, 3.5%; 0.009). In the Republic of the Congo, there were no differences in bacteria carriage between gorillas (n=38) and humans (n=38) who cohabited the same area as gorillas (gorillas%, humans%; p-value) for Leptospira spp. (65.8%, 81.6%; 0.186), Mycobacterium spp. (52.6%, 31.6%; 0.096), Rickettsia spp. (13.2%, 0.0%; 0.055), Wolbachia spp. and Mycoplasma spp. (7.9%, 0.0%; 0.229), S. aureus (7.9%, 10.5; 1.000), Salmonella spp. (0.0%, 10.5%; 1.113), and T. whipplei (2.6%, 10.5%; 0.349). Significant differences were observed between gorillas and humans for Treponema spp. (100.0%, 78.9%; 0.005), Acinetobacter spp. (34.2%, 73.7%; 0.010), H. pylori (2.6%, 23.7%; 0.012).

All the other bacteria, also listed in the Table S1 (Appendix A), that were searched for using the PCR method, were ultimately not found.

Furthermore, we performed PCR/sequencing for positive samples in qPCR for all of Mycobacterium spp., Leptospira spp. (pathogenic), Acinetobacter spp., Treponema spp., and Rickettsia spp. Sequencing did not succeed for Mycobacterium and Leptospira spp. despite obtaining specific bands using standard PCR, and superposed peaks were obtained for the same nucleotide which made it impossible to conclude about sequences. Two good quality sequences of 700 bp length of gltA gene of Rickettsia were obtained, one from an Algerian macaque and the other from a gorilla from Republic of the Congo. The sequences were identical in each other and were 99% identical to R. africae isolate QtHyaegR9 (MN306555) detected from a spur-thighed tortoise (Testudo graeca) sold in a Qatar live animal market, and isolate EgyRickHm-Raas detected on an adult male Hyalomma marginatum (camel tick) in Egypt (KX819298) (Figure 3).

Figure 3

Phylogenetic tree for Rickettsia spp. in detected in African NHPs. The evolutionary history, based on 700 bp gltA partial gene, was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.10688518 is shown. The confidence probability (multiplied by 100) that the interior branch length is greater than 0, as estimated using the bootstrap test (1000 replicates is shown next to the branches). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of base substitutions per site. The differences in the composition bias among sequences were considered in evolutionary comparisons. The analysis involved 19 nucleotide sequences. All positions containing gaps and missing data were discarded. There were a total of 682 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

We obtained 24 sequences of 322-365 bp length for rpoB gene of Acinetobacter spp. including three sequences obtained from macaques, five others obtained from gorillas, and 16 humans. One sequence obtained from gorilla feces (G9) showed 98% identity with A. baumannii strain MS14413 (CP054302). Two human genotypes (Mbo033, Mbo064, Ibou02) and one gorilla genotype (G06B) were almost identical and they highlighted 95%-99.5% of identity to A. berezinae strain YMC79 (JF302886). Two other human isolates (Mbo047 and Mbo057) were closely similar and exhibited 92% and 93% similarity with A. berezinae. They constitute a potential new species. Three other sequences obtained from humans constitute potential new species. One of them (Mbo056) showed 88% of similarity with A. berezinae, while the two others had 86% (Mbo058) and 93% (Mbo054) of identity with A. genomosp. (EU477133 and KT997528). Sequence Mbo003 from a human constituted a separate branch (Figure 4) and showed 92% similarity with A. tandoii strain LUH 13385 (KU961639). In addition, two identical isolates (Mbo040, Mbo054) from human stools created a separate branch and showed 91% identity to A. bohemicus strain ANC4315 (KJ124827). Another sequence (Mbo028) showed quasi-identity (98.5%) with Acinetobacter sp. strain WCHAc060041 (MH190065) found in wastewater in China. It also showed 90% similarity with the official A. defluvii strain WCHA30 (KY435935). Mbo036 and Ibou001, two almost similar genotypes from humans, were close (93% of identity) to the species A. venetianus. Two others, one (Mbo062) showed 91% similarity to A. nosocomialis (KX444511), the other one (Mbo001) was 93.5% identical to A. seifertii (KJ956464). Three sequences from gorilla feces and one other from a macaque constituted a separate clade (G05, G05A, G06, and CC37). They were almost similar and were 92-93% identical to A. wuhouensis strain WCHAc060049 (MK518338). Finally, two sequences from macaques (GC05 and CC10) close to Acinetobacter sp. ‘isolate 30Bi’ (FJ157977) were detected in a dog (Figure 4; Table S2 (Appendix A).

Figure 4

Phylogenetic tree for Acinetobacter spp. detected in African human and NHP feces. The evolutionary history based on rpoB partial gene was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 1.17635033 is shown. The confidence probability (multiplied by 100) of the interior branch length is greater than 0, as estimated using the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of base substitutions per site. The differences in the composition bias among sequences were considered in evolutionary comparisons. The analysis involved 50 nucleotide sequences. All positions containing gaps and missing data were discarded. There were a total of 260 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

We succeeded in obtaining at least 52 sequences of 760-870 bp of Treponema spp. 23S gene on NHP feces including six from chimpanzees (accession numbers: MT257111-MT257117), 13 sequences from gorillas (MT257084, MT257085, MT257088-MT257098), one from a green monkey (MT257101), five from Guinea baboons (MT257102-MT257106), four from hamadryas (MT257107-MT257110), and 23 others from Barbary macaque samples (MT257228-MT257250) (Figure 5). In addition, four good quality sequences were obtained from feces of humans from the Mbomo locality (MT257099, MT257100, MT257086, MT257087) (Figure 6). None of the identified Treponema species have been reported as clearly pathogenic. A specific DNA search for T. pallidum spp. gave negative results.

Figure 5

Phylogenetic tree for Treponema spp. detected in NHP feces. The evolutionary history based on 23S rRNA gene was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 2,23995197 is shown. The confidence probability (multiplied by 100) of the interior branch length is greater than 0, as estimated using the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of base substitutions per site. The differences in the composition bias among sequences were considered in evolutionary comparisons. The analysis involved 81 nucleotide sequences. All positions containing gaps and missing data were discarded. There were a total of 594 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

Figure 6

Phylogenetic tree of Treponema spp. detected in gorilla and human feces living in the same area in Mbomo, Republic of Congo. The evolutionary history based on 23S rRNA partial gene was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 2,51751187 is shown. The confidence probability (multiplied by 100) that the interior branch length is greater than 0, as estimated using the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of base substitutions per site. The differences in the composition bias among sequences were considered in evolutionary comparisons. The analysis involved 36 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 361 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

In human samples, we obtained two sequences (MT257099, MT257086) that were perfectly identical to T. succinifaciens DSM 2489 (CP002631). The two others seem to be new species, one of them (MT257100) was 83% identical to T. succinifaciens DSM 2489, while the second (MT257087) displayed 87% similarity with T. pedis str. T A4 (CP004120). Three gorilla genotypes, four genotypes from hamadryas, three others from baboons, one from a green monkey and one from a Barbary macaque were almost similar and close to T. succinifaciens official strains to which they highlighted >99% of similarity. Three other similar gorilla genotypes constituted a separate cluster, they had 83.5% of resemblance with T. pectinovorum strain Marseille-1-CSURP6641 (UOUI01000011) and 82.5% with T. brennaborense strain DD5/3 (NR076878). One other gorilla (MT257091) sequence split off separately, it was 81.5% identical to T. brennaborense. Three sequences from gorilla feces, two from baboons, and 16 from macaques were almost similar and exhibited most similarity 97-99.5% to T. berlinense strain ATCC BAA-909 (FUXC01000026) recovered from Swine feces in Berlin. A distinct isolate from gorilla (MT257084) shared 86% of similarity with T. berlinense. Four chimpanzee sequences constituted a new species and showed a 93.5% identity with T. berlinense strain ATCC BAA-909. Two other chimpanzee sequences (MT257115, MT257116) were almost similar to each other and showed 88-89% similarity to T. berlinense strain ATCC BAA-909, for which another gorilla isolate (MT257093) showed 84.5% identity and one other isolate showed 95% of similarity (MT257095).

Discussion

This study identified pathogenic bacteria in human and NHP fecal samples from Africa. The technique used, qPCR/sequencing in feces, presented no ethical requirements. PCR was used in the diagnosis of gastrointestinal infections [16,17]. Using large specificity qPCR, we detected pathogenic bacteria, such as Leptospira spp. and nonpathogenic bacteria, such as Treponema spp., including several potential new species.

We found many Treponema spp. nonpathogenic despite the existence of the syphilis in NHPs. Most of the Treponema spp. detected (non-pathogenic) such as T. succinifasciens are part of the normal flora of primates, and all species in this study shed treponemes in their feces. The presence of Spirochaetes has been reported in the gut microbiota of NHPs [18]. High prevalence rates were observed in all primate species of Treponema spp. (not pallidum), T. succinifasciens, T. berlinense, and at least six potential new species. T. succinifasciens and T. berlinense were highly prevalent species in the microbial genomes from NHPs gut metagenome [19]. It had been reported that rural individuals were enriched with Spirochaetes, especially Treponema succinifasciens and T. berlinense being the most prevalent species identified. This may be due to the rare use of antibiotics [20]. Special attention should be drawn to endemic Treponema pallidum infection with genital stigmata in NHPs from Guinea, Senegal, and Tanzania. Many NHPs in Africa were found to suffer from treponematoses [21-24]. During fieldwork in Senegal, an epizootic of venereal disease was directly observed in green monkeys (Chlorocebus sabaeus) due to infection with T. pallidum subsp. pertenue, then an epizootic was observed in Senegal which spread to baboons one year later [25].

We agree with the conclusion drawn by Manara et al. 2019 [19], the overlap (20% of microbial candidate species in NHPs also found in the human microbiome) occurs mainly between NHPs and non-Westernized human populations and NHPs living in captivity, suggesting that host lifestyle plays a significant role comparable to host speciation in shaping the primate intestinal microbiome. Several NHP-specific species are phylogenetically related to human-associated microbes, such as Treponema, and could be the consequence of host-dependent evolutionary trajectories. Gut Treponema have been found in NHPs and all rural peoples studied to date, suggesting that they are lost symbionts in urban-industrialized societies [26].

Potentially new species of Acinetobacter were detected in humans, gorillas, and macaques. In humans, Acinetobacter is an organism of questionable pathogenicity as an infectious agent of importance to hospitals worldwide—it easily infects wounds [27]. The organism has the ability to accumulate diverse mechanisms of resistance, leading to the emergence of strains that are resistant to all commercially available antibiotics [28]. Acinetobacter is one of the main genera detected in primate gut microbiota. They might play a significant role in breaking down plant exudates [29]. Very high diversity was observed in humans, gorillas, and macaques.

Tuberculosis is rare in wild NHPs, but animals carrying M. tuberculosis could infect humans. Conversely, humans are the source of most NHP infections. M. tuberculosis and M. bovis can be acquired from infected humans or ruminants [30]. In this study, M. tuberculosis has not been detected and great apes were found to be more carriers of Mycobacterium spp. than monkeys and humans. Natural infections with M. leprae was reported in chimpanzees and sooty mangabeys (Cercocebusatys) [31]. Recently, different strains of M. leprae have been isolated from NHPs, including chimpanzees, sooty mangabeys, and cynomolgus macaques [32]. Mycobacterium orygis was isolated from captured rhesus monkeys [33]. It will be necessary to isolate the mycobacteria detected in this study to investigate their species diversity and identify their roles.

In addition, high prevalence for pathogenic Leptospira spp. has been observed in both humans and gorillas, which remains difficult to understand. All humans are apparently healthy and gorilla stools do not reflect any sign of diseases. Unfortunately, we were not able to genotype these Leptospira and the sequences obtained were unclear and suggested possible co-infection by more than one species. NHPs could be sensitive to Leptospira infection, an outbreak of severe leptospirosis was reported in capuchin (Cebus) monkeys [34]. The presence of Leptospira in the feces of wild NHPs could be due to environmental contamination because the samples were collected from the soil, which is not the case in humans, leading to the weakness of this hypothesis. Inappropriate breeding of NHPs could create new reservoirs and transmission routes for Leptospira, threatening conservation efforts and public health. Furthermore, the extent of Leptospira transmission between humans and NHPs remains unknown.

Surprisingly, Rickettsia and Wolbachia had been detected in great apes and monkeys in the present study. Wolbachia is assumed to be Wolbachia eaten together with their insect hosts by monkeys. For Rickettsia, it is R. africae, a pathogenic species and widespread in the continent, but very common in ticks. It is either R. africae eaten together with their tick hosts or a natural infection of NHPs (very unlikely).

Enteric bacteria, such as Salmonella spp., including S. typhi/paratyphi, were detected in humans only, and pathogenic H. pylori, T. whipplei, and S. aureus were detected in both humans and gorillas sharing the same living area, and humans were found to be more infected. H. pylori and T. whipplei are a priori anthroponotics and their origin remains unknown. These results suggest a transmission of pathogens from humans to gorillas. The threat is a direct function of the pathogens’ mode of transmission and their ability to survive in aerosols, soil, water, food, or feces [9]. In Uganda, it was observed that the number of gorillas carrying human gut Salmonella or Campylobacter had doubled in 4 years, and Shigella was isolated for the first time in this group of apes, probably because of ecotourism [35]. In addition, the fecal-oral transmission of Shigella flexneri and S. sonnei, enteropathogenic Escherichia coli, Salmonella enteritidis, S. typhimurium, Campylobacter fetus, C. jejuni, Helicobacter pylori, and many other infections are common in NHPs [36]. For T. whipplei, two important but as yet unresolved issues are the natural habitat and the route of infection. Analysis of stool samples by PCR has detected T. whipplei DNA in patients with Whipple disease. Recovery of T. whipplei from culture of stool from a patient with Whipple disease has highlighted the presence of viable bacteria, suggesting that the disease could be linked to fecal-oral transmission [37]. Asymptomatic carriage in stool was found in humans (ranging from a prevalence of 4% in the control group, to 12% among a subgroup of sewer workers), but not in monkeys and apes [37]. Thus, T. whipplei identified here in the gorilla most likely originates from humans.

Also, the samples were negative for the other bacteria, including the sexually transmitted pathogens in the current study (Chlamydia spp., T. pallidum, N. gonorrhea). Obtaining samples from different origins would also broaden the range of pathogens not found in stools and provide a clearer diagnostic vision in NHPs. It would therefore be interesting, in further studies, to use other types of excrement and biological fluids from these animals, especially for those in contact with humans, in order to assess the potential risks of interspecies transmission.

The prevalence of extra-intestinal infections might be underestimated in this study. Despite this, this method (search for pathogens in the feces) nevertheless made it possible to discover the origins of Plasmodium and HIV.

The study presents certain limits that were required by the difficulties encountered in the field and the study does not represent all African countries. The sample size of humans in Congo and some NHP species (such as hamadryas) are not important. This required larger investigations in the other regions with larger sample size and other NHP species. Unfortunately, we did not characterize all the detected microorganisms, such Leptospira, Treponema, and Mycobacterium spp., nor isolated them. If it was performed, this could give more information on their transmission and pathogenesis. Feces are not the ideal sampling method to search for pathogens, such as blood or sexually transmitted pathogens.

This work has contributed to the expansion of knowledge on NHP threatening bacteria in Africa by using a molecular stool technique. In addition, we have also shown that humans and gorillas in the same ecosystems share pathogens, indicating a real interspecies transmission. Constant monitoring is highly recommended to prevent any overflow of these pathogens. This effort entails a combination of epidemiology, molecular ecology, behavioral ecology, social and clinical survey, and spatially explicit modeling. The end outcomes are achievable plans to protect the health of humans and wildlife, while ensuring the sustainability of the ecosystems in which they live. In addition to better understanding the role of human-induced habitat change on pathogen dynamics, this work allows for the early detection of emerging pathogens that may pose a threat to global health and/or wildlife conservation.