Investigation of Some Antibiotic Resistance Genes of Indigenous Lactobacilli Isolated From Traditional Yogurt In Zanjan Province of Iran

Purpose: Given the increasing use of antibiotics on humans and livestock for treatment or as a growth stimulant, antibiotic resistance has become a general concern. The food chain and specially fermented foods could be a source of antibiotic-resistant bacteria and resistance genes. Lactic Acid Bacteria (LAB) and Lactobacilli are considered safe to use as starter culture or probiotic strains. Recently, however, antibiotic-resistant genes isolated from LABs showed the necessity of setting international regulations to reduce the risk of antibiotic resistance genes transmission via the food chain. The current study aimed to investigate the antibiotic resistance of Lactobacilli isolated from traditional yogurt samples from Zanjan province in Iran. Methods: Lactobacilli characterization and identication were carried out through biochemical and molecular methods. The disk diffusion method was applied to determine phenotype resistance using 13 antibiotic disks resistance genes presence were investigated in the isolates to determine transferability risk, respectively. Results: Based on biochemical and molecular methods, 24 isolates have been identied as Lactobacilli with multiple antibiotic-resistant phenotypes. Vancomycin resistance was a typical phenotype and genotype among isolates. On investigated Lactobacilli chromosome, Tetracycline resistance genes Chloramphenicol (cat), beta-lactam, aminoglycosides (aph (3’)-III), and aadA resistance genes have been detected. While the examined resistance genes have not been detected on the plasmids, they were all on the bacterial chromosome. Conclusion: The results showed that the investigated isolates did not carry the resistance genes on their plasmids. It, therefore, would be a good point since they probably do not transfer resistance genes to other bacteria, and they would be proper candidates to do more investigation for introducing new safe starter culture or probiotic strain to food industries. al.2017).All samples were tested twice. Positive control was the cat gene-positive Lactobacillus. Using 1% agarose in TAE buffer (40 mMTris-acetate, 1 mM EDTA, and pH 8.2), agarose gel electrophoresis was conducted, and staining was done with ethidium bromide and then visualized by a gel documentation device. bacterial plasmid. resistance genes bacterial plasmid. results agarose Ampicillin Belleti Čanžek et al. (2007), Flórez et al. (2005)Temmerman et al. (2003). In this research, resistance to penicillin in eight isolates 3, 4, 9, and resistance to ampicillin in (37.5%) 9 isolates 1, 6, 7, 8, 14, 17, 19, 22, and 23) were observed. Four isolates, including Nos. 1, 17, 22, and 23, shown resistance to penicillin and ampicillin together. The bla Z gene can be responsible for penicillin, and in some cases, it is resistant to Ampicillin and found in isolates Nos.4, 3, 1, 22, 9, and 23. Phenotype and genotype correspondence 75% in penicillin-resistant isolates and in Ampicillin. et al. 17% of


Introduction
Antibiotics are among those extensive groups of therapeutic agents commonly used in bacterial infection treatments. The term "antibiotic" was rst used by Sales Waksman for small molecules produced by microorganisms with antagonistic effects on other species (Ribeiro et al.2019). The antibiotic turning point was in 1928 when Sir Alexander Fleming discovered Penicillin. During World War II, Penicillin was used successfully to treat bacterial infections (Ventola 2015). Fleming was the rst to warn of the dangers of Penicillin resistance due to its overuse (Rosenblatt 2009). Different types of antibiotics can trigger bacterial cell membrane, cell wall, protein, genetic material, and other biologic compounds synthesis (Tortora et al. 2015). Bacteria, however, have exibility against environmental stresses such as antibiotics. They can resist these molecules through different mechanisms (Munita and Arias 2015) including, altering the antibiotic, permeability changes in the bacterial cell wall, e ux pumps and active transport of the antibiotic, change in antibiotic target structure, antibiotic degradation, and target enzyme overproduction. Resistance to some speci c antibiotics in bacteria could be intrinsic, or they could resist antibiotics through mutation or acquiring resistance genes via plasmids or other mobile genetic elements (Peterson and Kaur Antibiotic resistance studies have focused mainly on clinical isolates of bacterial species, but it should be noted that food bacteria might also act as reservoirs of antibiotic resistance genes (Franz et al. 2005). Fermented foods and fermented dairy products may be considered vehicles for transferring antibioticresistant bacteria because of their high microbial population (Mathur and Singh 2005). Though Lactobacilli have generally known as safe, there are some reports about intrinsic and acquired resistance to antibiotics in some of them like L.plantarum, L. paracasei, L.reuteri, and L.acidophilus (D' Aimmo et al.2007;Liu et al;2009Rojo-Bezares et al.2006, so showing that more investigation on antibiotic resistance in Lactobacilli is necessary.

Antibiotic Susceptibility Test
In this research, the disc diffusion method was applied to determine the antibiotic susceptibility of isolated Lactobacilli. Isolates have been cultured on nutrient agar for 18-24h, at 37 C under microaerophilic condition transferred to sterile physiologic serum to compare the turbidity of bacterial suspension with Mac Farland standard solution according to Iran National Standard No. 13560 in 625 nm wavelength. The dried surface of the Muller Hinton agar plate was inoculated by bacterial suspension using a sterile swab. Antibiotic disks including, Tetracycline 30µg, Erythromycin 15µg, Kanamycin 30µg, Gentamycin 10µg, Vancomycin 30µg, Penicillin G 10µg, Nalidixic acid 30µg, Rifampin 5µg, and Cipro oxacin 5µg Chloramphenicol 30µg, Trimethoprim, 5µg Ampicillin 10µg Streptomycin 10µg (Rosco, England, and Fan Azma Iran) were placed on the Muller Hinton agar plates using sterilized forceps. The plates were incubated 16-18-hours at 37°C. For each disk, the apparent zone diameter was measured three times, and for all of them, according to Iran national standard No. 13560, standard deviation was calculated.

Molecular Characterization
By ready-to-use Yakhteh Saba Arena Company in Iran, as constructor instruction, Chromosomal DNA was extracted. Molecular characterization of Lactobacilli carried out by speci c 16S rDNA sequence as follows: R16-1: (5 -CTTGTACACACCGCCCGTCA − 3 , LbLMA1-rev: 5 -CTCAAAACTAAACAAAGTTTC-3 ). PCR reaction was carried out in Biorad 1000 thermal cycler using Sina clone master mix 25µl, template DNA 1µl, forward primer 1µl, reverse primer 1µl, and sterile distilled water to a nal concentration of 50µl (Saavedra et al.2004). PCR reaction cycles were carried out with the following condition, denaturation at 95˚C for 5 minutes, followed by 30 cycles for 40 seconds at 95˚C. Annealing was done at 57.6˚C for 40 seconds. Elongation was carried out at 72°C for 40 seconds, followed by nal elongation at 72°C for 5 minutes (Hudzicki2009,Ghobadi Dana 2018). Then, gel electrophoresis was carried out with agarose 1% in TAE buffer (40 mMTris-acetate, 1mM EDTA, pH 8.2) and stained with ethidium bromide visualized by a gel documentation device.

Antibiotic Resistance Genes Detection
By ready-to-use Yakhteh Saba Arena Company in Iran, chromosomal DNA was extracted. To extract plasmid DNA, PishgamanEnteghal Gene 50 reaction kit was used based on alkaline lysis using RNase to minimize RNA and genomic DNA pollution. Plasmid extraction steps were following according to kit instructions. According to Table 3, the polymerase chain reaction was carried out to detect antibiotic resistance genes in chromosomal DNA and plasmid DNA separately, using speci c primers synthesized by Dena Zist Asia, Iran, and Sina Clone Company. The PCR reaction was carried out in Bio-Rad 1000 thermal cycler with the respective conditions, described for each resistance gene separately. Agarose gel electrophoresis was carried out for each resistance gene, with agarose 1% in TAE buffer (40 mMTris-acetate, 1 mM EDTA, pH 8.2) and stained with ethidium bromide then visualized by gel documentation device. Primers and amplicons sizes for each are listed in Table1. Tetracycline Resistance Genes PCR Ampli cation Two common genes studied, including tet (M) and tet (W), were used to evaluate Tetracycline resistance. The polymerase chain reaction was done to detect these genes using speci c primers for tet (M): F: 5 GTG GAC AAA GGT ACA ACG AG 3 , R: 5 CGG TAA AGT TCG TCA CAC AC 3 , and tet (W): F: 5 GAG AGC CTG CTA TAT GCC AGC 3 R: 5 GGG CGT ATC CAC AAT GTT AAC 3 . PCR carried out using Sinaclon master mix 25µL, forward primer 1µL, reverse primer 1µL, 1µL of template DNA, and DNA/ RNA free distilled water to reach the nal volume of 50µL. The polymerase chain reaction was carried out as following ampli cation program: Initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 45s, annealing at 55°C for 1 min, (Aminov et al. 2007) and extension at 72°C for 1 min. A nal extension was performed at 72°C for 5 minutes (Hedayatianfard et al. 2014). All samples were tested twice.
Erythromycin Resistance Gene (erm (B)) PCR Ampli cation Polymerase chain reaction carried out using speci c primer for erm (B) gene: F: 5 TGG TAT TCC AAA TGC GTA ATG 3 and R: 5 CTG TGG TAT GGC GGG TAA GT 3 . Using 12.5µL Sina Clone company PCR master mix, forward primer 1µL, reverse primer 1µL, 1µL of template DNA, the polymerase chain reaction were done and DNA/ RNA free distilled water to reach the nal volume of 25µL (Aminov et al.2004 The PCR reaction, in this research, was carried out to determine the resistance to Penicillin and Ampicillin among isolates, PCR reaction using speci c primer for (bla Z) gene: F: 5 ACT TCA ACA CCT GCT GCT TTC 3 and R: 5 TAG GTT CAG ATT GGC CCT TAG 3 . The reaction was conducted using Sina clone master mix 25µL, forward primer 1µL, reverse primer 1µL, 1µL of template DNA, and DNA/ RNA free distilled water to reach the nal volume of 50µ.
Cycling conditions for bla Z gene ampli cation were as follows: denaturation at 95°C for 5min, followed by 35 cycles of 94°C for 30 s, annealing temperature at 60°C for 30 s, extension at 72°C for 60 s and nal extension of 72°C for 10 min (Guo et al.2017). All samples were tested twice. Positive control was the bla Z gene-positive Lactobacillus. Using 1% agarose in TAE buffer (40 mMTris-acetate, 1 mM EDTA, pH 8.2) to detect the β-lactam resistance gene, agarose gel electrophoresis was done and stained with ethidium bromide, and then visualized by a gel documentation device.
Aminoglycoside resistance genes including aac (6″) -aph (2″), aph (3″)-III, and aad A were detected as the following condition: initial denaturation at 95°C for 5 minutes followed by 30  Vancomycin Resistance Gene (vanA) Detection The PCR reaction was conducted with a speci c primer for van(A) gene to investigate Vancomycin resistance: F: 5 GGGAAAACGACAATTGC 3 and R: 5 GTACAATGCGGCGTTA 3 , using ready to use master mix 25µL, forward primer 1µL, reverse primer 1µL, 1µL of template DNA, and DNA /RNA free distilled water to reach the nal volume of 50µL.
Vancomycin resistance gene PCR ampli cation program was as follows: denaturation step at 94°C for 5 minutes, followed by 30 cycles of 45 seconds at 94°C, 45 seconds at 59°C, and 45 seconds at 72°C. A nal extension step was performed at 72°C for 5 minutes (Tenorio et al.2001). Agarose gel electrophoresis was then carried out with 1% agarose in TAE buffer, including (40 mMTris-acetate, 1 mM EDTA, pH 8.2), and then stained with ethidium bromide, nally visualized using a gel documentation device.

Lactobacilli Isolation
Lactobacillus colonies were white, circular, and they were gram-positive as well as rod-shaped, in microscopic evaluation.

Biochemical Identi cation
The Biochemical tests, including catalase, oxidase, indole production, and motility growth at different temperatures (15-37-45), furthermore, sugar fermentation tests were carried out. Consequently, Lactobacilli isolates were catalase and oxidase negative, and did not produce indole, non-motile, moreover, could grow on 15, 37, and 45˚C. The sugar fermentation tests were carried out using sucrose, glucose, lactose, maltose, trehalose, galactose, arabinose, mannitol, fructose, and ribose. All of the isolates could ferment glucose and lactose; 75% of isolates were fermented galactose, 62.5% fermented fructose, 45% could ferment sucrose, 33% arabinose, and 29% of our isolates fermented maltose, and any of our isolates could ferment ribose, 8% mannitol, and trehalose.The results of the sugar fermentation test for the isolates are shown in Table 2.
Zone diameter charts are listed in Table 3. To determine antibiotic sensitivity and resistance according to CLSI 2018 (Performance Standards for Antimicrobial Susceptibility Testing 2018). Antibiotic susceptibility test was performed by the disk diffusion method and the results are mentioned in Table 4. DNA Extraction D value for DNA extracts determined using a spectrophotometer at 260 nm and 280 nm. The ratio of (260/280) indicated the concentration of DNA in the samples, which was about 1.9 for investigated samples.

Plasmid DNA Extraction
The ratio of absorbance at 260 nm to 280 nm was calculated. The ratio for plasmid extracts was 1.8 in this research, which shows the acceptable purity of plasmid DNA.

Molecular Characterization
Identi cation of Lactobacilli carried out by polymerase chain reaction using 16S rDNA sequence. Finally, according to the 16S rDNA sequence, 24 isolates were identi ed, as shown in Fig. 2.

Antibiotic Resistance Genes Detection
Tetracycline Resistance Genes Detection

Detection of tet (W)
Among the investigated Lactobacilli, only one (isolate No. 22) had the tet (W) gene on its chromosome. Electrophoresis results for chromosome DNA extracts are shown in Fig. 3. Amplicon sizes for tet (W) were168 bp, and it is one of the main Tetracycline resistance determinants. Plasmid extracts were also examined to identify this gene, but none was found. Using PCR, the van (A) gene on the bacterial plasmid was detected and was not found in any isolates. The agarose gel electrophoresis results to detect the van (A) gene in seven isolated plasmids are shown in gure 5.

Discussion And Conclusion
Traditional fermented dairy products are a good source for the isolation of Lactic acid bacteria, especially Lactobacillus (Soleimanifard et al. 2015). Since fermented dairy products could act as a reservoir of antibiotic resistance genes, they can transmit resistant genes to commensal or pathogenic bacteria (Erginkaya et al. 2018). Though Lactobacilli are commonly not recognized as pathogens, the main concern concerns transferring resistance genes to commensal or pathogenic bacteria. Antibiotic resistance genes expression and transferring resistance determinants to the regular human or pathogenic bacteria are essential components of the food enterprise's safety evaluation and should be considered (EUCAST 2019). The prevalence of antibiotic resistance in Lactobacillus isolated from dairy products has been studied in different regions. Using the disk diffusion method, Wang  Aminoglycoside resistance is a common feature in Lactobacillus species. It is usually inherent, resulting from a lack of an electron transfer system for antibiotic uptake, alteration of cell permeability, and the use of enzymatic modi cation (Charteris et al. 2001; Jaimee and Halami2016).In this research, the presence of genes related to enzyme modi cation could be transferable (Turnidgeand Bell2005) in isolates was investigated. The results showed resistance to Kanamycin, Gentamycin, and Streptomycin.
Gentamicin resistance was observed in 25% of isolates (1, 3, 8, 20, and 23  Vancomycin resistance is a typical phenotype in Lactobacillus and is often an inherent trait. In the present study, Vancomycin resistance (Dec et al.2017) was the most common, and 11 of the isolates ( 45%), including Nos. 4,5,9,10,11,12,13,14,15,16 In conclusion, ndings suggest that more studies are needed to investigate traditional and industrial starter cultures to reduce the risk of antibiotic resistance gene dissemination among symbiotic pathogens and normal ora. As isolates did not carry out resistance genes on their plasmid, it seems that they could be good candidates for more safety evaluation to introduce safe probiotic or starter culture strains.

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