In this study, Lactobacillus species carried ARGs for a variety of antibiotic classes, though resistance genes were not distributed evenly among species. Any surveillance of gut bacterial populations must take into account the distinction between resident and transient bacterial species. In comparison to transient bacteria, resident members have the ability to attach and colonize intestinal tissue and have a significant impact on the host's health status. However, the transient inhabitants would have an impact on the resident members through the transmission of virulent factors such as antibiotic resistance genes, which would change the characteristics of this community . Horizontal transfer of ARGs to this permanent community creates a resistance reservoir known as the resistome, which has a major effect on the distribution of acquired mobile resistance genes to various environments, such as transferring these genes to pathogenic bacteria in the GIT or other environments due to fecal contamination . L. plantarum, L. rhamnosus , L. brevis, L. acidophilus, L. casei, L. crispatus, L. delbrueckii, L. fermentum, L. fructivorans, L. gasseri, L. paracasei, L. ruminis, L. sakei, L. salivarius, L. vaginalis are the permanent residence of human GIT . The most common species in the current study were Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus paracasei, and Lactobacillus fermentum, all of which are classified as resident microbiota except for Lactobacillus paracasei. Although the diversity of the carried genes was not high in each species, these species had a high abundance of resistance gene determinants.
The majority of studies revealed that Lactobacillus species are generally susceptible to a variety of antibiotic drug classes, including penicillin, chloramphenicol, tetracycline, quinupristin-dalfopristin, macrolide (erythromycin antibiotic), lincosamide (clindamycin antibiotic), oxazolidinone (linezolid antibiotic), and rifampicin [19–21]. Antibiotic resistance in this group of bacteria is classified as intrinsic and acquired as a result of their natural resistance to a wide range of antibiotics. Intrinsic resistance is caused primarily by efflux pumps, the absence of drug targets, antibiotic inactivation mechanisms, and cell wall impermeability. Intrinsic resistance is innate and is unrelated to prior antibiotic exposure . Acquired resistance, on the other hand, is caused by chromosomal gene mutation or horizontal gene transfer (HGT). Because HGT is the primary mode of resistance transmission, intrinsic and chromosomal mutated genes are unlikely to be transferred to other bacteria [23, 24] .
Most Lactobacillus species have been found to have high intrinsic resistance to the antibiotic diaminopyrimidine (trimethoprim). Trimethoprim resistance is generally conferred by dfr-genes that encode dihydrofolate reductase (DHFR)  . Although in silico studies have reported dfr genes related to trimethoprim resistance in lactobacilli, MIC reports have shown that trimethoprim resistance in lactobacilli is related to the folate auxotrophy [26, 27]. In the current study, nearly 58 percent of the assemblies were resistant to the diaminopyrimidine (trimethoprim) antibiotic via the dfrA42 gene, indicating the complexity of this type of resistance and implying the possibility of additional mechanisms for trimethoprim resistance in Lactobacillus species. L. johnsonii, L. acidophilus, L. salivarius, L. brevis, L. casei, L. gasseri, L. rhamnosus, L. delbrueckii, L. fermentum, L. helveticus, L. plantarum, L. reuteri, L. sakei, and L. crispatus were previously identified as auxotrophic species . In this study, the drfA42 gene was found in twenty-nine species, which included all of the previously reported auxotrophic Lactobacilli except L. acidophilus, L. rhamnosus, and L. fermentum. In addition, L. ruminis, L. paragasseri, L. mucosae, L. amylolyticus, L. amylovorus, L. hilgardii, L. hominis, L. saniviri, L. senioris, L. ultunensis, L. agilis, L. animalis, L. buchneri, L. curvatus, L. gallinarum, L. murinus, L. parafarraginis and Lactobacillus spp, were identified harboring dfrA42 gene.
Lactobacillus species are also intrinsically resistant to quinolones and the majority of aminoglycoside antibiotics [21, 29]. Aminoglycoside resistance was not prevalent in this study, although a variety of resistance genes were identified, their abundance was only around 1%. According to the findings of a recent study, despite resistance to aminoglycosides, none of the related resistance genes were found . Several mechanisms are known to contributed to aminoglycoside resistance including the activity of aminoglycoside modifying enzymes, increased efflux and/or decreased permeability and target modification (30s ribosomal subunit) . As a result, it is possible to conclude that high MIC values for aminoglycoside antibiotics do not indicate the presence of well-characterized resistance genes.
Lincosamides inhibit protein synthesis by inhibiting peptidyltransferase on the 50S subunit of the ribosome. There is cross resistance to lincosamide, macrolide and streptogramin B due to their same mechanism of action . In this study, two gene families were found to contribute to lincosamide drug resistance: ATP-binding cassette (ABC) antibiotic efflux pump (lmrB) and lincosamide nucleotidyltransferase (lnuA, lnuC, lnuG). Furthermore, Erm 23S ribosomal RNA methyltransferase (ermB, ermT, ermA, ermY, erm46, and ermR) caused cross-resistance to lincosamide, streptogramin, and macrolide antibiotics, while non-erm 23S ribosomal RNA methyltransferase (myrA) caused resistance to both lincosamide and macrolide. lmrB was the most prevalent gene associated with lincosamide resistance (17%) found in L. rhamnosus, with the others occurring at a frequency of around 1%. In Streptomyces lincolnensis, lmrB is one of three resistance genes (lmrA, lmrB, and lmrC) that are responsible for lincomycin biosynthesis . In agreement, previous studies have reported the lincosamide resistance associated with lmrB gene in lactobacilli, as well as erm and lnu genes related to lincosamide resistance and/or cross-resistance with macrolide and streptogramin [33–35]. It has been demonstrated that lmrB is located on a plasmid that contributes to bacteriocin production. Because Lactobacilli are naturally susceptible to lincosamide, plasmids containing lmrB indicate that this acquired resistance is being transmitted. Previously, it was discovered that L. gasseri UFVCC 1091 has a plasmid pTRK1024 that contains the lmrB gene [36, 37]. ARGs were also studied on Lactobacillaceae-associated plasmids, with the results showing that lmrB was present on plasmids derived from L. plantarum, L. paraplantarum, L. buchneri, L. sakei, L. curvatus, and L. brevis . In the study, the lmrB gene in L. rhamnosus species was found in their genome mostly next to ISLrh2 and ISLrh4. Because these insertion sequences are from L. rhamnosus, it is possible that lmrB transferred from a plasmid to the genome and spread among L. rhamnosus strains .
Lactobacillus species are mostly susceptible to the carbapenem class of antibiotics; some genomic studies show lactobacilli carrying carbapenem resistance genes; however, Lactobacillus phenotypic resistance to this class of antibiotic has been published as case reports [39, 40]. BJP-1 is a new Subclass B3 metallo-lactamase (MBL) that was discovered in Bradyrhizobium japonicum, a nitrogen-fixing bacteria used in agriculture, whose genome was recently sequenced [41, 42]. BJP-1 is a chromosomally encoded intrinsic MBLs that may not acquire through horizontal gene transfer [43, 44]. In this study, the BJP-1 gene was found in nearly 11% of the assemblies, implying that it was transferred from soil microbiota to humans. Interestingly, all of the detected genes were carried by L. rhamnosus, which is, to the best of our knowledge, the first report of BJP-1 in Lactobacillus species. According to the findings of a new study, Firmicutes bacteria have the potential to be the future recipients of ARGs . The genome of L. rhamnosus contains a large number of insertion elements, which contribute to the organism's genomic instability . The presence of insertion elements in the vicinity of ARGs, on the other hand, could potentially be the reason for the receipt and transfer of those resistance genes. As a result, L. rhamnosus's high density IS elements establish it as a successful organism in receiving and transferring ARGs to new bacterial families in the gut. The presence of the insertion sequence ISLrh2 upstream and downstream of the BJP-1 in this study suggests the possibility of transmission to other bacterial families in the gut.
Tetracycline is a broad-spectrum antimicrobial drug class that is widely used in clinics to treat Gram-positive and Gram-negative bacterial infections. Tetracycline resistance is mainly caused by three general mechanisms: ribosomal protection, efflux, and enzymatic inactivation . The majority of these resistance determinants were acquired by MGEs such as transposons and plasmids [48, 49]. The most common tetracycline resistant genes found in this study were tetW and tetC, which were found in 6 and 9 genomes, respectively. TetW has been repeatedly identified as the most phenotypic and genotypic tetracycline resistance gene in environmental, veterinary, and human microbiome samples [50–52]. A variety of MGEs, including integrative conjugative transposons, have been reported to flank this large-sized gene (1.9 kb) [53, 54]. A recent in silico study reported a list of MGEs involved in the transposition of the tetW gene in lactobacilli and bifidobacteria . The abundance of tetC, a tetracycline efflux pump gene, was higher in the current study than in the other tetracycline resistance genes. TetC was previously discovered in a variety of gram-negative bacteria, mostly in plasmids . PoxtA was also found in 24% of the genomes and was linked to cross-resistance to multiple antibiotics, including tetracycline. PoxtA is a transferable resistance gene that belongs to the ABC-F ATP-binding cassette ribosomal protection protein family. This is a new resistance gene found in Methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus that confers cross-resistance to oxazolidinone, phenicol, and tetracycline [56, 57]. Furthermore, this gene has been identified in animal-derived samples, and analysis has revealed that it is located in IS1216 elements . Previous dairy product studies identified the poxtA gene frequently in Lactobacillus species, implying that the widespread use of phenicols and other related antibiotic agents is the cause of the prevalence of this gene among lactobacilli [59, 60]. Numerous IS elements were found upstream and downstream of the poxtA gene in this study, indicating the potential mobilization of this gene among Lactobacillus species.
Lactobacillus species are generally susceptible to the phenicol drug class (chloramphenicol), although some studies have reported lactobacilli resistance to this class of antibiotics [26, 27]. In this study, several genes related to chloramphenicol resistance were discovered with less than 1% frequency, the majority of which belonged to the chloramphenicol acetyltransferase (CAT) gene family.
Lactobacillus species are the most important component of the microbiome that promotes health benefits. Antimicrobial resistance is also a significant threat to human health. According to the findings of this study, Lactobacillus could be regarded as a reservoir for the potential spread of acquired resistance genes in the gut environment from commensal to pathogen bacteria.