The Role of Plasmids in Antibiotic Resistance in Pasteurella multocida Isolated from Various Sources

doi:10.21203/rs.3.rs-1665746/v1

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The Role of Plasmids in Antibiotic Resistance in Pasteurella multocida Isolated from Various Sources

https://doi.org/10.21203/rs.3.rs-1665746/v1

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Antimicrobial resistance transmitting agents present in animal products and antibiotic use contributed to its occurrence and aggravation. The emergence of resistance toward several antibiotics groups among pathogenic bacteria is a growing global threat, and Pasteurella multocida is no exception in this case. The role of plasmids in creating different patterns of AMR on anyone is not hidden. One of the easiest ways to reduce antibiotic resistance is to stop the plasmid from replicating, dividing, and transferring by removing the plasmid. In this study, we investigated the role of P. multocida plasmids in the occurrence of antibiotic resistance. From different geographical regions of Iran, 131 definite isolates of P. multocida have been isolated from apparently healthy and sick animals. After plasmid DNA extraction, five isolates with evident plasmid were selected. Restriction mapping is used to determine the size of plasmids, so the restriction enzymes BglII, SspI, AluI were used to cleave the plasmids.Plasmid curing was performed, anddisk diffusion method was utilized to compare the antibiotic resistance pattern before and after plasmid curing. We found that the samples that contain plasmids and exhibited extensive antibiotic resistance did indeed lose their ability to resist antibiotics after their deletions and became sensitive to them. Indeed, this observation confirms the role and importance of plasmids in antimicrobial resistance in P. multocida. The study indicates that the ability of these isolates to retain plasmids has a significant effect on their ability to develop resistance to various antibiotics.

Pasteurella multocida

Antibiotic resistance

Antibiogram

Plasmid Curing

Pasteurella multocida is a gram-negative, immobile bacterium that can infect a wide range of animals. P. multocida can also be transmitted to humans through animal bites or scratches (Peng et al., 2017). Louis Pasteur first identified this gram-negative bacterium as the cause of avian cholera in 1881. In recent years, it has been proven to be responsible for several other important economic diseases (Harper et al., 2006). Poultry cholera, hemorrhagic septicemia in chickens, atrophic rhinitis in pigs, and lower respiratory tract infections in cattle and pigs are among these diseases (Harper et al., 2017). In humans, this pathogen can cause pneumonia, meningitis, urinary tract infections, sepsis, and peritonitis (Townsend et al.,1998). Antimicrobial agents most effectively control such infections. The increasing transmission of resistance genes and the occurrence of mutations mediated by overuse of antibiotics significantly reduce the effectiveness of antimicrobial agents (Michael et al., 2012).

Plasmid-mediated multi-resistance and integrative conjugative elements-ICE have been repeatedly reported in P. multocida (Ujvár et al., 2018). Research indicates that plasmids and ICE play a key role in developing multi-resistance (Michael et al., 2012) and must be considered in the development of AMR. Plasmids are extrachromosomal genetic elements responsible for causing antimicrobial resistance in bacterial populations. Plasmids are extrachromosomal genetic elements accountable for causing antimicrobial resistance in bacterial populations. The epidemiological-molecular study of resistance plasmids has been of great interest since scientists discovered the role of plasmids in AMR (San Millan et al., 2009). Plasmids have also been used in several studies to better understand P. multocida pathogenicity and the mechanism of pathogenesis and AMR. These extrachromosomal elements act as a source of resistance genes and transporters of transposons that include resistance genes in their sequences. Through the horizontal gene transfer process, plasmids can be exchanged between members of a species and between bacteria belonging to different species. Plasmids are highly flexible elements, and there are a variety of ways to recombine, fuse, or partially or entirely integrated into chromosome DNA or other plasmids. Likewise, plasmids will play an essential role in transmitting AMR genes (Niemann et al., 2019). In this respect, it is clear that the horizontal spread of antibiotic resistance genes among bacteria is primarily facilitated by bacterial plasmids (San Millan et al., 2018).

In various studies, several isolates of P. multocida were isolated from different sources, and most contained plasmids, which were usually harboring antimicrobial resistance genes (Liu et al., 2012). Studying plasmid profiles and capabilities is a valuable tool in epidemiological studies. There have been a variety of studies performed on the plasmid P. multocida during the last few decades; similar to the study we conducted on the plasmid of P. multocida, few studies have explored how plasmids contribute to antibiotic resistance in recent decades.

In bacterial strains, plasmid curing is a way to removing plasmids, determine the mediator of antibiotic resistance, and analyze the effect and role of the plasmids. To plasmid curing, various techniques have been developed, including chemical and physical agents (Letchumanan et al., 2015). By combining culture medium with Ethidium Bromide and culture at 42°C, we isolated the plasmid from P. multocida successfully. Our study aimed to test the relationship between the existence of plasmids responsible for the development of antibiotic resistance in P. multocida and the incidence of antibiotic resistance to understand why resistance occurs. We tested antibiotic susceptibility before and after plasmid curing.

Bacterial strains, purification, and molecular validation:

A total of 131 P. multocida isolates were examined, isolated from Sick or seemingly healthy animals from diverse geographical regions of Iran. Among them are cattle (11), sheep (44), goats (24), cats (21), dogs (16), and poultry (15). From February 2014 to September 2014, these isolates were collected.Isolated from cattle, sheep and goats from Fars province and Shiraz city with pneumonic involvement, as well as from the oral cavity and tonsils of cats and dogs in Tehran and Shiraz, also obtained isolates from blood samples and liver samples of suspicious birds, including ducks, chickens, and turkeys from Tehran and Shiraz, as well as cattle blood samples from Tehran and Varamin that had septicemic symptoms. In aseptic and ideal conditions, samples were taken to the laboratory in sampling containers. They are stored as standard in the freezer at a temperature of minus 80°C. Blood agar plates (Oxoid, United Kingdom) supplemented with 5% defibrinated fresh sheep blood were used to culture and purify these isolates and were incubated aerobically for 24 hours at 37°C. For confirming morphological and biochemical properties, standard bacteriology methods like Munir et al.'s work were used (Munir et al., 2006). After DNA extraction using Evers et al.'s boiling method (Ewers et al., 2006). Perform special PCR tests for P. multocida according to the method of Tausand et al.'s (Townsend et al., 1998), KMT1T7, and KMT1SP6 (Sinacolon, Iran) were used as reverse and forward primers. (that the PCR program shown in Table <link rid="tb1">1</link>–1). Electrophoresis was performed on 1% agarose gel with safe staining to detect the amplification products. Products were then photographed under UV light. As a positive control, P. multocida strain 85020 (wild type, type B:2) was used.

Detecting the presence of plasmid DNA

We used alkaline lysis to isolate plasmid DNA from bacteria and to assess plasmid presence or absence in isolates. Using this method, user can analyze their plasmid products by gel electrophoresis,andthismethod enabled us to select five isolates with definite plasmids. This method relies on alkaline denaturation; basically, the alkaline conditions of this method lead to the denaturation and linearization of chromosomal DNA and plasmid DNAs. As soon as the neutral condition is formed, the plasmid DNA renatures faster than chromosomal DNA because of its small size, creating the initial loop state. The chromosomal DNA, as a result of its high molecular weight, will not have the opportunity to be renatured, and so it will be selectively deposited and removed from the reaction.

We were inspired by Sambrook et al., which was developed in 1989 (Sambrook et al.,1989). but we modified it slightly. A single bacterial colony was cultured in 5 ml of LB or BHI broth culture medium to extract plasmid DNA. Overnight, the tube was placed in a shaker incubator at 37°C. An Eppendorf tube was filled with approximately 1.5 ml of the culture medium and Microfuge for 1 minute in order to obtain a bacterial pellet. Repeat this until the entire volume of the culture medium has been spun down in one tube. The pellet was dissolved in 100 µl of Alkaline lysis solution I, and then 20 µl of 10 mg/ml lysozyme enzyme was added to the suspension and mixed well. After this, the tube was left at room temperature for 2 min. Then, 200 µl of Alkaline lysis solution II was added and gently mixed (by turning the tube upside down), then placed on ice for 5 minutes. The next step was to add 150 ml of alkaline lysis solution III to the suspension, gently mixed to form small white clumps. After that, place it on ice for 5 minutes. Ideally, we should incubate on ice for no more than 5 minutes so that we don't introduce chromosomal DNA into our final sample. Genomic DNA and plasmid DNA are renatured under these neutral conditions and thus appear in the final sample. It should be noted that turning the tube upside down very slowly will reduce the risk of plasmid DNA fragmentation.

Next, the suspension was centrifuged for five minutes at 4°C. Then the supernatant is transferred into a clean tube. After adding 400 µl of phenol-chloroform mixture and mixing, it was centrifuged for 2 minutes. Once the microfuge is complete, transfer the aqueous phase into a new microtube. Afterward, 1 ml of pure ethanol (EtOH) was added, mixed, and allowed to stand at room temperature for two minutes. Next, centrifuged for 5 minutes using a refrigerated microfuge. A white pellet will form at the end of the eppendorf tube after centrifugation. Removed all ethanol and was allowed the ethanol residue to evaporate at room temperature. In the end, the end pellet containing the plasmid is dissolved in 50 µl of TE solution and stored at minus 20°C for later use. 100 ml of alkaline lysis solution I contains 25.0 ml of 1 M Tris-Cl at 0.8 pH, 2.0 ml of EDTA 0.5 M, 5.0 ml of 1 M glucose, 68.0 ml of sterile distilled water (SDW). 1 ml of 10 mg/ml lysozyme solution contains 0.01 g lysozyme in 1 ml of 0.250 M Tris pH 8.0. 1 ml of alkaline lysis solution II contains 50.0 µl of 20% SDS, 20 µl NaOH 10N, 930.0 µl of SDW. 100 ml of alkaline lysis solution III contains 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, 28.0 ml of SDW (5 M potassium acetate: 49.075 g potassium acetate fill to 100.0 ml with SDW).

Gel electrophoresis can be conducted to determine if plasmid is present in your bacterial sample. Nano-Drops were performed on the plasmid sample to determine the concentration of dsDNA.

Plasmid Curing

During plasmid curing, the bacteria that contain the plasmid will lose the plasmid, and the colony will subsequently be plasmid-free. Caro et al., 1984, used the same plasmid curing method as we do with a few modifications (Caro et al., 1984). As part of this method, a known plasmid-removing agent, ethidium bromide (at final concentration is 25 µg/ml), was added to 10 ml of BHI broth. In this medium, a single colony of the bacterium was cultured and incubated for 18 hours in a shaker incubator at 42°C. Following incubation, ten-fold diluted in fresh BHI broth, 500 µl each of the dilutions 6 to 10 were spread on BHI agar plates with the help of a sterile spreader. Colonies were screened for loss of plasmid DNA after overnight incubations at 37°C. In the end, colonies without plasmids were stored (-20°C) for use in later stages of the study. In 2005, Jamal et al. successfully removed the plasmid of the P. multocida using a similar process to ours (Jamal et al., 2005).

Restriction-Enzyme analysis

We tested the ability of endonuclease restriction enzymes to digest plasmids derived from our five samples. The restriction endonucleases BglII, SspI, and AluI were used under conditions recommended by the supplier (Thermos Scientific, USA). Electrophoresis of restricted DNA was performed in 1.2% agarose gel.

Disk-Diffusion test for antibiotic susceptibility:

The work process and measurements were performed in accordance with CLSI guideline M45-2015 and EUCAST-Version 10.0. Five plasmid-containing and five plasmid-free organisms were sub-cultured on blood agar plates supplemented with 5% defibrinated fresh sheep blood and incubated at 37°C overnight. After making a bacterial suspension equal to 0.5 McFarland standard, it was used to inoculate plates containing Mueller-Hinton blood agar with 5% defibrinated sheep blood. After the antibiotic discs were placed on the inoculated plates, they were incubated at 37°C for 18 hours; the antibiotic discs (Padtanteb, Iran) are: amoxicillin (25µg), chloramphenicol (30µg), trimethoprim-sulfamethoxazole (1.25/23.75µg), erythromycin (15µg), oxytetracycline(30µg), gentamycin (10µg), streptomycin (10µg), tylosin(30µg), enrofloxacin(5µg), imipenem(10µg), cephalothin(30µg), knamycin(30µg), cefalexin(30µg), florfenicol(30µg), cefotaxime(15µg) and tetracycline (30µg). We measured and recorded the diameter of the no-growth zone. As in some cases, when the areas overlapped and were very large, it is necessary to measure each area's radius and double it to determine the diameter of each area.

As quality control organisms, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used (Following the articles of association).

Morphological, biochemical and molecular validation properties of bacterial strains

Based on biochemical and morphological analysis and PCR test results, all isolates were clearly confirmed as P. multocida.

After purification, gram-negative short rods of coccobacillus-shaped can be found singularly or in pairs and in short chains as a result of gram stains. Colonies of P. multocida ranged in size from 0.5 mm to 1.0 mm. The results of our study were consistent with the report of Itoh and colleagues who described P. multocida as a gram-negative, oval bacillus in 1980 (Itoh et al., 1980). P. multocida oxidase has been detected in all purified specimens of the bacterium because the enzyme oxidase oxidizes p-Phenylenediamine (PPD). Observation of rapid and persistent bubbles (foam) during the P. multocida catalase test indicated that the test was positive. There were no black deposits in the SIM medium, which indicates the lack of hydrogen sulfide production. On top of the SIM medium, the reaction of indole with the aldehyde agent of the red complex becomes evident after the addition of the coaxial reagent.It is clear that tryptophan is converted to indole when the bacteria possess tryptophanase and the indole is positive. In addition, no movement was observed in the SIM environment, which indicated that the bacterium was immobile. These isolates have been shown to ferment glucose and sucrose but not maltose. Neither sample showed evidence of urease. Munir et al. (2006) also found similar results in their study of the biochemical properties of P. multocida (Munir et al., 2006).

Based on the results of PCR, all isolates had an optimal band of 460 bp, as shown in Fig. 1.

Detecting the presence of plasmid DNA

It was studied whether plasmid DNA was present in confirmed isolates of P. multocida. From the available samples, we isolated 5 plasmid-containing samples using the alkaline lysis method, as shown in Fig. 2.

Plasmid Curing

As part of this study, it was necessary to lose the plasmids from selected samples during the plasmid curing process via ethidium bromide and culture at 42°C in order to evaluate and compare antibiotic resistance properties, as well as investigate the incidence of antibiotic resistance properties in both the absence and presence of plasmids. During the cooking method, the plasmid of five selected isolates was successfully removed. This can be seen in Fig. 3, which shows no band for the presence of plasmids on the agarose gel. Jamal et al in Malaysia also removed the plasmids of P. multocida samples with an ethidium bromide procedure (Jamal et al., 2005), which confirms the process we employed.

Restriction-Enzyme analysis

In order to confirm the plasmids and determine their sizes, restriction mapping was performed on five extracted samples. The AluI detection sequence is rich in guanine (G) and cytosine (C) bases, cross-sectioning adjacent C and G bases, and SspI and BglII recognize a DNA sequence that is adenine-rich adenin (A) and thymine (T) more than G and C. According to Fig. 4, which shows samples of plasmids obtained from enzyme digestion with each enzyme separately, one cut band was obtained following digestion with AluI enzyme, and two cut bands were obtained following digestion with SspI and BglIIenzymes. As a result of these results, P. multocida plasmid DNA contains more A and T than G and C. Similarly, researchers Berman and Hirsh found that the contents of DNA of P. multocida contained 40.3% cytosine (C) and guanine (G) (Berman and Hirsh, 1978). P. multocida plasmid DNA is therefore rich in A-T. The results of restriction endonuclease analysis (REA) are shown in Table 1.The analysis of these samples showed that three samples had approximately the same plasmid pattern, and two of the samples had a similar pattern. The results are strongly suggestive that P. multocidaisolates descended from different ancestors. Sample D5 is approximately 4.65 Kb in size, sample B1 is approximately 3.1 Kb in size, and samples S3, P1 and D3 are approximately 10.8 Kb in size.

Table 1

Analysis of the enzymatic digestion (REA)
		No. ofcuttingsites					Size of fragments(kb)
Sample	B1	D3	D5	P1	S3	B1	D3	D5	P1	S3
BglII	1	2	2	2	2	1.1	3,1.5	1.1,0.65	3,1.5	3,1.5
AluI	1	1	1	1	1	1	1.7	1.8	1.8	1.8
SspI	1	2	1	2	2	1	3,1.5	1`.1	3,1.5	3,1.5
Approximate size of the plasmid genome(kb)						3.1	10.7	4.65	10.8	10.8

BglII, AluI and SspI are restriction enzymes

Antibiotic susceptibility

As a result of assessing antibiotic susceptibility by disk diffusion methods on 5 isolates, we were able to determine the general pattern of antibiotic resistance before and after plasmid curing. Table 2 shows the general pattern of antibiotic resistance before and after plasmid removal in five isolates. After plasmid curing, samples B1 and D5, each resistant to 3 antibiotics, sample S3 resistant to 6 antibiotics, and samples P1 and D3, both resistant to 7 antibiotics erythromycin, oxytetracycline, streptomycin, tylosin, cefalexin, tetracycline, cefotaxime, showed a resistance reaction. This extensive resistance was eliminated after plasmid curing and only resistance to streptomycin in D3 and tetracycline in samples S3, D3, and P1 remained. Table 2 also shows that there was an intermediate reaction for a number of samples, regardless of plasmid presence or absence. Plasmids play a crucial role in developing antibiotic resistance, as these observations demonstrate and a strong correlation exists between the presence of plasmids and antibiotic resistance. Plasmids play a crucial role in the development of antibiotic resistance, as these observations demonstrate, and a strong correlation exists between the presence of plasmids and antibiotic resistance. Before and after plasmid removal, none of the five isolates tested were resistant to penicillin, gloramphenicol, trimethoprim, sulfamethoxazole, gentamicin, imipenem, cephalothin, kanamycin, and fluorophenicol. In this study, no resistance to florfenicol was observed and all isolates were susceptible to this antibiotic. In a 2009 study by Tang et al., no resistance to this antibiotic was reported (Tang et al. 2009).

Table 2

Pattern of antibiotic resistance before and after plasmid curing in 5 isolates for which Antibiotic susceptibilitytesting was performed.
Antibiotics						After Plasmid Curing
	B1	D3	D5	P1	S3	B1	D3	D5	P1	S3
Amoxicillin	S	I	I	I	I	S	I	I	I	I
Chloramphenicol	S	S	S	S	S	S	S	S	S	S
Trimethoprim-sulfamethoxazole	S	S	S	S	S	S	S	S	S	S
Erythromycin	R	R	R	R	R	S	S	S	S	S
Oxytetracycline	S	R	S	R	R	S	S	S	S	S
Gentamycin	S	S	S	S	S	S	S	S	S	S
Streptomycin	R	R	I	R	R	S	R	I	S	S
Penicillin	S	S	S	S	S	S	S	S	S	S
Tylosin	S	R	R	R	R	S	S	S	S	S
Enrofloxacin	S	I	S	I	I	S	I	S	I	I
Imipenem	S	S	S	S	S	S	S	S	S	S
Cephalothin	S	S	S	I	S	S	S	S	I	S
Knamycin	S	S	S	S	S	S	S	S	S	S
Cefalexin	R	R	R	R	S	S	S	S	S	S
Florfenicol	S	S	S	S	S	S	S	S	S	S
Cefotaxime	S	R	S	R	R	S	S	S	S	S
Tetracycline	S	R	S	R	R	S	R	S	R	R
S3, P1, D5 and B1 are Test samples, R: Resistant, S: sensitive, I: intermediate susceptibilitytesting was performed.

In most gram-negative bacteria, multiple antibiotic resistance is mediated by large and small plasmids that collect and carry large counts of antibiotic resistance genes and transmit them to other bacteria (San Millan et al.,2009). Plasmids have been used in several studies to obtain more information about the pathogenicity and mechanism of pathogenesis of P. multocida, as well as the multiple antibiotic resistance of this bacterium. The extensive antibiotic resistance in P. multocida may be a result of the use of antibiotic additives in animal feed and the overuse of antimicrobial agents following the development of various infections in various animals in veterinary medicine (Arashima et al., 2005). The treatment of infections caused by P. multocida is usually blindly performed by veterinarians with a broad-spectrum antibiotic (Tang et al., 2009). In general, following our experiments, we found that samples with plasmids that showed high levels of antibiotic resistance lost their ability to develop resistance upon removing the plasmids and became sensitive to antibiotics. This observation highlights the importance and implication role of plasmids in developing antibiotic resistance in P. multocida and possibly other bacteria and confirms the presence of genes involved in multiple resistances, usually in plasmid sequences. Following the analysis of our results, we observed that plasmid samples with large genomic sizes (about 11 Kb) had higher levels of resistance (about 7 antibiotics) than plasmid samples with smaller genomic sizes (about 3 antibiotics), observation has shown, for example, that plasmid sample B1, being the smallest plasmid, has the least antibiotic resistance.

We cannot ignore this point that mobile elements such as integrative conjugative elements (ICEs) can also enter bacteria by different mechanisms and become insoluble in plasmids. This trend resulted in the spread of antibiotic resistance through horizontal gene transfer. Moreover, these sequences can be moved and integrated on a bacterial DNA chromosome through site-specific recombination and a bacterial conjugation system such as T4SS, which allowing them to be transferred between bacteria and even between plasmids and chromosomes (Johnson et al. 2015). ICEs are rarely detected in P. multocida, and further studies are needed, but sequence analysis of several antibiotic-resistant strains of the bacteria in 1012 led to the identification of the first ICE in in P. multocida (Michael et al., 2012). Plasmid, these extra chromosomal DNA elements can easily be passed between species and occasionally even genera. Even without direct selection, the spread and scatter of a plasmid carrying a multi-resistance gene cluster poses a risk for the transmission and persistence of resistance genes (Kehrenberg et al., 2003). Despite this plasmid containing antibiotic resistance genes and the presence of these antibiotic resistance transfer elements, it can be stated that over time and in the coming years, multiple antibiotic resistance is potentially transferrable among livestock species on an increasingly. From 2003 to 2007, the percentage of P. multocida isolates with at least three antibiotic resistances was constantly high, The prevalence of isolates resistant to more than five antimicrobials has increased over time, increasing from 47.8% in 2003, 54.1% in 2004, and 57.6% in 2005 to 81.6% in 2006 and 97.1% in 2007 (Tang et al., 2009). Plasmids that contain multi-resistance genes are particularly important. These genes in the bacterium make it resistant to several different antibiotics or antibiotic groups, such as sulfonamides, chloramphenicol, tetracyclines, and streptomycin (Kehrenberg et al., 2003). Taking action against antibiotic resistance at all possible levels around the world may be a necessary action. It is still one of the concerns of scientists working to combat the emergence and spread of antibiotic resistance to find ways to manipulate plasmids, eliminate them, and prevent them from spreading.Our study showedthat resistance to erythromycin, oxytetracycline, streptomycin, tylosin, cefalexin, tetracycline, cefotaxime occurs when plasmids are present in bacteria, In this study, we investigated the effect of plasmids on antibiotic resistance events, a plasmid removal procedure developed for the P. moltecida bacterium was developed. It can be used with selective inhibition of plasmid replication and transfer to fight antibiotic resistance.

In three samples, resistance to tetracycline was observed both in the presence and absence of plasmids, it seems that tetracycline resistance genes have dried up on bacterial chromosomes. Interestingly, this condition can also be caused by the presence of plasmids and integrative conjugative elements, which have been transferred from the plasmid to the chromosome over time and in previous generations. During the presence of plasmid, the resistance to streptomycin in one sample was of the resistance type, whereas in the other sample it was of the intermediate reaction type, which, after removal of the plasmid, only one sample remained resistant and the others ceased to be resistant (The sample with the Intermediate reaction remained unchanged). An event such as this indicates the existence of a streptomycin resistance gene on chromosomes of one sample, andIt can be concluded that that plasmid containing the streptomycin resistance gene of the bacterium, like the observed resistance to tetracycline, transferred the resistance sequence to the chromosome, Researchers observed streptomycin (strA) and tetracycline (tetB) resistance mechanisms on chromosomal DNA in the study by Ujvári and colleagues in 2018, The authors note that the genes entered the genomic content over time (Ujvári et al., 2018). In a study by Alhamami et al., Tetracycline resistance genes were detected on plasmids and chromosomal DNA in P. multocida isolates. Resistance to erythromycin in the presence of plasmids was widespread among the isolates. Therefore, we are referring to Citron's 2005 study, in which many isolates tested resistant to erythromycin following disk diffusion. The MIC range of erythromycin was also the highest (Citron et al., 2005). This can be a confirmation of our results.

It can be used to design drugs that effectively prevent the spread and development of antibiotic resistance if the synergistic interaction of antibiotics and resistance modifiers is examined and substances and compounds involved in the removal and prevention of the transfer and proliferation plasmids in vitro.

All that was said was confirmation that plasmids are most definitely responsible for widespread antibiotic resistance. The number of plasmids containing resistance genes is expected to grow in the future. Our study findings may further investigate whether researchers can provide solutions and methods to combat the occurrence and transmission of antibiotic resistance by plasmids. Extensively eliminating or preventing plasmids from spreading through bacterial populations can be addressed. The most straightforward way to reduce antibiotic resistance is removing the plasmid entirely, since removing the plasmid stops the plasmid replicating, dividing, and spreading. Hence, concomitant administration of antibiotics and resistance modifiers can help treat serious infections resistant to one or more drugs and inhibit bacteria's plasmid transport systems. A major goal is preventing this spread of resistance among bacteria so that diseases with footprints of pathogenic bacteria can be treated with no trouble.

Conflict of interest: On behalf of all authors, the corresponding author states that there is no conflict of interest.

Authors’ contributions

This research was conducted as a part of a MSc thesis by Seyed Mehdi Joghatayi in the Faculty of Veterinary medicine, Shiraz University, supervised by Mohammad Tabatabaei. MT Conceived the experiments and SMJ performed the experiments. The first draft of the manuscript was written by SMJ and edited and evaluated by MT. All the authors have read and approved the final version.

Ethics approval and consent to participate: Not applicable.

Availability of data and materials:

All data generated or analyzed during this study are included in this published article.

Funding:

This work was supported by Shiraz University and Ministry of Science, Research and Technology [Grant number 9830985].

Consent to participate: 'Not applicable'

Consent for publication: 'Not applicable'

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The Role of Plasmids in Antibiotic Resistance in Pasteurella multocida Isolated from Various Sources

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