TRACING THE CRIMINAL Part three: More G+ criminals Institute of Microbiology shows: Survey of G+ bacteria Contents Clinical characteristics - enterococci Story 1 n Lucy 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 n Enterococcus faecalis n As the „entero-“ in his name tells us, it is a bug normally present in the intestine. Nevertheless, it is also a common UTI pathogen. n The 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. n Maybe, mother, her too, missed something, if she forgot to teach Lucy a corect „intime hygiene“ – to clean herself from front to back More about enterococci n There are tens of species of them today n All of them may be found n in stool (as a normal flora) n in the urinary bladder (as pathogen) n in vagina (both symptomatically and asymptomatically) n sometimes in other sites (wounds, bloodstream) n Among two most common species, E. faecalis rather tends to be a pathogen, E. faecium is more often part of normal intestinal flora n One of Enterococci, found in Brno, is named Enterococcus moraviensis Clinical characteristics – G+ rods Story 2 n European comission had to solve one more problem. French agriculturers protested againts several EU-members, that do not want to inport some delicatess french cheese specialitis to their area. n German officials stated, that one pregnant woman, Mrs. Hildegarda Messerschmidt, after having eaten the cheese had enlarged lypmphonodes and after delivery, her baby was heavily infected. Criminal No 2 Strory 3 n Mr. Ulcerous, chronical diabetic, treated for ulcerations on his legs. He is often infected by various pathogens. What will be the newest one? Criminal No 3 n Corynebacterium jejkeium, the worst among non-diphtheria coryneforms. Its name is derived from „corynebakterium group JK“. n Corynebacteria are grampositive rods club-shaped (koryné = club), sometimes pleomorphic. n In the same genus, we have also diphtheria causative agent, rare in Europe, because of vaccination – C. diphtheriae. Diphtheria More about non-diphtherical corynebacteria n Part of normal flora of skin, together with staphylococci and yeast. Pathogens in wounds What are „coryneform rods“ n „Coryneform rods“ (eventually „diphtheroids“) are various rods that share simillar morphology (although size of rods may vary considerably). n All of them are rare causative agents of various human infections. n Arcanobacterium haemolyticum is a rare causative agent of pharyngitis n Other genera: Dermatophilus, Rhodococcus*, Turicella etc. Story 4 n Nurse 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… And she was very glad: n it 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. But some Bacilli are important n Bacillus anthracis was popularized by Mr. Osama & Co. n Bacillus 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) n Bacillus stearothemophillus & Bacillus subtilis are able to survive hot temperatures à we use them as control organisms for hot air and steam sterilisers. Enterococci and G+ rods: therapy Therapy of infections caused by enterococci and G+ rods n No 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 Diagnostics of enterococci and G+ rods (+ pictures) Description of criminals (diagnostics 1) Enterococci – colonies Description of criminals (diagnostics 2) Enterococci n Biochemical tests: catalase negative, possible biochemical determination, arabinose splitting (E. faecalis does not split, green medium, E. faecium makes it yellow) n Antigen analysis used rarely. (Originally „group D streptococi“ according to Lancefield, as genus Enterococcus did not exist in time of Lancefield research) n Atb testing on common MH agar. There exist also screening media for VRE. Description of criminals (diagnostics 3) G+ rods n Biochemical 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) n Growth at low temperatures, high NaCl concentrations etc. used in Listeria dg. n Biochemical dg. and atb testing are also a part of the diagnostics n Antigen analysis – e. g. searching diphteria toxin Photos of criminal database 1 Enterococci Photos of criminal database 2 Rods I Photos of criminal database 3 Rods I – corynebacteria, forms Photos of criminal database 4 Rods III Differential diagnostics of enterococci and G+ rods Differential diagnostics - enterococci n Gram staining differenciates Gram + cocci, Gram + rods and other bacteria. n Catalase of NaCl differentiates staphylococci n Slanetz-Bartley / Bile-aesculin, PYR test differentiates enterococci from streptococci n Arabinose test/other biochemical tests – mutual differentiation of Enterococci Differential diagnostics – Bacillus n Bacillus, Listeria & coryneforms = G+ rods n Bacillus: n culture: large, flat, dry, felt-like colonies, „spreading“ through the agar surface, sometimes with a massive haemolysis, sometimes with no haemolysisat all n microscopy: 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 n Listeria n culture: colourless to greyish colonies, very simillar to those of Enterococcus, with or without haemolysis n it does grow on bile aesculin (but not Slanetz-Bartely) agar; it also does grow on BA at 4 °C n microscopy: tinier than Bacillus, not arranged in pallisades, rather in short chains n Corynebacterium (and related genera): n culture: greyish or whitish colonies simillar to those of Staphylococcus, but less or more smaller, usually ahaemolytical n microscopy: rather smaller than previous, but club-shaped and arranged in palisades Bile-aesculin agar Differentiation of Enterococcus n EnCoccus test has only 8 reactions, but otherwise it is like other similar tests Listeria growth at 4 °C n Among Gram positive rods, only Listeria is able to grow in low temperatures. This enables it to spread in cheese factories n Among other bacteria (not being G+ rod), there are some more species able to grow at such low temperatures (Yersinia, some Pseudomonas sp.) A chromogenous medium A chromogen (colourles) is changed to a blue dye by a specific enzyme of Listeria. Pathogenous listeriae perform a halo due to some more enzymes. Elek test It 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. The end