TRACING THE CRIMINAL Part one: Straphylococcus Institute of Microbiology shows: L [USEMAP] Overview Clinical characteristics: Staphylococcus aureus Clinical characteristics: CONS (coagulase-negative staphylococci) Diagnostics of staphylococci Differential diagnostics of staphylococci I Differential diagnostics od staphylococci II Antibiotic susceptibility testing and antibiotic treatment Clinical characteristics: Staphylococcus aureus Story One nMrs. J. K., cook in students canteen, has a blister on her hand, full of white-yellow pus. She is not aware. She takes dumplings by her hand, although the dumplings are already cooked (and now they ill be only slightly heated, not cooked). nStudent Rashid and his girl-friend eat the dumplings. In the afternoon, they should have a rendez-vous … BUT… half an hour before the rendez vous, Rashid started to have abdominal pain, vomitting and diarrhoea. The girl-friend, called, says she has the same problems… So, no romantic afternoon… 34 Furuncle1500 www.brooksidepress.org Who is guilty? nIt is Staphylococcus aureus nname from greek staphylé = grape nThis „golden staphylococcus“ often cases pyogene infections of skin, hairs, nails etc. nSome strains produce enterotoxins, that act as so named superantigens nIntoxication by a bacterial toxin usually starts quickly and finishes quickly, unlike a bacterial infection (e. g. salmonelosis) nOf course, the cook, that prepared meals despite her blister, is guilty, too! 01 10b-Staphylococcus-sp_mediu http://www.biology.ualberta.ca/facilities/microscopy/uploads/gallery/ESEM/10b-Staphylococcus-sp_med iu.jpg 17a staph zvětšení 50 000krát emu.arsusda.gov/typesof/pages/staph.htm nGolden staphylococcus, 10 000 × magnified in an electron microscope Story Two nStudent P. Z. is nervous: again, she has „her days“. Luckily, she has the cheap tampons, that she bought several days ago… nSuddenly, she started to have shaking, faintness, fever. The room-mate found her lying on the floor, and called 155 (or maybe 112?). A rash emerged. The students is hospitalized on emergency unit of infectological clinic… 07a Staphylococcus_aureus_01 http://www.answers.com/topic/staphylococcus-aureus-01-jpg Who is guilty now? nThis toxin, too, is a superantigen nIt causes toxic shock, typically in users of menstruation tampones nAgain, it is Staphylococcus aureus, now a strain called TSST-1 (toxic shoc syndrome toxin) 32 tsst_pic http://www.cbs.umn.edu/bmbb/ohlen_lab/new/web/research_interests/superantigens.htm Superantigens 25 superantigeny www.zuova.cz/informace/nrlpab16.php Immunity response Antigens Superantigens Cytokins Overproduction of cytokins T and B lymphocytes proliferation TNF production PRODUCTION OF ANTIBODIES SEPTICAL SHOCK Staphylococcus aureus (golden staphylococcus) nThe only one routinely important for humans among so named coagulase positive staphylococci nCauses skin, hair, nail infections, otitis externa, conjunctivitis, respiratory infections nSometimes also causes abscessi in tissues nSome strains with uncommon virulence factors cause serious, but rare, diseases nOn the other hand, the microbe may be often found even on skin of healthy persons There exist plenty of virulence factors found in S. aureus… 03 Virulence_factors Staphylococcus http://www.ratsteachmicro.com …but only some of them are present in nearly 100 % strains; other are produced just by one strain among one thousand! Abscessi nUnlike streptococci, producing in tissues mostly uncoated phlegmonae, staphylococci form mostly coated abscessi. Formation of an abscessus (using so named clumping factor and plasmacoagulase – see further!) is in the next scheme, from a german website 45 jak vznikne furunkl http://www.autovaccine.de/abscess_formation/Abszess_b.jpg Examples of infections caused by staphylococci: Impetigo… 15 stafylokokové impetigo http://pathmicro.med.sc.edu/fox/staph-impetigo.jpg …bulous impetigo… 37 BullousImpetigo3 …otitis externa with a furuncle… 38 otitis externa with furuncle www.merck.com/mmpe/print/sec08/ch088/ch088c.html. …or skin infection with crusts… 05 Infection_staphylococcus_crusting_chin_closeup http://www.dermatology.co.uk/media/images/Infection_staphylococcus_crusting_chin_closeup.jpg …but also brain abscessi 36 got10354_fm-2 [USEMAP] http://www.mja.com.au/public/issues/176_12_170602/got10354_fm.html Clinical characteristics: Staphylococcus sp. (coag.-neg.) Story Three nYoung man F. B. recovers after a severe traffic accident. He has two venous cateters for infusion nutrition and blood taking. nSuddenly, his status worsened acutally, high and quickly changing fevers – the ward doctor has suspicion for septicaemia and takes blood for blood culture nAfter catether change and antibiotic treatment the status improved again 19 aureus v hemokultuře fluorescence http://www.zuova.cz/informace/pic/ompovabac20b.jpg And who is guilty now? nThe guilty is Staphylococcus epidermidis, the most common among coagulase negative staphylococci nCoagulase negative staphylococci beong to the same genus as „golden staphylococcus“ nThe are much less pathogenous nIn last decades, they started to be very important causative agents of infections in weekened persons, mostly as hospital infections nOften forms biofilm on venous catheters nWhy „coagulase negative staphylococci“? See later… 03 staphylococcus_epidermidis Staphylococcus epidermidis 06a staphylococcus_epidermidis 12 Staphylococcus epidermidis SideView http://www3.niaid.nih.gov http://www.difossombrone.it http://www.microbelibrary.org Coagulase negative staphylococci nCoagulase negative staphylococci (Staphylococcus epidermidis, S. hominis, S. haemolyticus and about fourty other species and subspecies) are the main parts of the common skin microflora. nNevertheless, they may cause UTI (mostly S. saprophyticus), wound infections, catheter septicaemiae etc. nSo, the finding has a different meaning e. g. in nasal cavity (or in stool), in the urine, and of course, in blood culture. There are many species of staphylo- cocci today nE. g. S. simiae was found by Moravian scientists in rectal swabs of Saimiri sciureus monkey in a ZOO on Saint Hill at Olomouc http://www.szu.cz/cem/zpravy/zpr0905/sse_soubory/image005.gif 21a strom druhů stafylokoků inverze [USEMAP] Diagnostics of staphylococci Description of criminals (diagnostics) 1 nMicroscopy: grampositive cocci nCultivation: on BA colonies 1–2 mm, slightly convex, butter consistence, white, or (mostly in golden staphylococcus) goldish nBiochemical tests: catalase positive, oxidase negative, it is possible to differenciate individual species biochemically nAntigen analysis and special tests maybe very helpful at the diagnostics Photos from Criminal Database S2 S13 [USEMAP] www.medmicro.info (our webpage) Differential diagnostics of staphylococci I: from „unknown bacterium“ to „Staphylococcus“ Survey of microbiological diagnostics of a staph infection n(Microscopy of SPECIMEN (e. g. sputum) nMicroscopy of isolated STRAINS nNow, we are able to distinguish G+ cocci from others n(Description of colonies on blood agar) nCatalase test (Staphylococci × other G+ cocci) nGrowth on BA with 10 % NaCl nNow, we have differenciated staphylococci from the other G+ cocci nDifferenciation of "golden" Staphylococcus from coagulase negative species nSpecies diagnostics of Staphylococcus nAtb susceptibility testing (when Staph is a pathogen) Searching for criminal microscopically in the specimen nWe observe a Gram stained microscopic sputum preparation nWe search for Gram-positive cocci in clusters, but also for leucocytes (polymorphonuclears mainly), typical for bacterial inflamation) sputumgpkoUPR Photo: O. Z. leucocytes cluster of G+ cocci Singling out of other suspects (differencial diagnostics 1) nGram stain differenciates gram-positive cocci from other shape/cell wall type bacteria nPositive catalase differenciates staphylococci from streptococci and enterococci nThe same (and even better in a mixture) is cultivation on BA with 10 % NaCl nFor orientation we can also use the fact that colonies of other G+ cocci are neither white nor goldish, and in microscopy, there do not have clusters Gram stain (repeating) nGram stain: we make a smear (using a small drop of saline), we let it dry, we fixate by a flame, then we stain: Gram 30 s, Lugol 30 s, alcohol 15 s, water, safranin 60 s, water, dry, imersion obj. 100× magnifying) nNow, we can exclude all object that are gram negative and/or rods, eg. that do not belong into group of „G+ cocci“ Catalase test (for remembering) 48 katalase http://memiserf.medmikro.ruhr-uni-bochum.de Catalase test 40 CatalaseResults1 http://www.telmeds.org Survey of diagnostics (simplified) http://www.ratsteachmicro.com/Staphylococci_Notes/HCOE_CAI_Review_Notes_Staphylococci.htm 29 Gram_Positive_Flow_Chart zmenšeno a inverze Enterococcus or (or other tests) Description of colonies on BA nDescription of colonies on blood agar does not have a specific place in differential diagnostics of staphylococci. Nevertheless, it is usefull, as it can lead us to certain suspicion (e. g. stapyhlococci have rather whitish/yellowish collonies, unlike grey/colourless streptococci) Discrimination between Staphylococcus and Streptococcus/Enterococcus nIn a bacterial mixture, a Staphylococcus may be selected using growth on BA with 10 % NaCl; other G+ cocci do not grow. nIf a pure strain is available and we require a quick diagnostics, catalase test catalase test may be used (a colony is mixed with a drop of hydrogen peroxyde, bubbles = positivity). nAttention! By jumping over the previous steps, we would do a mistake. Positive catalase test is common in many bacteria. Only in a known G+ coccus it is possible to use it for diagnostics! p [USEMAP] Differential diagnostics of staphylococci II: steps inside genus Staphylococcus Singling out of other suspects (differencial diagnostics 2) nFree plasmacoagulase is positive in „golden staphylococcus“, negative in coagulase negative ones (here the origin of their name) nClumping factor or bound plasmacoagulase is used in the same situations, but is worse nComercial tests based on antigen analysis are very good on the other hand (but expensive) nHyaluronidase is not only good, but cheap, too S3 Less sure tests: useful in searching, but cannot be used as a proof for court! nHemolysis: Coagulase negative staphlococci may produce delta haemolysin, „Golden“ staphylococci may produce alfa, beta and delta haemolysin, so their haemolysis uses to be stronger. nGoldish colour of colonies and their larger diameter may be useful, too. nLarger clusters in microscope n are also typical n for „golden“ staphylococci www.medmicro.info Clumping factor or bound plasmacoagulase – quick nColonies are mixed with a drop of rabbit plasma on a slide nPositivity is formation of „clusters“ in plasma drop (see next screen) nIn fact, it is not a COAGULATION, but AGLUTINATION of plasma nThe test is not very sure 47 clumping http://memiserf.medmikro.ruhr-uni-bochum.de Free coagulase – classic nThe most classical among diferenciation test for "golden" Staphylococcus (the coagulase positive Staphylococcus) nColonies, taken by a loop, are mixed with rabbit plasma in a test-tube nWhen the plasma coagulates (gel consistence), the strain is n coagulase n positive 41 koaguláza www.hardydiagnostics.com More pictures of plasmacoagulase 44 coagulase-1 http://microbiology.scu.edu.tw 43 coagulase Comercial tests, e. g. Staphaurex (not in the practical) nThe way of using them is the same as in the clumping factor test, but they are even more sure than free coagulase nUnfortunatelly, they are quite expensive 49 stafaurex www.microbes-edu.org Staphaurex kit and results 50 staphaurex 51a murex1 51b murex2 http://www.pathologyinpractice.com www.microbes-edu.org Hyaluronidase (decapsulation) nAn elegant test, its principle is the fact, that the hyaluronidase, produced by S. aureus (but not coagulase negative staphylococci) breaks the capsula of encapsulted bacteria. We use Streptococcus equii, a streptococcus that is not pathogenous for humans nLack of a capsulla is seen as change of feature of streptococcus (no „mucosity“) Hyaluronidáza P1020006a P1020006ax Foto: O. Z. Yellow – Streptococcus equi (mucous) White – tested staphylococci Results for this example: C and D are positive (S. aureus) A, B and E are negative (coagulase negative staphyl.) P1020007a P1020007b Foto: O. Z. Survey of methods distinguishing S. aureus from CONS (coagulase negative staphylococci) nClumping factor test: a drop of plasma is mixed with a tested strain on a slide nPlasmacoagulase test: strain is mixed with rabbit plasma in a test tube. Prelimnary reading is done after 4 h and deffinitive reading after 24 h. Coagulated liquid = positive nHyaluronidase test: Positive strain dissolves the mucosity of an encapsulated strain (a horse streptococcus Streptococcus equi is used mostly for this test) It is not „The Golden“. What now? nUsually we simply say „it is a coagulase negative one“ and we do not insist on species diagnostics nWhen species would be important (e. g. in blood cultures), it can be performed biochemically nIn Czech conditions, mostly STAPHYtest 16 (Erba-Lachema) is used Mutual differenciation of staphylococci nSTAPHYtest 16 is the most typical Czech variant of a biochemical testing systém of staphylococci. It should be done according to guidelines. It certifies the identity of "golden" Staphylococcus and it identifies the other. nNormally it is useless to diagnose „golden staphylococcus“ by STAPHYtest 16 , tests of tasks 6a, b and c, or comercial tests are rather used for this nSo the test is used for diagnostics of coagulase negative staphylococci STAPHYtest 16 – how to read it nAttention – despite its name, there are 17 reactions in it. We start by reading VPT test in a test tube. Red fluid in the test tube = positive VPT, colorless fluid = negative nFirst row of the STAPHYtest = 2nd–9th reaction nSecond row of the STAPHYtest = 10th– 17th r. nCount the code and compare with the codebook nThe code consistis of six numbers. Five of them are based on triplets of test, the sixth is based on the last two tests (16 + 17) An example of a result (703 651 = S. aureus, 99.8 %, Tin=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 First row of panel Second row of panel + 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 7 0 3 6 5 1 Another example of a result (703 241 = S. epidermidis, 97.95 %, Tin=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 First row of panel Second row of panel + 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 7 0 3 2 4 1 Api Staph – in some countries used equivalent of STAPHYtest 16 apisapro nNot regarding the producer, the principle is the same – combination of many enzymatic reactions, that can be seen as colour change http://www.microbes-edu.org Another variant of a API-Staph nThe previous one was an API-Staph for automatic reading in a photometer. This one is for „ocular“ reading http://www.microbes-edu.org 56 api ruční 1 57 api ruční 2 [USEMAP] Antibiotic susceptibility testing and antibiotic treatment of staphylococcal infection Susceptibility testing nTo check secondary resistances, we mostly use a diffusion disk test – we measure the inhibition zones and compare with reference zones nAmong used antibiotics we use commonly e. g. OX = oxacilin, VA = vankomycin, RD = rifampicin, KF = cefalotin, MY – linkomycin CN = gentamicin nOf course, antibiotic testing is only performed for pathogens (= not for staphylococci belonging to common microflora) Reference zones for the most common antibiotics Antibiotic Abbr. Refer. zone Oxacilin (protistaf. penic.) OX 13/18 mm Vankomycin (glycopept.) VA 12 mm Rifampicin (rifamycine) RD 20 mm Cefalotin (cephalo. 1. g.) KF 18 mm Linkomycin (lincosamid) MY 21 mm Gentamicin (aminoglyk.) CN 15 mm OX: 13 mm S. aureus, 18 mm coagulase negative st. atbstau17 www.medmicro.info (stránky ústavu) Ilustration photo Quantitative and qualitative tests nAs mentioned, usually we use a qualitative test (diffusion disc test). Nevertheless, it is also possible to use quantitative tests (microdilution test, E-test) 53 ab1 55 etest staf http://www.microbes-edu.org According to situation, we use either ßqualitative, or quantitative tests Usual law: worse pathogen – better susceptibility nYou will probably see, that a worse patogen (S. aureus) uses to be more susceptible than the milder pathogen (coagulase negative stafylococcus). It is logical: milder pathogenicity shows better adaptation, ability of a microbe to coexist without causing a disease à being used to common antibiotics nIt is not absolute! There are nicely susceptible S. epidermidis strains, and MRSA. Anti-styphylococcal drugs nIn staphylococci, the drug of choice is oxacilin, in UTI cefalosporins of first generation. Often used macrolids are good in allergic persons only, lincosamids are good in locomotor system infections and aminoglycosides in combination only. Glykopeptidic antibiotics (vankomycin and teikoplanin) are a reserve. They are used in strains resistant to oxacilin, so named MRSA and MRSKN. In strains resistant even to glycopeptices, or in patients that has contraindications, newer antibiotic linezolid can be used. MRSA and their detection nMethicilin resistant staphylococci (MRSA) are epidemiologically important strains, often causing serious hospital infections nThey are caused by change of so named membrane penicillin binding proteins (PBP) nProblem is seen by a small zone in oxacilin. But it is not a clear proof. nThe proof is, when the zone is small not only in oxacilin, but also cefoxitin The End 24 oběť stafylokoků [USEMAP] www.osel.cz A victim of a staphylococcal infection