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 nutrient-rich 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 1
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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 nutrient-
rich 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
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nutrient limiting media (also known as oligophilic conditions). One might be tempted to
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.
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Review of Literature
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
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Serological classification (Wolff and Broom):
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.
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Leptospiraceae
Turneria Leptospira
L.interrogans
serogroups
(>25)serova
rs (>250)
L. biflexa
serogroups (38)
serovars
(>60)
Leptonema
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
- 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).
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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
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
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- 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
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).
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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.
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.
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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).
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 antigen-
antibody 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
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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,
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).
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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
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