Microbiology Lecture

Group 4:
Leosala, Jamila Anne
Lin, Tim Lawrence
Lugue, Ma. Cecilia
Lupac, Bernadette
Macuha, Ninya
Manrique, Aldric Mari
Introduction:

Order: Spirochaetales

Family: Leptospiraceae Family: Spirochaetaceae

Genus: Leptospira
Genus: Borrelia Genus: Treponema
- slender, flexuous, helically shaped,
unicellular
- 0.1 - 0.5 um wide, 5-20 um long
- have flexible cell wall with several
fibrils (periplasmic flagella)
- Periplasmic flagella (a.k.a. axial fibrils,
axial filaments, endoflagella, periplasmic
fibrils) – responsible for motility
- chemoheterotrophic – utilize CHO, amino
acids, long-chain fatty acids, long-chain fatty
alcohols as carbon and energy sources
- anaerobes, facultative anaerobes, or aerobes
-Reproduction:
 Treponema – transverse fission
 Leptospira and Borrelia – binary fission
Because of the vigorous motility,
spirochetes spread rapidly over agar
plates, forming colonies with sharp
defined edges, and penetrate into the agar
 A. General Characteristics
Pathogenic organisms:
Leptospira interrogans
Saprophytes:
Leptospira biflexa
 
- tightly coiled, thin, flexible spirochetes
- have Gram negative-like cell envelope
- spirals are very close together
- one or both ends have hooks
- motion: rapid and rotational
- long axial filament covered by a very fine
sheath
- all species have two periplasmic flagella
- not readily stained, but can be impregnated
with silver
- obligate aerobes
- Culture media: Fletcher’s semisolid, Stuart
liquid, Ellinghausen-McCullough-Johnson-
Harris (EMJH)semisolid media
- has diaminopimelic acid without ornithine
(exhibit normal growth)
Colony Morphology:
EMJH media
1. L. biflexa - characteristic linear disk
2. L. interrogans serovars grippotyphosa
and hardjo, L. biflexa serovar patoc, L.
illini, L. interrogans serovar Pomona, L.
interrogans serovar ballum - colonies
appear as discrete, opaque, or diffuse milky
forms, varying in size from pinpoint to 2 to
3 mm in diameter.
FIG. 2. Twenty-one-day-old culture of L. interrogans serovar
hardjo grown in EMJH medium gelled with gellan gum.
Virulence factors:
1. Hemolipins – haemolytic agents
2. Endotoxin – causes fever, necrosis
3. Sphingomyelinase C - promotes
intracellular proliferation by mediating the
disruption of the phagocytic vacuole and
the release of bacteria into the host cell
cytosol
4. Fibronectin binding protein - adhesion
and invasion
5. LPS and outer membrane proteins
Factors that play a role in pathogenicity:
- reduced phagocytosis in the host
- soluble hemolysin
- cell-mediated sensitivity to leptospiral
antigen by the host
- small amounts of endotoxins
(endotoxemia)
Cultivation:
- Leptospires grow well on a pair of fatty
acids (one saturated, the other unsaturated)
if they contain at least 15 carbon atoms.
- vitamins B1 and B12
- recommended cultivation: media of pH
7.4 at 30°C
- average generation time is about 12
hours
GROWTH REQUIREMENTS OF LEPTOSPIRES 

Peptone Protein source

Asparagine Can replace peptone

Beef extract Protein source

Fatty acids Leptospira growth depends on long chain fatty acids

for energy and carbon source

Sodium-pyruvate Addition of 50-400 ug/ml sodium pyruvate is optimal

for  reducing the lag periods seen with lower inoculum

Glycerin Addition of 50-400 ug/ml of glycerol decreases the

generation times and increases growth rates;
Remark: Acetate permits full utilization of fatty acids
of Tween, i.e. 200 ug/ml medium of each sodium
pyruvate, sodium acetate and glycerol
Salts Higher salt concentrations might be essential for
membrane stabilization and growth of cells

Na/K Stimulatory effects of higher levels of Na and K. The

requirements for higher K could be replaced by equimolar
amounts of Na although the use of K permitted shorter
generation times

Ca/Mg Virulent cells respond to higher levels of Mg. Increased

levels of Ca did not replace the requirements for higher
levels of Mg
Fe 2+ Iron is known to be active in oxidation-reduction functions
and may contribute to the reduction of peroxides which
results from the accumulation of organic peroxides
resulting from auto-oxidation of long-chain, unsaturated
fatty acids. Fe sulfate solution should be freshly prepared.
Iron is not only an adequate substitute for hemoglobin but
also retains its effectiveness longer
Ammonium Ammonium salts are an effective source of cellular
nitrogen

Rabbit serum Schuffner noted that growth was often improved when
rabbit serum was slightly haemolysed. Rabbit sera used
in media: Increasing percentages of serum up to and
including 12% results in a progressively steeper growth
curve. This stimulation is seen in the first four days and
is significant in attainment of high early growth levels.
6-8% of rabbit serum-enrichment results in total growth
response is linear up to 6%

5 Fluoro-uracil Uracil is a pyrimidine, pyrimidine is lethal to various

micro-organisms, but not to leptospires. Add a smaller
amount of 5 FU when continued subcultures are made.
Concentration of 200-500 ug/ml
Diseases produced:
Leptospira interrogans
 Serovar icterohemorrhagiae – Weil’s
disease / icteric leptospirosis
 Serovar hebdomadis – 7 day fever
 Serovar canicola – infectious jaundice
 Serovar mitis – Swineherd disease
 Serovar grippotyphosa – March fever
 - Weil’s disease (severe systemic disease) –
includes renal failure, hepatic failure,
intravascular disease
Jaundice
References:
Cultivation of Leptospires: Fatty Acid Requirements.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC380495/pdf/
applmicro00046-0211.pdf>

Leptospiral Colonial Morphology.

< http://www.ncbi.nlm.nih.gov/pmc/articles/PMC278460/?page=5 >

Leptospiral Media.
<http://jcm.asm.org/cgi/reprint/23/3/500.pdf>

Lehman, D., Mahon, C., Manuselis, G. (2010). The Spirochetes.

Diagnostic Microbiology. Singapore: Elsevier , p540-541.