Plasmid-Mediated Antibiotic-Resistant Pattern of Lactobacillus spp. Isolated From Dairy Products

Background: Microorganisms have potent activity for transferring antibiotic-resistant genes with either chromosomallyor plasmid-mediated characteristics. The purpose of this study was to isolate Lactobacillus from different commercial products and evaluate their potential in antibiotic-resistant development. Chromosomallyor plasmid-mediated resistant genes were investigated as well. Methods: In total, Lactobacillus strains were isolated from 20 commercial dairy product samples such as cheese and yoghurt. The isolates were phenotypic and molecularly identified and their antibiotic-resistant properties were assessed by the disk-diffusion method. Finally, the plasmid-mediated antibiotic resistant characters of the isolates were evaluated by plasmid curing via evaluated temperatures and acridine orange methods. Results: Five strains Lactobacillus paracasei, L. rhamnosus, L. casei, L. plantarum, and L. fermentum were isolated different products. The results of the antibiotic susceptibility assay indicated that all strains were susceptible to amoxicillin and imipenem and resistant to ciprofloxacin and vancomycin. Furthermore, different responses were observed among the isolates against streptomycin and gentamicin. The evaluation of plasmid-mediated antibiotic resistance in the isolates revealed that streptomycin and gentamicin-resistant characters were of plasmid-mediated type in L. rhamnosus and L. plantarum strains. Conclusions: In general, our finding demonstrated that some commercial Lactobacillus strains harboured antibiotic-resistant genes. These genes can be located either in chromosome or plasmid group. Hence, the frequency of antibiotic-resistant pathogenic bacteria might be increased after consuming some dairy products because of the horizontal transfer of antibiotic-resistance genes among the bacteria.

vulnerable to chloramphenicol, penicillin G, erythromycin, clindamycin, and oleandomycin although none proved hemolytic activity. Accordingly, the increase of several probiotic microorganisms in dairy products and pharmaceutical formulations, and limited studies performed on the safety of probiotics should not carry any genes for antibiotic resistance transfer.
Lactobacilli spp., via OPA11 and OPD20 primers, which are used to amplify the isolated colonies by random amplified polymorphic DNA-polymerase chain reaction (PCR), has proven to be effective in discrimination, classification, and delineation of the bacterial culture and its origin clustering (9). Therefore, the present study aimed to evaluate antibiotic-resistant patterns in Lactobacillus spp. and determine the type of antibiotic resistance of chromosome-or plasmid-mediated isolates.

Isolation of Lactobacillus spp. From Dairy Products
Twenty samples, from commercial dairy products viz., cheeses, and yoghurts were collected during the first six months of 2019. To perform the experiment, 1 g of the samples was added into the liquid De Man, Rogosa, and Sharpe (MRS) broth and the suspension was incubated at 37 °C in microaerophilic conditions for 24 hours. Then, a loopful of the MRS broth was streaked on the MRS agar and incubated at 37 °C for 48 hours. The isolates were identified by phenotypical tests such as catalase, oxidase, oxidative/fermentative, nitrate, and fermentation of different sugars (10).

Antibiotics Susceptibility of Lactobacillus Isolates
The disk-diffusion method was used to determine the antibiotic susceptibility of the isolates. To perform the test, the overnight growth cultures of the isolates were diluted with sterile normal saline (equal to the turbidity of a 0.5 McFarland tube). Then, the suspensions were cultivated on Mueller-Hinton agar (MHA) plates and the antibiotic disks (Patanteb, Iran) of erythromycin (15 μg), tetracycline (30 μg), vancomycin (30 μg), gentamicin (15 μg), streptomycin (10 μg), chloramphenicol (30 μg), ampicillin (10 μg), amoxicillin (25 μg), cefotaxime (30 μg), imipenem (10 μg), and ciprofloxacin (5 μg) were placed on the seeded MHA. The plates were incubated at 37°C for 48 hours and the diameters of the inhibition zones around each antibiotic disk were measured and recorded after this period (11). Antibiotic susceptibility testing results were interpreted using the Clinical Laboratory Standards Institute (CLSI) guidelines 2019.

Plasmid Isolation
The plasmids from Lactobacillus strains were isolated by the DNA plasmid extraction kit and then verified by 2% agarose gel electrophoresis in a submerged horizontal gel apparatus. The electrophoresis apparatus adjusted to 80 V for 45 minutes. Having finished the electrophoresis, the visible bands were evaluated by the alpha imager gel documentation system (Hidolgh, Germany) under ultraviolet at 260 nm (10).

Plasmid Curing
In the plasmid curing study, isolated Lactobacilli strains showing resistance to most antibiotics and a harbored plasmid band were selected for plasmid curing and put into effect. Plasmid curing was performed using acridine orange and elevated temperature (44°C) as curing agents.
To perform the experiment, 20 µL of the overnight growing culture of five antibiotic-resistant Lactobacillus strains was subjected to plasmid curing, each of which was inoculated into the tube containing 1 mL MRS broth, prepared and supplemented with different concentrations (i.e., 0.01, 0.005, 0.0025, and 0.00125 g/mL) of the acridine orange. The samples were then incubated at 37°C for 48 hours. After 48 hours of incubation, the minimal inhibitory concentration (MIC) of the acridine orange was determined, and then the sub-minimum inhibitory concentration (SIC) was considered as plasmid curing.
The elevated temperature for plasmid curing was done by the inoculation of the overnight growing culture of Lactobacillus strains into a tube containing 5 mL MRS broth and incubated at 44°C for 24 hours.
The culture with the SIC of curing agents for each Lactobacillus strain and the MRS broth incubated at 44°C were inoculated on the MRS agar and incubated at 37°C for 48 hours (10).

Antibiotic Assay of Plasmid Curing Lactobacillus Isolates
The antibiotic resistance pattern of non-cured Lactobacillus strains was selected for evaluating the antibiotic susceptibility of cured Lactobacillus isolates. Four antibiotics of vancomycin (30 μg), gentamicin (15 μg), streptomycin (10 μg), and ciprofloxacin (5 μg) were selected to perform this experiment. Simultaneously, single colonies were picked up by sterile toothpicks and inoculated on MHA plates with and without antibiotics. Each MHA plate contained only one antibiotic concentration. Then, the plates were incubated at 37°C for 48 hours. A growth colony on MHA w/wo antibiotic was considered chromosomally mediated antibiotic resistance. However, the growth colony on MHA containing no antibiotics and no growth of a given bacterium on MHA containing antibiotic was considered plasmid-mediated antibiotic resistance.

Isolation of Lactobacillus spp.
The five isolated strains with desirable probiotic features were identified as Lactobacillus. These strains were L. rhamnosus, L. paracasei, L. casei, L. plantarum, and L.

Conflict of Interest Acknowledgement
Ethical Approval Authors' Contribution Funding/Support fermentum.

Antibiotics Susceptibility Test
The results of the antibiotic susceptibility of the isolates by the disk-diffusion method were evaluated by CLSI guidelines 2019 and expressed as sensitive (S), marginally susceptible (I), and resistant (R) as per the recommended reported standards ( Table 1).

Plasmid Isolation
As shown in Figure 1, the result of plasmid isolation indicated that all Lactobacilli strains were harbored plasmids.

Plasmid Curing
Plasmid curing experiments on tested Lactobacilli strains were performed using both the curing agent of the acridine orange and elevated temperature (44°C). The results of the MIC and SIC of acridine orange for Lactobacilli strains (Table 2) revealed that Lactobacillus plantarum was relatively more resistant to acridine orange.

Isolates
The obtained results from antibiotic susceptibility after plasmid curing represented that all L. isolates, L. rhamnosus, and Lactobacillus plantarum lost resistance phenotypically to streptomycin and gentamicin. Hence, streptomycin and gentamicin-resistant properties were of plasmid-mediated type in these two strains. However, vancomycin and ciprofloxacin-resistant properties were of chromosomally-mediated type.

Discussion
Although several microorganisms such as Lactobacillus, Bifidobacteria, Enterococcus, and Streptococcus are introduced as probiotic bacteria, (12,13), information on their antibiotic-resistant characters is not widely available yet (14). In this study, five Lactobacillus strains were isolated from commercial products and assessed for their susceptibility to different antibiotics. The isolated strains were resistant to vancomycin and ciprofloxacin (100%) in addition to gentamicin (93%) and streptomycin (87%) while they were susceptible to amoxicillin and imipenem (100%), as well as chloramphenicol (93%) and ampicillin (73%). However, all the isolates harbored DNA plasmid although the plasmid-mediated resistant character was found only in two strains. This means that streptomycin and gentamicin resistance genes in L. rhamnosus and L. plantarum are located on the plasmid, and therefore,  horizontal gene transfer among probiotic strains might be expectable. Comunian et al (15) isolated 197 strains of lactobacilli from Italy's fermented products. They verified the results by observing their resistance genes through the PCR method (13) and found that all strains were resistant to erythromycin and tetracycline. Zheng et al reported that 88% of lactic acid bacteria isolates were resistant to chloramphenicol and tetracycline. In addition, they found that the isolated strains could transfer antibioticresistance gene tet(M) to pathogenic bacteria in the human gut (16). Furthermore, several reports demonstrated that many probiotics such as lactobacilli were resistant to vancomycin, gentamicin, streptomycin, and ciprofloxacin, and most were of plasmid-mediated type (16,17).
On the other hand, the findings of several studies in Italy and Spain showed that some probiotic bacteria such as L. rhamnosus and L. jensenii have potent activity for causing bacteremia (15). Therefore, the possibility of transferring antibiotic-resistant genes and pathogenic potential of some probiotics should be considered as a critical option for selecting probiotic microorganisms. Hence, as mentioned above, the antibiotic resistance character in probiotic bacteria has some advantages and disadvantages.
Survival in people on oral antibiotic therapy is an advantage while acquired resistance genes within the bacterial species of gut microflora or to pathogenic commensals is a disadvantage. Therefore, to introduce commercial probiotic bacteria, several standard screenings including their evaluation for carriage antibiotic resistance genes should be done to eliminate the distribution of the genes among various gut microflora and potentially pathogenic bacteria.

Conflict of Interests
None.