Antimicrobial Susceptibility and Detection of Genes for Antimicrobial Resistance of Mycoplasma bovis, Staphylococcus aureus and Escherichia coli

Mycoplasma bovis (Gram-positive bacteria) belongs the class Mollicutes and to the family Mycoplasmataceae (Maunsell and Donovan, 2009). It is a cell wall-less bacterium and are instead enveloped by a complex plasma membrane (Lysnyansky and Ayling, 2016; Maunsell and Donovan, 2009). Mycoplasmas are the smallest microorganisms capable of self-replication (diameter of approximately 0.2 to 0.3 μm) with the (smallest genomes 500 to 1,000 genes)  (Gautier Bouchardon, 2018).
         
The mycoplasma cell contains the minimum set of organelles important for growth and replication: a plasma membrane, ribosomes and a genome consisting of a double-stranded circular DNA molecule (Lysnyansky and Ayling, 2016). In cattle, M. bovis is widely known causes various diseases, such respiratory disease, mastitis, arthritis and otitis (Malmberg et al., 2020; Bhaladhare et al., 2018). 
         
It is now known that this mycoplasma species is the most important etiological agent of bovine respiratory disease and often it is the only causing agent implicated in the chronic forms of respiratory disease for bovine (Goswami et al., 2019; Caswell et al., 2010).
         
In cattle, control of M. bovis infections is inherently difficult. The resulting clinical signs and disease are increasingly recognized as having a significant adverse impact on animal welfare and the economy of cattle farming around the world (Dudek et al., 2020; Barua et al., 2017). Chronic infections of M. bovis are responsible for economic losses in cattle and milk production. The only approaches that can be used in attempts to control M. bovis infections are healthy control measures and antimicrobial treatment. However, M. bovis is resistant to b-lactams and to all antimicrobials that target the cell wall (cell-wall-less bacterium). In addition, mycoplasmas are also naturally resistant to polymyxins, sulfonamides, trimethoprim, nalidixic acid and rifampin (Lysnyansky and Ayling, 2016).
         
There is worldwide concern about the appearance and rise of bacterial resistance to commonly used antibiotics (Aslam et al., 2018). 16S rDNA genes are coding for the small ribosomal subunit have great important in the phylogenetic analysis of bacteria because of their universal distribution and because mutations occur at a slow and constant rate (Alsanie et al., 2018).
         
M. bovis has an elaborate genetic system encoding (Vsps) that consider an antigenic repertoire for this pathogen each (Vsp) was shown to possess the following features: (i) independent high-frequency phase variation between ONand OFF expression states, (ii) membrane anchorage via the N-terminal domain, (iii) independent high-frequency size variation and a C-terminal region which is surface exposed, (iv) extensive repetitive domains over the full length of the Vsp molecule, and (v) regions of shared epitopes (Bürki et al., 2015; Nussbaum et al., 2002; Lysnyansky et al., 1999).
         
Tetracyclines are considered broad spectrum antibiotics used against Gram-positive and Gram-negative. Tetracyclines inhibit protein synthesis through bacterial ribosome from associating with the aminoacyl-tRNA (Ullah et al., 2012). TetK, tetM, tetO and tetL genes related with Tetacycline resistance genes which prevent tetracycline from accumulating within the cell in Gram-positive bacteria (Ullah et al., 2012).
         
Comprehensive efforts are needed to reduce the pace of resistance by studying emergent microorganisms, mechanisms of resistance and antimicrobial drugs (Aslam et al., 2018). The present study aimed to identify the molecular mechanisms of resistance in M. bovis, S. aureus and E. coli against antimicrobial drugs by phenotyping and genotyping method.

Microbial culture
 
The study was conducted of 2019-2020 at the research in Eskil Vocation of High School, Laboratory Veterinary Science, Aksaray University, Turkey. Microorganisms which that have been used in this study consisted of Mycoplasma bovis PG45 obtained from ATCC S. aureus and E.coli isolates identified. Inhibitory concentration (MIC) of each antimicrobial agent was determined by applying an agar dilution method with some modifications and in accordance with the recommendations of Hannan (2000) and Kolarević et al., (2016).
         
Twenty antimicrobial agents (Table 1) were included in the testing which listed in Table 1. Based on Hannan (2000) using 104 -105 CCU/ml of each strain. In brief, the 96-wells microtiter plates were designed to contain Mycoplasma broth medium. Antimicrobial agent concentrations ranged from 0.39 to 100 μg/ml for all antibiotics. The procedures were performed as described by Hannan (2000). All antimicrobial agents were dissolved in the optimum diluent at 100 µg/ml. The MIC value of each isolate was defined as the lowest concentration of the antibiotic that completely inhibits the growth in the broth after a one week incubation period. MIC₅₀ and MIC₉₀ values were defined as the lowest concentrations that inhibit 50% and 90% of bacterial tested.
 

 
DNA extraction and primers design
 
DNA was extracted from identified M. bovis PG45, E. coli and S. aureus cultures using DNA minikit (QIAGEN QIAmp DNA mini kit, Germany). Primer set A was designed on the basis of 16S rRNA nucleotide sequences, as an effective tool for amplification of mycoplasma DNA. Set B specifically targets a variable initial portion of the vspA gene of M. bovis PG45^T annealing to its more conserved flanking regions and was used for molecular characterization. S. aureus strain was tested for the presence of following antibiotic resistant gene Tetracycline (tetK), E. coli strain was tested for the presence of following antibiotic resistant gene Tetracycline (tetA). Amplifications were performed in a programmable thermal cycler:
1. For the Set A (16SrRNA) encoding gene (Kojima et al., 1997), 94°C (7.5min) of initial denaturation and then 30 cycles of denaturation at 94°C (30s), annealing at 56°C (30s), elongation at 72°C (60s) and final extension in 72°C (5min).
         
For Set B (Vsp_A) an initial denaturation step (94°C for 5 min), followed by 35 cycles of denaturation (94°C for 1 min), annealing (55°C for 1 min) and extension (72°C for 1.5 min). A final extension step at 72°C for 10 min was included.
2. For tetK gene in S. aureus an initial denaturation step (94°C for 5 min), followed by 35 cycles of denaturation (94°C for 1 min), annealing (57°C for 1 min) and extension (72°C for 1.5 min). A final extension step at 72°C for 10 min was included.
3. For tet (A) gene in E. coli an initial denaturation step (94°C for 5 min), followed by 30 cycles of denaturation (94°C for 20 sec), annealing (55°C for 1 min 15 sec) and extension (72°C for 15 sec). A final extension step at 72°C for 2 min was included.
         
All the PCR amplicons were visualized using an ultraviolet light box after electrophoresis on a 1.5% agarose gel containing 0.5 µg/ml ethidium bromide.

Evaluation of antibiotics activity
 
The results of antibacterial susceptibility testing are presented in (Table 2) for all the bacterial pathogens, M. bovis, S. aureus and E. coli. M. bovis, S. aureus and E. coli showed higher resistance to most tested antibiotics (Cefotaxime, Cefuroxime, Cefaclor Cefalexin, Ofloxacin, Norfloxacin, Nalidixic acid, Amikacin, Ampicillin, Oxacilin, Amoxyclav, Rifampicin, Penicillin G and Tylosin) with MIC₅₀<100 MIC₉₀<100.
 

         
In Table 3. M. bovis showed only sensitive to Enrofloxacin with MIC₅₀ 0.39 and MIC₉₀ 1.56, but S. aureus and E. coli showed higher resistance with MIC₅₀ 100 and MIC₉₀>100. M. bovis showed resistance to Gentamicin with MIC₅₀>25 MIC₉₀>50, but S. aureus and E. coli showed higher resistance with MIC₅₀>100 and MIC₉₀>100.
 

 
PCR results showed that all tested bacteria (M. bovis, S. aureus and E. coli) were positive for 16SrRNA gene as shown in (Fig 1). M. bovis was positive for Vsp_A gene but negative for tetA and tetK genes (Fig 2). E. coli was positive for tetA gene but negative for VspA and tetK genes (Fig 3), but S. aureus was positive for but negative for Vsp_A and tetA genes (Fig 4). The spread of bacterial infection is a major concern worldwide because infections caused by these pathogens may lead to prolonged hospital stays, higher medical costs and increase rates of morbidity and mortality (Cho et al., 2019).
 

 

 

 

         
Sequencing analysis of 16SrRNA gene fragments has proved to be one of the most significant tools for phylogenetic studies and for many diagnostic applications (Sahar et al., 2013). Many studies have proved that the evolution of antibiotic resistance is due to gene mutations (Szacawa et al., 2014). A number of recent studies indicate that the efficacy of tetracyclines, lincosamides and macrolides against M. bovis has diminished. So it is needful to search for alternative compounds that could effectively inhibit these bacteria (Szacawa et al., 2014).
         
In this study M. bovis, showed highly resistance to most of antibiotics and positive for VspA gene. The possible explanation for this Mycoplasma possesses a complex system capable of creating large repertoires of cell surface phenotypes which may impact the various interactions of this organism with its host and dictate its potential as a successful infectious agent and pathogen (Droesse et al., 1995).
         
In this study M. bovis, showed highly resistance to tetracycline MIC50 was 100, MIC90 was 100. Similar results were obtained by (Siugzdaite et al., 2012) that proved the MICs for oxytetracycline of all isolates of M. bovis were higher than the breakpoint (4-16 µg/ml). In veterinary medicine, raises in tetracycline resistance have been described for M. bovis, M. hyopneumoniae, M. bovirhinis and M. alkalescens (Thomas et al., 2003).
         
Resistant Mycoplasmas exhibit a reduced uptake of tetracycline into cells, lowering the initial concentration, and have acquired the ability to excrete the drug out of the cell (Siugzdaite et al., 2012; Boothe 1998). The frequent use of oxytetracycline for the treatment of respiratory disease infection is a possible explanation for the high prevalence of oxytetracycline resistance in M. bovis. In the present study, all M. bovis isolates were highly resistant to beta-lactam antimicrobials (penicillin G, Ampicillin, Oxacilin, Cefotaxime) and this due to the lack of a cell wall (cell-wall-less bacterium) makes the Mycoplasma resistant to b-lactams and to all antimicrobials that target the cell wall. These results were similar to the findings of (Siugzdaite et al., 2012) that confirmed penicillin G can be used in antimicrobial susceptibility testing only as a negative control against Mycoplasma. S. aureus isolate in this study showed higher resistance to tetracycline with (MIC₅₀<100 MIC₉₀<100) and in genotype results was positive for tetK gene. These results were similar to the findings of (Ullah et al., 2012) that found the majority of the S. aureus strains either carried tetK or tetL genes and highly resistance to tetracycline. The presence of tetK prevents the accumulation of tetracycline within bacterialcells by the synthesis of a cytoplasmic membrane protein which pumps tetracycline out of the cell at a quicker rate than it enters (Ullah et al., 2012).
         
E. coli in this study showed higher resistance to tetracycline with (MIC₅₀<100 MIC₉₀<100) and in genotype results was positive for tetA gene. These results were similar to the findings of (Ullah et al., 2012) that found E. coli strains were phenotypically resistant to tetracycline and showed all tetracycline-resistant strains carried at least one of the tetA, tetB and tetc genes. In conclusion, our results demonstrated the presence of Vsps, tetK and tetA in M. bovis, S. aureus and E. coli respectively make this strains multi-drug resistant So it is needful to search for alternative compounds that could effectively inhibit these bacteria. Comprehensive efforts are needed to minimize the pace of resistance by studying emergent microorganisms, resistance mechanisms and antimicrobial agents.

In conclusion, our results demonstrated the presence of Vsp_A, tetK and tetA in M. bovis, S.aureus and E. coli respectively. The tested bacteria also showed resistant to different antimicrobials so it is needful to search for alternative compounds that could effectively inhibit these bacteria. Comprehensive efforts are needed to minimize the pace of resistance by studying more number of emergent microorganisms, and antimicrobial agents.