Detection of virulence factors of Pseudomonas aeruginosa in different animals by using bacteriological and molecular methods

The aim of this study was to detect the presence of virulence factors of Pseudomonas aeruginosa in different animals. For this purpose 120 samples were collected and examined to detect fourteen virulence factors by using biochemical and molecular methods. The results showed that the highest isolation rate was recorded in doges (29.6%) among studied animals, and highest isolation rate was recorded in milk samples (26.8%) among the studied samples. The virulence factors were detected in different ratio, and highest of them were capsule detected in 50% from skin isolates, amylase enzyme detected in 28.5% from milk isolates, hemolysin enzyme detected in 75% from wound isolates, protease detected in 100% from skin isolates, phospholipase enzyme detected in 56.1% from milk isolates, urease enzyme detected in 50% from skin isolates, gelatin liquefaction detected in 100% from skin and ear isolates, β -lactamase production detected in 100% from skin and wound isolates, pigments production detected in 100% from skin and ear isolates, oprI, oprL and exoT detected in 100% from skin and wound isolates, exoS detected in 100% and 85.7% from skin and milk isolates respectively. We conclude from his study that the dogs are more sensitive in compare with studied animal, while the milk sample is more susceptible to contamination by Pseudomonas aeruginosa. Regarding the virulence factors we noticed that the appearance of it basis on infection state.

Pseudomonas aeruginosa: is a saprophytic bacteria in soil, water and plants, in addition to its apart of the normal flora in animals and human (4).In animals, it causes mastitis, metritis, pneumonia, dermatitis and enteritis (1).
Pseudomonas aeruginosa possess many virulence factors including: Protease: its two types: Elastase and Alkaline protease; that causes tissue necrosis, distraction of immunoglobulin and inhibition of tumor necrosis factors and gamma interferon (5).Ureases: its play a role in urea hydrolysis and ammonia releasing; these events which lead to increase of urine pH (6).Lipases and phospholipases: distraction of lipids surfactant and phospholipids of host cell membranes (7).Haemolysin: play role in pulmonary infection, its involved two types: Heat-labile (Phospholipase C) and Heat-stable (Glycolipid) (8).Motility : Pseudomonas aeruginosa motile by single polar flagellum, the bacterium can adhere to host epithelial cells through the binding of its flagellum to the asialyated glycolipid (9).Pigments: its causes host oxidative stress, damage of host catalase enzyme and mitochondrial electron transport (10).Also so it apple to cause inhibition of chemokine production and intra-phagocytic killing (11).Outer membrane proteins of Pseudomonas aeruginosa (OprI and OprL): play important roles in the interaction of the bacterium with the environment as well as the its inherent resistance to antibiotics, where the consequence of the presence of these specific outer membrane proteins that have been implicated in efflux transport systems that affect cell permeability (12).As these proteins are found only in this organism, they could be a reliable factor for rapid identification of Pseudomonas aeruginosa in clinical samples (13).Exoenzyme S: encoded by the exoS gene, is an ADP ribosyltransferase that is secreted by a type-III secretion system directly into the cytosol of epithelial cells (14).

Materials and methods
This study was designed in Salahaldeen province on animals with different age, sex and species in period extended from February to May 2017.The samples were collected and transported immediately for bacterial culturing then molecular technique done in Tikrit university, laboratory of Veterinary Medicine College.

Samples
Skin scraps, swabs from wound and ear, and samples from digestive system and milk were collected.The species of animals and number of samples as in table 1.

Bacterial isolation
it was performed by using (Pseudomonas agar base -Himedia), with nalidixic acid (500 mg dissolved in 100 ml of distilled water then add of 3 ml of suspension to each 1L of medium) and tetrazolium bromide (3g dissolved in 100 ml, then add of 10 ml of suspension to each 1 L of medium) (1).

Biochemical tests which include
Oxidase test, catalase test, Urease test, nitrate reduction test, H2 S production test, Lysine decarboxylase test, indole test and Oxidation -fermentation test (1).Serotyping By used monoclonal antibodies (Sanofi Diagnostics Pasteur-France) which are anti O-poly saccharide (1P-16p).appearance of agglutination between bacteria and monoclonal antibodies refer to its serotype.

Detection of virulence factors
Capsule detection: by using of Indian ink, Motility activity detected by used of Triple Sugar Iron sugar iron and according to (15).Production of amylase enzyme detected by using of Starch medium and according to (16).Production of hemolysin enzyme detected by using of blood agar.Production of protease enzyme detected by using of skim milk agar and according to (17).Production of phospholipase enzyme detected by using of nutrient agar, Nacl and egg yolk and according to (18).Production of Urease enzyme detected by using of urea media Production of DNAase detected by using of DNA media.Gelatin liquefaction test: by used of gelatin media and according to (15).Detection of β -lactamase production applied according to (19).

DNA extraction
For genetic methods performed by reactivation of Pseudomonas aeruginosa by culturing in trypton soya agar at 37c for 24 hours.and bacterial DNA extracted according to methods describe by (20).And by using of Genomic DNA Mini Kit (blood/cultured cell) (Geneaid).

Compounds used in preparation of reaction mixture
The component that used in genetic included: Taq PCR Master Mix KIT (20µl), Forward primer (1.4µl), Reverse primer (1.4µl),DNA Template (2µl) and DNA free water (15.2µl).

Thermocycler programs
Thermocycler were set in three steps: First Denaturation step with 95°C for 5mints, Denaturation step with 95°C for 30 second, DNA extension step with 72°C for 30seconds and Primer-annealing step with 55°C, 58°C, 60°C and 55°C for 30seconds for, oprI, oprL and exoT respectively.

Results
The current study showed difference in isolation rate of Pseudomonas aeruginosa according type of samples, the highest isolation rate was recorded in milk and ear samples which are 26.8% and 25.9% respectively and lowest isolation rate in wound infection (11.4%).
The isolation rate differs according to species of animals the higher infection rate recorded in doges was 29.6%.and lowest rate recorded in horses (16.6%).Table 3 describe the infection rate in different samples for each animals spp.

Genetic methods used in detection of virulence factors
The OprI and exoT genes were detected in about 88% of pseudomonas isolates, exoS gene detected in 76.9% while OprL detected in 69.2% from total number of pseudomonas isolates, as in table 6 and figure 1-4 shows result of PCR test.

Discussion
In this study, P. aeruginosa isolated from all animal samples with different levels.This result was in agreement with the study of AL-hadithi (22) who isolated P. aeruginosa from different animal samples in Baghdad.The highest isolation rate was recorded in samples of dogs ear and this was in agreement with Doge et al. (23).
P. aeruginosa can causes otitis externa in canines it cause inflammation and ulceration within the external ear canal (13).The isolation of P. aeruginosa from milk in the current study, may be referred to either ability of P. aeruginosa to live in wide range of temperature or its one the causes of mastitis also might be due to contamination after milking.
The present study also showed difference in the serotyping of the isolation.The dominance of one serotype in compare with others may be due to geographic distribution, resistance to host immunity and environmental prevalence, and antibiotics resistances (23,24).Some isolates appeared to have capsule.This result differ from result recorded by Al-Mashhadani (25) were 0%, and result recorded by AL-Salihi and Hasan (26) were 42.8%, that's may be due to different in type of infection.Capsule play important role in prevention of phagocytosis so that its protect bacteria from anther types of immunological invasion (27).
Hemolysin enzyme detection in 61.5%, that agreement with (25).Hemolysin associated with necrotoxicity and cytotoxicity of the cells.It can form pores in the erythrocytes plasma membrane (28).All isolates were motile, DNAase negative, this agreement with (25).Most isolate were positive to pigments production and gelatin liquefaction.and that's was agreement with (25).Pigments play important role in pathogenicity of P. aeruginosa and reduce of host immunity (10,11).
Ureases enzyme detected in 19.2% of total isolates, this enzyme able to distract urea into CO 2 and NH 3 and increase pH which enhance bacterial growth (29).
Further more the rsults study showed many bacteria gave positive results in protease detection test.Protease enzyme enhance infection by damage of host cells and break down of immune defense mechanism like skin and mucous membranes (29,30).
In current study extracellular toxin (exoS and exoT ) were detected in most isolates.The extracellular toxin can causes necrosis, edema and hemorrhage (14).
The outer membrane proteins OprI and OprL were detected in different rate according to isolate source.The outer membrane proteins effect in cellular permeability and increase antibiotic resistance (14).

Table 1 :
species of animals and number of samples

Table 2 :
primers used in study

Table 3 :
Pseudomonas aeruginosa infection ratio in different animals and simplesSerotyping of Pseudomonas aeruginosa:Table4shows serotypes of Pseudomonas aeruginosa that isolated in current study.

Table 4 :
Serotype of P. aeruginosa isolated from different animal

Table 5 :
Virulence factors of P. aeruginosa isolated from different animal samples

Table 6 :
virulence factors of Pseudomonas aeruginosa isolated from different animal samples