Evaluation of toxin-antitoxin genes, antibiotic resistance, and virulence genes in Pseudomonas aeruginosa isolates

SUMMARY

OBJECTIVE:

Toxin-antitoxin genes RelBE and HigBA are known to be involved in the formation of biofilm, which is an important virulence factor for Pseudomonas aeruginosa. The purpose of this study was to determine the presence of toxin-antitoxin genes and exoenzyme S and exotoxin A virulence genes in P. aeruginosa isolates and whether there is a relationship between toxin-antitoxin genes and virulence genes as well as antibiotic resistance.

METHODS:

Identification of the isolates and antibiotic susceptibilities was determined by a VITEK 2 (bioMérieux, France) automated system. The presence of toxin-antitoxin genes, virulence genes, and transcription levels were detected by real-time polymerase chain reaction.

RESULTS:

RelBE and HigBA genes were detected in 94.3% (82/87) of P. aeruginosa isolates, and exoenzyme S and exotoxin A genes were detected in all of the isolates (n=87). All of the isolates that harbor the toxin-antitoxin and virulence genes were transcribed. There was a significant increase in the RelBE gene transcription level in imipenem- and meropenem-sensitive isolates and in the HigBA gene transcription level in amikacin-sensitive isolates (p<0.05). There was a significant correlation between RelBE and exoenzyme S (p=0.001).

CONCLUSION:

The findings suggest that antibiotic resistance may be linked to toxin-antitoxin genes. Furthermore, the relationship between RelBE and exoenzyme S indicates that toxin-antitoxin genes in P. aeruginosa isolates are not only related to antibiotic resistance but also play an influential role in bacterial virulence. Larger collections of comprehensive studies on this subject are required. These studies should contribute significantly to the solution of the antibiotic resistance problem.

KEYWORDS:
Pseudomonas aeruginosa ; Toxin-antitoxin systems; Virulence; Anti-bacterial Agents

INTRODUCTION

Pseudomonas aeruginosa (P. aeruginosa) is one of the major pathogens causing hospital-acquired infections, particularly affecting patients with immunocompromised or prolonged stay in the intensive care unit¹. As a pathogen, P. aeruginosa is of growing clinical significance as a result of its inherent resistance to multiple antimicrobials and its ability to develop high-level multidrug resistance (MDR) due to the presence of a lot of virulence factors expressed in its genome².

These virulence factors allow P. aeruginosa to easily reproduce and live in both the host cell and the environment. Virulence factors can cause a number of harmful effects, including damage to tissues, the spread of infection to blood and tissue, the escape of bacteria from the host cell defense, and disease progression. In addition, they can induce antibiotic resistance in P. aeruginosa, making treatment difficult³.

In recent years, studies on toxin-antitoxin (TA) genes have shown that they are associated with virulence regulation, biofilm formation, plasmid maintenance, and antibiotic resistance⁴–⁶. TA genes are small operons composed of both a growth-inhibitory toxin and an antitoxin that regulates toxin activity by direct inhibition. This antitoxin also plays a role in cell physiology by acting as a regulator of transcription⁷–⁹. Previous studies have shown that TA genes play several important physiological roles and, therefore, may be able to treat infections caused by MDR bacteria⁶.

It has been indicated that TA genes are involved in the formation of biofilm, which is an important virulence factor for P. aeruginosa¹⁰–¹². The purpose of this study was to determine the presence of RelBE and HigBA TA genes and exoenzyme S (ExoS) and exotoxin A (ToxA) virulence genes in P. aeruginosa isolates. In addition, we aimed to investigate whether there is a relationship between TA genes and virulence genes as well as whether TA genes are associated with antibiotic resistance.

METHODS

Bacterial isolates and antimicrobial susceptibility testing

This study included 87 P. aeruginosa isolates from various samples sent to the Microbiology Laboratory at Tokat Gaziosmanpasa University Training and Research Hospital between January 2016 and March 2017. Identification and antibiotic resistance profile of P. aeruginosa isolates were determined by a Vitek 2 (bioMérieux, France) automated system according to Clinical and Laboratory Standards Institute criteria¹³. The total number of susceptible and resistant antibiotics is not equal to all antibiotics because not all antibiotics have been studied in every isolate. Only one isolate was obtained from each patient. P. aeruginosa ATCC 27853 isolates were used as quality controls.

Genomic DNA isolation of RelBE, HigBA, ExoS, and ToxA genes in Pseudomonas aeruginosa isolates by real-time polymerase chain reaction

A volume of 10⁵ McFarland bacterial suspension (1.5 μL) was centrifuged at 12,500 × g for 5 min. Then, 200 μL of lysozyme was added to the pellet and incubated at 37°C for 30 min. To degrade the RNA, 4 μL of RNase A (50 mg/mL) was added to the sample, which was vortexed for 10 min at room temperature. In addition, 40 μL of proteinase K was added, and the DNA isolation was completed according to the manufacturer's recommendations using a Magnesia 16 isolation device (Anatolia Geneworks, Turkey).

Total RNA isolation from Pseudomonas aeruginosa isolates

The prepared bacterial suspension (1.5 μL) was centrifuged at 12,500 × g for 5 min and 200 μL of RB buffer prepared with mercaptoethanol was added to the pellet, and RNA isolation was performed by using the Magnesia 16 Cultured Cell and Tissue Total RNA Extraction Kit. To degrade the genomic DNA, 20 μL of 10′ reaction mix, 7.5 μL of DNase I, and 172.5 μL of water were added to each sample, and pure RNA was obtained. RNA isolation was performed using a Magnesia 16 isolation device (Anatolia Geneworks, Turkey).

Preparation of cDNA from total RNA in Pseudomonas aeruginosa isolates

The cDNA mixture was prepared by adding 10 μL of water, 8 μL of the reaction mix, and 2 μL of reverse transcriptase (RT) to the final volume of 20 μL. The cDNA was prepared using a Montania 4896 real-time PCR device (Anatolia Geneworks, Turkey) for a total of 40 min as follows: 5 min at 22°C, 30 min at 42°C, and 5 min at 85°C. The activity of the gene region was proven by the detection of the cDNA using SYBR green dye.

Detection of RelBE, HigBA, ExoS, and ToxA genes expression in Pseudomonas aeruginosa by real-time polymerase chain reaction

All of the genes were prepared by mixing 12.5 μL of Super SYBR Mix, 0.5 μL of forward and reverse primers, 6.5 μL of water, and 3 μL of cDNA for a total volume of 20 μL. The gene expression levels were detected by a Montana 4896 real-time PCR device (Anatolia Geneworks, Turkey).

The amplification programs for RelBE, HigBA, ExoS, and ToxA were as follows: 3 min of denaturation at 95°C and 45 cycles of 15-s denaturation at 95°C; for the RelBE primer, binding at 56°C for 45 s; elongation at 72°C for 30 s, followed by a final elongation step in which the temperature was increased from 60 to 90°C; for the HigBA, ExoS, and ToxA primers, binding at 52°C for 45 s; elongation at 72°C for 30 s, followed by a final elongation step in which the temperature was increased from 60 to 90°C. The primers were used in the PCR step according to the previous study¹²,¹⁴.

Statistical analysis

Statistical analysis was performed by using commercial software IBM SPSS Statistics version 20 (SPSS Inc., an IBM Corp., Somers, NY, USA). The differences between antibiotic resistance in the P. aeruginosa isolates and the transcription levels of the TA and virulence genes were investigated with independent samples t-test and Mann-Whitney U test. The relationship between RelBE and HigBA genes and ExoS and ToxA genes was investigated with the Pearson's correlation test. The values of p≤0.05 were considered significant.

Ethics

This study was approved by the Ethics Committee of Tokat Gaziosmanpasa University (number 17/KAEK/022).

RESULTS

The isolates were obtained from respiratory samples (40.3%, n=35), wound samples (26.4%, n=23), urine (20.7%, n=18), blood (11.5%, n=10), and sterile body fluid samples (1.1%, n=1). The P. aeruginosa isolates had the highest rates of antibiotic resistance to aztreonam 64.4% (47/73), piperacillin-tazobactam 64% (55/86), imipenem 42.7% (35/82), and meropenem 36.8% (32/87). While the RelBE and HigBA genes were detected in 94.3% (82/87) of the P. aeruginosa isolates (n=87), the ExoS and ToxA genes were detected in all of the isolates (n=87). It is shown that TA genes (82/82) and virulence genes (87/87) are involved.

There was a significant increase in the RelBE gene transcription level in imipenem- and meropenem-sensitive isolates (p<0.05). There were no correlations between RelBE and HigBA gene transcription levels with any of the other antibiotics (p>0.05). Antibiotic susceptibility rates of isolates and the relationship between antibiotic susceptibilities and transcription levels of RelBE and HigBA TA genes are shown in Table 1. There was a significant correlation between RelBE and ExoS (p=0.001); none of the other correlations were significant.

Table 1
Antibiotic susceptibility rates of isolates and the relationship between antibiotic susceptibilities and expression levels of RelBE and HigBA Toxin-antitoxin genes.

Antibiotic		n	%	RelBE gene p	HigBA gene p
Piperacillin-tazobactam	Sensitive	31	36	0.436a	0.723a
Seftazidime	Sensitive	62	71.3	0.154a	0.636a
Sefepime	Sensitive	55	66.3	0.331a	0.431a
Aztreonam	Sensitive	26	35.6	0.449a	0.664a
Imipenem	Sensitive	47	57.3	0.002 a	0.365a
Meropenem	Sensitive	55	63.2	0.043 a	0.367a
Amikacin	Sensitive	80	92.0	0.627a	0.050a
Gentamycin	Sensitive	75	86.2	0.756a	0.181a
Netilmicin	Sensitive	64	87.7	0.848a	0.228a
Tobramycin	Sensitive	67	93.1	0.406b	0.086b
Siprofloxacin	Sensitive	67	77.9	0.251a	0.144a
Levofloxacin	Sensitive	52	72.2	0.236a	0.323a
Colistine	Sensitive	82	100	-	-

• TA: toxin-antitoxin. The total number of susceptible and resistant antibiotics is not equal to all antibiotics because not all antibiotics have been studied in every isolate. Differences in antibiotic resistance in P. aeruginosa isolates and transcription levels of TA genes were investigated with the independent samples t-test and Mann-Whitney U test. The relationship between RelBE and HigBA genes and ExoS and ToxA genes was investigated with a Pearson's correlation test.
• ^a
  Independent samples t-test.
• ^b
  Mann-Whitney U-test. Bold indicates statistically significant p-value.

DISCUSSION

The MDR P. aeruginosa caused 32,600 estimated infections among hospitalized patients and 2,700 estimated deaths in the United States. Some types of MDR P. aeruginosa are resistant to nearly all antibiotics, including carbapenems, which means that several classes of antibiotics including aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems may not cure these infections¹. In recent years, the increase in carbapenem-resistant frequency among P. aeruginosa is becoming a major challenge. The reported rates of carbapenem resistance seem to be considerably different (12–67%) in various regions¹⁵–¹⁷. In this study, it was observed that the carbapenem's resistance rates were consistent with the previous studies. It is seen that carbapenems still maintain their importance among the antibiotics used in the treatment of P. aeruginosa infections.

Pseudomonas aeruginosa have virulence factors including biofilm, ToxA, ExoS, pigments, mucoid exopolysaccharide, lipopolysaccharide, protease, leucocidin, and hemolysins. ToxA is secreted outside the cell and causes cell death and cell damage as well as suppression of host response by inhibiting protein synthesis¹⁸. Nikbin et al. indicated ExoS and ToxA genes existed in wound samples at rates of 62 and 90% and in respiratory system at rates of 47.4 and 46.6%, respectively. They indicated that the prevalence of ToxA gene was significantly higher in the pulmonary tract and burn isolates. In addition, the difference between ExoS prevalence in isolates from the pulmonary tract and burn isolates was statistically significant¹⁹.

Faraji et al. in 2016 reported that ExoS and ToxA genes were detected in cystic fibrosis isolates at rates of 70.8 and 63.1%, respectively²⁰. Wolska et al. indicated that the ExoS gene was present in 78.5% of 62 P. aeruginosa isolates, while the ToxA gene was found in 88.7%¹⁴. In this study, all the P. aeruginosa isolates had ExoS and ToxA genes. Therefore, the statistical distribution of virulence genes according to the samples has not been studied. However, most isolates were isolated from respiratory tract samples. The results of this study determined that ExoS and ToxA virulence factors are found at high rates in P. aeruginosa isolates, which is consistent with previous studies.

Hemati et al. observed biofilm formation in 87.5% of 140 P. aeruginosa isolates; furthermore, the TA genes MazEF, RelBE, HigBA, CcdAB, and MqsR were found at rates of 85.71, 100, 1.42, 100, and 57.14%, respectively. In addition, they reported a relationship between biofilm formation and TA gene expression⁹. In 2016, Wood et al. detected the HigBA gene in P. aeruginosa PA14 isolate and investigated the biofilm formation by using crystal violet, pyocyanin production by using acetic acid, and dichloromethane and pyoverdine production by using chrome azurol S agar plate method. They indicated that the HigBA TA gene is effective not only on biofilm formation but also on pyoverdine production¹⁰.

Previous studies have determined that P. aeruginosa isolates have RelBE and HigBA TA genes⁹,¹³,²¹,²². Guo et al. demonstrated the antitoxin HigA regulates virulence in P. Aeruginosa by binding especially to the promoter region of the MvfR gene that regulates pyocyanin synthesis⁵. Song et al. indicated that HigA-mediated transcriptional inhibition on stress stimulation could affect virulence genes and also take attention to the potential of the HigBA TA system as an antibacterial treatment target⁶. In 2022, Zadeh et al. determined that ciprofloxacin and colistin may induce persister cell formation by enhancing the expression of type II TA systems during stationary and exponential phases²³. Also, it was shown that there was a strong correlation between the mazEF TA gene and resistance against gentamicin, meropenem, and amikacin⁹.

Even though TA genes and virulence genes were considered to be associated with antibiotic resistance, we observed the level of RelBE gene expression was higher in imipenem- and meropenem-sensitive isolates, and the level of HigBA gene expression was higher in amikacin-susceptible isolates. In our previous study, we investigated the relationship between toxin genes and antibiotic resistance in a different bacterial collection consisting of 92 P. aeruginosa and 148 staphylococci isolates. It was found that in P. aeruginosa, the level of RelBE TA gene expression is increased in isolates sensitive to aztreonam compared to those resistant to aztreonam. Also, in staphylococci, the levels of mazEF gene expression were found to be higher in isolates sensitive to gentamicin, ciprofloxacin, levofloxacin, clindamycin, phosphomycine, nitrofurantoin, fusidic acid, and cefoxitin compared to those resistant to the above antibiotics²¹. In the present study, we observed that toxin genes were associated with antibiotic resistance, and RelBE TA gene expression was associated with the exoS virulence gene in P. aeruginosa isolates.

CONCLUSION

The fact that TA genes are expressed more in strains sensitive to carbapenems should draw attention to these strains, which may cause serious infections that are difficult to treat in the future. The relationship between RelBE and ExoS indicates that TA genes in P. aeruginosa isolates are not only related to antibiotic resistance but also play important roles in bacterial pathogenesis and virulence. Further studies including larger numbers of genes are necessary to illustrate the role of TA genes in the pathogenesis of P. aeruginosa and to elucidate their connection with antibiotic resistance. These studies should make a significant contribution to the solution of the antibiotic resistance problem.

• ETHICAL APPROVAL
  This study was financially supported by Tokat Gaziosmanpasa University Scientific Research and Projects Unit (project number 2015/25). This study was presented in the 4th National Clinical Microbiology Congress (Abstract Paper/Poster) (publication number: 3825136). All the protocols were performed under the supervision of the Ethics Committee of Tokat Gaziosmanpasa University (number 17/KAEK/022).
• Funding: none.

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Publication Dates

• Publication in this collection
  17 Feb 2023
• Date of issue
  2023

History

• Received
  29 Aug 2022
• Accepted
  05 Oct 2022