TRACING THE CRIMINAL Part three: More G+ criminals Institute of Microbiology shows: L Survey of G+ bacteria Story Shape In police evidence written as P01 Medically important Cocci Staphylococci (S. aureus, CONS) P02 Streptococci (viridating, haemolytical) 1. Enterococci (E. faecalis, E. faecium) 2. Medically important Rods Listeriae (L. monocytogenes) 3. Corynebacteria 4. Bacilli Listeriae a coryneforms do not sporulate, unlike Bacilli 15 Corynebacterium 25 BASU http://vietsciences.free.fr http://web.fccj.org [USEMAP] Contents Clinical characteristics – enterococci Clinical characteristics – G+ rods Enterococci and G+ rods: therapy Diagnostics of enterococci and G+ rods (+ pictures) Differential diagnostics of enterococci and G+ rods Clinical characteristics - enterococci Story 1 nLucy has problems with urination. Doctor prescribed Zinnat, but problems did not change. On the next visit, he let Lucy to urinate in a glass and sent her urine sample to microbiology. But the specimen could not be examined: urine was contaminated. Finally, it was possible to take urine asseptically and to change the therapy. Criminal No 1 nEnterococcus faecalis nAs the „entero-“ in his name tells us, it is a bug normally present in the intestine. Nevertheless, it is also a common UTI pathogen. nThe doctor is guilty, too – prescribed antibiotics before knowing microbial susceptibility. Enterococci are resistant to all cephalosporin atb. And he did not perform aseptic urine sampling in the first phase. nUTI (urinary tract infections) are mostly bacterial, and many pathogens are primarily or secondarily resistant to some atb-s. So bacteriological examination of urine is recommended, althoug often not done in practice. 02 Ec faecium http://www.lbl.gov More about enterococci nThere are tens of species of them today nAll of them may be found nin stool (as a normal flora) nin the urinary bladder (as pathogen) nin the vagina (both symptomatically and asymptomatically) nsometimes in other sites (wounds, bloodstream) nAmong two most common species, E. faecalis is slightly more often a pathogen, E. faecium is more often part of normal intestinal flora nVancomycin-resistant enterococci (VRE) are dangerous nOne of Enterococci, found in Brno, is named Enterococcus moraviensis [USEMAP] Clinical characteristics – G+ rods Story 2 nEuropean comission had to solve one more problem. French agriculturers protested againts several EU-members, that do not want to import some delicatess French cheese specialities to their area. nGerman officials stated, that one pregnant woman, Mrs. Hildegarda Messerschmidt, after having eaten the cheese had enlarged lypmphonodes and after delivery, her baby suffered newborn meningitis 13 těhotná listeria 08 Baby%20with%20Listeria http://womansday.ninemsn.com.au http://www.leighday.co.uk 07 Listeria%20bacteria Criminal No 2 nListeria monocytogenes is a G + rod, able to grow at low temperatures and high NaCl concentrations nAdult infections are rare, except lymfonode syndrom. Nevertheless, it is dangerous for pregnants, or rather fot their future children nIn newborns, septicaemia, pneumonia and meningitis is typical. nAs it is rare, is it hardly a reason for true closing borders for camions. Nevertheless, of course, always a momentary situation is to be judged. http://www.leighday.co.uk 09 Listeria 10 listeria http://microbewiki.kenyon.edu http://www.territoire-belfort.gouv.fr Strory 3 nMr. Ulcerous, chronical diabetic, treated for ulcerations on his legs. He is often infected by various pathogens. What will be the newest one? 19 bércák www.zilniklinika.cz Criminal No 3 nCorynebacterium jejkeium, the worst among non-diphtheria coryneforms. Its name is derived from „corynebakterium group JK“. nCorynebacteria are grampositive rods club-shaped (koryné = club), sometimes pleomorphic. nIn the same genus, we have also diphtheria causative agent, rare in Europe, because of vaccination – C. diphtheriae. http://microbewiki.kenyon.edu 14 Corynebacteriumdiphteriae Diphtheria 16 záškrt 17 záškrt www.emedicine.com More about non-diphtherical corynebacteria nPart of normal flora of skin, together with staphylococci and yeast. Pathogens in wounds xPalisáda nIn microscopy, they form „palisades“ – like the early medieval wooden fortifications What are „coryneform rods“ n„Coryneform rods“ (eventually „diphtheroids“) are various rods that share simillar morphology (although size of rods may vary considerably). nAll of them are rare causative agents of various human infections. nArcanobacterium haemolyticum is a rare causative agent of pharyngitis nOther genera: Dermatophilus, Rhodococcus*, Turicella etc. Rhodococcus jostii was found on the body of the Moravian Marques and uncrowned Roman Emperor Jodocus (Jošt), that died 1411. The body is burried in St. Thomas church in Brno. Story 4 nNurse Eileen was shocked: microbiology examination of ward furniture, week ago taken by hospital epidemiologists, showed some BACILLI! Yes, it is here – Bacillus sp. Eileen was worying all the night about it. In the morning, she asked microbiologists… 23 lůžko www.dahlhausen.cz 20 Bacillus%20cereus And she was very glad: nit is no criminal! Usual members of Bacillus genus are harmless microbes from external environment. When found in clinical material, it is usually a contamination. So, the finding was not a problem – problem would be only when a Bacillus would be found from a site that is supposed to be sterile. www.waterscan.co.yu But some Bacilli are important nBacillus anthracis was popularized by Mr. Osama & Co. nBacillus cereus is causative agent of intoxications coming from cereals (one Swiss let a remaining spaghetti oudside fridge, then he decided to eat it, and then he died) nBacillus stearothemophillus & Bacillus subtilis are able to survive hot temperatures à we use them as control organisms for hot air and steam sterilisers. 21 bacillus 22 Genre%20Bacillus aa [USEMAP] www.cropsoil.uga.edu http://membres.lycos.fr Enterococci and G+ rods: therapy Therapy of infections caused by enterococci and G+ rods nNo cephalosporins to fight enterococci and listeriae! In E. faecalis, ampicillin, is good, but in E. faecium there is a primary resistance. More atb‘s are co-trimoxazol, doxycyklin, and, as a reserve, vankomycin. In haematooncological pacients we can find epidemiologically serious vancomycin resistant strains – VRE. In such strains, only a new atb – linezolid – is effective Antibiotics used for enterococci and G+ rods Antibiotic Abbr. Reference zone Ampicilin (aminopenicilin) AMP 17 mm Co-amoxicilin (aminopnc*) AMC 18 mm Co-trimoxazol (mixt. 2) SXT 16 mm Tetracycline TE 15 mm Chloramphenicol C 21 mm Vancomycin (glykopeptid) VA 17 mm *potentiated by a beta-lactamase inhibitor nEnterococci are tested on MH. G+ rods are tested on MH + red blood cells. [USEMAP] Diagnostics of enterococci and G+ rods (+ pictures) Description of criminals (diagnostics 1) Enterococ. Listeria Coryneform Bacillus G+ cocci short chains G+ rods chains or palisades G+ rods palisades G+ robust rods, sporulating (sometimes non visible) greyish, as large as that of S. agalactiae, various hamolysis like enterococci of Strep. agalactiae very tiny colonies, like flour large colonies, sometimes intensive haemolysis 01 Enterococcus http://textbookofbacteriology.net Enterococci – colonies 03 ECFM VRE normální morfo http://microbiology.mtsinai.on.ca Description of criminals (diagnostics 2) Enterococci 06 ECFS nBiochemical tests: catalase negative, possible biochemical determination, arabinose splitting (E. faecalis does not split, green medium, E. faecium makes it yellow) nAntigen analysis used rarely. (Originally „group D streptococi“ according to Lancefield, as genus Enterococcus did not exist in time of Lancefield research) nAtb testing on common MH agar. There exist also screening media for VRE. http://www.morgenwelt.de Description of criminals (diagnostics 3) G+ rods nBiochemical tests: catalase positive in all three of our genera. But e. g. genus Arcanobacterium (not member of Corynebacterium genus, but nevertheless a coryneform) is CAT neg. Biochemical detection possible (API Coryne, Remel) nGrowth at low temperatures, high NaCl concentrations etc. used in Listeria dg. nBiochemical dg. and atb testing are also a part of the diagnostics nAntigen analysis – e. g. searching diphteria toxin Photos of criminal database 1 Enterococci Ent mikro Ent že Ent SB Microscopy Bile-aesculin Slanetz-Bartley Photo: www.medmicro.info both left pictures made by Prof. MVDr. Boris Skalka, DrSc. Photos of criminal database 2 Rods I Listeria – BA, Gram limo1 diftmik Corynebacterium Gram diftmik1 12 LIMO mikr 1, 2, 3 www.medmicro.info 4 http://medinfo.ufl.edu 1 2 3 4 Photos of criminal database 3 Rods I – corynebacteria, forms diftmik2 diftmik2a www.medmicro.info, Photo O. Z. raven wings palisade Photos of criminal database 4 Rods III Bacillus subtilis arhe basu6 Arcanobacterium haemolyticum Bacillus cereus Bace [USEMAP] www.medmicro.info Differential diagnostics of enterococci and G+ rods Differential diagnostics - enterococci nGram staining differenciates Gram + cocci, Gram + rods and other bacteria. nCatalase of NaCl differentiates staphylococci nSlanetz-Bartley / Bile-aesculin, PYR test differentiates enterococci from streptococci nArabinose test/other biochemical tests – mutual differentiation of Enterococci Differential diagnostics – Bacillus nBacillus, Listeria & coryneforms = G+ rods nBacillus: nculture: large, flat, dry, felt-like colonies, „spreading“ through the agar surface, sometimes with a massive haemolysis, sometimes with no haemolysisat all nmicroscopy: very robust rods, sometimes with finding of central or subterminal spores, that may, but must not be larger then the diameter of the rod. Differential diagnostics – Listeria and coryneforms nListeria nculture: colourless to greyish colonies, very simillar to those of Enterococcus, with or without haemolysis nit does grow on bile aesculin (but not Slanetz-Bartely) agar; it also does grow on BA at 4 °C nmicroscopy: tinier than Bacillus, sometimes arranged in pallisades, sometimes in short chains nCorynebacterium (and related genera): nculture: greyish or whitish colonies simillar to those of Staphylococcus, but less or more smaller, usually ahaemolytical nmicroscopy: rather smaller than previous, but club-shaped and arranged in palisades Bile-aesculin agar 11 bileazideagar http://www.geocities.com Differentiation of Enterococcus nENCOCCUStest has only 8 reactions, but otherwise it is like other similar tests p Green negative E. faecalis Yellow positive E. faecium nArabinose test: colonies are mixed with arabinose and indicator, and let to incubate Listeria growth at 4 °C nAmong Gram positive rods, only Listeria is able to grow in low temperatures. This enables it to spread in cheese factories nAmong other bacteria (not being G+ rod), there are some more species able to grow at such low temperatures (Yersinia, some Pseudomonas sp.) 26 Chromogenní půda na listerie A chromogenous medium nA chromogen (colourles) is changed to a blue dye by a specific enzyme of Listeria. Pathogenous listeriae perform a halo due to some more enzymes. ALOA HALO www.oxoid.com Elek test nIt is a detection of a toxin of Corynebacterium diphtheriae. We use a paper with specific antitoxin, that is put on the surface of the agar, then tested strains are inoculated. Positive result = precipitation lines. Elekův test The end 24 Banthracissporeandvegatativeform-gramstain [USEMAP] http://www.cdphe.state.co.us Bacillus anthracis