5th Micro Seminar & Lab: Gram-positive Cocci PDF

Katedrai Zakład Mikrobiologii Lekarskiej wydziału Nauk Medycznych w Katowicach. e -e. -. e “ - -... -...

Katedrai Zakład Mikrobiologii Lekarskiej wydziału Nauk Medycznych w Katowicach. e -e. -. e “ - -... - '.. '" ".'- I!.’.’' "“ - “ - “ " M k rob”.loafL karsk. - - - - \ - - ” - ' ” Most spesies are facultative anaerobes, and some grow only inatmosphere enhanced Previously classified as Lancefield groupD with carbon dioxide (capnophilic growth — streptococci, because they share the group Biology — grow ina variety of conditions —aerobically and about 8f6of all pneumococcal isolates are D cell wall antigen,a glycerol teichoic acid anaerobically, in the presence of high concentration of salt capnophiles that require CO - enriched Enterococi grow both aerobically and (7.5-10%) halophytes (mannitol salt agar useful diagnostic growth conditions), their nutritional anaerobically ina wide pH range (4.6 to 9.9) test) requirements are complex, necessitating the and in presence of high concentrations of use of blood or serum — enriched media for sodium chloride (6.59t) and bile salts (4096) isolation (fastidious bacteria) Catalase (useful diagnostic test) positive (catalases are enzymes that convert hydrogen peroxide into water and Catalase-negative Catalase-negative oxygen gas— bubbles appear when theoxygen gas is formed with bacterial colony and peroxide solution) Arranged in clusters or grapes, however, organisms in Arranged inpairs or chains (when grow in Arranged in pairs or short chains clinical specimens may also appear as single cells liquid media) The genus Staphylococcus was differentiated from Micrococcus species on the basis of the oxidation and fermentation of glucose, bacitracin resistance (0.04U) Enterococci are positive for PYR and grow indicated by the absence of an inhibition halo or formation intryptic soy broth containing 6.59¢ NaCI ofa halo fi9mm, and susceptibility to furazolidone (100yg) characterized by an inhibition halo measuring >15mm in diameter M:kra og::L'ka:she‘ The enzyme Catalase is used to neutralize the bactericidal effects of hydrogen peroxide. The Catalase accelerates the decomposition of hydrogen peroxide (H2O2) into water and oxygen (2H2O +O2). 2 H2O2 4 2H2O + 0, $' Detection of the presence of catalase in bacteria is essential to differentiate: 0 catalase-positive Staphylococcaceae and Micrococcaceae from catalase- negative Streptococcaceae and Enterococceae 0 species belonging to the same genus such as Campylobacter fetus, Campylobacterjejuni and Campylobacter coli (catalase positive) from other Campylobacter species (catalase negative)  This method is used forthe differentiation of staphylococci and micrococci (genera Staphylococcus and Micrococcus). Micrococci are resistant to furazolidone. Susceptibility to furazolidone can be determined by using the filter paper-disk diffusion method.A plate of Mueller-Hinton agar is inoculated and a filter paper disk, impregnated with 100 kg of furazolidone, is placed on the plate. The plate is then incubated for 16—18h at 35°C. Zones of inhibition havinga diameter> 15 mm show that the tested specimen is susceptible to furazolidone, indicating that further tests need to be runthat will lead to the identification of Staphylococcus. Staphylococcus aureus —„golden” - colonies can havea yellow color because of Staphylococcus epidermidis, carotenoid pigments Staphylococcus saprophyticus Staphylococcus lugdunensis Staphylococcus haemolyticus The most virulent Relatively avirulent, although production of the extracellular slime layer S. aureus can producea range oftoxins including enterotoxins (food (biofilm) can allow adherence to foreign bodies (catheters, grafts, poisoning), cytotoxins (general systemic toxins), and toxic shock superantigens. prosthetic valves and joints, shunts) Normal human flora on skin, large intestine, vagina Normal human flora on skin and mucosal surfaces e.g. S. capitis is found Colonizes the anterior nares where sebaceous glands are present and S. haemolyticus and S. hominis are found in areas where apocrine glands are present - axilla Can survive on dry surfaces for long periods Can survive on dry surfaces for long periods Person-to-person spread through direct contact or exposure to contaminated Person-to-person spread through direct contact or exposure to fomites, although most infections are with the patient's own organisms contaminated fomites, although most infections are with the patient's own organisms S. aureus is able to cause many superficial pyogenic (pus-forming) infections of coagulase-negative staphylococci (CNS) are much less frequently found the dermis and underlying tissues as well as serious systemic infections. as pathogens but are occasionally associated with endocarditis, prosthetic joint infections,and wound infections  Katedrai zaktad M ikrob oio9 iiL ukarskiaq Mannitol salt agar or MSA isa commonly used selective and differential growth medium in microbiology. It encourages the growth ofa group ofcertain bacteria while inhibiting the growth ofothers. It containsa high concentration (about 7.5-10%) of salt (NaCI) which is inhibitory to most bacteria — making MSA selective against most Gram-negative and selective for some Gram-positive bacteria (Staphylococcus, Enterococcus and Micrococcaceae) that tolerate high salt concentrations. It is alsoa differential medium formannitol-fermenting staphylococci, containing the sugar alcohol mannitol and the indicator phenol red,a pH indicator for detecting acid produced https://oI.wikipedia.org/ by mannitol-fermenting staphylococci. Staphylococcus otzreus produces yellow colonies with yellow zones, whereas other An MSA plate with: coagulase-negative staphylococci produce small pink or red Micrococcus sp.(1) colonies with no color change tothe medium. If an organism can ferment mannitol, an acidic byproduct is formed that causes the Staphylococcus epidermidis (2) phenol red in the agar to turn yellow. It is used forthe selective S. aureus colonies (3) isolation of presumptive pathogenic Staphylococcus species. Capsule and slime layer Protects the bacteria by inhibiting phagocytosis and chemotaxis Cell wall Peptydoglycan Makes thecell wall more rigid and provides osmotic stability (in salt environment), stmulates production of endogenous pyrogen, leucocyte chemoattractant (abscess formation) Teichoic acid — species-specific, phosphate-containing polymers that Binds to fibronectin are bound covalently to N-acetylmuramic acid residues of the peptydoglycan or to the lipids in the cytoplasmic membranr (lipoteichoic acid) Surface proteins (microbial surface components recognizing adhesive Important foradherence to host matrix matrix molecules MSCRAMM) e.g. fibronectin-binding proteinsA and B proteins (fibronectin, fibrinogen, elastin, and clumping factor (CF bind fibrinogen and convert it to insoluble collagen) fibrin, causing the staphylococci to clump oraggregate) ProteinA Binds to the Fc of Ig G1-2 and IgG4 and inhibits phagocytosis phaeocvtosis  Enzymes Coagulase Converts fibrinogen to fibrin Hyaluronidase Hydrolyzes hyaluronic acids in connective tissue, promoting the spread of staphylococci in tissue Fibrinolysin (staphylokinase) Dissolves fibrin clots Lipases Hydrolyzes lipids Nucleases Hydrolyzes DNA Proteases Hydrolyzes proteins Long chain Staphyloxanthin The golden pigment (shield bacteria 2. Osteomyelitis carotenoid from the oxidative stress inside 3. Septic arthritis neutrophil phagosomes — hasan 4. Pneumonia and empyema antioxidant effect) 5. Endocarditls/Catheter-related bloodstream infections 6. Breast abscess in puerperium 7. Hordeolum „Styes”: an infection of the glands on the eyelid These appendages come from the epidermis: Skin appendage includes: 1. Cutaneous glands: 0 Sebaceous glands, 0 Sweat glands: 0 Eccrine sweat glands (Location and distribution: most areas of the body. Absent in lips, ear canal, clitoris, labia minora, and glans penis. Function: Secretion of sweat-thermoregulation) 0 Apocrine sweat glands (Location and distribution: Mostly axilla, perineum, areola of the nipple, and external ear. Function: Modified apocrine cells produce ear wax or breast milk) 2. Hair follicles 3. Nails  Infectious diseases of skin appendages: Staphylococcus aureus can cause: Furuncles Boils: An abscess within the skin, also Carbuncles calleda furuncle. Folliculitis Furuncles are common ontheneck, Paronychia breasts, face, and buttocks. Carbuncles are clusters of furuncles that are subcutaneously connected. They may be accompanied by fever and prostration. Folliculitis: An infection of hair follicles. Paronychia: An infection of the skin. O O, 0 folds of the nails. 0 FoIi ! I'” I m ’i Boils. The most common type of staph infection is the boil. This isa pocket of pus that develops ina hair follicle or oil gland. The skin over the infected area usually becomes redand swollen. Boils occur most often under the arms oraround the groin or buttocks. https://europepmc.org/ Cellulitis is an infection of the deeper layers of skin. It causes redness and swelling on the surface of skin. Cellulitis is most commonly caused by Streptococcus and Staphylococcus bacteria. Streptococci spread rapidly in the skin because they produce enzymes that hinder the ability of the tissue to confine the infection. Cellulitis that is caused by staphylococci usually occurs around open wounds and pus-filled pockets (skin abscesses). A hordeolum, commonly calleda „stye“, is an infection of an oil gland atthe edge of the eyelid. Some people are more prone to developa hordeolum, but it is also associated with: https://prestigemedicaI.pl/ Z Contact lens wear. 0 Poor hygiene. Symptoms: U Using eye makeup that is old or 0 Swelling, redness, pain or tenderness of contaminated. the eyelid. 0 Blepharitis, an inflammation or infection 0 Feeling like there is something in your of the eyelids. eye. U Systemic conditions such as rosacea, 0 Excessive tearing. seborrheic dermatitis, or diabetes. 0 Crusting of the eyelid. Toxins Cyotoxins (a, g, 6, y=Panton- Cytolytic or membrane-damaging a toxin is believed to be an important mediator of tissue damage and Valentine=leukocidin) — toxic for erythrocytes disrupts also the smooth mustle in blood vessels and it becomes (hemolysin), fibroblasts, integrated in the hydrophobic regions of host cell membrane, leading leukocytes, macrophages, to formation of 1-2nm pores platelets § toxin=sphingomyelinaseC 6toxin acts asa surfactant disrupting cellular membranes bymeans of a detergent-like action ytoxin and Panton — Valentine=leukocidin can lyse neutrophils and macrophage and PV is present in MRSA strains associated with community-acquired infections (skin) Exfoliative toxins (ETA-heat stable, and Serine proteases that split the SSSS (Ritter syndrome) is seen mostly in neonates and young children — the gene is phage associated, ETB-is intercellular bridges in the slight pressure displaces the skin (a positive Nikolsky sign) and large heat labile and located ona plasmid) stratum granulosum epidermis bullae or cutaneous blisters (with no organisms or leukocytes) form (destroy desmogIein-1) Enterotoxins (A-R) stable to heating at Superantigens — stimulate release A-most commonly associated with food poisoning — rapid onset (3-8h 100’C for 30min and resistant to of inflammatory mediators in after eating) of severe vomiting, diarrhea and abdominal tramping hydrolysis by gastric and jejunal mast tells, increasing intestinal C andD — in contaminated milk products enzymes peristalsis and fluid loss, as well as The most commonly contaminated foods are processed meats such as nausea and voiziiti ng a ham and salted pork, Custard-filled pastries, potato salad, ice cream Toxic shock syndrome toxin-1 Superantigen — stimulate release Menstruation associated TSS fever, hypotension, diffuse, macular of inflammatory mediators erythematous rash Stimulate proliferation ofT cells and release of cytokines. Superanfigens are not processed, but the intact molecule binds to the major histocompatibility complex — MHC class II molecules outside the peptide-binding groove and cross-links the MHC II molecule and MHC classI (enterotoxin B) with the variable region of the TCR §- chain expressed by CD4 Th. Each suprantigen binds toT cells expressinga particular V§ TCR gene product. The systemic effects of the toxin activating so manyT cells can have clinical consequences, including fever and cardiovascular shock, and can be fatal. WNM w Katowicach Animl assoñdted dhd0Cher S. ro£0OSUG subsp. S. ep/dermid/8 S. fioemoI}tiru8 S. capitit 6. hoininis, 1 penenitofer/, S. simu/ans S. warren andothes Clin Microbiol Rev. 2014 Oct; 27(4): 87&926. 2 Urinary tract infecñons — dysuria and pyuria in young sexually acñve women (urease positive S. saprophyticus), in patients with urinary catheters 0 Catheter and shunt infections — chronic inflammatory response to bacteria coatinga catheter (the major nosocomial pathogens, with S. epidermidis and S. haemolyticus — they account substantially for foreign body-related infections and infecñons in preterm newborns) S Prostet1c joint infections — chronic infect1on with pain and mechanical failure of the joint 2 Endocardit?s (S. lugdunensis — but mainly S. aureus)  UTI Symptomatic Asymptomatic Asymptomatic Complicated Uncomplicated becteriuria Acute Acute Catherer Uncomplicated complicated uncomplicated associared UTI pyelonephritis cystitis cystis Complicated pyelonephritis their morphology and specific color, creamy bright white colonies types of haemolysis on agar with 5% sheep blood non-hemolytic colonies Mannitol-salt Agar yellow-colored colonies indicating mannitol fermentation catalase g0SÏtiV9 coagulzse testing or clumping factor testing negativa novobiocin resistant 3 novobiocin (Sig) resistance is defined as the presence of en novobiocin susceptibility inhibition halo ñ12mm ortheabsence ofa halo, and susceptibility was defined as the presence of en inhibition halo >16mm  Novobiocin isa member ofthe Result and interpretation of Novobiocin Susceptibility Test for aminocoumarin antibiotic class produced by MHA (Kirby Bauer Disk Diffusion Method) According to CLSI Streptomyces niveus. Novobiocin binds with the GyrB subunit of Novobiocin Susceptibility Test Results the DNA gyrase enzyme and inhibits the DNA transcription process. It is also found to Novobiocin Antibiotic inhibit the action of DNA topoisomerase IV. Novobiocin is not used for treatment, due to its toxicity and lower effectiveness, but it is Plate wlth bacteria still used in clinical laboratories in the Novobiocin Susceptibility Test for the Zone size ofc 16 mm identification and differentiation Resistant to noYobiOCif\ Sensitive to novobiocin Staphylococcus saprophyticus from other + fi›hr! occus sap fi›hyticus, fitaphr! S aureus,S. epidermidis, S. £/oosi/, S. cohnii S. haemolyticus, S. hominis, S. capitis CNS (coagulase-negative Staphylococci| https://microDenotes.com/novobiocin-susceptibility-test especially Staphylococcus epidermidis. Mikrodiolo9 iiL ekarskie Alpha toxin (g-hemolysis) + (most strains) Acid from mannitol + (most strains) (Chapman agar) Coagulase reaction Clumping factor Pigment production Usually golden Usually white Usually white DNase production + (usually) Sensitivity to novobiocin sensitive sensitive resistant  Kwa i zaklad * Responsible for suppurative Asymptomatic colonization Most infections are caused 0 Dental caries Colonizes the and nonsuppurative (normal human flora) of by endogenous spread 0 Subacute endocarditis gastrointestinal tract infections: the upper respiratory tract from the colonized associated with S. mifis (normal flora) 0 Suppurative infections: and genitourinary tract, nasopharynx or and S. salivarius groups Are one ofthe most 1. Pharyngitis at increased risk for GBS oropharynx Septicemia in common causes of 2. Scarlet fever infection are: 3 Lobar pneumonia neutropenic patients nosocomial infections: 3. Pyoderma O Neonates and pregnant 3 Meningitis associated with S. mifis 0 UTI 4. Erysipelas women with genital 3 Sepsis group 0 Endocarditis: infection 5. Cellulitis colonization 3 Sinusitis Meningitis associated of the heart 6. Necrotizing fascitis 0 Men and nonpregnant O Otitis media with S. gallolyficus endothelium or valves, 7. Streptococcal toxic women with diabetes subsp. pasteurianus, associated with shock syndrome mellitus, cancer, or S. suis, S. m/tis group persistent bacteremia, 8. Puerperal sepsis alcoholism (sepsis, skin Abscess formation in can present acutely or 9. Lymphangitis and soft-tissue deep tissue associated chronically 0 Nonsuppurative infections, bone and with S. anginosus group 0 Sepsis infections: joint infections) 0 Rheumatic fever 0 Acute glomerulonephritis M ik rash incl“ !i Le ka*rsk ie 0 Complete (§) — GAS, GBS 0 Incomplete (a) green pigment formed by the partial hemolysis of blood agar — S. pneumoniae, viridans streptococci. The greenish color is caused by the presence of biliverdin, which isa by-product of the breakdown ofhemoglobin. 0 No hemolysis (y) — some Enterococci Blood agar is made with 5% sheep 0 Staphylococcus is usually either beta hemolytic or blood. It isa common medium used to not hemolytic at all (called gamma hemolysis). culture bacteria because: 0 It isa great enrichment medium for 0 To know thetype of hemolysis, the blood agar plate fastidious bacteria. must be held up toa light source and observed with 0 Hemolysis of blood cells can be very the light coming from behind (transmitted light). useful as an identification test.  §-hemoIytic streptococci (GAS, GBS) produce hemolysins that lyse the sheep RBCs (poorly lyse human RBCs too), resulting ina clearing of the blood agar plate surrounding the colonies. More intense §-hemolysis is noted in areas where the medium has been “stabbed,” pushing some ofthebacteria under the surface of the medium. The §-hemoIysis in these areas is due to the combined act1on of both hemolysins of Streptococci (Streptolysin0 and Streptolysin S). The surface §-hemoIysis is largely due to streptolysinS (oxygen stable hemolysin), as Streptolysin0 which is oxygen-labile does not show maximal activity on the surface of the agar. U There are now over 100 recognised species of Streptococcus, many ofwhich are pathogens or commensals in humans and animals. Z Beta hemolytic streptococci are arranged into twenty Lancefield groups A-V withoutI and J (group D composed ofalpha haemolytic organisms). U Though there are many groups of streptococci, only3 are known tocommonly cause disease in immune-competent human beings: group A, group B, both members ofgroupD (Streptococcus gallolyticus and Streptococcus infantarius, both members oftheStreptococcus bovis group) 0 Group K-V contains streptococcal species of limited virulence which can cause infections in immunocompromised individuals. 0 The basis of Lancefield grouping is the antigenic differences inC carbohydrates,a group-specific antigen.C carbohydrate is located in the cell wall of streptococci. 0 The original Lancefield precipitin test is now rarely performed in clinical laboratories. Now, it has been replaced by either latex agglutination or coagglutination. 0 Lancefield serotyping is useful in identification of Streptococcus; however, it should be used in conjunction with other tests to get accurate identification.  colonizes the genital tract of some women andcantause neonatal meningitis and sepsis. It can cause septic abortion and puerperal or gynecological sepsis and occasionally urinary tract infections include enterococci (e.g., £nterococcus Enterococci are members ofthenormal flora of the colon and they tan D faecalis and Enteracaccus faecium) cause urinary, biliary, and cardiovascular infections and nonenterotocci (S. bovis) S. agalactiae B Beta Enterococci cells are Gram-positive with an ovoid shape. Enterococci form neither spores nor capsules, but some species may be capable of movement bytheflagellum. These motile species are Enterococcus casseliflavus and Enterococcus gallinarum. Their colonies are milky white when grown on usual agar plates, but e specieS p roduce carotenoid pigmens which color them yellow SOITI )E. sulfureus, E. casseliflavus and E. mundfii). They arechemoorganotrophic, catalase negative and some species can show haemolytic activity. omp ex media, rich with nutrients, are required for cultivation in laboratory conditions. Enterococci are facultative anaerobic bacteria. wNM w Eatowica‹h PYR (Pyrrolidonyl Aminopeptidase) Test is used for the detection of pyrolidonyl arylamidase (also called pyrrolidonyl aminopeptidase) activity in Streptococcus pyogenes (groupA strep), Enterococcus spp. (Enterococcus faecalis and Enterococcus faeciurn|, some coagulase- negative staphylototti (S. hemolyticus, S. lugdunensis, S. schleiferi), and some Enterobacteriaceae (Enterobacter, Citrobacter, Klebsiella, Yersinla and Serratia) and Aerococcus, Gamella, Lactococcus, most Corynebacterium (Arcanobacteriurn) hemolyficum. The enzyme L-pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl- §-naphthyIamide substrate to producea §-naphthyIamine. The §- naphthylamine can be detected in the presence of N,N-methyIaminocinnamaIdehyde reagent by the production ofa bri$ht red precipitate. Enterococcosel Agar,a Bile Esculin Agar with Azide, is used for the rapid, seIe&ve detection and enumeration of enterococci. Enterococci and GroupD streptococci hydrolyze the glycoside esculin to esculeñn and dextrose. Esculeñn reacts with an iron salt, ferric ammonium citrate, to forma dark brown orblack complex. Oxgall is used to inhibit Gram-positive bacteria other than enteroco«i. Sodium azide is inhibitory for Gram- negative microorganisms. WNM w Katocicach Viridans streptoco¢¢i comprise» v»st collecñon of0ram-posiñve commensalstreptoco«aI bactefia th»t are hemolyñc and produce» green hue on blood plates (hence the name „viridans”, from Latin „viridis”, green). The greenish color is caused by the presence of bilivefdin, which is» by-product of the breakdown of hemoglobin. They do not possess Lancefield anñgens. In general, the risk of p»thogenicity is very low. Viridans streptoco¢¢i can be dist1nguished from Streptococcvs pneumoniae by an optochin test. This is because virid»ns streptococci are resistant to optochin end are also devoid of the polys»ccharide-based capsule that is typical fors. pneumoniae, or the Lancefield antigens that are found in the Pyogenic members of the Genus. The most prevalent organisms are in the mouth -S.mutant and S. sanguinis»re responsible for dental c4ries. If they enter the circulation of blood, they are at the potential to c»use subacute endocardiñs. This is especially true for those with heart valves that ale damaged. Viridans streptoco¢ri are found in the c»se of neonatal infecñons.  M”krooiologii Lekarskie WNM w Katow ca‹h Cell wall M protein M — protein is the major type-specific protein and interfere with 0 Pharyngitis proteins F protein phagocytosis by blocking the binding of the C3b, lipoteichoic 0 Pyoderma (impetigo) — purulent lipoteichoic acid acid andF protein facilitate binding of host cells by tomplexing infection of the skin with fibronectin, whith is present on the host cell surface 0 Erysipelas is an atute infection of the shin C5a peçtidase Serine protease inactivates CSa,a chemoattractant of PMNs 0 Cellulitis Enzymes StreptolysinS and0 S - nonimmunogenic, cell-bound hemolysin that can lyse 0 Puerperal sepsis erythtocytes, leukocytes, platelets 0— immunogenic hemolysin, Ab — antistreptolysin 0— ASO are useful for documenting recent GAS infection StreptokinaseA andB The enzymes mediate the clezvzge of plasminogen, releasing the protease plzsmin that, in turn, cleaves florin and fibrinogen thus can lyse blood clots and fibrin deposits and facilitate the rapid spread of GAS in infected tissues DNAses Can depolymerize free DNA present in pus TOXÎïl Streptococcal pyrogenic Are produced by lysogenic strains and are similar to the toxic Scarlet fever (cirtumoral pallor, exotoxins (Spe A, B, C, F)= produced in Co/y0e6orterii/m diphtheriae, the toxin act as strawberry tongue, Pastia lines) erythrogenic toxins superantigens, interacting with both macrophages and Th, with Necrotiiing fascitis the enhanced release of tytotines Streptotoccal toxic shots syndrome Identification through: 0 Hemolytic paRerns — §-hemoIysis 0 Catalase negative 2 The demonstration of the group-specific carbohydrate — LancefieldA group 0 Suscept1bility to bacitracin S Biochemical properñes This test is used for presumptive identification and differentiation of beta-hemolytic groupA streptococci |Streptococcuspyogenes — susceptible) from other beta-hemolytic streptococci (GBS). It is also used to distinguish staphylococci species (resistant) from micrococri (susceptible). The antibiotic batitratin inhibits the synthesis of bacterial cell walls.A disk (7axoA) impregnated witha small amount of bacitracin (0.04 units) is placed on an agar plate, a!lowing https://www.researthgate.net the antibiotic to diffuse into the medium and inhibit the growth ofsusceptible organÍSlTl5. Positive: Any zone of inhibition greater than 10 mm; After incubation, the inoculated plates are examined for zones of inhibition surrounding susceptible the disks. Negative: No zone of inhibition; resistant Streptococcus pneumoniae growing on sheep blood agar as “draughtsman” colonies. The disk contains optochin and is surrounded bya zone of inhibition.  J 8 -- e3' ”“* ” e o I- tJ' o“ o ' * a j I a '“ https://www.mdcalc.com/calc/104/centor-score-modified-mcisaac-strep-pharyngitis Erysipelas — isa superficial form ofcellulitis, (localized skin infection with pain, inflammation, lymph node enlargement, and systemic symptoms). Erysipelas predominantly affects the skin of the lower limbs, but when it involves the face, it can havea characteristic butterfly distribution on the cheeks and across the bridge of the nose. Erysipelas recurs in up to one-third of patients due to: 0 Persistence of risk factors 0 Lymphatic damage (hence impaired drainage of toxins) https://dermnetnz.org/  Katedrai Zakfad ¥ikrooiologii Ltkarskiej W NM w E at0wicarh Scarlet fever is an tomplitation of streptototcal pharyngitis-GAS strains with erythrogenit toxins. Also known as scarlatina, starlet fever featuresa dright red rash that covers most of the body. Scarlet fever almost always includesa sore throat enda high fever. Scarlet fever is most common in children5 to 15 years of age. The signs and symptoms that give scarlet fever its name include: 1. Red rash. The rash looks likea sunburn and feels like sandpaper. It typically begins on the face or neck and spreads to the trunk, arms and legs. Pushing on the reddened skin makes it turn pale. 2. Pastia red lines. The folds of skin around the groin, armpits, elbows, knees and neck usually becomea deeper red than the other areas with the rash. Flushed fate. The face may appear flushed witha pale ring around the mouth. 4. Strawberry tongue. The tongue generally looks red and bumpy, end it's often covered witha white coating early in the disease. Rheumat1c fever characterized by inflammatory changes of the heart (pancarditis), joints (arthralgias to arthritis), blood vessels, subcutaneous tissues Acute glomerulonephrit1s: acute inflammation of the renal glomeruli with edema, hypertension, hematuria, and proteinuria Erythema nodosum: isa hypersensiñvity reactionis,a type of panniculitis —an inflammatory disorder afiecñng subcutaneous fat. It presents as tender red nodules on the anterior shins. Less commonly, they afiect the thighs and forearms  M ik robi olo9 iiL eka rskie The CAMP test is used to idenñ§ Streptococcus agalacfiae (group B-GBS) - CAMP positive and to differentiate it from Streptococcus pyogenes (group A-GAS) - CAMP negative and nongroupB Streptococcus-CA/ /lP negative. The §-Iysin produced by §-hemoIytic Staphylococcus aureus acts synergistically with the CAMP factor produced by GBS. This synergisñc reaction results in an enhanced and very visible zone of hemolysis in the region between thetwo cultures. The synergistic zone is not observed in group A, C, and G Streptococcus. 0 Early — onset neonatal disease (sepsis, pneumonia). 0 Late — onset neonatal disease (sepsis with meningitis). The primary risk factor for neonatal GBS early-onset disease (EOD) is maternal colonization of the genitourinary and gastrointestinal tracts. Approximately 50% of women who arecolonized with GBS will transmit the bacteria to their newborns. Vertical transmission usually occurs during labor or after rupture of membranes. Other risk factors include gestational age of less than 37 weeks, very low birth weight, prolonged rupture of membranes, intraamniotic infection, young maternal age. The American College of Obstetricians and Gynecologists recommends performing universal GBS screening between 36 0/7 and 37 6/7 weeks ofgestation. All women whose vaginal-rectal cultures at 36 0/7-37 6/7 weeks ofgestation are positive for GBS should receive appropriate intrapartum antibiotic prophylaxis (penicillins). Puerperal sepsis (with endometritis, wound infection, UTI).  Antiphagocyting polysaccharide capsule — currently 90 serotypes are recognized — purified capsular polysaccharides from the most commonly isolated serotypes are used ina polyvalent vaccine IgA protease Pneumolysin (cytotoxin similar to the streptolysin O) can destroy the ciliated epithelial cells and phagocyt1c cells Surface protein adhesins Pneumococci are unique in that the wall teichoic acid and lipoteichoic acid contain the same unusually complex repeating units decorated with phosphoryl choline residues, which anchor the choline-binding proteins. S Gram-posiñve cocci in pairs facultat1ve anaerobic do not form spores and are non movie 2 Catalase negative 2 a-hemolysis (Alpha hemolysis or incomplete hemolysis or partial hemolysis is caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin to green methemoglobin) 0 Suscept1bility to optochin S Solubility in bile (bile solubility test) v^ La rv° Kated raI Zaklad * M kr olognL e kars ej Most infect1ons are caused by endogenous spread from the colonized nasopharynx or oropharynx: 2 Lobar pneumonia 2 Meningiñs 2Sepsis 2 Sinusitis 00tiñs media Lobar or segmental consolidation There aretwo kinds of pneumococcal vaccines: 0 Pneumococcal conjugate vaccine or PCV13 0 Pneumococcal polysaccharide vaccine or PPSV23 0 CDC recommends PCV13 forall children younger than2 years old and people2 years or older with certain medical conditions. Adults 65 years or older also can get PCV13. CDC recommends PPSV23 forall adults 65 years or older, people2 through 64 years old with certain medical conditions, and adults 19 through 64 years old who smoke cigarettes. The pneumococcal polysaccharide vaccine most commonly used today consists of purified polysaccharides from 23 serotypes (1, 2, 3, 4, 5, 6b, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F). Immunity is induced primarily through stimulation of B-cells which release lgM without the assistance ofT cells. The pneumococcal conjugate vaccine consists of capsular polysaccharides covalently bound tothe diphtheria toxoid CRM197, which is highly immunogenic but non-toxic. This combination provokes a significantly more robust immune response by recruiting CRM197-specific type2 helperT cells, which allow for immunoglobulin type switching (to produce non-IgM immunoglobulin) and production of memoryB cells. Z aztreonam N temocylin Z polimyxinB N colist1n Z quinolones: 1-st generation e.g. nalidixic acid Mikrobiolo9iiL ekarskie Intrinsic resistance Acquired resistance 3 §-Lactams (particularly cephalosporins and penicillinase- 0 High concentrations of §-Iactams, through resistant penicillins) - In E. faecalis, susceptibility to alteration of PBPs or production of §- ampicillin, amoxicillin and piperacillin (with and without beta- lactamase lactamase inhibitor) is the expected phenotype, while in E. High concentrations of aminoglycosides faecium, resistance is common. Isolates resistant to (HLAR) ampicillin can be reported resistant to ampicillin, amoxicillin g Glycopeptides: vancomycin, teicoplanin and piperacillin (with or without inhibitor). (VRE) Low concentrations of aminoglycosides —aminoglycoside Tetracycline monotherapy is ineffective. There is likely to be synergy Erythromycin between aminoglycosides and penicillins or glycopeptides Fluoroquinolones against enterococci without acquired high-level aminoglycoside resistance. All testing is therefore to Rifampin distinguish between intrinsic and high-level acquired Chloramphenicol resistance. Fusidic acid Clindamycin Nitrofurantoin Fluoroquinolones — Moxifloxacin has been used in oral follow- up treatment of endocarditis caused by Enterococcus spp. Trimethoprim-sulfamethoxazole The transferred DNA can be integrated into the recipient chromosome orstably maintained as an extrachramosomal element (plasmid) ora bacterial virus (bacteriophage) and passed on to daughter bacteria as an autonomously replicafing unit. Mechanisms ofgenetic transfer between cells: 0 Conjugation (results in one-way transfer of DNA froma donor cell toa recipient cell through the sex pilus — Escherichia coli), Transduction (is mediated by bacterial viruses — bacteriophages, which pick up fragments of DNA and package them into bacteriophage particles, the DNA is delivered to infected cells and becomes incorporated into the bacterial genomes), Transformation (is the process by which bacteria: Haemophilus influenzae, Streptococcus pneumoniae, Bacillus species, Neisseria species take up fragments of naked DNA and incorporate them into their genomes). Transposition — transfer of transposomes, „jum ping genes”. Kated rai Zaklad M ikrodiolo9ii Lekarskie Macrolides area class of natural or semisynthetic products comprisinga large macrocyclic lactone ring to which one or more deoxy sugars are attached. The lactone rings can be either 14-membered (clarithromycin, dirithromycin, erythromycin, and roxithromycin), 15-membered (azithromycin), or 16-membered (josamycin, kitasamycin, spiramycin, and tylosin). Macrolides, lincosamides and streptograminB bind to the POS ribosomal subunit, which blotks polypeptide elongation. In general, modification of ribosomal targets (due to methylation of the 23S rRNA) and drug efflux confer cross-resistance to macrolides. The erm gene, which is usually plasmid coded and produces the methylation of adenine at position 2058 of the SOS rRNA, causing resistance to macrolides (erythromycin and many others), lincosamide, and streptogramin of group B, the MLS phenotype. The double-disk diffusion test (D-test with erythromycin and clindamycin disks) is used to determine the MLSB resistance type. Cross-resistance to macrolides, lincosamides and streptograminB (MLSB) antibiotics can be either constitutive or inducible. Inducible clindamycin resistance can be detected by antagonism of clindamycin activity bya macrolide agent. If detected, comment on resistance should be reported: „Clindamycin may still be used for short-term therapy of less serious skin and soft tissue infections as constitutive resistance is unlikely to develop during such therapy”. M ik?ââi“oIo' !.'L ka'r.ki. Other resistance mechanisms, e.g., the msr(A) and msr(B) “ macrolide efflux pumps and enzymic modification (by esterases, phosphorylases, glycosidase) encoded by the ere(A) and ere(B) genes, have also been described in 5. aureus. Erythromycin can be used to screen for macrolide resistance in staphylococci and in Streptococcus groups A, B,C and G. Isolates categorised as susceptible can be reported susceptible to azithromycin, clarithromycin and roxithromycin.  0 PRP (Penicillin Resistant Pneumococci) — penicillin (MIC ofatleast 2yg/mL) resistance among Streptococcus pneumoniae is also associated witha decreased affinity of the ant1biot1c for the PBP present in the bacterial cell walla similar to PRP mechanism of penicillin resistance was also found in Neisseria meningitidis EW "L,JJ“"%L.:..\,/" Methicillin resistance is conferred by mecA ormecC gene, which is located on the staphylococcal chromosomal casseRe, and encodes penicillin-binding protein 2A (PBP2A or PBP2’) or PBP2ALGA, the enzymes that are responsible for crosslinking the pepndoglycans of bacterial cell wall. Both enzymes showa lowaffinity for §-Iactams, thereby leading to resistance to this category of antibiotics i.e. penicillins, cephalosporins (V-th generation may be act1ve) and carbapenems. Kated rai zaktad MR — Methicillin Resistant SA — Staphylococcus aureus SE — Staphylococcus epidermidis CNS — coagulase-negative Staphylococci Vancomycin (Glycopeptides) or daptomycin arethe agents of choice fortreatment of invasive MRSA infections. Alternañve agents that may be used forsecond-line or salvage therapy include telavancin, ceRaroline (V generations of cephalosporins), and linezolid. Kated rai zaktad VRE vancomycin-resistant strains of enterococci become prevalent in a hospital environment, colonize the pañents, and cause infections that are difficult to treat. Study of the resistance mechanism showed that the end of the pentapeptide, d-Ala-d-AIa, where vancomycin binds, was replaced in the resistant strain (e.g. by an ester structure, d-AIa-d-Iactic acid, which is not bound by vancomycin).  Vancomycin resistance is classified into several gene clusters based on the DNA sequence ofthe ligase van gene homologues that encode the k ey enzymef or the syn th esis of d-alanyl-d-lactate (d-AIa-d-Lac) or d- alanyl-d-serine (d-AIa-d-Ser). At least 11 van gene clusters that confer vancomycin-resistance, responding for VanA, VanB, VanD, Van F, Vam, VanM, VanC, VanE, VanG, VanL, and VanN phenotypes, have been described to date. The genes that encode d-Ala:d-Lac ligases, such as vanA, vanB, vanD, van F vanl, and vanM, often result in high-level vancomycin resistance with MlCs >256mg/mL, while the genes that encode d-AIa:d-Ser ligases, including vanC, vanE, vanG, vanL, and vanN, generally result in low- level resistance with MlCs of8-16mg/mL. Althougha dozen Enterococcus species have been identified, only two \E.]'aecaIis and E.]'aecium)*” "" are responsible for the majority of human infections. Other Enterococcus species (E. gallinarum, E. casseliflavus, E. durans, E. avium, and E. raffinosis) are isolated much less frequently and account forless than 5% of clinical isolates. Glycopeptide resistance in enterococci is mediated by von gene clusters, nine operons (vanA, vanB, vanC, vanD, vanE, vanG, vanL, vanM andvanN), each with disfinguishing resistance characteristics, have been described. Clinically, the vanA (most frequent inE./oecium, E. foecalis) and vanB (most frequent in E. faecium, E. faecalis\ operons are the two most dominant and relevant vancomycin resistance factors. VanA-type resistance is characterized by high-level resistance to both vancomycin and teicoplanin, whereas VanB-type strains are resistant to variable levels of vancomycin but susceptible to teicoplanin. VanD-type strains are characterized by resistance to moderate levels of vancomycin and teicoplanin. VanC (in E. gallinarum, E. casseliflavus and E. flavescens), VanE, and VanG isolates exhibit low-level resistance to vancomycin only. Antimicrobial therapy — options include: 0 Linezolid — may be preferred over teicoplanin: 0 greater efficacy, o better tissue pen etration (it is poorly protein bound, so volume ofdistribution approximates to total body water), 0 no dosage reduction is necessary in renal or hepatic failure, 0 Van A resistance is common, U Teicoplanin (if VRE strains are sensitive), 0 Daptomycin (daptomycin and tigecycline generally regarded as third line drugs), 0 Tigecycline, 0 Quinupristin-dalfopristin, 0 Ceftaroline. 1. HLAR (High-level Aminoglycoside Resistance) —aminoglycosides are inactivated by modihcafions that reduce the net positive charges on these polycationic antibiotics. There are now many dozens of aminoglycoside-modifying enzymes known, 2. Resistance caused bya §-Iactamase; §-Lactamases are classihed into several phylogenetic families. ClassA includes both the Staphylococcus aureus penicillinase and TEM enzymes, whereas ClassC represents chromosomally coded enzymes (e.g., AmpC) that are present in many Gram-negative bacteria. These two classes are both similar to serine proteases in their mechanism, whereas ClassB enzymes aremetalloenzymes that hydrolyze carbapenems efficiently. Most S. aureus are penicillinase producers and some aremethicillin resistant. No currently available method can reliably detect penicillinase production in all species of staphylococci. For S. aureus, disk diffusion with benzylpenicillin is more reliable than MIC determination for detection of penicillinase producers. Chromogenic cephalosporin-based beta-lactamase tests do not reliably detect staphylococcal penicillinase. Methicillin resistance can be detected with tefoxitin as described. Either mechanism renders them resistant to benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, piperacillin and ticarcillin. Isolates that test susceptible to benzylpenicillin and cefoxitin can be reported susceptible to all penicillins. Isolates that test resistant to benzylpenicillin but susceptible to cefoxitin are susceptible to §-Iactam §- lactamase inhibitor combinations, the isoxazolylpenicillins (oxacillin, cloxatillin, dicloxacillin and flucloxacillin) and nafcillin. For agents given orally, care to achieve sufficient exposure at the site of the infection should be exercised. Isolates that test resistant to cefoxitin (MR) are resistant to all penicillins, cephalosporins and carbapenems. Some methicillin-resistant S. aureus are susceptible to ceftaroline and ceftobiprole.  clavulanic acid (in combination with amoxicillin or ticarcillin) sulbactam (in combination with ampicillin — e.g. against Acinetobacter spp. 2g ampicillin and 1g sulbactam iv or with cefoperazone (III generation of cephalosporin) — Sulperazon tazobactam (in combination withz piperacillin — Tazocin or with ceftolozane — Zebraxa against Pseudomonas aeruginosa and Gram-negative ESBL-positive enteric rods avibactam (in combination with ceftazidime — Zavicefta against ESBL and KPC-positive enteric rods vaborbactam (potent activity against ESBL, KPC i Amp C) in combination with meropenem — Vabomere durlobactam (approved in combination with sulbactam (Xacduro) to treat hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia (HABP/VABP) pneumonia caused by Acinetobacter baumannii-calcoaceticus complex), relebactam, used in combination with imipenem/cilastatin (Recarbrio) Enzymatic modification is the most common type of aminoglycoside resistance. Enzymatic modification results in high-level resistance (HLAR). The genes encoding foraminoglycoside modifying enzymes areusually found on plasmids and transposons. There arethree types of aminoglycoside modifying enzymes: 0 N-Acetyltransferases (AAC) — catalyzes acetyl CoA-dependent acetylation of an amino group 0 O-AdenyItransferases (ANT) — catalyzes ATP-dependent adenylation of hydroxyl group 0 O-Phosphotransferases (APH) — catalyzes ATP-dependent phosphorylation ofa hydroxyl group The isolates of S. aureus with susceptibility or reduced susceptibility to vancomycin are classified into three groups (bythe Clinical and Laboratory Standards Institute): VSSA — vancomycin-susceptible S. aureus (with MIC s 2 kg/mL), VISA — vancomycin-intermediate S. aureus (with MIC of 4-8 yg/mL), VRSA — vancomycin resistant S. onretfs (with MIC ? 16 yg/mL). In confirming whether an isolate belongs to VRSA, the presence of vanA orother van resistance determinants should be demonstrated by molecular methods.