Genus Staphylococcus - General Features (Main Species, Habitat, Morphological Features)

SUBJECTS FOR THE FINAL EXAM
SECOND TERM
ADEL ABBAS
1. Genus Staphylococcus – general features (main species, habitat, morphological features)
• Gram positive cocci with grape like arrangement, colonize normal skin, but may be
involved in invasive, suppurative infections or toxinoses of medium or high, life
threatening severity
• 0.5-1.5 micrometers
• Non Motile
• HETEROTROPHIC - grow only on organic substrates
• Non Spore Forming 
• Non Capsulated after 2-3 laboratory passages 
• Non Fastidious – cultivate on common growth media
• Facultative Anaerobes
• Fermentative (ferment glucose)
• Catalase positive
• Halophile – tolerate high concentration of salt (Sodium Chloride)
• Habitat
o Ubiquitous
o Nose vestibule
o Mucosal surface of cavitary organs that communicate with external environment:
colon, vagina
o Skin – in high humidity zones - armpits, perineum, interdigital spaces
• Species
o s. aureus: opportunistic pathogen, commensal in nose
o s. epidermidis: non pathogenic, commensal in nose and skin – opportunistic
§ can cause infection in immunocompromised people or by foreign body
introduction
o s. saprophyticus: UTI in females, sometimes commensal on skin
§ opportunistic pathogen
• 49 species and 25 isolated from humans
2. Genus Staphylococcus – cultural and biochemical features, antibiotic susceptibility

• Aerobes, facultatively anaerobes
• mezophilic (cultivate between 10-42 C), with an optimal at 37 C
• cultivate on usual bacteriological media, forming round, convex, frequently pigmented
colonies, with yellow colour (S. aureus), frequently causing a complete hemolysis (S.
aureus)
• Cultivate on salt mannitol agar – containing 10% Sodium Chloride
o S epidermidis grows on chapman, does not ferment mannitol
o S aureus grows on chapman and ferments mannitol
• Coagulase positive: s aureus
o Enzymes that coagulates oxalated plasma, rest of staphylococci are coagulase
negative
• Thermostable dezoxyribonuclease in s aureus
• Protein A in s aureus wall
• Thermonuclease – method using Hydrochloric acid which produce a clarification of the
medium around the positive colony, dissolving depolymerized nucleic acids
• Thermonuclease – method with BromThymol Blau: modification of spatial position in
the nucleic acid- BTB complex, followed by change of the colour around the positive
colony from purple to rose
• ID using kits
o API 20
o API ID 32 staph
• Rapid test for s aureus
• Antimicrobial susceptibility
o Natural susceptibility to almost all antimicrobials families
o Natural resistant to polymixins (colistin) first generation quinolones (nalidixic
acid)
o Resistance
§ beta-lactamases production – codified by plasmidic genes (inhibited by
-lactamase inhibitors as clavulanic acid (in augmentin),
sulbactam, tazobactam, which inactivate the enzymes
o Methicillin Resistant Staphylococcus Aureus (MRSA) strains – involved in
hospital acquired (nosocomial) infections, usually also resistant to other
antimicrobials in Romania  Methicillin, Oxacillin and Cloxacillin = Penicillins
resistant to penicillinases  
§ MRSA strains spread also in the community, but usually community
acquired S. aureus are more susceptible to other antimicrobials
Antimicrobial susceptibility testing of S. aureus
• For methicillin resistance – screening by using disc diffusion test to cefoxitin + a second
test: PLP2a or PCR for mecA gene  
• D Test – for detecting inducible resistance to MLSb (Macrolides, Lincozamides,
Streptogramines b): Erythromycin and Clyndamycin at a distance of 10 mm:  Erythro R
and flattening of the inhibition zone to Clindamycin = MLSB inducible resistance  
3. Genus Staphylococcus – pathogenicity factors and main diseases produced

• Pathogenicity factors
o S. aureus
§ Proteins and or surface mucopolisaccharides
• Adherence and or promote host colonization
• Inhibit phagocytosis
• Protein A- coagulase, capsule
o Fixes antibodies by their Fc segments
§ Carotenoid pigments
• Survival inside phagocytes
• S. aureus: yellow pigment with polyunsaturated molecules,
reduce toxic oxygen radicals
§ Catalase
• Survival inside activated phagocytes by reducing peroxide
radicals
§ Coagulase
• Use plasma clotting to protect themselves against antibodies,
phagocytes and antimicrobials, binds fibrinogen and clumps
plasma
§ Invasins
• Promote invasion of staphylococci inside host tissues
• Leucocidin and hyaluronidase
§ Cytolytic/cytotoxic toxins
• Hemolysins, leukotoxins, leucocidin
§ Exotoxins
• Staphylococcal enterotoxins, TSST1, epidermolytic toxin
§ Antimicrobial resistance: constitutive or acquired
• Diseases caused by s. aureus
o Suppurative (Pyogenic) inflammation:
§ Localized superficial infections
• Impetigo
o face, arms, legs; mostly in small children
o maculae, pustules, vesicles and crusts on erythematous
background
o may associated with group A streptococcus
• folliculitis: hair follicle infection
• Orgelet/stye: eyelid infection
• Furuncle/boil: sebaceous gland
• Carbuncle: many furuncles and black tissue from dermonecrotic
toxin
o Resembles lesion from anthrax
• Deep face cellulitis: danger of deep venous cavernous sinus
thrombophlebitis
o Give higher dose of oxacillin on first day -> 4g/day then
2 g/day
• Hydrosdenitis: sweat glands – armpits, perineum, genital areas
• Mastitis: mammary gland, 1-3% of breast feeding mothers
• Wound infections: post trauma/ surgical
o Local edema, erythema, pus and pain
§ Septicemia/bacteremia: 50% hospital acquired, after surgery, catheters
§ Deep infections: osteomyelitis, endocarditis, meningitis
• Endocarditis: from bacteremia -> secondary septic dissemination
§ Pneumonia and pulmonary empyema: extreme ages
§ Osteomyelitis and septic arthritis: secondary infection after
dissemination from primary infection or post traumatic; involves long
bone metaphysis in kids
§ Septic arthritis after intra articular injections
§ CNS infections in adults
§ Post antibiotic enterocolitis: fever, diarrhoea, dehydration, after using
antibiotics
o Toxinoses
§ Food borne staphylococcal toxinoses
• In summer more frequently
• Heat can destroy bacteria but not enterotoxins
• Incubation: 1-8 hours, sudden onset, short evolution: 12-24 hours
§ Mixed staph diseases: infection and poisoning
• Staphylococcal scalded skin syndrome
o Newborns
o Perioral erythema, extends to whole body in 2 days
o Vesicles w/clear liquid, without microorganisms or
leukocytes inside
o Intraepidermic cleavage, epithelium peeling, recovery in
10 days
• Toxic shock syndrome
o Children, fever, low BP
o Scarlet like erythema
o May be lethal
o Young women
§ TSST-1 because vaginal secretions, blood, high
cellulose
• Goes into blood stream : stimulate
macrophages; produce interleukin 1,
prostaglandins and TNF-alpha
o Hospital acquired infections
§ Methicillin resistant s.aureus (MRSA)
• Direct contact transmission
• Resistant to beta lactamins
• Susceptible to vancomycin
4. Steps of the laboratory diagnostic of staphylococcal infections

• Sampling:
o pus, CSF, nose/throat exudates, skin biopsies, articular fluid, conjunctival
secretions, sputum etc.
• Transport
o Triple package, rapid
• Direct examination
o Macroscopic: yellow pus
o Bacterioscopical
§ Stains: gram positive, methylene blue, MGG
• Isolation
o Simple nutritive media; 24-48 hours
o Blood supplement for evaluating haemolytic characters
o Non selective media for normally sterile samples – blood csf, articular fluid
§ Nutrient, blood or McConkeys
• Nutrient: colonies: large, circle, convex, smooth, shiny, easily
emulsifiable
• MacConkeys: small/pink colonies
• Blood: most produce beta hemolysis
o Selective media for normally contaminated samples – feces, food
§ Mannitol salt ager, chapman
• Identification
o Clinical purpose
§ Microscopy  
§ Culture aspect  
§ Diagnostic of the Genus: Catalase + (# Streptococcus)  
§ Diagnostic of the species: Coagulase (S. aureus # SCN)
o Epidemiological purpose – differentiation at the sub-species level = clonal or
strain identification: typing - phage typing, molecular typing  
5. Genus Streptococcus – cultural features and classification according to the type of

hemolysis and antigenic structure
• Cultural features
o Cultivate on simple nutritive media with blood enrichment
o On blood agar
§ Show different forms of hemolysis
o Heterotrophic: grow only in presence of organic substances
o Facultatively anaerobes
o Less resistance than staphylococcus to cold, dryness and pH variations
• Classification according to hemolysis
o Beta hemolysis: complete hemolysis
§ Group A, C and G
o Alpha
§ Partial hemolysis
§ Greenish colour – also called viridans hemolysis
§ Str. Pneumonia, str viridans (oral)
o Alpha Prime
§ Partial hemolysis
§ Group D
o Gamma
§ Absence of hemolysis
6. Genus Streptococcus – antigenic structure and classification according to it

• Lancefield groups
o Classification by group specific cell wall polysaccharide antigen
o Groupable
§ A-W except I and J
§ Have c polysaccharide
§ A, B and D are most important
o Group A
§ Major classes: M and T antigens
§ Minor classes; R and F
• Non groupable
o Don’t have c polysaccharide
o Str. Pneumonia
o Str. Mutans
o Str. Viridans – dental caries
7. Genus Streptococcus -pathogenicity factors and main diseases produced

• Pathogenicity Factors
o Cell wall antigens
§ Lipoteichoic acid/ F protein in str pyogenes
• Fimbria
• Attach to epithelium wall
§ M proteins
• Anti-phagocytic
• Involved in natural and adaptive immunity
• Several M type: reinfection is possible with different M strain
§ Capsule
• Antiphagocytic
• Pathogenesis
o Colonize skin and mucosal surfaces
o Invasion and infectious process
o Toxins: erythrogenic pyrogenic
o Structures to avoid immune response
§ Capsule
§ Protein M
• Suppurative infections, toxinoses and poststreptococcal complications
• Group A streptococcus
o Sore throat
o Scarlet fever – acute pharyngitis
o Necrotizing fasciitis
o Post streptococcal diseases
§ Acute rheumatic fever
§ Rheumatic heart disease
§ Acute post streptococcal glomerulonephritis
• Group B streptococcus
o Can colonize female genital tract and infect newborn during delivery: newborn
meningitis
• Streptococcus pneumonia
o Pneumonia, meningitis, recurrent otitis
• Enterococcus
o Wound infections, UTIs , sepsis
o Nosocomial infections
8. Post-streptococcal diseases – enumeration, mechanisms

• Acute rheumatic fever
o 2-4 weeks after throat infection, light arthritis – big articulations, moving arthritis
without sequalae
o involves joints, heart valves, nervous system
o mechanism
§ streptococcal M protein – cross reacts with myosin; autoimmunity
§ cell wall antigens – difficult to be cleared in vivo, persist indefinitely
• Rheumatic heart disease
o After several episodes of ARF
o Heart damage, fibrillation, infection, heart failure, thrombotic accidents
• Acute poststreptoccal glomerulonephritis
o Inflammation of renal glomeruli; edema
o Immune complexes- type III hypersensitivity
9. Steps of the bacteriological diagnostic of streptococcal infections

• Sampling and transport
o samples: nose and pharynx exudate
§ wound secretion
§ blood – in streptococcus viridans endocarditis, pneumonia, sepsis
§ CSF: in newborn meningitis or Str. Pneumonia meningitis
§ Sample before antimicrobial therapy
o Gram positive cocci grouped in chains
§ 0.6-1 micrometer
• Cultivation of Samples
o Cultivate on simple nutritive media with blood enrichment
o Heterotrophic: grow only in presence of organic substances
o Less resistant than staphylococcus to cold, dryness, and pH variation
• Isolation and identification
o Beta haemolytic colonies
§ Complete hemolysis is caused by streptolysin O
§ Larger colonies and more transparent than staphylococcus
o Because they’re oxygen sensitive – inoculate by introducing loop inside the medium
– helps to protect haemolytic activity from oxygen
o Catalase negative – no bubbles
o Oxidase negative
o Bacitracin test
§ Inhibition of growth : streptococcus group A
o Serologic ID using group specific sera
o Commercial agglutination test
§ Group A agglutinates
o Rapid strep Test
o Indirect diagnostic – serological
§ Groupable streptococci – ABD most important
§ Non groupable: str pneumoniae, mutans and viridans
§ Antistreptolysin O (ASO)
• Normal is under 200-250 UI
§ Beta hemolysis
• Complete
• A, C, G
§ Alpha hemolysis
• Partial – greenish colour – also called Viridans hemolysis
• Str. Pneumoniae, and viridans
§ Alpha prime hemolysis
• Partial
• Group D
§ Gamma
• No hemolysis
o Group B streptococcus
§ Alpha prime hemolysis
§ Hippurate test positive
§ CAMP test: incomplete group B streptococcus hemolysis and incomplete
hemolysis of s.aureus = complete hemolysis
o Group D streptococcus
§ Grows on agar with bile and esculin – black aspect
• Enterococcus grows too
§ On medium with 6.5% salt
• Group D doesn’t grow but enterococci grow
o Streptococcus with viridans hemolysis
§ Oral species
• Negative for optochin and other tests
§ Str pneumonia
• Shoes viridans hemolysis and is POSITIVE for other tests
o AST
§ For penicillin allergy: test macrolides and cephalospirins
§ Pneumococci usually susceptible to penicillin, macrolides
• Penicillin resistant strains – test caphalosporins
• Cephalosporin resistant strains- give Vancomycin
§ All group A are susceptible to penicillin
10. Serological diagnostic of streptococcal infections – ASO method(principle, interpretation,

use)
Anti Streptolysin O (ASO)
• Important for monitoring of strep A elimination and poststreptococcal diseases

• Normal values: under 200 – 250 International Units (IU) (for Romania)
• Principle : ASO reaction is a seroneutralization reaction, based on the capacity of anti- ASO
antibodies found in the patient serum to neutralize the haemolytic activity of ASO
Working protocol :
- Treat patient serum for inactivating non-specific inhibitors of reaction:
0,2 ml patient serum + 0,7 ml rivanol 0,4% ® agitation, 10 min. at room temperature;
1.1 ml isotonic saline solution (ISS) are then added
- Centrifugation ® the supernatant represents the 1 / 10 dilution of the patient serum
- Prepare the 1 / 100 dilution : 0,5 ml 1/10 dilution + 4,5 ml ISS
- Prepare 1 / 500 dilution: 1 ml 1/100 dilution + 4 ml ISS
- Distribute 1/100 and 1/500 dilutions in tubes and dilute with isotonic saline, according
to the provided table
- Add SLO and keep the tubes at 370 C for 15 minutes, with agitation
- Add rabbit red blood cells and keep at 370 C for 45 minutes, with agitation
- Keep at +40C over night (ON)
- Read and interpret the results: the titer is the higher dilution of serum which totally
inhibited the haemolytic activity of SLO (no hemolysis)
•
11. Streptococcus pneumoniae – general features, habitat, morphology and cultural features
• General features
o Oxidase and catalase negative
• Habitat
o Nose and pharynx
o Rate is increased in cold seasons
• Morphology
o Gram positive, 1 micrometer
o Slightly elongated cocci, with one end broad and other pointed
o Occur in pairs with broad ends opposing each other
o Capsule encloses each pair
o Non motile and non sporing
• Cultural feature
o Grow only in enriched media
o Aerobes and facultative anaerobes
o Optimum temperature: 37 degrees Celsius
o pH 7.8
o growth is improved by 5-10% CO2
o blood agar
§ after 18 hours the colonies are small, dome shaped and glistening, with an
area of alpha hemolysis
§ after further incubation the colonies become flat with raised edges and
central umbonation ->draughtsman or carrom coin appearance
12. Streptococcus pneumoniae – biochemical features and antigenic structure

• Biochemical features
o Catalase negative
o Bile solubility test positive
o Ferments inulin
• Antigenic structure
o Has no group specific antigen
o Capsular polysaccharide:
§ It is the most important antigen & type specific.  
§ since it diffuses into infective tissue & culture medium it is called as
specific soluble substance(SSS).  
§ Pneumococci are classified into types based on the nature of capsular
polysaccharide & more than 90 serotypes are recognized & named
1,2,3....  
o M protein: It is not associated with virulence.
o ‘C’ Carbohydrate antigen:
§ It is present in all pneumococci so species specific.
§ an abnormal protein(β-globulin) that precipitates with ‘C’ carbohydrate
antigen of pneumococci, appears in the acute phase sera of cases of
pneumonia but disappears during convalescence. It also detected in sera
of patients with some other illness. This is known as the C-Reactive
Protein(CRP). It is an ‘acute phase’ substance, produced in hepatocytes.
Its production is stimulated by bacterial infections, inflammation,
malignancies & tissue destruction.
13. Streptococcus pneumoniae –pathogenicity factors and main diseases produced

o Capsule: antiphagocytic
o Pneumolysin
§ Membrane damaging toxin with cytotoxic and complement activating
properties
• Main diseases produced
o Otitis media
o Pneumonia
§ Lobar pneumonia
§ Bronchopneumonia
o Tracheobronchitis
o Meningitis
o Other infections: empyema, pericarditis, conjunctivitis, suppurative arthritis,
peritonitis
14. Steps of the laboratory diagnostic of pneumococcal infections
• Sampling
o Sputum, CSF, blood, synovial fluid , in children can take laryngeal swab if
sputum cannot be collected
o Direct microscopy
§ Gram stained smears reveal gram positive lanceolate shaped diplococcic
with numerous pus cells
o Antigen detection
§ Capsular polysaccharide antigen in blood csf and urine detected by
• Passive agglutination
• Counter immunoelectrophoresis
• coagulation
o Quelung reaction
§ On a slide the sputum is mixed with type specific antiserum against
capsular antigen & a loopful of methylene blue solution. The capsule
becomes swollen & refractile.  
o Optochin positive
§ Differentiate from oral species of streptococcus
o Viridans hemolysis
o Lysed by bile
o Inulin positive
o AST
§ Pneumococci are usually susceptible to penicillin, macrolides
• Penicillin resistant strains: test cephalosporins
• Cephalosporin resistant: give vancomycin
15. Neisseria meningitidis – general features (habitat, morphology, cultural features, antigenic
structure)
• Gram negative cocci: bean or kidney shaped
• 0.6-1 micrometers – in pairs (diplo)
• oxidase positive
• catalase positive
• fastidious
o sensitive to temperature variations – optimal is 37 degrees Celsius
o need special media: chocolate agar
§ Thayer martin transparent colonies
• Resist decolaration with alcohol
• Non sporulated, non mobile
• Strictly aerobic, carboxylic, relatively reduced sacchorolytic spectrum
• Must be kept at 37 degrees: mezophilic
• Heterotrophic metabolic type
• Habitat
o In healthy carriers
o 5-30% carriage rate in general population – in nose and throat
o carriage rate in close communities can be up to 80% during an epidemic
o Fimbria – adhere to mucosal epithelia and serous surfaces
o Capsule
§ Inhibits phagocytosis
§ 12 groups: B, C, A, Y 95%
o outer membrane proteins
§ types Pclass 2,4 subtypes P1
o LPS antigens
§ Lipid A and oligosaccharides core
§ Hyper produced during multiplication
§ Intravascular disseminated coagulation syndrome: IVDC Waterhouse
Frederickson
o IgA proteases
§ Destroy local defence factors
16. Steps of the laboratory diagnostic of meningococcal infections

o CSF, blood, nose and throat exudate, skin lesion biopsy, articular fluid,
conjunctival secretions, sputum etc.
o Transport
§ 37 degrees, do not refrigerate
§ for samples other than blood: direct inoculation in biphasic medium or
on plates with suitable enriched medium and transport in CO2 enriched
atmosphere
§ Amies or Stuart transport medium
§ WHO trans isolate medium
• Solid phase is black -> charcoal; takes out toxic substances and
absorbs inhibitors of bacteria
o Bacterioscopic
§ Tiny dots
§ Stains: gram, methylene blue, MGG
o rapid test: latex or coagglutination, immune electrophorese
o Isolate
§ Special media, 24-48 hours, 37 degrees
• Most atmosphere with 5-10% CO2
• Selective with antimicrobial supplements: for contaminated
• Non-selective: for blood CSF, Etc.
o Chocolate agar – cant see hemolysis, flat
o Oxidase positive
o Kovas oxidase positive : purple
o Saccharolytic spectrum (biochemical)
§ Glucose and maltose positive: yellow
o Serotyping and molecular typing
o Antimicrobial resistance:
§ Penicillin resistance is rare
17. Neisseria gonorrhoeae – general features (habitat, morphology, cultural features, antigenic
structure)
• Gram negative cocci: bean/kidney shaped
• 0.6-1 micrometers in pairs/diplo
• oxidase positive
• catalase positive
• fastidious
o sensitive to temperature variations: optimal is 37 degrees
o need special media - chocolate agar
• Habitat
o In urogenital tract
§ Via fimbria
o Rectal carriage in healthy individuals
o Typically seen in pus cells
o Found intracellularly in polymorphonuclear leukocytes
o Only in humans
o Can also be in mucous and serous tissues: conjunctiva, vagina, urethra, rectum
o Heterotrophic; cultivate on nutritive media
o Thayer martin chocolate agar
§ S colonies, greyish colour
o Fimbria: proteic adhesion
o Capsule
o LPS antigen
o Iron chelator: ensure multiplication in local sideropenia conditions
18. Steps of the laboratory diagnostic of gonococcal infections

o Endocervival secretion, urethral secretion, anal swab, pharyngeal swab,
conjunctival swab, blood, articular fluid
o Transport
§ 37 degrees, do not refrigerate
§ for samples other than blood: direct inoculation in biphasic medium or
on plates with suitable enriched medium and transport in CO2 enriched
atmosphere
§ amies or stuart transport medium
§ WHO trans isolate medium
• Solid phase is black -> charcoal; takes out toxic substances and
absorbs inhibitors of bacteria
o Bacterioscopic
§ Tiny dots
§ Stains: gram, methylene blue, MGG
o rapid tests: latex or coagglutination, immune electrophorese
o Isolate
§ Special media, 24-48 hours, 37 degrees
• Most atmosphere with 5-10% CO2
• Selective with antimicrobial supplements: for contaminated
• Non selective: for blood CSF, Etc.
o Thayer martin agar
o Oxidase positive
o Kovas oxidase positive : purple
o Saccharolytic spectrum (biochemical)
§ Glucose positive and maltose negative
o Serotyping and molecular typing
o Antimicrobial resistance:
§ Penicillin resistance is frequent
o Serological diagnostic search for specific antibody by CFR
§ In chronic gonococcal infections
19. Microscopical features of medical important cocci

o
20. General features of Enterobacteriaceae(main genera / species, habitat, morphology, cultural

and biochemical features)
• Glucose fermenters
• Gram negative rods
• Cytochrome oxidase negative
• Oxidase negative
• Catalase positive
• Reduce nitrates to nitrites
• Ubiquitous, spreading from humans or animals intestine; rarely found in other anatomical
zones, may be involved in extra-intestinal infections
• Non motile or motile by peritrichous flagella
• 7 tribes, 20 genera and over 100 species
o tribes
§ escheriaschiae
§ proteae
§ yersineae
§ erwineae
§ edwardsiellae
§ salmonellae
§ klebsiellae
• pathology
o invasive infections
§ recognized pathogens
• salmonella, shigella, Yersinia
§ opportunistic pathogens
• e. coli, klebisiella, enterobacter, proteus
o toxinoses
§ endotoxins: all
§ exotoxins: e. coli, salmonella, shigella dysenteriae, yersenia enterocolitica
• site of infections
o enterocolitis, UTI, abscesses, pneumonia, meningitis, wound infections, septicaemia
21. Antigenic structure of Enterobacteriaceae (types of antigens, chemical structure,

importance for diagnostic, examples)
o In salmonellae
o Somatic antigen O : lipopolysaccharide or endotoxin
§ Thermostabile – resists two hours of boiling
§ Immunogenic: determines production of antibodies
• Mostly IgM
§ Produces stabile agglutination in fine clusters, occurring late
o Antigen H: flagellar
§ Protein thermolabile immunogenic
§ Determines production of antibodies mainly IgG
o Vi surface antigen
§ In salmonella typhi
o O and H are used for serological identification
o Capsulated enterobacteriaecae (Klebsiella and some strains of e.coli)
o Antigen K : capsule
22. Genus Escherichia – main features (main species, habitat, morphology, cultural and
biochemical features)
• Main species
o Diarrheigenic e coli
§ Enteropathogenic e coli (EPEK)
§ Enterotoxic e coli (ETEK)
§ Entero invasive e coli (EIEK)
§ Enterohemorrhagic e coli (EHEK)
o Uropathogenic E. coli
§ Type 1
§ P
o Extraintestinal infections
§ K1- in newborn meningitis
• Habitat
o Urogenital tract. Intestinal tract, respiratory tract
o Surgical wound infections
o Post traumatic infections
o Can be found in CSF in newborn meningitis
• Morphology
o Gram negative rods
• Cultural and biochemical features
o Cytochrome oxidase negative
o Reduce nitrates to nitrites
o Produce gas
o Ferment glucose
o Usually mobile
§ There are some shiga-like immobile strains
o Indole positive
o Urease negative
o Doesn’t produce H2S
o Culture
§ Blood agar
• Grey colonies, bit mucous, sometimes hemolysis
§ MacConkey
• Lactose positive: pink/red
§ ABTL
§ Aerobic, facultatively anaerobic
§ Smooth colonies; rough when they’re capsulated strains
• opportunistic
23. Escherichia coli pathotypes involved in intestinal infections (enumeration, main features)
• Enteropathogenic e.coli
o Serotypes with diarrhoea in children
o Adherence to epithelium and destruction of microvilli without invasion
o Fever, diarrhoea, vomiting, nausea, usually without blood in stool
• Enterotoxigenic e.coli
o Cholera like diarrhoea but much milder
o Travellers diarrhoea
o Thermolabile toxin
§ Choleragen-like, activates adenylate cyclase, AMPc synthesis
§ Affects water and ion secretion
o Thermostable toxin
§ Activates guanylate cycles, GMPc synthesis, affects water and ions
reabsorption
• Entero invasive e.coli
o Dysentery
§ Fresh blood in small quantity of stool
§ Similarities with shigellosis
• Enterohaemorrhagic e.coli
o Usually e.coli 0157:H7
o Has flagella
o Ingestion of under cooked meat
§ Haemorrhagic stool
• Large quantity of blood
• Few leukocytes
• Absence of fever
§ Toxins
• Vero toxins
• Hemolysins
24. Pathogenicity features of Enterobacteriaceae

o In e.coli
o Capsule
o Endotoxin
o Exotoxins
o Klebsiella
o Capsule
o Adhesins
o Aerobactin: iron binding protein
o Endotoxin
o ESBL
o Carbapenemases
o Proteus
o Endotoxin
o Motility
o Plasticity
o Adhesins
o Antimicrobial resistance
o Salmonellae
o Somatic antigens
§ Immunogenic
o Capsular antigens
§ Antiphagocytic
o Flagellar antigens
o Antibiotic resistance
o Exotoxins
o Enzymes
o Shigella
o Capacity to invade and produce verotoxin
o Shiga toxin
o Yersinia
o Capacity to adhere and invade
o Produce exotoxin
o Complex antigenic structure
25. Genus Escherichia – pathogenicity features, main diseases in which it is involved

o Pathogenicity features
o Diseases
o Diarrhoea in children: enteropathogenic e.coli
o Travellers diarrhoea/cholera like but much milder: enterotoxic diarrhoea
o Dysentery: enteroinvasive e.coli
o Hemolytic uremic syndrome: enterohemorrhagic e.coli
o Haemorrhagic stool: enterohemorrhagic e.coli
o UTI: uropathogenic strains
§ Can travel from contaminated perineum or from permeable inflamed
intestine
o Extra intestinal infections
§ Newborn meningitis: e. coli K1
§ Bacteremia, septicaemia
§ Respiratory tract infections
§ Surgical wound infections
§ Post traumatic infections
26. Steps of the laboratory diagnostic of Escherichia coli infections

• Sample
o Urine, blood, stool, csf, surgical wounds, etc.
o Cultivation
§ Blood agar
• Grey colonies, bit mucous. Sometimes hemolysis
§ MacConkey
• Lactose positive: pink red
§ ABTL
§ Lactose positive on differential media
o Biochemical tests
§ TSI, MILPH, urea
• Lactose positive
• Doesn’t produce hydrogen
• Indole positive
• Mobile (rarely immobile)
• Urease negative
§ Expanded biochemical kit : API E
o Serological ID using antigen O and H
o AST
§ Growing beta lactamin resistance
o Pulsed field gel electrophoresis for molecular typing
27. Steps of the laboratory diagnostic of Escherichia coli urinary tract infections: quantitative
urine culture and interpretation
o Urine culture
o Dilutions method
§ Make 3 dilutions of urine: 1/10, 1/100 and 1/1000
§ By using a calibrated pipette, inoculate 100 microliters of each dilution on a
different Petri dish, spreading the inoculum on the appropriate
bacteriological medium: Brom Thymol Blue Agar for Enterobacteria, blood
agar for Gram positive cocci etc.
§ incubate ON (over night) at 35-370 C.
§ Enumerate colonies grown on the appropriate dilution that lead to obtain
isolated colonies and calculate the number of colonies/mL.
§ Use the formula: CFU/mL urine = n x DF x VF
• Where CFU/mL urine = Colony Forming Units/mL urine (number of
bacteria/mL urine)
• n = numbered colonies on the plate
• DF = dilution factor = reverse of dilution (e.g. for 1/100 dilution, DF
= 100)
• VF = 1 mL/inoculated volume (e.g. if the inoculated volume was
100 microliters, VF = 1000 microliters/100 microliters = 10)
o Calibrated loop method

o Inoculate non diluted urine by using calibrated disposable loops of 1, 5 or 10
microliters
o Incubate ON (over night) at 35-370 C.
o Enumerate colonies grown on the plate you find isolated colonies and
calculate the number of colonies/mL.
o Use the formula: CFU/mL urine = n x VF
o Where CFU/mL urine = Colony Forming Units/mL urine (number of
bacteria/mL urine)
§ n = numbered colonies on the plate
§ Interpretation:
o Number of colonies usually considered significant for urinary tract infection
(UTI) is 105 CFU/mL
o However, if there are clinical symptoms of UTI and/or leukocytes are
present, the significant number may be lower.
o If two bacterial species have been isolated, repeat the test recommending
strict local hygiene procedures. If you obtained the same result, report both
bacteria.
o If three bacterial species have been isolated, consider contamination and
repeat recommending strict local hygiene procedures.
o If you obtain ≤ 103 or no growth report negative urine culture, except
Listeria, Salmonella, Leptospira, and/or other bacteria in the same category
o If UTI is confirmed (or when needed pending on the number of CFU/mL) the diagnostic
continues with:
o Identification of isolated bacteria:
o Macroscopic morphology - aspect of growth (Smooth colonies, non coloured or
coloured, transparent or semitransparent on Brom Thymol Blue medium etc.)
o Microscopic morphology – e.g. gram negative rods
o Biochemical identification (TSI = Triple Sugar Iron), MILPH (Motility, Indole,
Lysine, Phenylalanine), Urea, Simmons citrate
o Serological identification: slide agglutination – see provided materials
o Antimicrobial Susceptibility Testing
o Clonal identification: biochemotyping, antibiotyping, phagetyping, molecular
tests
28. Genus Salmonella – general features (main species, habitat, morphology, cultural and
o Species
o Pathogenic for humans
§ S. typhi
§ S. paratyphi A, B and C
o Salmonellae adapted to animals
§ S cholera suis: in pigs, may colonize and give rise to illness in humans
§ Dublin: cattle4
§ Galinarum pullorum : chickens
§ Abortus equi: horses
§ Abortus ovis: sheep
o Habitat
o Ubiquitous
o Vertebrates, flies, cockroaches fleas. Ticks, etc.
o Morphology
o Various length, most species are motile
o Cultural and biochemical features
o Non fastidious, growing on usual culture media
o Selective differential media: Lactose negative colonies
o Glucose fermentative with gas production
§ (s. typhi doesn’t produce gas)
o Lactose negative
o Majority are mobile
o Oxidase negative
o Produce hydrogen sulphate through metabolism of amino acids and sulphur
o Indole negative
o Use citrate as a carbon source (except s. typhi)
o Urease negative
o Reduce nitrates to nitrites (Voges-Proskauer positive)
29. Genus Salmonella – antigenic structure

• Somatic antigens (o)
o Polysaccharides in the cell wall
o Thermostable (resists 2 hours of boiling)
o Immunogenic: determines production of antibodies (mostly IgM)
o Produces stable agglutination in fine clusters, occurring late
§ 1-3 per slide, 18-24 hours in tube at 52 degrees Celsius
• capsular antigens
o polysaccharide or proteic antigens
o in salmonella typhi: Vi antigens
• flagellar antigens (H)
o proteic, thermolabile, immunogenic- determines productions of antibodies
(mostly IgG)
o produce less stable agglutinates, occurring early:
§ immediately on slide
§ in 2 hours in tube at 52 degrees Celsius
• classification based on antigenic structures
o Kauffman white scheme
§ Serogroups: based on O antigen
§ Serotypes : based on H antigen
30. Genus Salmonella – pathogenicity features, main diseases in which it is involved

o Pathogenicity factors
o Polysaccharide antigens: responsible to toxic shock
o Capsule: antiphagocytosis
o resistance to antibiotics
o exotoxins
o enzymes
o digestive entry
o Number sufficiently high to survive after passing through the stomach: infecting dose is
10^6-10^9
o For s typhi: under 10^3-10^2
o Survival in the stomach is possible after antacids and gastric resections
o Small intestine colonization
o Adhesion to ileal mucosal surface microvilli, to receptors containing mannose (by
adhesins)
o Attachment-destruction of microvilli : mucosa breaks and bacteria invade cells
o Multiplication in cells and macrophages of peyers patches
o Submucosa – lymph nodes
o Bacteremia
o Secondary colonization
o Liver, spleen, kidney, bone marrow, lymphoid intestinal tissue: multiplication,
bacteremia: resume cycle
§ About 2 weeks until bacteremia: incubation
o Reservoir with intermittent release: gallbladder
o Typhoid fever
o Systemic disease
o Infected mononuclear cells aggregate and form basic lesions: typhoid nodule-> in
peyers patches, lymph nodes, mesenteric nodes, liver, spleen, bone marrow
o Obstruction of small vessels: necrosis, ulcerations, massive bleeding, lenticular
spots on skin
o Intestinal perforation: from peyers patches ->peritonitis-> death
o Main diseases
o Salmonellosis
§ Enteric fever = typhoid fever
§ From bacterial invasion of blood
§ 1-2 weeks after incubation
§ onset: gradual increasing body temperature, tiredness, muscle aches,
insomnia, anorexia, gradual enlargement of spleen
§ typhic state: apathy, indifference, intense headache, sometimes rash –
lenticular spots
§ variable intestinal transit
§ hepato/spleno megaly, bradycardia, leukopenia, lymphocytosis
o Acute gastroenteritis
§ Food borne disease/toxinoses
§ 24-48 hours after ingestion of contaminated food/liquids
§ watery diarrhoea, headache, malaise, nausea, vomiting
§ symptoms caused by toxin resembling cholera toxin
o septicaemia
§ complication in newborn/small children: meningitis
§ abscesses in organs
31. Steps of the laboratory diagnostic of Salmonella infections – general scheme
• Sampling
o Typhoid fever
§ Blood cultures in 1st and 2nd week of illness, +/- bone marrow cultures
§ Urine cultures in 2nd week
§ Blood for widal reaction
• 2 probes at 7-10 days in the 2nd and rid weeks of the illness
interval
o gastroenteritis
§ stool cultures starting in 1st week
o septicemia
§ repeated blood cultures
o carriers
§ sampling of bile by duodenal intubation or T-tube drainage
§ blood for Vi seroreaction
• direct examination
o gram negative rods
• isolation and identification
o blood culture: media for anaerobic bacteria work better
o urine and stool
§ enrichment media: selenite of tetrathionate broth
§ selective or differential media
• ADCL: favors salmonella and allows differentiation from other
enterobacteria
o Black in center because of citrate
o Biochemical
§ Ferments glucose with gas production (except s typhi)
§ Lactose negative
§ produce hydrogen sulphate
§ indole negative
§ use citrate as unique carbon source (except s typhi)
§ urease negative
§ reduce nitrates to nitrites
o Serological diagnosis
§ Widal reaction or sera qualitative analysis
• For antibodies against antigen O and H, as well as anti Vi
• Dynamics of serum antibodies in typhoid or paratyphoid fever
• Antibodies start to appear in 2nd week and rise progressively
• Acute infection: o titers equal to or greater than 1/250 and riding
• Past infection or vaccination: H titres greater or equal to 1/2500
• Vi titers greater or equal to 1/40 in carriers of s.typhi
32. Bacteriological diagnostic of typhoid fever
o Sample
o Blood cultures: 1st and 2nd week of illness: +/- bone marrow cultures
o Urine culture in 2nd week
o Stool cultures in 2nd and 3rd weeks
o Direct examination
§ Flagella, capsule, endotoxin, pili
o Variable length, most species are motile
o Cultivation of samples
o blood culture: media for anaerobic bacteria work better
o urine and stool
§ enrichment media: selenite of tetrathionate broth
§ selective or differential media
• ADCL: favors salmonella and allows differentiation from other
enterobacteria
o Black in center because of citrate
o Non fastidious, grow on usual culture media
o Selective differential media: lactose negative colonies
o Identification
o Biochemical testing
§ Ferments glucose with gas production (except s typhi)
§ Lactose negative
§ produce hydrogen sulphate
§ indole negative
§ use citrate as unique carbon source (except s typhi)
§ urease negative
§ reduce nitrates to nitrites
o Antimicrobial susceptibility testing
33. Serological diagnostic of typhoid fever

Salmonella spp. bacterial cell possess two types of antigens:
• Somatic Ag (O antigens, group antigens):
For example: TO = S. typhi; AO = S. paratyphi A; BO = S. paratyphi B
• Flaggelar Ag (H antigens, type antigens):
For example: d = S. typhi; a = S. paratyphi A; b = S. paratyphi B
Serological identification steps:
1. Firstly put in contact on a glass slide Salmonella strain culture isolated from patient blood culture with
anti-O multivalent serum. Multivalent serum is a mixture of several monovalent specific antibodies
against several O antigens (group antigens)
If there is a positive reaction (appearance of agglutination on the slide) check the non-specific
agglutination capacity of the Salmonella bacterial culture by putting in contact the bacterial culture with
isotonic saline solution (ISS). You may encounter the following situations:
a. If there is agglutination with ISS, there is strain autoagglutinability and the serological identification
can not be interpreted.
b. If there is not agglutination with ISS, proceed to the second step.
2. Secondly put in contact on a glass slide Salmonella strain culture isolated from patient blood culture
with each anti-O monovalent antiserum.
The group of the Salmonella strain isolated from the patient will be indicated by the monovalent serum
that will agglutinate with the bacterial culture.
Serological identification principles:

1. There is an agglutination reaction based on the antigen-antibody reaction between specific sera
prepared in the laboratory and Salmonella bacterial cells antigens.
2. It is an agglutination reaction because the antigen is of corpuscular nature (a bacterial cell).
II. About bloodculture
Bloodculture (BC) is a medical emergency:
Sampling: asepsia; before antimicrobial treatment starts; sampling/inoculating systems
directly near the patient bed
When to sample?
Two-three times in 24 h:
- Acute endocarditis: 3 BC from 3 different venous puncture sites in 24 h
- Subacute endocarditis: minimum 2 BC from 2 different venous puncture sites
- Septicemia – 3 BC from 3 different venous puncture sites, in 10 minutes
- Unknown origin fever: 2-3 BC, from different venous puncture sites, at 45-60 min
interval
- Salmonellosis: enteric fevers (typhoid in case of S. typhi), with invasion of blood; Blood
cultures in week 1 (w1) and 2 (w2) of disease
Ideally, sampling to be done within 30 min before fever pick

How much to sample?
20 ml blood in adults; 1-5 ml blood in new born, baby and little child (concentration of
bacteria is higher)
Microorganisms frequently isolated:
Staphylococcus aureus, enterobacteria (E. coli, Klebsiella), enterococci, anaerobes, less
frequently Pseudomonas aeruginosa; bacteria involved in infectious diseases with typical
Neisseria meningitidis, Brucella spp.; fungi etc.
Salmonella typhi and Salmonella paratyphi A and B may be isolated from bloodculture in
enteric fevers with invasion of blood, in week 1 and week 2 from disease onset.
If you suppose inhibitors of bacterial growth being present in the bloodstream (e.g.
antimicrobials) you may add different antagonists of these inhibitors as polianetolsulphate,
activated charcoal etc.
Incubation and media for presumed microorganisms, taking into account the clinical and
epidemiological data:
Examination three times/day in the first 3 days, then daily, for at least 7 days (may be 14-to
21 days according to the supposed pathogen).
Targeted elements: turbidity, gas bubbles, pellicle, deposit under the erythrocytes layer from
the bottom of the bottle, colonies on the biphasic medium etc.
Blind passages (even in the absence of the bloodculture bottle modifications)
Next steps: Identification, antimicrobial susceptibility testing (AST).
Automated BC equipment – BacT-Alert, BACTEC etc. – hundreds of readings; sensors; light
or audio signal if positive
Quality control of media: sterility, biological efficiency (using reference strains)
Interpreting: correlated with clinical aspects; in order to separate real positives from
contaminations during sampling
Results communicated as fast as obtained, in steps: smear results, turbidity, identification,
AST results etc.
34. Genus Shigella – general features (habitat, morphology, cultural and biochemical features)
• Habitat
o Enterobacteria found in the intestinal tract of human and primates
• 4 groups: flexneri, dysenterae, boydi, and sonnei
• morphology
o gram negative rods, sometimes coccobacilli
o non capsulated and non motile
• cultural features
o aerobic, facultatively anaerobic
o convex or slightly flattened colonies, smooth surface, regular edges,
approximately 2mm diameter at 24 hours
o non motile
• biochemical
o ferments glucose without gas
o s. dysenteriae don’t ferment mannitol but other species do
o lactose negative
o TSI: G+, L-, Z-
o H2S –
o MIU: U-, I variable , M-
o Urea: - 
o Simmons: -
35. Antigenic structure and classification of Shigella

• Over 40 serotypes, depending on polysaccharide types
• Rapidly inactivated by heat
• Resist in dust for 10 days, in water for 1-16 days
• S.dysenteriae (13 serotypes), mannitol negative: group A
• B. S.flexneri (6 serotypes) group B
• S.boydii (18 serotypes) Group C
• S. sonnei (1 serotype)  group D
• The genus Shigella is comprised of four species:
36. Genus Shigella – pathogenicity features, main diseases produced
• Digestive entry, limited generally to intestinal tract: rarely bacteremia/septicemia
• Infecting dose <10^3 CFU
• Resist acid pH
• Reach terminal ileum and intestine
• Phases
o Multiplication in intestinal lumen
o Adhesion to polypeptidic adhesion to epithelial cell receptors
o Penetrate by endocytosis
o Intracellular multiplication
o Cell distribution and propagation by near by, followed by necrosis, ulceration
and bleeding, stools with fibrin, leukocytes, bacteria
• Double action
o Initially exotoxin then bacterial invasion with necrotic bleeding lesions
• Severe cases with haemolytic uremic syndrome: haemolytic anemia, thrombocytopenia,
YVDC
• Non protective antibodies in serum
• Protective action: IgA protective antibodies
• Shiga toxin
o Codified in chromosome
o Neurotoxic
o Enterotoxic – causes diarrhoea
o Cytotoxic
o Toxin inhibits protein synthesis – acts on 60s ribosome unit and lyses 28s rRNA
• Diseases
o Bacillary dysentery
§ Diarrhoea with small quantities of fresh blood because bacteria acts on
lower GI tract
37. Laboratory diagnostic in bacterial dysentery caused by Shigella

• Samples
o Stool: zones with mucous
o Blood: 1st sampling 10 days after onset
o Leukocytes and erythrocytes
• Cultivation
o ADCL
• Identification
o Cultural and microscopic aspect
o Multitest biochemical test
o Serological ID
• Serological identification of a bacterial strain isolated from a stool culture in a patient
with dysentery syndrome
o I. Dysentery syndrome definition and bacterial etiology
§ 1. Dysentery syndrome is characterized by the presence of small
quantities of fresh blood in stool.
• II. Shigella spp. antigenic characters
• The genus Shigella is comprised of four species:
o Shigella dysenteriae (also referred to Group A),
o Shigella flexneri (also referred to Group B),
o Shigella boydii (also referred to Group C),
o Shigella sonnei (also referred to Group D).
• Among these organisms, there is the most frequent etiologic agent of epidemic dysentery,
S. dysenteriae serotype 1.
• With the exception of S. sonnei, each species may be further divided into serotypes on
the basis of reactivity with hyperimmune serum:
o S. dysenteriae (15 serotypes),
o S. flexneri (6 serotypes and 2 variants),
o S. boydii (20 serotypes).
• Shigella group and serotype are identified by slide agglutination using anti sera.
• Both polyvalent and monovalent Shigella antisera are available.
• Polyvalent antisera are used to determine the group and contain antibodies to multiple
serotypes of Shigella. For example, most commercially available polyvalent antisera for
Shigella flexneri will react with all serotypes of S. flexneri
• Monovalent antisera are used to determine serotype. For example, S. dysenteriae Type 1
antiserum will only react with isolates of S. dysenteriae Type 1.
• III. Serological identification steps:
o Firstly put in contact on a glass slide Shigella spp. strain culture isolated from
patient stool with Shigella polyvalent sera. Polyvalent sera are used to identify
the species or the group of Shigella (i.e. S. dysenteriae – Group A, S. boydii –
Group B, S. flexneri - Group C, or S. sonnei – Group D).
o If there is a positive reaction with polyvalent sera (appearance of agglutination on
the slide) check the non-specific agglutination capacity of the Shigella bacterial
culture by putting into contact the bacterial culture with isotonic saline solution
(ISS). You may encounter the following situations:
§ If there is agglutination with ISS, there is strain autoagglutinability and
the serological identification can not be interpreted.
§ If there is not agglutination with ISS, proceed to the second step.
o Secondly put in contact on a glass slide Shigella strain culture isolated from
patient stool with individual monospecific sera found in the positive polyvalent
antiserum.
o The type of the Shigella strain isolated from the patient will be indicated by the
monospecific serum that will agglutinate with the bacterial culture.
• IV. Serological identification principles:
o There is an agglutination reaction based on the antigen-antibody reaction
between specific sera prepared in the laboratory and Shigella bacterial cells
antigens.
o It is an agglutination reaction because the antigen is of corpuscular nature (a
bacterial cell)
38. Genus Klebsiella – general features(main species, habitat, morphology, cultural and
biochemical features, antigenic structure)
• Main species
o Frequently isolated: K. pneumonia, K. oxytoca, K. ozenae, K, rhinoscleromatis
• Habitat
o Can be found in normal flora of upper respiratory tract and intestinal tract
• Morphology
o Gram negative rod
o Very well represented capsule – honey drop/mucous aspect on non selective
media
§ 77 capsular types
o Glucose fermenter, oxidase negative
o Reduces nitrates to nitrites: Voges-Proskauer positive (k oxytoca)
o Lactose positive on MacConkey: pink/red
o Non motile
o K.pneumoniae: indole negative (K. oxytoca isn’t)
§ Doesn’t produce Hydrogen sulphide
§ Produces gas in large quantities (also oxytoca)
§ Urease positive (also oxytoca)
§ Malonate positive (not ozenae)
§ Lysine decarboxylate positive (also oxytoca)
§ Phenylalanine deaminase negative
o TSI: G+, L+, Z+
o SIM: H2S-, indole variable, M-
o Simmons citrate positive
39. Genus Klebsiella – pathogenicity features, main diseases produced

• Capsule
• Adhesins
• Aerobactin: non binding protein
• Endotoxin
• Antimicrobial resistance
o ESBL: extended spectrum beta lactamases: hydrolyze all beta lactamases,
including cephalosporins
o Carbapenemases:
o Colistin resistance emerged in the last years
• Opportunistic pathogen
o Upper respiratory tract and intestinal tract
o Pneumonia
§ Septicemia, meningitis
§ Enteritis
§ Wound infections
§ UTI
§ Infections in burns
o Autoimmune diseases: ankylosing spondilitis
40. Laboratory diagnostic in Klebsiella pneumoniae infection

• Sample: sputum, blood, stool, CSF, urine
o Lactose positive on MacConkey
o Biochemical tests
§ TSI: G+, L+, Z+
§ SIM: H2S-, indole variable, M-
§ Simmons citrate positive
§ Urease positive
§ Vogues proskauer positive
o Expanded set kit: API E
o Serological ID
§ Kauffman Orscov Scheme
• Agglutination, coagulation, latexagglutination
o AST
§ ESBL double disc test
• Clavulanic acid inhibits penicillinases
• Centre: amoxicillin and clavulanic acid
• Periphery: cephalosporins and azthreonam
• At clavulanic disk diameter increases if bacteria has an enzyme
that isn’t affected by clavualnic acid: extended spectrum beta
lactamase
• cefpodoxime best for K.pneumonia
§ has carbapenamases : growing resistance to beta lactamases and
carbapenems
§ most resistant in hospitals: nosocomial infections
o Phage typing
o Molecular typing: pulsed field gel electrophoresis
41. Genus Proteus - general features(main species, habitat, morphology, cultural and
• Main species
o Vulgaris, mirabilis and penneri
• Habitat
o Normal microbiota: intestinal tract
o UTI
o Gastroenteritis
§ Septicemia, wound infections, UTI, infections in burn patients
• Morphology
o Gram negative rods, glucose fermenter
o Lactose negative on MacConkey
o Motile
§ Swarming or invasion of bacteriological media
§ Dienes phenomenon: lines of demarcation between different strains:
waves
§ Climbing: after 24 hours can find on top of agar slant a bacteria
inoculated at bottom of slant
o H2S positive
o Ferment glucose
o Produce gas from glucose in small quantities
o Urease positive
o Lysine decarboxylase negative
o Proteus vulgaris: ornithine decarboxylase and simmons citrate usually positive
o P mirabilis and p. penneri: maltose positive
o Endotoxin
o Motility
o Plasticity
o Antigenic relativeness with rickettsia
§ Weil Felix reaction with proteus serovars OX19, OX2, OXK for
detecting rickettsiosis
o Antimicrobial resistance
o Adhesins
42. Genus Pseudomonas - general features(main species, habitat, morphology, cultural and
• Main species
o Pseudomonas aeruginosa is the main species in human infections
• Habitat
o Ubiquitous: Largely spread in nature but mainly found in water
o 10% of population is colonized at colon level
o in hospitals
§ in aerosols, flowers, fluids, surfaces
§ water, heparin, antiseptic solutions and or disinfectants
§ equipment: respiratory equipment, aspiration
§ frequently in nosocomial infections
o human
§ skin
§ urinary tract
§ lower respiratory tract
§ endocarditis, meningitis, otitis, ocular infections
§ septicemia
• Morphology
o Gram negative rods- thin rods, slightly curbed
o 0.5-0.8/1.5-3 micrometers
o isolated, in pairs or short chains
o Non sporulated
o Aeruginosa: pyocyanin pigment: blue pus
o Non fermentative, strictly aerobic
o Oxidase positive
o Non fermenters on TSI
o Grow easily with scarce nutritional resources
o Lime/acacia smell
o Pigments that spread into medium
§ Pyocyanin: greenish blue – King A media
§ Pyoverdin, fluorescein: green/yellow: p aeruginosa and others – King B
media
§ Erythrine: red pigment
§ Pyomelanin: black pigment –rare
§ Pigments are synthesized anaerobic atmosphere
o Blood agar: grey metallic sheen, beta hemolysis – better at room temperature
o MacConkey: small lactose negative colonies- no colour
o Nutritive broth/peptone water: uniform turbidity, vail on medium surface, grey
pigment
o TTC: dark red colonies
o Somatic structures
§ Polysaccharide capsule
§ Pilli, flagella
§ Lipopolysaccharide – endotoxinic activity
43. Pseudomonas aeruginosa–pathogenicity features, main diseases produced, antibiotic

susceptibility
• Pathogenicity features
o Somatic structures
§ Polysaccharide capsule
§ Pilli, flagella
§ Lipopolysaccharide – endotoxic activity
o Extracellular products
§ Enzymes:
• neuraminidases - involved in cell adherences
• proteases
• elastases
• hemolysins: letinase and phospholipase C
§ toxin
• exotoxin A, exotoxin E
• Endotoxin (LPS) – pyocyanine – mediate tissue damage
• Exotoxin A: specific for P. aeruginosa, acts like diphtheria toxin
• Pathogenicity steps
o Adherence to endothelial cells, with the help of pilli and proteins involved in
adherence
o Persistence at local level through formation of a biofilm that protects that bacteria
from antibiotics etc
o Exotoxin A inhibits protein synthesis
• Main diseases
o Skin infections: surgical wounds, burns etc
o UTIs- catheters
o Lower respiratory tract infections: ventilation pneumonia
o Chronic respiratory infections: cystic fibrosis
o Endocarditis, meningitis, otitis, ocular infections
o Septicaemia: main transmission by lymph vessels
§ Blood cultures are rarely positive
• Antibiotic susceptibility
o Very resistant to innate and acquired mechanisms
o 22 serogroups
o often resistant to beta lactamins, aminoglycosides and quinolones
44. Steps of the laboratory diagnostic in Pseudomonas aeruginosa infections

• Sampling
o Sputum. CSF, tracheal aspirations, liquid from puncture
o Blue pus: pyocyanin pigment
o Microscopy
§ Thin rods, straight or slightly curbed
§ 0.5-0.8/1.5-3 micrometers
§ non sporogenous and non capsulated
• capsulated variants only in severe infections
§ Young cultures: filamentous
§ Old cultures, selective media
o Grow very easily with scarce nutritional resources
o Simple agar: colonies with blue green pigment that diffuse into the medium
o Blood agar: grey colonies, metallic sheen, type B hemolysis –stronger at room
temperature
o MacConkey: small colonies, lactose negative, no colour
o Lime acacia smell, metallic glistening, green/yellow
o Pigments that spread into medium
§ Pyocyanin: greenish blue
o AST
§ Very resistant to innate and acquired mechanisms
o Analysis of microbio markers for public health interventions
§ Types of P. aeruginosa sources, tracking of transmission
§ By
• Phage typing
• Serotyping: 22 serogroup
• Molecular typing: pulse field gel electrophoresis
45. Genus Vibrio - general features(main species, habitat, morphology, cultural and
• Main species
o Vibro cholera
§ Serogroup 01: etiological agent of cholera – secretory mechanism, non
invasive
§ Serogroup non 01: intestinal milder forms and extraintestinal infections
o Vibrio parahaemolyticus: invasive colitis and extraintestinal infections
• Habitat
o Most common microorganisms in surface sweet or saline waters
o Colonize aquatic invertebrates and vertebrates
§ Some bioluminescence species establish mutualistic relationships with
fish and other marine life
§ Other species can be pathogenic for fish
o Survive for days in surface waters and can multiply in absence of fecal
contamination
o Survive several days on vegetables, fresh fruit, fresh fish, kitchen utensils
o Live for 1-2 weeks in milk, ice, butter
• Morphology
o Asporogenous, non capsulated, mobile, polar flagellum
o Phase contrast microscope – mobile vibrios
o 2-4 micrometers
o Oxidase positive
o Grow in slightly alkaline pH – 7.4-9.6
§ Very sensitive to pH changes
o Enrichment in alkaline peptone waters
o Selective media with bile salts and sugars
o Small shiny colonies on thyosulphate citrate bile salts
o On liquid media they form a film on the surface which can fall to become a
deposit
o Glucose positive without gas production
o Maltose positive
o Metabolic activity on amino acids and proteins
46. Vibrio cholerae – pathogenicity features, diseases produced, antibiotic susceptibility

• Somatic, structural factors
o Polar flagellum – antigen H – ensures mobility
o LPS from external membrane of cell wall: antigen O
§ Group 01 vibrions: 12 somatic antigens – A to M
§ Group 01 – 2 biotypes: classic and ElTor
§ Non 01 Bengal strain: special O phenotype
• V. cholera enterotoxin
o Daisy with 5 petals
§ Petals: B unit, centre is A unit
• A1 is active unit
• A2 is connecting piece of disulphide brides
o Systemic effect
§ Severe hydroelectric and pH imbalance, possibly driving towards death
within a few hours if not corrected
• Extracellular enzymes
o Mucinase, protease, neuraminidase
o Role in adherence to ciliary epithelium and in fixation of toxin by activated
receptors
• Fimbria
• Adhesins: hemoagglutinins and outer membrane proteins
o Chemotactiscm for intestinal mucus
• Hemolysins
• Bile salts resistance
• Diseases produced
o Vibrio cholera serogroup 01 is the etiological agent of cholera
§ Secretory mechanism: non invasive
o Serogroup non 01: in intestinal milder forms and extra intestinal infections
• Antibiotic susceptibility
o Interruption in abnormal cAMP activation cycle by tetracycline
§ Some resistant strains
§ Only shortens evolution
47. Steps of the laboratory diagnostic of cholera

• Samples: feces
• Transport: alkaline peptone water
• Direct microscopy
o Asporogenous, non capsulated, mobile, polar flagellum
o Phase contrast microscope – mobile vibrios
o 2-4 micrometers
• Cultivation
o Enrichment in alkaline peptone water
o Small, shiny colonies on thyosulphate citrate, bile salts
• Identification
o Oxidase positive
o Slide agglutination with 01 antiserum
o Grow in slightly alkaline pH – 7.4-9.6
§ Very sensitive to pH changes
o Enrichment in alkaline peptone waters
o Small shiny colonies on thyosulphate citrate bile salts
o On liquid media they form a film on the surface which can fall to become a
deposit
o Glucose positive without gas production
o Maltose positive
o Metabolic activity on amino acids and proteins
48. Haemophilus influenzae -general features(habitat, morphology, cultural features, antigenic

structure, diseases produced)
• Gram negative coccobacilli and or bacilli
• Facultatively anaerobic, non mobile, non sporogenous
• Virulent strains re capsulated
• Growth
o Enriched media: supplemented with peptones glucose etc.
o Fastidious: need growth factors from blood for cultivation
§ X factor: haemin or other iron containing porphyrins
• thermostable
§ V factor: NAD
• Thermolabile
• Inactivated by pyrophosphatase and nicotine amine
deoxyribonuclease
§ Alternatives to X and V
• Heating blood agar to obtain chocolate agar
• Supplements: X and V factors, 5% blood, Fildes or Levinthal
supplements
• Staphylococcus streak or satellite test
o Will grow on haemolytic zone of s.aureus on blood agar
plates
o Hemolysis of cells by s. aureus releases factor V which
is needed for growth
o capnophilic species: X ducreyi, aphrophylus and certain H. parainfluenzae
o horse or rabbit blood
o need humidity
o prolonged incubation: 48-72 hours after antibiotic treatment and 5 days for H.
ducreyi
o selective media for highly contaminated samples
• Morphology
o Polymorphism pending on growth conditions and age of culture
o Species specific aspects
o Borderline gram negative
§ Decolorate harder
§ Counter stain harder: better to use fucsin not safranin
o Capsule
o Endotoxin with lipooligosaccharide in external membrane
o Outer membrane proteins P1 and P2
o Fimbria increase adherence to mucosal surface
o All virulent strains produce a neuraminidase and IgA protease
o Receptor for fimbria on erythrocytes: antigen anton
• Types of infections
o Respiratory tract and neighbouring sites: pneumonia, meningitis, acute
epiglottitis, otitis media, mostly encapsulated strains
o Cellulitis
o Bacteremia, septicemia
o Arthritis, pericarditis, endocarditis, osteomyelitis
o Infections of genitals, infections in pregnant women, women in childbed, and
fetus
o Infection of wounds
o Digestive and biliary infections
• Habitat
o Humans, mammals, fish, reptiles
§ Normal microbiota of respiratory tract, genitals, intestine, dental plaque
§ Exception: H aegyptius and h. ducreyi: always pathogens
49. Genus Bordetella - general features(main species, habitat, morphology, cultural and
• Main species
o Bordetella pertussis and parapertussis: only in humans: etiological agents of
whooping cough
o B. bronchiseptica: in animals – rarely in humans
o B. holmesii: isolated from hemocultures in humans
• Habitat
o Obligate parasite for humans and animals
o Tropism for respiratory mucosa
o Very short survival (2 hours) in exterior medium
o Destroyed by antiseptics and disinfectants, UV and temperature 55 degrees
Celsius for 30 minutes
• Morphology
o Small coccobacilli: 0.2-0.3 micrometers to 0.5-1 micrometers
o Gram negative
o Isolated pairs, rarely in short chains
o Strictly aerobic
o Cultivate only on media supplements with amino acids, vitamins, mineral salts,
neutralizing substances (stark, charcoal, potato extract)
§ Don’t need factor X or V
o Optimal temperature: 35-37 degrees Celsius
o Pertussis and parapertussis are mobile
• Biochemical feature
o Metabolically inactive
§ Don’t ferment carbs, don’t produce gas, indole and H2S
§ Don’t liquefy gelatin
§ Alkalize milk
o O: genus specific
o K: capsular
• Pathogenicity
o Adhesins
§ On fimbria
• Filamentous hemagglutinin (FHA)
o Adheres on ciliated cells, lymph cells and macrophages
o Immunogenic
• Agglutinogens
o Adheres to ciliated cells
• On cell surface
o Perlactin
o Toxins
§ Pertussis toxin
§ Adenylate cyclase
§ Dermonecrotic toxin
§ Tracheal cytotoxin
§ Lipopolysaccharide
50. Laboratory diagnostic of Bordetella pertussis infections

• Sampling
o Perinasal charcoal swab
o shortly after disease onset before administering antibiotics
o nose pharynx aspirate in hospitals
o nose pharynx swab – Dacron of calcium alginate
§ not cotton swabs that may inhibit the bacteria
o cough plate
• transport
o bringing media near patient
o transport medium: Regen-lowe semisolid, selective medium
o universal transport medium – amies, stuart
• cultivation
o bordet gengou selective/ non selective
o rich medium with blood, potato extract (for neutralizing inhibitors) and glycerol
• incubation
o 35-37 degrees Celsius, aerobically, humid atmosphere, 3-4 up to 7 days
o colonies on bordet gengou agar: small, smooth, dome shaped, grayish white,
glistening like mercury drops
• indirect
o serological diagnostic
§ specific antibodies serum titer followed in dynamics on 2 sera; at onset
and after 2 weeks
§ methods: in tube agglutination, ELISA
51. DTP vaccine – composition, administration scheme

o 3 inoculations starting at 2 months at 2 month intervals in case of contact with a
confirmed case
o for diphtheria, tetanus and poliomyeltis
o diphtheric anatoxin
o tetanic anatoxin
o corpuscular pertussis vaccine
o can also have acellular pertussis vaccine that appears to be less active
52. Mycobacterium tuberculosis - general features (habitat, morphology, cultural features)

o Habitat
o Humans are the only natural reservoir
o Many non-pathogenic mycobacteria are part of normal human flora especially in
fatty or high humidity anatomical zones
o Bacteria arrive in the lung only if they’re in very small particles with 1-3 bacteria
which remain in the alveoli
o The small drops called Pflugge drops aren’t eliminated from the respiratory tree
o Some zoonotic species exist and can only infect humans
o Can resist dry environment for months and years because of their cell wall
structure
o Morphology
o 2-4 micrometers/0.2-0.5 micrometers: large bacilli
o immobile
o non sporulated
o strict aerobe
o facultatively intracellular
o slow multiplication
o special structure of cell wall
§ impermeable to dyes : no gram staining
§ resistance to many antibiotics
§ resistance to alkaline or acidic chemical agents
§ resistance to complement
o Cultural features
o Complex media Categories:
§ Liquid media: (e.g. 7H9 broth)
§ Media with coagulated eggs (e.g. Lowenstein-Jensen): egg, potato, salts,
asparagine, glycerin; malachite green, certain antimicrobials for
selectivity; adding 0,4% Natrium pyruvate favors developing of M.bovis,
M. africanum, M. tuberculosis resistant to Isonicotinic Acid Hydrazide
(INH)
o Other solid media (e.g.. Middlebrook) Incubation in 8-10% CO2 favors primary growth
Incubation temperature: 35-37 o C
o Examination: Daily – for 1 week Weekly for 6-8-12 weeks (generation time:
12-27 hours)
o Rapid culture systems: MB-Bact, Bactec – definitive results in 4-25 days; could help in
antimicrobial susceptibility testing
53. Mycobacterium tuberculosis – chemical composition and antigenic structure

• Mycobacterium tuberculosis has unique cell wall structure among prokaryotes – major
pathogenicity determinant:
o Peptidoglycan (similar with Gram positive microorganisms)
o Complex lipids – more than 60% from the cell wall – 3 major components:
§ Mycolic acids (present also in Nocardia, Corynebacterium, but different):
involved in permeability, virulence, acid fast staining, resistance to
antimicrobials and alkalis (the last used to decontaminate samples)
§ Cord factor – toxic for mitochondria; inhibits PMN  
• A glycolipid found in the cell wall structure = trehalose dimycolate
• Virulent Mycobacterium tuberculosis – grows in “cords” = coiled
structures – called “cord factor”
• Acid Fast rods, arranged in parallel rows  
• Detection of “cord factor” – used for preliminary, rapid differentiation
of M. tuberculosis  
§ D wax – major component of complete adjuvant Freund used for increasing the
immunogenicity of other antigens  
•
54. Immune response in tuberculosis infection, ways of assessment

• Stages of TB
• Stage 1: inhalation of Flugge droplets (most effective 5 µ m in  diameter)  
• Stage 2: 7-21 days after the initial colonization – multiplication in macrophages and
activation of macrophages; other macrophages go outside the blood vessels; they
can’t destroy Mycobacterium tuberculosis.  
• Stage 3 – beginning of lymph cell infiltration, especially T cells, their activation,
delivery of cytokines, including Interferon (IFN) γ. Delivery of IFN triggers
activation of macrophages, which become able to destroy M. tuberculosis ; IDR turns
positive; pathology: progressive setting up of the TB tubercle, with formation of a
caseous centre  
• Stage 4 – punching of bronchia – miliary TB: exudative or granulomatous lesions  
• Stage 5 – liquefaction of the tubercle centrum (cazeum), extracellular multiplication,
resulting in cavities formation (TB caverns), lung dissemination; calcification (Ghon
complex) and active remaining centre (Simon focus)  
• pathogenesis
o respiratory form in most cases (but also bone, kidney, genital, nervous
system TB etc. foci)
o at the macrophage level
o facultative intracellular pathogen – inhibits the phagolysosome fusion –
resists lysosomal enzymes
• immunity
o inflitration 
§ macrophages
§ lymph cells
o granuloma
o tubercles
55. Mycobacterium tuberculosis – drug susceptibility

• Multiple methods to test antibiotic sensitivity
o Method of resistance fraction
§ Binary dilutions of antibiotic
§ Fraction of resistance is MIC for the tested strain/MIC for control strain
o Proportion test
§ Test 2 dilutions of 1/100 successively of the tested strain
§ % resistance= number of colonies per medium without
antibiotics/number of colonies on the medium with antibiotic X 100
o radiometric methods:
• Tuberculostatic treatment
o long intervals (e.g. 9 months) 
o The microorganism grows slowly or is dormant 
o Two or more antibiotics ex. Rifampicin and isoniazid 
o The risk of resistance is diminishing
56. Laboratory diagnostic in tuberculosis

o Direct microbiological diagnosis
o Sampling and transport –
§ sputum, bronchial lavage fluid, CSF, urine; articular, pleural, pericardic,
peritoneal puncture fluid; biopsies, purulent discharges etc.)
§ General rules: close to the onset, before antimicrobial therapy, aseptic, rapid
and technically correct sampling, aiming to protect the involved personnel
etc.)
§ Processing of samples may be required:
• Sample centrifugation for concentration  
• Sample decontamination - killing of bacteria that are not
Mycobacterium (4% Na OH and sample in equal parts, neutralized
with acids before testing) – only Mycobacterium resists to alkaline
environment, due to the cell wall structure  
• Direct microscopy – relevant for prescribing tuberculostatic treatment:
o At least 3 smears: Methylene Blue, Gram, Ziehl Nielsen
o Immersion objective Description of smear:
§ Presence of the respiratory cells or inflammatory cells (macrophages,
PMN)  
§ Presence of Acid Fast rods: thin, straight rods, 0.4/3 µm, acid fast red on
blue background  
§ non capsulated  
§ non sporogenous  
§ All bacteria and cells which are not acid fast are stained in blue by
Ziehl-Neelsen stain!!  
o Complex media Categories:
o Liquid media: (e.g. 7H9 broth)
o Media with coagulated eggs (e.g. Lowenstein-Jensen): egg, potato, salts, asparagine,
glycerine; megahit green, certain antimicrobials for selectivity; adding 0,4% Natrium
pyruvate favors developing of M.bovis, M. africanum, M. tuberculosis resistant to
Isonicotinic Acid Hydrazide (INH)
o Other solid media (e.g.. Middlebrook) Incubation in 8-10% CO2 favors primary
growth Incubation temperature: 35-37 o C
o Examination: Daily – for 1 week Weekly for 6-8-12 weeks (generation time: 12-27 hours)
o Rapid culture systems: MB-Bact, Bactec – definitive results in 4-25 days; could help in
antimicrobial susceptibility testing
o Grows very slowly on Lowenstein-Jensen medium
o Several weeks
o Non pigmented colonies
o Production of niacin
o Differentiation from other mycobacteria
o Identification
o Microscopy: Acid Fast Rods on smear from isolated colonies
o Culture:
§ M. tuberculosis and M. bovis (BCG = Bacille Calmette Guerin) – R (rough)
colonies, cauliflower appearance, hardly emulsified, non-pigmented  
§ M. tuberculosis resistant to NIH (Nicotinic Acid Hydrazide) may grow as S
colony  – Biochemistry:  
• Production of niacin: positive M. tuberculosis; negative M.  bovis  
• Nitrates reduction to nitrites: positive M. tuberculosis;  negative M. bovis  
• Susceptibility to tiophene-2 carboxylic acid: inhibits M. bovis  
• Other tests:
o Molecular phenotyping (fatty acids from the cell wall - chromatography)
o Genotyping and PCR for resistance or virulence genes  
o Phagetyping etc.  
o rapid diagnosis
o PCR
o AST
o Required
o National program – special methods: - absolute concentrations method - proportions
method, - resistance rates method
o radiometric methods etc.
o immunobiologic diagnosis
o IDR (Intra Dermo Reaction)
o delayed hypersensitivity
o antigen = prepared from tuberculin (a protein extracted from Mycobacterium)
o PPD = Purified Proteic Derivative
o Indicates there is exposure to the mycobacteria  
o In Romania, infiltration > 10 mm (do not quantify the rash, but the infiltration or
induration)  
• Does not indicate active, acute illness  
Serological diagnosis
o Seric antibodies titer by ELISA •
57. Corynebacterium diphtheriae - general features(habitat, morphology, cultural, biochemical
and metabolical features)
• Habitat
o mucous surfaces, human and certain animals ( horses, dogs, monkeys) skin:
illness or carrier status
• Morphology
o Borderline gram positive in old cultures with strong gram positive granulations
o Straight or slightly curved, club shaped rods, with metachromatic inclusions =
metachromatic granulations of poly metaphosphate which stain red with
methylene blue  
o Sometimes thin, slightly sharpened bacilli, other times oval or round shaped  
o Arrangement in sharp shaped letters: V,Y,L, Chinese letters or palisades, rarely
parallel cells  
o On Loeffler medium(characteristic):
§ Borderline Gram positive rods, 1-8 µ length; 0.3-0.5 µ thick
§ Accentuated polymorphism : impairment of murein synthesis in media
with seric proteins, iron and phosphates and/or in old cultures
§ Typical arrangement: X, Y, V, L  
§ Tinctorial heterogeneity – Babes-Ernst inclusions1888 – polyphosphate
granules (Gram stain: violet on red background, Methylen Blue stain: red
on blue background); mitis - long rods, with plenty of inclusions;
intermedius – long rods, moderate number of inclusions; gravis - shorter
rods, with few inclusions
o Coccoidal or ovoidal aspect on other media: Gundel- Tietz, Tinsdale  
o Structure
§ Cell wall
• muropeptid containing meso α,ε-diaminopimelic acid;
• peptidoglican, arabinogalactan  
• A corynemycolic and corynemycolenic component (glycolipid) –
cord factor involved in virulence and invasivity, determined by
the local action of the bacteria on the tissues  
• surface proteins considered thermolabile type specific antigens  
• surface receptors for bacteriophages  
§ Cytoplasmic membrane–actively participating in diphtheric toxin
production  
§ Polyphosphate granules(Babes-Ernst metachromatic inclusions Babes-
Ernst) – inside bacterial cell; in a polar or subpolar position; appear
especially in anaerobic conditions and are useful for differentiating C.
diphtheriae from common habitat species  •
§ Genetic material–sometimes inside the bacterial genome there is inserted
the genetic material of a temperate phage, causing the lysogeny state and
the toxigenesis  
• Cultural, biochemical and metabolic features
• Cultural
o On solid media - Loeffler:
§ C.diphtheriae: semi-creamy culture
o Gundel-Tietz (blood -agar-cystine-telurite)
§ C.diphtheriae var. gravis: R type, opaque, plate colonies, difficult to
emulsify, black-grey, Ø = 1.5 – 2 mm
§ Resemble the shape of a daisy;
§ C.diphtheriae var. intermedius: S type, opaque, umbilicated, black or
grey colonies, with transparent edges, Ø = 1.5 – 2 mm (but dimensions
may vary)
§ C.diphtheriae var. mitis: S type colonies, black or grey, translucent, Ø =
0,5 - 1 mm\; may turn in R type by ageing
o Blood agar:
§ Morphology similar with Gundel-Tietz colonies; characteristic, colonies
have white-grey pearl appearance:
• gravis - daisy colonies, granular, (friabil) surface
• intermedius - plate colonies, sometimes acuminated, with
circular margins, granular surface, semi-creamy consistency
• mitis – circular, convex, glancy colonies, creamy consistency
o modified Tinsdale (agar-serum-cystine-telurite- thyosulfate)
§ All types of C.diphtheriae grow as small, black-grey colonies, with a
brown, well delimited halo; the halo turns stronger colour after 48 hrs.
This medium allows differentiation of C.diphtheriae, C.ulcerans and
C.pseudotuberculosis from other Corynebacterium non-pathogenic
species that are inhibited on this medium, or grow without halo
o On liquid media
§ C.diphtheriae var. gravis grows and forms a pellicle and a deposit, the
medium remaining clear
§ C.diphtheriae var. intermedius forms a ring at the medium surface and a
deposit, it is slightly modifying the clarity of the medium
§ C.diphtheriae var. mitis is uniformly modifying the clarity of the
medium and forms a deposit
• Metabolic and enzymatic
o C.diphtheriae develops on simple culture media; growth is favors on media
enriched with animal proteins: cow serum, egg  
o Growth temperatures varies between 15 -40oC with optimum 37oC.  
o It is growing in aerobic conditions, but it is also facultatively anaerobic  
o Characteristic carbohydrates fermentation pattern  
o Reduces Theluric salts; metallic Thelur incorporated as crystals inside the
bacterial cell gives the black color of colonies on Thelurite media  
o Cystinase–deliversH2S (valuable differential character on Tinsdale medium with
serum, cysteine, thelurite- thyosuplphate – black colony; thelur sulphate gives the
brown halo;  
o Urease, phosphatase and pyrasinamidase are negative in C. diphtheriae, urease
and phosphatase are positive in C. ulcerans and C. pseudotuberculosis  
o Hemolysin active on Guinee apig, rabbit, sheep erythrocytes, for gravis and
mitis types; it is not active for intermedius type  
o Nitrate reductase–negative for belfanti biotype; positive for the rest of
Corynebacterium species: C. diphtheriae, C. ulcerans and C. pseudotuberculosis
 
o Neudaminidase,catalase  
o Old cultures:coproporphyrine  
58. Corynebacterium diphtheriae – biochemical and antigenical features, pathogenicity
§ Diphtheric toxin
o It is an exotoxin produced by toxigenic C.diphtheriae, delivered in the
environment; it is delivered without bacterial cell lysis  
o Strains of C.diphtheriae belonging to all biochemical and culture growth types
produce a unique toxin, which is neutralised by anti- toxic diphtheric serum,
prepared by using a unique antigen = diphtheric toxoid, obtained by detoxifying
the diphtheria toxin after heat and chemical formaldehyde treatment  
o It is a strong toxin, with specialised toxic properties: - dermonecrotic  - produces
degenerative lesions at the miocardial level (mytochondial lesions): impairment
of contractility, and under endocardic vascularisation,  - produces adrenal
degenerative lesions with capillary stasys and haemorrhage at the adrenal level  -
degenerative lesions of nervous system  
o If a non toxigenic C.diphtheriae strains gets infected with a temperate
bacteriophage, the last one may integrate in the bacterial chromosome, and the
bacterial cell becomes  lysogenic;  
o By lysogenisation the cell Turns resistant to the activity of the phage it is carring
on, or to any phage which is antigenically related with this phage.  
o If the bacteriophage carries the tox+ gene, the replication of the bacteriophage
DNA inside the bacteria is accompanied by the production of a protein which
will be delivered extracellulary = diphtheric toxin (reversible lysogenic
conversion)  
o Toxin production is initiated 3 minutes after the phage infected the bacteria and
extracellular delivery of the toxin is initiated before first phage particles are
delivered by the newly lysogenised cell  At a reduced iron concentration, the
virus multiplication cycle is prolongued, thus increasing the diphtheric toxin
production  
§ other pathogenicity factors
o Invasivity factors
§ adhesins
§ cord factor (corynemycolic and corynemycolenic) acids acting at the
mytochondrial level – cell respiration and phosphorylation functions
impared
§ Neuraminidase and hyaluronidase – necrotic and diffusion factors;
neuraminidase is present both in toxigenic and in non-toxigenic strains of
C. diphtheriae
o pathogenesis
§ Adherence 
§ Multiplication at the entry portal (throat or wound) - Toxemia - the
pathogenic agent is the toxigenic C.diphtheriae
§ The evolution of the disease is dependent on the quantity and the rapidity
of toxin diffusion in the host organism;
§ Only rapid therapy with diphtheric anti-toxin may favorably influence
the evolution of the disease.
59. Laboratory diagnostic of diphtheria
• Sampling
o From sick/suspect person:
§ in tonsillar diphtheria it is better to sample 3 swabs from the false
membrane level (by detaching a fragment of the false membrane or from
tonsillar adherent deposits) and 1 swab with nasal exudate
§ in the other clinical forms, swabs are sampled from the primary lesions:
exudate from diphtheric rynithis or pus from ocular conjunctivitis, vulvar
or otic diphtheria etc. To avoid confusion with common pyodermitis, it is
recommeded to sample every skin purulent lesion and to mandatory
investigate in parallel nasal cavity and pharynx in diphtheria suspected
cases
o From carriers: it is recommanded to sample both nasal and pharingeal exudates
§ Sampling has to be done simultaneously from all contacts in the
collectivity; as for suspected persons
• Transport
o Inoculation on specific culture media has to be as fast as possible. If not
available, transport has to be ensured to the appropriate laboratory within 2 hours
from sampling
o Enrichment media: EST (Egg Serum Tellurite) for maintaining the
microorganisms viability for 4-5 hrs, or other transport media (Amies, Stuart)
• Direct diagnosis
o Done for the ill person:
§ Swabssampledfromthefalsemembrane: – Spread on Tinsdale and blood
agar media – Enrichment by passing on EST medium – Gram stained
smear
§ SwabfromthenoseisintroducedinESTmedium
o Direct microscopy:
§ Smear:evidenceofsuspectborderlineGrampositive rods, polymorphic
microbiota or presence of spirilli and fusiform bacteria (Plaut Vincent
pharyngitis); it may support presumptive diagnostic of C. diphtheriae
suspicion
• cultivation
o Inoculated media are incubated for 24-48 hrs at 37o C
o a. Blood agar – value:
§ Morphology of bacterial colonies
§ Differential diagnostic with streptococcal or mixed pharyngeal
infections.
§ Certain strains of C.diphtheriae are susceptible to Potassium Tellurite,
and may be inhibited on media containing this ingredient
o b. Tinsdale medium: black colonies with brown, clearly delimited halo
• smear from culture is mandatory for eliminating the confusion
with certain Staphylococcus species
o c. Inoculation of nasal swab from EST enrichment medium on Tinsdale medium
o Done for the carrier person:
§ Swabs prelevated from nose and throat are inoculated on EST
enrichment medium  
§ Incubation for 24 hrs at 37oC,  
o Spread on Tinsdale medium – growth of black colonies with brown, clearly
delimited halo after incubation for 24-48 hrs at 37oC, indicates suspicion of
C.diphtheriae.  
• identification
o Morphological and staining characters
o Culture characters
o Biochemical tests: - cystinase, - urease, - toxinogenesis
o Phage typing
o toxigenesis
§ Current “in vitro” testing of toxinogenic properties of the isolated strains
by Elek test – rapid, simple, precise immune diffusion test> it is a
precipitation, double diffusion test performed inside of a solid medium
§ Results at 24-48 hrsof incubation at 37oC.
§ Positive test: precipitation lines in the corner between the inoculated
culture and anti-toxin (either a band or a dig with anti-diphtheric
antibodies =Toxigenic C.diphtheriae/C.ulcerans
o Reference strains: toxigenic, weekly toxigenic and non-toxigenic C.diphtheriae
o Other tests used for checking for toxinogenesis: - “in vivo” test on Guineea pigs
– only for unclear Elek results, only in the national reference laboratory -
molecular methods for detecting the tox+ gene (PCR)
•
60. Bacillus anthracis - general features(habitat, morphology, cultural and biochemical

features, antigenic structure)
§ Habitat
o Most species are opportunistic  
o Endospores resistant to hostil environment  physical and chemical conditions
o Unusual physiological peculiarities which allow their survival in unfavorable
media: desert sand, arctic soils, sweet waters, sea sediment  
o species survive to a large spectrum of temperatures, pH, Na Cl concentrations:
§ Thermophylic 
§ Pshichrophylic 
§ Acidophylic 
§ Alkalophylic, 
§ Halotolerant and halophylic etc.  
§ Morphology
o Gram positive rods, may turn Gram negative in old cultures  
o Big rods  
o Mobile by peritrichous flagella (B. anthracis is  not mobile)  
o Sporgeneous,  
o Capsule may be present  
o Aerobic, facultatively anaerobic  
o
§ Cultural
o On blood agar at 37 o C: R colonies, gray or white, non-haemolytic, resembling
crushed glass; at least 3 mm in diameter, sometimes compared with a comet or
jelly fish (medusa) head
o On selective media (PLET = Polymyxin, Lysosyme, EDTA, Thalium acetate): S
colonies, small; alcohol or heat treatment for separating spores from mixed
cultures
§ Biochemical
o Catalase + 
o Some species haemolytic 
o Active on several organic substrates
o
§ Antigenic
o Polypeptidic capsule (polyglutamic acid)
o antiphagocytic protection,
o Toxin produced in the logarithmic grow phase - 3 proteins:
§ protective antigen (PA) – a cell- binding protein
§ lethal factor (LF), 
§ edema factor (EF) LF and EF are enzymes
§ Pathogenesis
o Activation of protective antigen (PA) by  host blood and/or eukaryotic cell
surface proteases - cutting a fragment and thus making possible the exposure of
the binding site for LF and EF  
o PA + LF complex or PA +EF complex are internalized by endocytosis  
o EF is responsible for the anthrax characteristic edema – it is a Calcium dependent
adenylate cyclase  
§ clinical
o Cutaneous anthrax - over 95% of cases: edema preceding skin lesions (sore,
ulcer, eschar), haemorrhagic black colour lesions – by manipulation of infected
material, flies bite etc.; incubation 1-3 days; initial stage - papule, intense edema;
late stage – eschar; hystory of exposure to animals or their product;
o Oropharyngeal or intestinal anthrax – by ingestion of infected meat
o Inhalational (respiratory) anthrax – by inhalation of infected material (dust with
spores)
o Septicemic anthrax
o initial phase: lack of symptoms or mild symptoms: malaise, low fever, gastro-
intestinal symptoms in intestinal anthrax
o Multiplication phase– delivery of toxin in lymph nodes and spleen and
impairment of gastro-intestinal or respiratory function, organ impairement,
mostly of the spleen function, then liver organ failure
o Sudden onset of hyper-acute disease: dyspnoea, cyanosis, high fever,
disorientation, shock, coma and death whithin hours. This phase is highly
bacteremic, but sometimes bloodculture may remain negative
o Protective immunity – antibodies against toxic components, mainly Protective
Antigen  
o Immunisation: Acellular vaccins for humans and vaccins with living spores  
o Poly-D-glutamic capsule of B. anthracis is weakly immunogenic. Antibodies
against the polysaccharide and other cell wall components are not protective  
o Immune response in the intestinal anthrax is still less known  
§ epidemiology
o Transmission way: direct contact with infected herbivores or their products;
herbivores = primary hosts, by ingesting spores from fodder
o Spores are coming from soil or dust or are deposited on plants by insects which
eated meat from a contaminated animal carcass
o Risk groups: persons coming in contact with infected animals or their products
o
61. Laboratory diagnostic of anthrax infection

§ Samples
o Smears from vesicular fluid, eschars, blood, lymph nodes or splenic aspirate or
CSF (in meningitis)  
§ Direct microscopy
o Polychrom Methylene Blue staining (Mc'Fadyean staining): typical Gram
positive, straight cut ends, big rods (1-1.5 µm/3-10 µm), arranged in long chains;
sporogenous (elyptic, central spore, smaller than the size of the rod) surrounded
by a pink capsule  
§ cultivation
o On blood agar at 37 o C: R colonies, gray or white, non-haemolytic, resembling
crushed glass; at least 3 mm in diameter, sometimes compared with a comet or
jelly fish (medusa) head
o On selective media (PLET = Polymyxin, Lysosyme, EDTA, Thalium acetate): S
colonies, small; alcohol or heat treatment for separating spores from mixed
cultures
§ identification
o Susceptible to gamma phage
o Susceptible to Penicillin  
o Patogenicity: capsulogenesis on nutrient agar with Sodium bicarbonate in
anaerobiosis – Polychrom blue stain, or pathogenicity for mouse  
o Biochemical identification:
§ Catalase positive  
§ Lecitinase positive
§ Susceptible to Penicillin(R:B.cereus,B. subtilis)
§ Molecular testing
§ antimicrobial susceptibility
o Usually susceptible to Penicillin, Eryhtromycin, Tetracycline  
o Antimicrobial Susceptibility Testing not needed in routine practice  
o Strains resistant to Penicillin may occur  
62. Enumerate the sporulated anaerobic microorganisms and the infections they determine
• Clostridium tetani
o Tetanus
• Clostridium botulinum
o botulism 
• C. perfringens
o Gas gangrene
o Food poisoning
o Cellulitis
o necrotizing fasciitis
o septicaemia
o surgical infection
• Clostridium difficile
o Diarrhea
o Post antibiotic enterocolitis
• C. sordellii
o Gas gangrene, cellulitis
63. Clostridium tetani - general features(habitat, morphology, cultural and biochemical

§ Habitat
o are widely distributed in soil and in fresh water
o it is the only genus of spore-forming anaerobes found in humans, either in
nonpathogenic habitats or in infected sites
o several species inhabit the lower intestinal tract of humans and other animal as
part of the normal flora (most are harmless saprophytes)
o
§ Morphology
o It currently contains 203 species and 5 subspecies, with only
o a few species being pathogenic to humans
o are Gram positive (can decolorise easily and appear as Gram negative or
variable), sporeforming (form oval or spherical spores that distend the cell),
catalase-negative, anaerobic bacilli
o many are motile many produce potent exotoxins
o Gram stain appearance: Gram positive rods, which may possess a single
endospore.
o Spore stain: is used to determine the shape and position of the spore (e.g.
malachite green + safranine)
o Drum stick like rods – because of terminal endospore
o
§ Cultural
o Media: blood agar, egg yolk agar (+ antibiotics for selectivity)
o Incubation: anaerobiosis
o Fine film of bacteria
§ Biochemical
o Nagler test – determines the ability to produce the enzyme lecithinase (zone of
opalescence surrounding the culture)
o C.perfringens lecithinase (alpha toxin) is inhibited by the antitoxin C. perfringens
type A
o Positive result: zone of opalescence surrounding the streak and inhibition due to
antitoxin
o Identification – biochemical tests Reverse CAMP test – alternative to Nagler test
o (Alpha toxin producing C. perfringens and group B, β- haemolytic streptococci
grow in a characteristic pattern on blood agar)
o Lipase test (e.g. C. botulinum is positive ) Indol test Urease test, etc.
o Commercially identification systems Nucleic acid amplification tests (PCR)
o Catalase negative
o Very active metabolically
§ Produce butyric acid
§ Acetic acid
§ Butanol
§ Acetone
§ Foul smell from aminoacids and fatty acids
o Form large quantities of gas: CO2 and H2 from fermenting sugars
§
§ Antigenic
o Clinically relevant Clostridium species produce a variety of toxins which lead to
the distinctive clinical features of the disease they produce
o The toxins are antigenic in nature and can therefore be neutralised by specific
Abs (antisera)!
o Detection of particular toxins directly from some clinical samples may render the
isolation of the organism unnecessary for primary investigation (e.g.C. difficile)
o Culture is required for typing (in outbreaks) and susceptibility testing
o C. tetani – produces tetanospasmin (neurotoxin) and tetanolysin (lyses RBCs)
64. Clostridium tetani infection – pathogenic mechanism, preventive measures

§ Pathogenic mechanism
o produces tetanospasmin (neurotoxin) and tetanolysin (lyses RBCs)
o tenanic toxin blocks the release of inhibitory neurotransmitters at the level of the
synapse: causing muscular spasms
o When favoring factors, affecting Oxygen exchanges are present anaerobic
bactria from exterior or from endogenous flora multiply at the ecological niche
created in the host
§ Deep wounds or anatomic sites near cavities heavily colonized with
anaerobe microorganisms  
§ Human bite wounds  
§ Tissue distruction by tumours, trauma, complex  surgery  
§ Diabetes/acidosis  
§ Aspiration pneumonia; endocarditis  
o Antimicrobial therapy with antibiotics which are inactive on anaerobe
microorganisms (Aminoglycosides, Quinolones, Sulphamides)  
o Bacteria manifest several pathogenicity factors: toxins, enzymes, adhesins,
capsule etc.
o Multiply at the entrance port where they can arrive in case of accidental
contimation of wounds with soil
§ Preventative measures
o Wound cleaning  
o Hydrogene peroxide washing  
o Vaccination for tetanus  
65. Clostridium botulinum - general features(habitat, morphology, cultural and biochemical
• Habitat
o are widely distributed în soil and in fresh water
o it is the only genus of spore-forming anaerobes found in humans, either in
nonpathogenic habitats or in infected sites
o several species inhabit the lower intestinal tract of humans and other animal as
part of the normal flora (most are harmless saprophytes)
o Every ecological niche meeting nutritional and anaerobiosis requirements:
§ Humans and animals: natural cavities – comensal and/or symbiotic (e.g.
Propionibacterium/Vit. B12 source)
§ Clostridia 10-100:1 aerobe microorganism in upper respiratory tract,
vagina, pylosebaceous units  
§ Anaerobes 1000: 1 aerobe microorganism in colon
§ Food  
§ Environment
• Morphology
o Gram positive, sporulated, mobile
o Gram stain appearance: Gram positive rods, which may possess a single
endospore.
o Spore stain: is used to determine the shape and position of the spore (e.g.
malachite green + safranine)
o Subterminal endospore
o Anaerobic
§ For cultures
§ If possible, for manipulation
§ If possible, for media:
• PRAS = Prereduced Anaerobically Sterilised
• Other media may be prereduced by boiling and other procedures
• Anaerobiosis Jar  
• Plate anaerobiosis: reducing chemical mix  
• Plastic bag culture (food bacteriology)  
• Deep inoculation in a semisolid medium column  
o Strict anaerobiosis conditions
§ Adequate media, preferably prereduced; with selective and nutritional
supplements if samples are contaminated with mixed microorganisms
(aerobe and anaerobe)
o Translucid colonies, beta hemolysis on blood agar
• Biochemical features
o Catalase negative
o Very active metabolically
§ Produce butyric acid
§ Acetic acid
§ Butanol
§ Acetone
§ Foul smell from aminoacids and fatty acids
o Form large quantities of gas: CO2 and H2 from fermenting sugars
o Production of exotoxins
66. Clostridium botulinum infection – pathogenic mechanism, diagnostic

• Pathogenic mechanism
o produces seven toxin types (A-G), all neurotoxins (cause paralysis)
o Botulin toxin is a neurotoxin that blocks the release of acetylcholine which
determine an impossibility to contract and progressive muscular paralysis with
the risk of respiratory arrest
o When favoring factors, affecting Oxygen exchanges are present
§ Deep wounds or anatomic sites near cavities heavily colonized with
anaerobe microorganisms  
§ Human bite wounds  
§ Tissue destruction by tumors, trauma, complex  surgery  
§ Diabetes/acidosis  
§ Aspiration pneumonia; endocarditis  
o Antimicrobial therapy with antibiotics which are inactive on anaerobe
microorganisms (Aminoglycosides, Quinolones, Sulphamides)  
o Bacteria manifest several pathogenicity factors: toxins, enzymes, adhesins,
capsule etc.
o Can manifest after consumption of the toxin that is released into food by the
bacteria or by multiplication at the level of the intestine, especially in small
children, or through contamination of wound with C. botulinum
• diagnostic
o Gas inside the tissues, fetid leaks, blackened blood  
o Presence of suggestive aspect on smears examined  in direct microscopy  
o Red fluorescence in UV of fetid leaks  
o Absence of aerobe growth of organisms that are visible on the smear  
o Growth only in the anaerobe zone of a solid or semisolid bacteriological medium
o Growth in anaerobiosis on selective media with Kanamycin or Neomycin 75-100
µg/mL or  Vancomycin 7,5 µg/mL (anaerobe Gram Negative Rods)  
o Direct examination
§ Macroscopic
• Necrosis, putrid small, lysed blood with dark colour
§ Gram positive bacilli
§ Can sometimes see subterminal spores
§ Must used prereduced media for anaerobiosis
§ Can use VF culture
o Principles
§ Step wise algorithm:
§ Confirmation of anaerobe character (by double inoculation and
cultivation in aerobe/anaerobe conditions – anaerobes show lack of
growth in aerobiosis!!!)  
§ Identification of microscopy category (sometimes difficult);  
§ Differentiation inside the microscopy category (e.g. Clostridia, Gram
negative rods etc)  
o Biochemical identification
§ Rapid/Presumptive identification techniques:
• Galeries, kits: 
o Of first generation: incubation in anaerobiosis 
o Of second generation: incubation in aerobiosis (4 h)
Definitive identification techniques – biochemical and enzymatic
• Fermentation of carbohydrates  
• Production of phospholipases-lipases  
• Degradation of amino acids and proteins:  - Urease - Indol - Gelatinase  
• Degradation of turnesol milk (proteolysis + fermentation of lactose)  
• Reducing nitrates to nitrites  
• Reducing sulfites  
• antimicrobial susceptibility
o Only in Gram negatives ,sometimes Peptostreptococci (G+ always susceptible to
Penicillin!!!)  
o In anaerobiosis conditions  
o Concentration of CO2 Max.5%  (otherway we obtain false results for Macrolides
and Tetra cyclines)  
o Anaerobiosis must be ensured immediately  
o Need to use growth controls in anaerobiosis  
o Mueller – Hinton Medium supplemented with hemin and menadione, if needed
Wilkins-Chalgren; BBL (Blood Bile Lactose)  
o Reference technique: dilution in solid medium  
o Other methods currently accepted:
§ E-test  
§ Broth dilution  
§ Micro dilution  
o Disputable methods:
§ Disk-diffusion  
§ Disks in broth method (unique concentration)
o last generation techniques
§ Do not require isolation and identification by classical methods  
§ Based on use of molecular probes and nucleic acid amplification: PCR  
§ Disadvantages:
• Do not use data on viability  
• Do not provide data on expression of detected genes  
§ Importance:
• Rapid orientation of diagnostic  
• Taxonomy studies  
• of virulence genes, antimicrobial resistance genes, genes coding
for toxins etc.  
• Epidemiological studies  
• Ecological studies  
§
67. Gas gangrene clostridia - general features(main species, habitat, morphology, cultural and
§ Main species
o Gram positive
§ Perfringens  - most common
§ Oedematiens
§ Septicum 
§ Histoliticum
§ Sporogenes
§ Fundiliformis  
§ Habitat
o Found in soil, skin, cavities, digestive tract
o Every ecological niche meeting nutritional and anaerobiosis requirements:
Humans and animals: natural cavities – comensal and/or symbiotic (e.g.
Propionibacterium/Vit. B12 source)
o Clostridia 10-100:1 aerobe microorganism in upper respiratory tract, vagina,
pylosebaceous units  
o Anaerobes 1000: 1 aerobe microorganism in colon
o Food
o environment  
§ Morphology
o Gram positive rods
o Spores
o Sometimes in diplo, sometimes disposed as capital letters or small palisades
§ Cultural and biochemical features
o Perfringens has double hemolysis on blood agar
§ Antigenic structure
68. Gas gangrene clostridia – pathogenicity features

§ When favoring factors, affecting Oxygen exchanges are present
o Deep wounds or anatomic sites near cavities heavily colonized with anaerobe
microorganisms  
o Human bite wounds  
o Tissue distruction by tumours, trauma, complex  surgery  
o Diabetes/acidosis  
o Aspiration pneumonia; endocarditis  
§ Antimicrobial therapy with antibiotics which are inactive on anaerobe microorganisms
(Aminoglycosides, Quinolones, Sulphamides)  
§ Bacteria manifest several pathogenicity factors: toxins, enzymes, adhesins, capsule etc.
§ Non sporogenous bacteria may cause infections only if both conditions are met:
o only in immunosuppresed microorganisms,  
o if local anaerobiosis is present  
§
69. Treponema pallidum - general features(habitat, morphology, cultural features, antigenic

structure)
o Habitat
o Humans are the only hosts – infection with venereal transmission
o Morphology
o Very thin with dimensions: 0.3/0.8-15 micrometers
o Helicoidal shape
o Motile – rotation around the axis
o Cell wall structure similar to gram negative
o Cultural features
o NOT cultivatable
o Antigenic structure
o Cardiolipids or Wasserman lipid hapten
§ Along with treponemic protein it determines production of antibodies
o Antigens specific for each grou0p
o Poliozidic antigen used in immunoflouresence reactions
o Somatic treponmic antigen
70. Pathogenicity features of Treponema pallidum. Immunity in syphilis.

o causes syphilis
o an infection with venereal transmission (humans are the only hosts)
o an infection that can be divided into more stages which has implications for diagnostic
and treatment (early stage/ primary syphilis, late stages/secondary and tertiary syphilis)
o in some stages the disease may be asymptomatic (latent syphilis)
o there are problems with the diagnostic of very early syphilis, neurosyphilis,
asymptomatic congenital syphilis
o cannot be grown in culture!
o Factors
o External protein membrane has a role in adherence to the host cell
o Hyuluronidase, lytic enzymes and factors that give mobility for penetraction into
cells and blood vessels
o Bacteria may stay in latent stage intracellularly for years and certain
circumastances can spread them in other anatomical regions
o Pathogeny
o Most often, t. pallidum penetrates an organism through intact membranes or
small cutaneous lesions, then quickly arrive in lymphatic circulation or blood
circulation causing a systemic infection
o Interval between exposure and the first lesions is 10-90 days with an average of 3
weeks
o Immunity
o 3rd stage lesions are caused by a late cellular response.
§ This response presents T lymphocytes and macrophages which can result
in formation of ulcerations and necrosis
o Humoral immune response causes formation of antibodies for treponema and non
specific regains
o Immunity isn’t complete, humoral and cell mediated responses can stop
formations of new primary lesions but its not sufficient in eliminating treponema
from the organism
71. Laboratory diagnostic of syphilis

o Sampling
o Direct detection
o in fluids/tissues from lesions of primary or secondary syphilis
o dark-field microscopy (wet mount – characteristic morphology and motility of live
treponemes)
§ advanatage is simplicity, speed and sensibility of approximately 80%
o direct fluroescent antibody testing (detects Ag) *PCR
o Serological tests (detection of Abs against T. pallidum)
o Nontreponemal tests (detects antiphospholipid/anticardiolipin Abs) - use
§ Become positive 1-4 weeks ager the appearance of the primary chancre and 6
weeks ager the exposure .
§ Use the basic antigen formula of Venereal Disease Research Laboratory
(VDRL) test (standardized amounts of cardiolipin, cholesterol, and lecithin).
§ Are rapid, simple, and inexpensive.
§ Are recommended to monitor the course of infections during and ager
treatment and to detect reinfection.
§ Limitations:
• have reduced sensitivity in primary and late latent syphilis
• false reactions (positive – cross-reactivity; negative-prozone
reaction)
§ Bordet-Wasserman test (complement-fixation test)
§ VDRL test (flocculation test)
§ RPR (Rapid Plasma Reagin) test, etc.
o Treponemal tests 
§ TP-PA (T. pallidum particle agglutination)
§ Are used mainly as confirmatory tests to verify reactivity in nontreponemal
tests.
§ In population with low disease prevalence can be used for screening!
Limitations:
§ May remain reactive for years with or without treatment and the Ab titres
correlate poorly with disease activity -> should not be used to evaluate
response to therapy, relapse or reinfection in previously treated patients.
§ Are technically more difficult to perform and more expensive.
§ False positive reactions can occur (less common than for former tests).
§ False positive reactions due to other medical conditions than syphilis (e.g.
some viruses/mononucleosis, hepatitis, drugs, pregnancy, autoimmune
disorders/rheumatic fever, rheumatoid arthritis, lupus, etc).
§ False negative reactions due to the prozone phenomenon
§ TPHA (Treponema pallidum hemagglutination assays) FTA-ABS
(fluorescent treponemal antibody absorbtion test)
§ EIA
72. Genus Leptospira - general features (main species, habitat, morphology, cultural features,
main diseases produced)
o Main species
o L. interrogans – the pathogenic species 
o L. biflexa – the nonpathogenic species (exist in water and soil)
o Habitat
o Sites of entry for pathogenic leptosires mucosa and broken skin (no lesions
appear) and direct or indirect contact with urine containing virulent leptospires.
o Morphology
o flexible helical rods that are actively mobile
o have 2 periplasmic flagella, one originating at each end of the cell with the free
ends extending toward the center of the cell and not overlapping
o Cultural features
o Obligate aerobes
o isolation in culture (grow slowly) e.g. various culture media (contain long chain
fatty acids, vitamins B1 and B12, ammonium salts) specimens: blood, urine, CSF
o Antigenic classification
o L. interrogans is divided into approx. 250 serotypes (arranged in serogroups)
Immunity is serotype specific!
o Diseases produced
o Leptospirosis
§ Zoonotic disease with broad spectrum of animal hosts
o The central nervous system (meningitis), kidneys (nephritis, renal failure), and
liver (hepatitis/icteric leptospirosis) are the organ systems most frequently
involved in infection due to bacteremia (during acute, leptospiremic phase of the
disease)
73. Genus Rickettsia - general features(main species, habitat, morphology, cultural features,
antigenic structure, diseases produced)
o Main species
o spotted fevers group: R. rickettsii, R. conorii, R. akarii, R. sibirica, R.
australis, R. japonica
o typhus group: R. prowazekii, R. tiphy
o morphology
o Dimensions : 0.3 – 0.6 µ m / 0.8 -2 µ m  
o Protective capsule – resistance in the environment  
o General
o Susceptible to tetracyclines, macrolides, quinolones  
o Intracelular parasitism
o both DNA and RNA
o Possess cytoplasmic membrane 
o Multiply by binary division 
o Possess independant metabolic activity
o Possess rigid cellular wall
o Habitat
o reservoir : animals, arhtropods (ticks)
o transmission : tick bite
o geographycal areal : Mediterranean basin, Africa, India, few cases in the
south-eastern part of Romania
o Transmitted from animals to humans by arthropods (except Coxiella burnetii)
o Rickettsioses are “zoonoses” – diseases in animals which may affect humans
o Broadly spread worldwide
o Rickettsia are genetically related (except Ehrlichia, Bartonella and Coxiella
burnetii)
o Cultural features
o Cultivable only on eukariotic cells – celular linea, embrinated eggs,
laboratory animals (except B. quintana)
o capsule
o Diseases produced
o Rickettsia conorii – boutonneuse fever
§ Clinical manifesations: - macular-papular skin rash
§ eschar (“tache noire”) at the tick bite site - 50% of cases
§ fever
§ microvascular damage (periferal vasculitis)
§ structurally and clinically very close to Rocky Mountain spotted
fever (R. rickettsii).
o Rickettsiosis
§ Encephalitis
§ Pneuomonitis
§ Rash: pox and eschar
§ Nausea and vomiting
§ Kidney failure
74. Genus Chlamydia - general features(main species, habitat, morphology, cultural features,
o Main species
o C. trachomatis
o C. suis
o C. muridarum
o Habitat
o High humidity zones – in humans and animals: preference for mucosa of
urogenital tracts, respiratory tracts and ocular conjunctive
o Obligate intracellular
o Morphology
o Obligate inctracellular bacteria that appear as cellular inclusions
o 0.3 micrometers
o Cultural features – Giemsa/IF
o detecting elementary bodies (EB)
o the only test accepted in forensics 
o transport in special medium at 2-4°C 
o allowed keeping time until examination: 24-48 hrs
o specificity : 100% 
o reduced sensitivity: approx. 55% 
o only in reference laboratories 
o time consuming
o
o Main diseases produced
o C trachomatis
§ Trachoma
§ Inclusion conjunctivitis
§ Non gonoccocal urethritis
§ Salpingitis
§ Cervicitis
§ Lymphogranuloma venereum
§ In pregnant woman infection with C. trachomatis may cause:
• spontaneous abortion - ectopic (extrauterine) pregnancy -
postpartum endometritis
• In new-borns infection with C. trachomatis may cause : -
conjunctivitis (at 5-12 days after birth)
o pneumonia (acute pneumonia without fever at 1-3
months after birth)
o oro-pharyngeal infections and infections of the genital
tractus
§ Pregnant women affected by C.trachomatis may be treated with
erythromycin, azithromycin or amoxicillin and need to be retested after 3
weeks from completion of treatment
§ Recommendation: all people who suffered from a C. trachomatis
infection have to be retested after 3-4 month
§ C. trachomatis is the most frequent cause of non-gonococcal urethritis in
men.
§ C.trachomatis infections are asymptomatic, especially in women, in
variable proportions (after several authors as much as 85%) → important
to early detect infection.
§ Yearly screening of women in the 20-25 age group is recommended in
USA (aprox. 2000000-3000000 new cases yearly).
•
o C. suis
o C. muridarum
75. Genus Mycoplasma - general features (main species, habitat, morphology, cultural features,
o Main species
o Mycoplasma pneumoniae
o Mycoplasma genitalium
o Mycoplasma hominis
o Habitat
o Humans and animals
o Ubiquitous
o prefer humidity
o Morphology
o 0.3 micrometers in coccoid forms
o 100/0.4 micrometers in filmentous forms
o fried egg appearance
o no cell wall
o has cell membrane with adhesins
o Cultural features
o several species need cholesterol - need fatty acids, amino acids and nucleic acids
precursors - produce free radicals: Hydrogen peroxides, superoxides etc  
o mycoplasma pneumoniae
§ culture on solid and liquid media
o urogenital mycoplasma
§ culture on liquid media with pH indicators
§ urease positivve
o Main diseases produced
o Mycoplasma pneumoniae
§ Atypical pneumonia  
§ Vasculitis  
§ Arthritis  
§ Anemia etc.  
o Uro-genital Mycoplasma  
§ Non-gonococcal urethritis  
§ Salpingitis  
§ Abortion  
§ Infertility  
§

Genus Staphylococcus - General Features (Main Species, Habitat, Morphological Features)

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Genus Staphylococcus - General Features (Main Species, Habitat, Morphological Features)

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SUBJECTS FOR THE FINAL EXAM

2. Genus Staphylococcus – cultural and biochemical features, antibiotic susceptibility

3. Genus Staphylococcus – pathogenicity factors and main diseases produced

4. Steps of the laboratory diagnostic of staphylococcal infections

5. Genus Streptococcus – cultural features and classification according to the type of

6. Genus Streptococcus – antigenic structure and classification according to it

7. Genus Streptococcus -pathogenicity factors and main diseases produced

8. Post-streptococcal diseases – enumeration, mechanisms

9. Steps of the bacteriological diagnostic of streptococcal infections

10. Serological diagnostic of streptococcal infections – ASO method(principle, interpretation,

• Important for monitoring of strep A elimination and poststreptococcal diseases

12. Streptococcus pneumoniae – biochemical features and antigenic structure

13. Streptococcus pneumoniae –pathogenicity factors and main diseases produced

16. Steps of the laboratory diagnostic of meningococcal infections

18. Steps of the laboratory diagnostic of gonococcal infections

19. Microscopical features of medical important cocci

20. General features of Enterobacteriaceae(main genera / species, habitat, morphology, cultural

21. Antigenic structure of Enterobacteriaceae (types of antigens, chemical structure,

24. Pathogenicity features of Enterobacteriaceae

25. Genus Escherichia – pathogenicity features, main diseases in which it is involved

26. Steps of the laboratory diagnostic of Escherichia coli infections

o Calibrated loop method

29. Genus Salmonella – antigenic structure

30. Genus Salmonella – pathogenicity features, main diseases in which it is involved

33. Serological diagnostic of typhoid fever

Serological identification principles:

Ideally, sampling to be done within 30 min before fever pick

35. Antigenic structure and classification of Shigella

37. Laboratory diagnostic in bacterial dysentery caused by Shigella

39. Genus Klebsiella – pathogenicity features, main diseases produced

40. Laboratory diagnostic in Klebsiella pneumoniae infection

43. Pseudomonas aeruginosa–pathogenicity features, main diseases produced, antibiotic

44. Steps of the laboratory diagnostic in Pseudomonas aeruginosa infections

46. Vibrio cholerae – pathogenicity features, diseases produced, antibiotic susceptibility

47. Steps of the laboratory diagnostic of cholera

48. Haemophilus influenzae -general features(habitat, morphology, cultural features, antigenic

50. Laboratory diagnostic of Bordetella pertussis infections

51. DTP vaccine – composition, administration scheme

52. Mycobacterium tuberculosis - general features (habitat, morphology, cultural features)

53. Mycobacterium tuberculosis – chemical composition and antigenic structure

54. Immune response in tuberculosis infection, ways of assessment

55. Mycobacterium tuberculosis – drug susceptibility

56. Laboratory diagnostic in tuberculosis

60. Bacillus anthracis - general features(habitat, morphology, cultural and biochemical

61. Laboratory diagnostic of anthrax infection

63. Clostridium tetani - general features(habitat, morphology, cultural and biochemical

64. Clostridium tetani infection – pathogenic mechanism, preventive measures

66. Clostridium botulinum infection – pathogenic mechanism, diagnostic

68. Gas gangrene clostridia – pathogenicity features

69. Treponema pallidum - general features(habitat, morphology, cultural features, antigenic

70. Pathogenicity features of Treponema pallidum. Immunity in syphilis.

71. Laboratory diagnostic of syphilis

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