Microbiology Lecture

Group 4:Leosala, Jamila AnneLin, Tim LawrenceLugue, Ma. CeciliaLupac, BernadetteMacuha, NinyaManrique, Aldric Mari

Introduction:Introduction:

Order: Spirochaetales

Family: Leptospiraceae Family: Spirochaetaceae

Genus: LeptospiraGenus: 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 CharacteristicsPathogenic 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

disk2. 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 serovarhardjo grown in EMJH medium gelled with gellan gum.

Virulence factors:1. Hemolipins – haemolytic agents2. Endotoxin – causes fever, necrosis3. 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: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.