Detection of Exotoxin A gene in Pseudomonas aeruginosa from Clinical and Environmental samples
PCR was used to detect Pseudomonas aeruginosa from clinical and environmental samples by amplifying a 396 bp region of the exotoxin A (ETA) structural gene sequence. Specific primer amplified ETA positive P. aeruginosa DNA, whereas other species of Pseudomonas and GC rich bacteria did not yield any 396 bp fragment. The specificity of the assay was 1% (just one isolate from 100 isolates used in this study(,this gene is not present in these isolates which means that these isolates do not have the ability to produce the Exo A toxin. With this PCR method, ETA positive P. aeruginosa was detected only in one water sample in comparison with the use of the 16SrDNA gene which yielded 100% positive results of all tested isolates. This PCR method is rapid and more accurate than other diagnostic methods for the identification of P. aeruginosa strains, and it can be used to detect a low level of P. aeruginosa from different samples without using a selective medium or additional biochemical tests. Introduction Pseudomonas aeruginosa is an opportunistic pathogen capable of infecting both humans and animals. P. aeruginosa is an important cause of bacteremia in patients receiving organ transplants and is responsible for about 28% of most bacteremia episodes [1] Pulmonary colonization with mucoid P.aeruginosa is also a major cause of morbidity and mortality in patients with cystic fibrosis [2] Correa et al. 1991[3] have reported that vegetables were the main source of P. aeruginosa in hospitals even though 1% hypochloride solution is used for sanitizing the vegetables. P. aeruginosa has a wide arsenal of virulence factors at its disposal. Among these virulence factors are a variety of secreted factors, such as proteases, phospholipases, and the exotoxin A. P.aeruginosa strains also posses a type III secretion system that allows them to deliver toxins (effectors) directly into the cytoplasm of a host cell [4] Exotoxin A which causes tissue necrosis since it blocks protein synthesis [5] P. aeruginosa strains producing relatively large amounts of exotoxin A (ETA) and proteases at the level of 107 cells per ml in drinking water of mice which can cause endogenous bactermia in few days [6;7] Exotoxin A (ExoA, toxA) is a 66 kDA protein acts as a major virulence factor of P. aeruginosa, analogous in action to that of diphtheria toxin. ExoA is a highly virulent protein, exhibiting and LD50 of 2.5 mg/kg in mice, it has been shown that DtoxA mutants are less virulent than wild type strains, and that vaccination against ExoA confers partial immunity to P. aeruginosa infection in animals [8] Injection of purified ExoA results in leucopaenia, hepatic necrosis, hypotension and shock when injected into test animals. On a microscopic level, collagen is disrupted, proteoglycan ground substance is lost and widespread endothelial and epithelial cell death is observed [9] P. aeruginosa produces two different ADP ribosyltransferase toxins: ETA and exoenzyme S [10; 11; 8; 12] Exoenzyme S causes significant tissue damage in lung, burn and wound infections [12] The highly toxic ETA is produced by the majority of P. aeruginosa strains and can inhibit eukaryotic protein biosynthesis at the level of polypeptide chain elongation factor 2, similarly to diphtheria toxin [12] ETA consists of two subunits; fragment A is catalytic, and fragment B is responsible for interaction with eucaryotic cell receptors. ETA is cytotoxic to numerous mammalian cells [13] Wathiq Abas Al Daraghi 168 stimulates in vitro production of interleukin 1 in murine peritoneal macrophages [14] and induces murine cytotoxic T lymphocytes [15] Gray et al. 1984 [16] have cloned and sequenced the ETA structural gene from a P.aeruginosa strain overexpressing ETA. Because P. aeruginosa is medically important, various methods have been developed to rapidly and accurately identify P.aeruginosa species. Although conventional microbiological methods for identifying P. aeruginosa from environmental samples are reliable, they require several days to complete. PCR has the potential for identifying microbial species rapidly by amplification of gene sequences unique to a particular organism [17] In this paper, PCR technique was used to detect P. aeruginosa strains in clinical and environmental samples by amplifying ETA structural gene. Materials and Methods Bacterial isolates and growth conditions Fifty environmental isolates and fifty clinical isolates of P. aeruginosa were obtained from different sources (Table (1) All isolates were maintained in MacConky broth (CDH, NewDelhi) containing 20% glycerol at 20degC. Organisms were grown overnight at 37degC in MacConky broth or on citrimide agar (Microgen, New Delhi) plates. P. aeruginosa was isolated from water samples was isolated by plating on cetrimide agar and incubated at 37degC for 48 hrs. Isolated colonies were identified by using the oxidase test, and using Api 20 system [18] A positive strain was obtained from the general health laboratory, Ministry of Health.