Molecular characterizations of antibiotic resistance, biofilm formation, and virulence determinants of Pseudomonas aeruginosa isolated from burn wound infection

Abstract Background Burn injuries result in disruption of the skin barrier against opportunistic infections. Pseudomonas aeruginosa is one of the main infectious agents colonizing burn wounds and making severe infections. Biofilm production and other virulence factors along with antibiotic resistance limit appropriate treatment options and time. Materials and Methods Wound samples were collected from hospitalized burn patients. P. aeruginosa isolates and related virulence factors identified by the standard biochemical and molecular methods. Antibiotic resistance patterns were determined by the disc diffusion method and β‐lactamase genes were detected by polymerase chain reaction (PCR) assay. To determine the genetic relatedness amongst the isolates, enterobacterial repetitive intergenic consensus (ERIC)‐PCR was also performed. Results Forty P. aeruginosa isolates were identified. All of these isolates were biofilm producers. Carbapenem resistance was detected in 40% of the isolates, and bla TEM (37/5%), bla VIM (30%), and bla CTX‐M (20%) were the most common β‐lactamase genes. The highest resistance was detected to cefotaxime, ceftazidime, meropenem, imipenem and piperacillin, and 16 (40%) isolates were resistant to these antibiotics. The minimum inhibitory concentrations (MIC) of colistin was lower than 2 μg/mL and no resistance was observed. Isolates were categorized to 17 MDR, 13 mono‐drug resistance, and 10 susceptible isolates. High genetic diversity was also observed among the isolates (28 ERIC types) and most carbapenem‐resistant isolates were classified into four main types. Conclusion Antibiotic resistance, particularly carbapenem resistance was considerable among the P. aeruginosa isolates colonizing burn wounds. Combining carbapenem resistance with biofilm production and virulence factors would result in severe and difficult‐to‐treat infections.


| INTRODUC TI ON
Normal and intact skin is a barrier against infective agents, such as Pseudomonas aeruginosa. 1 Burn injuries destroy the skin protection against infection and disrupt the physiologic function of the immune system, and burn patients are at high risk of acquiring hospitalassociated infections. 2 P. aeruginosa is an aerobic Gram-negative bacilli and accounts for opportunistic or nosocomial infections in burn patients, cystic fibrosis, and immunocompromised individuals. 3 Pseudomonas aeruginosa possesses a wide range of virulence factors such as elastase, exoenzymes, and exotoxin A which are regulated by cell-to-cell signaling systems. The main virulence factor produced by isolates of P. aeruginosa is exotoxin A which has an important role in the pathogenesis of this microorganism. 4 Also, flagella and pili have a key role as virulence factors independently. 5 P. aeruginosa is able to enhance the excretion of virulence determinants in the cytoplasm of target cells through a type III secretion system. These factors are associated with higher mortality, especially in burn patients. 6 Moreover, biofilm formation is a basic and critical virulence factor that improves bacterial survival in harsh circumstances such as dryness or the presence of antiseptics. 7 Biofilm also is one of the main strategies for antibiotic resistance that increases horizontal gene transfer between susceptible and resistant strains. 8 It is a complex aggregate of bacteria encased in alginate polysaccharides and encoded by the algD gene. 7 Biofilm also makes a barrier between bacterial cells and antibiotics or immune responses. 8 P. aeruginosa destroys natural structures of skin or mucous membranes using protease (such as elastase or Las), phospholipase (Plc), neuraminidase (Nan), and exotoxins. They are among those virulence factors that destroy connective tissue proteins, cytokines, cell membranes, and antibodies and modulate P. aeruginosa infections in proper sites such as burned skin or cystic fibrosis lungs. 9 In burn injuries, the natural defense of skin is destructed, and exposed matrix proteins and inflammatory factors accelerate the colonization of P. aeruginosa and infection. 10 Besides these virulence factors that make microorganism a destructive pathogen, antibiotic resistance also complicates the treatment of P. aeruginosa infections. Antibiotic resistance is mediated by various strategies such as β-lactamases, efflux pumps and mutations, and multi-drug-resistant (MDR) isolates harbor several mechanisms for antibiotic resistance. 11 In P. aeruginosa different β-lactamases like extended spectrum β-lactamases (ESBLs) and metalloβ-lactamases (MBLs) cause resistance to β-lactam antibiotics. 11 The combination of β-lactamase-producing phenotype and virulence factors creates a highly human pathogen, especially in burn patients. 10 Characterization of local epidemiology and determination of genetic relatedness of the drug-resistant isolates is necessary to control their dissemination in healthcare setting. 12 To determine the genotypic relationship amongst P. aeruginosa isolates, various genotyping methods including, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) have been used. 13 Furthermore, polymerase chain reaction (PCR)-based techniques such as enterobacterial repetitive intergenic consensus (ERIC)-PCR are rapid, cost-effective, reproducible, and reliable typing methods with acceptable discriminatory power for non-fermenting Gramnegative bacilli. 13,14 In the current study, we aimed to assess virulence factors, biofilm formation ability, β-lactamase associated genes, and the genetic relationship amongst P. aeruginosa isolates, obtained from in burn wound infections.

| Bacterial isolates
In this study, clinical isolates of P. aeruginosa were isolated between March 2020 and September 2020 from burn wound samples in the selected hospitals in Tehran, and Ahvaz, Iran. All patients or their legal guardians provided informed written consent, and this study was approved by the Ethics Committee of Ilam University of Medical

| Phenotypic tests for ESBL, carbapenemase, and metallo β -lactamase
All isolates were screened for the production of ESBL and MBLs enzymes using the combination disc method. In brief, an overnight incubated suspension of each isolate was inoculated on Muller-Hinton agar media. Then, ceftazidime and ceftazidime/clavulanic acid discs were used to determine ESBL enzymes. Imipenem and EDTA discs were also used to detect MBLs enzymes. Carbapenemase activity was assessed using the carba-NP test method, as described previously. 16

| Biofilm assay
Biofilm formation assay was performed as described previously. 17 In brief, P. aeruginosa isolates were inoculated in 5 mL trypticase soy broth (TSB) and overnight incubated at 37°C. Then a concentration equal to 0.5 McFarland standard was prepared in TSB and each well of a flat-bottomed polystyrene 96-well microtiter plate was inoculated with 100 μL of these dilutions. After 24 h incubation at 37°C, the supernatant was removed and wells were rinsed with normal saline solution (0.9% NaCl). Adherent biofilms were fixed with 99% ethanol. The solutions were removed, and the plate was air-dried, and stained with crystal violet (1.5%) for 20 min after that the unbound stain was rinsed with water. The dye was solubilized in 150 μL of 30% (v/v) acetic acid. The optical densities (OD) of the wells were measured by a microplate reader at 550 nm. The whole process was performed in triplicate for each isolate, and P. aeruginosa ATCC 27853 and sterile broth were used as a positive and negative control. A cut-off value (ODc) was determined and it is defined as three standard deviations (SD) above the mean OD of the negative control: ODc = average OD of negative control + (3 × SD of negative control). The isolates were categorized into the four following groups based on the OD: non-biofilm producer (OD < ODc); weak-biofilm producer (ODc < OD <2 × ODc); moderate-biofilm producer (2 × ODc < OD <4 × ODc); strong-biofilm producer (4 × ODc < OD). 17,18

| Molecular detection of virulence and resistance
The whole genomic DNA was extracted from pure colonies of iso- PCR method using the specific primers (Table 1). [19][20][21][22] Then, 1% agarose gel electrophoresis and gel staining (stain load dye (CinnaGen Co, Iran)) were conducted for the analysis of PCR products.

| Enterobacterial repetitive intergenic consensus (ERIC-PCR)
To characterize the genetic relatedness among the isolates, ERIC-PCR was performed using followed primers, ERIC1 5′-ATGTA AGC TCC TGG GGA TTCAC-3′ and ERIC2 5′-AAGTA AGT GAC TGG GGT GAGCG-3′, as described previously. 13 The PCR protocol consisted of a pre-denaturation step at 95°C for 5 min, followed by 30 cycles of 60 s at 95°C, 50 s at 59°C, and 60 s at 72°C. A final extension step was done at 72°C for 10 min. PCR products were separated by electrophoresis in 1.5% agarose gels with 0.5× TBE (Tris/Boric acid/ EDTA) buffer. DNA bands were visualized using UV light after staining with safe stain load dye. The GelJ software version 2.0 was used to analyze ERIC patterns 23 and the isolates with a similarity coefficient ≥90% were clustered in the same genotypes. In other words, the isolates with equal or more than 90% similarity in their banding patterns were considered the same ERIC type.

| Statistical analysis
The SPSS version 22.0 (SPSS, Inc.) was used to analyze the data.
Pearson Chi-Square test was used to determine the statistically significant correlation between the existence of genes and antibiotic resistance or biofilm production. In addition, p-value <0.05 was considered as a significance level. The results are presented as descriptive statistics in terms of relative frequency.

| Phenotypic assessment of ESBL, metalloβlactamase, and carbapenemase
While the ESBL activity was not detected in any of the isolates, 12 (30%) isolates were positive for MBL, and 16 (40%) isolates had carbapenemase activity.

| Biofilm formation
All the isolates (100%) were positive for biofilm production.

| ESBL and carbapenemase-related genes
Among the ESBL genes, bla TEM , bla CTX , and bla SHV genes were positive in 15 (37.5%), 8 (20%), and 6 (15%) isolates, respectively. MBL and Carbapenemase genes were less frequent and only bla VIM gene was present in the isolates (30%), and bla IMP and bla NDM genes were not detected. Moreover, bla OX A-48 and bla OX A-23 genes were found in 7 (17.5%) and 1 (2.5%) isolates and no isolate possessed bla OX A-11 gene ( Table 4). The co-occurrence of different types of β-lactamase was seen in 15 isolates and the details are shown in Table 5.
Although we did not find any correlation between the virulence and β-lactamase genes, the co-existence of virulence genes (lasB, exoA, plcH, exoS, and nan1) was observed among the isolates. The algD gene was present in 17 (43%) isolates and all of them were strong biofilm producers.

| ERIC-PCR typing
ERIC-PCR typing indicated high genetic diversity among the isolates.
The results of genotyping showed that 36 isolates were classified into 28 ERIC types according to a 90% cut-off ( Figure 1). No band was detected following ERIC-PCR in four isolates, and thereby they were non-typeable. According to our analysis, 12 isolates were clustered in four main genotypes (A-D). The predominant type was type A, and it contained five isolates, followed by B (three), C (two), and D (two). Other 24 isolates possessed different banding patterns and they were distributed in 24 single types ( Figure 1).

| DISCUSS ION
The growing rates of antibiotic resistance in P. aeruginosa neutralize antibiotic efficacy against infections caused by this opportunistic agent. In this study, we isolated P. aeruginosa from burn wounds TA B L E 3 Distribution of biofilm formation among P. aeruginosa isolates and correlation between biofilm production and antibiotic resistance patterns or co-presence of virulence factors. Interestingly, meropenem resistance has been demonstrated to be higher in P. aeruginosa isolates from cystic fibrosis patients. 29 It is speculated that a complicated environment and a chronic infection in cystic fibrosis lungs are responsible for the higher resistance rates. 29 Local studies from Iran have reported higher carbapenem resistance in P. aeruginosa isolates from burn wounds. Moreover, the imipenem resistance rate was found to be 58% and 94% in Shahrekord and Isfahan, respectively. 30,31 Also in a report from India, 61% of P. aeruginosa isolates from burn wounds were imipenem-resistant. 11 Altogether, it seems that the prevalence of carbapenem resistance depends on the geographical area of studies. MBL and carbapenemase enzymes are considered to be the main underlying carbapenem resistance.
In the current study, 40% of isolates were positive for encoding at least one of the MBL or carbapenemase enzymes, and coexistence of ESBL, MBL, and carbapenemase genes was observed in 37.5% of isolates. The co-existence of these enzymes resulted in high levels of β-lactam resistance, and as shown in Table 5, the co-presence of these genes was related to the formation of the MDR phenotype. Also, bla TEM (37.5%), bla VIM (30%), and bla CTX-M (20%) were the most common β-lactamase genes among the isolates. In other study by Peymani et al., the bla TEM-1 (26.7%) and bla CTX-M-15 (17.3%), were the most common genes. 32 The prevalence rate of ESBL in the study performed by Senthamaria et al., 33 Begum et al., 34 and Mirsalehian et al., 35 40 have demonstrated that the existence of toxA and toxS genes is related to high antibiotic resistance in P. aeruginosa isolates, we did not find any significant correlation between the presence of these virulence factors and high antibiotic resistance rate.

Although Khosravi and colleagues
The plcH gene is a source of hemolytic phospholipase C in P. aeruginosa. 42 This virulence factor has a link to the high growth rate and pathogenicity, and mutant isolates have attenuated pathogenicity and slow growth rate. 42 We found the plcH gene in 92.5% of the isolates, and this factor as well as toxA, lasB, and toxS could be related to the high pathogenicity of the studied isolates.
We investigated the genetic relatedness of the P. aeruginosa isolates using ERIC-PCR fingerprinting, and the results showed high genetic diversity. Most carbapenem-resistant isolates (12/16) were classified into four ERIC types (A-D). ERIC patterns of other carbapenem-resistant isolates were also comparable with type A (lanes no. 14 and 19) and type B (lanes no. 31 and 32) (Figure 1). It seems that these genotypes are circulating strains among hospitalized patients in various wards of the hospitals. Notable antimicrobial resistance and biofilm formation ability were identified in these types (Table 6), and these factors are associated with long-term persistence in a medical setting. 43,44 According to the cut-off, most of the isolates (n = 24) showed high-level heterogeneity. These isolates, classified into 24 single types, were susceptible or did not show high-level antimicrobial resistance. This diversity could be due to environmental or exogenous sources of the isolates. Based on the ERIC-PCR method, four isolates were nontypeable; therefore, 90% (36/40) efficiency was calculated for this method in this study.

| CON CLUS ION
Antibiotic resistance of P. aeruginosa is considerable among the burn wound samples. Biofilm production is a synergistic factor that amplifies antibiotic resistance in these isolates, and alternative treatment for the elimination of biofilm could help decrease the antibiotic resistance rate in the life-threatening burn infections by P. aeruginosa.
Also, the high prevalence of virulence factors such as toxA, plcH, toxS, and lasB in our isolates shows that these factors are important in the pathogenesis of these bacteria in burn wounds. Innovation of new strategies for the inhibition of these virulence factors could be also beneficial for the treatment of burn infections by P. aeruginosa.

AUTH O R CO NTR I B UTI O N S
SGH, HH, SKH, SGH, and MKZ substantially contributed to the conceptualization, methodology, validation, and investigation of the work. EK and HK have been involved in data curation, supervision, writing, and acquisition of data or revised the review article for intellectual content. All authors agreed and confirmed the manuscript for publication.

ACK N OWLED G M ENTS
We would like to thank the Clinical Microbiology Research Center, Ilam University of Medical Sciences, for their cooperation. This study was supported by Ilam University of Medical Sciences (Project no. 1188).

CO N FLI C T O F I NTE R E S T
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. The authors report no conflict of interest in this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article. TA B L E 6 Characteristics of the predominant genotypes.