Uncultivable bacteria and recent trends towards their identification

1. Speaker- Dr. Abhishek Yadav
Moderator- Dr. Rohit Chawla
1

2.  Uncultivable bacteria: Bacteria that cannot be
grown on any artificial -non living conditions.
 Relevance of non-culture based approaches -
molecular genetic methods.
 Koch’s Postulates
2

3. Koch’s Postulates
 Isolation in pure culture
 Identification
 Testing for viability
 Basic biology
 Antibiotic sensitivity testing
 Production of antigens
 Production of vaccines
3

4.  Does bacterial uncultivability represent an intrinsic
property of bacteria…????
OR
 Does it reflect the deficiency of our knowledge about
bacterial growth requirements?
OR
BOTH
4

5.  Uncultivable bacteria: bulk of global microbial
diversity
 0.4% of the total number of bacteria in the world
identified
 Majority of environment microorganisms resist
cultivation
 These represent 99-99.99% of microorganisms in
nature & 40% of all bacteria in oral cavity
5

6. Primary causes
 Absence of key metabolic pathways preventing
bacteria from utilizing essential nutrients in vitro
which are available in vivo.
6

7.  Deficiency of essential nutrients
 Deficiency of atmospheric requirements
 Inappropriate pH
 Inappropriate temperature of incubation
 Insufficient time of incubation
 Prior treatment with antibiotics
7

8.  Limitations of Koch’s postulates
 Molecular Koch’s postulates
 Development of
 Cell lines
 Embroyonated eggs
 Laboratory animals
 Co cultivation
 Search for new pathogens
8

9.  Treponema pallidum
 Mycobacterium leprae
 Rickettsia spp.
 Chlamydia spp.
 Spirillum minus
 Ehrlichia chaffeensis
 Anaplasma phagocytophilum
 Trophyrema whippelii
9

10. 10

11.  Caused by Treponema pallidum.
 Motile spiral-shaped gram –ve bacteria
 Characteristic cock-screw motility
 Inability to survive outside in an animal host
 Cannot be cultured in vitro
 Size : approx 10–14 μm in length and 0.1–0.2 μm in
diameter, 10 regular spirals at interval of about 1 μm
 Transmission: sexual; maternal-fetal, and rarely by
other means
11

12. 12

13.  Animal Inoculation-
 Most sensitive method for detecting infectious
treponemes and is used as the gold standard for
measuring the sensitivity of methods such as the PCR
 Dark Field microscopy
 Most productive during 1˚, 2˚, early relapsing, and
early congenital syphilis when lesions contains large
numbers of treponemes
 Direct fluorescent antibody test (DFA-TP)
 Nucleic acid amplification methods – PCR –
 Highly sensitive, able to detect as low as 1 to 10
organisms per specimen with high specificity
13

14.  Antitreponemal antibody response
 IgM antibodies are produced ∼2 weeks after
exposure, followed by IgG antibodies 2 weeks after
IgM production
 T.pallidum infection produces antibodies to more
than 20 different polypeptide antigens.
 Antibodies are of two types :
 1) Non specific antibodies (reagins) : directed
against lipoidal antigen of T. pallidum as well as
mitochondrial & nuclear membranes of human cells
 2) Specific anti-treponemal antibodies : directed
against T.pallidum
14

15.  Early latent syphilis : Faint to moderate IgM and
strong IgG reactivity are evident
 Late Latent syphilis : Faint IgM & variable IgG
 IgM antibodies decrease rapidly, becoming
undetectable within 6–12 months after treatment
 Several studies suggest that decreasing IgM levels
indicate adequacy of treatment.
 In contrast, IgG1 and IgG3 antitreponemal
antibodies can persist for years despite therapy
15

16.  Four nontreponemal tests are currently considered
standard tests:
 All these non treponemal tests measure anti lipoidal
IgM and IgG antibodies
 These tests are used for initial screening and for follow
up after treatment
Microscopic tests Macroscopic tests
VDRL RPR
USR TRUST
16

17. They can be performed as a :
 Qualitative test (to check for presence or absence of
antibodies)
 Quantitative test (to check the amount of
antibodies present in the serum)
 Except for VDRL & RPR tests, most lipoidal
antigen tests are not used
 Limitations : Reduced sensitivity in primary
syphilis and late latent syphilis- False positives and
False negatives
17

18.  Fluorescent Treponemal Antibody Absorption (FTA-
Abs) test
 Treponema pallidum Haemagglutination Assay
(TPHA)
 Treponemal Enzyme Immunoassay (EIA)
 Fluorescent Treponemal Immobilisation (TIA)
18

19.  Primary syphilis
 Dark field microscopic examination : - Most specific
and sensitive
 Non treponemal tests: - Positive in 80% cases
 Treponemal tests: - Positive in 80 - 90% cases
 Secondary syphilis
 Dark field microscopic examination: - fluid from moist
wet lesions and lymph node aspirate
 Non treponemal tests: - Always positive, usually at a
high dilution
 Treponemal tests: - Always positive
19

20.  Early Latent syphilis
 Non treponemal tests: - positive in 95-98% cases
 Treponemal tests: - positive in 97-100% cases
 Diagnosis  based on reactive serological tests-
treponemal and non treponemal in absence of any
apparent signs of disease
 Late Latent syphilis
 Non treponemal tests: - positive in 34 - 94% cases
 Treponemal tests: - positive 94 - 96% cases
20

21. CSF examination is done in :
 Patients with neurosyphilis
 In patients with syphilis of more than 2 years
duration to exclude asymptomatic neurosyphilis
 Before retreatment of patients who have had relapses
after any form of treatment
 As a follow up procedure for patients who have been
treated for neurosyphilis
 In all infants suspected of prenatal syphilis
21

22. CSF sample is taken and a cell count is made
It is further checked for protein abnormalities and
subjected to VDRL test
Diagnosis of neurosyphilis is indicated by
1. Increased cell count (> 10 lymphocytes per
mm3)
2. Increased proteins (> 40 mg% in the CSF)
3. REACTIVE VDRL test
Serum VDRL test is reactive in about 2/3rd of the
cases
22

23. Cardiovascular syphilis -
 Serological tests - usually reactive, esp. if extensive
involvement
 Negative reaction may accompany a localized lesion
Congenital syphilis :
 Demonstration of T. pallidum by direct examination
from nasal discharge or from early lesions
 Positive treponemal test in a titre, higher than mother
or serially rising
 FTA-IgM test is more specific with infection
23

24. Diagnostic Algorithm
Reverse
24

25.  Now widely available for use, where they expand
the range of settings in which STI testing can be
under- taken, thus facilitating earlier diagnosis and
access to rapid treatment and support
 POCT also offer the unique ability to offer
immediate testing and treatment in a single
encounter to mitigate further transmission of
syphilis, making this an attractive alternative to
standard testing in populations such as men who
have sex with men (MSM) and sex trade workers
25

26.  (i) Immunochromatographic strip (ICS) tests
 (ii) Particle agglutination tests (PATs)
 Because a positive treponemal POCT result may
indicate new or old infections, a quantitative
nontreponemal test is often helpful. (dual tests are
commercially available)
26

27. 27

28.  Family- Mycobacteriaceae
 Appear as straight or curved rods
 Size is 1 – 8 microns x 0.5 microns
 Acid fast but less resistant only 5 % H2So4
 Live bacilli, solid uniform structure.
 Dead appear as fragmented with granules
 Armadillo’s used for obtaining M leprae
28

29. Leprosy - Case Definition
Cardinal Signs of Leprosy
 Definite loss of sensation in a skin lesion
consistent with leprosy
 Skin smears positive for acid fast bacilli
 Thickening of one or more peripheral nerves

30. Leprosy Classifications
 Ridley-Jopling – Referral centres/Research
TT - BT - BB - BL - LL
– Skin lesions
– Bacterial load
– Histology
WHO Classification- Operational
– Paucibacillary (2-5 skin lesions)
– Multibacillary (>6 lesions)

32.  Skin smear microscopy
 It is performed using the Ziehl Neelsen staining
technique, which consists of staining bacilli with red
dyes and makes it possible to assess the
morphological index (MI) and the bacterial index
(BI)
 Smear is positive in the multibacillary group (MB),
which helps establish a definite diagnosis of leprosy,
but sensitivity is low in the paucibacillary group
(PB)
32

33.  Gold standard
Full-thickness skin biopsy sample
Histological pattern in H & E stained smears
- involvement of cutaneous nerves
- identification of acid-fast bacilli within
nerves (Fite-Faraco modification of
carbol fuchsin)

34.  Not possible
 Can be propagated in Foot pads of Mice
 Granulomas develop at the site of inoculation.
 Nine banded armadillo highly susceptible.
 Chimpanzees
 Generation time 12 -13 days.
 Average may be 8- 42 days.
Dr.T.V.Rao MD 34

35.  PGL-1, which has an anti- genically specific
trisaccharide of M. leprae
 PGL-1 was synthesized as mono-, di- and trisaccharide
compounds, and currently it is used by means of the
 Immunoenzymatic assay (ELISA),
 Passive hemagglutination test,
 Hemagglutination in gelatin particles,
 Dipstick
 Rapid lateral flow test (ML flow).
Limited to Multibacillary group
Role in Monioring
35

36.  Currently, studies has been focusing on serological
diagnosis based on the cell immune response to
candidate antigens of M. leprae (recombinant
proteins and peptides) as assessed by the
measurement of the production of gamma interferon
(IFN-γ), an indirect indicator of protective cellular
immunity.
 This method may detect earlier evidence of infection
by M. leprae and diagnose PB cases
36

37. Slit-skin smear
 For semi quantitative enumeration of AFB
 Useful in follow-up
 At least seven sites
1. Smears from 4 skin lesion
2. Smears from both ear lobules
3. Nasal swab Smear
 Bacteriological Index
 Morphological Index
 Fluorescin diacetate-ethidium bromide stain
( live bacilli) ( Dead Bacilli)
( GREEN) ( RED)

38. Molecular identification of M. leprae bacillus
PCR may allow :
 Confirming cases of initial, PB and pure neural
leprosy
 Demonstrating subclinical infection in contacts
 Monitoring treatment
 Determining patients’ cure or their resistance to
MDT drugs
38

39. Tissue sample Gene
Skin smears hsp18
Nasal smears ag36
Skin biopsies groEL1
Paraffin-embedded skin biopsy samples 16S rRNA
Nerve lesions RLEP

40. 40

41.  Family Rickettsiaceae
 Small (0.3 to 0.5 μm by 1 to 2 μm), obligately
intracellular bacteria
 Divided into Typhus Group (TG) and Spotted fever
group (SFG)
41

42. 42

43.  DIRECT DETECTION
 Immunologic Detection
 Fig: Direct immunofluorescence staining of skin biopsy
specimens with anti-SFG Rickettsia antibodies
facilitates rapid diagnosis. Rickettsiae are present in
the vessel wall
43

44.  Serologic assays for the diagnosis of rickettsial
infections focus on the “gold standard,” -the indirect
IFA.
 Other approaches include indirect
immunoperoxidase assay, latex agglutination,
enzyme immunoassay (EIA), Proteus vulgaris OX-
19 and OX-2 and Proteus mirabilis OX-K
agglutination (Weil-Felix febrile agglutinins), line
blotting, Western immunoblotting, and rapid lateral
flow assays
 Serologic tests, such as indirect hemagglutination,
microagglutination, and complement fixation, are no
longer in general use
44

45.  WEIL FELIX TEST
45
OX 2 OX 19 OX K Diagnosis
- + -
Epidemic,
Endemic
Typhus
+ + -
Spotted fever
group
- - + Scrub Typhus

46.  Micro-immunofluorescent Technique (MIF) –
 The test is performed by placing microdot solutions on a
microscope slide, drying the solution and adding
dilutions of patients’ antisera, followed by fluorescein
labelled anti-human antibodies.
 The microdots are, thereafter, viewed for specific
fluorescence.
 Multiple species can be tested simultaneously.
46

47.  Isolation:
 Identifying the species of rickettsial isolates by
microimmunofluorescence serotyping requires
intravenous inoculation of mice with large doses of
rickettsiae on days 0 and 7 and collection of sera on
day 10
47

48. 48

49. 49
 Family - Chlamydiaceae
 Nonmotile, obligate intra- cellular bacteria
 Cannot be grown on artificial media

50. 50

51.  Antigen Detection Technique
 Direct Fluorescent Antigen (DFA) Detection
Technique- Monoclonal antibodies, labelled with
fluorescein isothiocyanate - apple green elementary
bodies and reticulate bodies
 Enzyme Immunoassay (EIA) - colour change as an
indicator of positivity rather than visualization of Ebs
(immunofluorescence) or Rbs (cell culture).
52

52.  Antibody Detection Tests
 Complement Fixation (CFT)
 Radio-Immunoprecipitation Test (RIP)- radiolabeled
antigen
 Micro-immunofluorescent Technique (MIF) - The test
is performed by placing microdot solutions of each of
14 serovars on a microscope slide, drying the solution
and adding dilutions of patients’ antisera, followed by
fluorescein labelled anti-human antibodies. The
microdots are, thereafter, viewed for specific
fluorescence
53

53.  Tissue Culture
 A number of cell lines such as Hela 229, Hep-2, baby
hamster kidney cells (BHK 21) and McCoy cells, are
susceptible to infection with C. trachomatis
 Nucleic Acid Amplification Tests (NAATs)
 Polymerase Chain Reaction test
 Ligase Chain Reaction test
 GEN - PROBE PACE 2 - is a non- isotopic, DNA probe
test which uses the technique of nucleic acid
hybridization for the detection of C. trachomatis based
on chemiluminescence
54

54. 55

55.  S. minus also causes rat-bite fever in humans and is
referred to as sodoku.
 Common symptoms of RAT BITE FEVER -acute onset of
chills, fever, headache, vomiting, and often severe joint
pains.. In the first few days of illness, patients develop a
rash on the palms, soles of the feet, and other
extremities
 The clinical signs and symptoms are similar to those
caused by Streptobacillus moniliformis, except that
arthritis is rarely seen in patients with sodoku and
swollen lymph nodes are prominent; febrile episodes are
also more predictable in sodoku.
 The bite wound heals spontaneously, but 1 to 4 weeks
later, it re-ulcerates to form a granulomatous lesion; at
the same time, the patient develops constitutional
symptoms of fever, headache, and a generalized, blotchy,
purplish, maculopapular rash.
56

56.  Differentiation between rat-bite fever caused by S.
minus and that caused by S. moniliformis is usually
accomplished based on the clinical presentation of the
two infections and isolation of the latter organism in
culture.
 The incubation period for S. minus is much longer than
that for streptobacillary rat-bite fever, which has
occurred within 12 hours of the initial bite.
 Common symptoms of RAT BITE FEVER -acute onset of
chills, fever, headache, vomiting, and often severe joint
pains.
 In the first few days of illness, patients develop a rash on
the palms, soles of the feet, and other extremities.
57

57.  Samples- Blood or joint fluid is extracted
 Microscopy:
 Direct visualization of characteristic spirochetes in
clinical specimens using Giemsa or Wright stains.
 Dark-field microscopy- S. minus appears as a thick,
spiral, gram-negative organism with two or three
coils and polytrichous polar flagella.
58

58.  Isolation
 Diagnosis is definitively made by injection of lesion
material or blood into experimental white mice or
guinea pigs and subsequent recovery 1 to 3 weeks after
inoculation.
 Serology:
 No serological tests available yet
59

59. 60

60.  Ehrlichia chaffeensis, the etiologic agent of human
monocytotropic ehrlichiosis (HME)
 HME is an emerging zoonosis that causes clinical
manifestations ranging from a mild febrile illness to
a fulminant disease characterized by multi-organ
system failure.
 Fatality rate – 2-3%
61

61.  A. phagocytophilum causes human
granulocytotropic anaplasmosis (HGA)
 Symptoms of which are similar to HME
 Both zoonotic diseases
62

62.  Direct Examination
 Microscopy by Romanowsky Staining of Peripheral
Blood- leukocytes examined for the presence of
morulae. – low Sn
 Antigen Detection by Immunohistology
63

63. C- A. phagocytophilum morula (arrow) is present in a neutrophil
D- Wright stain (original magnification, × 1,000) of A. phagocytophilum from
the blood of an infected patient cultured in the human promyelocytic cell line
HL-60
64

64.  Nucleic Acid Detection Techniques
 PCR targeting 16s rRNA gene
 Isolation
 The most frequently used cell for primary isolation is
the canine histiocytic cell line DH82
65

65.  Serologic Tests
 The gold standard for the diagnosis of HME is
demonstration of a 4-fold rise in IgG titer or seroconversion
by examination of paired (acute- and convalescent-phase)
sera
 The most frequently used serologic method is the IFA. Other
methods include enzyme-linked immunosorbent assay
(ELISA) or enzyme immunoassay and protein (Western)
immunoblotting, but none have been well validated.
 Currently, there is little standardization for any method of
Ehrlichia serology, and cutoff titers are dependent upon
validation in individual laboratories that perform these
assays or are per manufacturer instructions
66

66.  Direct Examination
 Microscopy by Romanowsky Staining of Peripheral
Blood
 Immunohistology for Antigen Detection
 Nucleic Acid Detection Techniques (PCR)
 Isolation:
 Serology
 Anaplasma phagocytophilum IFA
67

67. 68

68.  Gram positive bacteria causing Whipple’s
disease(WD)
 Transmission- feco-oral
 Immunological predisposition?
 T. whipplei can be detected in saliva, dental plaque,
intestinal biopsy, gastric juice, and stool specimens
of healthy individuals without signs of Whipple’s
disease.
 2 types- Classical(systemic) and Isolated
69

69. 70

70.  DIRECT EXAMINATION
 Microscopy – PAS-positive, diastase- negative granular
inclusions of intracellular or ingested bacteria
 Antigen Detection- Immunohistochemical staining (IHC)
 Nucleic acid detection-
 FISH –
 fluorescently labeled oligonucleotide probes to target T.
whipplei 16S rRNA
 Correlated to the activity of bacteria,
 Localise organism in extra- intestinal tissues
71

71. 72

72.  Polymerase chain reaction
 Tropheryma whippleii specific quantitative
polymerase chain reaction (qPCR) is a reliable and
specific method for diagnosing infections with T.
whipplei, if confirmed by sequencing of multiple T.
whipplei target genes to avoid false positive results.
 PCR analysis from cerebrospinal fluid is highly
recommended in all WD cases, because
asymptomatic T. whippleii infection of the CNS has
been described in up to 40% of all patients with
gastrointestinal manifestation of WD and
represents a complication with a high mortality
73

73.  ISOLATION PROCEDURES, IDENTIFICATION
 Tropheryma whipplei can be cultured by the
centrifugation-shell vial technique and a human
fibrobroblast cell line (human erythroleukemia cell
line- HEL)
 However, this approach is a time consuming method
due to the generation time of 18 days of T. whipplei
74

74. 75

75.  SEROLOGY
 Serological antibodies play a minor role in the
current routine diagnosis of WD. The determination
is only possible in designated reference centers.
 Studies have shown that antibodies against T.
whippleii do not only occur in patients with WD but
also in healthy subjects and asymptomatic carriers.
They can even miss entirely in patients with classic
WD.
 However, newly developed Western-Blot methods
seem to differentiate serological antibodies between
patients with classic WD and asymptomatic
carriers 76

76.  T. whipellii Western blot
 In one of the studies-
 Overall, total immunoglobulin detection against the
antigenic membrane proteins discriminated
between patients with classic Whipple disease
and T. whipplei carriers on the basis of difference in
reaction to glycosylated and deglycosylated proteins.
77

77. 78

78.  Clinically important microbial pathogens remain
unrecognized
 Molecular methods novel agents
 Traditional techniques obsolescent
 Undoubted value of novel molecular methods -
crucial role of traditional technique
79

79.  Are “sterile cultures” a true picture of diseased
tissue or a reflection of the inadequacy of our
methods ?
 No human bacterial pathogen is uncultivable so far:
the real issue is whether we are able to determine
the environmental conditions required by
prokaryotic agents for growth
80

80.  Establishment of criteria for disease causation
 Reconsideration of Koch’s Postulates
 Further characterization of human microbiome
during state of health
81

81. 82