Biotechnological
Communication
Biosci. Biotech. Res. Comm. 9(1):
Comparison of genetic and drug resistance patterns in Pseudomonas aeruginosa isolated from the burn wound patients using primers 272 and 277
Z. Mahmmudi1, A. A. Gorzin*2 and A. Emami3
1Islamic Azad University, Kazeroon, Iran
2School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
3Department of Microbiology, Shiraz Burn Research Center, Shiraz University of Medical Sciences, Shiraz, Iran School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
ABSTRACT
Pseudomonas aeruginosa is one the main causes of opportunistic infections, especially in subjects with weakened immune systems such as patients with burns. Carbapenems such as imipenem (IMP) and meropenem (MEM) are among the most important anti- microbial drugs which are used to treat P. aeruginosa isolates resistant to penicillin and aminoglycosides. Given that identify the source of contamination and the resulting pattern of isolates resistant transmission in health centers play an important role in the control of hospital infections, the aim of this study was to determine antibiotic susceptibility and genetic patterns of P. aeruginosa bacteria isolated from patients admitted to Qutb
KEY WORDS: CARBAPENEMS, P. AERUGINOSA, BIOCHEMICAL TESTS, GENETIC PATTERNS, BURN WOUNDS,
ARTICLE INFORMATION:
*Corresponding Author: Received 5th February, 2016
Accepted after revision 30th March, 2016 BBRC Print ISSN:
Online ISSN:
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INTRODUCTION
One of the important problems in global health care is burn lesions, which puts patients at risk of hospital infec- tions. Nosocomial infections are one of the most impor- tant medical problems worldwide, leading to outbreaks of infectious diseases in the community and are a major cause of morbidity and mortality, increased length of hospitalization, increased medical care cost and health problems. Hospital infections are created as limited or disseminated due to the pathogenic reactions associated with the infectious agents or their toxins in the hospi- tal. The majority of nosocomial infections are caused by opportunistic agents such as P. aeruginosa. These bacte- ria have been identified as the most important bacteria that cause hospital infections. New methods of hospi- tal cares have reduced the burn wound infections, but there is still the risk of deadly infections in severe burns, especially in developing countries and may result in the death of several thousand people annually.These cylin- drical bacteria are mobile and classified accordance with the similarity of rRNA and common characteristics of culture. They smell like grape and grow easily on many media at 37 to 42 °C. These oxidase positive microor- ganisms do not ferment carbohydrates and are regarded as the second most common
These bacteria cause damages to the first line of defense and lead to dangerous infections in the skin of patients suffering from burns. The genetic patterns of P. aeruginosa by
Maki et al., in 1997 isolated and identified 85 iso- lates P. aeruginosa during a year from patients in ICU with routine bacteriological tests. Then their antibiotic susceptibility was determined with different disks; 50%,
Mahmmudi, Gorzin and Emami
70.5% and 17.6% of isolates were resistant to ceftazi- dime, cefotaxime and imipenem, respectively (Maki et al., 1997). Another earlier study was done by Pritt et al., in 2007, on blood, urine and fluids samples using MacConkey agar and blood agar media as well as anti- biogram test according to NCCLS standards by Kirby - Bauer method. During 1 year, 89 isolates of P. aeruginosa were isolated from samples and the most prevalence was observed in urine samples about 51%. These organisms were resistant to ceftazidime and imipenem, respectively, 56% and 24% (Pritt et al., 2007).
Ekrame et al.,(2011) conducted at burn injury hospi- tal of Ahwaz, Iran; 70 isolates suspected to P. aeruginosa were collected, of which 44 isolates were confirmed using biochemical tests,
According to studies mentioned in many countries, resistance among strains of P. aeruginosa is increasing relative to
The emergence of
Mahmmudi, Gorzin and Emami
ties (Dariouche, 2001).Group A can hydrolyze penicillin and cephalosporin antibiotics that have been identified in bacteria such as Escherichia coli and Klebsiella pneu- moniae (Trautner et al., 2004).Group B is able to hydro- lyze carbapenem in bacteria such as Pseudomonas aer- uginosa and Serratia marcescens (Trautner et al., 2004). Group C, generally, leads to the kind of chromosomal resistance and especially in Enterobacter (MacFaddin , 2000). Group D hydrolyze antibiotics such as oxacillin and cloxacillin with high strength (MacFaddin, 2000).
Since Pseudomonas aeruginosa is an important fac- tor for nosocomial infection, especially in burn patients, so knowing how to release the special strains of bacte- ria has epidemiological importance to find the sources of infection, propagation mechanism and quick control, and because there is apparently particular genetic pat- terns of bacterial infections, the aim of this study was to evaluate the genetic patterns of resistant and susceptible strains of P. aeruginosa isolated from burn patients with RAPD - PCR technique to determine the diversity and genetic differences among patients admitted to a center as well as the origin will be identified.
MATERIAL AND METHODS
This experimental
Drug susceptibility testing: susceptibility of bacteria to different antibiotics was determined based on disk dif- fusion using the
Differential and biochemical tests for P. aeruginosa: the tests were performed as follows:
Test SIM: A colony of bacteria was inoculated by needle in sterile medium, production of H2S, indole and motil- ity of bacteria were characterized after adding Kovac’s reagent.
Test TSI: a colony of bacteria was taken by sterile nee- dle and cultured in depth and on the surface the TSI
medium. Red color of the medium still remains due to lack of fermentation of glucose, lactose and sucrose on the surface and in depth.
Citrate test: A colony of bacteria was inoculated by nee- dle in sterile medium. These bacteria due to the use of citrate as a carbon source change the color of medium from green to blue.
Growth at 42 °C: a colony of bacteria was cultured in two tubes containing Tryptic Soy Agar as sloping. For
Oxidase test: 1 to 3 drops of reagent oxidase was placed on a piece of paper inside a
Catalase test: Up to 2 drops of reagent catalase were poured onto a glass slide and some of the colonies were taken and solved in it. The positive reaction is character- ized by the formation of oxygen bubbles, otherwise is considered as
MOLECULAR METHODS
To prepare buffer X50, tris base (242 g), glacial acetic acid (57.1 ml), EDTA 0.5 M (100 ml) were poured in a balloon and reached to a total volume of 1 lit by adding distilled water. The buffer was diluted 50 times using distilled water in order to provide a buffer X1 (for using in electrophoresis tank or gel). To prepare buffer X10, tris base (54 g), Boric acid (27.5 g), EDTA 0.5 M (30 ml) were poured in a balloon and reached to a total vol- ume of 0.5 lit by adding distilled water. The buffer was diluted 10 times using distilled water in order to provide a buffer X1 (for using in electrophoresis tank or gel).
Bacteria DNA extraction (Kit of
was repeated once more. In the next phase, filter pipes was centrifuged without adding any substance at the same speed. In continuing, dry filters were removed and placed in sterile microtubes 1.5. In the final step, 50 μl of dilution buffer, which had been already subjected inside thermoblock at 65 °C for
DNA extraction by boiling: a
INVESTIGATION OF P. AERUGINOSA ISOLATES BY
IDENTIFYING THE OPTIMAL TEMPERATURE OF
In this study, two primers (Bioneer Germany) (ten nucle- otides) 272 and 277 were considered for
First, master mix was prepared based on the compositions listed in the table above, which had not the genomic DNA isolates studied and 45 μl of it was divided into microtubes
2.0ml. 5 ml of genomic DNA extracted (50 ng) was added to each microtube to reach the final volume of genomic
Mahmmudi, Gorzin and Emami
amplification reaction to 50 μl. All the preparation steps of master mix and distribution were performed on ice. Negative controls were used to infer the probable con- tamination, which consisted of all components of genomic amplification without template DNA. And also a positive control was used included components of genomic ampli- fication reaction with DNA of P. aeruginosa ATCC27853 as a representative of the pseudomonas complex. Following program to the number of 35 cycles were used for genomic amplification reaction. After electrophoresis and emerging bands, the right temperature for each of the three prim- ers used in
Statistical analysis: The data was analyzed using software SPSS version sixteenth, Excell, (Microsoft office 2012), analysis of variance and T test (p <0.05).
Cluster analysis: clustering of results obtained from the electrophoresis of PCR products was carried out by soft- ware NTSYSpc (Numerical Taxonomy and Multivariate Analysis System). For each primer was drawn a dendro- gram as well as an overall dendrogram derived from all the bands amplified by the primers. Finally matrix obtained from Jaccard similarity coefficients and UPGMA method (Unweighted Pair Group Method with Arithmetic Mean) was used to calculate the genetic distance.
RESULTS AND DISCUSSION
Of 200 burn patients, 117 (58%) were males and 83 (42%) were females; 83 (41%), 54 (27%), 35 (17%) and 28 (14%) samples were respectively related to men’s ward, women’s ward, children’s ward and the ICU. Percentage of drug resistance in 50 isolates of P. aeruginosa has been shown in Figure 1.
All 50 isolates of P. aeruginosa DNA was extracted using kits, and was confirmed using 16srRNA gene. The genetic patterns of isolates were determined using RAPD- PCR technique with two primers and the results were com- pared. Primer 272 in 18 genetic patterns of P. aeruginosa demonstrated altogether 86 bands and primer 277 in 15 genetic patterns of P. aeruginosa showed 65 bands. The numbers of polymorphism bands, which are based on the variation in the number of base pairs of bands, were 22 and 17 in the primers of 272 and 277, respectively. Per- formance of primers 272 and 277 were as follows: 45.3 and 34.2, so the present study demonstrated higher perfor- mance of primer 272 than the other primer and results of this research were consistent with other studies on primer
272.Discriminatory power and detection of primer per- centages were 43.1% and 33.4% in the primers 272 and 277, respectively. In the present study it was found that discriminatory power and detection related to primer 272
Mahmmudi, Gorzin and Emami
FIGURE 1: The antibiotic susceptibility of Pseudomonas aeruginosa isolates from the Iranian Burn patients using the disk diffusion method.Amikacin (AN), Ceftazidime(CAZ), Chloramphenicol (CK), Erythromycin (E), Nalidixic acid (NA), Cefotaxime (CTX), Ciprofloxacin (CP), Gentamycin (GN), Imipenem (IPM), Meropenem (MEM), Piperacillin (PIP),and Piperacillin (PIP) were used.
were more than the other primer and this confirms same thing we expected. Data analysis showed that over 95% of carbapenem antibiotic resistance was observed in Pseu- domonas aeruginosa strains isolated from patients hospi- talized at burn ward of Qutb
This experimental study was performed on 200 sam- ples taken from patients by using standard microbiologi- cal tests to identify isolates of P. aeruginosa that 50 sam- ples were identified as P. aeruginosa isolates. Antibiotic susceptibility of isolates was used in accordance with
A study conducted by Costerton et al., in 2005, showed that 72% of the samples were infected by P. aeruginosa and in the next places were Escherichia coli, Staphylo- coccus aureus, Candida and Proteus, respectively (Cos- terton et al., 2005). The results of the study were not con- sistent and infection of Pseudomonas aeruginosa isolates was much more than this study, this could indicate a lack of hygiene and inappropriate sterilization and other factors in the study of Costerton . In investigation of
Maki et al (2006), the most common causative organism of burn wound infections were, respectively, staphylo- coccus, klebsiella and P. aeruginosa (Maki et al., 2006). The prevalence of P. aeruginosa isolates in this study (25%) was similar to present work and it was shown that, unlike previous years, the prevalence of these bac- teria in burns patients has been decreased.
The prevalence of the bacterium was 80% in a study conducted by Mahenthiralingam et al., in 2009 in the USA, actually more than our research (Mahenthiral- ingam et al., 2009). In a study by Pugashetti et al., (2008) in the Netherlands, the prevalence of bacteria involved in infections over a period of two years was as follows: Staphylococcus aureus (70%), Pseudomonas aeruginosa (15%), Klebsiella (9%), Candida (5 %) and Proteus (1%) (Pugashetti et al., 2008). The preva- lence of P. aeruginosa isolates in this study was lower than ours, which represents the difference of bacteria involved in infections in various parts of the world. In a study, 89 cases of P. aeruginosa were isolated from different clinical specimens, which the highest preva- lence was found in urine samples by 51.5% and were resistant to Ceftazidime (56%) and imipenem (24.7%), representing the annual increase (Akanji et al.,2011).
This study showed the increased resistances to imipe- nem and ceftazidime antibiotics are consistent with sev- eral studies conducted in the regions and neighboring countries, unlike industrialized countries; this indicates the importance of the proper use of these antibiotics in developed countries.
A study was done by Hunter et al., in 2005 in Brazil. In this study, 140 isolates of P. aeruginosa were collected from clinical sources, the resistance patterns of strains to different antibiotic were as follows: 49.3% gentamicin, 99.3% cephalotin, 100% thienamycin, 79.3% ceftizoxime, 97.7% cotrimoxazole, 35% amikacin, 67.1% carbenicillin, 65.7% ceftriaxone, 58.6% ciprofloxacin, 52.8% pipera- cillin, 1.4% imipenem, 65.7% kanamycin, 72.1% ofloxa- cin and 100% ampicillin (Hunter et al., 2005). This study shows reduced antibiotic resistance in isolates of P. aerugi- nosa rather than present study, but concerning gentamicin was similar. Study of
A study was conducted to investigate polymorphism for P. aeruginosa isolated from cystic fibrosis patients through
Some points should be considered to ensure the power of amplification in
1.Samples should be evaluated by two different primers to confirm the differences among isolates;
2.All reactions should be repeated twice;
3.It is very important to standardize the PCR reaction mixture and unify the conditions for amplification;
4.Several dilution of DNA should be used for PCR reaction and the best dilution should be applied with gel analysis.
Now with regard to the importance of infection in burn patients and taking into account the high prevalence of P. aeruginosa isolates in the world and spread the bacte- ria resistance, quick identification and determination of therapeutic resistance pattern are very important. Today, carbapenems such as imipenem and meropenem are the main choice for treatment of infections caused by oppor- tunistic
Mahmmudi, Gorzin and Emami
ance in P. aeruginosa strains isolated from patients at Qutb
According to studies, molecular techniques have higher power detection and repeatability of phenotypic tests and this progress is due to their ability to distinguish small genomic differences and high molecular stability com- pared to the phenotypic profiles of same strains. Advanced molecular typing methods such as
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