DETAILED BACTERIOLOGY

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, Stenotrophomonas, 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 o C 30 min), drying, salt (9% NaCl) survive in hospitals!

Pigment production Staphylococcus aureus: golden pigment (staphyloxanthin) Staphylococcus epidermidis: white pigment

-hemolysis S. aureus (+ S. haemolyticus + S. lugdunensis): strong -hemolysis

Catalase test Catalase +: Staphylococci Catalase -: Streptococci Aim: to differentiate Staphylococci and Streptococci performance: adding a few drops of H 2 O 2 to the culture, strong bubbling if positive H 2 O 2 H 2 O + O 2

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 Coagulase +: Staphylococcus aureus Coagulase -: all other Staphylococci ( CNS ) Tube coagulase test: inoculating the bacteria into citrate treated rabbit plasma, coagulation occurs in a few hours (exocoagulase) - +

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)

S. aureus virulence factors III. Cytotoxins 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 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 o C, 30 min) C, D: dairy products

Staphylococcus aureus skin infections Folliculitis folliculitis If on eye: hordeolum Furuncle Carbuncle bacteremia, fever Impetigo hordeolum furuncle impetigo

Staphylococcus aureus skin infections Cellulitis infection of the deeper layers of skin and the tissues beneath Bullous impetigo Localised form of SSSS Bullous impetigo

Staphylococcus aureus skin infections Wound infections burns, postoperational, trauma from the skin flora

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

A prosthetic joint with the presence of antibiotic beads TO PREVENT INFECTION Removal of an infected prosthetic joint shows a bacterial biofilm on an infected arthroplasty.

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 erysipelas phlegmone impetigo cellulitis severe impetigo

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 rush oll over the body toxin of S. pyogenes in the throat Raspberry tongue Peeling after scarlat fever

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 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 Str. agalactiae St. aureus

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% CO 2 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 normal intestinal flora microscope: in pairs or short chains culture: non-fastidious resistant to bile (40%), salt, high T (60 o C 30 min) hemolysis: might be α, β or γ esculin hydrolysis Enterococcus on E67 medium

Enterococcal infections Nozokomial infections! cystitis (chateter-associated) septis, endocarditis (venous canule) peritonitis, abdominal abscess 2 nd 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