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Identification of different faces of Pseudomonas aeruginosa isolates in burn patients by genetic fingerprinting
Gene Reports 15 (2019) 100377
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Gene Reports journal homepage: www.elsevier.com/locate/genrep
Identification of different faces of Pseudomonas aeruginosa isolates in burn patients by genetic fingerprinting
T
Amir Emamia, , Abdolkhalegh Keshavarzib, Neda Pirbonyeha, Mahrokh Rajaee Behbahania ⁎
a b
Burn & Wound Healing Research Center, Microbiology Department, Shiraz University of Medical Sciences, Shiraz, Iran Burn & Wound Healing Research Center, Surgery Department, Shiraz University of Medical Sciences, Shiraz, Iran
ARTICLE INFO
ABSTRACT
Keywords: Pseudomonas aeruginosa RAPD-PCR Antibiotic resistant Burn
Pseudomonas aeruginosa, as a prevalent infection in hospital settings, has a different form of performance and response to anti-bacterial agents. Categorizing of infections based on their genome structure may be helpful in control and treatment of prevalent infections. Total of 278 burn patients were evaluated (March 2016 to March 2017) at Amir-Al-Momenin burn hospital (SUMS) for P. aeruginosa by standard microbiological tests. Confirmed isolates were categorized by RAPD-PCR technique following the evaluation for antibacterial susceptibility tests. RAPD finger print results were analyzed by Gelj software (V.2). Based on the experiment results total of 47 (33.1%) P. aeruginosa were isolated. According to the antibiogram results, > 38% were resistant to all available antimicrobials except Colistin, while 32% were multi-drug resistant. In RAPD results, 8 patterns were identified with primer 272 and 5 with 277. Patterns 4, 2 in primer 272 and 4, 3 in primer 272 were the most prevalent patterns. Results showed that primer 277 had more power in categorizing the isolates for defining antibiotic sensitive pattern. In this category, sensitive isolates (> 95%) were in patterns 2 and 3, while more resistant isolates (> 90%) were in patterns 4 and 5. Based on the results of the study, it was found that fingerprinting with primer 272 had the ability to categorize P. aeruginosa isolates for introducing antibiotic pattern, while this result must be confirmed by more different species and techniques in other centers.
1. Introduction Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous gram-negative bacterium which is one of the top three causes of infections in hospital settings (Nanvazadeh et al., 2013; Ramos et al., 2013). Nutrient and moisture environments are suitable media for the growth of this bacterium. These conditions have caused the bacterium to be able to play a significant role in nosocomial infections; both the need of the bacterium and specifications of burn wounds are the leading cause of P. aeruginosa infections in burns (Emami et al., 2015; Hardalo and Edberg, 1997). Based on different studies, P. aeruginosa is a bacterium responsible for about > 20% of nosocomial infections which are more prevalent in immunosuppressed patients such as burns, cystic fibrosis, acute leukemia, organ transplants and intravenous drug addicts (Ramos et al., 2013; Emami et al., 2015). In a recent study in a burn center in the southwest of Iran (Amir-al-Momenin burn and wound healing center), it has been proved that P. aeruginosa is the most prevalent infectious agent in the center with the most prevalence in the wound
samples (Pirbonyeh et al., 2017). In another study performed in the same center on the isolates, it has been proved that different mechanisms of resistance works in P. aeruginosa isolates, which makes it more difficult to treat the infection in this category (Pirbonyeh et al., 2016). Although in many health centers, laboratories report infections in the genus, it has been proved that bacteria have species-specific function either. Because bacteria come from different sources in hospital environments, certainly they have different aspects and functional properties which are mainly related to their genomes. Today, there are molecular techniques used for genetic pattern detection of microorganisms which are less affected by environmental factors compared to phenotypic methods. These techniques play crucial roles in tracking the routes of pathogen transmission and different activities of a kind of species infection (Sing et al., 2006). Although there are different molecular techniques used for comparison and systematic epidemiological typing, such as Pulsed-field gel electrophoresis (PFGE) and Amplified fragment length polymorphism (AFLP) analysis, there is a low cost technique which has high discriminatory power and reproducibility
Abbreviations: RAPD, Random Amplified Polymorphic DNA; PFGE, Pulsed-field gel electrophoresis; P. aeruginosa, Pseudomonas aeruginosa; NNIS, National Nosocomial Infection Surveillance System; EMB, Eosin Methylene Blue ⁎ Corresponding author. E-mail address: [email protected] (A. Emami). https://doi.org/10.1016/j.genrep.2019.100377 Received 5 December 2018; Received in revised form 4 February 2019; Accepted 5 February 2019 Available online 07 February 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
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microbiology department of the research center (Burn and Wound Healing Research Center-SUMS) immediately. Cold chain condition was carried out for transferring of the samples. According to the previous results for this center (Emami et al., 2017) and the main aim of recent study, we focused on the most prevalent infectious agent (Pseudomonas aeruginosa) in our center. To this end, the collected samples were processed for aerobic Gram-negative bacteria on Eosin Methylene Blue (EMB) agar. According to the Pseudomonas aeruginosa specifications, after incubation time (37 °C for 20–24 h), colorless colonies were isolated and pure on new nutrient plates. For primary detection of the isolates, standard bacteriological tests were performed following Gram staining procedure. Gram-negative Rod bacteria with Alkaline/Alkaline result in triple sugar iron agar (TSI), Oxidase and catalase positive results and growth at 42 °C were screened as P. aeruginosa. For more species detection, the isolates were tested either by API 20E system according to the manufacturer's protocol (bioMerieux®SAfrance). To confirm the isolates as P. aeruginosa, molecular diagnostic technique for 16S rRNA with specific primers introduced in a previous study was performed (Emami et al., 2015).
named as Random Amplified Polymorphic DNA (RAPD) which is preferred in such studies (Rynga et al., 2015). RAPD reaction is a polymerase chain reaction (PCR) with some differences. In this kind of PCR, short (8-12mers) single primers are used which are randomly attached to several DNA sequences in the bacterial genome. This kind of amplification creates a repetitive electrophoresis pattern which can be used for genotypic differentiation of different isolates of a bacterial species. In this technique, if any changes or differences take a place in template DNA (especially at the site of primer annealing), the PCR product will change, resulting in a different pattern of amplified segments on the gel (Nanvazadeh et al., 2013; Abou-Dobara et al., 2010). While in many studies, particularly, numerous studies have been carried out at burn and wound healing research center affiliated with Shiraz University of Medical Sciences (SUMS) (Emami et al., 2015; Pirbonyeh et al., 2017; Pirbonyeh et al., 2016; Emami et al., 2017), it has been proved that although there are the same species of an infectious agent isolated from burn wounds, their activity in presenting aspects of infectious, antibiotic resistant pattern and resistant mechanisms is different. These different abilities of a kind of infection species make different appearances of an infectious agent; this has made us have different treatment policies for patients hospitalized in the same center. In this regard, knowing these aspects of microorganisms which are reflected from their genome structure will be helpful for infectious control in such centers. According to this view, the most prevalent infectious agent (P. aeruginosa) in Amir-Al-Momenin burn hospital was evaluated and gene mapping was performed with RAPDPCR technique. This study was performed to find different aspects of a kind of infection in order to investigate their relationship with drug resistance pattern, their transmission pathway and the source of infection. The results of the study could help to find the homology pattern of infection in different wards and have a group aspect for prevalent infection in our studied burn center with a practical antibiotic pattern.
2.4. Antibiogram test For anti-bacterial susceptibility tests, different groups of antibiotics were used. Antibiotics were selected from Carbapenems (Imipenem (10 μg), Meropenem (10 μg)), Aminoglycosides (Gentamicin (10 μg), Amikacin (30 μg)), Quinolones (Nalidixic acid (30 μg), Ciprofloxacin (5 μg)), Cephalosporins (Ceftazidime (30 μg), Macrolides (Erythromycin (15 μg)), Chloramphenicol (30 μg), and Polymyxin (Colistin (10 μg)). The antibiogram was performed by Kirby-Bauer (disk diffusion) method recommended by CLSI, 2016 manual ((CLSI) CaLSI, 2015). For this purpose, the surface of Muller-Hinton agar plates were inoculated with 0.5 McFarland suspensions of each isolate. Then, the studied disks were placed on plates with standard distances as mentioned in the manual. After the incubation time (22 h/35 °C), the inhibition zone diameters of each disc were measured in millimeters and reported as sensitive or resistant according to the guideline for the company. For Quality control Pseudomonas aeruginosa ATCC® 27853 were used as susceptible strain.
2. Material and methods 2.1. Sample collection
2.5. DNA purification and RAPD-PCR assay
This experimental study was performed in one year period (March 2016 to March 2017) at Amir-Al-Momenin burn hospital affiliated with SUMS. This study included patients from all 5 sections (Men, Women, Pediatrics, Surgery and Intensive Care Unit) of the hospital who had sustained deep burn with 2 or more degrees. Totally, 278 patients were admitted and hospitalized to the center during the study time. Considering that Amir-AlMomenin hospital in the south of Iran is the center of burn, the patients are referred from different regions of the south of our country. Demographic data of each patient including sex, age, burn degree and section of hospitalization were obtained from admission sheets of each patient.
To increase the efficiency of the extracted DNA's, phenol/chloroform extraction method was performed on overnight Tryptic Soy Broth (TSB) cultures of each confirmed isolate. Briefly, each sample was cultured in 1 ml of TSB (18 h/37 °C), incubated at 72 °C for 10 min and then cooled down to 4 °C for 5 min in 200 μl proteinase K (200 μg/ml). Following phenol/chloroform extraction (1:1), the bacterial DNA was precipitated with ethanol and the pellet was re-dissolved in DNase/RNase free, deionized water and stored at −20 °C for further tests. To perform the RAPD-PCR reaction, the following reaction was designed: The reaction was carried out in a 50 μl mixture containing 1× PCR buffer, 1.5 mM MgCl2, 0.1 mM each dNTP's, 1U Taq DNA polymerase, 0.5 pmol/μl of primer and 5 μl of extracted DNA. In this study, two primers (272 and 277), which were introduced before, were considered in separate reactions for each isolate (Mahenthiralingam et al., 1996). The PCR cycling was performed with the following setting: (i) 1 cycle at 94 °C for 5 min, 4 cycles at 36 °C for 5 min, and 5 min at 72 °C for each cycle (ii) 30 cycles, at 36 °C for 1 min, with 1 cycle at startup in 94 °C for 1 min and following with 2 min at 72 °C for each cycle. At the end of the cycles, the reaction was followed by a final extension step at 72 °C for 20 min. For detection of amplicons, PCR products were placed in 2% agarose gel wells and electrophoresed. The electrophoresis was performed with 1× TBE buffer at 10 V/cm for 2.5 h. A molecular weight marker (100 bp, Fermentase) was run in all gels beside the samples. At the end of electrophoresis, the gels were stained with ethidium bromide and visualized under UV transilluminator. The RAPD fingerprint results were analyzed by Gelj software (V.2). The pattern of migration variation in different gels was corrected with the standard molecular size marker.
2.2. Inclusion and exclusion criteria To maintain the structure of the study and analysis of the results related to each patient, one sample was collected from each hospitalized patient 3 to 5 days after hospitalization. All of the patients with heat burns, from both genders and all ages with degree two and more burns were included. Based on the National Nosocomial Infection Surveillance System (NNIS) role (System CN, 2003), patients with any criteria of infection in the first 48 h of hospitalization were excluded. Sampling was performed in accordance with the consent form taken at the time of hospitalization and all of the study process was approved by ethics committee code 1396-01-63-15170 from Shiraz University of Medical Sciences. 2.3. Sample processing For sampling, broad Z-stroke method was used for main wounds. Inoculated swabs were put in normal saline tubes and sent to the 2
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Table 1 Ward isolates pattern according to primer 272. Ward
Primer 272 pattern
ICU Men Pediatrics Surgery Women Total
Total
1
2
3
4
5
6
7
8
2 0 0 0 0 2
1 7 0 0 2 10
1 8 0 0 0 9
3 5 3 1 1 13
0 0 0 0 1 1
0 0 0 0 1 1
0 0 0 0 2 2
4 4 1 0 0 9
% Similarity
RAPD-PCR pattern
11 24 4 1 7 47
Group No.
Fig. 1. The results of P. aeruginosa antibiotics resistant pattern.
1
3. Results
2
The results of the 278 burns patients were evaluated in the five main wards of the burn hospital: men's ward 133 (47.8%), women's ward 78 (28.1%), pediatrics 21(7.6%), surgery 28 (10.1%) and intensive care unit (ICU) 18 (6.5%). The sex differentiation of patients was as follows: 170(61.2%) males and 108 females (38.8%). Patient's age ranged from 2 to 68 years with an average of 34.9 ± 14.6 years. From the total of 278 samples (one sample from each individual), 142(51.1%) were growth positive on EMB, while 47 (33.1%) of them and 16.9% from the total were confirmed as Pseudomonas aeruginosa in phenotypic and molecular tests. Out of 47 isolates of P. aeruginosa, the isolation result of each ward was: 24 (51.1%) from Men, 7 (14.9%) from Women, 11 (25.6%) from ICU, 4 (8.5%) from pediatrics and 1 (2.3%) from surgery wards. The results for the antibacterial resistant pattern are shown in Fig. 1. According to the antibiogram results, it has been shown that 18 (> 38%) isolates of P. aeruginosa were resistant to almost all available antimicrobials except for Colistin, while 15 (32%) isolates were known as multidrug resistant. Genetic fingerprinting of the isolates was performed by two RAPD primers. RAPD analysis was performed to compare the genetic background of P. aeruginosa isolates from different samples from different wards. A total of 8 patterns were identified with primer 272. RAPD patterns with > 90% identity were grouped. Due to space limitation, just one sample from each group is shown in the related dendrogram (Fig. 2), but the number of isolates for each pattern is written in the related table (Table 1). In another fingerprinting step by primer 277, a total of 5 patterns were identified. In this step, RAPD patterns with > 90% identity were grouped. In this step, either just one sample from each group was shown in the related dendrogram (Fig. 3). But the number of isolates for each pattern has been written in Table 2. According to the results for primer 272; patterns 4, 2, (3 and 8) are the most prevalent patterns respectively while according to the primer 277, patterns 4, 3, 5 are the most prevalent patterns, respectively. These results indicate that although P. aeruginosa isolates in our center are % Similarity
RAPD-PCR pattern
3 4 5 Fig. 3. RAPD-PCR gel electrophoresis analysis of P. aeruginosa clinical isolates with Primer 277. The dendrogram was generated by Gelj software with arithmetic mean using Dice coefficients (band tolerance 1.0%; optimization 1.0; cut-off value 90%). Table 2 Ward isolates pattern according to primer 277. Ward
Primer 277 pattern 1
ICU Men Pediatrics Surgery Women Total
0 4 1 0 0 5
2 5 0 0 0 0 5
Total 3 0 9 0 0 4 13
4 4 5 3 1 1 14
5 2 6 0 0 2 10
11 24 4 1 7 47
grouped in one species, they are categorized in different genotypes. According to the results of antimicrobial susceptibility tests, only in three groups of Carbapenems, Aminoglycosides, and Polymyxins, both resistant and sensitive isolates were seen and we were able to compare the results with genotyping groups. Based on Chi-square test a clear relation was found between primers typing and antibiotic pattern (P < 0.01) and analysis of the results for primer 272 typing, it was found that the isolates sensitive to antibiotics were more in genotype groups 2, 4, 3 and 1, respectively. The analysis of the results for primer 277 showed that sensitive isolates were more in genotype 3, 2 and 4, respectively. According to the total results, it was indicated that primer 272 had more power of genetic differentiation in P. aeruginosa isolates, but analysis of the isolates with primer 277 will be more helpful in antibiotic-resistant pattern. According to the sensitive results for the studied antibiotics and comparison of the results with primer 277 pattern, it was shown that more sensitive isolates (> 95%) were categorized in patterns 2 and 3, while more resistant isolates (> 90%) were categorized in patterns 4 and 5. Less than 5% of resistant and sensitive isolates were categorized in pattern one (Table 3).
Group No. 1 2 3 4 5 6 7 8
4. Discussion
Fig. 2. RAPD-PCR gel electrophoresis analysis of P. aeruginosa clinical isolates with Primer 272. The dendrogram was generated by Gelj software with arithmetic mean using Dice coefficients (band tolerance 1.0%; optimization 1.0; cut-off value 90%).
Based on current results and other studies in Iran it can be deduced that molecular typing can be used for finding different variations and examine the epidemiology of the infectious agents source and the 3
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used in the burn victims for source-tracking of P. aeruginosa, it can be used as supplementary test along with usual methods for categorizing the isolates in more limited patterns for using antibiotics more effectively and purposefully Considering the fact that in the present study, P. aeruginosa isolates had different antibiotic resistant pattern and different resistant mechanisms, and according to the studied molecular classification, we were able to classify them in more limited groups with a specific drug sensitivity pattern. According to the importance of antibiotic stewardship in the world and use of anti-microbial agents more targeted with sufficient outcome in infectious control in health systems, change in policies, especially in detection and categorization of the same microbial species for antibiotic treatment, is essential. Early detection of antimicrobial drugs revealed that there were effective compounds in controlling the incidence of infections that can be used in the treatment of infectious diseases.
Table 3 Antibiotic sensitive isolates pattern according to the primer 277. Antibiotic
Number of sensitive (%)
Primer 277 pattern 1
Gentamicin Amikacin Imipenem Meropenem Nalidixic acid Ciprofloxacin Ceftazidime Chloramphenicol Erythromycin Colistin
7 (14.9) 0 (0) 4 (8.5) 5 (10.6) 0 (0) 0 (0) 0 (0) 0 (0) 1 (2.1) 40 (85.1)
1 – – – – – – – – 1
2 4 – 3 4 – – – – – 32
3 2 – – 1 – – – – 1 6
4 – – – – – – – – – –
5 – – 1 – – – – – – 1
possibility of cross infection among patients (Nanvazadeh et al., 2013; Eftekhar et al., 2009; Vaez et al., 2015). Either molecular methods can be a proper candidate for evaluating the antibiotic resistant pattern of prevalent nosocomial infections in clinical settings for managing the Antibiotic stewardships. Base on different studies we checked the isolates with two routine primers 272 and 277. According to the results it was indicated that primer 272 had more power of genetic differentiation in P. aeruginosa isolates, while analysis of the isolates with primer 277 will be more helpful in antibiotic-resistant pattern. In the early detection of antimicrobial drugs, it was revealed that there were effective compounds in controlling the incidence of infections which can be used in the treatment of infectious diseases (Discovery and development of penicillin [internet], 1999). However, over the years, the inappropriate use of anti-microbial drugs has led to a selective pressure on resistance of microbial infections (Kolář et al., 2001). As we all know, while anti-microbial agents are used, only sensitive microorganisms are affected and resistant forms remain. It has been proved that selective pressure of anti-microbial agents differentiates different forms of microbial species according to their ability in response to such agents. While different anti-microbial agents are used in different infections, this pressure has occurred extensively and forms different aspects of microbial species by different abilities (Tello et al., 2012). One of the important points in the ability of microorganisms in dealing with drugs is their genetic reservoir. A microorganism is able to resist against a drug, while it has been able to produce an enzyme to degrade the drug or change the active site of drug attachment or pump the drug to the environment. These mechanisms almost depend on the genetic potency and genetic diversity of microorganism genome. Nowadays, rapid molecular methods such as RAPD-PCR are useful techniques for genetic system studies in order to conduct epidemiological studies concerning the distribution of infectious agents like P. aeruginosa isolates in certain hospital settings (Nanvazadeh et al., 2013). Utilization of this technique in tracking the source of nosocomial infections has shown great specificity and sensitivity. Previously, RAPDs technique was used for source tracking of P. aeruginosa in burn individuals, and it has been emphasized that this technique is helpful in routine microbiology laboratories combined with other detection techniques for controlling hospital-acquired infections, especially in burn units (De Vos et al., 1997; Speert, 2002; Salimi et al., 2010). In various studies in different years, there have been several reports of antibiotic-resistant pattern differences in P. aeruginosa isolates (Pirbonyeh et al., 2017; Raja and Singh, 2007; Javiya et al., 2008); in some other studies for evaluating resistant mechanisms in P. aeruginosa isolates, it has been shown that several mechanisms play a role in the development of resistance of the isolates (Emami et al., 2015; Livermore, 2002; Pirbonyeh et al., 2016). Due to the major differences in the results of these tests for isolates of one species at a specific center, this has led to a poor outcome in determining the pattern of antibiotic treatment for certain infections in a clinical center. According to the results of the recent study, using molecular methods such as RAPDs technique, which have previously been
5. Conclusion Creation of a new attitude on the use of antimicrobial agents and their target can, in addition to timely treatment of patients and control of the infections, lead to faster treatment and reduce related costs for patients and health systems in the countries. According to the fact that the modern science and techniques like molecular methods can be used as a supplementary test along with common tests, this may be more effective in evaluating the infections for categorizing and designing treatment policies. Either in some circumstances like in the crisis of epidemic diseases, such methods will be very helpful in antimicrobial treatment decisions before detection of the infectious agent at the species level. Based on the comparative results of the recent study, it was found that fingerprinting with primer 272 was more useful for genetic differentiation in P. aeruginosa isolates, but primer 277 will be more helpful in defining antibiotic resistant pattern than other primers. However, it is highly recommended that if a center intends to set up such techniques, it should evaluate the isolates with other primers and also have their own specific method. Acknowledgment The performance of the research was funded by Vice Chancellor of Research affiliated with Shiraz University of Medical Sciences with grant No. 15170. We thank Burn & Wound Healing Research Center and Amir-Al-Momenin Burn hospital for their kind corporation. References Abou-Dobara, M., Deyab, M., Elsawy, E., Mohamed, H., 2010. Antibiotic susceptibility and genotype patterns of Escherchia coli, Klebsiella pneuomoniae and Pseudomonas aeruginosa, isolated from urinary tract infected patients. Pol. J. Microbiol. 59 (3), 207–212. (CLSI) CaLSI, 2015. Performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. 32 (3). De Vos, D., Lim Jr., A., Pirnay, J., Duinslaeger, L., Revets, H., Vanderkelen, A., 1997. Analysis of epidemic Pseudomonas aeruginosa isolates by isoelectric focusing of pyoverdine and RAPD-PCR: modern tools for an integrated anti-nosocomial infection strategy in burn wound centres. Burns 23 (5), 379–386. Eftekhar, F., Hosseinkhan, N., Asgharzadeh, A., Tabatabaii, A., 2009. Genetic profiling of Pseudomonas aeruginosa isolates from Iranian patients with cystic fibrosis using RAPD-PCR and PFGE. Iran. J. Basic Med. Sci. 12 (3), 126–132. Emami, A., Bazargani, A., Mohammadi, A.A., Zardosht, M., Jafari, S.M.S., 2015. Detection of blaPER-1 & blaOxa10 among imipenem resistant isolates of Pseudomonas aeruginosa isolated from burn patients hospitalized in Shiraz Burn Hospital. IJM 7 (1), 7–11. Emami, A., Kazempour, A., Pirbonyeha, N., Keshavarzi, A., Zardosht, M., 2017. Hospitalization length survey and relation with distribution of LasA protease and type III secretion system encoding-genes in multi-drug resistant Pseudomonas aeruginosa isolates from burn wounds in southwest of Iran. Gene Rep. 9 (2017), 81–85. Hardalo, C., Edberg, S., 1997. Pseudomonas aeruginosa: assessment of risk from drinking water. Crit. Rev. Microbiol. 23 (1), 47–75. Discovery and development of penicillin [internet]. http://www.acs.org/content/acs/en/ education/whatischemistry/landmarks/flemingpenicillin.html. Javiya, V.A., Ghatak, S.B., Patel, K.R., Patel, J.A., 2008. Antimicrobial susceptibility pattern of Pseudomonas aeruginosa at a tertiary care hospital in Gujarat, India.
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A. Emami, et al. Indian J. Pharmacol. 40 (5), 230–234. Kolář, M., Urbánek, K., Látal, T., 2001. Antibiotic selective pressure and development of bacterial resistance. Int. J. Antimicrob. Agents 17 (5), 357–363. Livermore, D., 2002. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare? Clin. Infect. Dis. 34, 634–640. Mahenthiralingam, E., Campbell, M.E., Foster, J., Lam, J.S., Speert, D.P., 1996. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis. J. Clin. Microbiol. 34 (5), 1129–1135. Nanvazadeh, F., Khosravi, A.D., Zolfaghari, M.R., Prhizgari, N., 2013. Genotyping of Pseudomonas aeruginosa strains isolated from burn patients by RAPD-PCR. Burns 39 (2013), 1409–1413. Pirbonyeh, N., Zardosht, M., Emami, A., Rostampour, S., Moattari, A., Keshavarzi, A., 2016. Emergence of storm resistant mechanisms in Pseudomonas aeruginosa isolated from burn patients hospitalized in Ghotbeddin Shirazi Burn Hospital. Nova J. Med. Biol. Sci. 5 (1), 2–7. Pirbonyeh, N., Bazargani, A., Emami, A., Anvar, Z., Hosseini, S.M., Zardosht, M., et al., 2017. Cross sectional study of burn infections and antibiotic susceptibility pattern for the improvement of treatment policy PSQI. 5 (2), 535–541. Raja, N., Singh, N., 2007. Antimicrobial suceptibility pattern of clinical isolates of Pseudomonas aeruginosa in a tertiary care hospital. J. Microbiol. Immunol. Infect. 40 (1), 45–49. Ramos, G.P., Rocha, J.L., Tuon, F.F., 2013. Seasonal humidity may influence Pseudomonas aeruginosa hospital-acquired infection rates. Int. J. Infect. Dis. 17
(2013), e757-e61. Rynga, D., Sharif, M., Deb, M., 2015. Phenotypic and molecular characterization of clinical isolates of Acinetobacter baumannii isolated from Delhi, India. Ann. Clin. Microbiol. Antimicrob. 14 (40), 1–8. Salimi, H., Owlia, P., Yakhchali, B., Rastegar, L.A., 2010. Characterization of Pseudomonas aeruginosa in burn patients using PCR-restriction fragment length polymorphism and random amplified polymorphic DNA analysis. IJMS 35 (3), 236–241. Sing, A., Goering, R., Simjee, S., Foley, S., Zervos, M., 2006. Application of molecular techniques to the study of hospital infection. Clin. Microbiol. Rev. 19 (3), 512–530. Speert, D., 2002. Molecular epidemiology of Pseudomonas aeruginosa. Front. Biosci. 7 (1), 354–361. System CN, 2003. National Nosocomial Infections Surveillance (NNIS) system report, data summary from January 1992 to June 2003. Am. J. Infect. Control 2003 (31), 481–498. Tello, A., Austin, B., Telfer, T.C., 2012. Selective pressure of antibiotic pollution on bacteria of importance to public health. Environ. Health Perspect. 120 (8), 1100–1106. Vaez, H., Faghri, J., Nasr Esfahani, B., Moghim, S., Fazeli, H., Sedighi, M., et al., 2015. Antibiotic resistance patterns and genetic diversity in clinical isolates of Pseudomonas aeruginosa isolated from patients of a Referral Hospital, Isfahan, Iran. Jundishapur J. Microbiol. 8 (8), e20130-e.
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Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
Identification of different faces of Pseudomonas aeruginosa isolates in burn patients by genetic fingerprinting
T
Amir Emamia, , Abdolkhalegh Keshavarzib, Neda Pirbonyeha, Mahrokh Rajaee Behbahania ⁎
a b
Burn & Wound Healing Research Center, Microbiology Department, Shiraz University of Medical Sciences, Shiraz, Iran Burn & Wound Healing Research Center, Surgery Department, Shiraz University of Medical Sciences, Shiraz, Iran
ARTICLE INFO
ABSTRACT
Keywords: Pseudomonas aeruginosa RAPD-PCR Antibiotic resistant Burn
Pseudomonas aeruginosa, as a prevalent infection in hospital settings, has a different form of performance and response to anti-bacterial agents. Categorizing of infections based on their genome structure may be helpful in control and treatment of prevalent infections. Total of 278 burn patients were evaluated (March 2016 to March 2017) at Amir-Al-Momenin burn hospital (SUMS) for P. aeruginosa by standard microbiological tests. Confirmed isolates were categorized by RAPD-PCR technique following the evaluation for antibacterial susceptibility tests. RAPD finger print results were analyzed by Gelj software (V.2). Based on the experiment results total of 47 (33.1%) P. aeruginosa were isolated. According to the antibiogram results, > 38% were resistant to all available antimicrobials except Colistin, while 32% were multi-drug resistant. In RAPD results, 8 patterns were identified with primer 272 and 5 with 277. Patterns 4, 2 in primer 272 and 4, 3 in primer 272 were the most prevalent patterns. Results showed that primer 277 had more power in categorizing the isolates for defining antibiotic sensitive pattern. In this category, sensitive isolates (> 95%) were in patterns 2 and 3, while more resistant isolates (> 90%) were in patterns 4 and 5. Based on the results of the study, it was found that fingerprinting with primer 272 had the ability to categorize P. aeruginosa isolates for introducing antibiotic pattern, while this result must be confirmed by more different species and techniques in other centers.
1. Introduction Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous gram-negative bacterium which is one of the top three causes of infections in hospital settings (Nanvazadeh et al., 2013; Ramos et al., 2013). Nutrient and moisture environments are suitable media for the growth of this bacterium. These conditions have caused the bacterium to be able to play a significant role in nosocomial infections; both the need of the bacterium and specifications of burn wounds are the leading cause of P. aeruginosa infections in burns (Emami et al., 2015; Hardalo and Edberg, 1997). Based on different studies, P. aeruginosa is a bacterium responsible for about > 20% of nosocomial infections which are more prevalent in immunosuppressed patients such as burns, cystic fibrosis, acute leukemia, organ transplants and intravenous drug addicts (Ramos et al., 2013; Emami et al., 2015). In a recent study in a burn center in the southwest of Iran (Amir-al-Momenin burn and wound healing center), it has been proved that P. aeruginosa is the most prevalent infectious agent in the center with the most prevalence in the wound
samples (Pirbonyeh et al., 2017). In another study performed in the same center on the isolates, it has been proved that different mechanisms of resistance works in P. aeruginosa isolates, which makes it more difficult to treat the infection in this category (Pirbonyeh et al., 2016). Although in many health centers, laboratories report infections in the genus, it has been proved that bacteria have species-specific function either. Because bacteria come from different sources in hospital environments, certainly they have different aspects and functional properties which are mainly related to their genomes. Today, there are molecular techniques used for genetic pattern detection of microorganisms which are less affected by environmental factors compared to phenotypic methods. These techniques play crucial roles in tracking the routes of pathogen transmission and different activities of a kind of species infection (Sing et al., 2006). Although there are different molecular techniques used for comparison and systematic epidemiological typing, such as Pulsed-field gel electrophoresis (PFGE) and Amplified fragment length polymorphism (AFLP) analysis, there is a low cost technique which has high discriminatory power and reproducibility
Abbreviations: RAPD, Random Amplified Polymorphic DNA; PFGE, Pulsed-field gel electrophoresis; P. aeruginosa, Pseudomonas aeruginosa; NNIS, National Nosocomial Infection Surveillance System; EMB, Eosin Methylene Blue ⁎ Corresponding author. E-mail address: [email protected] (A. Emami). https://doi.org/10.1016/j.genrep.2019.100377 Received 5 December 2018; Received in revised form 4 February 2019; Accepted 5 February 2019 Available online 07 February 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
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microbiology department of the research center (Burn and Wound Healing Research Center-SUMS) immediately. Cold chain condition was carried out for transferring of the samples. According to the previous results for this center (Emami et al., 2017) and the main aim of recent study, we focused on the most prevalent infectious agent (Pseudomonas aeruginosa) in our center. To this end, the collected samples were processed for aerobic Gram-negative bacteria on Eosin Methylene Blue (EMB) agar. According to the Pseudomonas aeruginosa specifications, after incubation time (37 °C for 20–24 h), colorless colonies were isolated and pure on new nutrient plates. For primary detection of the isolates, standard bacteriological tests were performed following Gram staining procedure. Gram-negative Rod bacteria with Alkaline/Alkaline result in triple sugar iron agar (TSI), Oxidase and catalase positive results and growth at 42 °C were screened as P. aeruginosa. For more species detection, the isolates were tested either by API 20E system according to the manufacturer's protocol (bioMerieux®SAfrance). To confirm the isolates as P. aeruginosa, molecular diagnostic technique for 16S rRNA with specific primers introduced in a previous study was performed (Emami et al., 2015).
named as Random Amplified Polymorphic DNA (RAPD) which is preferred in such studies (Rynga et al., 2015). RAPD reaction is a polymerase chain reaction (PCR) with some differences. In this kind of PCR, short (8-12mers) single primers are used which are randomly attached to several DNA sequences in the bacterial genome. This kind of amplification creates a repetitive electrophoresis pattern which can be used for genotypic differentiation of different isolates of a bacterial species. In this technique, if any changes or differences take a place in template DNA (especially at the site of primer annealing), the PCR product will change, resulting in a different pattern of amplified segments on the gel (Nanvazadeh et al., 2013; Abou-Dobara et al., 2010). While in many studies, particularly, numerous studies have been carried out at burn and wound healing research center affiliated with Shiraz University of Medical Sciences (SUMS) (Emami et al., 2015; Pirbonyeh et al., 2017; Pirbonyeh et al., 2016; Emami et al., 2017), it has been proved that although there are the same species of an infectious agent isolated from burn wounds, their activity in presenting aspects of infectious, antibiotic resistant pattern and resistant mechanisms is different. These different abilities of a kind of infection species make different appearances of an infectious agent; this has made us have different treatment policies for patients hospitalized in the same center. In this regard, knowing these aspects of microorganisms which are reflected from their genome structure will be helpful for infectious control in such centers. According to this view, the most prevalent infectious agent (P. aeruginosa) in Amir-Al-Momenin burn hospital was evaluated and gene mapping was performed with RAPDPCR technique. This study was performed to find different aspects of a kind of infection in order to investigate their relationship with drug resistance pattern, their transmission pathway and the source of infection. The results of the study could help to find the homology pattern of infection in different wards and have a group aspect for prevalent infection in our studied burn center with a practical antibiotic pattern.
2.4. Antibiogram test For anti-bacterial susceptibility tests, different groups of antibiotics were used. Antibiotics were selected from Carbapenems (Imipenem (10 μg), Meropenem (10 μg)), Aminoglycosides (Gentamicin (10 μg), Amikacin (30 μg)), Quinolones (Nalidixic acid (30 μg), Ciprofloxacin (5 μg)), Cephalosporins (Ceftazidime (30 μg), Macrolides (Erythromycin (15 μg)), Chloramphenicol (30 μg), and Polymyxin (Colistin (10 μg)). The antibiogram was performed by Kirby-Bauer (disk diffusion) method recommended by CLSI, 2016 manual ((CLSI) CaLSI, 2015). For this purpose, the surface of Muller-Hinton agar plates were inoculated with 0.5 McFarland suspensions of each isolate. Then, the studied disks were placed on plates with standard distances as mentioned in the manual. After the incubation time (22 h/35 °C), the inhibition zone diameters of each disc were measured in millimeters and reported as sensitive or resistant according to the guideline for the company. For Quality control Pseudomonas aeruginosa ATCC® 27853 were used as susceptible strain.
2. Material and methods 2.1. Sample collection
2.5. DNA purification and RAPD-PCR assay
This experimental study was performed in one year period (March 2016 to March 2017) at Amir-Al-Momenin burn hospital affiliated with SUMS. This study included patients from all 5 sections (Men, Women, Pediatrics, Surgery and Intensive Care Unit) of the hospital who had sustained deep burn with 2 or more degrees. Totally, 278 patients were admitted and hospitalized to the center during the study time. Considering that Amir-AlMomenin hospital in the south of Iran is the center of burn, the patients are referred from different regions of the south of our country. Demographic data of each patient including sex, age, burn degree and section of hospitalization were obtained from admission sheets of each patient.
To increase the efficiency of the extracted DNA's, phenol/chloroform extraction method was performed on overnight Tryptic Soy Broth (TSB) cultures of each confirmed isolate. Briefly, each sample was cultured in 1 ml of TSB (18 h/37 °C), incubated at 72 °C for 10 min and then cooled down to 4 °C for 5 min in 200 μl proteinase K (200 μg/ml). Following phenol/chloroform extraction (1:1), the bacterial DNA was precipitated with ethanol and the pellet was re-dissolved in DNase/RNase free, deionized water and stored at −20 °C for further tests. To perform the RAPD-PCR reaction, the following reaction was designed: The reaction was carried out in a 50 μl mixture containing 1× PCR buffer, 1.5 mM MgCl2, 0.1 mM each dNTP's, 1U Taq DNA polymerase, 0.5 pmol/μl of primer and 5 μl of extracted DNA. In this study, two primers (272 and 277), which were introduced before, were considered in separate reactions for each isolate (Mahenthiralingam et al., 1996). The PCR cycling was performed with the following setting: (i) 1 cycle at 94 °C for 5 min, 4 cycles at 36 °C for 5 min, and 5 min at 72 °C for each cycle (ii) 30 cycles, at 36 °C for 1 min, with 1 cycle at startup in 94 °C for 1 min and following with 2 min at 72 °C for each cycle. At the end of the cycles, the reaction was followed by a final extension step at 72 °C for 20 min. For detection of amplicons, PCR products were placed in 2% agarose gel wells and electrophoresed. The electrophoresis was performed with 1× TBE buffer at 10 V/cm for 2.5 h. A molecular weight marker (100 bp, Fermentase) was run in all gels beside the samples. At the end of electrophoresis, the gels were stained with ethidium bromide and visualized under UV transilluminator. The RAPD fingerprint results were analyzed by Gelj software (V.2). The pattern of migration variation in different gels was corrected with the standard molecular size marker.
2.2. Inclusion and exclusion criteria To maintain the structure of the study and analysis of the results related to each patient, one sample was collected from each hospitalized patient 3 to 5 days after hospitalization. All of the patients with heat burns, from both genders and all ages with degree two and more burns were included. Based on the National Nosocomial Infection Surveillance System (NNIS) role (System CN, 2003), patients with any criteria of infection in the first 48 h of hospitalization were excluded. Sampling was performed in accordance with the consent form taken at the time of hospitalization and all of the study process was approved by ethics committee code 1396-01-63-15170 from Shiraz University of Medical Sciences. 2.3. Sample processing For sampling, broad Z-stroke method was used for main wounds. Inoculated swabs were put in normal saline tubes and sent to the 2
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Table 1 Ward isolates pattern according to primer 272. Ward
Primer 272 pattern
ICU Men Pediatrics Surgery Women Total
Total
1
2
3
4
5
6
7
8
2 0 0 0 0 2
1 7 0 0 2 10
1 8 0 0 0 9
3 5 3 1 1 13
0 0 0 0 1 1
0 0 0 0 1 1
0 0 0 0 2 2
4 4 1 0 0 9
% Similarity
RAPD-PCR pattern
11 24 4 1 7 47
Group No.
Fig. 1. The results of P. aeruginosa antibiotics resistant pattern.
1
3. Results
2
The results of the 278 burns patients were evaluated in the five main wards of the burn hospital: men's ward 133 (47.8%), women's ward 78 (28.1%), pediatrics 21(7.6%), surgery 28 (10.1%) and intensive care unit (ICU) 18 (6.5%). The sex differentiation of patients was as follows: 170(61.2%) males and 108 females (38.8%). Patient's age ranged from 2 to 68 years with an average of 34.9 ± 14.6 years. From the total of 278 samples (one sample from each individual), 142(51.1%) were growth positive on EMB, while 47 (33.1%) of them and 16.9% from the total were confirmed as Pseudomonas aeruginosa in phenotypic and molecular tests. Out of 47 isolates of P. aeruginosa, the isolation result of each ward was: 24 (51.1%) from Men, 7 (14.9%) from Women, 11 (25.6%) from ICU, 4 (8.5%) from pediatrics and 1 (2.3%) from surgery wards. The results for the antibacterial resistant pattern are shown in Fig. 1. According to the antibiogram results, it has been shown that 18 (> 38%) isolates of P. aeruginosa were resistant to almost all available antimicrobials except for Colistin, while 15 (32%) isolates were known as multidrug resistant. Genetic fingerprinting of the isolates was performed by two RAPD primers. RAPD analysis was performed to compare the genetic background of P. aeruginosa isolates from different samples from different wards. A total of 8 patterns were identified with primer 272. RAPD patterns with > 90% identity were grouped. Due to space limitation, just one sample from each group is shown in the related dendrogram (Fig. 2), but the number of isolates for each pattern is written in the related table (Table 1). In another fingerprinting step by primer 277, a total of 5 patterns were identified. In this step, RAPD patterns with > 90% identity were grouped. In this step, either just one sample from each group was shown in the related dendrogram (Fig. 3). But the number of isolates for each pattern has been written in Table 2. According to the results for primer 272; patterns 4, 2, (3 and 8) are the most prevalent patterns respectively while according to the primer 277, patterns 4, 3, 5 are the most prevalent patterns, respectively. These results indicate that although P. aeruginosa isolates in our center are % Similarity
RAPD-PCR pattern
3 4 5 Fig. 3. RAPD-PCR gel electrophoresis analysis of P. aeruginosa clinical isolates with Primer 277. The dendrogram was generated by Gelj software with arithmetic mean using Dice coefficients (band tolerance 1.0%; optimization 1.0; cut-off value 90%). Table 2 Ward isolates pattern according to primer 277. Ward
Primer 277 pattern 1
ICU Men Pediatrics Surgery Women Total
0 4 1 0 0 5
2 5 0 0 0 0 5
Total 3 0 9 0 0 4 13
4 4 5 3 1 1 14
5 2 6 0 0 2 10
11 24 4 1 7 47
grouped in one species, they are categorized in different genotypes. According to the results of antimicrobial susceptibility tests, only in three groups of Carbapenems, Aminoglycosides, and Polymyxins, both resistant and sensitive isolates were seen and we were able to compare the results with genotyping groups. Based on Chi-square test a clear relation was found between primers typing and antibiotic pattern (P < 0.01) and analysis of the results for primer 272 typing, it was found that the isolates sensitive to antibiotics were more in genotype groups 2, 4, 3 and 1, respectively. The analysis of the results for primer 277 showed that sensitive isolates were more in genotype 3, 2 and 4, respectively. According to the total results, it was indicated that primer 272 had more power of genetic differentiation in P. aeruginosa isolates, but analysis of the isolates with primer 277 will be more helpful in antibiotic-resistant pattern. According to the sensitive results for the studied antibiotics and comparison of the results with primer 277 pattern, it was shown that more sensitive isolates (> 95%) were categorized in patterns 2 and 3, while more resistant isolates (> 90%) were categorized in patterns 4 and 5. Less than 5% of resistant and sensitive isolates were categorized in pattern one (Table 3).
Group No. 1 2 3 4 5 6 7 8
4. Discussion
Fig. 2. RAPD-PCR gel electrophoresis analysis of P. aeruginosa clinical isolates with Primer 272. The dendrogram was generated by Gelj software with arithmetic mean using Dice coefficients (band tolerance 1.0%; optimization 1.0; cut-off value 90%).
Based on current results and other studies in Iran it can be deduced that molecular typing can be used for finding different variations and examine the epidemiology of the infectious agents source and the 3
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used in the burn victims for source-tracking of P. aeruginosa, it can be used as supplementary test along with usual methods for categorizing the isolates in more limited patterns for using antibiotics more effectively and purposefully Considering the fact that in the present study, P. aeruginosa isolates had different antibiotic resistant pattern and different resistant mechanisms, and according to the studied molecular classification, we were able to classify them in more limited groups with a specific drug sensitivity pattern. According to the importance of antibiotic stewardship in the world and use of anti-microbial agents more targeted with sufficient outcome in infectious control in health systems, change in policies, especially in detection and categorization of the same microbial species for antibiotic treatment, is essential. Early detection of antimicrobial drugs revealed that there were effective compounds in controlling the incidence of infections that can be used in the treatment of infectious diseases.
Table 3 Antibiotic sensitive isolates pattern according to the primer 277. Antibiotic
Number of sensitive (%)
Primer 277 pattern 1
Gentamicin Amikacin Imipenem Meropenem Nalidixic acid Ciprofloxacin Ceftazidime Chloramphenicol Erythromycin Colistin
7 (14.9) 0 (0) 4 (8.5) 5 (10.6) 0 (0) 0 (0) 0 (0) 0 (0) 1 (2.1) 40 (85.1)
1 – – – – – – – – 1
2 4 – 3 4 – – – – – 32
3 2 – – 1 – – – – 1 6
4 – – – – – – – – – –
5 – – 1 – – – – – – 1
possibility of cross infection among patients (Nanvazadeh et al., 2013; Eftekhar et al., 2009; Vaez et al., 2015). Either molecular methods can be a proper candidate for evaluating the antibiotic resistant pattern of prevalent nosocomial infections in clinical settings for managing the Antibiotic stewardships. Base on different studies we checked the isolates with two routine primers 272 and 277. According to the results it was indicated that primer 272 had more power of genetic differentiation in P. aeruginosa isolates, while analysis of the isolates with primer 277 will be more helpful in antibiotic-resistant pattern. In the early detection of antimicrobial drugs, it was revealed that there were effective compounds in controlling the incidence of infections which can be used in the treatment of infectious diseases (Discovery and development of penicillin [internet], 1999). However, over the years, the inappropriate use of anti-microbial drugs has led to a selective pressure on resistance of microbial infections (Kolář et al., 2001). As we all know, while anti-microbial agents are used, only sensitive microorganisms are affected and resistant forms remain. It has been proved that selective pressure of anti-microbial agents differentiates different forms of microbial species according to their ability in response to such agents. While different anti-microbial agents are used in different infections, this pressure has occurred extensively and forms different aspects of microbial species by different abilities (Tello et al., 2012). One of the important points in the ability of microorganisms in dealing with drugs is their genetic reservoir. A microorganism is able to resist against a drug, while it has been able to produce an enzyme to degrade the drug or change the active site of drug attachment or pump the drug to the environment. These mechanisms almost depend on the genetic potency and genetic diversity of microorganism genome. Nowadays, rapid molecular methods such as RAPD-PCR are useful techniques for genetic system studies in order to conduct epidemiological studies concerning the distribution of infectious agents like P. aeruginosa isolates in certain hospital settings (Nanvazadeh et al., 2013). Utilization of this technique in tracking the source of nosocomial infections has shown great specificity and sensitivity. Previously, RAPDs technique was used for source tracking of P. aeruginosa in burn individuals, and it has been emphasized that this technique is helpful in routine microbiology laboratories combined with other detection techniques for controlling hospital-acquired infections, especially in burn units (De Vos et al., 1997; Speert, 2002; Salimi et al., 2010). In various studies in different years, there have been several reports of antibiotic-resistant pattern differences in P. aeruginosa isolates (Pirbonyeh et al., 2017; Raja and Singh, 2007; Javiya et al., 2008); in some other studies for evaluating resistant mechanisms in P. aeruginosa isolates, it has been shown that several mechanisms play a role in the development of resistance of the isolates (Emami et al., 2015; Livermore, 2002; Pirbonyeh et al., 2016). Due to the major differences in the results of these tests for isolates of one species at a specific center, this has led to a poor outcome in determining the pattern of antibiotic treatment for certain infections in a clinical center. According to the results of the recent study, using molecular methods such as RAPDs technique, which have previously been
5. Conclusion Creation of a new attitude on the use of antimicrobial agents and their target can, in addition to timely treatment of patients and control of the infections, lead to faster treatment and reduce related costs for patients and health systems in the countries. According to the fact that the modern science and techniques like molecular methods can be used as a supplementary test along with common tests, this may be more effective in evaluating the infections for categorizing and designing treatment policies. Either in some circumstances like in the crisis of epidemic diseases, such methods will be very helpful in antimicrobial treatment decisions before detection of the infectious agent at the species level. Based on the comparative results of the recent study, it was found that fingerprinting with primer 272 was more useful for genetic differentiation in P. aeruginosa isolates, but primer 277 will be more helpful in defining antibiotic resistant pattern than other primers. However, it is highly recommended that if a center intends to set up such techniques, it should evaluate the isolates with other primers and also have their own specific method. Acknowledgment The performance of the research was funded by Vice Chancellor of Research affiliated with Shiraz University of Medical Sciences with grant No. 15170. We thank Burn & Wound Healing Research Center and Amir-Al-Momenin Burn hospital for their kind corporation. References Abou-Dobara, M., Deyab, M., Elsawy, E., Mohamed, H., 2010. Antibiotic susceptibility and genotype patterns of Escherchia coli, Klebsiella pneuomoniae and Pseudomonas aeruginosa, isolated from urinary tract infected patients. Pol. J. Microbiol. 59 (3), 207–212. (CLSI) CaLSI, 2015. Performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. 32 (3). De Vos, D., Lim Jr., A., Pirnay, J., Duinslaeger, L., Revets, H., Vanderkelen, A., 1997. Analysis of epidemic Pseudomonas aeruginosa isolates by isoelectric focusing of pyoverdine and RAPD-PCR: modern tools for an integrated anti-nosocomial infection strategy in burn wound centres. Burns 23 (5), 379–386. Eftekhar, F., Hosseinkhan, N., Asgharzadeh, A., Tabatabaii, A., 2009. Genetic profiling of Pseudomonas aeruginosa isolates from Iranian patients with cystic fibrosis using RAPD-PCR and PFGE. Iran. J. Basic Med. Sci. 12 (3), 126–132. Emami, A., Bazargani, A., Mohammadi, A.A., Zardosht, M., Jafari, S.M.S., 2015. Detection of blaPER-1 & blaOxa10 among imipenem resistant isolates of Pseudomonas aeruginosa isolated from burn patients hospitalized in Shiraz Burn Hospital. IJM 7 (1), 7–11. Emami, A., Kazempour, A., Pirbonyeha, N., Keshavarzi, A., Zardosht, M., 2017. Hospitalization length survey and relation with distribution of LasA protease and type III secretion system encoding-genes in multi-drug resistant Pseudomonas aeruginosa isolates from burn wounds in southwest of Iran. Gene Rep. 9 (2017), 81–85. Hardalo, C., Edberg, S., 1997. Pseudomonas aeruginosa: assessment of risk from drinking water. Crit. Rev. Microbiol. 23 (1), 47–75. Discovery and development of penicillin [internet]. http://www.acs.org/content/acs/en/ education/whatischemistry/landmarks/flemingpenicillin.html. Javiya, V.A., Ghatak, S.B., Patel, K.R., Patel, J.A., 2008. Antimicrobial susceptibility pattern of Pseudomonas aeruginosa at a tertiary care hospital in Gujarat, India.
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