Project Report Dec 2008

Introduction There are many bacteria which use the human body as a host, some with negligible effects while others are more detrimental. Two of the bacteria which pose severe threat to humans are Leptospira and Helicobacter. Much research has been done and continues even today as it relates to the culturing of these bacteria. Most of these works have been basically centred on culturing these bacteria in conventional nutrientrich media which have yielded success. Therefore, the thought of seeking alternative media has never been seen as a priority. While it is a fact that the culturing of these bacteria in nutrient rich media has been relatively successful there are several factors which necessitate the procural of alternative methods. These factors include: the high cost factor involved in culturing, the time consuming element (4-6 months), inaccessibility of nutrient rich materials (rabbit serum in case of Leptospira), and the need for a more expeditious approach in combating the diseases caused by these bacteria (Wechter, 2007). Today an ever increasing number of people suffer from Leptospirosis (caused by Leptospira) and gastritis, stomach and peptic ulcers (caused by Helicobacter). As a result of these phenomena medical science is seeking to understand more about these bacteria in order that quicker diagnosis and treatment can be given to patients. It therefore means that by developing alternative methods of culturing the probability increases of controlling the diseases. While some have cultured Leptospira and Helicobacter in a serum free medium (yet nutrient rich), no seminal research has been done in the area of culturing them in a nutrient limiting media (also known as oligophilic conditions). One might be tempted to 1

ask how is it possible that bacteria that normally cultured in nutrient rich media can be grown in nutrient limiting media. The reality is both if these bacteria share similar physiological niche in that the environment in which they are adapted to are generally low in nutrients. For instance, Leptospira lives in the proximal convoluted tubules of the kidney where the available nutrients consist of water, sugar, salts, urea, soluble vitamins and minerals. In the case of Helicobacter, it occupies the lining of the stomach walls where it feeds off the nutrients provided by the dead white blood cells. In sum, alternative methods are needed for the reliable cultivation, detection, identification, and treatment of diseases caused by these bacteria. As stated before, the currently used media are very cumbersome, time-consuming, and require a high level of skill and experience to perform. This research therefore aims to fulfil the following objectives: •

To culture and successfully isolate Leptospira and Helicobacter under nutrient limiting conditions, using Poor Ravan medium.

•

To use a serum-free culture media capable of growing Leptospira and Helicobacter organisms.

•

To provide a cheap and easy method for detecting and characterizing Leptospira and Helicobacter in a sample.

Review of Literature

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Leptospira Discovery of Micro-organism The first description of Leptospira (although not called by that name then) was in 1812 by one of Napoleon’s troops while they were in war in Egypt. Later the illness came to be known especially throughout Europe as‘bilious typhoid’ (Matthew et. al). In 1886, Adolf Weil described Leptospirosis as a disease entity. As a tribute to his work the disease was since called Weil’s disease.It was not until the second decade of the 20th century that Leptospires were recognized by Inada and Ido in Japan and soon after, independently, in Germany by Uhlenhuth and Fromme as the cause of the disease that had been originally described by Weil (World Health Organization).

Taxonomy and Classification Taxonomic Status: Order: Spirochaetales Family: Leptospiraceae Genus: Leptospira Species: L. interrogans L. biflexa

Serological classification (Wolff and Broom): 3

Leptospira is divided into 2 species: L. interrogans and L.biflexa. L. interrogans is pathogenic and causes diseases whereas L. biflexa is saprophytic which is found in non-sterile envornoment and does not transmit diseases. The main difference between these two is the former grows at 130C in the presence of 8-azaguanine and the latter fails to form spherical cells in 1M NaCl. Both L. interrogans and L. biflexa are divided into numerous serovars based on their antigenic composition.

Genotypic Classification Leptospiraceae __________________________________  Leptospira

 Leptonema

Turneria

 L. borgpetersenii, L. interrogans, L. inadai, L. noguchi, L. Weillii, L. alexandri, L. Wolbachii, L. meyeri, L. biflexa, L. santarosai, L. faini, L. parva, L. kirchneri The above genotypic scheme distinguishes Leptospira based upon DNA relatedness (Yasuda et. al, 1987).

Morphological characteristics 4

– Helical rods 6-12μm in length and 0.1μm in diameter. – Flexible and corkscrew-shaped with each cell having 18 or more coils. – One or both ends are characteristically hooked. – Cell is encased in a 3-5 layer outer membrane or envelope. Beneath this outer membrane are the helical peptidoglycan layer and the cytoplasmic membrane. – Two flagella originating at each end of the cell lie between the outer membrane and the peptidoglycan layer. The free ends extend toward the centre of the cell but do not overlap. – Basal bodies resemble that of Gram negative bacteria (Penn, 1990).

Movements of Leptopsira According to Cox and Twigg, Leptospira undergoes at least four types of motility: 1. Nontranslational: The extremities move in a cyclical motion while the other parts of the body stay stagnant. 2. Translational: One end moves like a coil while the other end moves in an inconsistent circular motion. Movement occurs towards the end showing helical motion. 3. Anchored: One end remains stationary while rest of body is in motion. 4. Shaking generally seen in semi-solid media (Cox and Twig 1974).

Epidemiology

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Mode of transmissions can either be indirect through contact with some form of contaminant in water, soil or urine of animals. (Turner et.al, 1967) Or it can be directly through the bites of animals or passed on from mother to offspring (Shaked et. al, 1993). Animals are often the primary host of Leptospira whereas human beings are the accidental hosts. The disease most affects people within the ages of 10-39 with higher prevalence in men and persons engaged in farming, sewage disposal, laboratory and veterinary work (Sanford, 1994). Conventional nutritional requirements and environmental conditions – Vitamin B1 and B12 – Long chain fatty acids bound to albumin – Animal serum – Nutrient rich supplements such as peptone, sodium pyruvate, glycerine, ammonium salts, Sodium or Potassium, Calcium or Magnesium and Iron – 5-fluoro-uracil for isolation from contaminated sources – Temperature of 28-30oC – Light protection – pH 7.2-7.6 – Oxygen – Amino acids such as L-asparagine (WHO)

Conventional forms of Media: 1. Liquid form

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Liquid media are essential for the isolation of leptospires and for growing cultures. Growth of leptospires in liquid media is indicated mainly by turbidity but sometimes by a granular appearance on the bottom of the tubes in which they are growing, both of which can be seen with the naked eye, but this should be confirmed by microscopic. observation. 2. Semi-solid form Semi-solid media contain 0.1–0.5 % agar (w/v). Such media are preferred for isolating the various strains and for medium-term maintenance (up to several years). Growth is readily initiated in these media and is usually easily visualized as one or more rings of dense growth several millimetres below the surface of the medium (Coghlan, 1966).

3. Solid form Solid media contain 0.8–1.3 % agar (w/v). The lower the concentration of agar, the greater the tendency for leptospires to swarm across the plate and through the medium; the higher the concentration, the smaller the colonies. (Johnson, 1964).

Types of conventional media containing sera: 1. Traditional media containing approximately 8–10% rabbit serum (Stuart, Korthof, Fletcher, Vervoort, Schüffner. Rabbit serum contains the highest concentration of bound vitamin B12, which is essential for the multiplication of leptospires.

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2. The Tween 80/bovine serum albumin (BSA) medium of Ellinghausen & McCullough and its modification by Johnson & Harris (EMJH). The BSA component of the medium is the most expensive ingredient. 3. Enriched media. To increase the growth of more fastidious leptospires such as serovar hardjo, media can be enriched by adding serum (e.g. 1–4% fetal calf serum (FCS) and rabbit serum) or other ingredients such as lactalbumin hydrolysate, superoxide dismutase and pyruvate (Ellis, 1986). EMJH medium is often enriched by adding 1% rabbit serum and 1% FCS. 4. Selective media with 5-fluorouracil (and/or other antimicrobials such as neomycin, nalidixic acid, actidione, sulfadiazol, rifampicin, amphotericin B). These additives may suppress the growth of contaminating bacteria in non-sterile clinical samples, while leaving leptospires unaffected but they may also cause some reduction in the growth of leptospires. This is particularly true of sulfadiazol.

Serum-free Media 1. Low-protein or protein-free media, often used for the preparation of vaccines (Coghlan, 1966). 2. A serum-free media for culturing spirochetes developed and patented by Wechter Stephen R. Diagnostic methods 1. Direct microscopy: Microscopy is performed on urine, and blood specimen and even bronchoalveolar lavage fluid. Since Leptospira cannot survive in acidic urine, the sample must be neutralized before microscopy (Babudieri, 1961). 8

2. Serological Tests The serological tests seek to detect antibodies and also serovars. Two of the more common tests which are done are Enzyme Linked Immunosorbent Assay (ELISA) and Microscopic Agglutination Test (MAT). ELISA involves the detection of antigenantibody system using enzyme linked antihuman antibody and a suitable substrate (Terpstra, 1985). MAT is carried out by using live cultures of various serovars of L. interrogans. Equal volume of antigen is added to serum dilutions and agglutination is observed under darkfield microscope (Babudieri, 1961). 3.

Molecular Methods The two more common molecular methods for detecting leptopsires are

Polymerase Chain Reaction (PCR), and DNA-DNA hybridization. PCR method involves in-vitro amplification of target DNA sequence brought about by thermostable DNA polymerase. There are several limitations of PCR: technique is expensive and complicated, contamination of test samples may lead to false results and also PCR is not able to identify the infecting serovar. Hybridization occurs when nucleotide sequence in a probe is used to detect a complementary sequence in a test sample (Terpstra, 1986 ).

Work done on growth of Leptospira in vitro In 1967, Russell and Harris attempted to identify the differentiating characteristics between pathogenic and saprophytic leptopsire by growing them at low temperatures using nutrient rich medium (rabbit serum). They tested the response of 20 pathogens and 30 saprophytes at temperatures of 13oC-30oC. At 30oC all organisms grew, however, only saprophytic grew at 13oC. They discovered that the pathogenic leptospira grows best at higher temperature unlike the saprophytic (Russell and Harris, 9

1967). In as much as these researchers have discovered that pathogenic leptospira grows better at higher temperatures than saprophytic, the media used for growth is still nutrient rich.

Helicobacter Discovery of organism The presence of spiral-shaped micro-organisms in the stomach mucosa was described almost 100 years ago. Their presence was not really taken seriously until the late 1970’s when John Warren, a pathologist in Perth, Western Australia, noted the appearance of spiral bacteria overlaying gastric mucosa and mostly over-inflamed tissue. Warren and Barry Marshall cultured these organisms in 1982 from eleven patients with gastritis. They were able to demonstrate a strong association between the presence of Helicobacter pylori and the finding of inflammation on gastric biopsy (Marshall, 1989). Originally called Campylobacter pyloridis, the name was changed to Campylobacter pylori and then later to Helicobacter pylori as specific morphologic, structural and genetic features indicated that it should be placed in a new genus(Marshall and Warren, 1984).

Taxonomic status and Classification The genus Helicobacter presently comprises 18 validly named species and two Candidatus species, a designation adopted by the International Committee on Systematic Bacteriology to record the properties of putative procaryotic taxa that are incompletely All Helicobacter species are characterized as fastidious, and most are

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associated with gastric or extragastric diseases. (Solnick and Vandamme, 2001-tax described of hel).

Morphological characteristics – 0.2 to 1.2 μm in diameter and 1.5 to 10.0 μm long – S-shaped bacterium with multiple, polar sheathed flagella(1-20). – The cellular morphology may be curved, spiral, or fusiform. – Periplasmic fibers or an electron-dense glycocalyx or capsule-like layer has been observed on the cellular surface of several species – The spiral wavelength may vary with the age, the growth conditions, and the species identity of the cells. In old cultures or those exposed to air, cells may become coccoid (Solnick and Vandamme, tax. of hel) – H. pylori in vivo and under optimum in vitro conditions is an S-shaped bacterium with 1 to 3 turns, 0.5 ×5 μm in length, with a tuft of 5 to 7 polar sheathed flagella Motility of Helicobacter Helicobacter cells are motile, with a rapid cork-screw-like or slower wave-like motion due to flagellar activity. They are often found within the lining of the stomach walls they have become acclimatized to the stomach’s acidity (Goto, 1998).

Nutrient requirements and environmental conditions – serum rich – Temperature of 30-37oC – Microaeropilic conditions (5-15% O2, 5-10% CO2, 85% N)

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– Nutrient rich supplements: ferrous sulphate, mucine, sodium pyruvate, whole blood, amino acids, sodium and potassium chloride, thiamine, hypoxanthine, zinc, magnesium, isovitale X, hemin, cyclodextrin and cholesterol. – pH 4.5-9 – Water activity (Aw)>0.98 (Battles, 1995).

Types of Medium containing serum -Columbia blood agar plates -moist Trypticase soy agar blood agar plates -brucella blood agar with TVP (trimethoprim, vancomycin, polymyxin)(helicobacter 1) Campylobacter agar (Becton Dickinson, Sparks, MD) containing 10% defibrinated sheep blood (Quad Five, Ryegate, MT) (CBA)(nutritional req)

Serum-free medium The growth of the gastric pathogen Helicobacter pylori in the absence of serum remains challenging, and nutritional requirements have only partially been defined. H. pylori grows in the chemically defined medium F-12, but not in other tissue culture media examined. H. pylori has surprisingly few absolute requirements for growth: 9 amino acids, sodium and potassium chloride, thiamine, iron, zinc, magnesium, hypoxanthine, and pyruvate. These data suggest that H. pylori and other Helicobacter species are not as particular as previously thought.

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The data also suggest that chemically defined media described herein could yield the growth of a wide range of Helicobacter spp., allowing a more detailed characterization of Helicobacter physiology and interactions with host cells. Nutritional requirements and antibiotic resistance patterns of several other Helicobacter species revealed that all except H. felis grew in serum-free, unsupplemented F-12. Identification and Diagnostic Methods: In taxonomic practice, the reference method for the delineation and identification of bacterial species is determination of the level of DNA-DNA hybridization. Strains are considered to belong to a single species if their whole genome DNA-DNA hybridization level is about 70% or greater. The fastidious growth characteristics of many Helicobacter species hamper the isolation of sufficient quantities of highly purified high molecular weight DNA required for these hybridization experiments. Yet, a number of DNA-DNA hybridization studies have been performed within and between Helicobacter species (Wayne, 1987).

Protein Electrophoresis It is not practical to implement DNA-DNA hybridizations in a routine laboratory or to use it for routine identification in a reference laboratory. The comparison of wholecell protein patterns obtained by highly standardized sodium dodecyl sulfatepolyacrylamide gel electrophoresis has proven to be extremely reliable to screen and identify large numbers of strains. Numerous studies revealed a correlation between high similarity in whole-cell protein content and level of DNA-DNA hybridization. However, this method is not appropriate for routine identification studies because it is laborious,

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time-consuming, and technically demanding to run the patterns in a sufficiently standardized way (Vandamme, 1996)

Cellular Fatty Acid Analysis The total cellular fatty acid methyl ester composition is a stable parameter provided that highly standardized culture conditions are used. Comparison of fatty acid profiles is of little value if different culture conditions or extraction procedures are used. However, it is a simple, inexpensive, and rapid method that is highly automated (Godwin, 1989).

DNA Probes and PCR Assays Specific oligonucleotide probes or PCR assays have been described for H. pylori and several other Helicobacter species. It should be stressed that, because of the constant developments in the taxonomy of Helicobacter species, none of these probes or PCR assays have been fully evaluated against all species presently described (Page, 1996).

Invasive techniques Histology Histological examination of biopsy samples taken during endoscopy is usually considered 'the gold standard'for the diagnosis of H.pylori. But owing to the patchy distribution of H.pylori in gastric mucosa, the biopsy-based tests may suffer from sampling error (Anderson, 1998). Furthermore, histological examination is highly dependent on the experience of the pathologist, and high inter-observer variation has been reported (Morris, 1989). 14

Rapid urease test (CLO test) Biopsies of gastric mucosa are placed in a gel containing urea, and the subsequent ammonia production causes a pH change, which is observed as a color change. Besides suffering from biopsy sampling error, the CLO test depends greatly on the pH of the media and the amount of the urea in the medium. These factors may vary in different products and thereby influence the results obtained with other tests (Thijs, 1996).

Culture Culture is the most specific diagnostic method for H.pylori infection but its sensitivity is low. The role of culture for primary diagnosis is limited but it is an important method as isolates for the traditional susceptibility testing are obtained. Although routine susceptibility testing for H.pylori is not recommended, increasing resistance rates to metronidazole and clarithromycin might make routine susceptibility more popular (7). Non-invasive techniques Serological tests Serological tests are based on the detection of specific anti-H.pylori IgG antibodies in a patient's serum. While serological tests are simple and easy to perform, they are not reliable tests for the diagnosis of H.pylori infection in elderly people because of poor antibodyproduction, or for determination of eradication ofH.pylori, since it remains positive for a long period despite adequate treatment (Newell, 1991). Serological tests are not able to distinguish between active infection and a previous 15

exposure to H.pylori. Different commercial kits also have different levels of diagnostic accuracy (range 68-82%) (Feldman, 1995).

Stool antigen test An enzyme immunoassay, which detects the presence of antigen in stool specimen, has recently become available. This assay has undergone extensive testing for the initial diagnosis of the H.pylori infection and in the confirmation of eradication after treatment. Several studies have suggested that polyclonal antibody test is comparable to the ure breath test in the initial diagnosis of H.pylori infection (sensitivity 93.2% and specificity (93.2%). It has been reported that stool antigen test isles accurate than UBT in the post-treatment setting. Recently it was been reported that monoclonal technique has higher sensitivity than the polyclonal one especially in the post-treatment setting (Bilardi, 2002). Urea breath tests H.pylori produces urease, an enzyme that splits urea into ammonia and carbon dioxide. The production of high amounts of urease by H.pylori has been used in the development of urea breath tests . Patients ingest urea labeled 13C or 14C. Hydrolysis of urea occurs within the mucous layer and results in the production of ammonia and labelled CO2. Labelled CO2 diffuses into the blood vessel and can be detected in the breath as a marker of infection (Christensen, 1992). Materials and Methods A total of fifteen reference serovars of leptospires will be used during this research which will be obtained from the Leptospira Department of B.J.Medical College. Out of these, eight serovars have been procured. The facilties at Abasaheb 16

Garware college will be used. The methodology follows a particular sequence: culturing of Leptospira on nutrient limiting media, microscopy investigation, subculturing onto nutrient limiting solid media, subculturing into conventional media, DNA isolation, PCR assays, sequencing and data analysis. Culturing Leptospira using Poor Ravan plates The following serovars were acquired from the Leptospira Department of B.J.Medical College: L. australis, L. autumnalis, L. icterohaemorrhagiae, L. Tarasorri and L. Bataviae. These organisms were streaked from conventional medium (EMJH see Appendix) directly onto Ravan medium agar plates in a 1:100 diluted or poor form (glucose 5g, peptone 5g, yeast extract 5g, sodium acetate 5g, sodium citrate 5g, pyruvic acid 2g, distilled water 11, pH 7-7.2, agarose 10g). These plates were all incubated at 37oC and growth was checked for each week using bright field microscopy.

Culturing Leptospira using a cyclic method Six serovars of Leptospira were used thus far from B.J. Medical college for using this technique. They are: L. patoc 1, L. patoc 2, L. autumnalis, L. Bataviae, L. icterohaemorrhagiae and L. pyrogenes. One loopful of each was placed into 3ml 1:100 diluted Ravan broth medium and incubated at room temperature. Wet mounts were prepared weekly for observing growth under dark field microscope. Morphology and motility were carefully documented and cell count was taken. When >15 organisms can be seen per field one loopful was subcultured onto diluted Ravan agar medium. These plates were incubated at 28oC.When colonies were visible with the naked eye, the colony morphologies were observed under bright field microscope. Wet mounts 17

were also prepared and the morphology and motility of the organisms recorded using dark field microscope. When >15 organisms can be seen per field one single colony was picked up and placed back into EMJH medium and incubated at room temperature. Morphology and motility was carefully documented. EMJH broth PR broth PR agar EMJH broth DNA isolation A loopful of cells were harvested and placed into PCR tube. 20ul of single colony lyses solution (SCL) was added to the tube and kept at 55oC for 1 hour. Enzyme activity was then inhibited at 85oC for 20 minutes. Contents were vortexed and centrifuged at 5000rpm for 1 minute.

Amplification of 16S rRNA gene Polymerase Chain Reaction amplification was conducted using the following combinations of primers: FOD2 and RPP2, 16R1525 and 16F27, and 16S-For-primers and 16S-Rev-. The following conditions were used: denaturation at 94oC for 3 mins, 94oC for 1 min, annealing at 60oC for 1 min and elongation at 72oc for 1 min. A total of 35 cycles will be performed followed by a further elongstion step at 72oC for 10 minutes. The purity of the amplified product was determined by electrophoresis in a 1% agarose gel. DNA was stained with ethidium bromide and viewed under shortwavelength UV light. Sequencing methods This method is yet to be conducted by the researcher. The purified DNA product will be sequenced using sequencer to confirm whether the isolate see is the desired strain. 18

Data analysis Growth curve will be plotted using cell count. C Results The organisms cultured on PR agar medium only were seen as tightly coiled appearing bumpy on the surface. Some had a motion which involved folding of elongated organism into a ball like structure and rotating in a haphazard motion. Colony morphology was also observed but only one type of each was seen. Contamination was the main problem initially on PR plates. The cyclic method was performed at the Leptospira Department in B.J. Medical college. The results are recorded in Figure 2. A sketch graph of L. patoc 1, L. patoc 2, L. autumnalis and L. Bataviae were plotted as shown in Figure 2. L. patoc 1, L. patoc 2 , L. autumnalis and L. Bataviae were subcultured onto PR agar plates and three distinct morphologies were recorded. Figure 1: Growth and Morphology of Leptospira serovars in PR broth medium. Serovar

Day

Organism

Morphology and motility

count L. patoc 1

6

st

L. patoc 1 (1 sub) L. patoc 2

L. autumnalis

1

Sluggishly motile

48 (subcultured) 4

Motile

59

3

Sluggishly motile

73

7

Non- motile

11

12

Motile; small and short

25

50

Motile

6

1

Sluggishly motile

47

6

Motile

63

5

Motile

6

15

Non-motile

47

11

Motile

19

L. Bataviae

73

3

Motile

30

15

Motile

43(subcultured)

L. Bataviae (1st sub) L. icterhaemorrhigaie L. pyrogenes

Autoclumping

57

10

Motile

25

15

Short and motile

11

15

Short and motile

25

15

11

2

Motile

25

2

Motile

11

1

Motile

25

0

20

Number of orgs./ field

Time (Days)

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Main type of morphology of colonies:

Colony type 1: – Translucent – no filaments inside – smooth egdes – brownish in colour – circular form which tend to spread along streak lines – most common in 1st and 2nd streak Colony type 2: – black colour – Rarely seen inside colony type 1 – hairy like projections – rough egdes – circular form – occurred only in 3rd and 4th streak mostly – very few in number compared to other two types

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Colony type 2: – translucent with filaments inside, usually along streak line and spreading – smooth edges – brownish in colour – occurs in all streak lines

PCR amplification from the initial batch grown on PR plates have only been successful with 16R1525 and 16F27. Good primers still need to be identified for improving PCR methods.

Discussion In the first culturing technique employing only PR agar medium, colonies did not seem to strive to well due to contamination problems. As compared to growth on agar plates in the cyclic method, small colonies are very much distinct and with very little contamination. This could have been due to simple difference in environmental conditions or maybe. Under bright field microscope, seem to be spreading more from observation but distinct colony types were not identified. This again could have been due to contamination since in both cases, morphology was observed using bright field microscopy. The morphology of the colony seem to change with time. Colony type 1 seem to precede before colony type 3. However , colony type 2 occurs most times isolated and its difficult to draw conclusions at this time as to whether it’s a contaminant or another morphological form of colony for Leptospira. 23

Rate of growth varies. Some strains grow well and multiply rapidly from the start; some appear to multiply fast for a few days and then become static and inert; in others small numbers of lively leptospires appear but seem to multiply very slowly. L. patoc 1 and L. patoc 2 seem to grow very slowly whereas L. autumnalis and L. Bataviae seem to grow faster as shown from graph. In broth, Leptospira serovars maintained there morphology and there motility clearly. Variations like short size and autoclumping was also seen when organisms was in adapting phase or probably in death phase. On plates, thus far all organisms seen on wete mount ate short and most are motile. The Soilid medium seem to inhibit there growth and also cause changes in morphology was seen under brightfield microscope from those grown on PR agar only. The reason to use the cyclic method is first to help the organism to adapt to nutrient limiting conditions while still maintain a fluid environment using PR broth. This is then followed by subculturing onto PR agar medium so the organisms will not be subjected to the shock of change in medium and form of medium but only form of media. Colonies are then subcultured from here back into conventional medium for just confirmation that it is indeed leptospira that is growing and not some contaminant. Other confirmation methods are also employed such as sequencing so that further confirmation can be made. Theoretically, if an organism grows in nature, it can be cultured if its physiological niche is perceived and duplicated under in vitro conditions. It is established that the nutrient concentrations in commonly used laboratory media are several-fold higher than those present in the natural environment, specially the aquatic habitat2. It is further revealed that a predominant group, i.e. oligophiles in the natural 24

bacterial population from both aquatic and terrestrial habitats does not grow on conventional media but forms distinct colonies on 100-fold diluted versions of such media3–8. Oligophilic ‘k-selected’ bacteria are adapted to grow in nutrient poor environments. These are characterized by slow growth and form small microscopic colonies. In their natural environments nutrients are limited, meaning that these bacteria cannot reproduce indefinitely.

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