The findings of this study showed that Firmicutes bacteria constitute the highest proportion (73.5%) of the bacterial population, which aligns with previous research on Colobine monkeys which have consistently identified Firmicutes as the predominant phylum in the gut microbiota of these primates [23–27]. The findings are also consistent with the research done on proboscis gut bacterial diversity in Sabah which found Firmicutes to be the most common phylum accounting for 16 to 82 percent of the bacterial composition [21–22]. Firmicutes is a group of anaerobic bacteria, primarily characterised by their gram-positive nature. Notably, several species within this group possess the ability to create endospores, enabling their survival in harsh and extreme environments.
Interestingly, the lowest Firmicutes percentage recorded by Hayakawa et al. [21] is only 16% of the individuals sampled in the semi-wild population (mangrove habitat, provisioned with artificial diet items). Meanwhile, this study documented consistent Firmicutes composition in all samples (including semi-wild population) which covers more than 60% of bacteria from every population. Even though the diet items supplied (vegetables cultivated for human consumption) for the semi-wild population comprised of high nutrient content, it drives the shift in bacterial diversity to human-like species composition [26, 28]. However, the comparison and interpretation of data must be done with caution as the study by Hayakawa et al. [21] had only one sample representative from the semi-wild population, while this study analysed a total of four samples from similar habitat types.
The Firmicutes bacteria in this study were represented by four major classes: Bacilli, Clostridia, Erysipelotrichia and Negativicutes. Nevertheless, class Clostridia dominated the Firmicutes composition covering 72% of the total frequency with family Ruminococcaceae and Lachnospiraceae as the most recorded families from that phylum. These two bacterial families are the bacteria that commonly occur in the mammalian gut microbiota, previously reported to have a high abundance in herbivorous animals and proven to have efficiency in the digestion of complex plant materials [29–31]. This study clarifies the abundance of Ruminococcaceae and Lachnospiraceae in proboscis monkeys, which is consistent with the findings of Jose et al. [22].
Ruminococcaceae and Lachnospiraceae both assist in the degradation of complex polysaccharides into short-chain fatty acids and are capable of breaking down plant-derived substrate which includes cellulose, hemicellulose, and starch [30]. Species from the family Lachnospiraceae (Roseburia sp., and Coprococcus sp.) produced high levels of butyrate significant as the energy source for the colon epithelium [32, 33]. On the other hand, the Eubacterium coprostanoligenes species (family Ruminococcaceae) that is found in this study carry an effective function as the cholesterol-reducing anaerobe, by converting cholesterol to coprostanol. The intestine is not able to absorb coprostanol and it will be excreted by the gastrointestinal system [34–36]. Thus, it is proven that the record of bacteria from these two bacterial families would be a good indicator in assessing the health status of proboscis monkey populations.
Not only Firmicutes, Bacteroidetes also provide similar purposes in maintaining the health of proboscis monkeys and become part of important gut microflora in animals. This study recorded the family Muribaculaceae as the main representative from phylum Bacteroidetes, which are also commonly found in the guts of homeothermic animals, e.g., birds and mammals [37]. Covering the highest bacteria percentage after Firmicutes, Bacteroidetes is much needed by the host as it is specialized in degrading high molecular weight organic matter [38, 39].
The primary driver of Muribaculaceae occurrence in the proboscis gut is due to its capability to degrade oxalate, complex carbohydrates (plant cell wall glycans), and encrypt elements of an electron transport chain for energy production. Oxalate and glycan are the main components of the plant cell wall that serve as the protection mechanism and support the energy metabolism of plants [40]. The proboscis monkey also benefited the plant glycan as a fundamental component of the protein folding process (glycosylation) for adaptive immune activation [41, 42]. Interestingly, the number of Muribaculaceae bacteria greatly decreased in the Lok Kawi population indicating captive population may have low varieties of food plant intake. The dietary sources of the proboscis monkey population in Lok Kawi solely depend on the food provided in the park. Thus, this finding is evidence of the fact that reduced natural food sources availability may account for the low abundance of gut bacteria observed in the captive population. Frankel et al. [43] also documented a comparable finding in which folivorous NHPs housed in captivity, display alterations in their gut microbiomes when compared to the wild population. These changes have been attributed to the differences in dietary intake amongst NHP populations.
Besides that, similar to the findings of this study, Hayakawa et al. [21] in their study of the semi-wild population reported the largest proportion of the genus Prevotella, which belongs to the phylum Bacteroidetes. In addition to the natural food resources present in their habitat, the proboscis monkeys were provided with supplementary food sources, including vegetables (i.e., carrots and long beans). Consequently, this has resulted in a significant prevalence of Prevotella species, which have been previously shown as the dominant members of the gut microbiota in both humans and captive primates [26, 28] The increase in Prevotella abundance signifies a decline in the monkeys' capacity to metabolise dietary fibre, as Prevotella primarily metabolises simple carbohydrates [27].
Other significant bacterial phyla detected in proboscis’ faecal samples are the Verrucomicrobia, Proteobacteria, Cyanobacteria and Actinobacteria with each phylum covering at least 1% of bacteria of the overall microbial diversity. Verrucomicrobia and Proteobacteria are well portrayed in this study even though they are rarely described as microbiome components of non-human primate gut [25]. The occurrence of Verrucomicrobia, Cyanobacteria, and Actinobacteria in the gastrointestinal tract of the proboscis monkey is commonly attributed to the animal's intake of water. Those bacterial communities are highly prevalent in various environmental settings, frequently observed in both soil and water sources [44, 45]. In addition to inhabiting habitats characterised by aquatic environments, the proboscis monkeys were observed to partake of water sources in close proximity to their feeding grounds. This observation potentially elucidates the presence of bacterial communities within the gastrointestinal tract of the proboscis monkeys. Meanwhile, the detection of Proteobacteria was primarily characterised by the prevalence of species commonly linked to diseases in animals and humans. Hence, it demonstrates the capacity of the proboscis monkey to serve as a reservoir for pathogenic microbes, which will be further discussed below.
Pathogen infection typically appears as asymptomatic, although it can lead to severe illness upon accumulation within the host organism. Previous research conducted on non-human primates has identified several pathogenic bacterial genera present in their gastrointestinal tract. These include Escherichia, Salmonella, Mycobacterium, Campylobacter, Helicobacter, Pseudomonas, Shigella, and Yersinia [46–50]. Likewise, the proboscis monkey is not exempt from this scenario and may potentially serve as a substantial reservoir for pathogenic microorganisms. This study identified various pathogens in the faecal samples obtained from the proboscis monkeys in different locations across Malaysian Borneo. Among the most significant findings are from the genera Campylobacter, Helicobacter, and Comamonas.
Campylobacter and Helicobacter are known to be pathogenic to both humans and animals and suppress the host immune system by toxin secretion [51]. Previously, McKenna et al. [48] recorded a high abundance of Campylobacter bacteria in symptomatic animals compared to healthy individuals. Due to the sensitivity of the bacteria to the stomach's normal production of hydrochloric acid, low levels of exposure may not result in illness. However, the number of bacteria can swiftly increase as a result. Both Campylobacter and Helicobacter are likely prevalent in the proboscis monkey population, as they are abundantly present in all populations across Malaysian Borneo.
On the other hand, the presence of Comamonas bacteria solely in the Lok Kawi region adds to the greater abundance of bacteria that are not shared with the semi-wild population. Comamonas is a prevalent pathogenic bacterium that is frequently encountered in many environmental settings and is known to be a causative agent of diseases in humans. The results obtained in the present study are consistent with previous research conducted by Amato et al. [52] on Colobine monkeys where the captive population exhibited a higher abundance of pathogenic bacteria which heightened susceptibility to gut microbial dysbiosis.
In addition to bacteria, the occurrence of archaea in the gastrointestinal tracts of humans, apes and ruminant animals has been documented in previous studies [28, 53, 54, 55]. However, there is a lack of comprehensive research on their metabolic roles. The methanogens are often encountered, and a comparable occurrence has been documented in proboscis monkeys, where 2% of the archaea population belongs to the families Methanobacteriaceae and Methanomethylophylaceae. The methanogens were able to endure in an environment devoid of oxygen and generate methane as a by-product through the process of reducing carbon dioxide using hydrogen or methanol. The archaea belonging to the Methanobacteriaceae family are frequently observed in the human gut and are commonly expelled from the body as flatus or by exhalation [54].
With a compartmentalised stomach, the digestive physiology of the proboscis monkey resembles that of ruminants, and it utilises archaea as part of an important microbial community in food fermentation. Hydrogen, as a fermentation intermediate, will not accumulate in the digestive organ and will instead be metabolised by methanogenic archaea, releasing methane as a by-product [56]. Even though archaea comprise a small portion of the proboscis gut microbiota, they play an important role in the complex metabolic pathways of dietary fermentation in the GI tract of the proboscis monkey.