Prevalence and Virulence of Commensal Pseudomonas Aeruginosa Isolates from Healthy Individuals in Southern Vietnam (2018–2020)

Understanding the colonization of Pseudomonas aeruginosa (P. aeruginosa) in healthy humans is useful for future prevention and treatment of P. aeruginosa infection. This study aimed to investigate the prevalence and risk factors of of P. aeruginosa colonization in healthy humans. At the same time, the virulence of the isolated P. aeruginosa was also studied. In the study, 609 Vietnamese volunteers (310 females and 299 males, age range of 2 to 73 years), who had no acute infection or disease symptoms participated at the time of sample collection. Samples were taken from the throat, nostrils, and outer ears. P. aeruginosa was found in 19 participants (3.12%, 95% CI: 0.017–0.045), mainly from the throat (11/19, 57.89%). Participants with a history of sinusitis were 11.57 times more likely to be colonized with P. aeruginosa than participants without a history of sinusitis (OR: 11.57, 95% CI: 4.08–32.76, p-value < 0.0001, Fisher’s Exact test). Age and sex were not significantly associated with P. aeruginosa colonization. Among 16 P. aeruginosa isolates used in virulence tests, 100% (16/16) were positive for the synthesis of biofilm, pyocyanin, and siderophores; 93.75% (15/16) isolates were positive for the synthesis of gelatinase and protease; and 50% (8/16) isolates were positive for lipase. There were no differences in the pattern and range of virulence factors of P. aeruginosa isolates taken from participants with and without sinusitis history. P. aeruginosa colonized 3.12% of participants, and its presence was associated with sinusitis history.


Introduction
P. aeruginosa is a Gram-negative bacterium popular in soil, water, and moist areas. It is also a part of the normal flora of humans, which is isolated on the skin (0-2%), in the throat (0-3.3%), and in the stool (2.6-24%) [1]. Under continuous pressure from the human immune system, commensal P. aeruginosa can transform into a virulent pathogen [2]. P. aeruginosa is classified as an opportunistic pathogen that can cause serious infections, particularly in immunocompromised patients, such as in cystic fibrosis, and patients with severe burns [3]. With numerous extracellular enzymes, this bacterium easily adapts to persist, replicate, and attack the host [4]. Extracellular enzymes such as protease, gelatinase, and lipase damage the proteins of host cells, interfere with the immune response, delay the wound-healing process, and are associated with motility, biofilm architecture, and rhamnolipid production [5][6][7][8].
Besides extracellular enzymes, this bacterium has many other virulence factors, such as biofilm, pyocyanin, siderophores, protease, gelatinase, and lipase [9,10]. Biofilm is a Biomedicines 2023, 11, 54 2 of 11 complex structure that contains many planktonic cells sticking together by extracellular polymeric substances [11]. This structure can form on virtually any moist surface, such as living tissues, medical devices, water pipes, and so on. Furthermore, biofilm contributes to the persistence of P. aeruginosa infection by protecting this pathogen against the host defenses and antimicrobial strategies [12]. Cells within the biofilm grow slowly, which makes antibiotics work ineffectively [13]. Pyocyanin is a blue phenazine pigment secreted by P. aeruginosa [14]. It damages the host by involving in oxidation-reduction reactions. The oxidants from pyocyanin cause cell respiration dysfunction, calcium homeostasis disruption, and so on [15]. Moreover, pyocyanin possesses antimicrobial properties, so P. aeruginosa can use it as a weapon to kill other microorganisms and become predominant at the site of the infection [16]. Siderophores are low molecular-weight peptides that have the ability to chelate and deliver iron to bacterial cells. Although iron is crucial for all living organisms, including bacteria, the insoluble iron (Fe 3+ ) is abundant in nature. This makes the uptake of iron by P. aeruginosa difficult [17]. Besides scavenging iron, they also stimulate biofilm formation [18]. In addition, siderophore-iron complexes can induce inflammation and oxidative damage, which increase the virulence of P. aeruginosa [19].
The colonization of commensal bacteria like P. aeruginosa on healthy humans deserves special attention because it can be a potential factor for subsequent disease [20]. Because it is opportunistic, it can cause an outbreak by being transmitted among healthy individuals before being detected [21]. So far, studies on P. aeruginosa colonization and its virulence, especially in the Vietnamese population, are limited. It is unclear whether sex, age, or any medical history affect the presence of P. aeruginosa in our body. Our study was the first study to analyze the prevalence of commensal P. aeruginosa in Southern Vietnam, its ability to produce common virulence factors, and the associated factors with P. aeruginosa colonization.

Commensal P. aeruginosa Isolation
From 2018 to 2020, throat, naris, and outer ear swab samples of Vietnamese people living in the Southeast area were collected and cultured on Pseudomonas selective Cetrimide media. This study was approved by the Ethics Committee of Vietnam National University of Ho Chi Minh City (Date 6 June 2019, No 1007/DHQG-KHCN).
These areas were chosen for sample collection because of their humidity and sampling convenience with less discomfort for the participants. All volunteers or volunteers' guardians gave their signed informed consent. Only people with no declared current acute infection or disease symptoms were included in the study. Colonies obtained on Cetrimide agar were further characterized using a Gram-staining, polymerase chain reaction (PCR) with oprL primers [22] as a screening step, and 16S rRNA sequencing for confirmation. For further experiments, all the P. aeruginosa isolates were stored in Luria Bertani broth containing 30% glycerol at −80 • C.

P. aeruginosa Identification oprL-Specific Polymerase Chain Reaction and 16S rRNA Sequencing
The specific oprL primers were used to primary detect P. aeruginosa (forward: 5 -ATGGAAATGCTGAAATTCGGC-3 and reverse: 5 -CTTCTTCAGCTCGACGCGACG-3 ) (PHUSA GENOMICS, Can Tho City, Vietnam) and followed the previous study [22]. All positive isolates for oprL were sent to NAM KHOA Biotek Company (Ho Chi Minh City, Vietnam) for 16S rRNA sequencing. The confirmed P. aeruginosa isolates were used for further experiments.

Virulence Testing
Biofilm, pyocyanin, siderophores, lipase, protease, and gelatinase of the isolated P. aeruginosa isolates were analyzed. P. aeruginosa ATCC 9027 (American Type Culture Collection, Manassas, VA, USA) was used as a positive control for all the producing virulence tests because it was a non-virulent strain isolated from the human outer ear by the American Type Culture Collection and it has ability to produce tested virulence factors in this study [23][24][25][26].

Pyocyanin
Pyocyanin was extracted and measured using the chloroform method [20]. P. aeruginosa isolate was cultured in glycerol-alanine (Gly-Ala) (HiMedia Laboratories, Kennett Square, PA, USA; Xilong Scientific Co., Ltd., Shantou, China) broth to maximize the yield of pyocyanin production in commensal isolates. When the OD 600 nm bacteria reached 0.08-0.1, 1% of the culture was inoculated in 5 mL of Gly-Ala broth at 37 • C for 24 h with shaking condition. After that, the supernatant was collected (centrifuge 6000 rpm for 15 m). A quantity of 3 mL of supernatant was mixed vigorously with 1.8 mL of chloroform (VN-CHEMSOL Co., Ltd., Ho Chi Minh City, Vietnam). Then, blue layer was collected and mixed with 0.2 N HCl (2:1). The top red color was collected and measured at 520 nm using BioTek Synergy HTX Multimode Reader (Agilent, Santa Clara, United States). The concentration of pyocyanin (µg/mL) was estimated by multiplying the OD at 520 nm by 17.072 (the molar extinction coefficient of pyocyanin 520 nm) [28,29].

Siderophores, Lipase, Protease, and Gelatinase
Before agar plate test, all tested isolates and controls were cultured in Luria-Bertani broth for 24 h at 37 • C. After the OD 600 nm reached the range 0.08-0.1, 5 µL of the cultures spotted on CAS blue agar [30], tributyrin agar (1% tributyrin), skim milk (3% skim milk, Brain Heart Infusion (BHI) medium), or gelatin agar (8% gelatin, BHI) for detecting siderophores, lipase, protease, and gelatinase, respectively. The enzymatic activity (EA) was estimated by measuring the halo zone size: EA = (D − d)/2, where D was the diameter of clear zone (mm) and d was the diameter of a colony (mm). It was categorized as negative (0 mm), weak (<2 mm), moderate (2-4 mm), and strong (>4 mm) activity [31]. Besides P. aeruginosa ATCC 9027, Staphylococcus aureus (S. aureus) was used a positive control for protease and lipase tests, while Vibrio cholerae (V. cholerae) was used for gelatinase. In case of siderophores, E. coli was also used as a positive control, while S. aureus was a negative control because it was Gram-positive and could not survive on CAS blue agar [30]. All commercial materials for these tests were purchased from Sigma-Aldrich, St. Louis, Missouri, United States; HiMedia Laboratories, Kennett Square, United States; Xilong Scientific Co., Ltd., Shantou, China.

Data Analysis
Each experiment was performed in triplicate. IBM ® SPSS ® Statistics 20.0 (IBM, Armonk, New York, NY, USA) was used to analyze the data. Chi-square, Fisher's Exact and Ordinal Regression tests were used to determine the risk factors for P. aeruginosa colonization. In addition, an ANOVA analysis was used to show the association between sinusitis history and commensal P. aeruginosa virulence. The p-value was set to be <0.05.

Prevalence of Commensal P. aeruginosa Isolates in Vietnamese Population
There were 612 volunteers who signed informed consent and provided samples. Three were excluded due to their declared health condition and 609 participants were finally included in the study. The number of male and female participants was similar, 299 males (49.10%) versus 310 (50.90%) females. Because the age of participants was not variety, it could only be grouped into three categories: young persons (0-17), adult , and older persons (≥60). It is well-noted that having sinusitis is a common health problem in the population. More than 1/5 of healthy declaring people experienced sinusitis. The summary of all participants and P. aeruginosa carrier characteristics was described in Table 1. From 609 participants, 35 P. aeruginosa-like isolates were obtained. These isolates grew on cetrimide selective media, being Gram-negative rod shaped and positive for oprL. The oprL gene encodes the outer membrane peptidoglycan-associated lipoprotein, which has an important role in the interaction between bacteria and environment, is a common and useful target for Pseudomonas detection [22,32,33].
A total of 20 out of 35 isolates positive for oprL (57.14%) were confirmed as P. aeruginosa via 16S rRNA sequencing (Supplementary Table S1). Among 20 P. aeruginosa isolates, two isolates came from one volunteer (from throat and nostrils) and 18 others came from a single site on each participant. In summary, 19 out of 609 participants (3.12%, 95% CI: 0.017-0.045) were colonized with P. aeruginosa (Supplementary Table S2). This was in agreement with previous data showing that P. aeruginosa was not commonly detected in healthy people, but under conditions of antibiotic exposure or hospitalization its prevalence, mainly in the throat and stool, was increased [34,35]. For example, in the case of bronchiectasis, which is a chronic respiratory disease associated with P. aeruginosa, the prevalence of P. aeruginosa colonization in bronchiectasis patients ranged from 9% to 33% [36]. Additionally, Casetta et al. also found that 17.5% of pregnant women were colonized by P. aeruginosa after more than 48 h hospital admission [37]. Importantly, colonization with P. aeruginosa represents a risk of infection: Gómez-Zorrilla et al. reported that 43% of colonized patients in their study developed infection [38].
From our findings, P. aeruginosa isolates were likely obtained from the throat (11/20 P. aeruginosa isolates), while P. stutzeri isolates were rather from the nostril area (9/12 P. stutzeri isolates). Furthermore, the throat was also primarily colonized by P. aeruginosa (11/13 throat-derived isolates from the throat, 84.62%). Only one P. azelaica, one P. nitroreducens isolate and no P. stutzeri isolate were found in this area (Supplementary  Table S3). It was consistent with previous studies in which P. aeruginosa is likely found in a mucoid and humid area and is the most common Pseudomonas causing infection in human [39,40].

Relationship between Sex and Age to P. aeruginosa Colonization
Among 19 commensal P. aeruginosa carriers, 63.16% (12/19) were female and 36.84% (7/19) were male. However, there were no associations between sex and P. aeruginosa colonization of healthy humans (OR: 1.68, 95% CI: 0.65-4.33, p-value = 0.29 (Fisher's Exact Test) and Chi-Square: p-value = 0.278). To our knowledge, there is still a lack of information about the effect of sex on P. aeruginosa colonization in healthy humans. In contrast to our finding, the study of P. aeruginosa colonization from wounds and burns swabs of patients showed the higher rate of P. aeruginosa among the male than female samples, 55.6% and 44.4%, respectively [41]. However, the presence of P. aeruginosa was associated with a higher morbidity rate in females with respiratory dysfunction, such as cystic fibrosis and bronchiectasis. The estrogen in females could regulate the conversion of P. aeruginosa from non-mucoid to mucoid forms, which are believed to have higher virulence [42]. The relationship between sex and P. aeruginosa colonization is described in Table 2. Although the group 18-59 years had the highest number of carriers (84.21%, 16/19 P. aeruginosa carriers), age was not a significant factor for P. aeruginosa colonization (0-17: 65 participants, 18-59: 514 participants, and ≥60: 30 participants) (p-value = 0.448, Ordinal Regression). It was because the 95% CI include number 1 in both adult (3.11%, OR: 2.02, 95% CI: 0.26-15.43, p-value < 0.05, Fisher's Exact test) and older person (6.67%, OR: 6.75, 95% CI: 0.59-77.55, p-value > 0.05, Fisher's Exact test) groups. Currently, the association between age and P. aeruginosa colonization in healthy humans is unclear, but age > 55 years is considered to be an independent predictor for P. aeruginosa colonization in bronchiectasis patients [43]. The incidence of infections caused by opportunistic pathogens seems to increase significantly in older ages [44] and colonization could be a risk factor for infection [45].

Relationship of Sinusitis History to P. aeruginosa Colonization
Our data indicated that sinusitis is quite common in the population with 129/609 (21.18%) participants having sinusitis history (Table 1). Interestingly, participants with sinusitis history are more likely to be colonized with P. aeruginosa (14/129, 10.85%) compared to the ones without sinusitis history (5/480, 1.04%). It is estimated that the participants with sinusitis history have a risk of P. aeruginosa colonization 11.57 times higher than that of participants without sinusitis history (OR: 11.57, 95% CI: 4.08-32.76, p-value < 0.0001, Fisher's Exact test, Table 2). Our data were somewhat in agreement with previous studies. For example, Niederman et al. indicated that the upper and lower respiratory tract was not colonized by Gram-negative bacteria under normal health conditions, but these sites could be harbored by these pathogens when illness developed [46]. In the case of cystic fibrosis patients, Shapiro et al. found that P. aeruginosa was detected in 38% of sinusitis-cystic fibrosis patients [47] while Kasper Aanaes et al., reported that the presence of P. aeruginosa in sinuses was associated with lung infection in cystic fibrosis patients [48]. However, data were also quite controversial as in some studies, P. aeruginosa was not a common pathogen in bacterial flora of chronic sinusitis patients, only present for 1% to 5% of cases [47][48][49].

Virulence Factors
Average All tested isolates showed the ability to produce biofilm (16/16). Among them, there were 25% (4/16) of weak, 56.25% (9/16) of moderate, and 18.75% (3/16) of strong biofilm producers. In addition, the biofilm production of commensal P. aeruginosa isolates was weaker than the P. aeruginosa ATCC 9027 (positive control). Some studies showed less dominance of strong biofilm producers in the clinical isolates [50] and isolates from cystic fibrosis patients [51]. Biofilm production did not associate with poor clinical outcomes [52]. Moreover, it was reported that P. aeruginosa often grew planktonically and did not form biofilm under laboratory conditions [53].
All tested commensal P. aeruginosa isolates had the ability to secrete phenazine pyocyanin and siderophores ( Figure 1, Table 3). High pyocyanin producers (>18 µg/mL) were positively correlated with septic shock [54]. In our study, the average pyocyanin concentration from commensal P. aeruginosa isolates was 0.61 µg/mL. For siderophore production, the ability of the isolates varied following the isolation sites depending on the availability of free iron. Clinical P. aeruginosa isolates from urinary tract infection produced more siderophores compared with burn skin [55]. In our study, the commensal isolates were unsurprisingly weak siderophore producers (Table 3).
While 93.75% of the tested commensal P. aeruginosa produced protease and gelatinase, their average enzymatic activity was 0.28 ± 0.09 (mm) and 0.29 ± 0.06 (mm), respectively. Only 50% of tested isolates were positive for lipase, for which the average halo size was 0.18 ± 0.04 (mm) (Figure 2, Table 3). Our data are comparable with previous studies on clinical isolates from chronic leg ulcers where 91.67% produced protease, 66.67% produced gelatinase, and 83.33% produced lipase [56], indicating the common presence of these extracellular enzymes in all P. aeruginosa isolates.
There were no significant differences in virulence between P. aeruginosa isolates from carriers with and without sinusitis history (p-value > 0.05, ANNOVA test) (Supplementary Table S7). Sharna's study showed that P. aeruginosa isolates from cystic fibrosis lung increased biofilm, alginate, and some virulence gene expression [57]. Furthermore, elastase was associated with chronic rhinosinusitis severity [58]. So far, there was no evidence on the increased virulence of commensal P. aeruginosa isolated from people with sinusitis history. However, people with a sinusitis history were more likely to be P. aeruginosa carriers than others, which is a potential risk of subsequent infection with P. aeruginosa [59], and P. aeruginosa infections were known to be particularly dangerous for patients with respiratory diseases [60,61]. cyanin and siderophores (Figure 1, Table 3). High pyocyanin producers (> 18 μg/mL) were positively correlated with septic shock [54]. In our study, the average pyocyanin concentration from commensal P. aeruginosa isolates was 0.61 μg/mL. For siderophore production, the ability of the isolates varied following the isolation sites depending on the availability of free iron. Clinical P. aeruginosa isolates from urinary tract infection produced more siderophores compared with burn skin [55]. In our study, the commensal isolates were unsurprisingly weak siderophore producers (Table 3).    0.29 ± 0.06 0.22 ± 0.03 While 93.75% of the tested commensal P. aeruginosa produced protease and gelatinase, their average enzymatic activity was 0.28 ± 0.09 (mm) and 0.29 ± 0.06 (mm), respectively. Only 50% of tested isolates were positive for lipase, for which the average halo size was 0.18 ± 0.04 (mm) (Figure 2, Table 3). Our data are comparable with previous studies on clinical isolates from chronic leg ulcers where 91.67% produced protease, 66.67% produced gelatinase, and 83.33% produced lipase [56], indicating the common presence of these extracellular enzymes in all P. aeruginosa isolates. There were no significant differences in virulence between P. aeruginosa isolates from carriers with and without sinusitis history (p-value > 0.05, ANNOVA test) (Supplementary Table S7). Sharna's study showed that P. aeruginosa isolates from cystic fibrosis lung increased biofilm, alginate, and some virulence gene expression [57]. Furthermore, elastase was associated with chronic rhinosinusitis severity [58]. So far, there was no evidence on the increased virulence of commensal P. aeruginosa isolated from people with sinusitis history. However, people with a sinusitis history were more likely to be P. aeruginosa carriers than others, which is a potential risk of subsequent infection with P. aeruginosa [59], and P. aeruginosa infections were known to be particularly dangerous for patients with respiratory diseases [60,61].

Conclusion
For the first time, we revealed that the prevalence of commensal P. aeruginosa in the Southern Vietnamese population was 3.12%. Sinusitis could be a potential factor contributing to the colonization of P. aeruginosa. The commensal P. aeruginosa isolates can produce multiple virulence factors, including biofilm, pyocyanin, siderophores, lipase, protease, gelatinase and lipase. Further studies on the transition of P. aeruginosa from commensal to pathogenic state and its persistence in the carriers could give us deeper understanding on the host-pathogen interaction, which would be useful for prevention and treatment of P. aeruginosa infections.
Supplementary Materials: The following supporting information can be downloaded at:

Conclusions
For the first time, we revealed that the prevalence of commensal P. aeruginosa in the Southern Vietnamese population was 3.12%. Sinusitis could be a potential factor contributing to the colonization of P. aeruginosa. The commensal P. aeruginosa isolates can produce multiple virulence factors, including biofilm, pyocyanin, siderophores, lipase, protease, gelatinase and lipase. Further studies on the transition of P. aeruginosa from commensal to pathogenic state and its persistence in the carriers could give us deeper understanding on the host-pathogen interaction, which would be useful for prevention and treatment of P. aeruginosa infections.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines11010054/s1, Table S1: Pseudomonas species were isolated from three body sites (1-throat, 2-naris, 3-outer ear). They were conforming by 16S rRNA sequencing. The bold fonts indicate P. aeruginosa isolates; Table S2: List of 19 P. aeruginosa carriers; Table S3: oprL-positive Pseudomonas isolates and their colonization sites: Table S4: 16 commensal P. aeruginosa isolates were used in virulence tests; Table S5: Biofilm formation, pyocyanin, and siderophores in commensal P. aeruginosa isolates after inoculation at 37 • C for 24 h. The values were expressed as mean ± standard deviation; Table S6: Lipase, protease, and gelatinase values in commensal P. aeruginosa isolates after incubation at 37 • C for 24 h; Table S7: The production of tested virulence factors in commensal P. aeruginosa isolates from participants with and without sinusitis history. Average ± standard deviation. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Author
Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files.