TRACING THE CRIMINAL Part two: Streptococcus Institute of Microbiology shows: L Most important streptococci Story On BA Name of the criminal 4. viri- dation (alpha) Streptococcus pneumoniae 5. Group of „oral streptococci“ 1. (beta) hemo- lysis* Streptococcus pyogenes 2. Streptococcus agalactiae 3. Group of „non-A-non-B“ streptococci – none Ahemolytical streptococci *in S. agalactiae partial haemolysis only [USEMAP] Survey of topics Clinical characteristics: Haemolytical streptococci Clinical characteristics: Viridating streptococci Therapy of streptococcal diseases Diagnostics of streptococci Differential diagnostics of streptococci Late sequellae of streptococcal diseases Clinical characteristics: (b-)haemolytical streptococci (with partial or total haemolysis) Story One nMr Hobby likes to work with wood. He worked at his workshop, when a large wood has fallen on his foot. A large lacerated wound emerged, and even dirty. Mr Hobby was taken to a hospital. The wound was sewed by a surgeon, but high fever and signs of sepsis were found. At reoperation, necrotizing inflamation of fascia with necrosis was found. Unfortunatelly, the care did not help: the leg had to be taken away. 99 small-fosny-01 www.rezivo-drevo.cz Necrotizing fasciitis http://www.jyi.org/articleimages/463/originals/img0.jpg 03a strep Who is guilty? nIt is Streptococcus pyogenes nstrepto = in chains, pyo-genes = making pus nStreptococcus pyogenes is known as causative agent of accute tonsilitis. Nevertheless, it causes pyogene tissue inflamations, too. Unlike staphylococci, causing abscesi, here flegmonae are rather common. nBesides tonsilitis, it causes also scarlatina, scarlatiniform tonsilitis and erysipelas. There are strains producing erythrogenous toxin (erythros = red) nWhen the bacterium itself is infected by a bakteriophage, it is even more virulent and becomes a „meat eating bug“ – our case. http://www.uni-tuebingen.de Necrotising fasciitis („flesh eating bacteria“) nIn fact, it is extremely rare,only in streptococcal strains infected by a phagus. Other infections are much more common. 43 flesh eating bacteria http://people.tribe.net 46 tonsillitis bac vs vir http://www.medicalook.com Scarlet fever 52 jazyk2 53 scarla4 http://www1.lf1.cuni.cz Scarlet fever = haevy tonsillitis + exanthema (on skin) + features on mucous membrane. Streptococcus must produce an erythrogenous toxin. Erysipelas 57 erysipel02r 58 erysipel01r www.infektionsnetz.at It is a superficial skin infection that characteristically extends into the cutaneous lymphatics Erysipelas with phlegmona 59 erysipel http://homepage.univie.ac.at 60 erysipefascit http://www.megru.unizh.ch More complications: Repeated erysipelas may also damage lymphatic vessels, leading to chronical lymphedema. Story Two nYoung lady Erika was not too often present at preventive controls during pregnancy. Few days before delivery she found herself in a birthhouse. Delivery itself did not bring any complications. Soon the child started to have signs of sepsis and respiratory failure. Quick treatment saved the child‘s life, and also prevented progression to meningitis that is, unfortunatelly, quite common here. nLater Mrs. Erika was shown to be a carrier of a bacteria, that was shown to be guilty. 63 Str agalactiae http://home.cc.umanitoba.ca/~soninr/Dylan%20in%20hospital.JPG Who is guilty now? nClever students should mention species name a-galactiae, i. e. „milk-less“. This bacterium really causes also milk gland inflamation with dammaged milk production; these features, nevertheless, are seen in cattle, not humans nBacterium Streptococcus agalactiae is a Streptococcus, too. In humans, it rather infects lower parts of body (urogenital infections) with risk of newborn infection SRAG krávy Scheme of transmission of S. agalactiae in cattle (from a veterinary website) http://www.infocarne.com Story Three nHarry the boy has a sore throat. It looks like tonsilitis, but he already subdued both adenectomia and tonsilectomia. nParents went with Harry to see a doctor, to prescribe him some antibiotics. But the doctor said – first throat swab, and then maybe antibiotics. She invited Harry in three days. After thath, she prescribed penicillin, and it started to have effect very soon. 47 tonsillitis2 http://www.medicalook.com 49 kidthroatexam 50 girldoc51 http://www.stronghealth.com http://www.childrenshospital.org Who caused Harry‘s problems? nSo named „non-A-non-B“ streptococci are called so as they do not belong neither to A group (in which Streptococcus pyogenes is the only one) nor to B group (where S. agalactiae is the most important one). nThey do not cause so often tonsilitis, but rather pharyngitis – inflamations of pharynx. Nevertheless, they are often present in healthy persons‘ throats. nThe same as in tonsilitis, in susceptible strains the first antibiotic to be used is penicilin; macrolids in allergic persons only. [USEMAP] Clinical characteristics: viridating (a-haemolytical) streptococci Story Four nMissis Evelyn, retreated, has her spleen let extracted long ago after a car accident. nSeveral days ago, she caught a „common cold“, she did not pay attention to this, but later her status worsened, so her daughter drove her to a hospital, where she was hospitalized on infectious diseases department with suspicious meningitis nGrace to soon antibiotic treatment her status became better and she got back her health. 19 Streptococcus%20pneumoniae http://contanatura.net/arquivo/Streptococcus%20pneumoniae.jpg This time criminal is: nStreptococcus pneumoniae, or „pneumococcus“. It was also called Diplococcus pneumoniae, as it does not form chains, but couples. Its shape is not perfectly spherical, but rather lancet shaped. (Remember this, examinators might ask you this the examination. J) nIn small amount, it is present in healthy persons‘ phrarynx. On the other hand, it causes pneumoniae, sinusitis, otitis media and even sepsis and meningitis. Healthy tympanon (left), otitis media (right) 74 normal_TM_and_AOM www.pedisurg.com Pneumococcal meningitis 04 Streptococcus_pneumoniae_meningitis,_gross_pathology_33_lores 14 pneumokoková meningitis http://www.meningitis.com.au http://commons.wikimedia.org This is how the criminal looks like: crosssection_streptococcus pneumo pneumokokken2 http://www.uni-tuebingen.de http://www.uni-tuebingen.de www.students.stedwards.edu 36 streptococcus_pneumonia050217 http://www.cbc.ca/gfx/pix/streptococcus_pneumonia050217.jpg Story Five nMr. Hearty has long durating heart problems. Even the artificial heart valvula had to be installed into his body. nOne month ago, he ad an awful dental carries, and it durated long time before he came to see a stomatologist. nNow his heart problems worsened so that he had to be hospitalised. Diagnosis endocarditis lenta was set down. plaque1c http://www.vcu.edu Who is the criminal in this crime? nOral streptococci, viridans streptococci, alpha streptococci, all these names describe streptococci viridating on blood agar; usually we mean „viridans streptococci, but not pneumococcus“ nThey are part of normal oral and pharyngeal flora. Even at physiological conditions, all the time some streptococci penetrate in small amounts into the bloodstream The problem starts, when they come there too many together, and when they meet a suitable terrain. Vegetation on a valve 66 endocarditis http://www.pathguy.com Diseased heart 68 endocarditis http://www.fao.org/docrep/003/t0756e/T0756E83.jpg Some possible criminals 16 str salivarius http://www.osel.cz S. salivarius 20 streptococcus sanguis http://microbewiki.kenyon.edu S. sanguis/sanguinis 22 Streptococcus_mutans [USEMAP] http://wishart.biology.ualberta.ca S. mutans Among „oral“ streptococci, S. mutans has probably the highest relation to dental caries. Therapy of streptococcal diseases Treatment: the criminal should be punished nGuilty Streptococci will be punished by a suitable antibiotic. In Streptococci the No. 1 drug is the classical Fleming‘s penicillin (either G-penicillin for parenteral use of V-penicillin for oral use). Macrolides shoudl be used in PNC-allergic persons only. Doxycyklin, co-trimoxazol, ampicilin and others might be used. Vancomycin is a reserve, 100% effective antibiotic (no zone = a mistake, it is not a streptococcus) Susceptibility of streptococci to atb 12 s_pyog_resis nThe picture demostrates a study in 1615 military addicts performed in 2003 nAs you can see, in S. pyogenes drug of choice remains penicillin http://www.nhrc.navy.mil Susceptibility testing nUsually we read the diffusion disk test by measuring the zones and comparing with the reference zones nAgain: the worse pathogen (pyogene streptococcus) is more susceptible than milder pathogens nThe tests are performed on MH agar with blood or on blood agar. On the MH agar without blood steptococci grow poorly, or do nt grow at all. nNevertheless, we cannot utilise this fact in diagnostic – some steptococci are able to grow there! Streptococcal susceptibility test Antibiotic Abbr. Reference zone Penicilin (basic penic.) P 28 mm Cefalotin (cefalosp. 1. g.) KF 18 mm Erytromycin (makrolid) E 23 mm Clindamycin (linkosamid) DA 19 mm Chloramphenicol C 21 mm Doxycyklin (tetracyclin) DO 19 mm Vancomycin (glycopeptid) VA 17 mm Clindamycin: Usually tested, but not in our practical session [USEMAP] Diagnostics of streptococci Description of criminals (diagnostics) 1 nMicroscopy: grampositive cocci nCultivation: on BA grey to colorless colonies, usually small, larger colonies has only Streptococcus agalactiae nHemolytical properties: some viridate, some partially or totally hemolyze nThey do not grow neither on BA with 10 % NaCl, nor on Slanetz-Bartley or Bile aesculin medium. nTogether with entorococci, they are resistant to aminoglykosides, so medium with amikacin is used as a selective medium. Description of criminals (diagnostics) 2 nBiochemical tests: both catalase and oxidase negative, biochemical differentiation of individual species possible especially in viridating streptococci nAntigen analysis helps rather in haemolytical streptococci. Lancefield system is used – theoretically all streptococci are involved, but many viridans streptococci have no antigen in this system. Groups are labelled by letters A, B, C, E, F, G etc. Photos of criminal database S21 S22 [USEMAP] www.medmicro.info Differential diagnostics of streptococci 48a katalase Differenciation from other suspects (diferencial diagnostics 1) nGram stain show all bacteria, that do not belong among grampositive cocci. nNegative catalase test differenciates streptococci from staphylococci nGrowth on SB and BE media differenciates enterococci. All of them are also positive in so named PYR-test, while among streptococci only one of them is positive, and that one is rarely confused because of its very strong haemolysis and other properties http://memiserf.medmikro.ruhr-uni-bochum.de Differenciation from other suspects (diferencial diagnostics 2) nHemolysis should be observed now – it clasiffies streptococci into haemolytical, viridating and others nPneumococcus vs. other viridans streptococci: Pneumococcus has positive optochin test, test of solubility in powder bile etc. nS. pyogenes vs. other haemolytic streptococci: n Both Bacitracin and PYR test are Å in S. pyogenes nS. agalactiaevs. other haemolytic streptococci: n CAMP test is Å in S. agalactiae nAbout all these tests – more info later Schematically: nUnknown bacterium Others G+ coccus Enterococcus Staphylococcus Streptococcus Virid. Streptococcus Haemol. Streptococcus Streptoc. no virid., no haem. Pneumococcus Oral streptococcus S. pyogenes S. agalactiae (SAG) Streptococcus non-A-non-B StreptococcusPyogenes mikroby.blox.pl nPneumococcus can be differenciated by the noptochin test – see following slide. nSuspicion maybe taken, when: nmicroscopically lancet-shaped diplococci can be seen ncultivation: colonies flat, coin-shaped to dish-shaped, sometimes with a central elevation non the other hand, sometimes the colonies are large and mucoid: those are strains with a strong capsulla production (usually highly virulent) Pneumococcus: How to become suspicious streptococcus_pneumonia050217 http://www.cbc.ca Optochin test nClassical test to differenciate pneumococcus from oral streptococci. Pneumococcus is susceptible to optochin (antibiotic), oral streptococci are resistant. (Optochin is not used therapeutically today, it remained in diagnostics only) nSometimes, the test of solubility in powder bile is used. Test of mouse pathogenicity is today considered to be historical 10 opto4a http://www.mc.maricopa.edu Species determination of oral Streptococcus nOnly someone mad (or a researcher – sometimes it is the same) would differenciate an oral streptococcus to species level, when the strain is from oral cavity of pharynx. Why to do it, when we consider it to be a part of normal flora? nOn the other hand, in strains from blood cultures, differenciation is logical. In viridating streptococci, it has no sense to attempt the antigen analysis, but, as we know already, biochemical tests are very useful. nIn Czech conditions, it is mainly STREPTOtest 16 STREPTOtest 16 – how to read it nFirst reaction is again VPT (D‘Artagnan!) n2nd to 9th reaction is again the first strip in the double-strip nSimilarly, 10th to 17th reaction is the second strip in the couble strip n. Three musketeers were four. STREPTOtest 16 (and STAPHYtest 16 and ENTEROtest 16) use 17 reactions. An example of result of Streptotest 16: Code 511 420 Streptococcus salivarius % probab. 97.19 Typicity index 1.00 1 2 H 3G 4 F 5 E 6 D 7 C 8 B 9 A 10 H 11 G 12 F 13 E 14 D 15 C 16 B 17 A Panel – first row Panel – second row + S l l l l l l l l l l l l l l l l - S l l l l l l l l l l l l l l l l ? S l l l l l l l l l l l l l l l l + - + + - - + - - - - + - + - - - 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 5 1 1 4 2 0 Escpecially dagerous criminal: the pyogene Streptococcus. What to do? nPYR test nPYR test is performed similarly as oxidase test. We touch by the strip (its reaction square) the colonies. Then we wait ten minutes. A reagent is added, one more minute of waiting follows. Red = positive nBacitracin test was used sooner. It had the same principle as the Optochin test, only an other antibiotic wass used. Bacitracin test spy_a http://www.mgm.ufl.edu And now the second: Streptococcus agalactiae – 1 nMany bacteria produce haemolysins nWhen two bacteria produce haemolysins, their co-operation may be synergic or antagonistic. nAn example of a synergism is CAMP factor of Str. agalactiae and beta lysin of Staph. aureus nIt is not possible to use it for Staphylococcus diagnostics – not all strains of Stapyhlococi produce the beta lysin! So, the test is used in Streptococcus diagnostics only. camp1 Streptococcus agalactiae – 2 CAMP test nTESTED strain of a Streptococcus and TESTING strain of beta-lysin procucing Staphylococcus are inoculated on the blood agar nIn case of positivity, we see stronger haemolysis in shape of two triangles, or, more poetically, butterfly wings dov8 dov8 Photo O. Z. Stre3 Photo O. Z. Haemolytical criminals – conclusion Bacitracin and PYR test CAMP test Streptococcus positive negative* S. pyogenes negative positive S. agalactiae negative negative non-A-non-B Streptococcus** positive positive a nonsens, a bad test, mix of two strains etc. *sometimes week synergism, not having the proper size and shape *eventually more detailed diagnostic using antigen analysis Latex agglutination Latex agglutination is used for detailed diagnostics of non-A-non-B streptococci, if necessary, according to Lancefield scheme. However, conclusion „it is a non-A-non-B strep“ is usually sufficient. The principle of latex agglutination is showed on the pictures. Aglutination of streptococci with the antibody is helped by latex particles 79a cap19_fig2a 79b cap19_fig2b http://www.seimc.org Remember: nStreptococci with haemolysis (total or partial), but aslo streptococci with no haemolysis at all can be usually determinated using latex agglutination (if necessary). Their biochemical activity uses to be poor. nStreptocooci with viridation (alpha-streptococci) can be usually determinated using biochemical testing (if necessary). Their antigen determinants use to be poor. Latex agglutination – practically nPractical test: the vessels with mixtures of antibodies and latex particles, result (positive in the first cicrle) 76 aglutinační set http://www.pro-lab.com 75 aglutinuje SRPY [USEMAP] http://www.medicine.uiowa.edu Late sequelae of streptococcal diseases Streptococcus pyogenes is even worse than we already knew nYou know that S. pyogenes causes tonsillitis, scarlatina, erysipelas. But the worst still waits: Even after being flown out from the organism, a terrible sequelae may occur! Antibodies circulate in the blood… and mistakenly, instead of being bound to streptococci, they bind to some structures of the organism. So, accute glomerulonephritis or rheumatic fever occurs. nYou may mention, that we have had this already once in the spring semester… Scandal clarification! Rheumatic Fever 62 arf http://mednote.co.kr ASO: how to see, if the risk exists nUsing ASO test you will see, if a normal antibody response is formed, or an autoimmunity over-response with risk of development of glomerulonefritis/rheum. fever nASO test is usually performed after a streptococcal infection. By the antibody detection, we do not try to detect the infection (we know about it), but to clarify, whetrer autoimmunity response is developped. So it is NOT an indirect diagnostic, although antibodies are measured. ASO: principle (repeating) nThe antibody blocates the haemolytical effect of the toxin (streptolyzin O) on a RBC. nIn ASO, we do not use the geometrical row. The values of dilution are in a table. nTitre over cca 250 means a risk of antibody response nIn Czech, abbreviation ASLO is used instead of ASO in English. ASLO How to read an ASO panel nEach patient has one row. The dilutions are here and in tables on your working tables. n n n n n nPanel has a positive control and five patients 2006-04-07 10 ASLOu Goodbye at the next part! plyšový streptokok [USEMAP] Soft toy-Streptococcus www.giantmicrobes.cz