Pseudomonas aeruginosa is an important cause of health care-related infections and high mortality, especially in patients with underlying diseases.^[1] Due to the production of various virulence factors and antibiotic resistance, this organism is classified as a critical pathogen by the World Health Organization.^[1–3]P. aeruginosa is distinguished by the production of various pigments, with more than 90% of the strains producing pyocyanin, which corresponds to a slower growth rate.^[4] Also, the exotoxin A (ETA), related to the bacterial pathogenesis in burn and wound infections, corneal ulcers, and keratitis, could be produced by more than 90% of the P. aeruginosa isolates.^[4]

Water-soluble pyocyanin (phenazine) is expressed only by P. aeruginosa.^[5] This phenazine damages several enzymes and DNA due to increased reactive oxygen species (ROS), leading to cell lysis, severity of disease, and decreased lung function.^[6] In addition, it slows the respiratory cilia movement, disrupts the function of epithelial cells, increases the secretion of respiratory mucus and colonization of bacteria, and increases the inflammation.^[6] Phenazine production in P. aeruginosa is controlled by the QS system which stimulates the operon phzA1B1C1D1E1F1G1 to produce phenazine-1-carboxylic acid (PCA), which is converted to 5-methylphenazine-1-carboxylic acid betaine by phzM and then to pyocyanin by phzS gene product.^{[7, 8]}

On the other hand, the most toxic virulence factor of P. aeruginosa is exotoxin A, an ADP-ribosyltransferase.^[9]ETA inhibits the host protein synthesis and IL-18 secretion, reduces the production of TNF-α, IL-6, IL-8, and IL-10, and induces the apoptosis of the host cell.^{[3, 9, 10]}

P. aeruginosa is one of the most important causes of nosocomial and community-acquired infections with high morbidity and mortality. The high pathogenicity of this organism is due to the abundance of virulence factors, significant metabolic flexibility, and the adaptation to various harsh conditions.^[1] Identification of the specific virulence factors in the pathogenesis of this bacterium can help design a vaccine and specific infection control measures in each region.

In this study, we aimed to investigate the prevalence of genes encoding exotoxin A (exoA) and pyocyanin (phzA1B1C1D1E1F1G1, phzM, and phzS) in P. aeruginosa clinical isolates.

Pseudomonas aeruginosa is a serious opportunistic pathogen and an infectious agent in a diverse group of organisms that is largely related to the ability of this bacterium to produce a variety of pathogens.^[17] The expression of many virulence genes in P. aeruginosa is controlled by quorum sensing, which affects the transcription of hundreds of downstream genes.^{[17, 18]} The production of a green color phenazine called pyocyanin (PCN) by this bacterium is also linked to several QS systems and is often used as a marker to assess QS behavior.^[17]PCN is a potent bacterial pigment that has five different derivatives produced by P. aeruginosa and has an inhibitory effect on pathogenic microbes.^[19]PCN secretion has been detected in sputum, ear secretions, wounds, and urine of patients following chronic infection by P. aeruginosa.^[20]

The biosynthesis of this phenazine begins with the induction of phenazine-1-carboxylic acid (PCA) production by the action of enzymes encoded by the conserved operon phzA1B1C1D1E1F1G1.^[21] The PCA produced can be converted to phenazine-1-carboxamide by phzH and to 1-hydroxyphenazine by phzS. Also, the phzM gene product can convert PCA to 5-methylphenazine-1-carboxylic acid betaine, whereas it can be converted to PCN by phzS.^{[21, 22]} Inactivation of phzM and phzS genes in P. aeruginosa prevents the production of pyocyanin pigment by this bacterium.^[7] However, we concluded in our study that the absence of any of the three genes phzM, phzS, and phzA1B1C1D1E1F1G1 operon could lead to a lack of pyocyanin production, so that among the 100 clinical isolates studied in the present study, 10 isolates lacked the ability to produce pyocyanin. In the genetic study of these strains, we observed that all isolates, which individually or in combination did not have genes related to the production of pyocyanin pigment, did not produce any pigment in the phenotypic test.

An Iranian study conducted on 80 P. aeruginosa isolates collected from burn wounds, soil, and plants showed that 3 of the 48 clinical strains were unable to produce pyocyanin on cetrimide and Müller Hinton agar, while two strains lacked both phenazine-modifying genes (phzM and phzS) and the other strain lacked the phzM gene. Also, all 32 environmental isolates had the studied genes (phzA1B1C1D1E1F1G1, phzM, and phzS), and out of 48 burn wound isolates, only 2 isolates did not produce pyocyanin. On the other hand, the isolates without phzM gene had yellow pigment and the one without phzS gene also had reddish brown pigment.^[16] In our study, 10 isolates were unable to produce pyocyanin, four of which lacked the phzS gene. Of these four isolates, three produced brown pigment and one produced reddish brown pigment on Müller-Hinton agar. In such isolates, the absence of the phzS gene prevents the production of 1-hydroxyphenazine and then pyocyanin. We also observed that the strains lacking the phzS and phzA1B1C1D1E1F1G1 genes produced the yellow pigment, while the strains lacking the phzS and phzM genes produced the brown pigment. However, 2 isolates without the phzA1B1C1D1E1F1G1 operon produced the black pigment. So, absence of any of these three genes lead to a lack of pyocyanin production, which was consistent with previous studies.^{[7, 16, 17, 23]} In another study published in 2020 in Brazil, a total of 54 clinical isolates of P. aeruginosa collected from the ICUs were evaluated, while 22 (78.5%), 12 (85.7%), and 7 (58.3%) isolates collected from ICU of adults, children, and infants contained phzM gene, and 20 (71.4%), 12 (85.7%), and 5 (41.6%) of the isolates carried the phzS gene, respectively. On the other hand, 18 (78.2%) and 8 (57.2%) isolates collected from respiratory and blood samples carried the phzM gene, respectively, while 17 (73.9%) and 6 (42.8%) isolates carried the phzS gene, respectively. Also, all isolates from rectal swab, eye, stomach, urethral secretions, and urine samples carried both the phzM and phzS genes. Besides, of 3 surgical wound isolates, 2 carried both genes and out of the 6 catheter isolates, 5 and 4 isolates carried the phzM and phzS genes, respectively.^[24] Compared to the above study, in which 75.92% and 68.51% of the total isolates carried the phzM and phzS genes, respectively, 98.11% and 88.67% of the ICU isolates in our study carried the phzM and phzS genes, respectively, which indicates a higher presence of these genes in our study. Another study in Iran showed that the frequency of phzS gene was 27.9% and 31.2% of the isolates had 8 virulence genes simultaneously.^[25] In our study, 90% of the isolates had all the genes studied simultaneously, and among the remaining 10 isolates, most of them carried at least 2 virulence genes simultaneously. In addition, the frequency of phzI (phzA1B1C1D1E1F1G1) operon in our study was 96%, which is consistent with the study of Haghi et al.^[25] These isolates may belong to the PA7 group (group 3) of P. aeruginosa that cannot be identified by PCR method due to their high genetic variability.^[26]

The pathogenesis of P. aeruginosa is due to the production of several cellular and extracellular virulence factors, whereas exotoxin A is a secreted extracellular enzyme encoded by the toxA gene that causes cell death, severe tissue damage, and necrosis in host cells.^[27] According to the literature, it has been found that 56.7% of P. aeruginosa isolates in bacteremia cases are able to produce exotoxins and isolates collected from acute infections express more virulence factors than chronic infections.^[9] In a study by Rodrigues et al.^[24] in Brazil, the toxA gene was detected in 49 (90.74%) isolates, which was almost consistent with our research. Meanwhile, 89.2%, 92.8%, 91.6%, 91.3%, 92.8%, 83.3%, 100%, 66.6%, 100%, 100%, and 100% of the isolates collected from adults, children, and neonate ICUs, and respiratory, blood, catheter, rectal swab, surgical wound, ocular secretion, gastric secretion, and urethral secretion samples carried the toxA gene, respectively.^[24] Also, similarly to our study, 95.7% of the isolates collected in a study conducted in southern Iran, carried the toxA gene.^[28] This data shows the approximate similarity of the strains in terms of production of this virulence factor in the North and South of Iran, although the presence of this gene in our study was not significantly related to the type of clinical specimens. Also, in another study conducted in northwestern Iran, as in our study, the toxA virulence gene was identified in 97.8% of P. aeruginosa isolates.^[25]

Zarei et al. evaluated P. aeruginosa isolates obtained from ICU clinical specimens, ICU environmental samples, and beetle exterior specimens, and found that the exoA gene was found in 57.5%, 72.5%, and 20% of them, respectively.‌^[29] Also, in a study published in Iraq in 2017, 100% of the P. aeruginosa isolates collected from ICU clinical specimens and hospital environmental samples carried the exoA gene.‌^[30] However, the 4% increase in the presence of the exoA gene compared to our study could be due to the fact that they also used hospital environmental samples, although their clinical samples confirmed the presence of 100% of this gene, too. Given that their clinical specimens were collected from the ICUs, these results indicate the higher prevalence of virulence genes in clinical isolates collected from immunocompromised patients. In other studies conducted in Pakistan, Bangladesh, and Poland, 33.33%, 55.56%, and 88.7% of P. aeruginosa clinical isolates, respectively, carried the gene encoding exotoxin A.^{[15, 27, 31]} Due to the fact that the expression of bacterial virulence factors are different in various conditions, this can be due to the differences in bacterial clones and the high diversity of clinical strains in these studies. Interestingly, in most of the studies on the frequency of P. aeruginosa virulence genes, we observed a high presence of these factors in samples such as feces, wounds, and blood, which may be related to the severe conditions in these samples.

One of the most important limitations in this study was the lack of financial resources and the small sample size for a prevalence study.

The high prevalence of genes encoding exotoxin A and pyocyanin in clinical isolates of Pseudomonas aeruginosa in Northern Iran indicates the significant role of these virulence factors in bacterial pathogenicity. Meanwhile, no significant difference has been observed in the prevalence of these genes in the isolates collected from different hospitals and different clinical samples. These results indicate that probably the source of the isolates is similar and they are spreading in the region. Therefore, maybe in the future, we can take an important step in controlling the severity of pathogenicity caused by this organism by designing compounds that can be used against these bacterial virulence factors.

We received the clinical samples without names from the laboratories of the hospitals affiliated to the Mazandaran University of Medical Sciences. This study was conducted in accordance with the Declaration of Helsinki; however, written informed consent form was provided by the patients or their close relatives, and any classifying information of each sample was kept secret. This study was approved by the Iran National Committee for Ethics in Biomedical Research with the national ethical code IR.MAZUMS.REC.1398.075.

We thank the laboratory staff of Zare, Razi, Bu-Ali Sina, Fatemeh Zahra, and Imam Khomeini Hospitals for providing patients’ information and the collection of the clinical isolates.

The participant has agreed to the submission of this article. Also, this manuscript has neither been published nor is currently under consideration and is approved by all coauthors.

The authors declare no conflict of interest.

This study is a report of a database from MSc student thesis registered and carried out in Sana Institute of Higher Education, Sari, Iran, but not funded by any organization.

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

Conceptualization: H.R.G.; data curation: H.R.G., Z.P., and M.O.; formal analysis: Z.P. and M.O.; investigation: H.R.G., Z.P., and M.O.; methodology: H.R.G. and Z.P.; project administration: H.R.G.; software: H.R.G and M.O.; supervision: H.R.G.; validation: H.R.G.; visualization: H.R.G., Z.P., and M.O.; writing – original draft: Z.P.; writing – review & editing: H.R.G., Z.P., and M.O.