Aboalfazl Jafari-Sales1 
, Zahra Ghahremani2 , Aylin Golestani2 , Mohadeseh Ghorbanpour Koulani Jadid2 , Kosar Hosseini-Karkaj2 , Kosar Soleymanpour2 , Mohammad Mahdi Salek Faramarzi2 , Mehrdad Pashazadeh3
, Zahra Ghahremani2 , Aylin Golestani2 , Mohadeseh Ghorbanpour Koulani Jadid2 , Kosar Hosseini-Karkaj2 , Kosar Soleymanpour2 , Mohammad Mahdi Salek Faramarzi2 , Mehrdad Pashazadeh3
1- Department of Microbiology, Kaz.C., Islamic Azad University, Kazerun, Iran; Infectious Diseases Research Center, TaMS.C., Islamic Azad University, Tabriz, Iran
2- Infectious Diseases Research Center, TaMS.C., Islamic Azad University, Tabriz, Iran; Department of Cellular and Molecular Biology, Ta.C., Islamic Azad University, Tabriz, Iran
3- Infectious Diseases Research Center, TaMS.C., Islamic Azad University, Tabriz, Iran; Department of Laboratory Sciences and Microbiology, TaMS.C., Islamic Azad University, Tabriz, Iran ,mehrdadpashazadeh85@gmail.com
2- Infectious Diseases Research Center, TaMS.C., Islamic Azad University, Tabriz, Iran; Department of Cellular and Molecular Biology, Ta.C., Islamic Azad University, Tabriz, Iran
3- Infectious Diseases Research Center, TaMS.C., Islamic Azad University, Tabriz, Iran; Department of Laboratory Sciences and Microbiology, TaMS.C., Islamic Azad University, Tabriz, Iran ,
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Results
In this study, 100 isolates of P. aeruginosa were examined. The mean age of patients was 45.9 ± 16.12 years, ranging from 20 to 78 years. There were 38 females (38%) with a mean age of 49.61 ± 16.33 years and 62 males (62%) with a mean age of 39.84 ± 16.52 years. Statistical analysis showed no significant difference in the distribution of isolates between different age and gender groups (P-Value > 0.05). Antibiogram results revealed a high prevalence of resistance to levofloxacin (97%), meropenem (92%), ciprofloxacin (88%), and tobramycin (87%), while the lowest rate of resistance was related to ceftazidime (69%) (Figure 1). The combined disk test identified 58 ESBL-positive isolates (58%), including 22 isolates from females (37.93%) with a mean age of 45.45 ± 15.15 years and 36 isolates from males (62.07%) with a mean age of 39.30 ± 14.11 years. Among 58 ESBL-positive isolates, 29 (50%) carried the blaTEM gene (Figure 2). No significant association was found between ESBL or blaTEM status and patients’ age or gender (P-Value > 0.05). Neither isolate distribution frequencies nor antibiotic resistance patterns were significantly associated with demographic variables. Nonetheless, the high frequency of antibiotic resistance and blaTEM positivity (50% of ESBL-positive isolates) highlights the necessity of ongoing surveillance for antibiotic resistance and infection control initiatives.
Discussion
One of the main sources of hospital-acquired infections in burn patients is the opportunistic Gram-negative bacterium P. aeruginosa. Since P. aeruginosa can acquire extensive antibiotic resistance, treating infections caused by this bacterium has become very challenging (11). Investigating the antibiotic resistance pattern of P. aeruginosa in the hospital setting and determining its susceptibility to commonly used antimicrobials can help establish initial treatment protocols and effectively manage drug-resistant infections caused by this bacterium (12). P. aeruginosa is naturally resistant to several antimicrobial agents, making its control difficult. Various mechanisms contribute to this resistance (13), including producing beta-lactamase enzymes, resulting in resistance to beta-lactam antibiotics (14). The significant increase in ESBL-positive P. aeruginosa strains among burn patients has posed therapeutic challenges (15,16). The rate of organisms producing blaTEM enzymes continues to rise, and treating infections caused by these bacteria remains highly problematic due to the antibiotic resistance conferred by these enzymes (17). In previous studies, samples had been obtained from various sources such as urine, blood, infected wounds, and urinary tract infections (18). However, in this study, all clinical samples were collected from patients admitted to the burn unit. Among 100 isolates obtained from patients, 87 were resistant to at least three types of antibiotics. According to the findings of Abdelrahim et al. (2023), blaTEM was reported as the second most prevalent gene (46.2%) in ESBL-producing P. aeruginosa strains (19). The present study also identified this gene as one of the most prevalent resistance genes, with a frequency of 50%. In a study by Peymani et al. conducted in Tehran, the frequency of ESBL-producing P. aeruginosa strains was reported as 28.6% (20), which is lower than the rate recorded in the present study.
In contrast to a study by Chen et al., where ESBL-positive P. aeruginosa strains showed the highest resistance to ceftazidime (30%) (21), we found the lowest resistance rate (69%) to this antibiotic. Bokaeian et al. in Zahedan reported that 25.86% of 116 isolates were resistant to at least one cephalosporin or aztreonam antibiotic (22). This finding differs from the results of the current study in Tabriz, which demonstrated the highest resistance rates to levofloxacin (97%), meropenem (92%), ciprofloxacin (88%), and tobramycin (87%); however, both studies confirmed a similar pattern of antibiotic resistance. In the present study, among 100 P. aeruginosa strains isolated from burn patients, 97 were resistant to levofloxacin, while the lowest resistance rate was observed against ceftazidime (69%). However, in a 2010 study by Fazeli et al. in Isfahan, 94% of P. aeruginosa isolates exhibited complete resistance to ceftazidime (23), a discrepancy with our results. Bahrami et al. (2018) in Bandar Abbas found 47% resistance to meropenem among P. aeruginosa isolates (24), whereas this rate increased to 92% in the present study. Additionally, Bahrami et al. identified blaTEM as the most common beta-lactamase resistance gene with a frequency of 56.3% by PCR, which was relatively consistent with the current study’s 50% (24). Saleh et al. (2022) reported a 41% resistance rate to meropenem (25), whereas our data revealed a dramatic rise to 92%, indicating a concerning expansion of carbapenem resistance over the past two decades. Adjei et al. (2018) reported an 82% resistance rate to meropenem (26), while Haghighifar et al. (2021) observed an increase to 91.3% (27). The results of the current study showed that meropenem resistance was quite prevalent in the populations under investigation, which was in close agreement with both above studies. The discrepancies between our findings and those of previous studies likely root in several factors, including variations in sample size, the genetic diversity of local microbial populations, and distinct regional epidemiological patterns. Importantly, these same factors, particularly our limited sample size and the specific geographical focus of our work on Tabriz population, represent the key limitations of the present study and should be considered when interpreting the results. Consequently, larger multi-center cohorts are warranted in the future to validate and generalize these findings.
Conclusion
This study revealed a concerning level of antibiotic resistance among P. aeruginosa isolates from burn patients in Tabriz, with over 90% resistance to meropenem and fluoroquinolones. Also, 58% of the isolates were identified as ESBL producers. Notably, half of ESBL-positive strains harbored the blaTEM gene, highlighting the high penetrance of this resistance mechanism. Despite the lack of demographic correlations, the high prevalence of multidrug-resistant strains represents a significant public health challenge. These findings underscore the urgent need for regular antibiotic resistance surveillance, strict infection control protocols, and evidence-based antimicrobial stewardship practices in burn units. Future research should focus on longitudinal monitoring of resistance trends and investigating other genetic elements of resistance, such as other ESBL and carbapenemase genes, to guide targeted interventions and reduce the spread of resistant bacterial strains.
List of abbreviation
P. aeruginosa: Pseudomonas aeruginosa, ESBLs: Extended-Spectrum β-Lactamases, AMR: Antimicrobial Resistance, ATCC: American Type Culture Collection, CLSI: Clinical and Laboratory Standards Institute, E. coli: Escherichia coli, PCR: Polymerase Chain Reaction.
Acknowledgement
The authors sincerely thank the Vice Chancellery for Research at the Islamic Azad University, Tabriz Medical Branch, for supplying the laboratory facilities required for this study.
Funding sources
The authors received no financial support for the research, authorship, and/or publication of this article.
Ethical statement
Ethical approval was obtained from the Clinical Research Ethics Committee of Islamic Azad University, Tabriz Branch (Code: IR.IAU.TABRIZ.REC.1403.209). All participants were informed about the study’s objectives and signed written informed consent before enrollment.
Conflicts of interest
The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.
Author contributions
Concept/Design: A.J.S, M.P; Data acquisition: A.J.S; Data analysis and Interpretation: Z.G, A.G; Drafting the manuscript: Z.G, A.G, M.G.K.J, K.H.K, K.S, M.M.S.F; Critical revision of the manuscript: AJS; Final approval and Accountability: MP; Technical or Material support: A.J.S, M.P; Supervision: MP; Securing funding (If available): N/A.
Data availability statement
The data supporting the findings of this study can be obtained from the corresponding author upon a reasonable request.
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Introduction
A rod-shaped, aerobic, Gram-negative bacterium with pili and a polar flagellum, Pseudomonas aeruginosa (P. aeruginosa) is a serious health concern in hospitals. Both pili and the flagellum are essential for the movement and host cell attachment of bacteria. Especially in burn wounds, P. aeruginosa is one of the most prevalent organisms causing nosocomial infections (1), including pneumonia and infections in immunocompromised patients or individuals with structural lung diseases such as cystic fibrosis (2). Burn patients face an elevated risk of serious infections and related mortality due to impaired skin barrier function and immune suppression (3). Eradication of P. aeruginosa infecting burn wounds is a daunting task because of the bacterium’s intrinsic and acquired resistance to antibiotics (4). Antibiotic-resistant P. aeruginosa has become a global problem, posing a serious public health threat associated with limited treatment options and increasing mortality rates (5). Antimicrobial resistance (AMR) is defined as the ability of pathogenic bacteria to resist prescribed drugs, such as β-lactams. In Gram-negative bacteria, this resistance often arises from the production of extended-spectrum β-lactamases (ESBLs), which can inactivate β-lactam antibiotics (6). Among the most extensively studied AMR enzymes are β-lactamases (7), which are classified based on their inhibitory responses and substrate spectra. ESBLs encode class A β-lactamases, leading to resistance against certain β-lactam antibiotics (8). These enzymes, encoded by bla genes in Gram-negative bacteria, are responsible for one of the most critical mechanisms of antibiotic resistance (i.e., decapitating β-lactam antibiotics through degrading the β-lactam ring). Aggravating this threat, bla genes exhibit a high potential for horizontal gene transfer and integration into multidrug-resistant plasmids, promoting the rapid spread of resistance in clinical settings (9). The clinical challenge of beta-lactam resistance among Gram-negative bacteria, largely driven by beta-lactamase production, has spurred significant pharmaceutical innovations. The development of novel compounds designed to inhibit or disrupt these enzymes has led to groundbreaking accomplishments in expanding therapeutic interventions (10). This study aimed to accurately identify P. aeruginosa strains, determine their antibiotic resistance patterns, and investigate the prevalence of the blaTEM gene in burn patients in Tabriz.
Methods
Sampling and bacterial isolation
A descriptive cross-sectional study was conducted utilizing a convenience sampling method to recruit 100 burn patients admitted to the burn units of Tabriz hospitals over the course of six months from April to September 2024. Using sterile swabs, samples were obtained from burn wounds and immediately placed into Cary-Blair transport media. To maintain optimal conditions for bacteria survival, samples were transferred to the laboratory on ice and processed within a strict 2-hour window from the time of collection.
Inclusion criteria in this study included being adult patients hospitalized due to burn wounds in medical centers across Tabriz, northwest Iran, and showing clinical signs of active infections (Such as purulent discharge, erythema, localized warmth, or systemic fever). Furthermore, enrollment was contingent upon obtaining written informed consent from patients or their legal guardians. The definitive microbiological confirmation of P. aeruginosa infection, isolated from burn wound samples collected in a sterile manner, through standard cultural characteristics (e.g., grape-like odor, pyocyanin production), biochemical profiling (Oxidase-positive), and automated systems was mandatory for inclusion in the final analysis. Samples were initially cultured on sheep blood agar (Containing 5% sheep blood) to recognize hemolytic patterns and on MacConkey agar to selectively isolate Gram-negative bacteria, followed by incubation at 37°C for 24-48 hours. Suspected P. aeruginosa colonies were selected based on characteristic morphology: large size, smooth surface, irregular margins, and distinctive green-blue pigmentation (Pyocyanin). Final identification was confirmed through a series of standard biochemical tests. These included a positive oxidase reaction, which is a key diagnostic trait for P. aeruginosa, the ability to oxidize but not ferment glucose in Oxidation-Fermentation (OF) medium, alkaline/alkaline (K/K) reaction in Kligler Iron Agar (KIA) indicative of non-fermentative metabolism, positive citrate utilization, pyocyanin pigment production, and the ability to grow at 42°C (A trait that helps differentiate P. aeruginosa from other pseudomonads). Additional tests, such as gelatin hydrolysis and urease activity, were performed for differentiation from similar species. Using P. aeruginosa American Type Culture Collection-ATCC: 27853 as a positive control, all processes were verified. Confirmed isolates were kept at -80°C in Tryptic Soy Broth with 15% glycerol.
Exclusion criteria included unwillingness to continue cooperation, unconfirmed laboratory detection of P. aeruginosa, and the presence of severe underlying diseases (e.g., uncontrolled diabetes, metastatic cancer, or advanced autoimmune disorders). The study was conducted in compliance with biosafety principles and ethical protocols and acquired the approval code of IR.IAU.TABRIZ.REC.1403.209.
Antibiotic susceptibility testing by disk diffusion method
The standard agar disk diffusion technique (Kirby-Bauer) was used according to clinical and laboratory standards institute (CLSI) protocols. Mueller-Hinton agar and antibiotic disks manufactured by Padten Teb (Iran) were employed in this study. Target antibiotics included β-lactams such as piperacillin (100 μg) and ceftazidime (30 μg); aminoglycosides, including gentamicin (10 μg), tobramycin (10 μg), and amikacin (30 μg); fluoroquinolones such as ciprofloxacin (5 μg) and levofloxacin (5 μg); and other groups, including cefepime (30 μg) and meropenem (10 μg). For quality control, standard strains of Escherichia coli (E. coli) (ATCC: 25922) and P. aeruginosa (ATCC: 27853) were used in each test batch. Results were interpreted based on CLSI guidelines.
Phenotypic identification of β-lactamase-producing strains
In this study, ESBL-producing isolates were identified using the standard Combination Disk test in accordance with CLSI protocols. The antibiotic disks of cefotaxime (30 μg), cefotaxime plus clavulanic acid (10 μg), ceftazidime (30 μg), and ceftazidime plus clavulanic acid (10 μg) (Mast Group, UK) were employed. Freshly prepared Mueller-Hinton agar was used as the culture medium. A bacterial suspension with a standard concentration of 0.5 McFarland (~1.5×10^8 CFU/ml) was prepared and uniformly inoculated onto the culture medium. ESBL production was recognized based on a ≥5 mm increase in the inhibition zone diameter for antibiotic disks used along with clavulanic acid compared to the antibiotic alone. Final confirmation required positive results for both cefotaxime and ceftazidime disk pairs. Klebsiella pneumoniae (ATCC 700603) served as the positive control and E. coli (ATCC 25922) as the negative control. All culture media were obtained from Merck (Germany).
Genotypic identification of blaTEM-positive strains
The expression of blaTEM genes was investigated using polymerase chain reaction (PCR). Bacterial DNA was extracted using a kit (Invitek Stratec Business, Canada) and quantified by a Nanodrop device, and its purity was assessed by calculating the OD260/280 ratio. Ratios between 1.8 and 1.9 indicated acceptable DNA purity. PCR reactions were carried out using 2x master mix reactions from Sinaclon (Iran), containing 12.5 µL master mix, 1 µL forward primer (10 pmol), 1 µL reverse primer (10 pmol), 3 µL template DNA, and 7.5 µL nuclease-free distilled water. Specific primers for blaTEM gene amplification were designed (Table 1). The primers were designed using nucleotide databases (National Center for Biotechnology Information) and Allele ID7 software, and their sequences were verified. PCR started with a 10-minute initial denaturation phase at 95°C. This was followed by 30 amplification cycles, encompassing 30 seconds of denaturation at 95°C, 60 seconds of annealing at 50°C, and 60 seconds of extension at 72°C. To ensure complete amplification, a final extension step was carried out for five minutes at 72°C. The negative control tube contained distilled water. A 1.5% agarose gel was used for electrophoresis of PCR products. The size marker included a 100-base pair (bp) ladder. V2 Safe Stain was applied to the gel, and a gel documentation system was used to visualize DNA bands. Associations between genotypic and phenotypic data were evaluated using the chi-square test implemented via Statistical Package for the Social Sciences (SPSS) software version 23 at a statistical significance threshold of P < 0.05.
A rod-shaped, aerobic, Gram-negative bacterium with pili and a polar flagellum, Pseudomonas aeruginosa (P. aeruginosa) is a serious health concern in hospitals. Both pili and the flagellum are essential for the movement and host cell attachment of bacteria. Especially in burn wounds, P. aeruginosa is one of the most prevalent organisms causing nosocomial infections (1), including pneumonia and infections in immunocompromised patients or individuals with structural lung diseases such as cystic fibrosis (2). Burn patients face an elevated risk of serious infections and related mortality due to impaired skin barrier function and immune suppression (3). Eradication of P. aeruginosa infecting burn wounds is a daunting task because of the bacterium’s intrinsic and acquired resistance to antibiotics (4). Antibiotic-resistant P. aeruginosa has become a global problem, posing a serious public health threat associated with limited treatment options and increasing mortality rates (5). Antimicrobial resistance (AMR) is defined as the ability of pathogenic bacteria to resist prescribed drugs, such as β-lactams. In Gram-negative bacteria, this resistance often arises from the production of extended-spectrum β-lactamases (ESBLs), which can inactivate β-lactam antibiotics (6). Among the most extensively studied AMR enzymes are β-lactamases (7), which are classified based on their inhibitory responses and substrate spectra. ESBLs encode class A β-lactamases, leading to resistance against certain β-lactam antibiotics (8). These enzymes, encoded by bla genes in Gram-negative bacteria, are responsible for one of the most critical mechanisms of antibiotic resistance (i.e., decapitating β-lactam antibiotics through degrading the β-lactam ring). Aggravating this threat, bla genes exhibit a high potential for horizontal gene transfer and integration into multidrug-resistant plasmids, promoting the rapid spread of resistance in clinical settings (9). The clinical challenge of beta-lactam resistance among Gram-negative bacteria, largely driven by beta-lactamase production, has spurred significant pharmaceutical innovations. The development of novel compounds designed to inhibit or disrupt these enzymes has led to groundbreaking accomplishments in expanding therapeutic interventions (10). This study aimed to accurately identify P. aeruginosa strains, determine their antibiotic resistance patterns, and investigate the prevalence of the blaTEM gene in burn patients in Tabriz.
Methods
Sampling and bacterial isolation
A descriptive cross-sectional study was conducted utilizing a convenience sampling method to recruit 100 burn patients admitted to the burn units of Tabriz hospitals over the course of six months from April to September 2024. Using sterile swabs, samples were obtained from burn wounds and immediately placed into Cary-Blair transport media. To maintain optimal conditions for bacteria survival, samples were transferred to the laboratory on ice and processed within a strict 2-hour window from the time of collection.
Inclusion criteria in this study included being adult patients hospitalized due to burn wounds in medical centers across Tabriz, northwest Iran, and showing clinical signs of active infections (Such as purulent discharge, erythema, localized warmth, or systemic fever). Furthermore, enrollment was contingent upon obtaining written informed consent from patients or their legal guardians. The definitive microbiological confirmation of P. aeruginosa infection, isolated from burn wound samples collected in a sterile manner, through standard cultural characteristics (e.g., grape-like odor, pyocyanin production), biochemical profiling (Oxidase-positive), and automated systems was mandatory for inclusion in the final analysis. Samples were initially cultured on sheep blood agar (Containing 5% sheep blood) to recognize hemolytic patterns and on MacConkey agar to selectively isolate Gram-negative bacteria, followed by incubation at 37°C for 24-48 hours. Suspected P. aeruginosa colonies were selected based on characteristic morphology: large size, smooth surface, irregular margins, and distinctive green-blue pigmentation (Pyocyanin). Final identification was confirmed through a series of standard biochemical tests. These included a positive oxidase reaction, which is a key diagnostic trait for P. aeruginosa, the ability to oxidize but not ferment glucose in Oxidation-Fermentation (OF) medium, alkaline/alkaline (K/K) reaction in Kligler Iron Agar (KIA) indicative of non-fermentative metabolism, positive citrate utilization, pyocyanin pigment production, and the ability to grow at 42°C (A trait that helps differentiate P. aeruginosa from other pseudomonads). Additional tests, such as gelatin hydrolysis and urease activity, were performed for differentiation from similar species. Using P. aeruginosa American Type Culture Collection-ATCC: 27853 as a positive control, all processes were verified. Confirmed isolates were kept at -80°C in Tryptic Soy Broth with 15% glycerol.
Exclusion criteria included unwillingness to continue cooperation, unconfirmed laboratory detection of P. aeruginosa, and the presence of severe underlying diseases (e.g., uncontrolled diabetes, metastatic cancer, or advanced autoimmune disorders). The study was conducted in compliance with biosafety principles and ethical protocols and acquired the approval code of IR.IAU.TABRIZ.REC.1403.209.
Antibiotic susceptibility testing by disk diffusion method
The standard agar disk diffusion technique (Kirby-Bauer) was used according to clinical and laboratory standards institute (CLSI) protocols. Mueller-Hinton agar and antibiotic disks manufactured by Padten Teb (Iran) were employed in this study. Target antibiotics included β-lactams such as piperacillin (100 μg) and ceftazidime (30 μg); aminoglycosides, including gentamicin (10 μg), tobramycin (10 μg), and amikacin (30 μg); fluoroquinolones such as ciprofloxacin (5 μg) and levofloxacin (5 μg); and other groups, including cefepime (30 μg) and meropenem (10 μg). For quality control, standard strains of Escherichia coli (E. coli) (ATCC: 25922) and P. aeruginosa (ATCC: 27853) were used in each test batch. Results were interpreted based on CLSI guidelines.
Phenotypic identification of β-lactamase-producing strains
In this study, ESBL-producing isolates were identified using the standard Combination Disk test in accordance with CLSI protocols. The antibiotic disks of cefotaxime (30 μg), cefotaxime plus clavulanic acid (10 μg), ceftazidime (30 μg), and ceftazidime plus clavulanic acid (10 μg) (Mast Group, UK) were employed. Freshly prepared Mueller-Hinton agar was used as the culture medium. A bacterial suspension with a standard concentration of 0.5 McFarland (~1.5×10^8 CFU/ml) was prepared and uniformly inoculated onto the culture medium. ESBL production was recognized based on a ≥5 mm increase in the inhibition zone diameter for antibiotic disks used along with clavulanic acid compared to the antibiotic alone. Final confirmation required positive results for both cefotaxime and ceftazidime disk pairs. Klebsiella pneumoniae (ATCC 700603) served as the positive control and E. coli (ATCC 25922) as the negative control. All culture media were obtained from Merck (Germany).
Genotypic identification of blaTEM-positive strains
The expression of blaTEM genes was investigated using polymerase chain reaction (PCR). Bacterial DNA was extracted using a kit (Invitek Stratec Business, Canada) and quantified by a Nanodrop device, and its purity was assessed by calculating the OD260/280 ratio. Ratios between 1.8 and 1.9 indicated acceptable DNA purity. PCR reactions were carried out using 2x master mix reactions from Sinaclon (Iran), containing 12.5 µL master mix, 1 µL forward primer (10 pmol), 1 µL reverse primer (10 pmol), 3 µL template DNA, and 7.5 µL nuclease-free distilled water. Specific primers for blaTEM gene amplification were designed (Table 1). The primers were designed using nucleotide databases (National Center for Biotechnology Information) and Allele ID7 software, and their sequences were verified. PCR started with a 10-minute initial denaturation phase at 95°C. This was followed by 30 amplification cycles, encompassing 30 seconds of denaturation at 95°C, 60 seconds of annealing at 50°C, and 60 seconds of extension at 72°C. To ensure complete amplification, a final extension step was carried out for five minutes at 72°C. The negative control tube contained distilled water. A 1.5% agarose gel was used for electrophoresis of PCR products. The size marker included a 100-base pair (bp) ladder. V2 Safe Stain was applied to the gel, and a gel documentation system was used to visualize DNA bands. Associations between genotypic and phenotypic data were evaluated using the chi-square test implemented via Statistical Package for the Social Sciences (SPSS) software version 23 at a statistical significance threshold of P < 0.05.
|
Table 1. Primers used in this study
.PNG) |
Results
In this study, 100 isolates of P. aeruginosa were examined. The mean age of patients was 45.9 ± 16.12 years, ranging from 20 to 78 years. There were 38 females (38%) with a mean age of 49.61 ± 16.33 years and 62 males (62%) with a mean age of 39.84 ± 16.52 years. Statistical analysis showed no significant difference in the distribution of isolates between different age and gender groups (P-Value > 0.05). Antibiogram results revealed a high prevalence of resistance to levofloxacin (97%), meropenem (92%), ciprofloxacin (88%), and tobramycin (87%), while the lowest rate of resistance was related to ceftazidime (69%) (Figure 1). The combined disk test identified 58 ESBL-positive isolates (58%), including 22 isolates from females (37.93%) with a mean age of 45.45 ± 15.15 years and 36 isolates from males (62.07%) with a mean age of 39.30 ± 14.11 years. Among 58 ESBL-positive isolates, 29 (50%) carried the blaTEM gene (Figure 2). No significant association was found between ESBL or blaTEM status and patients’ age or gender (P-Value > 0.05). Neither isolate distribution frequencies nor antibiotic resistance patterns were significantly associated with demographic variables. Nonetheless, the high frequency of antibiotic resistance and blaTEM positivity (50% of ESBL-positive isolates) highlights the necessity of ongoing surveillance for antibiotic resistance and infection control initiatives.
|
Figure 1. Antibiotic resistance of Pseudomonas aeruginosa isolates from burn patients in hospitals of Tabriz
.PNG) |
|
Figure 2. PCR product electrophoresis for the blaTEM gene. M: 100-bp marker, PC: positive control, NC: Negative control, 1 and 10: blaTEM positive samples
.PNG) |
Discussion
One of the main sources of hospital-acquired infections in burn patients is the opportunistic Gram-negative bacterium P. aeruginosa. Since P. aeruginosa can acquire extensive antibiotic resistance, treating infections caused by this bacterium has become very challenging (11). Investigating the antibiotic resistance pattern of P. aeruginosa in the hospital setting and determining its susceptibility to commonly used antimicrobials can help establish initial treatment protocols and effectively manage drug-resistant infections caused by this bacterium (12). P. aeruginosa is naturally resistant to several antimicrobial agents, making its control difficult. Various mechanisms contribute to this resistance (13), including producing beta-lactamase enzymes, resulting in resistance to beta-lactam antibiotics (14). The significant increase in ESBL-positive P. aeruginosa strains among burn patients has posed therapeutic challenges (15,16). The rate of organisms producing blaTEM enzymes continues to rise, and treating infections caused by these bacteria remains highly problematic due to the antibiotic resistance conferred by these enzymes (17). In previous studies, samples had been obtained from various sources such as urine, blood, infected wounds, and urinary tract infections (18). However, in this study, all clinical samples were collected from patients admitted to the burn unit. Among 100 isolates obtained from patients, 87 were resistant to at least three types of antibiotics. According to the findings of Abdelrahim et al. (2023), blaTEM was reported as the second most prevalent gene (46.2%) in ESBL-producing P. aeruginosa strains (19). The present study also identified this gene as one of the most prevalent resistance genes, with a frequency of 50%. In a study by Peymani et al. conducted in Tehran, the frequency of ESBL-producing P. aeruginosa strains was reported as 28.6% (20), which is lower than the rate recorded in the present study.
In contrast to a study by Chen et al., where ESBL-positive P. aeruginosa strains showed the highest resistance to ceftazidime (30%) (21), we found the lowest resistance rate (69%) to this antibiotic. Bokaeian et al. in Zahedan reported that 25.86% of 116 isolates were resistant to at least one cephalosporin or aztreonam antibiotic (22). This finding differs from the results of the current study in Tabriz, which demonstrated the highest resistance rates to levofloxacin (97%), meropenem (92%), ciprofloxacin (88%), and tobramycin (87%); however, both studies confirmed a similar pattern of antibiotic resistance. In the present study, among 100 P. aeruginosa strains isolated from burn patients, 97 were resistant to levofloxacin, while the lowest resistance rate was observed against ceftazidime (69%). However, in a 2010 study by Fazeli et al. in Isfahan, 94% of P. aeruginosa isolates exhibited complete resistance to ceftazidime (23), a discrepancy with our results. Bahrami et al. (2018) in Bandar Abbas found 47% resistance to meropenem among P. aeruginosa isolates (24), whereas this rate increased to 92% in the present study. Additionally, Bahrami et al. identified blaTEM as the most common beta-lactamase resistance gene with a frequency of 56.3% by PCR, which was relatively consistent with the current study’s 50% (24). Saleh et al. (2022) reported a 41% resistance rate to meropenem (25), whereas our data revealed a dramatic rise to 92%, indicating a concerning expansion of carbapenem resistance over the past two decades. Adjei et al. (2018) reported an 82% resistance rate to meropenem (26), while Haghighifar et al. (2021) observed an increase to 91.3% (27). The results of the current study showed that meropenem resistance was quite prevalent in the populations under investigation, which was in close agreement with both above studies. The discrepancies between our findings and those of previous studies likely root in several factors, including variations in sample size, the genetic diversity of local microbial populations, and distinct regional epidemiological patterns. Importantly, these same factors, particularly our limited sample size and the specific geographical focus of our work on Tabriz population, represent the key limitations of the present study and should be considered when interpreting the results. Consequently, larger multi-center cohorts are warranted in the future to validate and generalize these findings.
Conclusion
This study revealed a concerning level of antibiotic resistance among P. aeruginosa isolates from burn patients in Tabriz, with over 90% resistance to meropenem and fluoroquinolones. Also, 58% of the isolates were identified as ESBL producers. Notably, half of ESBL-positive strains harbored the blaTEM gene, highlighting the high penetrance of this resistance mechanism. Despite the lack of demographic correlations, the high prevalence of multidrug-resistant strains represents a significant public health challenge. These findings underscore the urgent need for regular antibiotic resistance surveillance, strict infection control protocols, and evidence-based antimicrobial stewardship practices in burn units. Future research should focus on longitudinal monitoring of resistance trends and investigating other genetic elements of resistance, such as other ESBL and carbapenemase genes, to guide targeted interventions and reduce the spread of resistant bacterial strains.
List of abbreviation
P. aeruginosa: Pseudomonas aeruginosa, ESBLs: Extended-Spectrum β-Lactamases, AMR: Antimicrobial Resistance, ATCC: American Type Culture Collection, CLSI: Clinical and Laboratory Standards Institute, E. coli: Escherichia coli, PCR: Polymerase Chain Reaction.
Acknowledgement
The authors sincerely thank the Vice Chancellery for Research at the Islamic Azad University, Tabriz Medical Branch, for supplying the laboratory facilities required for this study.
Funding sources
The authors received no financial support for the research, authorship, and/or publication of this article.
Ethical statement
Ethical approval was obtained from the Clinical Research Ethics Committee of Islamic Azad University, Tabriz Branch (Code: IR.IAU.TABRIZ.REC.1403.209). All participants were informed about the study’s objectives and signed written informed consent before enrollment.
Conflicts of interest
The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.
Author contributions
Concept/Design: A.J.S, M.P; Data acquisition: A.J.S; Data analysis and Interpretation: Z.G, A.G; Drafting the manuscript: Z.G, A.G, M.G.K.J, K.H.K, K.S, M.M.S.F; Critical revision of the manuscript: AJS; Final approval and Accountability: MP; Technical or Material support: A.J.S, M.P; Supervision: MP; Securing funding (If available): N/A.
Data availability statement
The data supporting the findings of this study can be obtained from the corresponding author upon a reasonable request.
Type of Article: Original article |
Subject:
Basic Medical Sciences
Received: 2024/07/10 | Accepted: 2025/02/28
Received: 2024/07/10 | Accepted: 2025/02/28
References
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