Molecular Characterization of Pathogenic and Non-pathogenic Pseudomonas aeruginosa with Special Reference to Phenazine Gene

Transcription

1 JOURNAL OF MODERN BIOTECHNOLOGY, VOL. 1, NO. 2, pp 70 74, November 2012 Copyright 2012, by Madras Institute of Biotechnology. All Right Reserved. Research Article Molecular Characterization of Pathogenic and Non-pathogenic Pseudomonas aeruginosa with Special Reference to Phenazine Gene Sundararaj Jamunadevi 1, Pannerselvam Balashanmugam 2 *, Gangatharan Muralitharan 1 and Puthupalayam Thangavelu Kalaichelvan 2 1 Department of Microbiology, Bharathidasan University, Tiruchirappalli , Tamil Nadu, India 2 Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai , Tamil Nadu, India *Correspondence Author biobala17@gmail.com Received 29 August 2012; Revised 15 September 2012; Accepted 26 September 2012 Abstract In the present study phenazine gene (Phz gene) amplification was carried out for the differentiation and comparison of Pseudomonas aeruginosa pathogenic form and commensal isolates. A total of 40 isolates were collected from different sources in different hospitals around Tiruchirappalli region. Antibiotic sensitivity of the pathogenic organisms was done with 10 different antibiotics and showed all the organisms were sensitive to imipenem and carbenicillin and resistant to ampicillin and tetracycline. Interestingly, all the commensal isolates were resistant to pipercilliln, cefotaxime. Polymerase chain reaction amplified Phz gene in pathogens and commensal revealed that phenazine gene was amplified only in pathogenic isolates but not in commensal. This study is shed light on the possible application of this phz gene for rapid differentiation and comparison of pathogenic and non-pathogenic strains of Pseudomonas aeruginosa from various samples. Keywords: Antimicrobial resistance, Pseudomonas aeruginosa, Phenazine pigment, Molecular characterization. INTRODUCTION: Pseudomonas aeruginosa is the epitome of an opportunistic pathogen of humans. It is a versatile gram negative bacterium that grows in soil marshes and coastal marine habitats, as well as on plants and animal tissue. It forms biofilms on wet surfaces such as those of rocks and soil (Costerton et al., 1999; Ahearn et al., 1999). P. aeruginosa is one of the principal causes of nosocomial pathogen particularly among immunecompromised patients. They induce a variety of human infections including bacteremia, respiratory infections, and genitor urinary tract infections and wound infections (Javis et al., 1992). As an opportunistic infections pathogen can offer lead to life threatening disease for example P. aeruginosa a is the main cause of mortality in cases of poly microbial bacteremia (Usher et al., 2002) and the second most common sepsis in the ICU (Vincent et al.,2006). P. aeruginosa is a common cause of nosocomial infections and the most important pathogen for cystic fibrosis (CF) patients. It plays an important role in the pathogenesis of CF pulmonary disease, critical for P. aeruginosa killing of Caenorhabditcs elegans and of mice in septicemia models (Mahajan et al., 1999). The major phenazine produced by the organism is pyocyanine (1- hydroxy 5 methyl 1 phenazine) and its physiological significance has long been a mystery (Weinberg, 1970). Pseudomonas aeruginosa produces a blue pigment pyocyanin. It is a redox active phenazine compound that kills mammalian and bacterial cells through the generation of reactive oxygen intermediates (Hassett et al., 1992). Pyocyanine was recently shown to inhibit T lymphocyte proliferation in humans and mice (Muhlradt et al., 1989). The Predominant phenazine pigment pyocyanin induces rapid apoptosis of human and once established in the airway of these patients is seldom eradicated (Woods et al., 1983; Hoiby, 1982). P. aeruginosa produces two types of soluble pigments Pyocyanin and Pyoverdin (fluorescent). The latter is produced abundantly in media of low content, and could function in iron metabolism in the bacterium. Pyocyanin refers to Blue Pus which is a characteristic of suppurative infections caused by P. aeruginosa. The most widely studied phenazine-producing fluorescent pseudomonad is P. aeruginosa, a gram-negative opportunistic pathogen of animals, insects, nematodes, and plants (Jander et al., 1998; Rahme et al., 1997). In humans, P. aeruginosa infects immune-compromised, burned, or injured patients and can cause both acute and chronic lung disease. Strains of P. aeruginosa produce a variety of redox-active phenazine compounds, including pyocyanin, phenazine-1- carboxylic acid (PCA), 1-hydroxyphenazine (1-OH-PHZ), and phenazine-1-carboxamide (PCN) (Budzikiewicz, 1993). P. aeruginosa generates highly diffusible pigmented toxic secondary metabolites known as phenazines that are neutrophils. This report describes a new specific primer pair targeting the Phz gene of P. aeruginosa for differentiation and easy identification of pathogenic isolates from environmental isolates. MATERIALS AND METHODS Collection of samples Totally 35 samples of pus, throat, cervical cells, burn wounds etc. were collected from different hospitals in Tiruchirappalli and 10 samples were collected from different environment (commensal) like soil, litter, sewage etc. 70 JOURNAL OF MODERN BIOTECHNOLOGY VOLUME 1 NUMBER 2 NOVEMBER 2012

2 Jamunadevi et al. Molecular characterization of pathogenic and non-pathogenic P. aeruginosa Identification of bacteria The bacteria were cultured on a selective medium cetrimide agar base (Himedia, Mumbai, India) and were incubated at 37 C up to 48 h and observed colonies were sub-cultured, identified by standard microbiological methods. Antibiotic sensitivity assay Antibiotic sensitivity was carried out by disc diffusion method (Baeur et al., 1966) for both pathogenic and commensal isolates. Ten different antibiotics such as gentamycin, ciproflaxin, piperacillin, amikacin, cefotoxime, imipenem, carbenicillin, ampicillin, tetracycline and tobramycin were used. Sensitivity test was analyzed from the identified strains by using Mueller Hinton agar (Himedia, Mumbai, India) and incubated at 37 C for 24 h. PCR amplification of phenazine gene DNA Extraction (Hassett et al., 1992). Bacterial culture were swabbed from culture plate, washed twice in a solution of 50 mm Tris HCl (ph 8.0) 5 mm EDTA (ph 8.0), 50 mm NaCl. To which, 1 mg/ml lysozyme was added and incubated at 55 C for 30 min followed by the addition of 10% SDS and 10 µl of 10 mg/ml proteinase K and again incubated for 10 min. This solution was extracted with equal volume of phenol: chloroform: Isoamyl alcohol (25:24:1). This organic extraction was repeated and supernatant was added to one volume of 4 M ammonium acetate solution and two volume of isopropanol and centrifuged at rpm. DNA was precipitated with cold ethanol, centrifuged, air dried and re-suspended in TE buffer (ph 8.0). The DNA purity and concentration was assessed at 260/280 nm in a spectrophotometer (Shimadzu, Tokyo, Japan). Primer Selection Phz gene (Phenazine) P. aeruginosa possess 3 types of phenazine pigment and phenazine compounds are biologically active metabolites that function in microbial competitiveness pyocyanine was the main phenazine pigment associated with this particular organism. Pyocyanine (1-hydroxy 5-methyl phenazine) was selected for further amplifications (Mavrodi et al., 2001). Phz -F (5'-TAAGGATCCGGTAGTTCCAA GCCCCAGAAAC-3') Phz -R (5'-CACATTTGATCTAGATGGGTCACGGCTATTCAG-3') PCR Condition PCR was performed in a 20 µl reaction mixture containing 2 µl of template DNA of the processed clinical samples, 1.5 µl of each primer 2 µl of DNTP mix, 5µl of Taq DNA buffer and 0.5 µl of 1U Taq DNA polymerase (Finzymes). Samples were subjected to the following thermo cycling process (ABI Thermal cycler gradient) 94 C for 5 min followed by 30 cycles consisting of 94 C for 1 min, 64 C for 1 min, 72 C for 1 min. A final extension step at 72 C was continued for another 5 min. A tube contained the reaction mixture and sterile water was included in all reactions as a negative control. RESULTS Isolation and Identification A total of 40 P. aeruginosa were collected from the hospitals in and around Tiruchirappalli, 30 samples were collected from clinical sources and 10 samples were collected from environmental sources. Collected clinical and non clinical isolates were subjected to microscopic examination in laboratory P. aeruginosa (Figure 1) was confirmed by using preliminary and selective biochemical test followed by Bergey s Manual of determinative bacteriology. Figure 1: Cetrimide Agar Plate (P. aeruginosa) Table 1: Antibiotic Sensitivity of Pathogenic Isolates Isolate Gen Cip Pip Ami Cft Imi Car Amp Tet Tob PA01 S S S S S S S R R S PA02 S R S S R S S R R S PA03 S S S S S S S R R R PA04 S R S S S S S R R S PA05 S S S S S S S R R S PA06 S S S S S S S R R R PA07 S S S S S S S R R S PA08 S S S S S S S R R S PA09 S R R S R S S R R R PA10 S S S S S S S R R R PA11 S S S S S S S R R S PA12 S R S S S S S R R S PA13 S S S S S S S R R S PA14 S S S S S S S R R S PA15 S R R S R S S R R S PA16 S R S S S S S R R S PA17 S S S S S S S R R S PA18 S S S S S S S R R S PA19 S R R S R S S R R S PA20 S R S S S S S R R S PA21 S S S S S S S R R S PA22 S S S S S S S R R S PA23 S R R S R S S R R S PA24 S R S S S S S R R S PA25 S S S S S S S R R S PA26 S S S S R S S R R S PA28 S S S S R S S R R S PA29 R R R R R S S R R R PA30 S S S S S S S R R S S-Sensitive; R-Resistant Gen Gentamycin, Cip Ciprofloxacin, Pip Piperacillin, Ami Amikacin, Cef Cefotaxime, Imi Imipenem, Car Carbenicillin, Amp Ampicillin, Tet Tetracycline, Tob Tobramycin Antibiotic Susceptibility All 30 pathogenic isolates were sensitive to Imipenem and Carbenicillin and resistant to Ampicillin and Tetracycline, of these 29 (96.6%) isolates were sensitive to Gentamycin and Amikacin. For Cefotaxime 20 (66.6%) isolates were sensitive 71 VOLUME 1 NUMBER 2 NOVEMBER 2012 JOURNAL OF MODERN BIOTECHNOLOGY

3 Molecular characterization of pathogenic and non-pathogenic P. aeruginosa Jamunadevi et al. and 10 isolates were resistant. Twenty one (70%) isolates were sensitive to Tobramycin and 9 isolate were resistant to same antibiotics. Piperacillin is sensitive for 24 (80%) isolates and resistant for 6 isolates (Table 1). Among 10 commensal isolates, all isolates are resistant to Piperacillin and Cefotoxime. Five isolates were sensitive and five were resistant to Ampicillin and tetracycline. Three were resistant to Imipenem, Carbenicillin, and Amikacin. Two were sensitive to Tobramycin and Cipraflaxin. Seven isolates were sensitive to Gentamycin and 7 were resistant to Ciproflaxin (Table 2). In this experiment all the pathogenic Isolates were sensitive to imipenem and carbenicillin, and resistant to Ampicillin and tetracycline, All Environmental isolates were highly resistant to piperacillin and 2 cefotoxime In addition the environmental isolates of P. aeruginosa exhibited higher antibiotic resistance than clinical isolates (Figure 2). stating the usefulness of this primer for rapid detection of pathogens from samples (Figure 3). Table 2: Antibiotic sensitivity of commensal isolates Isolate Gen Cip Pip Ami Cef Imi Car Amp Tet Tob PAC01 R S R R R R R S S S PAC02 R R R S R S S S S R PAC03 S R R S R S S S S R PAC04 S S R R R R R S S S PAC05 S S R S R S S S S R PAC06 S R R R R R R R S R PAC07 S R R S R S S R R R PAC08 S R R S R S S R R R PAC09 S R R S R S S R R R PAC10 S R R S R S S R R R S-Sensitive; R-Resistant Gen Gentamycin, Cip Ciprofloxacin, Pip Piperacillin, Ami Amikacin, Cef Cefotaxime, Imi Imipenem, Car Carbenicillin, Amp Ampicillin, Tet Tetracycline, Tob Tobramycin G Cf P Ak Ct I Cb Am Tc Tm ANTIBIOTICS Pathogenic isolates S Pathogenic isolates R Commensal isolates S Commensal isolates R G Gentamycin, Cf Ciprofloxacin, P Piperacillin, Ak Amikacin, Ct Cefotaxime, I Imipenem, Cb Carbenicillin, Am Ampicillin, Tc Tetracycline, Tm Tobramycin Figure 2: Antibiotic sensitivity pattern of Pseudomonas isolates PCR Characterization of phenazine gene Polymerase chain reaction was done for all the strains (both pathogenic and commensal) using primers flanking the phenazine region to differentiate them PCR was performed for the gene Phz (Phenazine Pigment) were produced in 30 Pathogenic Isolates. Surprisingly, as expected as there was no amplification of phenazine gene in environmental isolates, Figure 3: PCR Characterization of phenazine gene DISCUSSION P. aeruginosa is an important cause of nosocomial infections and this may result from its ability to colonize abiotic surfaces for prolonged periods of time (Grundmann et al., 1993; Cernohorska et al., 2008). P. aeruginosa is an opportunistic human pathogen most commonly affecting immunecompromised patients such as those with cystic fibrosis (Elkin et al., 2003). P. aeruginosa has become an important cause of infection. In a survey of community hospitals (Bodey et al., 1983) this organism was found to have 343 infections per 100,000 discharged patients, or 12% of all reported infections. P. aeruginosa reportedly caused 10% of urinary tract infection, 9% of surgical wound infections, 17% of lower respiratory tract infections and11% of bacteremias, as revealed by the hospital surveillance program of the centers for disease control (Bennet et al., 1974). P. aeruginosa is synthesizing a characteristic blue chloroform soluble compound pyocyanin (1 hydroxy-5 methyl-phenazine) and this pigment is one of the factors contribute to the pathogenicity of the organism. Pyocyanine is a redox compound produced by P. aeruginosa and may facilitate the persistence of the organism in colonized airways by showing ciliary beating and reducing mucociliary clearance (Munro et al., 1989; Pitt, 1986, Wilson et al., 1987). A number of pharmacological interventions were used to delineate the mode of action of pyocyanin. Antioxidants and inhibitors of cyclooxygenase had no effect on either ciliary beat frequency showing or the disruption of epithelium integrity. Many phenazine compounds are found in nature and are produced by bacteria such as Pseudomonas spp., Streptomyces spp., and Pantoea agglomerans. These phenazine natural products have been implicated in the virulence and competitive fitness of producing organisms. For example, the phenazine pyocyanin produced by P. aeruginosa contributes to its ability to colonize the lungs of cystic fibrosis patients. Similarly, phenazine-1-carboxylic acid, produced by a number of Pseudomonas, increases survival in soil environments and has been shown to be essential for the biological control activity of certain strains (Turner et al., 1986). The oxygen 72 JOURNAL OF MODERN BIOTECHNOLOGY VOLUME 1 NUMBER 2 NOVEMBER 2012

4 Jamunadevi et al. Molecular characterization of pathogenic and non-pathogenic P. aeruginosa dependent toxicity of pyocyanin was due to auto oxidation by reduced pyocyanin, leading to O 2 - and/or H 2 O 2 generation. However, pyocyanin appears to have no detrimental effect on P. aeruginosa, the organism from which it is derived (Kersulyt et al., 1995). The antimicrobial resistance properties of 968 P. aeruginosa isolates to various classes of antimicrobial agents (Harsan and Fridovich, 1980) were tested. More than 30% of P. aeruginosa isolates proved resistant to the currently utilized antimicrobial agents, which include the anti Pseudomonal β lactams, the third and fourth generations of Cephalosporins and the Fluroquinones. Resistant to Carpenems, would constitute the first choice for the treatment of multidrug resistant P. aeruginosa infection. In this investigation, isolation and molecular characterization of pathogenic and commensal P. aeruginosa was carried out. All the isolates were compared for the presence of Phz phenazine producing gene by PCR method. This gene was shown to be present only in pathogenic forms and not in the commensal / environmental isolates, thus confirming the applicability of this primer in the screening and characterization of pathogenic forms of P. aeruginosa from environmental / clinical samples. The present study, 30 isolates were isolated from clinical sources and the 10 isolates from other environmental sources. The clinical sources such as pus, wound, tracheal, urine etc were used for the isolation. Pus (43.3%) isolates give a high positive result when compared to other sources and also at the age group of range Pseudomonas growth is positive. In the present study, the isolates were identified as Pseudomonas based on the previous reports and confirmed with other biochemical test. The isolates were then undergone antibiotic sensitivity by using Baeur et al., (1966) method with 10 different antibiotics. This discrepancy can be attributed to the continuous development of MDR strains of P. aeruginosa in different parts of the world. In our study also 22 pathogenic isolates was sensitive to Ciproflaxacin. Antibiotic sensitivity pattern against Pseudomonas was given by many antibiotics based on that antibiotics Gentamycin, Ciproflaxacin, Piperacillin, Amikacin, Cefotaxime, Impenem, Carbenicillin, Tobramycin were sensitive to that organism. Ampicillin and Tetracycline was resistant to Pseudomonas. Kersulyt, et al., 1995; Lee et al., 2007) compared PFGE with a PCR based typing method arbitrarily primed PCR (AP-PCR), which was easier to perform. They found an excellent correlation between PFGE and AP-PCR for a similar result with equivalent discrimination, AP-PCR presented advantages of rapidly and simplicity and was recommended by the author for the initial screening with many strains of P. aeruginosa are to be typed. PCR amplification was made using the primer Phz (phenazine pigment) for both pathogenic and commensal organisms. The amplified product was obtained in the size range of 250 bp only in pathogens not in the commensal isolates. Our present study reveals that Phenazine producing gene present in pathogenic organism but not in the environment samples. CONCLUSION In the present study Phz gene amplification was carried out for differentiation and comparison of pathogens and commensal isolates. Totally 40 samples were collected from different sources in different hospitals in and around Tiruchirappalli. Antibiotic sensitivity of the pathogenic organisms was done with 10 different antibiotics. From this, all the organisms were sensitive to Imipenem and Carbenicillin and resistant to Amipicillin and tetracycline. All the commensal isolates were resistant to Pipercilliln, Cefotaxime. DNA was extracted from all isolates by using Phenol/chloroform method. Polymerase chain reaction was used for amplification of the Phz gene in pathogens and commensal. The results showed that Phenazine gene was present only in pathogenic forms but not in commensal. This study elucidate the presence of Phenazine, a blue redox dye producing gene present in pathogenic samples like pus, wound etc and it is absent in other environmental samples. This study also shed light on the possible application of this primer pair (phz) for rapid differentiation and comparison of pathogenic and non-pathogenic strains from various samples. REFERENCE Ahearn DG, Borazjani RN, Simmons RB and Gabriel MM Primary adhesion of Pseudomonas aeruginosa to inanimate surfaces including biomaterials. Methods in Enzymology 310: Baeur AW, Kirby W and Sherris TS Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Pathology 36: Bennet JV. Hospital acquired infections and the altered host Nosocomial infections due to Pseudomonas. International Journal of Infectious Diseases 130:S4 S7. Bodey GP, Olivar R, Fainstein V and Jadeja L Infections caused by Pseudomonas aeruginosa. Reviews of Infectious Diseases 5: Budzikiewicz H Secondary metabolites from fluorescent pseudomonads. FEMS Microbiology Reviews 104: Cernohorska L and Votava M Antibiotic synergy against biofilm forming Pseudomonas aeruginosa. International Journal of Folia Microbiologica 53: Costerton JW, Stewart PS and Greenberg EP Bacterial biofilms a common cause of persistent infections. Journal of Science 284: Elkin S and Geddes D Pseudomonal infection in cystic fibrosis. The battle continues expert rev. Expert Review of Anti-Infective Therapy 1: Grundmann H, Kropec A, Hartung D, Berner R and Daschner F Pseudomonas aeruginosa in a neonatal intensive care unit reservoirs and ecology of the nosocomial pathogen. International Journal of Infectious Diseases 168: Harsan HM and Fridovich I Mechanism of the antibiotics action of pyocyanine. Journal Bacteriology 141: Hassett JD, Charniga L, Bean K, Oman ED, and Cohen SM Response of Pseudomonas aeruginosa to pyocyanin: Mechanisms of resistance antioxidant defenses, and demonstration of manganese, co factored superoxide dismutase. Infection and Immunity 60: Hoiby N Microbiology of lung infections in cystic fibrosis patients. Acta paediatrica scandinavica 301: Jander G L, Rahme G and Ausubel FM Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects. Journal of Bacteriology 182: VOLUME 1 NUMBER 2 NOVEMBER 2012 JOURNAL OF MODERN BIOTECHNOLOGY

5 Molecular characterization of pathogenic and non-pathogenic P. aeruginosa Jamunadevi et al. Javis WR and Martone WJ Predominant pathogens in hospital infections. Journal of Antimicrobial Chemotherapy 29: Kersulyt D, Struelens MS, Deplano A and Berg DE Comparison of arbitrarily primed PCR and macro restriction (pulsed field gel electrophoresis and typing of Pseudomonas aeruginosa strains from cystic fibrosis patients. Journal of Clinical Microbiology 33: Lee JH, Choi CH, Kang HY, Lee JY and Kim J Differences in phenotypic and genotypic traits against antimicrobial agents between Acinnetobacter baumannii and Acinetobacter genomic species 13TU. Journal of Antimicrobial Chemotherapy 59: Mahajan SM, Tan MW, Rahme LG and Ausubel FM Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa Caenorhabditis elegans pathogenesis model. Journal of Cell Science 96: Mavrodi VD, Bonsall RF, Delaney SM, Soule MJ, Philps G and Thomshow LS Functional analysis of genes for biosynthesis of Pyocyanin and Phenazine carboximide from P. aeruginosa PA01. Journal of Bacteriology 183: Muhlradt PF, Tsai H and Conradt P Effects of pyocyanine a blue pigment from Pseudomonas aeruginosa on separate steps of T cell activation interleukin 2 (IL2) production and induction of cytolytic activity. European journal of immunology 16: Munro NC, Barker A, Rutman A, Tayler G, Watson D, Mc Donald WJ and Cole PJ The effect of pyocyanine and 1 hydroxy phenazine on in vivo tracheal mucus velocity. Journal of Applied Physiology 76: Pitt TL Biology of P. aeruginosa in relation to pulmonary infection in cystic fibrosis. Journal of Royal Society of Medicine. 76: Rahme LG, Tan MW, Le L, Wong SM, Tompkins RG, Calderwood SB and Ausubel FM Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proceedings of the National Academy of Sciences USA 94: Turner JM and Messenger AJ Occurrence, biochemistry and physiology of phenazine pigment production. Journal of Advances in Microbial Physiology 27: Usher LR, Lawson RA, Geary I, Taylor C.J, Bingle CD, Taylor GW and White MK Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: A potential mechanism of persistent inflection. Journal of Immunology 168: Vincent JL, Sakr Y and Sprung CL Sepsis in European intensive care units: Results of the SOAP study. Journal of Critical Care Medicine 34: Weinberg ED Biosynthesis of secondary metabolites, role of trace metals. Journal Advances in Microbial Physiology 4:1 44. Wilson R, Pitt T, Taylor G, Watson D, Mc Dermott J, Sykes D, Roberts D and Cole PJ Pyocyanin and 1 hydroxy phenazine produced by Pseudomonas aeruginosa inhibit human ciliary beating in vivo. The Journal of Clinical Investigation 79: Woods DE and Iglewski BH Toxins of Pseudomonas aeruginosa new perspectives. Reviews of Infectious Disease 5: JOURNAL OF MODERN BIOTECHNOLOGY VOLUME 1 NUMBER 2 NOVEMBER 2012