Medically important bacteria

Special bacteriology
Bacterial taxonomy

Domeniul
Tipul
Clasa
Ordinul
Familia
Genul
Specia
 Classification according to Gram staining
• Gram-positive cocci
• Gram-negative cocci
• Gram-positive rods
• Gram-negative rods and coccobacilli
• Bacteria not stained by Gram
– acid and alcohol resistant bacteria
– cell wall lacking bacteria
– spirochaetes
– strictly intracellular bacteria

3
Gram-positive cocci
Staphylococci and related Gram-positive
cocci (GPC)
= catalase-positive GPC
 Staphylococcus genus
• Gram-positive spherical cells
• arranged in grape-like irregular clusters (staphylos =
grape)
• several species (more than 40)

6
 Habitat

• present on the skin and mucous membranes of humans and

animals

• some staphylococcal species are named after the site they colonize:
– S. auricularis
– S. capitis

7
 Classification
according to the production of free coagulase:
– coagulase-positive staphylococci
• S. aureus
• S. hyicus-intermedius group
– coagulase-negative staphylococci (CNS) -
• S. epidermidis group
– S. epidermidis
– S. haemolyticus
– S. hominis
– S. capitis
– S. caprae
• S. saprophyticus group
– S. saprophyticus
– S. xylosus
– S. lentus
• S. simulans group
• S. lugdunensis

8
 Morphology
• spherical
• size: 0,8-1 µm
• no flagella, no spores
• capsule may be present

9
Staphylococcus spp.- Gram
stain
• Gram-positive
cocci:
– in clusters
– isolated cocci
– diplococci
– short chains

10
 Cultivation

• staphylococci grow readily on most media

• aerobic, facultative anaerobic bacteria
• optimal growth temperature: 37°C (10-42°C)
• incubation:18-24 hours
• resistant to elevated salt concentration (7,5% NaCl)
– selective isolation of staphylococci from specimens with mixed flora on mannitol salt agar

11
 Colony morphology

• relatively large, creamy S type colonies

• colonies might be pigmented due to a non-diffusible pigment

• some staphylococci produce β haemolysis on blood agar plate

12
Staphylococcus spp. on blood agar plate - β
haemolysis

13
Pigmented S colonies

14
Pigment production +/-

15
 Biochemical properties

• all staphylococci are catalase positive

• in case of medically important staphylococcal species free coagulase is produced only by S. aureus

• species differentiation among coagulase-negative staphylococci is based on sugar fermentation and

other tests

16
 Staphylococcus aureus
• most important species
• major pathogen for humans
• may be present as a colonizer - healthy carrier status
– typical sites of S. aureus colonization:
• anterior nares – reservoir in humans
• pharyngeal mucosa
• skin (axillary, perineal, groin region)
• carrier rate: 10-40%

17
 Pathogenesis
• Portal of entry
– skin lesions (including surgical wounds), hair follicles
– gastrointestinal tract
– respiratory tract
– vaginal mucosa

18
 Virulence factors
• Surface proteins
– clumping factor
• adherence of the organism to fibrinogen and fibrin (endocoagulase)
• when mixed with plasma on a slide, S. aureus forms clumps
• Enzymes
– Coagulase, fibrinolysin
– Hyaluronidase
– DN-ase
– Lipolytic enzymes
– others

19
 Factors that inhibit phagocytosis
• Microcapsule
• Protein A
– binds to the Fc portion of IgG molecules
– role in the diagnosis of S. aureus
– spa – gene encoding protein A – molecular typing
• Leukocidin
• Coagulase – forms a fibrin coat around the infectious focus that stop
phagocytic cells
• Clumping factor: masks surface antigens

20
 Exotoxins
• α-, β-, γ-, δ-toxins (haemolysins) – lysis of erythrocytes through pore
formation on their membranes
• leukocidins – destroy leukocytes (e.g. Panton-Valentin leucocidin - PVL)
• superantigens: non-specifically activate large numbers of T-cells → large
amounts of cytokins are released
– toxic shock syndrome toxin (TSST-1)
– enterotoxins (A-E, G-I, K-M) – heat-stable
– exfoliative toxins A, B – epidermolytic toxins (separation of the epidermis at the granular
cell layer)

21
 Types of infections:

• localized infections
• systemic infections
• toxic syndromes

22
 Diseases
Typical pyogenic infections: skin infections such as furuncle or other localized
abscesses (focal suppurations), hair follicle infection
- localized, painful inflammatory reaction

23
Furuncle (boil)
Carbuncle
 Diseases
– impetigo – skin disease
 Other infections caused by
S. aureus
• other soft tissue infections – mastitis, wound infections (most frequent cause
of surgical site infections)
• osteomyelitis - leading to necrosis of the bone and chronic suppuration
• arthritis
• pneumonia – HCAI, especially in ventilated patients
• sinusitis, otitis, mastoiditis
• meningitis
 Generalized diseases
• from any one infection focus organisms may spread via
the lymphatics and bloodstream to other parts of the body
– endocarditis
– sepsis – secondary suppurative foci in any organ = metastatic
abscesses with the typical signs of the affected organ
 Toxin-mediated staphylococcal diseases - 1
• Food poisoning – due to enterotoxin producing strains (growing in
carbohydrate and protein foods)
– enterotoxin: heat-resistant (not inactivated by short cooking)
– short incubation period
– violent nausea, vomiting and diarrhea
– rapid convalescence
– no fever
Toxin-mediated staphylococcal diseases - 2
• Toxic shock syndrome (due
to TSST)
– toxic shock: fever,
hypotension, diffuse rash
(desquamate), involvement of
liver, kidneys, central nervous
system, blood, etc.
• tampon-using menstruating
women
• wound infections
• nasal packing used to stop
bleeding from the nose
 Toxin-mediated staphylococcal diseases - 3
• Scalded skin syndrome – exfoliative toxin
– fever, large bullae, serous fluid exudes, electrolyte imbalance
– young children
 Nosocomial infections

• S. aureus is an important cause of nosocomial infections

– intravascular catheters, urinary catheters, prosthetic valves, prosthetic joint infections
– surgical site infections
– ventilation pneumonia

• HA-MRSA – healthcare associated methicillin-resistant S. aureus – efficient spread in

hospital environment via hands
– hospital epidemics

32
 Emerging MRSA strains:
community-acquired MRSA (CA-
MRSA)
• different genetic background
• higher virulence, better bacterial fitness
• PVL production – necrotizing pneumonia
• children, young adults may be affected (serious, life-threatening infections)
 Immunity in staphylococcal
diseases
• there is no long-lasting immunity
• reinfection is possible

34
 Epidemiology

• main sources of infections: human carriers, fomites

• transmission:
– contact spread
– from human to human via hands, fomites

35
 Prevention
• no vaccine
• hand hygiene, aseptic management of lesions
• high risk areas in hospitals:
– newborn nursery
– operating rooms
– intensive care units
– cancer chemotherapy wards
• high-risk patient and personnel screening
• isolation policies – infection control policies,
• “search and destroy” policy: newly admitted patients are quarantined until MRSA colonization
is excluded
– for this approach to be efficient:
• rapid diagnostic methods are to be used (PCR)
• low prevalence of MRSA

36
 Prevention
• screening for S. aureus colonization
– in patients for whom surgical interventions (mainly in orthopedics, thoracic
surgery) are planned
– in close communities in epidemiological context

– samples: at least 3 sites

• nasal swab +
– skin swabs (axillar, inguinal swabs)
– pharyngeal swab (detects long term colonization)
• swabs must be moistened with sterile saline
 Treatment

• According to antibiotic susceptibility testing results

• Infections due to methicillin-susceptible strains (MSSA) are well treated with
oxacillin (or cloxacillin), antistaphylococcal cephalosporins (I, II generation)

• empiric treatment of MRSA infections in high endemicity settings: according to

the susceptibility profile of the local strains
– vancomycin, linezolid for severe infections

• S. aureus is highly adaptable and able to develop resistance mechanisms

through mutations, plasmid acquisition

38
 Diagnosis
• rapid diagnosis of MRSA
– polymerase chain reaction based methods (results in a few
hours), chromogenic agar media
• conventional bacteriology:
– cultivation
– identification

39
Coagulase-negative staphylococci (CNS)

• part of the normal flora

• opportunistic pathogens
 S. epidermidis
• typical reprezentative of CNS
• disease: in case of risk factors
– immune deficiency
– presence of invasive medical devices (intravascular catheters,
prosthetic devices)
• slime production – biofilm on the surface of foreign materials

41
 S. saprophyticus
• inhabits the skin surrounding the genitourinary tract
• young women – acute cystitis (honeymoon cystitis)
• strong adhesion
• intense urease production: NH3 release, irritation - painful
• exoprotein (haematuria – presence of blood in urine)
• diagnosis: novobiocin resistance

42
Diagnosis of staphylococcal infections
Streptococci, enterococci and
streptococcus-like bacteria
Catalase negative group of Gram-positive
cocci
Streptococcus genus
 Streptococcus genus
• Gram-positive cocci in chains
• streptos=chain
• large and heterogeneous group of bacteria
• widely distributed in nature
– some are member of the normal flora
– others are associated with important human diseases
• due to infection with them
• due to sensitization to them
• Aerobic, facultative anaerobic (some species favor
microaerophilic conditions)
• some species are strictly anaerobic

47
 Classification
• according to group-specific C carbohydrate contained in the cell wall
(Lancefield classification)
– A-U
• medically important groups:
– group A streptococci (GAS) – Streptococcus pyogenes
– group B streptococci (GBS) – Streptococcus agalactiae
– group C, G streptococci (Streptococcus dysgalactiae and others)
– group D streptococci (Streptococcus bovis, Streptococcus gallolyticus)
• according to hemolysis
– β-hemolysis (GAS, GBS, GCS, GGC)
– α-hemolysis (Streptococcus pneumoniae, viridans streptococci)
– non-hemolytic streptococci (Streptococcus group D)
 Morphology
• spherical or oval
• Size: 1 µm
• no flagella
• no spores
• capsule (S. pneumoniae -
polysaccharide, S. pyogenes –
hyaluronic acid)

49
 Cultivation

• fastidious bacteria (of various degrees)

• 37°C, 18-24 hours
• blood agar plate
• small colonies, usually do not form confluent culture

50
 Biochemical properties

• catalase negative
• ferment different sugars
• resistant to bile (exception: S. pneumoniae)

51
 Antigenic structure
• C carbohydrate
– group specific
– Lancefield classification – precipitation or agglutination reaction
– diagnostic importance
• M protein
– type-specific
– GAS: includes 80 serotypes according to M protein
• protective antibodies are formed against M protein
– important virulence factor

52
 S. pyogenes (group A
streptococci)
• susceptible to bacitracin – specific to A group
• M protein – over 200 serotypes
– in certain diseases certain serotypes are predominant
• rheumatogenic
• nephritogenic

53
 Streptococcus pyogenes

Smear from pure culture: Gram- Blood agar plate: β-hemolytic

positive cocci in chains colonies, their diameter must be
greater than 0.5 mm
54
 Transmission
• droplet transmission – main route of transmission
• small airborne particles (e.g. dust) (airborne transmission)
• skin-to-skin contact (direct contact)
• surfaces (indirect contact)
• bedding and fabrics (indirect contact)
• food (vehicle)
• insects (biological vectors)

Both the infectious and carrier state may lead to transmission, dominated by the infectious state.
A few studies have reported Strep A transmission from carriers to uninfected individuals who have
subsequently become symptomatic.

Barth et al. Systematic Reviews (2021) 10:90

 Pathogenesis
• GAS cause disease by:
– pyogenic infections
– exotoxin production
– immunologic mechanism
 Virulence factors
• Inflammation related enzymes

– hyaluronidase – spreading factor, as it degrades hyaluronic acid, which is the ground

substance of subcutaneous tissue
– streptokinase (fibrinolysin): dissolves fibrin
– streptodornase (DN-ase) – antigenic; antibodies have diagnostic importance

57
 Virulence factors: toxins
• erythrogenic toxin (superantigen)
– rash – scarlet fever
– produced only by certain strains lysogenized by a phage containing the gene of the toxin
– antigenic – antibodies are protective
• streptolysin O (SLO)
– oxygen-labile hemolysin
– antigenic; antibodies: ASLO – diagnostic importance
• streptolysin S
– oxygen-stable
– not antigenic; responsible for hemolysis on blood agar plate
• pyrogenic exotoxin A - streptococcal toxic shock syndrome
• exotoxin B: preotease – involved in the pathogenesis of necrotizing fasciitis
 Virulence factors – surface proteins
• M protein - gives the streptococcus the ability to resist phagocytosis by polymorphonuclear leukocytes
in the absence of type-specific antibodies

Thin section electron micrograph of a chain of streptococci showing the

surface M protein. Magnification 50,000x

The appearance of the M molecule on the surface of the streptococcal cell wall.
Feretti JJ et al: Streptococcus pyogenes Basic Biology to
Clinical Manifestations, 2017
 Virulence factors – pili
• colonization of human tissues

• bind to salivary glycoproteins, which result in bacterial autoaggregation when

exposed to human saliva

• future targets for vaccine development

 Diseases – according to portal of entry
• Skin infections
– erysipelas
– impetigo
– cellulitis, myositis
– necrotizing fasciitis
– lymphangitis
Erysipelas
Cellulitis, flictens, necrotizing fasciitis
 Diseases – according to portal of entry
• respiratory tract
– pharyngitis – SGA is the most common bacterial cause of sore throat
• inflammation, exudate, fever, leukocytosis, tender cervical lymph nodes
• it may extend to
– otitis
– sinusitis
– mastoiditis
– retropharynx (retropharyngeal abscess)
– meningitis
• scarlet fever – erythrogenic toxin producing strains – rash
Streptococcal pharyngitis
Streptococcal pharyngitis
Scarlet fever
Rash

Strawberry tongue
Diseases – according to portal of entry

• Genital infections
– endometritis and sepsis after delivery
 Streptococcal diseases

• tendency for invasion,

spreading
• sepsis, meningitis
• toxic shock syndrome
 Postsreptococcal (nonsuppurative) diseases

• a localized streptococcal infection is followed weeks later by inflammation in an

organ which was not infected

• immunological response

• acute glomerulonephritis
• acute rheumatic fever
 Acute glomerulonephritis
• usually after skin infections

• symptoms
– hypertension
– edema of the face and ankle

• antigen-antibody complexes deposited on the glomerular basement

membrane (hypersensitivity type III)
Acute rheumatic fever
• 2-3 weeks after pharyngitis (skin infections do not cause
rheumatic fever)
• approximately 20% of young children recollect
pharyngitis

• symptoms:
– fever
– painful migratory polyarthritis
– carditis (myocardial and endocardial tissue are damaged –
mitral and aortic valve)
– chorea (uncontrollable movement of limbs)

• ASLO titers are high

• recurrences exacerbate the disease
• cross-reacting antibodies (hypersensitivity type II)
Erythema marginatum
Poststreptococcal reactive arthritis vs acute
rheumatoid fever
Reactive arthritis Acute rheumatoid fever
• polyarthritis whith a recent evidence of streptococcal • Jones criteria fulfilled
infection and no other major Jones criteria. – 2 major or
– 1 major and 2 minor criteria or
• develops within 10 days of streptococcal infection
– 3 minor criteria
• arthritis
• migratory polyarthratis
– non-migratory
• responds to NSAID
– non-responsive to aspirin/non-steroidal anti-
inflammatory drugs,
– longer duration (2 months)
• risk of carditis in children: approx. 5-8% (1-18 months after
arthritis)
• adults: no risk of carditis
• 1 year antibiotic prophylaxis
 Jones criteria
Major Minor (in low-risk population)
• Carditis, clinical and/or subclinical (ie, • Polyarthralgia
detected by echocardiography) • Fever ≥38.5°C
• Arthritis • Acute phase reactions: Erythrocyte
• Chorea sedimentation rate (ESR) ≥60 mm in
• Erythema marginatum the first hour and/or C-reactive protein
• Subcutaneous nodules (CRP) level ≥3.0 mg/dL
• Prolonged PR interval, after accounting
for age variability (unless carditis is a
• in low-risk populations arthritis must major criterion)
be polyarthritis
 Laboratory studies to confirm recent
streptococcal infection
• Elevated or rising streptococcal antibody titer; a rise in titer is better
evidence than a single titer result

• A positive throat culture for group A β-hemolytic streptococci

• A positive rapid group A streptococcal carbohydrate antigen test in a

child whose clinical presentation suggests a high pretest probability of
streptococcal pharyngitis
 Immunity against GAS
• antibacterial immunity
– type-specific

• antitoxic immunity
– TSS, erythrogenic toxin

76
 Epidemiology

• carrier state (pharynx, skin)

• S. pyogenes may be shed for a while – after cure

• loss of M proteins during long-term carriage: loss of virulence – no point for searching GAS
carriage (except close family members of a patient with rheumatic fever)

77
 Treatment of streptococcal
infections

• Pharyngitis: wait for the lab result and treat only if positive! this way antibiotic overuse may
be prevented;
• Penicillin (10 days needed) – there is no documented resistance to penicillin
• Cephalosporins (5 days)
• In case of allergy: macrolides (susceptibility should be tested)

78
 Diagnosis

• Rapid diagnosis from throat swab: antigen detection

• Conventional diagnosis
– cultivation (high sensitivity)
– identification

79
 S. agalactiae (group B
streptococcus)
• Morphology same as for GAS
• Colonies are larger, grayish, surrounded by hemolysis
• positive CAMP test
• virulence factors
– M protein
– Lipoteichoic acid
– hemolysin

80
 GBS
• Found in digestive tract and vagina (35% of women are carriers)
• Diseases during pregnancy
– abortion
– early membrane rupture
– Intrauterine infection
– puerperal fever
• Diseases of the neonate
– early onset sepsis, meningitis
– late onset sepsis
• Diagnosis
– cultivation – in case of infection
• Prevention
– screening of pregnant women – 36th gestational week (do not treat colonized women!) – give
penicillin or ampicillin prophylaxis during delivery to prevent neonatal infection
• Treatment of established infections: penicillin

81
 Group D streptococci
• bovis group, includes Streptococcus gallolyticus (involved in sepsis,
endocarditis – patients developing systemic infections should be searched for
colon cancer)
 S. pneumoniae
• Morphology
– Gram-positive diplococci, chains
– lance shaped cocci
– Capsule – (mainly in vivo)
• Cultivation
– Blood agar plate – 1-3 mm colonies (S, R, M)
– α hemolysis
• Identification
– susceptible to optochin, bile

83
Streptococcus pneumoniae

LL 84
Streptococcus pneumoniae
- immunofluorescent assay

85
Sputum – Gram stain

86
 Sputum – Gram stain

staphylococci

pneumococci

2005 LL 87
Neufeld reaction – quellung
reaction (antigen-antibody
reaction)

88
 Susceptibility to optochin

2005 LL 89
• Antigenic structure
– capsule: type specific polysaccharide
– over 85 serotypes

• Virulence factors
– Capsule – antiphagocytic, several serotypes defined according to
its structure
– Neuraminidase – toxic effect
– Pneumolysin - hemolyzin, cytotoxic
– Hyaluronidase

90
 Diseases

• present in the normal flora - 50-70% of population (IgA production enables colonization of the
upper respiratory tract mucosa)

• Pneumonia – most common cause of community acquired pneumonia (CAP)

• Bacteriaemia, sepsis
• Meningitis
• Otitis media, sinusitis
• Primary peritonitis (girls)
• Eye infections (ulcus serpens corneae)

91
 Streptococcus pneumoniae

• Prevention
– Polysaccharide vaccine – for adults (includes 7 or 23 serotypes)
• People older than 65 years
• Smokers
• Chronic underlying diseases
– Conjugate vaccines – for children
• (antigen + diphtheria toxoid)
• heptavalent (7), decavalent (10), triskaivalent (13)
– Herd effect!

92
 S. pneumoniae
• Treatment
– susceptibility to antibiotics must be tested
– decreased susceptibility to penicillin or even resistance may occur

• Diagnosis
– rapid diagnosis:
• detection of pneumococcal antigens from sputum, urine, LCR
• detection of nucleic acid from sputum
– conventional methods: cultivation, identification
 Viridans streptococci –
oropharyngeal streptococci
• Facultative/strict anaerobic
• α hemolysis
• Groups:
– group anginosus (S. anginosus, S. constellatus, S. intermedius)
– group mitis (S. mitis, S. oralis, S. sanguinis, S. gordonii and others)
– group salivarius (S. salivarius, S. vestibularis)
– goup mutans (S. mutans, S. sobrinus)

94
 Group anginosus
• involved in purulent lesions, deep abscesses of abdominal or oral origin, endocarditis
• cultures have a peculiar caramel smell
• S. anginosus, S. constellatus, S. intermedius
• most anginosus group isolates belong to the non-β-hemolytic oral streptococci, but β-
hemolytic strains are found in all 3 species.
• Some anginosus group strains carry a typeable Lancefield group antigen, which belongs to
group F, C, G, or A!
 Streptococcus mutans

– Caries (sugar fermentation – acid production – softening of

teeth structure, cavity formation)
– Infective endocarditis

96
 Streptococcus mutans

2005 LL 97
 Enterococcus genus
• normal flora of the gastrointestinal tract
• blood agar plate: grayish colonies
• α hemolysis +/-
• resistant to bile, decompose esculine
• low virulence
• high nosocomial potential
• difficult to treat infections – due to natural
resistance against several antibiotics

98
• Diseases
– urinary tract infections
– gallbladder infections
– wound infections
– bacteraemia – may indicate colon cancer
– endocarditis
• Diagnosis
– conventional methods are not enough for relible diagnosis and species level identification
• Treatment
– intrinsic resistance to cephalosporins, clindamycin, SXT + low-level resistance against
aminoglycosides (if high level resistance is excluded, gentamicin or streptomycin can be used
in association with other active antibiotics for their synergistic effect)

99
 Streptococcus-like bacteria
• Abiotrophia and Granulicatella
– “nutritionally variant streptococci”
– thiol-requiring, pyridoxal-requiring, and
“satelliting” streptococci, staphylococci or enteric
rods
– part of human microbiome
– occasionally involved in infections: sepsis,
endocarditis, CNS infections, early-onset
neonatal, ocular, lung infections…
– difficult to culture – cystein containing media can
support their growth
– more resistant than viridans group streptococci
– MIC is needed for AST
 Streptococcus-like bacteria
• Aerococcus spp
– found in the environment, including air, dust, soil, vegetation, meat products, and the hospital
environment
– can easily be confused with the viridans streptococci and enterococci
– tend to form tetrads when grown in broth
– Aerococcus urinae may colonize urinary tract – or cause infections (urinary and others)
• standardization in EUCAST

• Leuconostoc spp.
– use in the dairy and pickling industries and in wine making
– found in fermented sausages, vacuum-packed meat products, cereals, and dairy products (butter,
cream, fresh/raw milk, cheese)
– intrinsically resistant to vancomycin
– Leuconostoc bacteremia has been documented predominantly in patients with underlying malignancies
(acute myeloid leukemia, non-Hodgkin’s lymphoma, hepatocellular carcinoma) and as a complication
of solid-organ (e.g., liver) and stemcell transplantation.
 Streptococcus-like bacteria
• Pediococcus spp.
– may be found in beers and ales and are also used in foods for processing and preservation
– flavor enhancers in processed vegetables and soy products and are used in biotechnology
as indicator strains for vitamin bioassays
– may be isolated from various human clinical specimens, including stool, urine, wounds,
abscesses, and blood cultures.
– usually there are some underlying conditions - hematologic malignancies, cardiovascular
disease, chronic lung disease, pancreatitis, and diabetes
– previous abdominal surgery or nasogastric tubes or central venous catheters for total
parenteral nutrition in place for prolonged periods
– intrinsically resistant to vancomycin
 Streptococcus-like bacteria

• Gemella spp.
– characteristically appears as diplococci with
adjacent sides flattened, easily destained GPC
– Gemella species are infrequently isolated from
clinical specimens. Cultural similarity to viridans
streptococci has likely resulted in misidentifications
– G. haemolysans is part of the upper respiratory tract
flora, whereas G. morbillorum is found in the
respiratory and gastrointestinal tracts. Both
organisms have been isolated as occasional
causes of bacteremia and endocarditis involving
both native and prosthetic heart valves in pediatric
and adult patients
 Streptococcus-like bacteria
• Vagococcus spp. • Globicatella sanguinis
– motile Gram-positive cocci – animal species – =short chain composed of spherical cells
– Only V. fluvialis has been isolated from clinical – endocarditis, UTI and other infections (rarely
specimens obtained from humans. isolated)

• Alloiococcus spp.
– A. otitis – middle ear infections - middle ear
aspirates submitted for culture should be
incubated under microaerophilic conditions for at
least 5 days to facilitate detection of these
bacteria
– might be also isolated from blood, sputum