INTRODUCTION:

Bacterial collagenases produced by Clostridium histolyticum (C. histolyticum) are the first collagenolytic enzymes which are able to produce collagenase to digest collagen fibres. These are used widely as therapeutic enzyme due to their ability to disaggregate connective tissue and to isolate the different cells of interest from various tissues¹. Collagenases produced from the bacteria particularly from C. histolyticum are preferred till date cause of their ability to hydrolyze practically all collagen isoforms, including their native forms². Mandl and coworkers were the first who isolated the enzyme collagenase from C. histolyticum. These preparations were then referred as clostridiopeptidase³.

Although many microorganisms are known to produce collagenases, the terms bacterial collagenase and microbial collagenase have become synonymous with clostridium collagenase⁴. The culture filtrate of C. histolyticum contains collagenases and mixture of other proteinases that exhibit potent collagenolytic activity towards collagen and extracellular matrix⁵.

Clostridia are classified into three classes according to toxin they produced or pattern of tissue invasion and disease proliferation that creates neurotoxin, enterotoxin and histotoxin to that C. novyi belong to ‘histotoxic’ category⁶. This, bacterium is a cause for wide variety of diseases in man and animals. It divided into three different types, known as A, B, (both pathogenic) and a non-pathogenic type C. These are differentiated based on toxins they produce. Among these all, the α-toxin is characterised as lethal and necrotizing, the bacteria also claimed to produce collagenase enzyme⁷.

Clostridium novyi-NT (C. novyi-NT) is a modified attenuated strain of the obligate anaerobe C. novyi type A, with potential oncolytic and immunomodulating activities. The modification eliminated the secretion of α-toxin (lethal toxin from cpa gene)⁸. The bacterium named after the American bacteriologist F.G. Novy. Recently this microorganism also claimed to produce extracellular collagenase enzyme⁹.

The present paper describes identification, cultural characterisation and collagenase production from novel bacterial strain C. novyi-NT. It is believed that the purified form of this crude collagenase enzyme derived from this C. novyi-NT species would become the reagents of choice for collagen based fibro-proliferative disorders of skin and also for in vitro tissue dissociation experiments. In the present study, an attempt was made on presumptive identification and cultural characterization of novel bacterial culture C. novyi-NT. Hence, it would be prerequisite to understand the pathogenesis and its role in improving their application in pharmaceutical biotechnology. This study also illuminates light on detection and characterization of microbial collagenase to determine collagenolytic activity on agar plates.

MATERIALS AND METHODS:

Bacterial strain:

Clostridium novyi-NT spores were procured from Lokmangal Biotechnology Institute, Wadala, Maharashtra. Clostridium perfringens spores were procured from NCIM, Pune. These spores were first cultivated separately on reinforced clostridial medium (RCM, HiMedia, Mumbai) in an anaerobic atmosphere. About three to five days after cultivation, the grown colonies of vegetative bacteria were transferred into cooked meat medium (HiMedia, Mumbai) for sporulation. The aliquots of culture were preserved in glycerine buffered solution at -80^oC for future use.

Materials:

All chemicals and media components were analytical grade and procured from the Sisco Research Laboratory, Mumbai. Tributyrin agar, Blood agar media, Tryptophan broth and Urease broth were procured from HiMedia, Mumbai.

Safety considerations:

Biosafety level-2 (BSL-2) was maintained during the microbial research activities as advocated by NIH¹⁰.

Stab culture:

A stab culture was prepared by nutrient agar in microbial tube containing a deep column of medium, to which the inoculating loop was thrust to deep bottom of the tube. About one cm layer of sterilised olive oil was poured on the surface of the medium and incubated further for 24-48hrs at 37⁰C in anaerobic conditions followed by candle-jar technique¹¹.

Culture in broth:

Further, the culture was inoculated into nutrient broth and kept in a candle jar for incubation at 37ºC for 24-48 hours. Anaerobic condition was maintained by pouring 5 ml of sterilised olive oil (2-3 cm) was poured on surface of culture broth in 10 ml size test tube¹¹.

1. Presumptive identification and characterization:

The routine presumptive identification tests were carried out to confirm Gram nature, colonial appearances and growth characteristics and were investigated as follows.

1.1. Gram's staining of smear impression:

A thin smear impression from broth culture was prepared on presterilised glass slide and stained with Gram's staining method. These stained slides were further examined according to the procedure described elsewhere¹².

1.2. Endospore staining:

A thin smear film from the bacterial strain was dried, covered with malachite green was prepared and heated until the liquid get converted to form steam (aggressive staining). The steaming liquid was reinstated permanently with dye and water. The glass slide was washed thoroughly with tap water and counterstained with safranin dye solution followed with rinsing and drying¹³.

1.3. Motility test:

Investigation of the movement of live bacteria by microscope is possible with hanging-drop preparation. The motility was observed in young cultures (about 48 hours) as well as in old culture (more than 48 hrs old culture). A suspension of microbial broth culture is placed in the centre of a cover slip and turned over with a special glass slide with a hollow depression in the centre¹³.

2. Cultural characteristics of Clostridium novyi-NT

2.1 Hemolytic activity on blood agar media:

The stab culture was spread on blood agar media (sheep blood) and was incubated anaerobically at 35-37⁰C for 24-48 hours (11).

2.2 Nagler’s Test:

The half the egg yolk agar plate portion was inoculated with 60µL C. perfringens type-A specific antitoxin by spreading with a 'hockey stick’ spreader and allowed to absorb and dry. The test organism was streaked in a straight line from the antitoxin-free half across to the antitoxin side of the plate. The control organism was inoculated in the same manner on the same plate and plate incubated anaerobically at 35-37^oC for 24-48h. The plate was examined for opaque precipitate (opacification) due to interaction of lecithinase with α-antitoxin around the inoculums (14).

2.3 Lipase Test:

The previously sterilised petri plates with tributyrin agar medium was inoculated with single line streaking of organism and incubated anaerobically at 35-37^oC for 24 to 48 hours. The plates were examined for the clear halo zone around the bacterial growth (14).

2.4 Indole test:

About 2 ml of sterile tryptophan broth culture was inoculated with broth culture of organism under test and incubated for 48 hours at ambient temperature in anaerobic condition. About 0.5 ml Kovac’s reagent was added, shaken well and examined after 1 minute (11).

2.5 Urease Test:

Urease broth was inoculated with broth culture of organism under test and incubated for 48 hours in ambient temperature for 7 days in anaerobic condition. The development of colour for as long as 7 days was examined (14).

3. Screening for collagenolytic activity of C. novyi -NT:

The isolate colony from egg yolk agar media plate was streaked by zigzag method on plate containing collagenase specific medium (g/l) K2HPO4 2.0, glucose 1.0, peptone 5.0, collagen 15.0, and agars 15. The clear zone diameters were determined after 24-48 h of anaerobic incubation at 37°C by flooded the plates with mercuric chloride solution (15).

RESULTS:

1. Presumptive characterisation of Clostridium novyi-NT:

The organisms were appeared as numerous large, thick, straight gram-positive rods, with occasional spores were observed in impression smears. These gram positive bacilli later do become gram negative as culture become old. This is the one of the characteristics of C. novyi (16). Cells were observed in singly and also in pairs with 0.7 - 1.4 μm wide and 1.6 - 18.0μm long as in figure 1 (17).

Figure 1. Gram staining of C. novyi- NT

In endospore staining, the spores appeared as green ellipse within and outside of cells while the vegetative cells as pinkish red. The shape of endospore observed as oval with terminal or subterminal position and may cause the cell to swell as shown in figure 2. These mature, free endospores were not associated with the vegetative bacteria and seen as green ellipses.

The hanging drop technique unveils the highly motile nature of bacteria due to its loose propellar- like flagella.

Figure 2. Endospore staining of C. novyi- NT with subterminal spore appearance

2. Cultural characteristics of Clostridium novyi-NT

Identification of C. novyi-NT is very difficult since the bacterium is extreme anaerobic in nature. The bacterial growth is enhanced by employing fermentable carbohydrates, serum or peptic digest of blood in culture media. As shown in figure 3, on blood agar after overnight anaerobic incubation, the colonies appeared as small, raised, circular, greyish, rough, and translucent which became flatten and irregular as culture goes old. Colonies tend to fuse forming a spreading growth with zone of haemolysis (γ-haemolysis pattern) with 1 – 5 mm in diameter after incubation for 48-72 hr. Colonies appeared as coalesce forming a fine spreading growth that covered the entire plate as a continuous film (as the bacteria "swarms") (17).

Figure 3. C. novyi-NT appearance on blood agar media

Many Clostridium spp. produces phospholipases (lecithinases) enzyme. Nagler’s test differentiates perfringens group from other Clostridia. This test employs addition of a specific α-antitoxin for the neutralization of lecithinase-C. The phospholipase of C. perfringens is known as α-toxin (cpa, lethal toxin), which is the major characterization of several toxic clostridial phospholipases causing tissue necrosis and oedema (18). Similarly, C. novyi type A is well known to produces α-toxin to give phospholipase-positive, but not β-toxin (19). In case of C. novyi- NT, there is lacking of a cpa-related gene and hence it is unable to produce lethal α-toxin. Hence, no opaque precipitate (opacification) of α-toxin around the colonies was observed resulted in negative Nagler’s reaction (20) while control organism C. perfringens growth resulted in opacification by reaction with α-toxin as shown in figure 4.

Figure 4. Negative Neglar’s test shown by C. novyi – NT

The inoculation over egg yolk agar plates though C. novyi-NT produced opalescence around the colonies indicating the lecithinase activity but it is attributed to γ-toxin gene (21) similar to that of C. histolyticum. As shown in figure 5.

Figure 5. Opalescence around the C. novyi – NT colonies indicating the lecithinase activity

The lipase test is used to identify capability of bacteria to produce the exoenzyme lipase. Various lipid substrates are used to detect differential characteristics. Lipase-positive clostridium species growing on the medium hydrolyze the tributyrin oil and resulted in an opaque zone of suspension in the media. C. novyi-NT produces lipase and breaks down the tributyrin, a clear halo was observed the areas surrounds where it has grown. C. novyi-NT produced lipolysis can be seen on egg-yolk agar with “pearly layer” was observed over the growth and in its vicinity on the surface of medium. The control organism C. perfringens unable to produce lipase and hence resulted in negative lipase test as shown in figure 6. These results are supportive with the earlier observations by Steadtke et al. (8).

Figure 6. Positive lipase test given by C. novyi-NT on tributyrin agar media

Indole test is performed to investigate the ability of an organism to split an amino acid tryptophan to generate indole compound which is detected by Ehrlich’s or Kovac’s reagent containing 4-(p)-dimethylamino benzaldehyde, to produce a red coloured compound by reaction with indole. C. novyi- NT bacterium does not have ability to produce tryptophanase enzyme since tnaA gene is deleted to prevent tryptophan catabolism and hence hydrolysis of tryptophan not occurs. Thus, indole is not produced as possible end product and hence C. novyi- NT answered negative indole test (22) as shown in figure 7.

Figure 7. Negative indole test given by C. novyi-NT

Urease test is performed to detect either bacteria is capable to hydrolyzeg urea by the enzyme urease. The hydrolysis of urea generates into a weak base and ammonia as one of its products. This weak base results in increased pH of the media above 8.4 and the pH indicator, phenol red, converts suspension from yellow to pink. C. novyi-NT does not have ability to produce the enzyme urease and hence unable to split urea resulted negative urease test as shown in figure 8 (17).

Figure 8. Negative urease test given by C. novyi-NT

3. Screening for collagenolytic activity of C. novyi -NT

The collagenolytic activities of C. novyi - NT were assayed using collagen based agar, and exhibited as diameter of clear zone. Collagen agar was found effective agar for qualitative test of collagenase. As showed in figure 9, the culture showed collagenolytic activity production by means clearing zone formation around the bacterial growth in collagen ager plates (15).

(a) (b)

Figure 9. Collagenolytic activity on collagen agar plate a) before incubation and b) after incubation zone of inhibition was seen surrounding to C. novyi -NT

The overall results of cultural characterisations of C. novyi-NT in comparison with other pathogenic strains of clostridia are summarised in Table 1.

Table 1. Comparitive cultural charecteristics of collagenase producing clostridia.

Species	Toxin production	Spores	Lecithinase	Lipase	Indole	Urease
C. novyi-NT	γ-toxin	ST	-	+	V	_
C. histolyticum	β and γ-toxin	C/ST	-	+	_	_
C. novyi Type- I	α and γ-toxin	ST	+	+	V	_
C. perfringens	α, β, and γ-toxin	-	+	_	_	_

ST: Subterminal, V: Variable.

DISCUSSIONS:

This paper summarized the main findings regarding presumptive identification and cultural characterisation tests to confirm the nature and properties of procured microorganism C. novyi-NT. The vegetative, young culture shows Gram positive while the old culture shows Gram negative characters. Such dual and bias Gram nature behaviour confirms C. novyi-NT growth in culture. The endospores appeared as green ellipse within and outside of cells while the vegetative cells as pinkish red. The shapes of endospores were observed as oval with terminal or subterminal which confirmed typical clostridial nature.

It was also observed that C. novyi colonies were flat, circular and greyish coloured surrounded by zone of hemolysis after cultured anaerobically on blood agar media at 35-37°C for 48 hrs. The bacteria observed “swarms” as a continuous film over the entire plate, this nature is typically seen in C. novyi and since it produced γ-haemolysis pattern which ultimately caused due to absence of α- toxin gene (responsible to form α-hemolysis pattern) and hence it confirmed the growth of C. novyi-NT on blood agar media.

The results are further confirmed by Naglar’s test where due to lacking of a cpa- gene C. novyi-NT is unable to produce lethal α-toxin and hence does not produce lecithinase to give negative Nagler’s reaction. This test clearly differentiates its non-pathogenicicity over C. novyi type A.C. novyi-NT bacterium capable of producing the exoenzyme lipase to give clear zone surrounding its growth in tributyrin agar. C. novyi- NT does not hydrolyse the amino acid - tryptophan due to absence of tryptophanase and ultimately gives negative indole reaction. Similarly it answers negative urease test indicating inability to hydrolyze the urea.

C. novyi- NT had shown its collagenolytic activity as zone of collagenolysis was detected on the collagen agar plates. Hence it is confirmed that C. novyi-NT is involved in production of proteolytic enzyme-collagenase. The collagenase presence was detected by the clear zone formation surrounding zizag growth of bacteria while the rest area in plate shows cloudiness.

CONCLUSION:

Though a lot of researches are being carried on C. novyi- NT considering its oncolytic and proteolytic properties, till date no reports have been published indicating its presumptive identification and cultural characterisation. This paper summarized simple and specific identification tests to identify Cl. novyi-NT and its characterisation on specific culture to confirm its presence as soon as the bacterium culture procured and sub-cultured. The techniques in this article, provides a sensitive, convenient, and inexpensive methods for screening Cl. novyi-NT amongst different pathogenic clostridium species. This paper finding is the first ever claiming production of collagenase by Cl. novyi- NT. This article is an effort to diminish a gap in literature about identification, characterisation and collagenase production by Cl. novyi-NT with high specificity. It would be very practicable as further research scope to isolate, purify and detect the therapeutic applicability of collagenase from C. novyi- NT.

ACKNOWLEDGEMENTS:

The authors herewith gratefully acknowledge the financial support by University Grants Commission, New Delhi for UGC-BSR Fellowship (grant number: F-25/1/2014-15(BSR)/7-269/2009 (BSR) 7^th Oct 2015) during research tenure. Authors also express their sincere gratitude towards Dr. Jitendra S. Bajare, Professor and Head, Dept. of Microbiology, Lokmangal Science and Entrepreneurship College, Wadala; for his guidance and help during the work.

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Received on 17.04.2019 Modified on 25.05.2019

Research J. Pharm. and Tech. 2019; 12(10):5015-5020.

DOI: 10.5958/0974-360X.2019.00870.9