DETAILED

BACTERIOLOGY

Classification of bacteria

• Gram positive / Gram negative *

• Shape: coccus, rod (even or curved),

coccobacillus, spirochets

• Spore forming / non spore forming

• Aerobic / anaerobic

• Intracellular (maybe facultatively IC)

* Difficult to stain by Gram:

– Mycobacterium, Actinomyces (unevenly), Mycoplasma

(no cell wall!)

Taxonomy - I• Gram + coccus: Staphylococcus, Streptococcus

• Gram – coccus: Neisseria, Moraxella, Kingella

• Gram + rod:

spore froming: Bacillus, Clostridium

non spore forming: Corynebacterium, Listeria,

Erysipelothrix, Gardnerella

((Mycobacterium))

• Gram – rod:

enteral bacteria: E. coli, Salmonella, Shigella, Klebsiella,

Enterobacter, Serratia, Proteus, Morganella, Providencia,

Citrobacter, Yersinia

other fermenters: Vibrio, Aeromonas, Pesteurella

non fermenters: Pseudomonas, Acinetobacter, Steno-

trophomonas, Burkholderia

Taxonomy - II• Gram- coccobacillus: Haemophilus, Brucella, Francisella,

Bordetella, Legionella

• Intracellular (Gram - coccobacillus): Chlamydia (different cell wall); Mycoplasma, Ureaplasma (no cell wall!); Rickettsia, Coxiella, Ehrlichia, Bartonella

• Spiral: Helicobacter, Campylobacter;

Treponema, Borrelia, Leptospira

• Anaerobic:

Gram + rod: Clostridium (spore forming), Actinomyces, Mobiluncus, Lactobacillus (non spore froming)

Gram + coccus: Peptococcus, Peptostreptococcus

Gram – rod: Bacteroides, Fusobacterium, Prevotella, Porphyromonas

Gram – coccus: Veilonella

PYOGENIC COCCI

Gram + Gram –

Micrococcaceae family Streptococcaceae family Neisseriaceae family

Micrococcus Streptococcus Neisseria

Moraxella

S. pyogenes (A) Kingella

Staphylococcus S. agalactiae (B)

S. pneumoniae N. meningitidis

Enterococcus (D) N. gonorrhoeae

viridant group

S. aureus coagulase negative

staphylococci

(CNS)

Gram-positive cocci

Staphylococci,

Streptococci

Staphylococci

Staphylococcus aureus Coagulase-negative

staphylococci (CNS)

Staphylococcus epidermidis

St. saprophyticus

St. haemolyticus

St. hominis

St. lugdunensis

etc. (~40 species)

Gram staining of all species

• Most are harmless and reside

normally on the skin and

mucous membranes of

humans and other organisms

Cultivation

• very easy to grow

• facultative anaerobic

• big, round, slimy colonies

• resistant to

• heat (50 oC 30 min),

• drying,

• salt (9% NaCl)

• survive in hospitals !

Pigment production

Staphylococcus epidermidis:

white pigment

Staphylococcus aureus:

golden pigment

(staphyloxanthin)

-hemolysis

• S. aureus (+ S. haemolyticus + S. lugdunensis):

strong -hemolysis

Catalase test

• Aim: to differentiate Staphylococci

and Streptococci

• performance: adding a few drops of

H2O2 to the culture, strong bubbling

if positive

• H2O2 H2O + O2

Catalase +: Staphylococci

Catalase -: Streptococci

S. aureus virulence factors

I. cell-bound factors

• Polysaccharide capsule

• Protein A

– acts as an Fc receptor binding Fc portion of IgG

– inhibition pf phagocytosis

– biofilm formation!

• Clumping factor

– masking fibrin coat

• Teichoic acid (CWTA)

Protein A

Coagulase test -I

• Tube coagulase test: inoculating the bacteria

into citrate treated rabbit plasma, coagulation

occurs in a few hours (exocoagulase)

Coagulase +: Staphylococcus aureus

Coagulase -: all other Staphylococci (“CNS”)

-+

Coagulase test -II

• Clumping test - fibrinogen-coated particles

endocoagulase binds fibrinogen

(“Staphaurex”, „Pastorex”)

• performance: mixing bacteria with fibrinogen-bound

latex particles on microscope slide clumping of

bacteria (in a few seconds)

S. aureus virulence factors

II. Enzymes for invasion and spread

• Hyaluronidase

– breaks down proteoglycans in connective tissue

• Fibrinolysin (=staphylokinase)

– lyses fibrin clots (similar to streptokinase)

• Lipase

– degradation of fats and oils (facilitation of colonization of sebaceous glands)

• Nuclease (DNAse)

• Hemolysins (alpha, beta, gamma, delta)

– they are hemolytic toxins that destroy red blood cells, neutrophils, macrophages, and platelets

• Leukocidins

– destroy leukocytes (is the cause of necrotic lesions involving the skin or mucosa, including necrotic hemorrhagic pneumonia)

– Panton-Valentine leukocidin (PVL)

• present in all CA-MRSA

• no hemolytic activity

S. aureus virulence factors

III. Cytotoxins

S. aureus virulence factors IV. Exotoxins

• TSST-1 (toxic shock syndrome toxin)

– Superantigen!

– often associated with tampon use

– localised infection (vagina or wound), but toxin can

go through mucosa and can lead to MOF

– characteristic rash (sunburn like) usually seen at

onset

S. aureus virulence factors

IV. Exotoxins

• Exfoliative toxin

– Superantigen!

– SSSS = staphylococcal scalded skin syndrome (Ritter disease)

– protease activity of the exfoliative toxins causes peeling of the skin

– no inflammation (important in diagnosis!)

– mainly in small children

S. aureus virulence factors

IV. Exotoxins

• Enterotoxins (A-F)

– Superantigens!

– heat stable exotoxins which cause food

poisoning, resulting in nausea, vomiting and

diarrhoea

– resistant to cooking (100 oC, 30 min)

– C, D: dairy products

impetigo

furuncle

Staphylococcus aureus skin infections

• Folliculitis

– If on eye: hordeolum

• Furuncle

• Carbuncle

– bacteremia, fever

• Impetigo

folliculitis

hordeolum

Staphylococcus aureus skin infections

• Cellulitis

– infection of the deeper layers of skin and the tissues beneath

• Bullous impetigo

– Localised form of SSSS

Bullous impetigo

• Wound infections

– burns, postoperational, trauma

– from the skin flora

Staphylococcus aureus skin infections

Invasive infections

• Osteomyelitis– hematogenous spread or superinfection of trauma

• Endocarditis– following heart surgery (esp. valve replacement)

– 50% letality

• Pneumonia– predominantly affects people with underlying lung

disease including those on mechanical ventillation

• Meningitis– accounts for 1-9% of cases of bacterial meningitis

• Sepsis– half of all cases of inpatient sepsis

– in immunocompromised patients

Antibiotic therapy

• Skin infections: local treatment

• High resistance rates !!

• Antibiogram must be prepared

• Resistance to penicillin G: ~ 90%

• Methicillin resistance (MRSA):

– varies from country to country

– resistance to all beta-lactams & others

MRSA• Treatment of MRSA infections:

– Vancomycin !

– Others: linezolid, TMP-SMX, clindamycin, tertacycline, quinupristin/dalfopristin (Synercid), aminoglycosides

MRSA rates

MRSA % of invasive isolates in Hungary:

(data of the National Center for Epidemiology)

2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

4.7 9.0 14.9 16.7 19.9 25.1 23.6 23.0 28.6 30.1 27.0 25.1 24.0

Staphylococcus aureus carriage

• frequently found in normal flora of skin and nasal mucosa

• Carriage rates:– ~20% are long term carriers

– ~30% intermittently

– higher rates among • hospital staff and patients

• ekzema patients

• farmers

• where:– nasal and throat mucosa

– perinasal skin, hands

– perianal skin

Carrying MRSA

• Prevention of spread:

– Hand hygiene!

– Environmental cleaning / disinfection

• iodine, hexachlorophene

– Personal protective equipments

– Isolation of patients

– Regular screening of medical staff

• Decolonisation:

– Intranasal mupirocin

– Resistance is developing!

(Hungary, 2012: 4,5%)

Staphylococcus epidermidis

• Normal flora of the skin, facultative

(opportunistic) pathogen

• Mainly nosocomial infections!! Related to foreign

body:

– Prosthetic joints

– Prosthetic heart valves

– Sepsis from i.v. devices

– UTIs related to catheters

• Frequent skin contaminant in hemocultures!

• Th: vancomycin

Removal of an infected

prosthetic joint shows a

bacterial biofilm on an

infected arthroplasty.

A prosthetic joint with the

presence of antibiotic beads

TO PREVENT INFECTION

Other coagulase negative staphylococci

• S. saprophyticus:

– second frequent in UTIs after E. coli, esp. in young

women

– differentiation from S. epid: novobiocin resistance

– Th: ciprofloxacin

• S. hominis

– Skin flora; opportunistic pathogen

• S. haemolyticus

– Skin flora; opp. pathogen; inserted devices

• S. lugdunensis

– agressive endocarditis

S. epidermidis (S) and S. saprophyticus (R)

with novobiocin disc

Streptococci

Streptococcus pyogenes

Gram staining

Streptococcus pyogenes

Streptococci

• Streptococcus pyogenes

• Streptococcus agalactiae

• Streptococcus pneumoniae

• viridant streptococci

• Enterococcus genus

Grouping of streptococci

• Lancfield grouping (cell wall polysaccharide):

– S. pyogenes = A (GAS)

– S. agalactiae = B (GBS)

– Enterococci = D

• Based on hemolysis:

– β-hemolytic: S. pyogenes, S. agalactiae

– α-hemolytic: S. pneumoniae, viridant streptococci

Streptococcus pyogenes

• Gram-positive coccus,

arranged in chains

• smal, pin-point colonies

• fastidious!

• strong β-hemolysis

S. pyogenes infections

I. Respiratory tract

• pharyngitis („Strep throat”)

• tonsillitis follicularis

II. Skin infections

Erysipelas

severe impetigo

• erysipelas

• phlegmone

• impetigo

• cellulitis

II. Skin infections - 2

Flame-like spread

necrotising fasciitis

“flesh eating bacterium”

Other severe, acute,

pyogenic infections

• otitis media, sinusitis

• meningitis

• puerperal fever

Scarlat fever

Raspberry tongue

Peeling after scarlat fever

• rush oll over the body

• toxin of S. pyogenes in the throat

Post-streptococcal diseases

• rheumatic fever

• glomerulonephritis

• rheumatoid arthritis

ASO-titer!

• allergic origin

• a few weeks after the

acute infection

Treatment and prevention

• penicillin !!

• no vaccination

• scarlat fever chemoprophylaxis: penicillin

Streptococcus agalactiae

• Formerly only in animals (bovine mastitis)

• Since 1970’s: neonatal sepsis, meningitis No. 1

• In newborns:

– early manifestation (EOD)

• within 6 days

• risk groups (e.g. low weight)

• meningits, sepsis, pneumonia

– later manifestation (LOD)

• within 4 months

• meningitis, bacteremia, osteoarthritis

Str. agalactiae adult infections

• In pregnant women:

– 20-35% asymptomatic vaginal carriage

– newborns aquire in 50-75%

– screening in 35-37. week of pregnancy

– chemoprophylaxis: i.v. ampicillin during birth

• In non-pregnant adults: mainly >60 y

– skin- and bone infections, bacteremia, urosepsis, pneumonia

CAMP test

CAMP +: Streptococcus agalactiae

Str. agalactiae

St. aureus

• aim: Str. agalactiae diagnostics

• performance: Str. agalactiae and St. aureus

enhance each other’s β–haemolysis if

inoculated in a form of crossing lines

bacitracin sensitivity:

S. pyogenes: S

S. agalactiae: R

Streptococcus pneumoniae„pneumococcus”

• lobar pneumonia

• CAP (= comunity acquired pneumonia)

No.1. causative agent!

Other pneumococcal infections

• meningitis !!

• otitis media, sinusitis !!

• (peritonitis)

• ulcus serpens corneae

• Age specificity: <5 years and >60 years

• WHO: >1 million children die every year

Laboratory detection

• microscope: Gram + diplococci

• culture: blood agar, 5% CO2

incubator

– α-hemolysis, autolytic colonies

– optochin sensitive

– bile soluble

• capsule detection

– with antibody (capsule swelling test,

agglutination)

– capsular staining

Optochin sensitivity

Pneumococcal vaccines

• Pneumovax 23– Polysaccharide vaccine (MSD)

– for adults or >5y children

– 23 capsular antigens (=serotypes)

• Prevenar-13– < 2 y children (at 2, 4, 15 months)

– conjugated vaccine

– 13 serotypes:

4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F, 3, 6A, 19A

Total: 94 capsular antigens

(= 94 serotypes)

Pneumococcus asymptomatic carriage

• Carriers are the major sources of infection

• very frequent in small children attending DCCs,

cca. 30-40% carriage rate (peaks at ~3 y)

• decreases in adulthood

• seasonal changes

• different serotypes, sometimes multiple strains

• carriage duration: weeks -- months

• conjugate vaccines influence carriage as well

Enterococcus genus

Enterococcus on E67 medium

• normal intestinal flora

• microscope: in pairs or short chains

• culture: non-fastidious

• resistant to bile (40%), salt, high T (60oC 30 min)

• hemolysis: might be α, β or γ

• esculin hydrolysis

Enterococcal infections

• Nozokomial infections!– cystitis (chateter-associated)

– septis, endocarditis (venous canule)

– peritonitis, abdominal abscess

– 2nd most frequent Gram+ after staphylococci

• E. faecalis, E. faecium

• High-level resistance!

- vancomycin R (VRE)

- antibiogram must be prepared

- reservoir of resistance genes!!

Viridant streptococci

• S. mutans, S. mitis, S. sanguis, S. oralis, S.

salivarius

• α-hemolysis

• infections:

– subacute bacterial endocarditis (heart valves)

– due to major oral surgery, tooth extraction

– caries !

Caries (Streptococcus mutans)

Dental plaque

• Dental Caries is the destruction of the enamel, dentin or cementum of teeth due to bacterial activities.

• Caries are initiated by direct demineralization of the enamel of teeth due to lactic acid and other organic acids which accumulate in dental plaque.

• Lactic acid bacteria in the plaque produce lactic acid from the fermentation of sugars and other carbohydrates in the diet of the host.

• Streptococcus mutans and Streptococcus sanguis are most consistently been associated with the initiation of dental caries.

Peptostreptococci

• normal flora of oral cavity, intestine

andvagina

• obligate anaerobe!

• produce stinking gases

• infections: abscesses (lung, brain)

• treatment: metronidazole, clindamycin