RAPD CHARACTERIZATION OF PSEUDOMONAS SPECIES IDENTIFIED FROM CLINICAL SAMPLES

Pseudomonas aeruginosa is the most common dreadful pathogenic gram negative bacilli responsible for nosocomial infections among hospital patients especially in developing countries. In the present study P. aeruginosa isolated from various clinical samples and evaluated for their variability and genetic relationship using PCR based Randomly Amplified Polymorphic DNA (RAPD) technique. A total of 30 different samples from patients with wounds, ear infection, respiratory tract, nasal infection and urine infection were collected from various hospitals and diagnostic centers in Bangalore. The samples included pus swabs, wound discharge, sputum and blood. These samples were subjected to P. aeruginosa isolation using selective media and characterization using biochemical tests. Further genetic relationship was determined using RAPD technique. A total of 7 were isolated and characterized biochemically and identified belonging to P. aeruginosa. Cluster analysis and phylogenetic tree reveal close relatedness between P. aeruginosa strain P1, P3, P4, P5 and P6 but distantly related to the P2 and P7. This indicates that the infection due to P. aeruginosa is caused by diverse as well as closely related clones circulating in the study health care centers. This demands further investigation through prospective studies with a larger number of patients. This will aid suggesting efficient and sustained control measures and antibiotic policy in study area.


INTRODUCTION
Pseudomonas aeruginosa is one among the major opportunistic pathogens from the family Pseudomonaceae residing in part of human mycoflora and can cause many diseases [1].Nosocomial infections caused by P. aeruginosa are a major problem in hospitals due to its intrinsic capacity to acquire resistance to all effective antibiotics [2,3].P. aeruginosa is a leading cause of ventilator-associated pneumonia (VAP) and around 20% of pneumonia conditions in intensive care units (ICU) [4].Due to these reasons, it is significant to note the antibiotic resistance in the clinical specimens.
In humans the common source of P. aeruginosa isolation are blood, urine, pus, sputum and wounds samples of infected patients [5].The assessment of the clonal relationship is crucial for determining the epidemiology of nosocomial infections and designing of pathogen control methods [6,7].Previous studies have reported high rate of polymorphism and genetic diversity among P. aeruginosa isolates especially in different types of clinical specimens [8].In recent years PCR-based molecular genotyping of P. aeruginosa has been gained popularity over tedious phenotyping methods in both environment and clinical isolates [9].Although, different PCR-based genotypic techniques have been described for characterizing P. aeruginosa isolates, Random Amplified Polymorphic DNA (RAPD)-PCR has received considerable attention in recent years for studying genetic relatedness.It comprises high strain differentiation power and is a definitive method for molecular characterizing of bacteria [10].RAPD-PCR can be considered as a powerful tool to reveal extensive DNA polymorphism between the genomes of different species under the same genus [11].This rapid methodology has been shown to be as discriminatory as Pulsed-field gel electrophoresis (PFGE) for typing P. aeruginosa and was recommended for the primary screening of large numbers of isolates because of its efficiency [6,12].The present study was undertaken to isolate and determine the RAPD genotyping of P. aeruginosa strains isolated from different clinical samples from hospitals and various diagnostic centers in Bangalore.

Sample collection and isolation of Pseudomonas
Different 30 clinical specimens were collected, including wound discharge, ear swabs, nasal swabs, blood and urine from patients in hospitals.Blood culture was taken from patients who were suspicious of sepsis.Sputum samples were also collected from patients.The samples were immediately transferred to the laboratory.Pseudomonas was isolated using Cetrimide Agar (Hi-Media, India) a selective and differential medium for Pseudomonas spp.The colony morphology was noted and morphological characterization of bacterial culture was determined using Gram's Staining procedure.The isolates were further subjected to standard biochemical tests including catalase, oxidase, Indole-Methyl red-Voges-Proskauer-Citrate utilization test (IMVIC), casein hydrolysis, nitrate reduction, gelatin hydrolysis, and sugar fermentation [13].

Bacterial DNA extraction
The isolates were revived and freshly grown in Nutrient broth medium with optimum bacterial growth were subjected to DNA extraction as per the Phenol: Chloroform method described by Sambrook and Russel [14].The extracted DNA was dissolved in Tris EDTA (TE) buffer and stored at-20 °C for further experiments.The quality and concentration of extracted DNA was determined using NanoDrop™ 1000 Spectrophotometer from Thermo Fisher Scientific.

RESULTS AND DISCUSSION
Pseudomonas aeruginosa is the most common dreadful pathogenic gram negative bacilli responsible for nosocomial infections and also as the important cause for morbidity and mortality among hospital patients especially in developing countries [15].P. aeruginosa has an ability to acquire and harbor diverse resistance determinants.P. aeruginosa is a major microorganism which can be resistant to antibiotics causing Multi Drug Resistance (MDR).It remains one of the significant hospital acquired infection worldwide, despite efficient infection control policies [16].P. aeruginosa is the causing agent of 10%-20% of nosocomial infections [17].In India, prevalence rate of P. aeruginosa infection varies from 2-23% [18].Another study form India reports 66.07%(40/60) patients were found to be infected with P. aeruginosa during their stay in hospital, from among which 56 strains were isolated [19].
The present study deals with the determination of genetic diversity among Pseudomonas spp.isolated from clinical samples using RAPD markers.The source of the samples was from wound, pus, blood and urine from patient's in hospitals.The rationale in choosing these sample was based on published literature, on their frequency of P. aeruginosa isolation from these sample, some of them are discussed below.Although sputum is an ideal sample for detection of nosocomial infections, sputum often does not provide complete information about the progression of both acute and chronic lung diseases caused by P. aeruginosa viz.chronic obstructive pulmonary disease COPD, cystic fibrosis.Blood stream infections is associated with higher mortality rates depending on the patient's conditions [20].Similar study by Fazeli et al. [21] isolated P. aeruginosa strains from clinical specimens including urine (16%), tracheal (31.2%), wound (33.3%), and blood (33.3%) specimens.Often these isolates are Multi Drug Resistant (MDR).In an another study with a total of 932 clinical samples P. aeruginosa was isolated from pus (28.7%), urine (28.8%), blood (28.9%), and sputum (28.6) [22].
In the present study P. aeruginosa was isolated from various clinical samples from hospitals and diagnostic centers to study the genetic relatedness among the samples for determination of common source of infection.The Pseudomonas isolates were isolated and purified on Cetrimide Agar plates and was further maintained on Nutrient Agar slants.Totally 8 isolates were recovered from 4 different clinical samples viz, sputum, blood, pus and wound.All the isolates were gram negative, uniformly stained, straight or slightly curved rods, measuring 0.5 to 1.0 µm by 1.5 to 5.0 µm in length.They were aerobic, nonspore forming, motile bacteria.The isolates appeared fluorescent yellow or green color on Cetrimide agar plates (fig.1).Further the Pseudomonas strains were subjected to biochemical characterization (table 1).Seven out of eight isolates belonged to P. aeruginosa and for one isolate species could not be identified biochemically.
RAPD-PCR have shown to be useful technique to study the genetic variability between bacterial strains, due to its great specificity and sensitivity [23].This technique has been often used in the case of P. aeruginosa widely for different types of clinical and environmental isolates [24].Moreover, RAPD-PCR could be considered as valuable technique especially in developing countries as a source for tracking the infections since it is cost effective PCR based technique.
In this study, DNA fingerprinting was performed using two RAPD primers (OPA-11 and OP-D20).RAPD has been utilized to type a wide range of bacteria including P. aeruginosa and the method described here is proved very useful for discrimination among P. aeruginosa isolates.In this study, both the primers OP-A11 and OP-D20 produced clear banding patterns.All the observed bands were polymorphic and no monomorphic bands were observed.The bands obtained ranged between 500bp-2 kb.The use of molecular markers to study genetic diversity will help in characterizing the Pseudomonas spp.isolated from clinical samples.The cluster analysis and phylogenetic tree depending on genetic distance reveal the genetic relationship between the P. aeruginosa isolates subjected to RAPD-PCR analysis.This put to rest the fact that there is definitely a high rate of genetic recombination or mutations leading to a high level of genetic diversity among the P. aeruginosa isolates population.The UPGMA average cluster analysis of RAPD-PCR banding pattern using OP-A11 primer showed genetic distance values range from 0.23 to 0.15.The lowest genetic distance of (0.15) was found between P. aeruginosa strain 1 and 5, whereas the highest genetic distance (0.23) was observed between strain 7 and 3.The phylogenetic tree shows close relatedness between P. aeruginosa strain 4, 5, 6 and 1, but is distantly related to the other samples (fig.2).The UPGMA average cluster analysis of RAPD-PCR banding pattern using OP-A11 primer showed genetic distance values range from 0.24 to 0.14.The lowest genetic distance of (0.14) was found between isolate 3 and 4, whereas the highest genetic distance (0.23) was observed between isolate 6 and isolate 7. The phylogenetic tree shows close relatedness between P. aeruginosa strain 3 and 4 but distantly related to the other samples (fig.3).Earlier De Vos et al. [12] and Renders et al. [25] utilized this technique for typing and epidemiological characterization of P. aeruginosa strains and described usefulness of the technique in laboratory in combination with other techniques as a tool for managing nosocomial infections [12,25], which was discussed by Nanvazadeh et al. [24] in their paper on typing of P. aeruginosa isolates from burn wounds.The present was a primary attempt to isolate P. aeruginosa for clinical sample and determine the genetic relatedness among them.Moreover, this study also utilized RAPD-PCR as a reliable tool for typing P. aeruginosa in local setting.Further it is necessary to develop prospective studies with a larger number of patients to solve many uncertainties that still exist in such prevalent disease in hospitals.This will aid suggesting efficient and sustained control measures and antibiotic policy in study area.