Abstract
Background
Pseudomonas aeruginosa is a major cause of healthcare-associated infections (HAIs), particularly in immunocompromised patients, leading to high morbidity and mortality rates. This study aimed to investigate the antimicrobial resistance patterns, virulence gene profiles, and genetic diversity among P. aeruginosa isolates from hospitalized patients in Mazandaran, Iran.
Methods
From September 2021 to April 2022, 82 non-duplicate P. aeruginosa isolates were collected from diverse clinical sources. Identification was confirmed using API 20 NE (bioMérieux, Marcy l’Etoile, France). Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method according to CLSI guidelines to assess resistance to a range of antibiotics. The virulence profile (exoT, exoY, exoU, toxA, plcH, plcN, algD, aprA, lasB and exoS) of each P. aeruginosa isolate was determined by PCR. The genetic diversity among the strains was evaluated using the random amplification of polymorphic DNA (RAPD) technique. Clustering was based on a Dice similarity coefficient of ≥ 85%.
Results
Of the 82 total strains, P. aeruginosa exhibited the highest and lowest resistance toward ticarcillin-clavulanate (98.78%) and colistin (0%), respectively. Moreover, 100% of the P. aeruginosa isolates were MDR. The following prevalence of virulence factor genes was observed: aprA, lasB, algD, toxA, plcH, exoY, and exoT in 100% of isolates. The plcN, exoS, and exoU were identified 98.78%, 67.07%, and 45.12%, respectively. The RAPD patterns obtained with primers 272 and 208 had respectively 2–19 and 6–17 bands. According to the Dice similarity coefficient of higher than 85%, 56 and 39 clusters were recognized.
Conclusion
The high rate of multidrug resistance combined with the widespread presence of virulence genes in P. aeruginosa isolates highlights the potential for increased infection severity, morbidity, and mortality in hospitalized patients. The substantial genetic diversity observed among isolates suggests that P. aeruginosa in this region may rapidly evolve, necessitating ongoing surveillance and more targeted antimicrobial strategies.
Clinical trial number
Not applicable.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12866-024-03707-5.
Keywords: P. aeruginosa, Antibiotic resistance, Virulence gene, RAPD, Multidrug-resistant
Background
Among various community-acquired and nosocomial infections, Pseudomonas aeruginosa (P. aeruginosa) is a widespread gram-negative bacteria [1]. It is the main reason for nosocomial infections among non-fermentative Gram-negative bacteria (NFGNB) [2–5]. A major health problem in developing countries is the rising incidence of multidrug-resistant (MDR) P. aeruginosa [5]. The severity and duration of hospitalization associated with MDR P. aeruginosa infections are greater and so are costs, morbidity, and mortality [6]. During the past decades, the progressively frequent isolation of MDR strains has additionally intensified the issue of antimicrobial resistance (AMR) globally [7]. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins that increase the necessity of the proper use of antibiotics. Besides, the routine application of antimicrobial susceptibility testing to detect the antibiotic of choice as well as, the screening of the emerging MDR strains, is essential [8–10]. Prior investigations demonstrate that the MDR phenotype in P. aeruginosa clinical isolates was prevalent in Iran [4].
The pathogenicity of P. aeruginosa isolates depends on a variety of virulence traits (i.e., toxins, enzymes, and biofilm formation) that help the organism cause extensive tissue damage after colonization [11, 12]. Extracellular factors include phospholipases (PlcH and PlcN), hydrogen cyanide, alkaline protease, elastase (LasA), and zinc metalloprotease (LasB). Adhesion factor (pili type IV), lipopolysaccharides, exo-proteases (alkaline protease “AprA”, elastase, staphylolysin, protease IV), flagella, exotoxins “Exo A”, exoenzymes S, T, Y, and U, a type III secretion system and alginate, as well as sialidase [13–16]. P. aeruginosa is capable of producing exotoxin A through the toxA gene. This is one of the main elements of the type II secretion pathway (T2SS) that prevents protein synthesis via transferring adenosine diphosphate ribosyl moiety from nicotinamide adenine dinucleotide to elongation factor 2 [17–19]. In humans, this enzyme alone or in combination with other hydrolases causes necrosis, tissue disruption, and cell death [20, 21]. In the pathogenesis of serious infections caused by P. aeruginosa type III secretion system (T3SS) plays an important role. ExoS, ExoT, ExoY, and ExoU, are four type III-secreted effectors discovered in P. aeruginosa [20, 22]. It has been established that during infection, ExoS plays a vital role in colonization, invasion, and microbial dissemination [23, 24]. ExoU is a cytotoxin with phospholipase activity acting on epithelial cells and causing lung infections [25, 26]. The plcH and plcN genes encode two secreted phospholipases C that can hydrolyze phospholipids. It has been found that MDR strains of P. aeruginosa possess a high rate of the phospholipase C gene (plcH), which plays an important role in its pathogenicity [21]. The LasB elastase degrades the extracellular matrix component during the acute phase of P. aeruginosa infection [27]. A number of P. aeruginosa strains produce alginate, which forms a matrix for the biofilm and defends bacteria from the immune response. A protein called algD participates in the construction of alginate with two other proteins [27, 28]. Research shows P. aeruginosa strains do not necessarily share virulence factor genes [29, 30]. Molecular analysis of local P. aeruginosa isolates helps establish surveillance strategies, reduces outbreak risk, and increases knowledge on the role of resistance and virulence in infection. The number of comprehensive epidemiological surveys at the molecular level remains low, particularly in our region. So, this study aimed to investigate the antimicrobial resistance patterns, virulence gene profiles, and genetic diversity among P. aeruginosa isolates from hospitalized patients in Mazandaran province, Iran.
Methods
Bacterial isolates
In the present cross-sectional study, the isolates of P. aeruginosa were obtained from hospitalized patients in different educational hospitals located in Mazandaran, Iran, between September 2021 and April 2022. These strains were isolated from blood, bronchoalveolar lavage (BAL), sputum, wound, ear and eye exudates, urine, trachea, and catheter.
Bacterial identification test
All tested isolates were cultured on MacConkey agar and sheep blood agar plates, followed by incubation at 37 °C for 18–24 h. Presumptive colonies of P. aeruginosa were screened via standard biochemical tests such as oxidase, catalase, motility, oxidation-fermentation (OF), Kligler Iron Agar (KIA) tests, and growth on Mueller–Hinton agar medium at 42 °C [31, 32]. Confirmation of the P. aeruginosa isolates to species level was done using the analytical profile index (API) 20NE kit (bioMérieux, Marcy l’Etoile, France). The samples were kept in trypticase soy broth with 20% glycerol at − 80 °C for future use. All microbial culture media were purchased from Merck, Germany.
Antimicrobial susceptibility tests
The Standard Kirby-Bauer disk diffusion method determined the antimicrobial susceptibility of strains in accordance with Clinical and Laboratory Standards Institute (CLSI, 2021) breakpoints. Antibiotic disks used were piperacillin from penicillins class (100 µg), ticarcillin-clavulanate from β-lactam combination class (75/10µg), piperacillin-tazobactam from β-lactam combination class (100/10µg), ceftazidime from cephems class (30 µg), cefepime from cephems class (30 µg), aztreonam from monobactams class (30 µg), doripenem from carbapenems class (10 µg), imipenem from carbapenems class (10 µg), meropenem from carbapenems class (10 µg), tobramycin from aminoglycosides class (10 µg), gentamicin from aminoglycosides class (10 µg), amikacin from aminoglycosides class (30 µg), and ciprofloxacin from quinolones class (5 µg) (MAST, England). Susceptibility toward colistin was done by broth microdilution method according to the CLSI. Resistance to colistin was considered if the minimum inhibitory concentration (MIC) is ≥ 4 µg/mL. Isolates shown to be non-susceptibility to at least one agent in three or more antimicrobial groups were called MDR phenotype [33]. P. aeruginosa ATCC 27,853 and E. coli ATCC 25,922 were reference strains (RS).
Extraction of DNA
The boiling method was used to extract genomic DNA [34]. In order to measure DNA concentration (ng/μl) and purity (260/280), a QIAxpert spectrophotometer (Qiagen, USA) was used. DNA was saved at − 20 °C.
Molecular analysis of virulence-associated genes
Conventional PCR was used to detect the following virulence genes: GDP-mannose dehydrogenase enzyme for alginate (algD), elastase (lasB), alkaline protease (aprA), non-hemolytic phospholipase C (plcN), exoenzyme S, T, Y and U (exoT, exoS, exoY and exoU) and exotoxin A (toxA), and hemolytic phospholipase C (plcH). The amplification was performed in a 15µL volume, including 7.5 µL of PCR Master Mix (#Cat. No: A190301 AMPLIQON, Denmark), 0.5µL of the reverse and forward primers (5 pmol), 1µL of template DNA (300 ng), and nuclease-free water to reach the final volume. The primer sequences are presented in Additional File 1: Table S1 [35–37]. PCR products were observed after agarose gel (1%) electrophoresis by staining with a safe stain #Cat. No.: EP5082 (SINACLON, Iran).
Molecular typing based on RAPD-PCR
To characterize the genetic relatedness among the isolates, RAPD experiment was performed using arbitrary sequence 272 (5’-AGCGGGCCAA-3’), as described previously [38, 39]. Amplifications were conducted in a 15µL volume, containing 7.5 µL of PCR MasterMix (#Cat. No: A190301, Ampliqon, Denmark), 1.5 µL (15 pmol) of primer (BIONEER, Korea), 4 µL of nuclease-free water, and 2 µL (100 ng) of template DNA. The amplification conditions were as follows: 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 1 min at 45 °C, and 2 min at 72 °C. A final extension step was done at 72 °C for 10 min.
Assessments and dendrogram construction
Electrophoresis of the products was performed in a 1.5% agarose gel (Merck, Germany). The RAPD patterns were analyzed using the unweighted pair-group approach by arithmetic averages (UPGMA) clustering with Gel Compare II 5.10 (Applied Maths, Sint-Martens-latem, Belgium). Isolates characterized by 96% or more similarity were regarded as identical, and clustering was done using a cut-off value of 85% similarity. P. aeruginosa ATCC 27,853 was used as positive control in this test.
Statistical analysis
Chi-squared tests were performed using Statistical Package for the Social Sciences (SPSS) software, version 21.0 (SPSS Inc., Chicago, IL, USA). Descriptive statistical analysis was employed to characterize patient age, antibiotic resistance, and virulence gene profiling of the tested strains.
Results
This study aimed to investigate the antimicrobial resistance patterns, prevalence of virulence genes, and genetic diversity among P. aeruginosa isolates collected from hospitalized patients. Our hypothesis was that P. aeruginosa strains would exhibit significant multidrug resistance and possess a high diversity of virulence determinants. To achieve this, we collected 82 non-duplicate P. aeruginosa isolates from various clinical sources and assessed their antimicrobial susceptibility using the Kirby-Bauer disk diffusion method. We also performed PCR to determine the presence of specific virulence genes and employed RAPD PCR to evaluate the genetic diversity among the strains. The following sections present the detailed findings of our analyses, highlighting the resistance patterns, frequency of virulence genes, and the extent of genetic variability observed in the isolates.
Characteristics of the recovered isolates
A total of 82 P. aeruginosa clinical isolates were enrolled in our work. From these positive cases, the isolation rate of P. aeruginosa was the highest in sputum samples 20 (24.39%), followed by 19 (23.17%) in wound swabs, 19 (23.17%) in urine, and the lowest percentage was obtained from eye discharge and bronchoalveolar lavage (BAL). Analyses of isolates by the source of the specimen are shown in Table 1. The frequency of samples in hospital wards was as follows: intensive care units (ICUs) 47 (57.31%), burn 9 (10.97%), internal 8 (9.75%), surgery 7 (8.53%), emergency 6 (7.31%) and pediatric 5 (6.09%). Also, 48 patients (58.53%) were male and 34 patients (41.46%) were female. The prevalence of P. aeruginosa in our studied therapeutic centers was as follows: 47.56% in Imam Khomeini, 25.60% in Bu-Ali Sina, 14.63% in Zare, and 12.19% from Razi hospitals affiliated with the Mazandaran University of Medical Sciences.
Table 1.
Distribution of 82 MDR P. aeruginosa isolates according to sample source
| Type of specimen | Number of isolates | P. aeruginosa isolates, (%) |
|---|---|---|
| Sputum | 20 | 24.39% |
| Wound | 19 | 23.17% |
| Urine | 19 | 23.17% |
| Blood | 6 | 7.31% |
| Catheter | 6 | 7.31% |
| Ear | 5 | 6.09% |
| Trachea | 3 | 3.65% |
| Eye | 2 | 2.43% |
| BAL | 2 | 2.43% |
| Total | 82 | *100% |
*Percentages are rounded to two decimal places. Due to this rounding, the total may not sum to exactly 100%
Antimicrobial resistance profile
Antimicrobial resistance profile to 14 different antimicrobials showed that the highest resistance rate was related to ticarcillin-clavulanate (TIM = 98.78%), and all samples were vulnerable against colistin. Figure 1 shows the prevalence of resistance against other antibiotics. There were various antibiotic resistance patterns in samples: the highest resistance against aztreonam, imipenem, and amikacin was detected in wound and sputum specimens, and the highest resistance against ceftazidime was found in urine specimens. Table 2 shows the frequency of antibiotic resistance in samples.
Fig. 1.
The prevalence (%) of resistance to antibiotics in MDR P. aeruginosa isolates. *CS, colistin; TZP, piperacillin-tazobactam; AMK, amikacin; CPM, cefepime; IPM, imipenem; DOR, doripenem; PRL, piperacillin; TM, tobramycin; GEN, gentamicin; ATM, aztreonam; CIP, ciprofloxacin; CAZ, ceftazidime; MRP, meropenem; TIM, ticarcillin-clavulanate
Table 2.
Prevalence of antibiotic resistance in 82 MDR P. aeruginosa isolated from different clinical samples
| Antibiotics | PRL (%) | TZP (%) | TIM (%) | CAZ (%) | CPM (%) | ATM (%) | DOR (%) | IPM (%) | MRP (%) | GEN (%) | TM (%) | AMK (%) | CIP (%) | CS (%) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Samples | ||||||||||||||
|
Sputum (N = 20) |
13/20 (65) |
4/20 (20) |
20/20 (100) |
16/20 (80) |
9/20 (45) |
13/20 (65) |
11/20 (55) |
11/20 (55) |
18/20 (90) |
13/20 (65) |
12/20 (60) |
11/20 (55) |
15/20 (75) |
- |
|
Urine (N = 19) |
10/19 (52.63) |
5/19 (26.31) |
18/19 (94.73) |
18/19 (94.73) |
8/19 (42.10) |
11/19 (57.89) |
7/19 (36.84) |
9/19 (47.36) |
14/19 (73.68) |
11/19 (57.89) |
11/19 (57.89) |
7/19 (36.84) |
12/19 (63.15) |
- |
|
Wound (N = 19) |
10/19 (52.63) |
5/19 (26.31) |
19/19 (100) |
13/19 (68.42) |
12/19 (63.15) |
13/19 (68.42) |
13/19 (68.42) |
11/19 (57.89) |
17/19 (89.47) |
14/19 (73.68) |
14/19 (73.68) |
11/19 (57.89) |
16/19 (84.21) |
- |
|
Catheter (N = 6) |
2/6 (33.33) |
2/6 (33.33) |
6/6 (100) |
4/6 (66.66) |
5/6 (83.33) |
4/6 (66.66) |
4/6 (66.66) |
3/6 (50) |
4/6 (66.66) |
6/6 (100) |
6/6 (100) |
3/6 (50) |
4/6 (66.66) |
- |
|
Blood (N = 6) |
3/6 (50) |
1/6 (16.66) |
6/6 (100) |
3/6 (50) |
- |
4/6 (66.66) |
3/6 (50) |
4/6 (66.66) |
6/6 (100) |
3/6 (50) |
3/6 (50) |
1/6 (16.66) |
4/6 (66.66) |
- |
|
Ear (N = 5) |
- | - |
5/5 (100) |
5/5 (100) |
- | - | - | - |
5/5 (100) |
- | - | - | - | - |
|
Trachea (N = 3) |
1/3 (33.33) |
1/3 (33.33) |
3/3 (100) |
3/3 (100) |
1/3 (33.33) |
1/3 (33.33) |
- | - |
3/3 (100) |
- | - | - | - | - |
|
Eye (N = 2) |
1/2 (50) |
- |
2/2 (100) |
2/2 (100) |
1/2 (50) |
1/2 (50) |
2/2 (100) |
2/2 (100) |
2/2 (100) |
1/2 (50) |
1/2 (50) |
- |
1/2 (50) |
- |
|
BAL (N = 2) |
2/2 (100) |
1/2 (50) |
2/2 (100) |
2/2 (100) |
2/2 (100) |
2/2 (100) |
1/2 (50) |
- |
2/2 (100) |
1/2 (50) |
1/2 (50) |
1/2 (50) |
- | - |
*Abbreviations PRL, piperacillin; TZP, piperacillin-tazobactam; TIM, ticarcillin-clavulanate; CAZ, ceftazidime; CPM, cefepime; ATM, aztreonam; DOR, doripenem; IPM, imipenem; MRP, meropenem; GEN, gentamicin; TM, tobramycin; AMK, amikacin; CIP, ciprofloxacin; CS, colistin
Virulence-profiling
We assessed the frequency of virulence determinants in MDR P. aeruginosa nosocomial samples, and the frequency of exoT, exoY, plcH, toxA, algD, aprA, and lasB genes was 100%, also plcN, exoS, and exoU were 98.78%, 67.07% and 45.12%, respectively (Fig. 2). The studied genes were divided into three groups of 8, 9, and 10 genes and showed the five gene profiles shown in Table 3. 12 isolates had all virulence genes altogether, that 7 isolates (58.33%) related to ICU.
Fig. 2.
Frequency of virulence genes in MDR P. aeruginosa isolates. *exoT; exoenzyme T, exoY; exoenzyme Y, plcH; hemolytic phospholipase C, toxA; exotoxin A, algD; GDP-mannose dehydrogenase enzyme for alginate, aprA; alkaline protease, lasB; elastase, plcN; non-hemolytic phospholipase C, exoS; exoenzyme S, exoU; exoenzyme U
Table 3.
Co-existence of virulence genes among studied strains
| No. of Virulence Genes | Virulence Genes Profile | No. (%) of Bacterial Strain | Total No. (%) |
|---|---|---|---|
| 8 |
All genes except exoS and exoU :A All genes except exoU and plcN :B |
2.43%))2 1(1.21%) |
3(3.64%) |
| 9 |
All genes except exoU :C All genes except exoS :D |
42(51.21%) 25(30.48%) |
67(81.69%) |
| 10 | All Genes: E | 12(14.63%) | 12(14.63%) |
*Abbreviations exoS; exoenzyme S. exoU; exoenzyme U, plcN; non-hemolytic phospholipase C
Molecular typing
RAPD-PCR is a quick technique that characterizes the heterogeneity in the group using a cut-off value of 85% as the reference, and its results revealed 2 to 19 bands from 100 to 3000 bp using primer 272. RAPD 272 dendrograms of P. aeruginosa isolates indicated 54 various clusters (Fig. 3). Most isolates showed a unique fingerprint, but 18 cases showed 100% similarity with at least one isolate. RAPD-PCR assay performed genetic relatedness of P. aeruginosa isolates, which indicated 39–100% similarity. 36 isolates were either singleton (unrelated to any RAPD-type), which made up most of the isolates and indicates a dispersion in the genetics of the isolates.
Fig. 3.
The dendrogram shows the genetic relatedness of 82 MDR P. aeruginosa isolates using RAPD-PCR analysis. Clustering was performed using the unweighted pair group method with arithmetic averages (UPGMA). Gender: F) Female, M) Male; Hospitals: (1) Imam Khomeini, (2) Zare, (3) Razi, (4) Bu-Ali Sina. The horizontal axis represents the genetic similarity between isolates, with closer branches indicating higher similarity
Discussion
P. aeruginosa is one of the most prevalent Gram-negative bacteria that cause illnesses related to HAIs [40]. Infections and drug resistance are more prevalent among immunocompromised patients, such as those admitted to ICUs [23]. Globally, the prevalence of multidrug-resistant (MDR) P. aeruginosa strains is rising, which is restricting our options for treatment [41]. As the antibiotic-resistant pattern shows, all of the isolates were determined to be MDR (100%). This discrepancy between the frequency of MDR strains in this study and those in other studies may be due to either the source of strains or the infection control practices. Vaez et al. in their systematic review and meta-analysis revealed that the prevalence of MDR strains in different provinces of Iran is 58% on average [42], and varied in various provinces of Iran and the highest and lowest frequencies were found in Tehran (100%) [43, 44] and Zahedan (16%) [45]. The results of a comprehensive assessment of antimicrobial resistance in Europe indicated that the percentage of MDR P. aeruginosa isolates varied from 0% in Estonia and Iceland to 49.4% in Romania [46]. The highest and lowest resistance rates were related to TIM (98.78%) and CS (0%), respectively, in our research. This result is similar to studies in the southwest of Iran (Shiraz) and Germany, reporting all isolates susceptible to colistin [47, 48]. One of the few medications previously used to treat serious infections caused by Pseudomonas bacteremia was colistin, which exhibits good in vitro activity against a wide variety of gram-negative bacterium species. However, due to its considerable nephrotoxicity and neurotoxicity as well as its limited therapeutic efficacy, the use of colistin has been restricted. Nonetheless, there has been a rise in interest in using colistin to treat infections brought on by MDR organisms, as new anti-pseudomonal medications are unlikely to become accessible anytime soon [49]. In comparison with our data, Maleki et al. reported that doripenem, ertapenem, and meropenem antibiotics had the highest resistance (100%) [50]. Another study also showed disparity with our data, Azimi et al. where 76.12%, 75.64%, 74.63%,72.97%, 70.81%, and 2% of their isolates were resistant to GEN, TM, CAZ, PRL, AMK, and CS, respectively [3]. A combination of factors may explain these inconsistencies, such as the location of the study, hygiene level, type of samples, research date, the number of samples, and restriction on antibiotics consumption. In this study, all isolates showed a minimum of eight virulence genes with a maximum of 10 virulence genes detected in 13 isolates. The prevalence of seven virulence genes, i.e., exoT (encoding exoenzyme T), exoY (encoding exoenzyme Y), lasB (encoding elastase LasB), plcH (encoding hemolytic phospholipase C precursor), toxA (encoding exotoxin A), algD (encoding GDP-mannose 6-dehydrogenase) and aprA(encoding alkaline metalloproteinase) (100%), in P. aeruginosa was higher compared to other virulence-associated genes in this area. The exoS, exoT, exoU, and exoY are four known effector proteins produced by P. aeruginosa through the type III secretion system [51]. The exoT and exoY genes are carried by the majority of P. aeruginosa strains. However, there is a noticeable dissimilarity between the isolates in terms of exoS and exoU genes, and they appear to be mutually exclusive [52]. This possibly indicates that the genes that encode the cytotoxins exoS and exoU, are variable traits in P. aeruginosa and their existence is associated with the disease background or site [11]. Inconsistent with other reports indicating a low prevalence of exoT and exoY [3, 50], we observed a high prevalence of exoT and exoS (100%). In a similar study done by Bazghandi et al. (exoU: 56%; exoS: 51.2%) [53], and Bahador et al. (exoU:38.6%; exoS:55.7%) [54], the rate of exoU and exoS in our study was lower than other virulence genes (exoU:45.12%; exoS: 67.07%). The aprA is necessary for bacterial survival in tissues through a decrease in activating Toll-like receptor-5 (TLR-5) and degrading host proteins as complements and cytokines [55]. The aprA level was consistent with other reports and had a high prevalence [56, 57]. The GDP-mannose dehydrogenase enzyme encoded by algD was present among all in our samples, which is in line with the results by Izadi Pour Jahromi et al. and Mokhtari et al. indicating a very high frequency of algD in respiratory samples [58, 59].
In all of our isolates, we detected exotoxin A (one of the most toxic virulence factors). There is also an agreement between this study and that conducted by Khattab et al. and/or Nikbin et al., who have established that nearly all pulmonary isolates contained toxA [60, 61]. LasB is prevalent among our strains, similar to previous findings [58, 62].
It’s possible these two phospholipases (plcH and plcN) work together to degrade the phospholipids contained in pulmonary surfactants [56]. In this study plcN and plcH were detected, 98.78% and 100% respectively. Multiple studies on P. aeruginosa from hospitals have found a high incidence of both genes [53, 58]. But other studies have reported a lower prevalence of these genes [56, 57]. To identify common P. aeruginosa clones, various typing techniques, such as serotyping, biotyping, and pyocin typing, have been proposed. However, the limitations of each phenotyping technique can be overcome thanks to the genotyping of P. aeruginosa strains [63]. The epidemiology of P. aeruginosa has been investigated using a variety of gene-typing techniques, such as the enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), random amplification of polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) [38]. Due to their technical complexity, high expense, and lengthy turnaround times for findings, PFGE and MLST procedures are not widely used despite their great repeatability and good discriminatory power. However, RAPD and ERIC-PCR offer good levels of repeatability, are simple, inexpensive, and have quick turnaround times for results. Although it has been claimed that ERIC-PCR and RAPD analyses are equally successful at characterizing clinical isolates of P. aeruginosa, some researches have found that RAPD is more discriminatory than ERIC-PCR [38, 58]. RAPD-PCR has been shown to be valuable for conducting epidemiological surveys concerning the dissemination of P. aeruginosa strains within a particular setting. RAPD-PCR is a rapid molecular technique that exhibits high specificity and sensitivity in identifying and subtyping strains [64]. So far, RAPD-PCR has been carried out with a variety of primers, but 208 and 272 have been used most frequently. Among different primers used for RAPD-PCR, primer 272 was reliable because it can generate more reproducible profiles and genotypic patterns and has greater discriminatory ability [65].
All 82 samples were typed using RAPD-PCR. According to a Dice similarity coefficient of more than 85%, 54 groups were found in RAPD 272. In this study, the largest clusters consisted of 6 strains, one of which had a gene pattern C and the other had a gene pattern D. Also, 38 clusters were identified individually.
Different genotype distribution patterns have been reported in Iran. Nanvazadeh et al. found nine groups among 50 clinical P. aeruginosa isolates [64]. Vaez et al. found 39 clusters among 54 clinical isolates of P. aeruginosa [66]. Also, Hematzadeh et al. observed respectively 45 and 38 groups in RAPD 272 and RAPD 208 [38]. Genetic diversity was high among the isolates in our study. Possible reasons are dissimilar origins of P. aeruginosa that colonize the hosts. It is also possible for this genetic diversity to be linked to the quality of general hygiene as well. In this study 18 of the samples showed 100% similarity (RAPD 272). This percentage proximity between them displays that healthcare settings environments with high antibiotic pressure have usual sources of infections, despite the differences in expresses of virulence factors and isolation niches. During the hospital stay, P. aeruginosa clonal strains can transfer from patient to patient.
Limitations
The limitations of the current study include the lack of PCR-based identification of genes involved in antimicrobial resistance. Additionally, the correlation between phenotypic and genotypic multidrug resistance was not fully explored. The lack of investigation of biofilm production in the recovered isolates was another limitation of the study, as biofilm production is a key contributor to antibiotic resistance in P. aeruginosa infections. Finally, the lack of determine the quorum sensing genes among the strains was another limitation. These factors are crucial, and assessing them will lead to better and more accurate outcomes, providing a more comprehensive understanding of the topic.
Conclusion
This study contributes significantly to the understanding of P. aeruginosa resistance profiles, virulence factors, and genetic diversity in isolates from hospitalized patients in our region. Our findings reveal that the clinical isolates of P. aeruginosa exhibit high levels of resistance to common antimicrobial agents, underscoring the severity of the resistance issue. Furthermore, we observed a notable increase in the frequency of virulence determinants, which highlights a serious concern for infection control. The similarity in RAPD-PCR fingerprinting patterns among different isolates suggests the presence of transmissible and/or virulent strains within the studied hospitals. These results address the primary objective of our study by elucidating the resistance and virulence characteristics of P. aeruginosa and their potential implications for public health.
Electronic supplementary material
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Acknowledgements
Thanks to Department Microbiology and Virology of Mazandaran University of Medical Sciences and Molecular and Cell Biology Research Centre, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran for their contribution to this research.
Abbreviations
- RAPD
Random Amplification of Polymorphic DNA
- HAIs
Healthcare-Associated Infections
- MDR
Multidrug-Resistant
- AMR
Antimicrobial Resistance
- NFGNB
Non-Fermentative Gram Negative Bacteria
- PCR
Polymerase Chain Reaction
- CLSI
Clinical Laboratory Standards Institute
- RS
Reference Strains (RS)
- MIC
Minimum Inhibitory Concentration
- TSB
Trypticase Soy Broth
- API
Analytical Profile Index
- ICU
Intensive Care Unit
- TLR-5
Toll-Like Receptor-5
Author contributions
MG: Design of the study and supervision. GE: collected the data, cultured the samples and performed molecular experiments. HG and MS: Advisor in the study and contributed to the analysis of the data in collaboration with VSN. MG: drafting of the manuscript in collaboration with GE. All authors read and approved the final manuscript.
Funding
The authors declare that they did not receive any fund to conduct this study.
Data availability
All data generated or analyzed during this study are included in this article.
Declarations
Ethics approval and consent to participate
This study was performed in agreement with the Declaration of Helsinki. This study was also approved by the Iran National Committee for Ethics in Biomedical Research with the national ethical code (ref number: IR.MAZUMS.REC.1399.901) and informed consent was obtained from all participants and/or their guardian.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
The original online version of this article was revised: Following publication of the original article [1], the authors identified that the bibliographic details of references 38 and 67 are the same. Therefore, the authors would like to remove reference 38 from the reference list. The references 38 and 67 are shown in erratum 10.1186/s12866-025-03787-x.
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Change history
1/30/2025
The original online version of this article was revised: Following publication of the original article [1], the authors identified that the bibliographic details of references 38 and 67 are the same. Therefore, the authors would like to remove reference 38 from the reference list. The references 38 and 67 are shown in erratum 10.1186/s12866-025-03787-x.
Change history
2/1/2025
A Correction to this paper has been published: 10.1186/s12866-025-03787-x
References
- 1.Qin S, Xiao W, Zhou C, Pu Q, Deng X, Lan L, Liang H, Song X, Wu M. Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduct Target Ther. 2022;7(1):199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Kollef MH. Antibiotics for the critically ill: more than just selecting appropriate initial therapy. Crit Care. 2013;17(3):1–3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Azimi S, Kafil HS, Baghi HB, Shokrian S, Najaf K, Asgharzadeh M, Yousefi M, Shahrivar F, Aghazadeh M. Presence of exoY, exoS, exoU and exoT genes, antibiotic resistance and biofilm production among Pseudomonas aeruginosa isolates in Northwest Iran. GMS Hyg Infect Control. 2016;11. [DOI] [PMC free article] [PubMed]
- 4.Fazeli N, Momtaz H. Virulence gene profiles of multidrug-resistant Pseudomonas aeruginosa isolated from Iranian hospital infections. Iran Red Crescent Med J. 2014;16(10). [DOI] [PMC free article] [PubMed]
- 5.Strateva T, Yordanov D. Pseudomonas aeruginosa–a phenomenon of bacterial resistance. J Med Microbiol. 2009;58(9):1133–48. [DOI] [PubMed] [Google Scholar]
- 6.Gales AC, Castanheira M, Jones RN, Sader HS. Antimicrobial resistance among gram-negative bacilli isolated from Latin America: results from SENTRY Antimicrobial Surveillance Program (Latin America, 2008–2010). Diagn Microbiol Infect Dis. 2012;73(4):354–60. [DOI] [PubMed] [Google Scholar]
- 7.Alemayehu T. Prevalence of multidrug-resistant bacteria in Ethiopia: a systematic review and meta-analysis. J Global Antimicrob Resist. 2021;26:133–9. [DOI] [PubMed] [Google Scholar]
- 8.Al-Kadmy IM, Abid SA, Aziz SN, Al-Kadmy Z, Suhail A, Al-Jubori SS, Naji EN, Alhomaidi E, Yahia R, Algammal AM, Batiha GE. The secrets of environmental Pseudomonas aeruginosa in slaughterhouses: Antibiogram profile, virulence, and antibiotic resistance genes. Folia Microbiol 2023 Dec 13:1–8. [DOI] [PubMed]
- 9.Algammal AM, Eidaroos NH, Alfifi KJ, Alatawy M, Al-Harbi AI, Alanazi YF, Ghobashy MO, Khafagy AR, Esawy AM, El-Sadda SS, Hetta HF. Opr l gene sequencing, resistance patterns, virulence genes, quorum sensing and antibiotic resistance genes of xdr Pseudomonas aeruginosa isolated from broiler chickens. Infect Drug Resist. 2023 Dec;31:853–67. [DOI] [PMC free article] [PubMed]
- 10.Elbehiry A, Marzouk E, Aldubaib M, Moussa I, Abalkhail A, Ibrahem M, Hamada M, Sindi W, Alzaben F, Almuzaini AM, Algammal AM. Pseudomonas species prevalence, protein analysis, and antibiotic resistance: an evolving public health challenge. Amb Express. 2022;12(1):53. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Choy MH, Stapleton F, Willcox MD, Zhu H. Comparison of virulence factors in Pseudomonas aeruginosa strains isolated from contact lens-and non-contact lens-related keratitis. J Med Microbiol. 2008;57(12):1539–46. [DOI] [PubMed] [Google Scholar]
- 12.Firouzi-Dalvand L, Pooladi M. Identification of exoS, exoU genes in Pseudomonas aeruginosa. Archives Adv Biosci. 2014;5(4).
- 13.Goldufsky J, Wood S, Hajihossainlou B, Rehman T, Majdobeh O, Kaufman HL, et al. Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines. J Med Microbiol. 2015;64(Pt 2):164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Benie CKD, Dadié A, Guessennd N, N’gbesso-Kouadio NA, Kouame ND, N’golo DC, et al. Characterization of virulence potential of Pseudomonas aeruginosa isolated from bovine meat, fresh fish, and smoked fish. Eur J Microbiol Immunol. 2017;7(1):55–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Shaver CM, Hauser AR. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect Immun. 2004;72(12):6969–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Bricha S, Ounine K, Oulkheir S, Haloui N, Attarassi B. Facteurs de virulence et epidemiologie lies Au pseudomonas aeruginosa virulence factors and epidemiology related to pseudomonas aeruginosa. Revue Tunisienne d’Infectiologie-Oct. 2009;2:7–14. [Google Scholar]
- 17.Morgan RN, Saleh SE, Aboshanab KM, Farrag HA. ADP-ribosyl transferase activity and gamma radiation cytotoxicity of Pseudomonas aeruginosa Exotoxin A. AMB Express. 2021;11(1):173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Michalska M, Wolf P. Pseudomonas Exotoxin A: optimized by evolution for effective killing. Front Microbiol. 2015;6:151249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Morgan RN, Saleh SE, Farrag HA, Aboshanab KM. Gamma radiation coupled ADP-ribosyl transferase activity of Pseudomonas aeruginosa PE24 moiety. Appl Microbiol Biotechnol. 2023;107(5):1765–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Amirmozafari N, Mehrabadi JF, Habibi A. Association of the exotoxin A and exoenzyme S with antimicrobial resistance in Pseudomonas aeruginosa strains. Arch Iran Med. 2016;19(5):0. [PubMed] [Google Scholar]
- 21.Wolska K, Szweda P. Genetic features of clinical Pseudomonas aeruginosa strains. Pol J Microbiol. 2009;58(3):255–60. [PubMed] [Google Scholar]
- 22.Agnello M, Wong-Beringer A. Differentiation in quinolone resistance by virulence genotype in Pseudomonas aeruginosa. 2012. [DOI] [PMC free article] [PubMed]
- 23.Corehtash ZG, Ahmad Khorshidi FF, Akbari H, Aznaveh AM. Biofilm formation and virulence factors among Pseudomonas aeruginosa isolated from burn patients. Jundishapur J Microbiol. 2015;8(10). [DOI] [PMC free article] [PubMed]
- 24.Yousefi-Avarvand A, Khashei R, Ebrahim-Saraie HS, Emami A, Zomorodian K, Motamedifar M. The frequency of exotoxin A and exoenzymes S and U genes among clinical isolates of Pseudomonas aeruginosa in Shiraz. Iran Int J Mol Cell Med. 2015;4(3):167. [PMC free article] [PubMed] [Google Scholar]
- 25.Pobiega M, Maciąg J, Chmielarczyk A, Romaniszyn D, Pomorska-Wesolowska M, Ziolkowski G, et al. Molecular characterization of carbapenem-resistant Pseudomonas aeruginosa strains isolated from patients with urinary tract infections in Southern Poland. Diagn Microbiol Infect Dis. 2015;83(3):295–7. [DOI] [PubMed] [Google Scholar]
- 26.Magalhães MJTL, Pontes G, Serra PT, Balieiro A, Castro D, Pieri FA, et al. Multidrug resistant Pseudomonas aeruginosa survey in a stream receiving effluents from ineffective wastewater hospital plants. BMC Microbiol. 2016;16(1):1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Idris SNA, Desa MNM, Aziz MN, Taib NM. Antimicrobial susceptibility pattern and distribution of exoU and exoS in clinical isolates of Pseudomonas aeruginosa at a Malaysian hospital. Southeast Asian J Trop Med Public Health. 2012;43(1):116–23. [PubMed] [Google Scholar]
- 28.Tabatabaei M, Sohrabi N. Comparison of biofilm formation and antibiotic resistance pattern of Pseudomonas aeruginosa in human and environmental isolates. Microb Pathog. 2017;109:94–8. [DOI] [PubMed] [Google Scholar]
- 29.Aljebory IS. PCR detection of some virulence genes of Pseudomonas aeruginosa in Kirkuk city. Iraq J Pharm Sci Res. 2018;10(5):1068–71. [Google Scholar]
- 30.Garey K, Vo Q, Larocco M, Gentry L, Tam V. Prevalence of type III secretion protein exoenzymes and antimicrobial susceptibility patterns from bloodstream isolates of patients with Pseudomonas aeruginosa bacteremia. J Chemother. 2008;20(6):714–20. [DOI] [PubMed] [Google Scholar]
- 34.Pokharel K, Dawadi BR, Bhatt CP, Gupte S. Prevalence of pseudomonas aeruginosa and its antibiotic sensitivity pattern. J Nepal Health Res Counc. 2019;17(1):109–13. [DOI] [PubMed] [Google Scholar]
- 32.Banar M, Emaneini M, Satarzadeh M, Abdellahi N, Beigverdi R, Leeuwen, WBv, et al. Evaluation of mannosidase and trypsin enzymes effects on biofilm production of Pseudomonas aeruginosa isolated from burn wound infections. PLoS ONE. 2016;11(10):e0164622. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268–81. [DOI] [PubMed] [Google Scholar]
- 34.Gholami A, Majidpour A, Talebi-Taher M, Boustanshenas M, Adabi M. PCR-based assay for the rapid and precise distinction of Pseudomonas aeruginosa from other Pseudomonas species recovered from burns patients. J Prev Med Hyg. 2016;57(2):E81. [PMC free article] [PubMed] [Google Scholar]
- 35.Kaszab E, Szoboszlay S, Dobolyi C, Háhn J, Pék N, Kriszt B. Antibiotic resistance profiles and virulence markers of Pseudomonas aeruginosa strains isolated from composts. Bioresour Technol. 2011;102(2):1543–8. [DOI] [PubMed] [Google Scholar]
- 36.Lanotte P, Watt S, Mereghetti L, Dartiguelongue N, Rastegar-Lari A, Goudeau A, et al. Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patients compared with those of isolates from other origins. J Med Microbiol. 2004;53(1):73–81. [DOI] [PubMed] [Google Scholar]
- 37.Zhu H, Bandara R, Conibear TC, Thuruthyil SJ, Rice SA, Kjelleberg S, et al. Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection. Investig Ophthalmol Vis Sci. 2004;45(6):1897–903. [DOI] [PubMed] [Google Scholar]
- 38.Hematzadeh A, Haghkhah M. Biotyping of isolates of Pseudomonas aeruginosa isolated from human infections by RAPD and ERIC-PCR. Heliyon. 2021;7(9). [DOI] [PMC free article] [PubMed]
- 39.Schiavano GF, Carloni E, Andreoni F, Magi S, Chironna M, Brandi G, Amagliani G. Prevalence and antibiotic resistance of Pseudomonas aeruginosa in water samples in central Italy and molecular characterization of opr D in imipenem resistant isolates. PLoS One. 2017 Dec 6;12(12):e0189172. [DOI] [PMC free article] [PubMed]
- 40.Maraolo AE, Cascella M, Corcione S, Cuomo A, Nappa S, Borgia G, De Rosa FG, Gentile I. Management of multidrug-resistant Pseudomonas aeruginosa in the intensive care unit: state of the art. Expert Rev anti-infective Therapy. 2017;15(9):861–71. [DOI] [PubMed] [Google Scholar]
- 41.Tam VH, Chang KT, Abdelraouf K, Brioso CG, Ameka M, McCaskey LA, Weston JS, Caeiro JP, Garey KW. Prevalence, resistance mechanisms, and susceptibility of multidrug-resistant bloodstream isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2010;54(3):1160–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Vaez H, Salehi-Abargouei A, Ghalehnoo ZR, Khademi F. Multidrug resistant Pseudomonas aeruginosa in Iran: a systematic review and metaanalysis. J Global Infect Dis. 2018;10(4):212. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.MOAZAMI GS, Eftekhar F. Assessment of carbapenem susceptibility and multidrug-resistance in Pseudomonas aeruginosa burn isolates in Tehran. 2013.
- 44.Jafari M, Fallah F, Borhan RS, Navidinia M, KARIMI A, RAFII TS et al. The first report of CMY, aac (6’)-Ib and 16S rRNA methylase genes among Pseudomonas aeruginosa isolates from Iran. 2013.
- 45.Bokaeian M, Zahedani SS, Bajgiran MS, Moghaddam AA. Frequency of PER, VEB, SHV, TEM and CTX-M genes in resistant strains of Pseudomonas aeruginosa producing extended spectrum β-lactamases. Jundishapur J Microbiol. 2015;8(1). [DOI] [PMC free article] [PubMed]
- 46.Prevention, ECfD. Control. European Centre for Disease Prevention and Control. Antimicrobial resistance surveillance in Europe 2015. ECDC Stockholm; 2017.
- 47.Poorabbas B, Mardaneh J, Rezaei Z, Kalani M, Pouladfar G, Alami MH, et al. Nosocomial infections: Multicenter surveillance of antimicrobial resistance profile of Staphylococcus aureus and Gram negative rods isolated from blood and other sterile body fluids in Iran. Iran J Microbiol. 2015;7(3):127. [PMC free article] [PubMed] [Google Scholar]
- 48.Yayan J, Ghebremedhin B, Rasche K. Antibiotic resistance of Pseudomonas aeruginosa in pneumonia at a single university hospital center in Germany over a 10-year period. PLoS ONE. 2015;10(10):e0139836. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Hachem RY, Chemaly RF, Ahmar CA, Jiang Y, Boktour MR, Rjaili GA, Bodey GP, Raad II. Colistin is effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa in cancer patients. Antimicrob Agents Chemother. 2007;51(6):1905–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Maleki F, Tahmasebi H, Zandi M, Arabestani MR. Investigation of the relationship between the Presence of genes producing Toxin and Antibiotic Resistance Pattern in Pseudomonas aeruginosa strains isolated from burn wounds. Avicenna J Clin Med. 2018;25(2):112–20. [Google Scholar]
- 51.Roy-Burman A, Savel RH, Racine S, Swanson BL, Revadigar NS, Fujimoto J, et al. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J Infect Dis. 2001;183(12):1767–74. [DOI] [PubMed] [Google Scholar]
- 52.Lee VT, Smith RS, Tümmler B, Lory S. Activities of Pseudomonas aeruginosa effectors secreted by the type III secretion system in vitro and during infection. Infect Immun. 2005;73(3):1695–705. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Bazghandi SA, Arzanlou M, Peeridogaheh H, Vaez H, Sahebkar A, Khademi F. Prevalence of virulence genes and Drug Resistance profiles of Pseudomonas aeruginosa isolated from clinical specimens. Jundishapur J Microbiol. 2021;14(8).
- 54.Bahador N, Shoja S, Faridi F, Dozandeh-Mobarrez B, Qeshmi FI, Javadpour S, et al. Molecular detection of virulence factors and biofilm formation in Pseudomonas aeruginosa obtained from different clinical specimens in Bandar Abbas. Iran J Microbiol. 2019;11(1):25. [PMC free article] [PubMed] [Google Scholar]
- 55.Bardoel BW, van Kessel KP, van Strijp JA, Milder FJ. Inhibition of Pseudomonas aeruginosa virulence: characterization of the AprA–AprI interface and species selectivity. J Mol Biol. 2012;415(3):573–83. [DOI] [PubMed] [Google Scholar]
- 56.Badamchi A, Masoumi H, Javadinia S, Asgarian R, Tabatabaee A. Molecular detection of six virulence genes in Pseudomonas aeruginosa isolates detected in children with urinary tract infection. Microb Pathog. 2017;107:44–7. [DOI] [PubMed] [Google Scholar]
- 57.Hassuna NA, Mandour SA, Mohamed ES. Virulence constitution of multi-drug-resistant Pseudomonas aeruginosa in upper Egypt. Infect drug Resist. 2020;13:587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Izadi Pour Jahromi S, Mardaneh J, Sharifi A, Pezeshkpour V, Behzad-Behbahani A, Seyyedi N et al. Occurrence of a multidrug resistant Pseudomonas aeruginosa strains in hospitalized patients in southwest of Iran: characterization of resistance trends and virulence determinants. Jundishapur J Microbiol. 2018;11(4).
- 59.Mohktari K, Amini K. Molecular identification of virulence genes in Pseudomonas aeruginosa isolated from human and Animal Samples and their antibiotic resistance pattern. Iran J Med Microbiol. 2019;13:03. [Google Scholar]
- 60.Nikbin V, Aslani MM, Sharafi Z, Hashemipour M, Shahcheraghi F, Ebrahimipour G. Molecular identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins. Iran J Microbiol. 2012;4(3):118. [PMC free article] [PubMed] [Google Scholar]
- 61.Khattab M, Nour M, ElSheshtawy N. Genetic identification of Pseudomonas aeruginosa virulence genes among different isolates. J Microb Biochem Technol. 2015;7(5):274–7. [Google Scholar]
- 62.Pournajaf A, Razavi S, Irajian G, Ardebili A, Erfani Y, Solgi S, et al. Integron types, antimicrobial resistance genes, virulence gene profile, alginate production and biofilm formation in Iranian cystic fibrosis Pseudomonas aeruginosa isolates. Infez Med. 2018;26(3):226–36. [PubMed] [Google Scholar]
- 63.Stehling EG, Leite DS, Silveira WD. Molecular typing and biological characteristics of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Brazil. Brazilian J Infect Dis. 2010;14(5):462–7. [PubMed] [Google Scholar]
- 64.Nanvazadeh F, Khosravi AD, Zolfaghari MR, Parhizgari N. Genotyping of Pseudomonas aeruginosa strains isolated from burn patients by RAPD-PCR. Burns. 2013;39(7):1409–13. [DOI] [PubMed] [Google Scholar]
- 65.Saitou K, Furuhata K, Fukuyama M. Genotyping of Pseudomonas aeruginosa isolated from cockroaches and human urine. J Infect Chemother. 2010;16(5):317–21. [DOI] [PubMed] [Google Scholar]
- 66.Vaez H, Faghri J, Esfahani BN, Moghim S, Fazeli H, Sedighi M et al. Antibiotic resistance patterns and genetic diversity in clinical isolates of Pseudomonas aeruginosa isolated from patients of a referral hospital, Isfahan, Iran. Jundishapur J Microbiol. 2015;8(8). [DOI] [PMC free article] [PubMed]
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