MEDICAL

MICROBIOLOGY II

DR. KELI, MBChB.

Course objectives
1. Distinguish bacteria of medical importance
2. Distinguish viruses of medical importance
3. Differentiate fungi of medical importance
4. Demonstrate understanding of connection of parasites with disease
processes
 Bacteriology

Scope: Bacteria of medical importance; classification, morphology,

virulence factors, diseases caused, mode of transmission, preventive
measures
Bacterial Taxonomy
• Bacteria (and fungi, protozoa, helminths) are named according to the
binomial nomenclature system that uses genus and species,
• For example, regarding the name of the well-known bacteria
Escherichia coli, Escherichia is the genus and coli is the species name.
• Classification: Kingdom - Monera
• Phylum
• Class
• Order
• Family
• Genus
• Species
Families, Genera, Species/Strains
of medically important bacteria
A. Gram Positive Cocci
1. Micrococcaceae
• Staphylococcus spp
• S. aureus
• S. epidermidis
• S. saprophyticus
2. Streptococcaceae
• Streptococcus spp
• S. pyogenes
• S. pneumoniae
• S. agalactiae
• S. mutans
• S. salivarius
• S. sanguis
• S. bovis
• S. milleri
• S. mitis
• Enterococcus spp
• E. faecalis
• E. faecium
B. Gram negative Cocci
3. Neisseriaceae
• Neisseria spp
• N. gonorrhoeae
• N. meningitidis
• B. catarrhalis
• Acinetobacter calcoaceticus
4. Veillonellaceae
• Veillonella parvula
C. Gram positive bacilli/rods
5.Bacilliaceae 6. Mycobacteriaceae
• Bacillus spp • Mycobacterium spp
• B. anthracis • M. tuberculosis
• B. cereus
• M. bovis
• B. subtilis
• M. ulcerans
• Clostridium spp
• M. avium-intracellupare
• C. perfringens
complex
• C. tetani
• M. kansasii
• C. difficile
• C. botulinum
• M. leprae
• C. septicum
7. Actinomycetaceae 10. Lactobacillaceae
• Actinomyces israeli • L. acidophilus
• Listeria nonocytogenes
8. Propionibacteriaceae
• Eryspelothrix rhusiopathieae
• P. acnes
• Corynebacterium diphtheriae

9. Nocardiaceae
• Nocardia asteroides
• N. brasiliensis
• N. madurae (Actinomadura madurae).
D. Gram negative bacilli
11. Bacteroidaceae
• Bacteriodes fragilis
• B. melaninogeniucus
• Fusobacterium nucleatum
12. Vibrionaceae
• Vibrio spp
• V. cholerae
• V. parahaemolyticus
• Aeromonas hydrophila
13. Pasteurellaceae
• Pasteurella multocida
• P. haemolytica
• Francisella
(Pasteurella)tularensis
• Haemophilus influenzae
• H. ducreyi
• Gardnerella vaginalis
14. Brucellaceae 15.Campylobacteriaceae
• Brucella spp • Campylobacter spp
• B. abortus • C. coli
• B. melitensis
• C. jejuni
• B. suis
• C. fetus
• B. canis
• Bordetella spp • Helicobacter pylori
• B. pertussis
• B. parapertussis
• B. bronchiseptica
16.Pseudomonadaceae 17.Spirochaetaceae
• Pseudomonas spp • Leptospira interrogans
• P. aeruginosa • Borrelia spp
• P. fluorescens • B. vinicentii
• P. mallei • B. recurrentis
• P. psudomallei • B. burgdorferi
• B. duttoni
• Treponema pallidum
18. Enterobaeteriaceae

• Escherichia coli • S. choleraesuis

• Salmonella spp • S. typhimurium
• Salmonella typhi • Shigella spp
• S. paratyphi A • S. dysenteriae
• S. paratyphi B • S. flexneri
• S. paratyphi C • S. boydii
• S. enteridis • S. sonnei
• Proteus spp • Serratia spp
• P. vulgaris • S. marcescens
• P. mirabilis
• Yersinia spp
• Enterobacter spp
• Y. pestis
• E. aerogenes
• Y. enterocolitica
• Y. Pseudotuberculosis • E. cloacae
• Providentia spp
• P. stuartii
• P. rettgeri
• Citrobacter spp
• C. freundii
• C. intermedius

• Klebsiella spp
• K. pneumoniae
• K. ozaenae
• Edwardsiella tarda
• Morganella morgnii
19. Chlamydiaceae 20. Rickettsiaceae
• Chlamydia spp • Rickettsia spp
• C. trachomatis • R. tsutsugamushi
• C. psittaci • R. akari
• C. pneumoniae • R. prowazekii
• R. typhi
• Coxiella burnetii
• Rochalimaea quintana
21. Bartonellaceae 22. Mycoplasmataceae
• Bartonella bacilliformis • Mycoplama pneumoniae

• Legionella pneumophila • Ureaplasma urealyticum

Common Bacterial Infections
1. Staphylococcus: a gram-positive bacteria .
- S. aureus e.g. food poisoning, impetigo, and furuncles (boils).
- S. epidermidis. e.g. endocarditis.
2. Streptococcus. a gram-positive bacteria.
e.g. Pharyngitis, scarlet fever, and rheumatic fever.
- S. pneumonia is a common cause of bacterial pneumonia and meningitis.
3. Neisseria.
- N. gonorrhoea is the causative agent of gonorrhoea.
- N. meningitides is a leading cause of adult meningitis.

18
Common Bacterial Infections Cont’d
4. Escherichia. E. coli. e. g. gastroenteritis, UTIs and neonatal meningitis.
5. Salmonella. E.g. enteritis.
- S. typhi . e, g. typhoid fever.
6. Vibrio.
- V. cholera. E.g. cholera.
7. Clostridium.
- C. tetani e.g. tetanus (lockjaw)

19
1. Genus Staphylococcus
• Staphylococci: Gram-positive spherical bacteria that occur in microscopic
clusters resembling grapes.
• Bacteriological culture of the nose and skin of normal humans invariably
yields staphylococci.
• In 1884, Rosenbach described the two pigmented colony types of
staphylococci and proposed the appropriate nomenclature:
• Staphylococcus aureus(yellow) and Staphylococcus albus(white)now named
Staphylococcus epidermidis.
• S. aureus colonizes mainly the nasal passages, but it may be found regularly
in most other anatomical locales.
• S epidermidis is an inhabitant of the skin.
• Staphylococcus aureus forms a fairly large yellow colony on rich
medium, S. epidermidis has a relatively small white colony.
• S. aureus is often hemolytic on blood agar; S. epidermidis is non
hemolytic.
• Staphylococci are facultative anaerobes that grow by aerobic
respiration or by fermentation that yields principally lactic acid
• The bacteria are catalase-positive and oxidase-negative
• S. aureus can grow at a temperature range of 15 to 45 0C and at NaCl
concentrations as high as 15%.
• Nearly all strains of S. aureus produce the enzyme coagulase
(Coagulase positive): nearly all strains of S. epidermidis (Coagulase
Negative)lack this enzyme.
• S. aureus should always be considered a potential pathogen;
• Most strains of S. epidermidis are nonpathogenic and may even play a
protective role in their host as normal flora, but may be a pathogen in
the hospital environment.
 FIGURE 1. Gram stain of Staphylococcus aureus in pustular exudate

Gram stain of Staphylococcus aureus in pustular exudate.

FIGURE 3. Sites of infection and diseases caused by Staphylococcus aureus
Membrane-damaging toxins of Staph aureus
a-toxin (a-hemolysin)
•The best characterized and most potent membrane-damaging toxin of S. aureus is a-toxin.
•It is expressed as a monomer that binds to the membrane of susceptible cells. Subunits then
oligomerize to form heptameric rings with a central pore through which cellular contents leak.
ß-toxin
•A sphingomyelinase which damages membranes rich in this lipid.
•The classical test for ß-toxin is lysis of sheep erythrocytes. The majority of human isolates of S.
aureus do not express ß-toxin. A lysogenic bacteriophage is known to encode the toxin.
d-toxin
•A very small peptide toxin produced by most strains of S. aureus.
•It is also produced by S. epidermidis. The role in disease is unknown.
Virulence factors cont’d
Leukocidin is a multicomponent protein toxin produced as separate
components which act together to damage membranes.
• Leukocidin forms a hetero-oligomeric transmembrane pore composed
of four LukF and four LukS subunits, thereby forming an octameric
pore in the affected membrane.
•Leukocidin is hemolytic, but less so than alpha hemolysin.
•Only 2% of all of S. aureus isolates express leukocidin, but nearly 90%
of the strains isolated from severe dermonecrotic lesions express this
toxin, which suggests it’s an important factor in necrotizing skin infxns.
Virulence factors cont’d
Coagulase and clumping factor
•Coagulase is an extracellular protein which binds to prothrombin in the host to
form a complex called staphylothrombin.
•The protease activity characteristic of thrombin is activated in the complex,
resulting in the conversion of fibrinogen to fibrin.
•Coagulase is a traditional marker for identifying S aureus in the clinical
microbiology laboratory.
•However, there is no overwhelming evidence that it is a virulence factor, (although
it is reasonable to speculate that the bacteria could protect themselves from
phagocytic and immune defenses by causing localized clotting)
Virulence factors cont’d
Staphylokinase
•Many strains of S aureus express a plasminogen activator called
staphylokinase.
•This factor lyses fibrin. The genetic determinant is associated with
lysogenic bacteriophages.
•A complex formed between staphylokinase and plasminogen activates
plasmin-like proteolytic activity which causes dissolution of fibrin clots.
•The mechanism is identical to streptokinase, which is used in
medicine to treat patients suffering from coronary thrombosis.
Virulence factors cont’d
Other extracellular enzymes
• S. aureus can express proteases, a lipase, a deoxyribonuclease
(DNase) and a fatty acid modifying enzyme (FAME).
•The first three probably provide nutrients for the bacteria, and it is
unlikely that they have anything but a minor role in pathogenesis.
•However, the FAME enzyme may be important in abscesses, where it
could modify anti-bacterial lipids and prolong bacterial survival.
Virulence factors cont’d
•Enterotoxins and TSST-1 are superantigens (a class of antigens that
result in excessive activation of the immune system) that may cause
toxic shock.

•Staphylococcal enterotoxins cause emesis (vomiting) when ingested

and the bacterium is a leading cause of food poisoning.

•The exfoliatin toxin causes the scalded skin syndrome in neonates,

which results in widespread blistering and loss of the epidermis
Resistance of Staphylococci to Antimicrobial
Drugs
•The term MRSA refers to Methicillin resistant Staphylococcus aureus.

•Methicillin resistance is widespread and most methicillin- resistant strains are also multi-
drug resistant.
•A plasmid associated with vancomycin resistance has been detected in Enterococcus faecalis
which can be transferred to S. aureus in the laboratory.

•It is speculated that this transfer may occur naturally (e.g. in the gastrointestinal tract).

•In addition, S. aureus exhibits resistance to antiseptics and disinfectants, such as quaternary
ammonium compounds, which may aid its survival in the hospital environment.
2. Streptococcus
• Streptococcus pneumoniae
• Other Streptococcus spp
• Streptococcus pyogenes (Group A streptococcus)
• Streptococcus agalactiae (Group Bstreptococcus)
• Viridans streptococci
• Enterococci
•Streptococcus pneumoniae are Gram-positive, lancet-shaped cocci (elongated
cocci with a slightly pointed outer curvature).
•Usually they are seen as pairs of cocci (diplococci), but they may also occur
singly and in short chains.
•When cultured on blood agar, they are alpha hemolytic. They do not form
spores, and they are non motile.
•Like other streptococci, they lack catalase and ferment glucose to lactic acid.
•Unlike other streptococci, they do not display an M protein, they hydrolyze
inulin, and their cell wall composition is characteristic both in terms of their
peptidoglycan and their teichoic acid).
Gram Stain of a film of sputum from a case
of lobar pneumonia
Cultivation
• Streptococcus pneumoniae is fastidious bacterium, growing best in 5% carbon
dioxide. Nearly 20% of fresh clinical isolates require fully anaerobic conditions.
• In all cases, growth requires a source of catalase (e.g. blood) to neutralize the
large amount of hydrogen peroxide produced by the bacteria.
• In complex media containing blood, at 37°C, the bacterium has a doubling time
of 20-30 minutes.
• On agar, pneumococci grow as glistening colonies, about 1 mm in diameter.
• Two serotypes, types 3 and 37, are mucoid.
• Pneumococci spontaneously undergo a genetically determined, phase variation
from opaque to transparent colonies at a rate of 1 in 105.
• The transparent colony type is adapted to colonization of the nasopharynx,
whereas the opaque variant is suited for survival in blood.
•Streptococcus pneumoniae is a fermentative aerotolerant anaerobe.
•It is usually cultured in media that contain blood.
•On blood agar, colonies characteristically produce a zone of alpha (green)
hemolysis, which differentiates S. pneumoniae from the group A (beta
hemolytic) streptococcus, but not from commensal alpha hemolytic
(viridans) streptococci which are co- inhabitants of the upper respiratory
tract.
•Special tests such as inulin fermentation, bile solubility, and optochin (an
antibiotic) sensitivity must be routinely employed to differentiate the
pneumococcus from Streptococcus viridans.
Identification
• The minimum criteria for identification and distinction of
pneumococci from other streptococci are bile or optochin sensitivity,
Gram-positive staining, and hemolytic activity.
• Pneumococci cause alpha hemolysis on agar containing horse,
human, rabbit and sheep erythrocytes.
• Under anaerobic conditions they switch to beta hemolysis caused by
an oxygen-labile hemolysin.
Streptococcus pneumoniae A mucoid strain on blood agar showing alpha
hemolysis (green zone surrounding colonies). Note the zone of inhibition
around a filter paper disc impregnated with optochin. Viridans streptococci
are not inhibited by optochin.
 Serotyping
The quellung reaction
(swelling reaction) forms the
basis of serotyping and relies
on the swelling of the capsule
upon binding of homologous
antibody. The test consists of
mixing a loopful of colony
with equal quantity of specific Streptococcus pneumoniae Quellung (capsular
antiserum and then swelling) reaction can be used to demonstrate
examining microscopically at the presence of a specific capsular type of the
1000X for capsular swelling. bacterium.
Capsule
• A capsule composed of polysaccharide completely envelops the
pneumococcal cells. During invasion the capsule is an essential
determinant of virulence.
• The capsule interferes with phagocytosis by preventing C3b
opsonization of the bacterial cells. 90 different capsule types of
pneumococci have been identified and form the basis of antigenic
serotyping of the organism.
• Anti-pneumococcal vaccines are based on formulations of various
capsular (polysaccharide) antigens derived from the highly-prevalent
strains.
Cell wall
•The cell wall of S. pneumoniae is roughly six layers thick and is composed of
peptidoglycan with teichoic acid attached to approximately every third N-acetylmuramic
acid.

•Lipoteichoic acid is chemically identical to the teichoic acid but is attached to the cell
membrane by a lipid moiety.

•Both the teichoic acid and the lipoteichoic acid contain phosphorylcholine; two choline
residues may be covalently added to each carbohydrate repeat.

•This is an essential element in the biology of S. pneumoniae since the choline specifically
adheres to choline-binding receptors that are located on virtually all human cells.
Surface proteins
•On the basis of functional genomic analysis, it’s estimated that the pneumococcus
contains more than 500 surface proteins.
•Some are membrane-associated lipoproteins, and others are physically associated
with the cell wall.
•The latter includes five penicillin binding proteins (PBPs), Two neuraminidases, IgA
protease.
• A unique group of proteins on the pneumococcal surface is the family of choline-
binding proteins (CBPs).
• Twelve CBPs are non covalently bound to the choline moiety of the cell wall and
are used to "snap" various different functional elements onto the bacterial
surface.
Surface proteins cont’d
• The CBPs all share a common C-terminal choline-binding domain while
the N-termini of the CBPs are distinct, indicating their functions are
different.
• The CBP family includes such important determinants of virulence
such as PspA (protective antigen), LytA, B, and C (three autolysins),
and CbpA (an adhesin).
Bacterial Determinants of Virulence
1. Capsule
•The bacterial capsule interferes with phagocytosis by leukocytes, a
property dependent on its chemical composition. Apparently,
resistance to phagocytosis is brought about by interference with
binding of complement C3b to the cell surface.
2. Cell Wall Components
•The pneumococcal cell wall is a collection of potent inflammatory stimuli.
•Challenge with cell wall components alone can recreate many of the symptoms of
pneumonia, otitis media and meningitis in experimental models.
•The phosphorylcholine decorating the teichoic acid and the lipoteichoic acid is a
key molecule enabling invasion, and acts both as an adhesin and as a docking site
for the choline-binding proteins (CBPs).
•Other respiratory pathogens such as Haemophilus, Pseudomonas, Neisseria and
Mycoplasma also have phosphorylcholine on lipopolysaccharide, proteins or
fimbriae, suggesting a shared mechanism for invasion of the respiratory tract.
•Two host-derived elements that recognize choline are platelet
activating factor (PAF) receptor and the C-reactive protein.
•Since respiratory pathogens may be recognized and cleared by the C-
reactive protein response as part of the constitutive defenses,
respiratory pathogens may share this invasive mechanism to subvert
the signaling cascade of endogenous PAF.
•The peptidoglycan/teichoic acid complex of the pneumococcus is
highly inflammatory. Smaller components of peptidoglycan
progressively lose specific inflammatory activity.
 •The cell wall directly activates the alternative pathway of the
complement cascade, generating chemotaxins for leukocytes, and the
coagulation cascade, which promotes a "procoagulant state" favoring
thrombosis.
•In addition, peptidoglycan binds to CD14, a cell surface receptor
known to initiate the inflammatory response for endotoxin.
•This induces a cytokine cascade resulting in production of
interleukin-1, interleukin 6 and tumor necrosis factor from human
cells.
3. Choline Binding Proteins
•The CBP family includes such important determinants as PspA (protective
antigen), LytA, B, and C (three autolysins), and CbpA (an adhesin).
•The protective antigen (PspA) is a 65 kD protein with 10 choline-binding repeats.
•PspA appears to inhibit complement-mediated opsonization of pneumococci, and
mutants lacking PspA have reduced virulence.
•Antibodies against PspA confer passive protection in mice.

4. Neuraminidase and IgA protease – these thwart protective host immune

responses
5. Hemolysins
•In addition to surface-associated virulence determinants, pneumococci secrete exotoxins.
•Two hemolysins have been described, the most potent of which is pneumolysin.
•Pneumolysin is stored intracellularly and is released upon lysis of pneumococci by autolysin.
•Pneumolysin binds to cholesterol and thus can indiscriminately bind to all cells without restriction
to a receptor. This protein assembles into oligomers to form transmembrane pores which leads to
cell lysis.
•Pneumolysin can also stimulate the production of inflammatory cytokines, inhibit beating of the
epithelial cell cilia, inhibit lymphocyte proliferation, decrease the bactericidal activity of
neutrophils, and activate complement.
•In addition, pneumococci also produce hydrogen peroxide in amounts greater than human
leukocytes produce. This small molecule is also a potent hemolysin.
Diseases caused
• S. pyogenes (group A streptococcus) is the leading bacterial cause of pharyngitis and
cellulitis.
• It is an important cause of impetigo, necrotizing fasciitis, and streptococcal toxic
shock syndrome.
• It is also the inciting factor of two important immunologic diseases, namely, rheumatic
fever and acute glomerulonephritis.
• Streptococcus agalactiae (group B streptococcus) is the leading cause of neonatal
sepsis and meningitis.
• Enterococcus faecalis is an important cause of hospital- acquired urinary tract
infections and endocarditis.
• Viridans group streptococci are the most common cause of endocarditis.
• Streptococcus bovis also causes endocarditis.
• Most streptococci are part of the normal flora of the human
throat, skin, and intestines but produce disease when they gain
access to tissues or blood.
• Viridans streptococci and S. pneumoniae are found chiefly in the
oropharynx;
• S. pyogenes is found on the skin and in the oropharynx in
small numbers;
• S. agalactiae occurs in the vagina and colon; and both the
enterococci and anaerobic streptococci are located in the colon.
Lab Tests
• Blood culture
• Throat swab
• Microscopy – Gram stain of exudate
• ASOT – Anti-Streptolysin O titre
• Biochemical tests
Prevention
1. Prophylactic antibiotics
◦ Rheumatic fever can be prevented by prompt treatment of group A streptococcal pharyngitis
with penicillin.
◦ Prevention of streptococcal infections (usually with benzathine penicillin once each month for
several years) in persons who have had rheumatic fever is important to prevent recurrence of
the disease
◦ In patients with damaged heart valves who undergo invasive dental procedures, endocarditis
caused by viridans streptococci can be prevented by using amoxicillin peri-operatively.
◦ In patients with damaged heart valves who undergo gastrointestinal or urinary tract
procedures, endocarditis caused by enterococci can be prevented by using ampicillin and
gentamicin peri-operatively.
2. Pneumococcal vaccine - against S. pneumoniae
3. Pathogenic Neisseriae
• The family Neisseriaceae consists of Gram-negative aerobic bacteria
from fourteen genera (Bergey's2001), including Neisseria,
Chromobacterium, Kingella,and Aquaspirillum.
• The genus Neisseria contains two important human pathogens, N.
gonorrhoeae and N. meningitidis.
• N. gonorrhoeae infections have a high prevalence and low mortality,
whereas N. meningitides infections have a low prevalence and high
mortality.
• Both pathogens produce IgA proteases which promote virulence.
• Many normal individuals may harbor Neisseria meningitides in the
upper respiratory tract, but
• Neisseria gonorrhoeae is never part of the normal flora and is only
found after sexual contact with an infected person (or direct contact,
in the case of infections in the newborn).
Neisseria gonorrhoeae
• Neisseria gonorrhoeae : infections are acquired by sexual contact,
usually affect the mucous membranes of the urethra in males and the
endocervix and urethra in females, although the infection may
disseminate to a variety of tissues.
• The pathogenic mechanism involves the attachment of the bacterium
to non-ciliated epithelial cells via pili (fimbriae) and the production of
lipopolysaccharide endotoxin.
• Similarly, the lipopolysaccharide of Neisseria meningitides is highly
toxic, an it has an additional virulence factor in the form of its
antiphagocytic capsule.
Neisseria gonorrhoeae cont’d
• Neisseria gonorrhoeae is a Gram-negative coccus, 0.6 to 1.0 μm in diameter, usually
seen in pairs with adjacent flattened sides.
• The organism is frequently found intracellularly in polymorphonuclear leukocytes
(neutrophils) of the gonorrhea pustular exudate.
• Fimbriae, which play a major role in adherence, extend several micrometers from
the cell surface.
• N. gonorrhoeae is a relatively fragile organism, susceptible to temperature changes,
drying, UV light and other environmental conditions.
• Strains of N. gonorrhoeae are variable in their cultural requirements so that media
containing hemoglobin, NAD, yeast extract and other supplements are needed for
isolation and growth of the organism.
• Cultures are grown at 35-36 0C in an atmosphere of 3-10% added CO2
Neisseria gonorrhoeae cont’d
• Neisseria gonorrhoeae possesses a typical Gram-negative outer membrane
composed of proteins, phospholipids, and lipopolysaccharide (LPS).
• However, Neisseria LPS is distinguished from enteric LPS by its highly-
branched basal oligosaccharide structure and the absence of repeating O-
antigen subunits.
• For these reasons, neisserial LPS is referred to as lipo-oligosaccharide(LOS).
• The bacterium characteristically releases outer membrane fragments called
"blebs" during growth. These blebs contain LOS and probably have a role in
pathogenesis if they are disseminated during the course of an infection.
Infections caused by N. gonorrhoeae
• Urethritis
• Vulvovaginitis.
• Prostatitis,
• Orchitis
• Salpingitis,
• Ovaritis.
• Pelvic Inflammatory Disease (PID).
• Dermatitis-arthritis Syndrome, Endocarditis and Meningitis.
• Rectal Infections (Proctitis)
• Ophthalmia neonatorum)
Men; Dysuria, profuse, mucopurulent discharge…
Also; systemic and other sites.
Control
• There is no effective vaccine to prevent gonorrhea.
• Candidate vaccines consisting of PilE protein or Por are of little
benefit.
• The development of an effective vaccine has been hampered by the
lack of a suitable animal model and the fact that an effective immune
response has never been demonstrated.
• Condoms are effective in preventing the transmission of gonorrhea.
N. gonorrhoeae antimicrobial resistance
• The evolution of antimicrobial resistance in N. gonorrhoeae may
ultimately affect the control of gonorrhea.
• Strains with multiple chromosomal resistance to penicillin,
tetracycline, erythromycin, and cefoxitin have been identified.
• Sporadic high-level resistance to spectinomycin and fluoroquinolones
has been reported.
• Penicillinase-producing strains of N. gonorrhoeae were first described
in 1976.
• Five related ß-lactamase plasmids of different sizes have been
identified
Neisseria meningitidis.
• The bacterium Neisseria meningitidis, the meningococcus, is identical
in its staining and morphological characteristics to Neisseria
gonorrhoeae.
• However, at the ultrastructural level, N. meningitides has a prominent
antiphagocytic polysaccharide capsule.
• N. meningitides strains are grouped on the basis of their capsular
polysaccharides, into 12 serogroups, some of which are subdivided
according to the presence of outer membrane protein and
lipopolysaccharide antigens.
Neisseria meningitides cont’d
• Neisseria meningitides is usually cultivated in a peptone-blood base
medium in a moist chamber containing 5-10% CO2.
• All media must be warmed to 37 0C prior to inoculation as the
organism is extremely susceptible to temp above or below 37 0C.
• This trait is rather unique among bacteria.
• Also, the organism tends to undergo rapid autolysis after death, both
in vitro and in vivo.
• This accounts for the dissemination of lipopolysaccharide (endotoxin)
during septicemia and meningitis.
Neisseria meningitides cont’d
• The organism tends to colonize the posterior nasopharynx of humans,
and humans are the only known host.
• Individuals who are colonized are carriers of the pathogen who can
transmit disease to nonimmune individuals.
• The bacterium also colonizes the posterior nasopharynx in the early
stages of infection prior to invasion of the meninges.
• Most individuals in close contact with a case of meningococcal meningitis
become carriers of the organism.
• This carrier rate can reach 20% of the contact group before the first case
is recognized, and may reach as high as 80% at the height of an epidemic
Neisseria meningitides cont’d
Structure and Classification.
• The only distinguishing structural feature between N. meningitides and N.
gonorrhoeae is the presence of a polysaccharide capsule in the former.
• The capsule is antiphagocytic and is an important virulence factor.
Meningococcal capsular polysaccharides provide the basis for grouping
the organism.
• Twelve serogroups have been identified (A, B, C, H, I, K, L, X, Y, Z, 29E, and
W135).
• The most important serogroups associated with disease in humans are A,
B, C, Y, and W135.
Meningitis
• The term meningitis refers to inflammation the meninges of the brain or spinal
cord.
• Although a variety of cocci cause meningitis, the term meningococcus is reserved
for the Gram-negative, bean-shaped diplococcus, Neisseria meningitidis.
• Like its relative N. gonorrhoeae, the organism tends to occur intracellularly in the
cytoplasm of neutrophils which are attracted to the site of inflammation in the
meninges, so this type of infection is called pyogenic (pus-forming).
• Infection with N. meningitides has two presentations, meningococcemia,
characterized by skin lesions, and acute bacterial meningitis.
• The fulminant form of disease (with or without meningitis) is characterized by
multisystem involvement and high mortality.
N. meningitides Virulence Factors.
• For a time, the virulence of Neisseria meningitides was attributed to the
production of an "exotoxin" that could be recovered from culture
filtrates of the organism.
• But when studies revealed that antitoxin reacted equally well with
washed cells as culture filtrate, it was realized that the bacteria
underwent autolysis during growth and released parts of their cell walls
in a soluble form.
• Hence, the major toxin of N. meningitides is its lipooligosaccharide,
(LOS) and its mechanism is endotoxic.
• The other important determinant of virulence of N. meningitides is its
antiphagocytic polysaccharide capsule.
Virulence Factors cont’d
• The human nasopharynx is the only known reservoir of N. meningitidis.
• Meningococci are spread via respiratory droplets, and transmission requires
aspiration of infective particles.
• Meningococci attach to the non ciliated columnar epithelial cells of the
nasopharynx.
• Attachment is mediated by fimbriae and possibly by other outer membrane
components.
• Invasion of the mucosal cells occurs by a mechanism similar to that observed
with gonococci.
• Events involved after bloodstream invasion are unclear and how the
meningococcus enters the central nervous system is not known.
Control
• Groups A, C, AC, and ACYW135 capsular polysaccharide vaccines are available.
• However, the polysaccharide vaccines are ineffective in young children (in
children under 1 year old, antibody levels decline rapidly after immunization)
and the duration of protection is limited in children vaccinated at 1 to 4 years
of age.
• Routine vaccination is not currently recommended because the risk of
infection is low.
• The group B capsular polysaccharide is a homopolymer of sialic acid and is not
immunogenic in humans.
• A group B meningococcal vaccine consisting of outer membrane protein
antigens has recently been developed, but is not licensed.
• More than 10% of the population may be carrying the bacterium at
any one time on the mucosal surfaces of the nose and throat.
• The majority of these carriers will not have any symptoms of the
disease.
• But this continual exposure to the immune system puts pressure on
the bacterium to mutate its surface components in order to survive.
• Thus, natural selection is the driving force for the emergence of new
antigenic variants.
• Treatment - high-dose intravenous (IV) antibiotics, steroids etc
4. The Genus Bacillus
• The Genus Bacillus includes two bacteria of significant medical
importance, B. anthracis, the causative agent of anthrax, and B.
cereus, which causes food poisoning.
• Non anthrax Bacillus species can also cause a wide variety of other
infections, and they are being recognized with increasing frequency as
pathogens in humans.
• Pathogens of Animals: B. anthracis, and B. cereus.B. alvei, B.
megaterium, B. coagulans, B. laterosporus, B. subtilis, B. sphaericus,
B. circulans, B. brevis, B. licheniformis, B. macerans, B. pumilus,and B.
thuringiensis have been isolated from human infections.
Bacillus
• Bacillus, is placed in the family Bacillaceae.
• The family's distinguishing feature is the production of endospores,
which are highly refractile resting structures formed within the
bacterial cells.
• Bacillus are characterized as Gram-positive, rod-shaped, aerobic or
facultative, endospore-forming bacteria.
• Spore formation, universally found in the genus, is thought to be a
strategy for survival in the soil environment, where in the bacteria
predominate.
Surface Structure of Bacillus.
• Like most Gram-positive bacteria the surface of the Bacillus is
complex and is associated with their properties of adherence,
resistance and tactical responses.
• The vegetative cell surface is a laminated structure that consists of; a
capsule, a proteinaceous surface layer (S-layer), several layers of
peptidoglycan sheeting, and the proteins on the outer surface of the
plasma membrane.
Endospores
• Endospores were first described by Cohn in Bacillus subtilis and later by
Koch in the pathogen, Bacillus anthracis.
• Cohn demonstrated the heat resistance of endospores in B. subtilis, and
Koch described the developmental cycle of spore formation in B.
anthracis.
• Endospores are so named because they are formed intracellularly,
although they are eventually released from this mother cell or
sporangium as free spores.
• Endospores have proven to be the most durable type of cell found in
Nature, and in their cryptobiotic state of dormancy, they can remain
viable for extremely long periods of time, perhaps millions of years.
Anthrax
• Anthrax is primarily a disease of domesticated and wild animals,
particularly herbivorous animals, such as cattle, sheep, horses, mules,
and goats.
• Humans become infected incidentally when brought into contact with
diseased animals, which includes their flesh, bones, hides, hair and
excreta.
• The pathology of anthrax is mediated by two primary determinants of
bacterial virulence:
• Presence of an antiphagocytic capsule, which promotes bacterial invasion,
and;
• Production of a powerful lethal toxin, the anthrax toxin.
Anthrax cont’d
• The most common form of the disease in humans is cutaneous anthrax,
which is usually acquired via injured skin or mucous membranes.
• A minor scratch or abrasion, usually on an exposed area of the face or neck
or arms, is inoculated by spores from the soil or a contaminated animal or
carcass.
• The spores germinate, vegetative cells multiply, and a characteristic
gelatinous edema develops at the site.
• Infection may disseminate, giving rise to septicemia. Lymphatic swelling also
occurs within seven days.
• In severe cases, where the blood stream is eventually invaded, the disease is
frequently fatal.
Anthrax cont’d
• Another form of the disease, inhalation anthrax (woolsorters’
disease), results most commonly from inhalation of spore-containing
dust where animal hair or hides are being handled.
• The disease begins abruptly with high fever and chest pain.
• It progresses rapidly to a systemic hemorrhagic pathology and is often
fatal if treatment cannot stop the invasive aspect of the infection.
Anthrax cont’d
• Gastrointestinal anthrax is analogous to cutaneous anthrax but
occurs on the intestinal mucosa.
• As in cutaneous anthrax, the organisms probably invade the mucosa
through a preexisting lesion.
• The bacteria spread from the mucosal lesion to the lymphatic system.
• Intestinal anthrax results from the ingestion of poorly cooked meat
from infected animals.
• Gastrointestinal anthrax is rare, but may occur as explosive outbreaks
associated with ingestion of infected animals.
Bacillus cereus
• Bacillus cereus causes two types of food-borne intoxications.
• One type is characterized by nausea and vomiting and abdominal cramps and
has an incubation period of 1 to 6 hours.
• It resembles Staphylococcus aureus food poisoning in its symptoms and
incubation period. This is the "short-incubation" or emetic form of the disease.
• The second type is manifested primarily by abdominal cramps and diarrhea with
an incubation period of 8 to 16 hours. Diarrhea may be a small volume or
profuse and watery.
• This type is referred to as the "long-incubation" or diarrheal form of the disease,
and it resembles more food poisoning caused by Clostridium perfringens.
• In either type, the illness usually lasts less than 24 hours after onset.
B. cereus
• The short-incubation form of disease is caused by a preformed heat-
stable enterotoxin.
• The mechanism and site of action of this toxin are unknown.
• The long-incubation form of illness is mediated by a heat-labile
enterotoxin which activates intestinal adenylate cyclase and causes
intestinal fluid secretion.
5. Genus Clostridium
• The clostridia are relatively large, Gram-positive, rod-shaped bacteria.
• All species form endospores and have a strictly fermentative mode of
metabolism.
• Most clostridia will not grow under aerobic conditions and vegetative
cells are killed by exposure to O2, but their spores are able to survive
long periods of exposure to air.
• The clostridia are ancient organisms that live in virtually all of the
anaerobic habitats of nature where organic compounds are present,
including soils, aquatic sediments and the intestinal tracts of animals.
• Clostridia are able to ferment a wide variety of organic compounds.
• They produce end products such as butyric acid, acetic acid, butanol
and acetone, and large amounts of gas (CO2 and H2 ) during
fermentation of sugars.
• A variety of foul smelling compounds are formed during the
fermentation of amino acids and fatty acids.
• The clostridia also produce a wide variety of extracellular enzymes to
degrade large biological molecules in the environment into
fermentable components.
• Most of the clostridia are saprophytes but a few are pathogenic for
humans.
• Those that are pathogens have primarily a saprophytic existence in
nature and, in a sense, are opportunistic pathogens.
• Clostridium tetani and Clostridium botulinum produce the most
potent biological toxins known to affect humans. As pathogens of
tetanus and food-borne botulism, they owe their virulence almost
entirely to their toxigenicity.
• Other clostridia, however, are highly invasive under certain
circumstances.
Stained pus from a mixed anaerobic infection. At least three different
clostridia are apparent.
Clostridium perfringens
• Clostridium perfringens, which produces a huge array of invasins and
exotoxins, causes wound and surgical infections that lead to gas
gangrene, in addition to severe uterine infections.
• Clostridial hemolysins and extracellular enzymes such as proteases,
lipases, collagenase and hyaluronidase, contribute to the invasive
process.
• Clostridium perfringens also produces an enterotoxin and is an
important cause of food poisoning.
• Usually the organism is encountered in improperly sterilized (canned)
foods in which endospores have germinated.
Clostridium difficile
• Clostridium difficile causes antibiotic associated diarrhea (AAD) and more
serious intestinal conditions such as colitis and pseudomembranous colitis in
humans.
• These conditions generally result from overgrowth of Clostridium difficile in the
colon, usually after the normal flora has been disturbed by antimicrobial
chemotherapy.
• People in good health usually do not get C. difficile disease. Individuals who
have other conditions that require prolonged use of antibiotics and the elderly
are at greater risk of the disease.
• Also, individuals who have recently undergone gastrointestinal surgery, or have
a serious underlying illness, or who are immunocompromised, are at risk.
• C. difficile produces two toxins: Toxin A is referred to as an enterotoxin
because it causes fluid accumulation in the bowel. Toxin B is an
extremely lethal (cytopathic) toxin.
• Stool cultures for diagnosis of the bacterium may be complicated by
the occurrence and finding of non toxigenic strains of the bacterium,
so the most reliable tests involve testing for the presence of the Toxin
A and/or Toxin B in the stool.
• The toxins are very unstable. They degrade at room temperature and
may be undetectable within two hours after collection of a stool
specimen leading to false negative results of the diagnosis.
Clostridium tetani
• Clostridium tetani is the causative agent of tetanus. The organism is
found in soil, especially heavily-manured soils, and in the intestinal
tracts and feces of various animals.
• Carrier rates in humans vary from 0 to 25%, and the organism is
thought to be a transient member of the flora whose presence
depends upon ingestion.
• The organism produces terminal spores within a swollen sporangium
giving it a distinctive drumstick appearance.
• Although the bacterium has a typical Gram-positive cell wall, it may
stain Gram-negative or Gram-variable, especially in older cells.
Tetanus
• Tetanus is a highly fatal disease of humans.
• Mortality rates reported vary from 40% to 78%.
• The disease stems not from invasive infection but from a potent
neurotoxin (tetanus toxin or tetanospasmin) produced when spores
germinate and vegetative cells grow after gaining access to wounds.
• The organism multiplies locally and symptoms appear remote from
the infection site.
Tetanus Pathogenesis.
• Most cases of tetanus result from small puncture wounds or
lacerations which become contaminated with C. tetani spores that
germinate and produce toxin.
• The infection remains localized often with only minimal inflammatory
damage.
• The toxin is produced during cell growth, sporulation and lysis.
• It migrates along neural paths from a local wound to sites of action in
the central nervous system.
• The clinical pattern of generalized tetanus consists of severe painful
spasms and rigidity of the voluntary muscles
• Tetanospasmin initially binds to peripheral nerve terminals.
• It is transported within the axon and across synaptic junctions until it
reaches the central nervous system.
• There it becomes rapidly fixed to gangliosides at the presynaptic inhibitory
motor nerve endings, and is taken up into the axon by endocytosis.
• The effect of the toxin is to block the release of inhibitory
neurotransmitters (glycine and gamma-amino butyric acid) across the
synaptic cleft, which is required to check the nervous impulse.
• If nervous impulses cannot be checked by normal inhibitory mechanisms, it
produces the generalized muscular spasms characteristic of tetanus.
Tetanus Toxin.
• Tetanospasmin is encoded on a plasmid which is present in all toxigenic strains.
• Tetanus toxin is one of the three most poisonous substances known, the other
two being the toxins of botulism and diphtheria.
• The toxin is produced by growing cells and released only on cell lysis.
• Cells lyse naturally during germination the outgrowth of spores, as well as
during vegetative growth.
• After inoculation of a wound with C. tetani spores, only a minimal amount of
spore germination and vegetative cell growth are required until the toxin is
produced.
• The toxin is heat labile, being destroyed at 56 0C in 5 minutes, and is O2 labile.
• The purified toxin rapidly converts to toxoid at 0 0C in the presence of formalin.
Sir Charles Bell's portrait of a soldier dying of tetanus.
Neonatal tetanus
• Accounts for about half of the tetanus deaths in developing countries.
• Neonatal tetanus follows infection of the umbilical stump in infants
born to nonimmune mothers (therefore, the infant has not acquired
passive immunity).
• It usually results from a failure of aseptic technique during the
birthing, but certain cultural practices may contribute to infection.
Immunity.
• Unlike other diseases, such as diphtheria, recovery from the natural disease usually
does not confer immunity, since even a lethal dose of tetanospasmin is insufficient to
provoke an immune response.
• Prophylactic immunization is accomplished with tetanus toxoid, as part of the DPT
(DTP) vaccine or the DT (TD) vaccine. Three injections are given in the first year of life,
• Booster is given about a year later, and again on the entrance into elementary school.
• Whenever a previously-immunized individual sustains a potentially dangerous wound,
a booster of toxoid should be injected.
• Wherever employed, intensive programs of immunization with toxoid have led to a
striking reduction in the incidence of the disease.
Clostridium botulinum
• C. botulinum is a large anaerobic bacillus that forms subterminal
endospores.
• It is widely distributed in soil, sediments of lakes and ponds, and
decaying vegetation. Hence, the intestinal tracts of birds, mammals
and fish may occasionally contain the organism as a transient.
• Seven toxigenic types of the organism exist, each producing an
immunologically distinct form of botulinum toxin. The toxins are
designated A, B, C1, D, E, F, and G).
• Not all strains of C. botulinum produce the botulinum toxin.
Botulism
• Food-borne botulism is not an infection but an intoxication since it results
from the ingestion of foods that contain the preformed clostridial toxin.
• In this respect it resembles staphylococcal food poisoning.
• Botulism results from eating uncooked foods in which contaminating spores
have germinated and produced the toxin.
• C. botulinum spores are relatively heat resistant and may survive the
sterilizing process of improper canning procedures.
• The anaerobic environment produced by the canning process may further
encourage the outgrowth of spores. The organisms grow best in neutral or
"low acid" vegetables (>pH4.5).
Pathogenesis of Botulism.
• In food-borne botulism the botulinum toxin is ingested with food in
which spores have germinated and the organism has grown.
• The toxin is absorbed by the upper part of the GI tract in the
duodenum and jejunum, and passes into the blood stream by which it
reaches the peripheral neuromuscular synapses.
• The toxin binds to the presynaptic stimulatory terminals and blocks
the release of the neurotransmitter acetylcholine which is required
for a nerve to simulate the muscle-flaccid paralysis.
• Clinical symptoms of botulism begin 18-36 hours after toxin ingestion
with weakness, dizziness and dryness of the mouth. Nausea and
vomiting may occur.
• Neurologic features soon develop: blurred vision, inability to swallow,
difficulty in speech, descending weakness of skeletal muscles and
respiratory paralysis.
• Botulinum toxin may be transported within nerves in a manner
analogous to tetanospasmin, and can thereby gain access to the CNS.
• However, symptomatic CNS involvement is rare.
The Botulinum Toxins.
• The botulinum toxins are very similar in structure and function to the tetanus toxin, but
differ dramatically in their clinical effects because they target different cells in the
nervous system.
• Botulinum neurotoxins predominantly affect the peripheral nervous system reflecting
a preference of the toxin for stimulatory motor neurons at a neuromuscular junction.
The primary symptom is weakness or flaccid paralysis.
• Tetanus toxin can affect the same system, but the tetanospasmin shows a tropism for
inhibitory motor neurons of the central nervous system, and its effects are primarily
rigidity and spastic paralysis.
• The botulinum toxin is specific for peripheral nerve endings at the point where a motor
neuron stimulates a muscle. The toxin binds to the neuron and prevents the release of
acetylcholine across the synaptic cleft.
Prevention
• The most important aspect of botulism prevention is proper food handling and
preparation.
• The spores of C. botulinum can survive boiling (100 0C at 1 atm) for more than one
hour although they are killed by autoclaving.
• Because the toxin is heat-labile boiling or intense heating (cooking) of contaminated
food will inactivate the toxin.
• Food containers that bulge may contain gas produced by C. botulinum and should
not be opened or tasted. Other foods that appear to be spoiled should not be
tasted.
• A multivalent toxoid evokes good protective antibody response but its use is
unjustified due to the infrequency of the disease.
• An experimental vaccine exists for laboratory workers.
6. Genus Mycobacteria
• The genera Mycobacterium and Nocardia have been grouped into the
family Mycobacteriaceae within the order Actinomycetales based upon
similarities in;
• staining and motility,
• lack of spore formation, and
• catalase production.
• These genera are characterized by the presence of long-chain fatty
acids, called mycolic aids, whose side chains (R1 and R2) vary in length
according to the genus; C60 to C90 in Mycobacterium; C40 to C56 in
Nocardia.
• Several species produce disease in humans.
Mycobacterium Structure
• Mycobacteria are slender, curved rods in stained clinical specimens.
• The cell wall is composed of mycolic acids, complex waxes, and unique
glycolipids.
• The mycobacterial cell wall is acid-fast (i.e., it retains carbolfuchsin dye
when decolorized with acid-ethanol).
• Other important wall components are trehalose dimycolate (so-called cord
factor, as it is thought to induce growth in serpentine cords on artificial
medium) and mycobacterial sulfolipids, which may play a role in virulence.
• Another unique constituent which may contribute to pathogenesis is
lipoarabinomannan (LAM).
• This unusual cell wall structure endows mycobacteria with resistance
to dehydration, acids, and alkalis.
• They require enriched agar media containing bovine serum &
albumin, they can also grow on a chemically defined medium
containing asparagine, glycerol, and micronutrients.
• Even under ideal culture conditions M tuberculosis and M bovis grow
very slowly, with doubling times on the order of 18 to 24 hours.
• When they do appear after 4 to 6 weeks, the colonies are irregular,
waxy, and buff colored, with bacteria piled up into clumps or ridges.
mycobacterial cell wall
Classification and Antigenic Types.
• With the exception of M leprae, the mycobacteria are classified into
two broad categories- members of the;
• M tuberculosis complex (M tuberculosis, M bovis, M microtii, M
africanum) and,
• Nontuberculous mycobacteria (virtually all other species), which
often are described based on their growth rate and pigmentation
with and without exposure to light.
Tuberculosis
• Tuberculosis primarily affects the lower respiratory system and is
characterized by a chronic productive cough, low-grade fever, night
sweats, and weight loss.
• Clinical signs and symptoms develop in only a small proportion (5-10
percent) of infected healthy people.
• Tuberculosis may present with or also exhibit extrapulmonary
manifestations including lymphadenitis; kidney, bone, or joint
involvement; meningitis; or disseminated (miliary) disease.
Pathogenesis
• Virtually all M tuberculosis infections occur by airborne transmission of
droplet nuclei containing a few viable, virulent organisms produced by a
sputum-positive individual.
• The bacilli are deposited in the alveolar spaces of the lungs, where they are
engulfed by alveolar macrophages.
• A portion of the infectious inoculum resists intracellular destruction and
persists, eventually multiplying and killing the macrophage.
• The ability of virulent mycobacteria to survive within phagocytes justifies
their designation as facultative intracellular pathogens.
• In addition, some of the components of the mycobacterial cell wall (e.g.,
cord factor) may be directly cytotoxic to macrophages.
TB Pathogenesis cont’d
• Most of the tissue destruction associated with tuberculosis results from cell-mediated
hypersensitivity.
• Eventually, the accumulating mycobacteria stimulate an inflammatory focus which matures
into a granulomatous lesion characterized by a mononuclear cell infiltrate surrounding a
core of degenerating epithelioid and multinucleated giant (Langhans) cells.
• This lesion (called a tubercle) may become enveloped by fibroblasts, and its center often
progresses to caseous necrosis.
• Liquefaction of the caseous material and erosion of the tubercle into an adjacent airway
may result in cavitation and the release of massive numbers of bacilli into the sputum.
• In the resistant host, the tubercle eventually becomes calcified.
• Pri. site of infection- Ghon focus - upper part of lower lobe or lower part of upper lobe
Epidemiology
• Reactivation is usually associated with deterioration of the cell-
mediated immune response due to aging or to some associated
clinical condition.
• Exogenous reinfection also has been documented.
• Tuberculosis is particularly common in groups such as the elderly, the
chronically malnourished, alcoholics, and the poor.
• The most significant factor influencing the incidence of mycobacterial
disease since 1984 has been the HIV epidemic.
• HIV-infected individuals have a high incidence of tuberculosis,
characterized by frequent extrapulmonary disease.
Epidemiology
• 2017 year:
• 10 million new cases of active TB world wide
• 1.6 million deaths
• Estimated that 25% of the worlds population is infected with myc. TB
with new infections occurring in 1% of the population each year
• Approx. 95% of the cases are from the developing countries.
Diagnosis
1. Montux test - Intradermal PPD (purified protein derivative) skin test.
• Intradermal introduction of PPD into a previously infected, hypersensitive person
results in the delayed (48-72 hr) appearance of an indurated (raised, hard) reaction
with or without erythema
2. Smear microscopy
• Specimens (sputum, bronchial or gastric washings, pleural fluid, urine, or
cerebrospinal fluid) stained and cultured for acid-fast bacilli.
• Two types of stains are used specifically for detection of mycobacteria:
fluorochrome (recommended) and carbol fuchsin.
• In smears stained with carbol fuchsin, mycobacteria typically appear as red rods and
often are beaded or banded, but also may appear coccoid or filamentous but no
species identification on staining.
TB Dx cont’d
• Culture for mycobacteria involves inoculation of solid and broth media.
Cultures are incubated at 35°to 37°C in an atmosphere of 5 to 10% CO2 .
• All cultures should be examined weekly for 8 weeks (on L-J media).
• The major advantage of culture on solid media is that it allows
visualization of colony morphology and pigmentation, which is useful
diagnostically for distinguishing colonies of M tuberculosis from those of
some nontuberculous mycobacteria. However, they require 3 or 4 weeks.
• The more rapid broth systems (e.g. Bactec) require only 5 to 12 days, and
rely upon the detection of 14C-labeled CO2 produced by growing
mycobacteria.
TB Dx cont’d
• DNA probes/PCR,
• Gas-liquid chromatography, high-performance liquid chromatography,
and thin-layer chromatography allow identification of a few species of
mycobacteria within hours after sufficient growth is present on solid
or in a liquid medium.
• Molecular tests e.g gene xpert
• Urine Lipoarabinomannan (LAM) dipstick for urine. Detects M. Tb
through it’s cell wall antigen (LAM) that is excreted in urine.
• Other tests- imaging, blood tests
Prevention/Control
• (BCG) vaccine. - Viable, attenuated strain of M bovis, called Bacille
Calmette-Guérin. BCG vaccination prevents establishment of infection
and development of severe disease.
• Case detection and treatment using DOTS
• Public health interventions such as isolation, Contact tracing, prompt
treatment and household screening.
• Household ventilation
TB Treatment
Medication Formulation Weight in kg
<30 30-37 38-55 >55kg
FDC of Rifampicin 150mg + Isoniazid 75mg + Combo Tab mg/kg dosing 2 3 4
Pyrazinamide 400mg + Ethambutol 275mg (RHZE)
(Rifafour/Rihaz-E)
FDC of Rifampicin 120mg + Isoniazid 50mg + Combo Tab mg/kg dosing 2 3 4
Pyrazinamide 300mg (Rifater/Rihaz) (RHZ)
FDC of Rifampicin 150mg + Isoniazid 75- Combo tab mg/kg dosing 2 3 4
100mg (RH)
FDC of Ethambutol 400mg + Isoniazid 150mg Combo tab mg/kg dosing 1.5 2 2
(Ethizide) (EH)
Streptomycin IM 15-20 mg/kg 500 mg 750mg 1000mg
Mycobacterium leprae
• Leprosy (Hansen's disease) reflects variations in three aspects of the
illness: bacterial proliferation and accumulation, immunologic
responses to the bacillus, and the resulting peripheral neuritis.
• The disease affects peripheral nerves, skin, and mucous membranes.
Skin lesions, areas of anesthesia, and enlarged nerves are the
principal signs of leprosy.
Epidemiology
• More than 10 million cases of leprosy are estimated to exist
worldwide, predominantly in Asia (two-thirds) and Africa (one-third).
• Human-to-human transmission requires prolonged contact and is
thought to occur via intact skin, penetrating wounds or insect bites, or
by inhalation of M leprae and deposition on respiratory mucosa
• The source of the organism in nature is unknown;? MONKEYS.
Pathogenesis
• Since M leprae has never been cultured in vitro, it appears to be an
obligate intracellular pathogen that requires the environment of the
host macrophage for survival and propagation.
• Estimates of the replication rate in vivo are on the order of 10 to 12
days.
• The bacilli resist intracellular degradation by macrophages, perhaps
by escaping from the phagosome into the cytoplasm, and accumulate
to high levels (1010 bacilli/g of tissue) in lepromatous leprosy.
• The peripheral nerve damage appears to be mediated principally by
the host immune response to bacillary antigens.
7. Haemophilus influenzae
• Haemophilus influenza is a small, nonmotile Gram-negative
bacterium in the family Pasteurellaceae, on the level with the
Vibrionaceae and the Enterobacteriaceae
• Encapsulated strains of Haemophilus influenza isolated from
cerebrospinal fluid are coccobacilli, 0.2 to 0.3 to 0.5 to 0.8 um, similar
in morphology to Bordetella pertussis, the agent of whooping cough.
• Non encapsulated organisms from sputum are pleomorphic and often
exhibit long threads and filaments
• H. influenza is highly adapted to its human host. It is present in the
nasopharynx of approximately 75 % of healthy children and adults.
• It is rarely encountered in the oral cavity and it has not been detected in
any other animal species.
• It is usually the non encapsulated strains that are harbored as normal flora,
but a minority of healthy individuals (3-7%) intermittently harbor H.
influenzae type b (Hib) encapsulated strains in the upper respiratory tract.
• Pharyngeal carriage of Hib is important in the transmission of the
bacterium.
• The success of current vaccination programs against Hib is due in part to
the effect of vaccination on decreasing carriage of the organism.
• Haemophilus "loves heme", more specifically it requires a precursor of heme in order to
grow.
• Nutritionally, H.aemophilus influenza prefers a complex medium and requires preformed
growth factors that are present in blood, specifically X factor (i.e., hemin) and V
factor(NAD or NADP).
• In the laboratory it is usually grown on chocolate blood agar which is prepared by adding
blood to an agar base at 80 0C.
• The heat releases X and V factors from the RBCs and turns the medium a chocolate brown
color.
• The bacterium grows best at 35-37 0C and has an optimal pH of 7.6.
• Haemophilus influenza is generally grown in the laboratory under aerobic conditions or
under slight CO2 tension (5% CO2), although it is capable of glycolytic growth and of
respiratory growth using nitrate as a final electron acceptor.
• Nontypable (non encapsulated) strains are less invasive, but they are
apparently able to induce an inflammatory response that causes
disease.
• Outbreaks of H. influenza type b infection may occur in nurseries and
child care centers, and prophylactic administration of antibiotics is
warranted.
• Vaccination with type b polysaccharide (in the form of Hib conjugate
vaccines) is effective in preventing infection, and several vaccines are
now available for routine use.
• In infants and young children (under 5 years of age), H. influenzae
type b causes bacteremia and acute bacterial meningitis.
• Occasionally, it causes epiglottitis (obstructive laryngitis), cellulitis,
osteomyelitis, and joint infections.
• Nontypable H. influenza causes ear infections (otitis media) and
sinusitis in children, and is associated with respiratory tract infections
(pneumonia) in infants, children and adults.
Virulence
• H. influenzae does not produce any demonstrable exotoxins.
• The direct role of endotoxin in meningitis or bacteremia is unclear,
although the Gram-negative bacterium's outer membrane
lipooligosaccharide is thought to play a role in inflammation
associated with otitis media.
• All virulent strains produce neuraminidase and an IgA protease, but
the role of these extracellular enzymes in invasion is unproven.
• Fimbriae increase the adherence of bacteria to human mucosal cells
in vitro, and they are required for successful colonization of the
nasopharynx
• Virulence, at least in the case of bacteremia and meningitis, is directly related to
capsule formation.
• Virtually all of these infections are caused by the type b serotype, and its
capsular polysaccharide, containing ribose, ribitol and phosphate, is the proven
determinant of virulence.
• The capsule material is antiphagocytic, and it is ineffective in inducing the
alternative complement pathway, so that the bacterium can invade the blood or
cerebrospinal fluid without attracting phagocytes or provoking an inflammatory
response and complement-mediated bacteriolysis.
• For this reason, anticapsular antibody, which promotes both phagocytosis and
bacteriolysis, is the main factor in immune defense against H. influenza
infections
• Type b H. influenza is plainly the most virulent of the Haemophilus
species; 95 percent of bloodstream and meningeal Haemophilus
infections in children are due to this bacterium.
• In contrast, in adults, nontypable strains of H. influenza are the most
common cause of Haemophilus infection, presumably because most
adults have naturally acquired immunity.
Immunity.
• The age incidence of H. influenza meningitis is inversely proportional
to the titer of bactericidal antibody in the blood, whether passively
acquired from the mother or actively formed.
• Without artificial immunization, in children aged 2 months to 3 years,
antibody levels are minimal; thereafter antibody levels increase and
the disease becomes much less common.
• From this active immunization should begin at 2 months of age, when
nearly all passive immunity has waned, and the child enters a
vulnerable non immune period of life.
• The use of polyribosylribitolphosphate (PRP) vaccine and, more
recently, protein-conjugated PRP, has vastly reduced the frequency of
infection due to type b H. influenzae.
• The PRP vaccine consists of the type b capsular polysaccharide. Like
most bacterial polysaccharides, it elicits a strong primary antibody
response, but with little induction of memory.
• H. influenza type b Hib conjugate vaccines, which couple the
polysaccharide to a protein, induce memory type antibody responses
in children and are effective in younger infants who are at higher risk
for the disease.
• All of the vaccines are considered effective.
• The vaccines are given by injections.
• More than 90% of infants obtain long term immunity with 2-3 doses
of the vaccine.
• All children should have a vaccine approved for infants beginning at 2
months.
• Depending on the type used, the recommended schedule for infants
will vary.
8. Enterobacteriaceae
• The Enterobacteriaceae are a large, heterogeneous group of gram-
negative rods whose natural habitat is the intestinal tract of humans
and animals.
• Features common to all members of this heterogeneous family are their
anatomic location and the following four metabolic processes:
(1) they are all facultative anaerobes
(2) they all ferment glucose (fermentation of other sugars varies)
(3) none have cytochrome oxidase (i.e., they are oxidase-negative)
(4) they reduce nitrates to nitrites as part of their energy-generating
processes
Enterobacteriaceae cont’d
• These four reactions can be used to distinguish the
Enterobacteriaceae from another medically significant group of
organisms—the non fermenting gram-negative rods, the most
important of which is Pseudomonas aeruginosa
• Individual group includes
Klebsiella,
Escherichia coli,
Shigella,
Vibrio,
Anterobactor
Enterobactor.
Virulence factors/Surface antigens
1. The cell wall antigen (also known as the somatic, or O, antigen) is the
outer polysaccharide portion of the lipopolysaccharide..
2. The H antigen is on the flagellar protein. Only flagellated organisms,
such as Escherichia and Salmonella, have H antigens, whereas the non-
motile ones, such as Klebsiella and Shigella, do not.
3. The capsular or K polysaccharide antigen is particularly prominent in
heavily encapsulated organisms such as Klebsiella. The K antigen is
identified by the quellung (capsular swelling) reaction in the presence
of specific antisera and is used to serotype E. coli and Salmonella typhi
for epidemiologic purposes.
Diseases Caused by Members of the Enterobacteriaceae
Enterobacteria pathogenic group
• Klebsiella pneumoniae
• Causes pneumonia, sinusitis, otitis media, urinary tract infection.

• Escherichia coli.
• Causes urinary tract infection, travelers diarrhea and infantile diarrhea.

• Salmonella typhi.
• Causes typhoid fever,
• Salmonella Para typhii.
• Causes enteric fever which is milder than that caused by salmonella typhi.
9. Helicobacter pylori
• Helicobacter are curved gram-negative rods similar in appearance to campylobacters
• Strongly urease-positive, whereas campylobacters are urease-negative.

Pathogenesis & Epidemiology

• H. pylori attaches to the mucus-secreting cells of the gastric mucosa.
• The production of large amounts of ammonia from urea by the organism's urease,
coupled with an inflammatory response, leads to damage to the mucosa.
• Loss of the protective mucus coating predisposes to gastritis and peptic ulcer. The
ammonia also neutralizes stomach acid, allowing the organism to survive.
Epidemiologically, most patients with these diseases show H. pylori in biopsy
specimens of the gastric epithelium.
• The natural habitat of H. pylori is the human stomach, Probably acquired by ingestion.
• However, it has not been isolated from stool, food, water, or animals.
• Person-to-person transmission probably occurs, because there is clustering of
infection within families. The rate of infection with H. pylori in developing countries is
very high, a finding that is in accord with the high rate of gastric carcinoma in those
countries.

Clinical Findings
• Gastritis and peptic ulcer are characterized by recurrent pain in the upper abdomen,
frequently accompanied by bleeding into the gastrointestinal tract.
• No bacteremia or disseminated disease occurs.
Laboratory Diagnosis
• The organism can be seen on Gram-stained smears of biopsy specimens of the gastric
mucosa. It can be cultured on the same media as campylobacters. In contrast to C. jejuni,
H. pylori is urease-positive.
• Urease production is the basis for a noninvasive diagnostic test called the "urea breath"
test. In this test, radiolabeled urea is ingested. If the organism is present, urease will
cleave the ingested urea, radiolabeled CO2 is evolved, and the radioactivity is detected in
the breath.
• A test for Helicobacter antigen in the stool can be used for diagnosis and for confirmation
that treatment has eliminated the organism.
• The presence of IgG antibodies in the patient's serum can also be used as evidence of
infection.
• Treatment & Prevention - Treatment with antibiotics, triple therapy
10. Vibrio cholerae
• Gram-negative, motile, facultative anaerobe and comma-shaped bacteria.
• Epidemic cholera produces profuse watery diarrhea - If untreated rapidly
leads to dehydration and electrolyte imbalance.
• Pathogenesis is due to the action of cholera enterotoxin secreted by V.
cholerae in the bowel lumen
• Spread primarily by contaminated water under conditions of poor
sanitation. Has short incubation period (2 days)
• WHO declared cholera outbreak in Eastern and Southern Africa in Feb
2023, MoH- Kenya declared 6 endemic regions in Oct 2022
• Treatment- hydration, ORS, Abx – doxycycline etc
11. Pseudomonas aeruginosa
• is an aerobic, motile, encapsulated, gram-negative rod
• Pseudomonas species are most frequently seen as colonizers and
contaminants, but are able to cause opportunistic infections.
• Shows the most consistent resistance to antimicrobial agents of all
the medically important bacteria due to impenetrability of its outer
membrane.
• Commonest Healthcare Associated Infection
Gram-Negative Rods Related to Animal Sources (Zoonotic Organisms)
Rickettsia
• Two rickettsial diseases of significance: Rocky Mountain spotted fever, caused by Rickettsia
rickettsii, and Q fever, caused by Coxiella burnetii.
• Is a lower form of bacteria, a small parasitic organism thought to be related to virus due to small
size
• Rickettsiae are very short rods that are barely visible in the light microscope.
• Structurally, their cell wall resembles that of gram-negative rods, but they stain poorly with the
standard Gram stain.
• Rickettsiae are obligate intracellular parasites, because they are unable to produce sufficient
energy to replicate extracellularly.
• Rickettsiae divide by binary fission within the host cell, in contrast to chlamydiae, which are also
obligate intracellular parasites but replicate by a distinctive intracellular cycle.
• Transmits disease through bite or feaces of an infected insect vector e.g. fleas , lice, ticks.
Chlamydia
• Chlamydia are obligate intracellular bacteria.
• They lack the ability to produce sufficient energy to grow independently
and therefore can grow only inside host cells.
• They have a rigid cell wall but do not have a typical peptidoglycan layer.
• Their cell walls resemble those of gram-negative bacteria but lack
muramic acid.
• Disease caused by Chlamydia are: Urethriritis, cystitis, prostitis, pelvic
inflammatory disease, trichomoniasis, neonatal eye disease.
• Easily transmitted during sexual contact
Chlamydia/ Chlamydophila
• Questions?

• Clarification?
 REFERENCES
• Sherri’s Medical Microbiology 7th edition
• Medscape
• Jawetz, Melnick & Adelberg's Medical Microbiology. New York;
London: McGraw-Hill 24th edition
• W. John Spicer: Clinical Microbiology and Infectious diseases. Churchill
Livingstone Elsevier 2nd edition.
THANK YOU!