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Effect of mazEF, higBA and relBE toxin-antitoxin systems on antibiotic resistance in Pseudomonas aeruginosa and Staphylococcus isolates

Background A toxin-antitoxin (TA) system is a set of two or more closely linked genes that are encoded as a poison and a corresponding antidote on a protein. In typical bacterial physiology, an antitoxin binds to a toxin and neutralizes it, which prevents the bacterium from killing itself. We aimed to determine whether P.aeruginosa and Staphylococcus isolates have TA genes and to investigate whether there is a relationship between the expression levels of TA genes and resistance to antibiotics. Methods This study included 92 P. aeruginosa and 148 Staphylococcus isolates. RelBE, higBA genes were investigated in P.aeruginosa by multiplex polymerase chain reaction (PCR). The mazEF gene and the all TA genes expression were detected by real time PCR. Results RelBE and higBA genes were detected in 100% of P. aeruginosa. It was found that the level of relBE TA gene expression is increased in isolates sensitive to aztreonam compared to resistant isolates (p<0.05). The mazEF gene was detected in 89.1% of Staphylococcus isolates. In terms of MazEF gene expression level there was no significant difference between methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates (p>0.05) whereas there was a significant difference between MSSA and coagulase-negative Staphylococcus (CNS) isolates, MRSA and CNS isolates (p<0.05). The levels of mazEF gene expression were found to be higher in isolates sensitive to gentamicin, ciprofloxacin, levofloxacin, clindamycin, phosphomycine, nitrofurantoin, fusidic acid, cefoxitin compared to resistant isolates (p<0.05). Conclusion Studies on the prevalence and functionality of TA systems emphasize that it may be possible to have new sensitive regions in bacteria by activating TA systems. The results of this study lead to the idea that resistance to antibiotics can be reduced by increasing TA gene expression levels. But there is need for further studies to support and develop this issue.


Introduction
Bacterial infections are increasingly prevalent due to rapid changes in the patient population and increased number of chronic diseases and immunosuppressed patients. The increase in antibiotic resistance complicates the treatment of Pseudomonas aeruginosa (P. aeruginosa), coagulase-negative Staphylococcus (CNS) and especially S. aureus infections 1 . S. aureus is a pathogen that can cause invasive infections such as endocarditis, osteomyelitis and sepsis as well as skin and soft tissue infections that can be colonized in humans and animals 2 . Although the pathogenicity of CNS is lower than that of S. aureus, it has been observed to be more frequently isolated in invasive infections in recent years. In the United States, 80.461 invasive methicillin-resistant S. aureus (MRSA) infections were reported in 2011, of which 11.285 resulted in death. In the same study, it was found that approximately 51.000 hospital-acquired P.aeruginosa infections occurred, of which 13% were due to multi drug resistance (MDR) P. aeruginosa and 400 resulted in death. Along with a reduction in the number of antibiotics that can be used in the treatment of infections, infections that cannot be controlled indicate a universal danger 3 .
A toxin-antitoxin (TA) system is a set of two or more closely linked genes that are encoded as a poison and a corresponding antidote on a protein. In typical bacterial physiology, an antitoxin binds to a toxin and neutralizes it, which prevents the bacterium from killing itself. When the antitoxin is degraded or not functional, the toxin kills the bacterium; this is known as a programmed cell death 4 . TA systems are genes encoded on chromosomes and plasmids 5 that can be found in both Gram-negative and Gram-positive bacteria 6 . Studies over the past 30 years have revealed detailed information about the functions and movement mechanisms of TA systems as well as various interesting results regarding the importance of such systems for bacterial physiology 7,8 . Generally, toxin molecules act as negative regulators for cell life, whereas antitoxin molecules act as positive regulators. The interaction between toxin and antitoxin gene expression levels in stressful conditions is vital for the life of the bacteria. Therefore, studies are being conducted on the possibility that TA systems can be used to develop new antibiotics [9][10][11] . Bacteria often have more than one TA system in their genome 12 . The presence and type of TA systems and whether primer binding at 52°C for 45 seconds, and for the MazEF primer binding at 54°C for 30 seconds and then elongation at 72°C for 30 seconds, followed by a final elongation step by increasing from 60°C to 90°C.

Statistical Method
In study, Shapiro-Wilk's test was used to assess the data normality. When the data of the Staphylococcus isolates were evaluated, independent samples t test was used to compare the normally distributed independent variables between two groups and Mann-Whitney U test was used to compare the non normally distributed independent variables between two groups. One-way ANOVA test was used in three groups comparisons. For multiple comparisons, the Tukey HSD test was used. The difference between resistance to antibiotics in P. aeruginosa isolates and transcription levels of TA genes was investigated with an independent samples t-test. The statistical significance level of p was 0.05. Statistical analysis was performed using commercial software (IBM SPSS Statistics 20, SPSS Inc., an IBM Co., Somers, NY).

Results
P. aeruginosa and isolates were identified in respiratory tract specimens (36 endotracheal aspirates and 9 bronchoalveolar lavages) at a rate of 46.7%, whereas Staphylococcus isolates were most frequently identified in blood culture samples at a rate of 58.8%. The sample types in which the isolates were identified are shown in Table 2. In the P. aeruginosa isolates, aztreonam was identified as the most sensitive antibiotic type, whereas the highest resistance rates were detected against carbapenems, at a rate of 67% and 59.8% for meropenem and imipenem, respectively. The antibiotics

Total RNA isolation from P. aeruginosa and Staphylococcus isolates
The prepared 30± 5 ng/ µL bacterial suspension was centrifuged at 12.500 xg for 5 minutes. Then 200 μL of RB buffer (RB buffer, β-Mercaptoethanol) was added to the pellet and RNA isolation was performed according to the supplier's recommendation. In addition, 20 μL of 10X Reaction mix, 7.5 μL of DNase I and 172.5 μL of water were added for degradation of the genomic DNA during the protocol and pure RNA was obtained.

Preparation of cDNA from total RNA in P. aeruginosa and Staphylococcus isolates:
The cDNA was prepared by adding 10 μL of water, 8 μL of reaction mix and 2 μL of reverse transcriptase (RT), to a final volume of 20 μL. The cDNA was prepared for a total of 40 minutes with the amplification steps of 5 minutes at 22°C, 30 minutes at 42°C, 5 minutes at 85°C. The identity of the cell number of the resulting cDNAs was confirmed by measuring with NanoDrop spectrophotometer. The activity of the gene region was proven by the detection of the cDNA using SYBR green dye.

Detection of TA gene expression in P. aeruginosa and Staphylococcus isolates by real time PCR
The mazEF and relBE genes were prepared by adding 12.5 μL of Super SYBR Mix and for the relBE and higBA genes, 0.5 μL (+) and (-) of primer and for the mazEF gene, 0.25 μL (+) and (-) of primer, 6.5 μL of water, and 3 μL of cDNA, to obtain a total of 20 μL mix. The amplification program included 3 minutes of denaturation at 95°C and 45 cycles of 15 seconds denaturation at 95°C for the RelBE primer binding at 56°C for 45 seconds, for the HigBA they are encoded on a plasmid or on a chromosome varies between bacteria 9 . To our knowledge, there is no study showing the existence of TA systems in methicillin-sensitive Staphylococcus aureus (MSSA) and CNS isolates. The aim of this study was to determine whether P.aeruginosa isolates isolated from clinical specimens have relBE and higBA TA systems and whether Staphylococcus isolates have mazEF TA systems, and to investigate whether there is a relationship between the expression levels of TA genes and resistance to antibiotics.

Genomic isolation of relBE and higBA genes from P. aeruginosa isolates by multiplex PCR
RelBE, higBA genes were investigated in P.aeruginosa strains by multiplex polymerase chain reaction (PCR). A total of 1.5 ml (30± 5 ng/ µL) of bacterial cultures prepared for mold DNA isolation was placed into a 1.5 mL microcentrifuge tube and precipitated for 5 minutes at 13.000 xg. The upper part of the centrifuged liquid was discarded and washed with sterile distilled water. PCRs were performed on a final volume of 50 µL and included 5 μL of genomic DNA, 20 pM of each primer, 10 μL reaction buffer (Promega), 3 μL 25 mM MgCl2, 200 μM of each dNTP and 1.5 U of Taq Polymerase (Promega, USA). PCR amplification conditions were as follows: initial denaturation at 94°C for 3 minutes followed by 30 cycles of 25 seconds at 94°C, 40 seconds at 52°C and 50 seconds at 72 °C with a final extension of 5 minutes at 72°C. All PCR results were analyzed on 1% agarose containing 0.5 μg/mL ethidium bromide and were subsequently visualized under UV light. The primers used in multiplex PCR and real-time PCR are shown in (Table 1). The gel images of relBE and higBA TA genes are given in (Figure 1).

Genomic DNA isolation of mazEF gene from Staphylococcus isolates by real-time PCR
The prepared 30± 5 ng/ µL bacterial suspension was centrifuged at 12.500 xg for 5 minutes. Then, 200 μL of lysozyme was added onto the pellet and incubated at 37ºC for 30 minutes. For the degradation of RNA, 4 μL RNase A (50mg/ml) was added to the sample and was vortexed for 10 minutes at room temperature. Then 40 μL proteinase K was added and the DNA isolation was completed according to the supplier's recommendation (Anatolia Geneworks Turkey).  most resistant against Staphylococcus isolates were penicillin (95.5%) and erythromycin (76.5%). Resistance rates against antibiotics are shown in Table 3. HigBA and relBE genes were detected in 100% of the isolates (n=92). For each screened gene, the sample was displayed by running on the agarose gel ( Figure 1). The mazEF gene was detected in 132 (89.1%) of 148 Staphylococcus isolates. The resistance rates of isolates to antibiotics and the average cycle threshold (Ct) values for the mazEF gene are shown in Table 4. The mean Ct values were 23.9 ± 8.8 for the relBE gene and 18.24 ± 5.8 for the higBA gene. The RelBE TA gene expression level was found to be increased in isolates sensitive to aztreonam compared to resistant isolates (p<0.05). The distribution of MazEF values by groups is shown in Figure 2. In terms of MazEF gene expression levels, there was no significant difference between MSSA and MRSA isolates (p=0.181), whereas there was significant difference between the MSSA and CNS isolates (p<0.001) and MRSA and CNS isolates (p<0.05). The levels of mazEF gene expression were found to be higher in isolates sensitive to gentamicin, ciprofloxacin, levofloxacin, clindamycin, nitrofurantoin, fusidic acid and cefoxitin compared to resistant isolates (p<0.05). The level of gene expression was found to be higher in isolates without inducible clindamycin-resistance compared to thoswith inducible clindamycin resistance (p<0.05).

Discussion
Bacteria often have more than one TA system in their genome 12 . The presence and type of TA systems and whether they are encoded on a plasmid or on a chromosome varies between bacteria 4 . TA systems are thought to be encoded on chromosomes in the P. aeruginosa 15,16 . Based on bioinformatic data, four TA systems were identified on P. aeruginosa, including PAQ1, relBE, higBA, and parDE 17,18 . However, a study by Gang Li et al. in 2016 identified a new TA gene named hibAB in the P. aeruginosa 19 . The studies have also revealed the existence of new TA systems. The genes of seven known TA gene families for S. aureus have been identified, with the majority of the investigations involving the mazEF TA gene 17,18 . MazEF, an operon that is called a "plasmid addiction system" and which stabilizes plasmids, was first identified on the chromosome of E. coli in 1993. MazEF is the most studied TA system in E. coli and has been reported to be an irreversible mediator in cell death in stress conditions 20 as well as a modulator in the translation process 21,22 .
RelBE is also one of the most studied TA systems in E. coli 20 . It modulates the response induced in the case of amino acid starvation 22 , which leads to the inhibition of translation and consequently to bacteriosis 21 . In the literature, E. coli appears to be the most frequently investigated bacterium for TA systems. HigBA was first described on Proteus vulgaris 23 . In a study conducted in the United States, it was reported that TA genes were encoded on the chromosome in P. aeruginosa and S. aureus isolated from clinical samples collected from three centers and that 100% of the 78 MRSA isolates had higBA and relBE, whereas 30% had parDE and all 42 P. aeruginosa isolates had relBE and higBA TA genes. The authers of the study confirmed the PCR products by DNA sequencing, with the result that 97.8% to 100% of the PCR products were compatible with the DNA sequencing. In addition, they also emphasized that these genes are transcribed and that the activation of toxin genes would be an effective antibacterial strategy 15 . In a study conducted in 2016, 174 P. aeruginosa isolates isolated from clinical specimens were found to have relBE, higBA and parDE TA genes at a rate of 100%, 100% and 30%, respectively in Iran 16 . Another study conducted in Iran by Hemati et al. showed that P. aeruginosa isolates had mazEF TA genes at a rate of 85.7% and that the isolates were resistant to gentamicin (65%), meropenem (60%), piperacillin (59.28%), and amikacin (52.14%). The authors found a correlation between mazEF genes and resistance to gentamicin, meropenem, piperacillin and amikacin 24 . In this study, higBA and relBE genes were found in all (100%) of P. aeruginosa isolates, while the mazEF gene was found in 89.1% of the Staphylococcus isolates. The results of the study are consistent with the literature and show that TA genes are present at high rates in P. aeruginosa and Staphylococcus isolates. In Staphylococcus isolates, the levels of mazEF gene expression were found to be higher in isolates sensitive to gentamicin, ciprofloxacin, levofloxacin, clindamycin, phosphomycin, nitrofurantoin, fusidic acid and cefoxitin compared to resistant isolates (p<0.05). In P. aeruginosa isolates, in contrast to the study by Hemati et al. 24 , the relBE TA gene expression level was found to be increased in isolates sensitive to aztreonam compared to resistant isolates (p<0.05). For Staphylococcus isolates, the existence of TA systems has been proven only in MRSA isolates, while these systems remained unexplored in CNS and MSSA isolates.
In this study, we evaluated the presence of TA systems in MSSA and CNS and observed the transcription of the mazEF TA gene in MRSA and MSSA isolates is higher than in CNS (p<0.05). This suggests that the mazEF TA gene may be related to virulence in S. aureus. To our knowledge, this is the first study investigating TA genes in the CNS.
The determination of the prevalence of TA genes in strains with less virulence will also contribute to the studies investigating the possibility that TA systems are antibacterial targets. Bukowski et al. 25 suggests that TA systems induce bacteriosis, which in the long run result in the death of bacterial cells. The results of Bukowski et al. 25 and Hemati et al. 24 also point out that drugs that can activate silent toxins in TA systems can be used as antibiotics. The present study is consistent with the results of Bukowski et al. 25 and Hemati et al. 24 in terms of the finding that isolates with higher mazEF gene expression levels were more sensitive to gentamicin, ciprofloxacin, levofloxacin, clindamycin, phosphomycin, nitrofurantoin, fusidic acid and cefoxitin. In the present study, the toxin and antitoxin genes were evaluated together because they were located on the same operon. Bacteria were not exposed to any stress factors, such as heat, amino acid starvation, or antibiotic exposure. We investigated only the presence of these TA systems and the association between their presence and antibiotic resistance. Only the statistical determination of these data constitutes a limitation of this study. The studies on the prevalence and functionality of TA systems emphasizes that it may be possible to have new sensitive regions in bacteria by activating TA systems (by degrading antitoxins or increasing toxin expression) 6,18 . As a matter of fact the results of this study lead to the idea that resistance to antibiotics can be reduced by increasing TA gene expression levels. The most important step at this stage is to investigate the presence and functionality of TA genes in microorganisms that cause infections. Therefore, there is require to investigate the presence and frequency of TA genes in bacteria to support and develop this strategy and to determine which strategies are suitable for this target. Our study is the first study in Turkey investigating TA systems in P. aeruginosa and Staphylococcus isolates isolated from clinical specimens.

Conclusion
Studies on the prevalence and functionality of TA systems emphasize that it may be possible to have new sensitive regions in bacteria by activating TA systems. The results of this study lead to the idea that resistance to antibiotics can be reduced by increasing TA gene expression levels. But there is need for further studies to support and develop this issue.