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Year : 2008  |  Volume : 26  |  Issue : 1  |  Page : 65-67

Detection of extra-cellular enzymes of anaerobic gram-negative bacteria from clinically diseased and healthy sites

Department of Microbiology, Jawaharlal Nehru Medical College, Karnataka - 590 010, India

Date of Submission27-Jul-2006
Date of Acceptance15-Apr-2007

Correspondence Address:
J M Nagmoti
Department of Microbiology, Jawaharlal Nehru Medical College, Karnataka - 590 010
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.38862

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 ~ Abstract 

Anaerobic gram-negative bacteria (AGNB) produce enzymes that play a significant role in the development of disease. We tested 50 AGNB isolates, 25 each from clinically diseased and healthy human sites for in vitro production of caseinase, collagenase, etc. Majority of the isolates were Bacteroides fragilis and Porphyromonas gingivalis, which more commonly produced collagenase and haemolysin. Comparatively larger number of clinical AGNB produced collagenase (P = 0.004). No such difference was observed with other enzymes. Hence, collagenase is probably one of the key virulence markers of pathogenic AGNB, and the inhibitors targeting collagenases might help in the therapy of anaerobic infections.

Keywords: Anaerobic gram-negative bacteria, extra-cellular enzymes, virulence factors

How to cite this article:
Nagmoti J M, Patil C S, Nagmoti M B, Mutnal M B. Detection of extra-cellular enzymes of anaerobic gram-negative bacteria from clinically diseased and healthy sites. Indian J Med Microbiol 2008;26:65-7

How to cite this URL:
Nagmoti J M, Patil C S, Nagmoti M B, Mutnal M B. Detection of extra-cellular enzymes of anaerobic gram-negative bacteria from clinically diseased and healthy sites. Indian J Med Microbiol [serial online] 2008 [cited 2021 Feb 27];26:65-7. Available from:

Infections due to anaerobes are common and associated with considerable morbidity and potential mortality. Anaerobic gram-negative bacteria (AGNB) belonging to the genera Bacteroides, Prevotella and Porphyromonas are most commonly encountered in clinical infections. [1],[2] Clinical interests in these organisms are linked to the therapeutic problems usually encountered in treating mixed infections. Despite their clinical relevance, very little is known about the pathogenic mechanism of anaerobic infections.

Vast majority of anaerobic infections arise from the indigenous flora of the host. Although AGNB constitute a major portion of human microflora, very few exhibit marked pathogenic potential and are responsible for the majority of the infections. [3] There is some evidence that hydrolytic and proteolytic enzymes like chondroitinsulfatase, gelatinase, collagenase, caseinase and lipase secreted by AGNB play a role in the infectious process. [4] It has been observed that, the collagenases of P. gingivalis hallmark its presence at the infection sites. [4],[5]

In the light of the above facts and in view of detecting key virulence factors of AGNB and shedding light on the diagnostic and possible therapeutic approaches by targeting the virulence factors of AGNB, the present study has made an attempt for the detection of cellular and extra-cellular virulence markers of AGNB isolates from both infected and healthy human sites.

 ~ Materials and Methods Top

A total of 50 AGNB isolates from clinically diseased (25 in number) and healthy (25 in number) sites were tested for the production of extra-cellular enzymes. (collagenase, caseinase, catalase, chondroitin sulfatase, gelatinase, haemolysin, hyaluronidase and lipase). Clinical isolates were from periodontitis orofacial and neck infections, brain, intra-abdominal abscesses and others. Commensal AGNB isolates were from healthy mucosal sites of oral cavity, upper respiratory tract, intestine and vagina. The isolation of AGNB was done by using 'Internal gas generating system' and their identification was done as per the standard techniques. [6],[7]

Collagenase production was tested on an agar medium containing human collagen (Sigma C-9879); after inoculating the broth culture of the isolate and incubating anaerobically, the plates were stained with Coumassie brilliant blue. Collagenase (Sigma C-6885) served as a positive control. [8]

Caseinase producing AGNB on casein-containing medium produced zones of clearance beneath and around the areas of growth due to the digestion of casein. Protease (Sigma P-5130) was used as a positive control. [9] Catalase production was tested by the standard technique. [7] Hyaluronic acid (Sigma H-1876) and chondroitin sulphate (Sigma C-4134) containing plates were inoculated with the isolates and incubated for 7 days, after which they were flooded with a 2.0-N acetic acid solution which precipitated only intact mucopolysaccharide. Propionibacterium acnes, ATCC 11828, was used as the positive control. [10]

For gelatinase production test, the organisms were spot inoculated on 0.4% gelatin agar. After satisfactory growth, the cultures were flooded with mercuric chloride solution, which rapidly denatured and rendered opaque any unhydrolyzed gelatin. Protease (Sigma P-5130) served as positive control. [11] Lipolytic organisms produced a restricted opacity and pearly layer on egg yolk medium. Purified lipase (Sigma L-2253) served as the positive control. [12]

Hemolysin production was tested by adding 50 μL of culture supernatant to the red blood cell suspension. The results were read after incubation for 18 h. [11] Statistical analysis of the data was done by using 'Z' test, with the level of significance set at a value of P < 0.05.

 ~ Results Top

Of the 25 AGNB isolates from the clinical group, 10 were B. fragilis, 7 were P. gingivalis, 5 were P. melaninogenica and 3 were Fusobacterium nucleatum. Similar types and the number of isolates as mentioned above were obtained from commensal group as well. The AGNB were found to be highly variable with respect to the production of enzymes. Enzymes most often produced were collagenase (60%) followed by hemolysin (32%), Catalase (28%) and others by the clinical isolates, whereas commensal AGNB isolates less frequently produced the enzymes. In general, both clinical and commensal isolates of P. gingivalis and B. fragilis were more active in enzyme production [Table - 1],[Table - 2]. None of the AGNB produced all of the enzymes tested.

Interestingly, the collagenase production by clinical isolates was significantly higher (P = 0.004) as compared to the commensal AGNB isolates [Table - 3]. On the other hand, production of other enzymes did not differ significantly between clinical and commensal AGNB isolates (P = >0.05).

 ~ Discussion Top

Porphyromonas gingivalis and B. fragils are the commonest AGNB isolated from human anaerobic infections and were also found to produce more number of enzymes tested and hence, the most virulent species among AGNB.

Porphyromonas gingivalis expressed the highest enzymic activity, followed by B. fragilis and others, as has been shown by many other studies. [3],[12],[13],[14],[15]

A positive relationship appears to exist between the production of collagenase enzyme by a given strain and virulence of that strain. All the collagenase-producing organisms invariably produced other enzymes as well, suggesting it to be a key virulence marker of AGNB. It has been shown that P. gingivalis, B. fragilis and F. nucleatum disrupt the epithelial cells and degrade a range of plasma proteins: albumin, fibrinogen, immunoglobulins, complement components, etc. [1],[2],[3],[12]

Although some of the healthy AGNB isolates produced few enzymes, except for collagenase production there was no statistically significant difference in the production of other enzymes between these groups, which suggested that the commensal AGNB can exist in harmony with the host and the disease episodes ensuing from the shift in the ecological balance within the complex microenvironment.

It is concluded that AGNB produce a variety of hydrolytic and proteolytic enzymes, but that none of these enzymes are universally present in them. Bacteroides fragilis and P. gingivalis are the most virulent AGNB species. Collagenase is a key virulence marker of pathogenic AGNB and other enzymes being contributory for the pathogenesis of AGNB infections. Collagenase inhibitors might have a therapeutic role in AGNB infections. The study supports further investigations on qualitative and quantitative analyses of key enzymes, which help in the development of newer diagnostic tools.

 ~ References Top

1.Duerden BI. Virulence factors in anaerobes. Clin Infect Dis 1994;14:S253-9.  Back to cited text no. 1    
2.Botta GA, Arzese A, Minisini R, Trani G. Role of structural and extracellular virulence factors in gram negative anaerobic bacteria. Clin Infect Dis 1994;18:S260-4.  Back to cited text no. 2  [PUBMED]  
3.Bergan T. Pathogenicity of anaerobic bacteria. J Clin Pathol 1987;40:384-6.  Back to cited text no. 3    
4.Travis J, Potempa J, Bacterial proteinases as targets for the development of second generation antibiotics. Biochem Biophys Acta 2000;7:1477:35-50.  Back to cited text no. 4    
5.Teanpaisan R, Douglas CW, Eley AR, Walsh TF. Clonality of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens isolated from periodontally diseased and healthy sites. Oral Microbiol Immunol 1998;13:263-70.  Back to cited text no. 5    
6.Vaidyalingam K, Laxminarayana CS. Internal gas generator system suitable for creating anaerobiosis. Indian J Surg 1980;42:154-9.  Back to cited text no. 6    
7.Summanen P, Baron EJ, et al. editors. Wadsworth Anaerobic Bacteriology Manual. 5 th ed. Star Publishing: Belmont CA; 1993.  Back to cited text no. 7    
8.Fullmer HM, Gibson WA, Lazarus GS. Collagenase studies of gingivae of man. Trans Int Conf Oral Surg 1970;1:565-81.  Back to cited text no. 8    
9.Holdeman LV, Cato E, Moore WE, editors. Anaerobe laboratory manual 4 th ed. Verginia Polytechnic Institute and State University: Blaksburg; 1977.  Back to cited text no. 9    
10.Smith RF, Willet NP. Rapid plate method for screening hyaluronidase and chondroitin sulfatase producing organisms. Appl Microbiol 1968;16:1434-6.  Back to cited text no. 10    
11.Coque TM, Patterson JE, Steckelberg JM, Murray BE. Incidence of hemolysin, gelatinase and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from feces of hospitalized patients and community based persons. J Infect Dis 1995;17:1223-9.  Back to cited text no. 11    
12.Stafeen E, Hentges D. Hydrolytic enzymes of anaerobic bacteria isolated from human infections. J Clin Microbiol 1981;14:153-6.  Back to cited text no. 12    
13.Gazi MI, Cox SW, Clark DT, Eley BM. Characterization of protease activities in Capnocytophaga spp. Porphyromonas gingivalis , Prevotella spp, Treponema denticola and Actinobacillus actinomycetemcomitans . J Periodontal Res 1996;31:423-32.  Back to cited text no. 13    
14.Kuramitsu HK. Proteases of Porphyromonas gingivalis: What don't they do? J Biochem 2000;128:153-9.  Back to cited text no. 14    
15.Gharbia SE, Shah HN. Hydrolytic enzymes liberated by black pigmented gram negative anaerobes. FEMS Immunol Med Microbiol 1993;6:139-46.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]


  [Table - 1], [Table - 2], [Table - 3]


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