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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 2  |  Page : 191-193
Slime production a virulence marker in Pseudomonas aeruginosa strains isolated from clinical and environmental specimens: A comparative study of two methods


1 Department of Microbiology, Kasturba Medical College, Manipal, India
2 Department of Microbiology, Sikkim Institute of Medical Sciences, Gangtok Sikkim, India

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   Abstract 

Detection of slime in Pseudomonas aeruginosa can be useful in understanding the virulence of this organism. Here, comparative studies of two phenotypic methods using the tube method and the spectrophotometric method for slime production from 100 clinically and 21 environmentally significant isolates of
P. aeruginosa were performed. A total of 68 isolates were positive by either of the tests whereas only 34 were positive by both the tests. The tube method detected slime significantly in more number of isolates than the spectrophotometric method. The tube test was found to be superior to the spectrophotometric method in ease of performance, interpretation and sensitivity. Among the clinical isolates, systemic isolates produce less slime compared to wound, respiratory and urinary isolates. Isolates from the hospital environment produced more slime indicating that this virulence marker helps the organism to survive for longer periods and cause nosocomial infections.

Keywords: Psuedomonas, slime production, Virulence, environmental samples

How to cite this article:
Prasad S V, Ballal M, Shivananda P G. Slime production a virulence marker in Pseudomonas aeruginosa strains isolated from clinical and environmental specimens: A comparative study of two methods. Indian J Pathol Microbiol 2009;52:191-3

How to cite this URL:
Prasad S V, Ballal M, Shivananda P G. Slime production a virulence marker in Pseudomonas aeruginosa strains isolated from clinical and environmental specimens: A comparative study of two methods. Indian J Pathol Microbiol [serial online] 2009 [cited 2020 Nov 24];52:191-3. Available from: https://www.ijpmonline.org/text.asp?2009/52/2/191/48914



   Introduction Top


Pseudomonas is an opportunistic, often multidrug-resistant organism that commonly inhabits the soil and water areas of the environment. Pseudomonas aeruginosa is the preliminary pathogen among the Pseudomonas and is a common cause of nosocomial complications, such as burn wound infection, respiratory tract infection, urinary tract infection and acts as an opportunistic pathogen in immunocompromised patients.

P. aeruginosa contains extracellular slime, which may have originated from the capsular polysaccharide associated with the outer membrane complex, as in the capsular polysaccharide with other Gram-negative species. [1]

The polysaccharide isolated was found to be similar to the seaweed alginic acids, except that bacterial polymers contain O-acetyl groups absent in alginates. [2],[3] The outer membrane capsule forms an antiphagocytic capsule around the organism, equivalent to the capsules of Gram-negative bacilli. [4]

The biofilm formation allows P. aeruginosa to escape host defenses and resist the antimicrobial action of antibiotics. [5] Slime production in P. aeruginosa was tested by Christensen's tube adherence method and the spectrophotometric method. There is a considerable variation in sensitivity and specificity, ease of performance feasibility and cost effectiveness of these tests.

The present study was conducted to detect the slime production that is responsible for the production of biofilms in P. aeruginosa , an important virulence marker especially in chronic diseases like cystic fibrosis (CF), for the comparison of slime production in clinical and hospital environmental isolates and also to access the effectiveness of the tube and the spectrophotometric methods for slime production by the clinical and environmental isolates of P. aeruginosa .


   Materials and Methods Top


The study included 100 strains of P. aeruginosa isolated from clinical specimens, including sputum, urine, pus, systemic isolates and 21 isolates from the hospital environment. Identification of the organism was performed according to the standard procedures. [6] The organisms were stored in nutrient agar slants and preserved at 4C. P. aeruginosa ATCC 27853 was used as a positive control for the test.

Demonstration of slime production

Slime production by P. aeruginosa was tested by the tube method. [7] The slime was also detected in the polystyrene microtiter plates using the spectrophotometric method. [8]

Tube method

Two to three colonies were inoculated into 5mL of BHI broth in glass tubes. Cultures were incubated at 37C for 18-20 h and the culture contents were aspirated.

Tubes were stained with saffranin. The presence of a visible stained film on the wall of the tube was considered to be positive for slime production.

Formation of the ring at the liquid air interface was not considered as a positive test. If the wall of the glass tube remained unstained, the strain was considered as a non-slime producer.

Spectrophotometric method

This method was also used to demonstrate the production of slime by P. aeruginosa as per the method described by Christensen et al. Eighteen hour cultures were standardized by McFarland's standards. Two hundred microliters of standardized cultures were added to the flat bottom wells of sterilized polystyrene and incubated for 18 h at 37C. Following incubation, the contents of the plate were gently aspirated. The plates were washed with sterile phosphate-buffered saline four times at pH 7.2.

Slime and adherent organisms were fixed overnight with Bouins fixative. The fixative was removed by washing the wells three to four times with 50% ethanol.

The wells were stained with Hucker's crystal violet. Excess stains were removed by placing the plate under distilled water. The plates were dried. The optical density (OD) of the stained adherent films was read using an ELISA reader (ELX 800 Bio Tek instruments USA) at a wavelength of 630 nm.

The measurements were repeated in duplicates and the mean OD was calculated. OD value greater than 0.1 were considered positive for slime production. [9]


   Results Top


Of the 121 isolates of P. aeruginosa t ested for slime production, 68 isolates produced slime by the tube method and 40 isolates by the spectrophotometric method, as shown in [Figure 1]. A total of 34 isolates were positive by both the methods. Eight strains of P. aeruginosa isolated from sputum, five from pus, three each from blood and urine, one from bile, seven from burns wounds and five environmental strains were found to be positive for slime by the tube method. One strain each from sputum, pus, blood and urine as well as two strains of P. aeruginosa isolated from burns wounds specimens were found to be positive for slime by the spectrophotometric method only.

Twenty strains were negative by both the methods, four sputum isolates, six pus isolates, two burns wounds isolates, five isolated from systemic infections, one urine isolate and two environmental isolates, respectively [Table 1]. The tube method was found to be superior for the detection of slime production in P. aeruginosa than the spectrophotometric method. Statistical analysis was carried out for these two methods using the Mc Nemar test ( P < 0.001).

Among the clinical isolates, systemic isolates produce less slime compared with wound, respiratory and urinary isolates.


   Discussion Top


P. aeruginosa can be commonly encountered in the clinical specimens and considered as the normal bacterial flora of the pharynx, mucous membranes and skin. When this organism is isolated from the clinical sample, efforts should be made to substantiate the clinical relevance in a particular patient. Slime polysaccharide has been isolated from patients suffering from chronic infections like CF, respiratory tract disease and urinary tract infection. [10],[11]

In chronic pulmonary infections in CF patients, alginate acts as a virulence factor by contributing to antibiotic resistance in P. aeruginosa . Alginate provides the bacteria with selective advantages for colonization of the pulmonary tissue through increased resistance to opsonization and phagocytic engulfment as well as through increased protection from toxic oxygen radicals. [12],[13]

Evaluation of the prevalence of slime production among P. aeruginosa strains isolated from clinical samples is important as it helps in deciding the pathogenicity and to assess its diagnostic value as a virulence marker. Such type of investigation has already been reported in Staphylococci [7],[8],[14],[15] and in Gram-negative bacteria by staining methods, [16] but simple and reliable techniques are required to identify the slime producing strains of P. aeruginosa isolated from the clinical specimens as antibiotic resistance is commonly found in these strains. It was also observed that the environmental isolates from the hospital environment were producing more slime than the clinical isolates, which suggests that the organisms produces this virulence factor to tide over the unfavorable conditions of the external environment as an added survival advantage. The slime production may be one of the reasons why P. aeruginosa can thrive for longer periods in the hospital environments, acting as a potent source for nosocomial infections.

In the present study, two different methods of slime production were compared. The tube method detected slime in increased number (56.20%) than the spectrophotometric method (33.05%). A positive but weak correlation was noted between the two methods.

Biological and technical factors may have contributed to the observed differences in the results as the tube method was performed in the glass tube and the spectrophotometric method in the polystyrene microtiter plate. In the present study, we used BHI broth as the media in both the tests and Bouin's reagent as the fixative in the spectrophotometric method. Although the reagent is reliable, the fixative is dangerous for routine use as it is found to be potentially explosive. [17]

Technical factors influencing slime production depend on the type of medium, atmosphere of incubation and the nature of the solid surface. [18]

In the present study, differentiation of P. aeruginosa as slime producers and non-slime producers using the spectrophotometric method was based on OD measurements and was found to be less sensitive in detecting the slime production by P. aeruginosa , although the adherent bacterial films on the polystyrene wells serve as a quantitative model for the study of adherence of P. aeruginosa to medical devices. Because of the technical factors involved, the spectrophotometric method remains unsuitable to detect the slime production in P. aeruginosa .

In conclusion, the results of the study show that tube method is sensitive and superior in comparison with the spectrophotometric method. In addition, the tube test can be preferred due to the ease of interpretation and performance for the detection of slime production in P. aeruginosa . Slime production of the environmental strains was found to be more compared with the clinical isolates. This may help the organism to thrive for a longer period in the hospital environment.

 
   References Top

1.Anderson ES, Rogers AH. Slime polysaccharides of the enterobacteriaceae. Nature (London) 1963;198:714-5.  Back to cited text no. 1    
2.Brown MR, Foster JH, Clamp JR. Composition of Pseudomonas aeruginosa slime. Biochem J 1969;112:521-5.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.Bartell PF, Orr TE, Chudio B. Purification and chemical composition of the protective slime antigen of Pseudomonas aeruginosa . J Bacteriol 1970;92:56-62.  Back to cited text no. 3    
4.Schwarzmann S, Boring JR. Antiphagocytic effect of slime from a mucoid strain of Pseudomonas aeruginosa. Infect Immun 1971;3:762 - 67.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Livermore DM. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa- our worst nightmare. Clin infect Dis 2002;34:634.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Brown R, Poxton IR, Wilkinson JF. In: Collee JG, Duguid JP, Fraser AG, Marimion P, editors. Mackie and McCartney Practical Medical Microbiology. 13 th ed. London: Churchill-Living Stone; 1989. p. 245-6.  Back to cited text no. 6    
7.Christensen CD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime producing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 1982;37:318-26.  Back to cited text no. 7    
8.Christensen CD, Simpson WA, Younger JJ. Adherence of Coagulase negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 1985;22:996-1006.  Back to cited text no. 8    
9.Yassien M, Khardori N. Interaction between biofilms formed by Staphylococcus epidermidis and quinolones. Diagn Microbiol Infect Dis 2001;40:79-89.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Carlson DM, Matthews LW. Polyuronicacids produced by Pseudomonas aeruginosa . Biochemistry 1966;5:2817-22.  Back to cited text no. 10  [PUBMED]  
11.Linker A, Jones RS. A new polysaccharide resembling alginic acid isolated from Pseudomonads. J Biol Chem 1966;241:3845-51.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]
12.Simpson JA, Smith SE, Dean RT. Scavenging by alginate of free radicals released by macrophages. Free Radic Biol Med 1989;6:347-53.  Back to cited text no. 12  [PUBMED]  
13.Learn, DB, Brestel EP, Seetharama S. Hypochlorite scavenging by Pseudomonas aeruginosa alginate. Infect Immun 1987;55:1813-8.  Back to cited text no. 13    
14.Barker LP, Simpson WA, Christensen GD. Differential production of slime under aerobic and anaerobic conditions J Clin Microbiol 1990;28:2578-9.  Back to cited text no. 14    
15.Arciola CR, Campoccia D, Borreli AM, Donati ME, Montanaro L. Congo red agar plate method: Improved accuracy and new extended application to Staphylococcus aureus . New Microbiol 2001;24: 355-63.  Back to cited text no. 15    
16.Duguid JP. The demonstration of bacterial capsules and slime. J Pathol Bacteriol 1951;63:673-85.  Back to cited text no. 16  [PUBMED]  
17.Baldassarri L, Simpson WA, Donelli G, Christensen GD. Variable fixation of staphylococcal slime by different histochemical fixatives. Eur J Clin Microbiol Infect Dis 1993;12:866-8.  Back to cited text no. 17  [PUBMED]  
18.Leigh R, Evans, Alfred Linker. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa . J Bacteriol 1973;116:915-24.19.p  Back to cited text no. 18    

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Correspondence Address:
Mamatha Ballal
Department of Microbiology, Kasturba Medical College, Manipal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.48914

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