TRACING THE CRIMINAL Part Four: Enterobacteria (& Co.) Microbiological institute shows L A poem for beginning… nWe can check a healthy hen. nEverything OK. But then nPigeons start to fly to it nInfect others, that‘s their hit. nThen the egg is used for ice nLooking very, very nice. nBut salmonellas can hide ninvisible, still inside. nAnd then youngsters eat the ice… n…and their speed increases twice! Saen MAL Salmonella on MAL agar 32 eggs-and-salmonella http://www.uwec.edu www.medmicro.info, photo author: prof. Boris Skalka [USEMAP] Survey of topics Clinical description: Enterobacteriaceae Cl. descr.: Campylobacter, Helicobacter, Vibrionaceae Pictures of bacteria Diagnostics of Enterobacteriaceae Dg. of Campylobacter, Helicobacter, Vibrionaceae Clinical description: Enterobacteriaceae Story one nMiss Theresa ate a cake with egg cream after her lunch. In the afternoon, she started to vomit and to have diarrhoea. She visited a doctor and he made the anal swab. Several days later, officers of public health administration called her. Finally, although she did not believe, that cake was not guilty. Who is guilty now, then? nBacterial criminal is Salmonella enterica serovar Enteritidis, briefly Salmonella Enteritidis nCream cake cannot be the criminal! Incubation time does not correspond. nAnother cake, consumed two days before, was later proven to be the infection source. nAs to humans, probably someone in the „Hysterical Maid Cafee“ failed to do something. Public health officers do their monitoring just now. 30 salmonella 07 SAsp 33 SALMONELLA 34 Salmonella%20on%20XLD Salmonella http://www.microbiology.org.uk http://www.uwec.edu www2.mf.uni-lj.si 29 salmonella_ec http://www.fehd.gov.hk Salmonella endocarditis 31 salmonelová endokarditis http://www.som.tulane.edu 10 YEsp Primary pathogens among enterobacteria nEnterobacteriaceae is the clinically most important family among Gram– rods (not only clinically) nThe worst pathogens perform systemic infections: it is Yersinia pestis (causing plague) and so named anthropopatogenous serovars of Salmonella (serovar Typhi, Paratyphi A, Paratyphi B and C – causing typhoid fever – septicaemia with high fever and headache) nObligatory pathogens causing usually intestinal infections only are important, too. Even here, sometimes a systemic infection may occur. nWe are speaking about genera Salmonella, Shigella and Yersinia www2.mf.uni-lj.si Plague (Yersinia pestis) 98b black_plagueC_icon 98a bw13b 98 2 www.emedicine.com dermatology.about.com www.arrakis.es Note to Salmonella and Shigella nThe fact, that there are differences even between intestinal pathogens, can be shown on the example of Salmonella and Shigella. nSalmonella needs high infectious dose. They have to multiply in a food. So the infections are almost food-borne. nShigella, on the contrary, has a small infectious dose, so it is easily transmitted by dirty hands, WC handle or contaminated water. nThere exist also clinical differences (different character of diarrhoea etc.). For example, shigelosis has its specific name – bacillar dysenteria (do not confuse with amoeba dysenteria) Story Two nMrs. Wet is a pacient of a urological clinic. nShe has repeated problems with urination. After three children, he has dammaged muscles of pelvic region, she was treated even for urine incontinence. The doctors warned her, thet she is in an elevated risk of infection. And really – now, she got infected again. 03 _HSG%20ep%202%20Urine%20sample%2001 http://manganime.animeblogger.net/ Who is guilty now? nIt is Escherichia coli, more precisely, its uropatho- genous strain (uropathogenous E. coli – UPEC) nBut the same problems might be due to other opportune pathogenic enterobacteria (sometimes even obligatory pathogens, like Salmonella) nEscherichia coli is one of the most important components of intestinal microflora, and it is protective – it produces bacteriocins that do not enable other bacterie to colonize the mucous membrane. Escherichia coli even suplies our body by some vitamins (mostly E and K). nEscherichia coli was found by German-austrian professor Theodor Escherich (died 1911 – 100 years) 02 escherichia www.steve.gb.com Pathogenicity of Escherichia coli nIntra-intestinal pathogenicity: nEPEC (enteropatogenous E. coli) – mostly newborns, babies nETEC (enterotoxic E. coli) – mostly travellers nEIEC (enteroinvasive E. coli) nSTEC (shiga toxigenous E. coli; this group also contains EHEC – enterohaemorhagic E. coli) nEAggEC (enteroaggregative E. coli) nCombinations (STEC + EAggEC = strain O:104H:4, that caused a severe epidemic in Germany 2011) nExtraintestinal pathogenicity: nUPEC (uropathogenous E. coli) nStrains causing respiratory infections nStrains causing sepsis, wound infections, etc. n Urinary bladder with adhered escherichiae 19 Bladder epit cell with e coli http://microbewiki.kenyon.edu 20 velký Diagram_six_types 16 e http://microbewiki.kenyon.edu http://www.frankpasternak.com 13 Korneální vřed způsobený ESCO Even corneal ulcus may be caused by Escherichia coli www2.mf.uni-lj.si 01 Ecoli 03 escherichia_coli_1 14 ecoli 14 ESCO 04 ESCO E. coli http://www.biotox.cz res2.agr.ca my.opera.com my.opera.com www2.mf.uni-lj.si Some more oportune-pathogenous enterobacteriae nEnterobacter, Klebsiella, Pantoea – often enapsulated, mucous colonies. Especially Klebsiella is a common pathogen causing hospital infections (respiratory ways, UTI) nProteus, Providencia, Morganella – proteolytic bacteria (in diagnostic typical bad smell of their colonies). Occasionally causing UTI and different other infections nCitrobacter – biochemically simillar to Salmonella, but with positive ONPG test nHafnia – primary bee pathogen 24 KLPN plíce 25 KLPN Pneumonia rtg 23 KLPN opouzd 22 KLPN Action of Klebsiella http://zdsys.chgb.org.cn www.brown.edu http://microbewiki.kenyon.edu http://www.bact.wisc.edu Survey of enterobacteria Story Pathogen. Examples – Systemic Y. pestis, AP** Salmonella 1. Intestinal ZP* Salmon., shigella, Yersinia 2. Opportune E. coli, Klebsiella, Enterobacter, Serratia, Proteus, Providencia, Morganella, Citrobacter, etc. – Nearly zero Many, e. g. Pragia fontium and Budvicia aquatica *zoopathogenenous **antropopathogenous 15 SEMA pigment [USEMAP] A red pigmented strain of Serratia my.opera.com Clinical description: Campylobacter, Helicobacter and Vibrionaceae Story three nStudent Francis often visits fast-foods. Especially, he likes chicken meat. nThus, even public health officers were not able to source the food responsible for his diarrhoic problems. Francis thougth about salmonelosis. But it was not this. Salmonella comes mostly from eggs, this one rather from chicken meat. 36 CAJE Criminal‘s name nCampylobacter jejuni, Gram-negative curved rod. It does not belong in family of enterobacteria, but the infection is simillar that caused by Salmonella nNumber of cases in Czechia is about the same as that of salmonelosis. Formerly it was not so common, but maybe only the diagnostic was not so much developed. www.cdc.gov 35 10 http://biology.plosjournals.org Impotant notice: stool sampling nUnlike parasitology and virology, where a bit of stool is necessary, in bacteriology we are used to send an anal swab (but a bit of stool is not a mistake). nTodays method of sampling is use of a cotton swab with transport medium (usually Amies). This is especielly because of Campylobacter – Salmonella would survive even on a swab without transport medium. 97 editedulcer 38 helicobacter Story Four nMr. Acid has a problem: pyrosis. This is not the only problem – sometimes he even vomited blood. He is likely to have a peptic ulcus nHe comes very often to gastroenterology, and a fibroscope is often present in his esofagus. nAt last fibroscopy, the doctors took two samples endoscopically – one for histological, another for microbiological examination. nBothe methods showed the same: it is there. microbewiki.kenyon.edu www.mtc.ki.se 39 helicobacter%20pylori Only half-criminal… nPeptic (= gastric / duodenal) ulcus is caused by more causes. Such diseases are called multifactorial diseases. nThe part of Helicobacter pylori on ulceral disease is still discussed, not only among GPs, but even among specialists. Even healthy persons may have a helicobacter in their stomach. Nevertheless, certain and not negligeable role of this pathogen is sure. http://vietsciences.free.fr How can it survive at low pH? nIt adapts its microenvironment – in alcalizes it, by splitting urea nUrea is split into acid carbon dioxide, that is breathed out, and alcalic ammonia, that remains and alcalizes the environment: n nNH2-CO-NH2 + 3 H2O à CO2 + 2 NH4OH 44 helicoa Ureáza Once more the same (the difference bethween writing ammonia as NH4OH or NH3 is only fomral) www.univie.ac.at 40 Helicobacter 43 helicobacter%20pylori 42 helicobacter www.univie.ac.at www.univie.ac.at http://www.dpc-buehlmann.at 37 2631-helicobacter www.medizin.de Story Five nMr. Exotic was on his exotic holiday. He was used to drink water from local sources nSo, no surprise, he was attacked by a very severe watery diarrhoea. nThis one, neverhteless, was more heavy than usually. It was watery and very profound nOral ingestion of water was insufitient. Only infusion supplementation of missing liquids brought a help to him. Vibrionaceae nVibrio cholerae causes cholera, a profound diarhoeic disease in subtropic and tropic countries nOther members of genus Vibrio may accidentally perform either diarrhoea, too, or wound infections. They are called „halophilic vibrios“, as they prefere elevated NaCl concentrations nAeromonas, the second important genus, also causes wound infections, e. g. at preparing meals from fishes and seafood. [USEMAP] Pictures of bacteria saenmal3 Photographs from criminal database: Salmonella Saen Endo Endo Saen MAL MAL saenxld XLD www.medmicro.info PRVUka Proteus mirabilis, P. vulgaris (below) prmiKA2 PrmiKA Typical for Protei: they do not grow only in inoculation place, but they spread on the surface of agar (Rauss phenomenon) www.medmicro.info Proteus according to P. Ondrovčík peproteus2 „It is nice, colleague, that you are able to dekarboxylate ornithin; much more sad is, that you are not able to perform Rauss phenomenon as well as I do!“ 26 PRMI 27 PRMI 28 proteus1 Proteus – typical swarming growth http://www.infektionsnetz.at http://www.icbm.de http://faculty.smu.edu Klebsiella & Escherichia Colonie of Klebsiella on blood agar are more mucoid and more white than those of E. coli… klpn35 esco KA … but just THIS strain of E. coli is quite white and mucoid, too J www.medmicro.info Escherichia Haemolysis of Escherichia on blood agar is sometimes present, but it is not important for its diagnostics. esly KA www.medmicro.info Heli cinaedi Helicobacter cinaedi One less common Helicobacter www.medmicro.info, gift gf Rita Gander 47 vibrio_cholera_em http://www.cs.dartmouth.edu Vibrio cholerae 45 vibrio%20comma%20asiatic%20cholera 46 cholera [USEMAP] http://bepast.org Vibrio cholerae Diagnostics of Enterobacteriaceae Enterobacteria – methods nDirect nMicroscopy – not very important in real diagnostic, but we will use it in our practicals nCulture – many various media nBiochemical identification – very important nAntigen analysis – Salmonella, Shigella, EPEC nIndirect methods nWidal reaction in typhoid fever, antibodies agains yersinia Diferencial diagnostics nGram stain differenciates Gram– rods from other bacteria nEndo agar (I): among clinically important, only enterobacteria, Vibrionaceae and Gram negative non fermenters do grow nNon fermenters may be differenciated by not fermenting glucose (Hajna medium remains red, no change of colour). nVibrionaceae are oxidase positive, unlike enterobacteria Gram– rods: group differenciation of the three groups able to grow on Endo agar nEnterobacteria are oxidase negative and glucose fermenters nVibrionaceae are glucose fermenters, too, but allways oxidase positive nGram– non-fermenters never fermentate glucose. Oxidase may be positive or negative. They are sometimes cocobacilli. Diagnostic algoritm Unknown strain it grows on Endo oxidase „–“: enterobacteria, detailed diagnostics needed it does not grow on Endo other morphology G- rods HAJNA red: a non-fermenter HAJNA other than red oxidase „+“: Vibrionaceae Mutual differenciation of Enterobacteriaceae nEndo agar (II): orientation differeciation of obligatory pathogens (usually L-) and opportune pathogens (usually L+, L = lactose) nMore media: XLD, MAL, DC, WB and more for Salmonella, CIN for Yersinia, etc. nBiochemical tests: Hajna medium, MIU test, Švejcar plate, ENTEROtests etc. nAntigen analysis usually using slide agglutination nDiagnostics of Campylobacter, Helicobacter and Vibrio will be discussed at tasks 7, 8 and 9. Kult9 Lactose splitting nLactose positive bacteria have dark red surroundings on Endo agar. Lactose negative bacteria have pale colonies on the same medium. Photo O. Z. Culture characteristics of several enterobacteria nOn XLD agar: nSalmonella has pale colonies with black centre (the centre is like a yolk in a fried egg) nother bacteria do not grow at all, or grow in colonies of different morphology nOn MAL agar the results are simillar to those on XLD agar (slightly different colours of some colonies etc.) nOn CIN agar Yersinia would grow in tiny, dark pink colonies. If no bacteria do grow on the medium, then no one of your strains is a Yersinia. saenmal2 Salmonella on MAL agar www.medmicro.info Biochemical testing of enterobacteria nFor biochemical testing of Enterobacteriaceae, we use various tests. In Czechia, the most common ones are ENTEROtest 16 and ENTEROtest 24. We will use the first of them. nThe first reaction is ONPG test (a test tube with reagent strip, like in VPT test in StaphyTest and StreptoTest). First row in your panel is 2nd to 9th reaction, second row is 10th to 17th reaction. Antigen analysis nAntigen analysis is used in some situations only, so not very commonly. nThere are two main situations like this: nIn obligatory pathogens (Salmonella, Shigella, Yersinia) to make the diagnose more sure, and for epidemiological reasons nIn intestinal isolates E. coli in case of suspicion for EPEC or STEC (but usually not the other groups of E. coli) nBoth cases are demonstrated by examples E. coli agglutination for EPEC nWe try to detect any of 12 main serovars belonging to EPEC nIf nonavalent serum (I, II, III) is positive nwe continue with three trivalent sera (I, II and III) nwhen one of them is positive, we continue with corresponding monovalent sera nIf trivalent serum IV is positive, we continue with monovalent sera belonging the „IV“ group nUnderstand: there exist hundreds of serovars in E. coli species. So, the result „E. coli, EPEC excluded“ means „it is one of remaining cca 200 serovars“ Salmonella agglutination nAt agglutination of any motile enterobacterium, we evalate two types of antigens: body = O antigens, and flagellar = H antigens (exceptionally also capsullar K antigens). nSo, each Salmonella, too, has its specific antigenic structure. E. g. Salmonella of serovar Enteritidis has body (O) antigens type 9, 12 and flagellar (H) antigen type m. nSo, if we have a Salmonella Enteritidis, we should find presence of agglutination both for body and flagellar antigens. Tests of atb susceptibility nWe do not perform atb tests for stool strains. (Usually, use of antibiotics in bacterial diarrhoea leads paradoxically to longer time of presence of the pathogen in stool; dietary treatment and probiotics in reconvalescence are then recomended rather than anitibotics.) nSo, usually we test it in UTI origin strains, therefore also some of antibiotics are anti-UTI (like nitrofurantoin) atb = antibiotic Susceptibility zones table – example Antibiotic Abbrev. Reference zone Ampicilin (aminopenicillin AMP 17 mm Cefalotin (CS 1 gen) KF 18 mm Doxycyclin (tetracyclin) DO 16 mm Cefuroxim (CS 2 gen) CXM 23 mm Ciprofloxacin (chinol 3G) CIP 21 mm Ko-trimoxazol (mixture) SXT 16 mm Oxolinic acid*(ch1G) OA 19 mm *alternativelly norfloxacin (NOR) [USEMAP] Diagnostics of Campylobacter, Helicobacter and Vibrionaceae Diagnostics of Campylobacter nCampylobacter does not grow on common media; together with its typical morphology (curved rod) the diagnostic is possible nLook at culture results of campylobacter and write down its description nLook at oxidase tests result (the test will be performed by your teacher as a demonstration) Some notes to Campylobacter diagnostics nThey need four special conditions: nTheir special black medium – its name CCDA is not used commonly, so it is simply „Campylobacter medium“ nTemperature elevated to 42 °C. (Primarily, it is a bird pathogen, and bird body temperature is elevated if compared with others) nElevated pCO2 nProlonged culture period – not 24, but 48 hrs Urease test in diagnostics of Helicobacter nHelicobacter, too, does not grow on common media. It requires its own special medium, and it needs cca five days until any growth is visible. nUrea splitting is one of very specific test for Helicobacter. Unlike other biochemical tests used in microbiology, it can be used directly with the specimen (gastric tissue) and not with a strain. In task 8, you will see a difference between positive and negative result. 41 Helicobacter-Urease-Test Quick urease test http://de.wikipedia.org Urea breath test nThe patient gets a mixture with urea with heavy carbon isotope (13C) or radioactive carbon isotope (14C) nIn healthy person urea comes to the bowel and comes out of the body with stool nIn presence of helicobacter, it is split in the stomach and labelled CO2 is found in the air. The more CO2, the more helicobacter Vibrionaceae – diagnostics nSimilar to Enterobacteriaceae, but they are oxidase positive. nMicroscopically: motile, curved rods nWe use special media like alcalic pepton water or TCBS (Thioglykolate, cystein, bile salts) nWe use simillar biochemical tests as for enterobacteria nBut we have to choose „oxidase positive“ in computer system (there is no codebook for them) Differential dg. ofVibrionaceae nIn microscopy, Vibrio is a curved rod (see next picture, draw it to your laboratory report) nFor culture, we use TCBS medium (a solid medium) and alcalic pepton water (a liquid medium) nFor biochemical identification, we use the same Enterotest 16 as for enterobacteriaceae, but we need another codebook (or another programme in PC) nBy antigen analysis, we can find the major serotypes of Vibrio cholerae: O1 and O139. nMore precise differentiation inside O1 serotype (into biotypes Classic and El Tor) would require more biochemical tests 11 VIsp Vibrio sp. www2.mf.uni-lj.si The End 99 PRMI a ESCO - malůvka Drawn using Proteus and Escherichia www.sld.cu [USEMAP]