Molecular Analysis of Oxacillinase Genes and Identification of Drug Resistance Pattern in MDR Strains of Acinetobacter baumannii Isolated from Burn Wound Samples in Kermanshah, Iran
Correspondence Address :
Kamal Ahmadi,
Research Assistant, Department of Microbiology, Kermanshah University of Medical Sciences and Ph.D. Student of Medical Bacteriology, Pasteur Institute of Iran, Kermanshah, Iran.
E-mail: k_ahmadi@pasteur.ac.ir; kamal.ahmadi55@yahoo.com
Introduction: Carbapenem Resistant Acinetobacter Baumannii (CRAB) is a dangerous nosocomial pathogen that can cause high mortality in patients. This bacterium has a remarkable ability to acquire various resistance mechanisms due to this it is considered as one of the health priorities.
Aim: To investigate the prevalence of the OXA-23, OXA-24, and OXA-58 genes in Acinetobacter baumannii isolates collected from burn wound samples in Kermanshah, Iran.
Materials and Methods: A cross-sectional study was done during 11 months period from December 2018 to October 2019, 74 A. baumannii isolates were collected from those admitted to the Burns Unit of Imam Khomeini Hospital in Kermanshah, Iran. The 74 A. baumannii isolates were detected using particular bacteriological methods. Following determination of the antibiotic sensitivity of the specimens using the disk diffusion technique, polymerase chain reaction was performed to determine the frequency of the OXA-23, OXA-24, and OXA-58 genes using their specific primers. Data were analysed using Fisher’s-exact test and Chi-squared test in Statistical Package for the Social Sciences (SPSS) version 20.0. A p-value <0.05 was considered statistically significant.
Results: All the 74 A. baumannii isolates were Multidrug-Resistant (MDR) (41 from males and 33 from females). The highest drug resistance was against cefotaxime (100%) and piperacillin (98.6%), while all the isolates were sensitive to polymyxin B and colistin. Oxacillinase genes with the highest and lowest frequencies were OXA-23 (64.7%) and OXA-58 (3.5%), respectively. The highest frequency of isolates with two genes were related to OXA-23 and OXA-24. A significant relationship was observed among the existence of oxacillinase genes and resistance to some antibiotics.
Conclusion: The results of this study indicated the significance of OXA carbapenemase genes in burn patients. Due to the high drug resistance of A. baumannii isolates collected from wound samples, the identification of carbapenemase-producing A. baumannii isolates is paramount in developing prevention and control programs for these drug-resistant isolates.
Antibiotic sensitivity, Carbapenemase genes, Multidrug resistance
Acinetobacter is a gram negative, aerobic, forced aerobic coccobacillus capable of growing in a variety of environments (1). A. baumannii, as the most common species of this bacterium, can cause urinary tract infections, pneumonia, sepsis, skin and wound infections, meningitis, endocarditis, and peritonitis (2). Infections caused by this bacterium pose a serious challenge to the treatment process followed in burn patients due to the increased resistance to various antimicrobial agents (3),(4). In recent years, A. baumannii has been reported to be a major cause of nosocomial infections in burn patients. Burn cases are sensitive to infections because of damage to the skin and, subsequently, immune system disorders (5),(6). A.baumannii has been introduced as the second MDR bacterium causing nosocomial infections in burn patients (7).
Long-term hospitalisation, Intensive Care Unit (ICU) admission, surgery, burns, serious illness, immunosuppression, exposure to antimicrobial agents, use of central venous catheter, and other factors can lead to colonisation or infection caused by this bacterium (8). Acinetobacter baumannii has a high resistance to a variety of antibiotics; this resistance is either inherent or acquired through resistance genetic factors, including resistance genes present on mobile genetic elements such as transposons and integrons (9). Carbapenems are among the beta-lactam antibiotics with extensive activity in the treatment of bacterial infections, especially severe and life-threatening infections (10). In recent years, the presence of Carbapenem-Resistant Acinetobacter Baumannii (CRAB) isolates, including imipenem, has increased significantly, and most of these isolates are Multidrug-Resistant (MDR). The frequency of CRAB isolates is a serious problem in burn patients (11),(12). Lack of proper management in the antibiotic treatment of infections caused by these isolates can cause strains with Extensively Drug Resistant (XDR) and Pandrug Resistant (PDR). The ability of these drug-resistant isolates to hydrolyse carbapenems through carbapenemase enzymes is one of the most common and significant underlying mechanisms of their resistance to carbapenems, with Ambler class D enzymes called oxacillinases (OXA-type) being the most common among all A. baumannii isolates (5),(7),(13).
The OXA-type carbapenemases can be divided into eight subgroups or branches: OXA-23, OXA-24, OXA-40, OXA-58, OXA-143, OXA-235, and OXA-51, which are the most commonly identified subclasses of OXA in A. baumannii isolates (14),(15). Only OXA-51 is naturally present in A. baumannii, and other OXA genes are acquired by the bacterium (16). Thus, the identification of A. baumannii isolates producing carbapenemase genes is paramount in developing prevention and control programs for these drug-resistant isolates. Considering the importance of determining carbapenem-resistant A. baumannii isolates and the fact that in recent years no study has been performed in this field in Kermanshah, Iran to determine the frequency of OXA genes of this bacterium, the present study aimed at determining the frequency of the OXA-58, OXA-24, and OXA-23 genes and antibiotic-resistance pattern in MDR A. baumannii isolates separated from clinical samples of Imam Khomeini Hospital.
This cross-sectional descriptive study was conducted in a period of 11 months (December 2018 to October 2019), all burn wound samples (374 burn wound samples) were collected from patients admitted to the burn ward of Imam Khomeini Hospital in Kermanshah, Iran. Then, after microbiological studies and culture of the samples, 74 isolates of A. baumannii were isolated. Informed consent was obtained from patients in this study. The Ethics Committee of Kermanshah University of Medical Sciences, Kermanshah, Iran (approval code no.: 1395.621) confirmed this study.
Inclusion criteria: All MDR isolates of A.baumannii separated from patients who had not consumed any antibiotic a week before being hospitalised based on their report and file in given time period were included in the study.
Exclusion criteria: Other bacterial isolates separated from patient’s burn samples and other strains Acinetobacter separated from the samples were excluded from the study (17).
Procedure
All the samples in this study were the specimens of the burn wound. After collection, these samples were cultured on MacConkey agar and blood agar media (Indian media) and incubated for 1-2 days at 37°C under laboratory conditions. Then, to identify A. baumannii isolates, biochemical tests, including the growth of slant/alkaline butt pattern on Triple Sugar Iron (TSI) medium (Merck, Germany), oxidase and catalase negative test, immobility on Sulfide Indole Motility (SIM) medium (Merck, Germany), and no pigment production, were performed. Polymerase Chain Reaction (PCR) of the OXA-51 gene was used for the final confirmation of possible isolates of A. baumannii using its specific primer. A total of 74 A. baumannii isolates were identified. According to the Clinical and Laboratory Standards Institute (CLSI) instructions, antibiotic-sensitivity evaluation was performed by the disk diffusion method (Kirby-Bauer), by bacterial suspension equivalent to half McFarland turbidity (1.5×108 CFU/mL) and Müller-Hinton agar culture medium (media India) for antibiotic discs (MAST.UK), including amikacin (30 μg), gentamycin (10 μg), ceftazidime (30 μg), tobramycin (10 μg), ciprofloxacin (5 μg), meropenem (10 μg), levofloxacin (10 μg), imipenem (10 μg), cefepime (5 μg), polymyxin B (300 units), piperacillin (100 μg), colistin (25 μg), ampicillin-sulbactam (10 μg), and cefotaxime (30 μg). The suspension of isolated bacteria was first cultured on Müller-Hinton agar medium after comparison with the McFarland 0.5 standard. After placing the antibiotic disks and incubating at 37°C for one day, the diameter of their growth inhibition zone was evaluated and compared to that mentioned in the CLSI tables (18).
For quality control, standard strains of A. baumannii ATCC 19606 and Escherichia coli ATCC 25922 were used. Acinetobacter isolates with resistance to three or more groups of antibiotics were determined to be MDR strains. The PCR was performed to identify the presence of the OXA-58, OXA-24, and OXA-23 genes using specific primers (Takapou Zist Co., Iran) and according to (Table/Fig 1) (19). After that, the standard strains A. baumannii NCTC 13304, NCTC 13302 and NCTC 13305 were utilised as positive controls to detect the OXA-23, OXA-24 and OXA-58 genes, respectively. The boiling method was used to extract chromosomal Deoxyribonucleic Acid (DNA) of the isolates. In doing so, after culturing A. baumannii, we dissolved several pure bacterial colonies in 0.5 mL of sterile distilled water, and after 5 min of boiling and cooling in the next step, they were centrifuged at 7000 gm for 1 min. Afterward, the solution was transferred to new Eppendorf tubes as bacterial DNA to perform PCR. Then, using NanoDrop Synergy HTX (Bio Tek Instrument, Inc. Highland Park, USA), concentrations of DNA were measured at the Optical Density (OD) of 260 nm to be 33 pmol/μL, and DNA purity at the OD of 260/280 nm was calculated to be 1.85. PCR was performed with a final volume of 25 μL, including 12.5 μL of master mix, 1 μL of each primer, 3 μL of bacterial DNA, and sterile distilled water up to a volume of 25 μL. PCR reaction was performed separately for each of the oxacillinase genes. The PCR reaction temperature included primary denaturation at 94°C for 5 minutes, and followed by 35 main cycles, according to (Table/Fig 1) and the eventual extension at 72°C for 6 minutes (19). Finally, using 1.5% agarose gel and ethidium bromide staining under UV radiation in a gel doc device with a voltage of 80 V for 50 minutes, the PCR products were evaluated.
Statistical Analysis
Data were analysed using Chi-square test in Statistical Package for the Social Sciences (SPSS) version 20.0. A p-value ≤0.05 was considered statistically significant.
In this study, 74 isolates of A. baumannii were investigated {41 (55.4%) from males and 33 (44.6%) from females} in the age group of 8-71 years, with a mean age of 44.22±14.55 years. All isolates of this bacterium were collected from burn wound samples. According to (Table/Fig 2), the highest drug resistance of A. baumannii isolates was against cefotaxime (100%) and piperacillin (98.6%), and all samples were susceptible to polymyxin B and colistin (0). All the 74 A. baumannii isolates (100%) were found to be MDR. In addition, total 85 OXA genes were found from 74 isolates of A. baumannii. The highest and lowest frequencies of OXA genes were related to OXA-23 55 (64.7%) and OXA-58 3 (3.5%), respectively.
The frequency of the OXA-24 gene was 21.2% (18 isolates). The total number of isolates with two genes simultaneously was (9, 10.6%), among which the highest frequency was related to isolates with two genes OXA-23 and OXA-24 with a frequency of eight cases. None of the isolates had all the three genes present simultaneously (Table/Fig 3). The presence of OXA genes and resistance to some antibiotics, including the presence of the OXA-23 gene and resistance to carbapenems, amikacin, and ampicillin/sulbactam, as well as the presence of the OXA-58 gene and resistance to levofloxacin and amikacin, showed a significant relationship (Table/Fig 4). The PCR results for OXA genes are shown in (Table/Fig 5).
A.baumannii is a major hospital pathogens, found especially in burn patients, which causes a high mortality in these patients (16). The reason for this is the ability of this microorganism to survive in hospital environments and to acquire a mechanism of resistance against antimicrobial agents (20). The prevalence of MDR A. baumannii isolates has been regarded as a serious concern worldwide (9). In the present study, all 74 (100%) A. baumannii isolates were found to be MDR. In studies conducted in Iran, the prevalence of MDR in burn patients was reported to be between 97.9% and 100%, which was consistent with the current study findings (21),(22),(23),(24),(25),(26). Similar results related to these MDR A. baumannii isolates have been reported in other studies abroad. In 2020, a study was performed on burn samples by Mabrouk A et al., where in all 21 A. baumannii isolates were identified to be MDR, which was consistent with present study (27). However, in few other studies, the prevalence of MDR was found to be lower than the present study (28),(29),(30).
Among all MDR isolates examined in the current study, cefotaxime (100%) presented the maximum antibiotic resistance, followed by piperacillin (98.6%), while all samples were susceptible sensitive to colistin and polymyxin B. Other national and international research reported enhanced resistance in MDR samples to various antibiotics such as cefotaxime, piperacillin, imipenem, and meropenem (28),(29),(31). In the study by Sarhaddi N et al., all MDR isolates were found to be sensitive to colistin and polymyxin B (32). According to these results, it can be concluded that these antibiotics are still effective in the treatment of infections caused by this bacterium, so their use should be controlled.
Recently, the prevalence of carbapenem-resistant isolates, including imipenem, has increased significantly, and most of these isolates are MDR. A frequent and principal cause of resistance to carbapenem antibiotics is their ability to hydrolyse carbapenems through carbapenemase enzymes, with Ambler class D enzymes called oxacillin (OXA-type) being the most common among A. baumannii isolates (13).
The most common A. baumannii carbapenemase genes involved in carbapenem resistance are OXA-23, OXA-24, and OXA-58. The frequencies of the OXA-23, OXA-24, and OXA-58 genes among the 74 isolates of A. baumannii collected from burn samples were determined to be 64.7%, 21.2%, and 3.5%, respectively. In two studies, conducted in Iran, including the present study, the prevalence of OXA-23 was higher than OXA-24 and OXA-58 (6),(11),(21).
Tafreshi N et al., reported the prevalence of these three genes at 53.57%, 41.66%, and 30.59% in A. baumannii isolates collected from burn samples, respectively (22). Mohajeri P et al., reported the frequencies of 77.9%, 19.2%, and 0% for OXA-23, OXA-24, and OXA-58, respectively, indicating that the prevalence of OXA-23 was much higher than the other genes, which was consistent with the present study findings (33). The results of the current study were compared with other previous studies in Iran and other countries (Table/Fig 6) (11),(22),(23),(24),(28),(29),(31),(32),(34),(35),(36),(37). In the present study, the number of isolates carrying the two genes OXA-23 and OXA-24, which had the highest frequencies, was equal to 8 (9.4%) out of 85, and the combination of these two genes always showed resistance or reduced sensitivity to antibiotics (30).
Among the reasons for the differences in the reports on the frequency of these genes include the diversity in the pattern of antibiotic use and appropriate control strategies in different wards of hospitals. The OXA-58 gene produces a broad-spectrum class D beta-lactamase that can hydrolyse penicillin, oxacillin, and imipenem. The results of this study showed a significant relationship between the presence of the OXA-58 gene and resistance to levofloxacin and amikacin. Of the 55 strains resistant to imipenem and meropenem, all carried the OXA-23 gene. The OXA-23 gene produces a carbapenem-hydrolysing beta-lactamase that promotes resistance to imipenem and meropenem (14). Therefore, the reason for carbapenem resistance can be the high prevalence of these carbapenem genes. There is a significant link between the presence of the OXA-23 gene and resistance to carbapenems, amikacin, and ampicillin/sulbactam.
Limitation(s)
The limitations of this study was the sample small size examined and and lack of access to patient files.
In this study, A. baumannii showed resistance to most of the available antibiotics and it also appears that colistin and polymyxin B are currently the only antibiotics effective in treating infections caused by this bacterium. Therefore, it is necessary to pay more attention to controlling the use of these antibiotics in nosocomial infections. The results of this study show the importance of OXA-type carbapenemases in treating burn patients. Therefore, the identification of A. baumannii isolates producing carbapenemase genes is paramount in the development of prevention and control programs for these drug-resistant isolates.
Authors appreciate the support extended by the Deputy of Research and Technology and the Clinical Research Development Unit of Imam Reza Hospital affiliated to Kermanshah University of Medical Sciences, Kermanshah, Iran (No. 96082).
DOI: 10.7860/JCDR/2022/53232.16337
Date of Submission: Nov 09, 2021
Date of Peer Review: Dec 30, 2021
Date of Acceptance: Feb 04, 2022
Date of Publishing: May 01, 2022
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA
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