Research Article

Exploration of Anti-Microbial Resistant (AMR) Genes in the Gastrointestinal Microbiome of Birds of Paradise (Paradisaeidae) in Papua, Indonesia

Alif Rahman Rohim Puarada*, Safika, Agustin Indrawati

School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia.

Received | December 08, 2023; Accepted | January 06, 2024; Published | February 12, 2024

*Correspondence | Alif Rahman Rohim Puarada, School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia; Email: [email protected]

Citation | Puarada ARR, Safika, Indrawati A (2024). Exploration of anti-microbial resistant (AMR) genes in the gastrointestinal microbiome of birds of paradise (Paradisaeidae) in Papua, Indonesia. Adv. Anim. Vet. Sci., 12(3):539-545.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/13.2.539.545

ISSN (Online) | 2307-8316

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The avian gastrointestinal microbiome is a pivotal factor influencing host health, affecting metabolism and ecological interactions (Kohl, 2012). Within this intricate ecosystem, the prevalence and genomic characteristics of antimicrobial-resistant (AMR) genes within diverse microbiota components are gaining global attention (Grond et al., 2018). The consequences of AMR, including treatment failures and increased mortality, underscore its significance (Cao et al., 2020). Recent studies reveal the presence of antibiotic-resistant bacteria in wild bird species across the globe, even in remote habitats, suggesting the potential role of certain wild animal species as reservoirs for the emergence and spread of new AMR profiles (Martínez, 2008; Sjölund et al., 2008).

Understanding Antimicrobial Resistance (AMR) in wildlife is imperative for global health security, as wildlife serves as natural reservoirs and disseminators of AMR, shedding light on how resistance naturally arises in ecosystems and transfers between species (Djuwanto, 2021; Elsohaby et al., 2021). The urgency of this research is emphasized by the potential transmission of antimicrobial resistance from wildlife to humans, either through meat consumption or direct contact with animals, particularly relevant in Indonesia. Investigating antibiotic use in wildlife habitats contributes to developing effective management strategies. Engaging in environmental prevention strategies, such as waste management and responsible antibiotic use in agriculture, mitigates the risk of AMR and provides insights essential for maintaining global food security (McKernan et al., 2021; Olaru et al., 2023). The escalating concern over the rise of AMR necessitates a comprehensive understanding of these dynamics in diverse ecosystems such as birds of paradise (Hernández et al., 2012).

Birds of paradise, classified under the family Paradisaeidae, is an endemic species to the Indonesian islands of Maluku and Papua (Firth and Firth, 2009). This taxonomic group has 45 species, each characterized by intricate and highly specialized morphological adaptations. These birds present a fascinating intersection of ecology and microbial dynamics. Notable among these adaptations are the elaborate plumage and vibrant coloration, features that play a pivotal role in the elaborate courtship displays observed in these species (Gregory, 2020). The intricate mating rituals and specialized dietary habits of birds of paradise suggest potential symbiotic relationships with specific microorganisms, influencing nutrient metabolism and immune function could serve as a reservoir and mechanism for the spread of AMR genes (Culp and Goodman, 2023). Investigating the microbiome of these avifauna could provide valuable insights into the coevolutionary processes shaping both the microbial communities and the birds themselves.

Using the state-of-the-art genomic technique ‘Shotgun metagenomics, this study aims to unveil the genomic structure of AMR genes in the gastrointestinal microbiome of birds of paradise (Wang et al., 2022). This high-throughput sequencing method provides insights into complex interactions between the host and the microbiome.

Papua, dominated by tropical rainforests and divided into fifteen areas based on geographical differences, offers diverse habitats for birds of paradise. The lowland rainforest, a significant habitat, hosts species like the king bird-of-paradise, utilizing tall trees for various activities (Beehler and Pratt, 2016). The rich plant and wildlife diversity in these habitats provides a variety of food sources, including fruits, insects, flower nectar, seeds, and flowers (Frith et al., 1998). Understanding the genomic landscape of the gastrointestinal microbiome of birds of paradise in Papua is crucial to uncover the diversity of AMR genes in these unique microbial communities (Culp and Goodman, 2023).

MATERIALS AND METHODS

Sampling

Six fecal samples were collected in December 2022 from wild birds of paradise inhabiting the Rhepang Muaif Monitoring Site, Rhepang Muaif Village, Nimbokrang District, Jayapura Regency. The identification of the birds of paradise for sampling was conducted based on morphology. To ensure the samples truly represented a variety of individuals, a site survey was conducted to pinpoint the stopover locations most frequently visited by individual birds, facilitating the collection of fresh fecal samples. The fresh samples were obtained immediately after defecation to minimize surface contamination. Direct collection from wild avian specimens was implemented without human intervention or any form of invasive treatment. Each fecal sample was labeled at the time of collection to ensure accurate tracking of individual specimens.

To ensure the diversity of the sampled individuals, the collection process involved maintaining a minimum 1-meter radius between sampled feces. This precautionary measure aimed to guarantee that each sample originated from a distinct bird of paradise within their natural habitat. Following collection, all fecal samples were carefully placed in sterile containers within sterile tubes. The samples were transported to the laboratory using dry ice to preserve their integrity and were stored at -80°C until further processing. This approach ensures the reliability and representativeness of the collected samples from the wild birds of paradise in their native environment.

DNA extraction

Fecal samples were extracted using the DNeasy Power Soil Pro kit (Qiagen, Germany) according to the manufacturer’s procedures for the best performance (Shaffer et al., 2022).

Product qualification and sequencing

A total of 25 μl of total DNA was taken and then the DNA concentration was measured at the Advanced Research Laboratory (Bogor) using a Nano Photometer® N60/N50 (Implen, Germany). The A260/A280 ratio was recorded and calculated to determine the purity of the DNA. Qubit fluorometric (Thermo Fisher Scientific, USA) quantitation was then used to assess the extracted DNA quality. Total DNA concentration ≥15 ng/μL will proceed to the sequencing stage. A shotgun metagenomic sequencing process from the MGI DNBSEQ-G400 platform was carried out to obtain DNA sequences.

Analysis

Merging raw paired-end reads and quality control simultaneously using FLASH 1.2.11 (Fast Length Adjustment of SHort reads) (Magoč and Salzberg, 2021). This facilitates accurate sequence reconstruction while filtering out low-quality reads. Subsequently, Qiime2 (3.5.3 phyton version) is employed for further quality filtering to enhance data integrity by removing noise and artifacts (Bolyen et al., 2019). The criteria for quality control were a sequence was removed once its average quality within the window fell below Q20; sequences containing any N-bases were filtered out; reads that were below 50 bp in length were dropped; only paired-end reads were retained. The dataset undergoes dereplication and chimera removal using UCHIME, contributing to reduced redundancy and elimination of artificial sequences (Edgar, 2017).

Taxonomic classification of the processed sequences is performed using Diamond followed by MEGAN6 (Bağcı et al., 2021). These tools provide insights into the relative abundance of bacteria, offering valuable information on microbial community composition. If assembly is required, Qiime2 is utilized for this purpose (Culp and Goodman, 2023). Using MEGAN6, functional annotations (hits to SEED subsystem) were extracted (e value less than 1 × 10-5 and sequence match length greater than 50 nucleotides) to compare functional antimicrobial resistance gene attributes across the birds of paradise gastrointestinal metagenome (Overbeek et al., 2014). Finally, RStudio is utilized for custom scripting to quantify gene abundance and visualization (Rstudio Team, 2020).

RESULT and Discussion

Samples characteristic

Six of the initial samples failed to meet the required DNA concentration standard, which stipulated a minimum of 15.0 ng/μL. Two samples with the highest concentrations of 14.0 ng/μL and 14.6 ng/μL, with A260/280 ratios of 1.59 and 1.75, respectively, were used to continue the sequencing process using the shotgun metagenomics method of the MGI DNBSEQ-G400 platform. Although these concentrations are below the standard threshold, the A260/280 ratio and total DNA concentration being close to the optimal value indicates a fairly good relative purity of the DNA. This decision was based on the reliability of the MGI DNBSEQ-G400 platform in coping with samples with low DNA quantity and still producing accurate sequences. We believe that by selecting these two best samples, we can still obtain informative metagenomic data, despite the relatively low concentrations of the samples. The two samples were obtained from distinct bird of paradise species with varying dietary habits and display behaviors, as detailed in Table 1.

Gastrointestinal microbiome: Species abundance

The results of metagenomic analysis using the shotgun metagenomics platform on birds of paradise samples and feces found diverse species. A total of 104 species were successfully sequenced from the samples, 53 species from sample CDW2 (5 unspecified species) and 51 species from CDW6 (18 unspecified species) where 26 of the same species were obtained from both samples. Based on Figure 1, it can be seen that most of the microbiome abundance in the gastrointestinal of birds of paradise from both samples are Delftia sp., Pedobacter sp. (ASV17, ASV12, ASV8, ASV2), Klebsiella pneumoniae, Achromobacter sp., Bacillus pumilus, Rhizobium sp., and Brevundimonas sp. with some differences in the species Flavobacterium sp., Aquitaiea sp., and Acidobacterium sp., respectively.

 

SEED subsystems functional annotation

A total of 100,057 genes in the CDW2 sample and 225,830 from the CDW6 sample were successfully annotated into the SEED subsystem functional annotation. Annotation was carried out up to level 3 to determine the composition of AMR genes in the gastrointestinal of birds of paradise (Figure 2). Only 2379 genes from CDW2 and 8744 genes from CDW6 have functional genes of AMR (Figure 3).

 

Table 1: Birds of paradise sample information.

Sample ID	Date collected (dd/mm/yyyy)	Bird species	Diet	Display behavior	A2600/A280 ratio	DNA concentration (ng/μL)
CDW2	28/12/2022	Twelve-wired bird-of-paradise (Seleucidis melanoleucus)	50% fruits	Arboreal, solitary, dispersed, not territorial	1.59	14.0
CDW6	30/12/2022	Magnificent bird-of-paradise (Cicinnurus magnificus)	70% fruits	Terrestrial, solitary, dispersed, not territorial	1.75	14.6

 

 

 

The study reveals significant challenges in combating bacterial infections, particularly in light of the high resistance observed in specific mechanisms. Bacitracin resistance exhibits a formidable resistance level of 43.4%. Similarly, resistance to Daptomycin showcases a notable resistance level of 7.4%. In addition to antibiotic resistance, the diversity of antibiotic targets across various cellular processes is evident. Antibiotic targets encompass cell wall biosynthesis (3.9%), DNA processing (0.8%), metabolic pathways (3.5%), protein synthesis (0.6%), and transcription (5.1%). This diversity highlights the multifaceted nature of bacterial resistance. Bacteria exhibit not only antibiotic resistance but also resilience to environmental stressors. Mechanisms such as Resistance to chromium compounds (5.6%), Copper homeostasis: Copper tolerance (2.9%), and Arsenic resistance (2.5%) underscore the adaptability of bacteria to diverse challenges.

The study delves into the intricate world of signal transduction systems, exemplified by the Actinobacterial signal transduction system MtrAB-LpqB. These systems suggest complex regulatory networks influencing bacterial responses. While some resistance mechanisms exhibit lower values, such as tetracycline resistance, ribosomal protection type (0.005%), and macrolides, lincosamides, streptogramins, ketolides, oxazolidinones (MLSKO) resistance: Ribosomal protection (0.03%). The study also sheds light on regulatory systems contributing to bacterial tolerance, including VraTSR and LiaFSR three-component regulatory systems (0.1%) and Transcriptional regulators implicated in bedaquiline tolerance (0.005%).

This study revealed interesting findings regarding the high level of resistance to bacitracin in the gastrointestinal of birds of paradise. This resistance is most likely attributed to the abundance of Bacillus pumilus bacteria in the birds’ gastrointestinal. Bacillus pumilus, as a Gram-positive bacterial species, can produce a variety of bioactive compounds, including antibiotics, which may provide a competitive advantage in such environments (Iqbal et al., 2021).

High bacitracin resistance in the gastrointestinal of birds of paradise suggests that Bacillus pumilus, as a potential producer of bacitracin, may play a key role in developing bacterial resistance within the digestive system. Resistance to bacitracin is the result of evolutionary selection pressures that bacteria may face in competing for survival in an environment rich in antimicrobial compounds (Awais et al., 2007).

It has been discovered through recent research that bacitracin, a type of polypeptide antibiotic, has a positive role in supporting the growth of certain microorganisms, including Bacillus sp., and also promotes the intake of ion in the soil (Haavik, 1975, 1976). This discovery suggests that bacitracin not only has antimicrobial properties but can also act as a growth factor that could potentially benefit producer organisms during their growth process. This can also answer the higher abundance of Bacillus pumilus in terrestrial compared to arboreal birds of paradise.

Furthermore, the abundance of Bacillus pumilus may create a unique microecosystem within the gastrointestinal of birds of paradise. Factors such as the production of antimicrobial compounds, the capacity to form biofilms, and interactions with other bacteria may influence the dynamics of antimicrobial resistance in this environment (Uruén et al., 2020).

The metal and environmental resistance mechanisms observed in the gastrointestinal of birds of paradise reflect complex bacterial adaptations to specific ecological conditions. Arsenic resistance indicates the ability of bacteria to resist the toxic effects of arsenic, a metalloid found in soil and water. Cadmium resistance and chromium resistance indicate the ability of the bacteria to tolerate high concentrations of these metals. Copper homeostasis and tolerance indicate the ability of bacteria to maintain a balance of copper ions. This adaptation may be particularly important for birds of paradise as this resistance potentially originating from the bird of paradise environment, such as soil (especially in wetland) or food sources (Sánchez-Virosta et al., 2015; Łanocha-Arendarczyk and Kosik-Bogacka, 2019).

Resistance to chromium compounds further underscores the ability of bacteria to withstand certain chromium compounds present in the environment of paradise. Papua in Indonesia has chromium concentrations of stream sediment samples ranging from 3 ppm to 74,600 ppm, with a median value of 145 ppm, which was higher than the upper crustal abundance of chromium and the chromium geochemical baselines of Europe, Australia, North America, and China (Zhao et al., 2023). However, the sedimentation concentrations of chromium and arsenic did not exceed the Threshold Effect Level (TEL) (Hamuna and Wanimbo, 2021). Some possible factors that could influence this phenomenon involve ecological and biochemical aspects of the ecosystem. There may be genetic variation in bacterial populations on the island of Papua that accounts for the high abundance of chromium resistance genes. Bacterial populations in a given area can have different genetic profiles that develop in response to local environmental conditions. In addition, natural selection can promote the development of genetic resistance in bacterial populations (Santos-Lopez et al., 2021). Bacteria that have higher resistance genes have adaptation advantages and can reproduce better in certain environments. Also, certain microbiological conditions, such as soil pH, moisture, or certain types of vegetation, can favor the growth and proliferation of bacteria with chromium resistance genes (Abdul-Rahman et al., 2021).

Many AMR genes have been found in the gastrointestinal of birds of paradise. Most of these genes can appear naturally because antibiotic compounds can naturally exist in nature as a protective mechanism from other organisms (Andersson et al., 2020). However, some genes such as resistance to ethionamide and isoniazid (antibiotic to treat tuberculosis), as well as some resistance genes to floroquinolone mechanisms which are synthetic antibiotics can be found in this microbiome (Wolff and Nguyen, 2012; Gegia et al., 2017).

This is a concern because one of the possible causes of this resistance is continuous exposure to chemical compounds in their environment or even the possible transfer of genetic resistance from other microorganisms in their ecosystem (Larsson and Flach, 2022). The resistance gene can also come from genetic mutations in Mycobacterium tuberculosis (i.e. inhA double mutations) or even possibly from other microbes in the microbiome that have some similarity with this gen (Machado et al., 2013; Sandoval et al., 2019).

Of particular consideration is the role of antibiotics in agriculture and animal husbandry, which may result in antibiotic residues in the habitats of birds of paradise. This contamination can be a direct cause or contributor to antibiotic resistance in bird populations (Jechalke et al., 2014). It is crucial to note that, despite their primary diet consisting of fruits, birds of paradise can inadvertently be exposed to antibiotics through certain human activities in their habitat, such as agricultural runoff, disposal of pharmaceutical waste, or the use of antibiotics in nearby livestock or directly to these birds (Larsson and Flach, 2022). However, it is pertinent to highlight that these birds have not been actively administered any antibiotics and have limited interaction with humans, primarily occurring during monitoring purposes. Consequently, a comprehensive understanding of the factors contributing to antibiotic resistance in birds of paradise is imperative, with environmental pollution being one potential source among various factors.

CONCLUSIONs and Recommendations

This study shows the abundance of AMR genes in the gastrointestinal of birds of paradise. the highest abundance is shown in bacitracin resistance. This significant resistance is linked to the prevalence of Bacillus pumilus. This resistance is indicative of evolutionary selection pressures and suggests a potential role of Bacillus pumilus in bacterial resistance development. The presence of metal and environmental resistance mechanisms further underscores complex bacterial adaptations in the unique micro-ecosystem within the birds’ gastrointestinal. The abundance of antibiotic resistance genes, especially synthetic antibiotics, raises concerns about continuous exposure to environmental chemicals, including antibiotics used in agriculture, emphasizing the need for a comprehensive understanding of the factors influencing resistance in birds of paradise.

ACKNOWLEDGEMENT

The author is grateful to IPB University and PT. Saraswanti Gene Indotech for all the facilities to achieve this study. The author is also grateful to BKSDA Jayapura Mrs. Lusi, Mr. Danial, and other colleagues for their input in this research, especially in field monitoring and bird of paradise behavior, for Mr. Kholiq for the time and knowledge for this research and other people who contributed to this research.

Novelty Statement

This study uses shogun metagenomics sequencing tool to explore AMR gene of bird of paradise and functional annotation is performed using SEED subsystems.

AUTHOR’S CONTRIBUTION

ARRP, S, and AI: Study conception and design, contributed to writing, revising, and editing the manuscript, review, editing, and visualization of the manuscript. ARRP and S: Conducted the experiments and analyzed the data, acquisition data, review, and editing. All authors have read, reviewed, and approved the manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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