INTRODUCTION:

P. aeruginosa is one of the most common pathogen causing nosocomial infection in clinical setting.^[1] Hospital acquired infections caused by this organism often leads to significant morbidity and mortality^[2] as they are highly virulent and known to show resistance to wide variety of antibiotics.^[3] Many pathogenic bacteria are producing extracellular substances or enzymes that directly damage the host tissue cells. This diverse range of substances produced by pathogenic bacteria serve as virulence factors. They are involving in the pathogenesis of disease by facilitating spread of bacteria or toxins through host tissues. These are generally referred as “spreading factors”.^[4] Lipase are defined as hydrolases, which acts on carboxylic ester bonds to hydrolyse triglycerides into diglycerides, monoglycerides, fatty acids and glycerol.^[5]

Microbial lipases have been characterised for their role in virulence and their potential application in biotechnology.^[6] Lipase producing Pseudomonas are able to cause souring, spoilage of preprocessed dairy products thereby it causes infections in human beings.^[7]It involved in digestion of host cellular lipids for their

Nutrient acquisition, thereby it adheres and causes infections.^[8] Lipases are secreted by the following members of bacteria such as Pseudomonas spp , Burkholderia spp, Alcaligenes spp, Acinetobacter spp, Bacillus spp and Chromobacterium spp as well as fungal members such as Candida spp, Penicillium spp, Rhizopus spp, Aspergillus spp and act as a virulent factor to establish infections.^[9] Lecithinase producing bacteria can act on lecithin and produce phosphorous and choline with precipitation of fat.^[10] P.aeruginosa found to produce 2 phospholipases or phospho lecithinases such as PlcHR which is hemolytic and PlcN which is non-hemolytic.^[11] PlcN do not have any pathogenic activity, whereas PlcHR act as an important virulent factor .^[12]

MATERIALS AND METHODS:

Bacterial Isolates

A total of 20 non repetitive clinical isolates of P. aeruginosa were collected from Saveetha Medical College, Thandalam. They were processed for a battery of standard bio chemical tests and confirmed. Isolates were preserved in semi-solid trypticase soy stock and stored at 4ºC until for that use .^[13]

Antibiotic Susceptibility Testing

Antibiotic susceptibility testing was determined for this isolates to routinely used antibiotics such as to piperacillin- tazobactam, cefotaxime, ceftazidime, tetracycline, cotrimoxazole, aztreonam, gentamicin and imipenem by Kirby Bauer disc diffusion method as per CLSI guidelines .^[14]

Detection of Lipase

Lipase Base:

Trypticase soy broth - 8g

NaCl - 4g

Agar - 10g

Distilled water - 1000 ml

pH - 7.0

The medium was autoclaved and cooled in 31.25 ml of sterile triolylglycerol solution (HiMedia, Mumbai) and 10 ml of filter sterilized rhodamine D solution were incorporated in lipase base with vigorous stirring and 20 ml of media was poured over sterile petridishes. After solidification of media, spot inoculation of P. aeruginosa isolates were made and incubated at 37ºC for 24 hours. Substrate hydrolysis causes formation of orange fluorescent color visible upon ultra violet radiation was considered as positive for lipase production .^[15]

Detection of Lecithinase

Lecithinase detection was done by simplified plate method. Trypticase and agar with the addition of 0.11% CaCl2 and 5% egg yolk were used for demonstration of lecithinase activity at pH 7. Spot inoculation of P. aeruginosa isolates to lecithinase egg yolk was done. These inoculated plates were marked by an opaque zone extending from edge of colony.^[16]

RESULT:

Sample Wise Distribution of Clinical Isolates of P. aeruginosa.

Of the 20 isolates of clinical isolates of P.aeruginosa, 9/20 (45%) isolates were from sputum, 5/20 (25%) from blood, 3/20 (15%) from urine, 3/20 (15%) from pus.

Figure 1 : Sample wise distribution of clinical isolates of P. aeruginosa

Results of Antibiotic Susceptibility Testing

In our isolates, we have observed an increased percentage of isolates were shown to be resistant to most of the routinely used antibiotics. Only 2/20 (10%) isolates showed sensitivity to imipenem. Other than that, for all other antibiotics such as piperacillin-tazobactam, cefotaxime, ceftazidime, tetracycline, cotrimoxazole, aztrionum, gentamicin isolates showed complete reistance 20/20 (100%). The detailed resistant pattern of P. aeruginosa isolates were showed in table 1.

Table1: Results of antibiotic susceptibility pattern of P. aeruginosa

Antibiotics	Sensitivity (20) (%)	Intermediate (20) (%)	Resistant (20) (%)
Piperacillin-tazobactam	0(0)	0(0)	20(100)
Cefotaxime	0(0)	0(0)	20(100)
Ceftazidime	0(0)	0(0)	20(100)
Tetracycline	0(0)	0(0)	20(100)
Cotrimoxazole	0(0)	0(0)	20(100)
Aztreonam	0(0)	0(0)	20(100)
Gentamicin	0(0)	0(0)	20(100)
Imipenem	2 (10)	1 (5)	17 (85)

Results of lipase and lecithinase by P. aeruginosa

Out of 20 clinical isolates of P. aeruginosa , 4/20 (20%) were found to be strong lipase producer and 5/20 (25%) were shown to be moderate lipase producer of our isolates.

Figure 2: Representative picture showing lipase producing P. aeruginosa

By performing lecithinase plate assay 19/20 (95%) were found to be lecithinase producers (table 2)

Figure 3: Representative picture showing lecithinase producing P. aeruginosa

Table 2: Results of lipase and lecithinase of P. aeruginosa

P. aeruginosa	Lipase	Lecithinase
1	++	+
2	++	+
3	-	+
4	+	+
5	-	+
6	-	+
7	-	-
8	+	+
9	-	+
10	-	+
11	-	+
12	++	+
13	-	+
14	++	+
15	+	+
16	+	+
17	-	+
18	-	+
19	+	+
20	-	+

Thus, out of 20 isolates, 9 isolates (45%) showed both lipase and lecithinase positivity.

DISCUSSION:

Study done by Janda et al. in 1981 from New York reported that 81% of clinical and 9% of environmental isolates of P. aeruginosa were found to have lipase production ability.^[17] Yavankar and colleagues in 2007 also shown that 11.8% and 71.1% of commensal isolates of Acinetobacter spp were producing lipase and esterase respectively.^[18]

Lipase standardization assay done by Jagtap et al., in 2010 from Pune in Acietobacter haemolyticus strains of healthy human skin and proved its higher potential ability to produce lipase using different lipid substrates.^[19] Enzymatic profiling study conducted by Poh et al., in 1985 from Singapore demonstrated that 14% of A. calcoaceticus strains producing lipase and esterase.^[20] Finnan et al., in 2004 found that 82% of cystic fibrotic and hospital associated isolates of P. aeruginosa were shown to produce esterase activity.^[21] In the present study, we have observed 45% of clinical isolates of P. aeruginosa were appeared to produce lipase enzyme.

Enzyme profiling study on P. aeruginosa carried out by Janda et al., in 1981 from New York City found that around 57% of clinical and 64% of hospital environmental strains were found to produce lecithinase positivity.^[22] Yavankar et al., in 2007 from Pune carried out a study regarding physiological characterization of Acinetobacter spp from healthy human skin and observed that 87% of them were producing lecithinase enzyme.^[23] In contrast, we have observed an increase in percentage, 95% were producing lecithinase.

CONCLUSION:

A pile of literatures resolved and virulent determinants were produced by P. aeruginosa. We have seen a varied pattern of positivity in these two extracellular enzymes as virulent factors. We speculate that these two mechanisms exhibited by P. aeruginosa may associate with pavement and establishment of infections in hospitalized patients.

ACKNOWLEDGMENT:

We would like to thank Dr. Jeyalakshmi, Professor and Head of Dept of Microbiology, Saveetha Medical College, Chennai for kindly providing the isolates for conducting our research fruitfully.

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Received on 16.07.2016 Modified on 28.07.2016

Research J. Pharm. and Tech 2016; 9(11): 1909-1912.

DOI: 10.5958/0974-360X.2016.00391.7