Leptospira

Davood azadi

Taxonomy of leptospira

Domain:Bacteria

Phylum:Spirochaetes

Class:Spirochaetes

Order:Spirochaetales

Family:Leptospiraceae

Genus:Leptospira

THE GENUS LEPTOSPIRA

• The genus Leptospira comprises morphologically similar

thin helical bacteria (spirochetes)

• Trivial name used for all members of the genus is

leptospira or leptospire

• Similar morphologically and culturally, but can be grouped

serologically by agglutinating antigens into characteristic

serovars. 24 serogroups and 250 serovars

• 'Leptospira interrogans‘ major serological complex

suspected to be pathogenic

• 'biflexa complex'other major group, containing

nonpathogenic leptospire

• The genera Leptonema and Turneria were first recognized

because the type strains were morphologically different

from other leptospires

THE GENUS LEPTOSPIRA

Definitions

o genera Leptospira, Leptonema, and Turneria

o motile, flexible, helical aerobic bacteria, 6-12 pm long and 0.1 pm in diameter

o gram-negative but difficult to visualize, and oxidase-positive and chemoorganotrophicThe diamino acid in the peptidoglycan α -diaminopimelic acid and (G + C) ratio is 35-53 mol%.

o one or both ends are hooked, a pair of periplasmic flagella arising from it subterminally,

o Long chain fatty acids or fatty acid alcohols are used as carbon and energy source

• Phylogenetic analyses of 16S rRNA genes suggest that Leptospira

species cluster into three groups designated pathogenic,

saprophytic and intermediate

• Pathogenic group in include: alexanderi, borgpetersenii, fainei,

inadai, interuogans, kirschneri, meyeri, noguchii, santarosai, and

weilii

• Saprophytic('nonpathogenic): biflexa, hollandia, and wolbachla

Genetic classification and species

• A species comprises those leptospires whose DNA is 70%

related and whose related DNA sequences contain 5 % or fewer

unpaired bases (divergence)

• There are 12 named species with further groups awaiting

classification

• Analyzed by sequensing and other genetic methods

HISTORICAL PERSPECTIVE

• first described by Adolf Weil in 1886, it has been around for 100 of years with icteric form of the disease being reported in ancient China, India, and Europe.

• the first credited account of a leptospire being isolated was in 1916 by Inada et al

• Rats as a reservoir host were identified in Japan in early 1900s, and basic pathology as well as epidemiology was derived by 1940

• Outbreak being reported with, kayaking, windsurfing, swimming,, wading through puddles, white-water rafting, and other outdoor sports played in contaminated water

• Leptospirosis is a potentially fatal disease of humans or

animals caused by one of the pathogenic leptospires. It is

prevalent globally as an acute febrile,

• Sometimes congenital or chronic infection of wild or

domesticated animal

• Leptospires persist in the renal tubules of kidneys in carrier

animals, whence they are excreted in urine into the

environment to contaminate water and soil

• Humans are infected incidentally, affected by an acute

febrile, Sometimes the disease is subclinical. And do not

usually become chronic carriers or transmit infection

• Adult males are at more risk of leptospirosis due to higher

occupational and recreational exposure

HABITAT

• Natural habitat is surface waters, soil, and mud.

• They feed by attachment to surfaces where long-chain fatty

acids are available

• Halophilic leptospire has been isolated from estuarine

waters

• lyophilization can preserve Leptospires

Way of spread

• In the host they circulate and grow in an

aqueous milieu and, passing in urine into

surface waters

• leptospires adapt to environmental conditions

including the salinity and temperatures

• Leptospires infect humans only by accident of

contact with a contaminated environment

• spread from humans to other humans is

almost unknown

MORPHOLOGAY AN D VISUALIZATION

• helical gram-negative bacteria hard to see when

stained by conventional bacteriological stains

• Strong stains (carbol fuchsin) and stains that build up

on the surface (flagellar stains, Giemsa, silver stains,

immunostains) make them appear thicker and

improve visualization

• dark field microscopy The routine method of

observation.

• Leptospires pass through bacteriological filters of

average pore diameter 0.2 pm.

Shape and structure

• about 10-20 µm long

• peptidoglycan complex loosely referred

to as cell wall arranged as a flexible

hollow tubular right-handed helix

• amplitude of the coil is about 0.10-0.15

µm and its wavelength is about 0.5 µm

• Leptonema is generally longer (13-15

pm) and Turrneria is shorter (3.5-7.5 pm

long) and more tightly coiled

SPHEROPLASTS

• In some cultures leptospires appear in a

'granular' form. The granules are small cystic

structures, about 1.5- 2.0 mm in diameter

• Once thought to be a cystic stage of a life

cycle, they are really spheroplasts whose

formation can be induced with ethanol,

detergents, sodium deoxycholate, high salt

conditions, lysozyme or mild heat And also

be observed in tissues and in phagocytes

Flagella

• Leptospiral flagella originate in a disc rotor and

hooked proximal end structure in the cell wall,

similar to other gram-negative bacteria.

• with differential or gradient centrifugation can see

the leptospiral Flagella are seen as tight flat coils

• The isolated flagellum is composed of a helically

wound central core of proteins, surrounded by an

outer sheath

• Seven different flagellar proteins have been

recognized

• Theres a 34 kDa protein associated with

the core, of 11.3 nm, and a 36 kDa protein

associated with the sheath, measuring 27.5

nm

• 32 kDa FlaB protein leptospira similar in

sequence to FlaB proteins described in

Treponema pallidum

• The FlaB protein structure and its gene

sequences are highly conserved

throughout the species;

CULTURAL CHARACTERISATION

• TEMPERATURE:

optimum temperature range of 28-30'C, with extremes of 11-42C.

Growth at 11-13"C has been proposed as a phenotypic test for L.

biflexa

Pathogens grow in mammalian hosts at febrile body temperatures,

and in chick embryos and young chicks around 40-42C but do not

grow well in laboratory media at these temperatures

• pH:

Leptospira will grow in the range pH 6.5-8.4

• Osmolarity

Leptospira will grow in the range 0.05 – 0.8 M, molarity of salts

• OXIDATION-REDUCTIOPNO TENTIAL:

Leptospires are aerobic bacteria that do not tolerate reducing conditions or anaerobiosis, when the oxidationreductionpotential is less than about Eh - 0.250 mV at pH7.2.

Growth

• Leptospira strains grow slowly, colonies can take from 3-7 days to 3 weeks to appear.

• Leptospires are grown routinely in liquid media, Some strains may not grow at all on solid media

• leptospires cultures of liquid media at 30'C with a doubling time of 6-8 h under optimum conditions

• Leptonema grows rapidly in medium to reach maximum density on incubation for I8-:72h at 30'C.

Growth requirements• Leptospires require an oxygen source

• Pathogenic leptospires require long-chain (C12 –C18)

unsaturated fatty acids, as a carrbon source

• Leptospira biflexa strains can grow on long- or short-chain,

saturated or unsaturated fatty acids

• The only essential nitrogen source is ammonia probably

producing ammonia by deamination.

OTHER REQUIREMENTS FOR GROWTH

• all the pathogen group require pyrimidines.

• L. biflexa and other nonpathogens and Leptonema can

synthesize their own purines and pyrimidines. Turneria

parva requires purines.

• Phosphates, sulfates, ferric iron or hemoglobin (or

heme),calciuma nd magnesium, thiamine and

cyanocobalamin are essential

Cultur media

• media contain heat-labile proteins or other ingredients and

are sterilized by filtration

• used of rabbit or other animal serum

• Incubation in 28-30 c Cultures should be checked for

growth or contamination after 3-4 days and subcultured

after 7-21 days

culture media

• Oleic acid-albumin (OA) media and serum media based on 1 percent BSA and Tween 80.

• Special selective and indicator media: containingo one or more of cycloheximide , bacitracin, 5-fluorouracil nalidixic acid, polymyxin-B sulphate, polymyxin B ,rifampin (10 pglml) or vancomycin

Recommended to reduce contaminationon primary isolation or to purify contaminated culture

• Protein-free and low-protein media developed for vaccine production

The leptospiral genome and its

elements

• G + C content of 35-51 mol%

• genome size originally estimated to be 4500- 5000 kb

• Both pathogenic and saprophytic leptospires contain two

ribosomal 23S rRNA genes but only one 16S rRNA, for 5S

rRNA,

The leptospiral genome and its elements

no compound transposons have been

found in the genus Leptospira,

Genome sequencing has indicated

IS1533. An IS3-like element, designated

1S1500, was present only in pathogenic

leptospires

LPS biosynthetic locus is bounded by two

IS7533 elements

presence of these IS elements may

regulate gene expression leading to

differences in LPS structure

The leptospiral genome and its

elements

• Genetic analysis of Leptospira has been impeded by the

lack of genetic exchange systems

• isolated three bacteriophages, whose replication was limited

to the saprophyte L. biflexa.

• phage LE1, was shown to replicate as a plasmid in L.

biflexa and was used as the basis for the first l. biflexa- E.

coli plasmid shuttle vector, pGKLep4

• two report of gene inactivation by recombination-mediated allelic replacement in Leptospira.

1. The kanamycin resistance gene from pGKLep4 was used to inactivate the flaBgene, encoding the flagellar subunit protein, in L. biflexa

Mutants were nonmotile and lacked flagella and hooked ends, but retained their helical shape

2. Inactivation of recA in L. biflexa resulted in reduced growth rate and altered nucleoidmorphology

THE LEPTOSPIRAL SURFACE

Lipopolysaccharid

LPS comprises the major surface component of leptospires and is the

target for agglutinating and opsonizing antibodies thus important for

serological classiflcation of leptospires.

The structure of LPS similar to that of typical gram negative LPS (keto-

deoxy-octulonate (KDO) xylose ….)

rfb loci involved in the biosynthesis of the LPS O-antigen contains at

least 31 (ORF). Encode enzymes involved in the biosynthesis of

activated sugars, glycosyl transferases, and sugar processing and

transport proteins

Leptospiral LPS appears to be assembled via the classical Wzy (Rfc)

dependent pathway.

Protein and lipoprotein antigens

• Ompl-l: is a transmembrane protein with porin activity

which exists in a typical trimeric form in the leptospiral

outer membran

• lipoproteins, which are well conserved across the

pathogenic species(major OMP, LipLs, as well as OmpL1,

LipL41(synergystic), lipL31,48,…) but LipL36 is not

produced during infection

Other localized structures and

components

• A glycolipoprotein (GLP) with a high content of toxic lipids

(palmitoleic and oleic acids) is cytotoxic and lethal for laboratory

animals. produce antibodies to GLP

• GLP inhibits Na*, K*ATPase

• Peptidoglycan was cytotoxic, inducing the release of TNFα from

peripheral blood PMN

PATHOGENICITY AN D VIRULENCE FACTORS

• Pathogenic species are dermonecrotic and cytotoxic.

• L. biflexa and avirulent pathogenic leptospires kill with

immunoglobulins ,lysozyme and complement

• Virulent leptospires able to survive in macrophages, in

which they induce apoptosis

virulence

• LipL36 was synthesized at 30'C but not at 37"C

• LipL4l, LrpL32, and Ompl-l, are produced only at 37'C and during growth in an animal infection model

• Virulent leptospires survive because the antigens reacting with these opsonic immunoglobulins are not expressed or not available on the surface

• Opsonizing antibodies (LPS epitopes) (avirulentleptospira)appear, 3-10 days after inoculation lead to clearance by reticuloendothelial Phagocytosis

Virulence

• The primary lesions in leptospirosis consist of damage to the

endothelial cells of small blood vessels, leading to leakage of

plasma and hemorrhages.

• The consequences of the damage to blood vessels are ischemia to

the cells and organs dependent on the disturbed

• Renal tubular necrosis is common lead to localization of

leptospires on the luminal surface of the tubular cells, where

they grow and are excreted in urine

virulence

• Leptospira may have one or more toxins, one of which is a cytotoxic and antigenic GLP

unusualunsaturated fatty acids of leptospiral origin acting as competitive inhibitors

of the incorporation of normally occurring fatty acids in the target cell membrane

• LPS does not appear to play a significant part in pathogenes

• haemolysins( sphingomyelinases) produce holes in erythrocyte and probably other cell membranes

LABORATORY ISOLATION AND

IDENTIFICATION

• Leptospires are so hard to see and slow to grow that

conventional bacteriological diagnostic procedures are not

practicable

• Dark field microscopy rapid diagnostioc test . culture taking up

to 3-4 weeks for growth and days to weeks for identification.

• Immunofluorescent staining is rapid and speciflc if the serovar

or serogroup

• Molecular method

Identification of isolates; typing methods

• Colony type : typing by the morphology of colony

• The usual method for testing serological identity is by microscopic agglutination test (MAT).

• Serological identification should be done at serovar level if possible

• Molecular typing method: molecular typin based on sequencing of 16SrRNA, ITS, and RFLP , PFGE,(NotI or SgrAI)

• At subspecies level, the first widely

used method was restriction

endonuclease analysis (REA) of

whole genomic DNA

• insertion sequences; some of

these have been used as targets for

identification and molecular

typing schemes(diferent in

number like IS1500)

Treatment

• All classes of antibiotics except chloramphenicol and rifampin kill leptospires.

• Penicillin and doxycycline are used widely in therapy

• Resistance to any antibiotic has not appeared as a clinical problem, probably because there is no human-to-human transmission

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