IN SILICO EVALUATION REVEALS THE POTENTIAL RISK OF ANTIMICROBIAL RESISTANCE IN Bifidobacterium EVALUASI IN SILICO MENGUNGKAPKAN POTENSI RISIKO RESISTENSI ANTIMIKROBA

Bifidobacteria have beneficial health effects for their hosts. However, they may acquire antibiotic-resistance genes. They may transfer antibiotic-resistance genes to pathogenic microbes in the human intestine, resulting in antibiotic-resistant pathogens. This study aimed to predict their resistance to antibiotics by analyzing the whole genome sequence. The entire genome data of Bifidobacterium spp. were obtained from the National Center for Biotechnology Information (NCBI). This study included five Bifidobacterium strains of human origin, five strains of animal origin, and three strains isolated from the environment. The genomic sequences were analyzed using ResFinder and CARD web service. Antibiotic-resistance genes were detected in Bifidobacterium spp. from all sample sources. Bifidobacteria were potentially resistant to various antibiotics, such as tetracycline, rifamycin, chloramphenicol, macrolide, lincosamide, streptogramin, and mupirocin-like antibiotics. This study suggests the safety of applying Bifidobacterium spp. as a potential probiotic.


INTRODUCTION
Lactic acid bacteria (LAB) are known as safe bacteria that have beneficial health effects for their hosts, including Bifidobacterium.Various Bifidobacterium species reside in the gastrointestinal tract, mouth, non-human animal intestines, and dairy products (Hendrati et al., 2017;Kusharyati et al., 2020;Sakanaka et al., 2020;Sirilun et al., 2015).Bifidobacterium is one of the most commonly used probiotics because it produces various metabolites, such as lactic acid, H2O2, short-chain fatty acids, and bacteriocin.Consuming probiotics regularly as part of a healthy diet can help promote a healthy microbiome.A healthy microbiome is essential for many aspects of health, including digestion, immune function, cardiovascular health, and even mental health (Liu et al., 2016;Shreiner et al., 2015;Yan et al., 2017;World Health Organization, 2002).
Some LAB strains may acquire antibiotic-resistance genes through natural evolution or antibiotic exposure in the environment and food production chain.LAB can potentially develop and spread antibiotic-resistant genes (Duranti et al., 2017;Gueimonde et al., 2013;Zarzecka et al., 2022).Fermented foods can be a significant conduit for the spread of antibiotic-resistance genes from nonpathogenic microorganisms to humans.It results in the emergence of antibiotic-resistant pathogens.Therefore, EFSA suggested that only strains lacking acquired antibiotic-resistance genes (ARGs) can be used as starter cultures, probiotics, and feed additives (EFSA-FEEDAP Panel, 2018).
Bifidobacteria are intrinsically resistant to many antibiotics.Nunziata et al. (2022) reviewed that Bifidobacteria are typically regarded as susceptible to β-lactams, Gram-positive spectrum antibiotics, and broad-spectrum antibiotics at low doses.However, some Bifidobacterium were reportedly resistant to narrow and broad-spectrum antibiotics (Erginkaya et al., 2018;Rozman et al., 2020;Yasmin et al., 2020).This present study concerned the safety of applying Bifidobacterium spp. as a potential probiotic.
Genotypic methods have been explored as an alternative to phenotypic antimicrobial susceptibility testing (AST).However, phenotypic AST using disc diffusion and broth microdilution has many limitations.Some errors may arise during the culturing stage, inoculum preparations, and result interpretation (Florensa et al., 2022;Pedersen et al., 2018;Su et al., 2019).The findings between these two methods are not always equivalent, which may impede the results' repeatability (Rozman et al., 2020).Molecular-based resistance testing to antibiotics is usually done through PCR detection but with a limited number of genes.Whole genome sequences (WGS) based screening enhances the likelihood of discovering genes involved in antibiotic resistance.Understanding the resistance gene profiles of Bifidobacterium strains used in probiotics is crucial to ensure their safety.This study aimed to evaluate the genes associated with the probiotic properties of Bifidobacterium spp.

MATERIALS AND METHODS
The importance of studying antibiotic-resistance genes within Bifidobacterium strains from environmental, animal, and human samples cannot be understated.One of the critical reasons for studying ARGs in Bifidobacterium across diverse samples is to track the transfer of resistance.Bifidobacterium is known to engage in horizontal gene transfer, allowing resistance genes to move between different strains and species.The whole genome data of Bifidobacterium spp. was collected from The National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/).There are 107 available genomic data sets for Bifidobacterium spp.Only view data reported the source of Bifidobacterium isolates.Genome sequences are randomly selected from Bifidobacterium strains that have complete data and represent the source of the sample (Table 1).The data is downloaded in a fasta format (.fna).
This present study used five Bifidobacterium strains of human origin, five strains of animal origin, and three strains isolated from the environment.Thirteen whole genome sequences were analyzed for their resistance genes using ResFinder 4.1.It is available for free at the Center for Genomic Epidemiology's online platform at https://cge.food.dtu.dk/services/ResFinder/(Babatunde et al., 2022;McArthur et al., 2013).ResFinder detects acquired genes and chromosomal alterations that mediate antimicrobial resistance in the bacteria's complete or partial DNA sequence.The second analysis used the Comprehensive Antibiotic Resistance Database (CARD) web service at https://card.mcmaster.ca/analyze/rgi.It is a primary bacterial antimicrobial resistance knowledge resource and database that provides genotype analysis and phenotype prediction from curated publications and sequences (Alcock et al., 2023;Zankari et al., 2017).Using homology and SNP models, the Resistance Gene Identifier (RGI) can predict resistomes from protein or nucleotide data.

RESULTS
Scientists have significantly advanced in utilizing whole genome sequencing to screen probiotic properties.By analyzing the WGS, this present study reported that Bifidobacterium spp.contains antibiotic-resistant genes (86.97% ≤ ID ≤ 100%).For example, those against the classes of tetracycline, rifamycin, macrolide, lincosamide, streptogramin A, streptogramin B, and mupirocinlike antibiotics (Table 2).The CARD analysis showed that antibiotic-resistant genes were found in Bifidobacterium spp.from all sample sources, i.e., humans, animals, and the environment.Note: The antibiotic resistance ontology (ARO) describes antibiotic resistance genes and mutations, their products, mechanisms, and associated phenotypes, as well as antibiotics and their molecular targets (Alcock et al., 2023) Table 2 shows that only one genetic background was detected in Bifidobacterium spp.isolated from humans and the environment, i.e., the resistant gene of erythromycin (ErmX).Four genetic backgrounds associated with antibiotic resistance were detected in Bifidobacterium spp.from animal various commercial Bifidobacterium strains (Cao et al., 2020;Rozman et al., 2020).This raises concerns about the potential impact on human health and the effectiveness of antibiotic treatment when these resistant bacteria are introduced into the gut.
The most common erythromycin resistance gene in Bifidobacteria is erm(X) (Table 2).This gene encodes an enzyme that modifies the antibiotic's target site, rendering it ineffective (Cao et al., 2020).Other less frequent genes include erm(B) and erm(A).Meanwhile, several tetracycline resistance genes have been identified in Bifidobacteria, including tet(W), tet(M), and tet(S).These genes encode different mechanisms for tetracycline resistance, such as ribosomal protection or drug efflux pumps that remove the antibiotic from the cell (Duranti et al., 2017;Gueimonde et al., 2013).
Tetracycline is one of the most extensively utilized antibiotics in veterinary and clinical settings (Indrawati et al., 2021;Peiris et al., 2017).Tetracycline resistance genes can be transferred horizontally between different bacterial species through conjugation, transduction, and transformation (Ding et al., 2023).Although conjugative plasmids are uncommon in Bifidobacterium spp., other mobile genetic elements (e.g., transposons) must be considered because the tet gene appears to be positioned on the chromosome.Tetracycline resistance genes can be co-located on the same mobile genetic elements (e.g., plasmids) as genes conferring resistance to other antibiotics or stressors (Gueimonde et al., 2013).It is surrounded by transposase-coding genes or transposase target sequences in some Bifidobacterium strains (Duranti et al., 2017;Gueimonde et al., 2013).Therefore, tetracycline resistance in Bifidobacterium spp.warrants special consideration.
Bifidobacterium is not limited to the human gut but also inhabits various environmental niches, such as soil and water.Antibiotic resistance in environmental Bifidobacterium strains may contribute to the overall environmental resistors.It potentially affects the persistence and spread of tetracycline resistance genes in natural ecosystems (Larsson & Flach, 2022).ARGs within animal Bifidobacterium strains can directly impact food safety and public health.In humans, Bifidobacterium species are essential members of the gut microbiome, contributing to overall health and well-being.ARGs within human-associated Bifidobacterium strains raise concerns about the potential transfer of resistance to pathogenic bacteria.
The resistance of Bifidobacterium strains to various antibiotics complicates their potential use as probiotics.Tetracycline resistance in Bifidobacterium can also lead to failure to treat bacterial infections.Monitoring tetracycline resistance in Bifidobacterium strains is essential to prevent failure to treat bacterial infections and maintain digestive health.Industry can select strains with low or no ARGs, minimizing the risk of transferring resistance to the host or other gut bacteria.This discovery also emphasizes the need for prudent and responsible antibiotic use in clinical settings and agriculture.The spread of antibiotic resistance among bacteria, including beneficial ones like Bifidobacterium, underscores the urgency of minimizing unnecessary antibiotic use in healthcare and agriculture.
Bifidobacteria can acquire new genes from other bacteria in their environment through horizontal gene transfer (Larsson & Flach, 2022).This allows them to gain resistance genes not originally part of their genome.The widespread use of antibiotics in agriculture and human medicine creates a selective pressure, favoring the survival and propagation of bacteria with resistance genes.These genes can then be transferred to Bifidobacteria in the gut.If individuals consuming these probiotics become infected with bacteria susceptible to antibiotics, e.g., erythromycin or tetracycline, the presence of resistant Bifidobacteria in their gut could hinder the effectiveness of the antibiotics.The resistant Bifidobacteria could transfer their resistance genes to pathogenic bacteria in the gut, further complicating antibiotic treatment.

CONCLUSION
Antibiotic-resistance genes were detected in Bifidobacterium spp.used in this study from different sample sources, i.e., humans, animals, and the environment.Bifidobacterium spp. is potentially resistant to various antibiotic classes, including narrow-and broad-spectrum antibiotics.Tetracycline and erythromycin resistance warrants special attention since Bifidobacterium strains are increasingly used as probiotics in food products and dietary supplements.
In silico evaluation can be used to assess the potential risk associated with using Bifidobacteria strains isolated from various sources, such as food products or environmental samples.Combining in silico evaluation with traditional laboratory methods and ongoing research can develop strategies to mitigate the risks associated with antibiotic resistance in Bifidobacterium and harness the full potential of these beneficial bacteria for promoting gut health.

Table 1 .
Retrieved data for Bifidobacterium genome