TRACING THE CRIMINAL Part seven: Anaerobic criminals Institute for microbiology shows L 08 clostridium_tetani_1s Anaerobes www.biotox.cz [USEMAP] Survey of topics Pathogens with complicated diagnostics Clostridia – clinical characteristics Spore non forming anaerobes and lactobacilli – characteristics Relation of bacteria to oxygen (repeating from spring term) Diagnostics of anaerobic bacteria, anaerobiosis Pathogens with complicated diagnostics Before we start… •…it is something to think about. Until now (P01 to P06) we spoke mostly about bacteria, that do not need special approach. (Although, some bacteria from P06 did not match: gonococcus, brucella, legionella etc.) •Clinical doctor simply sends a specimen „for bacteriological culture“, and something would grow out of it. •Now, it is the END! Now, we will have bacteria that do not match to this system. 06 Clostridium_botulinum And so: •If the clinical doctor wants his specimen to be examined for presence of anaerobes, mycobacteria or actinomycetes, it should be written on the request for examination. Special approaches have to be used. •In other agents (e. g. mycoplasms or chlamydia) it is often necessary to take serum and to perform indirect diagnostics •Remember especially this for your practice! http://de.wikipedia.org nNot regarding the examination – this should be clear for you even after 20 years. [USEMAP] Clostridia – clinical characteristics Story one •Mrs. Cabbage was all the time seen working in the garden. It was her big hobby. Once she injured her hand, because a pointed remainder of a plant was hidden in the soil. She went to her general practicioner. •The GP used local treatment for the wound, and then recommended re-vaccination agaist one serious disease •If she would get the disease, it would be very dangerous, including spasms of her body Neurotoxic clostridia •The criminal that threatened Mrs. Cabbage was Clostridium tetani, causative agent of tetanus. The disease is typical by a small, local inflamation, and toxin action throughout the whole body. The toxin leads to spasms. •Another neurotoxic clostridium is Clostridium botulinum, causative agent of botulism. Here the agent does not enter the body at all. Only its toxin comes to the body (usually from badly prepared conserved meat) acting again as neurotoxin, but here producing pareses. 04 20-09a_Tetanustoxin_1 05 20-09b_Tetanustoxin_1 Tetanus www2.bc.cc.ca.us 03 20-08_Tetanus_1 Tetanus www2.bc.cc.ca.us 87 tetanický muž A tetanic man medinfo.ufl.edu Once more tetanus 65 tetanos2_pf http://pharmacie.univ-lille2.fr Trismus (spasm of chewing muscles) 66 Trismus-tetanos http://pharmacie.univ-lille2.fr Botulism 02 20-07_Botulism_1 www2.bc.cc.ca.us Typical tongue appearance in case of botulism 62 Botul-langue http://pharmacie.univ-lille2.fr Botox: use of Clostridium botulinum toxin to became younger 84 botoxZ www.fda.gov 63 G Story two •Mustafa, Kosovo Albanian, decided to visis his cousin in neighbouring village. The field he went through had to be mines-free. Nevertheless, one mine was still present. A particle of the broken mine, dirty of mud, came deeply into Mustafa‘s tigh. •Several days later, Mustafa came to one of field hospitals. His tigh was inflated and at knocking it was possible to hear breaking bubbles. Mustafa was operated immediatelly. http://pharmacie.univ-lille2.fr The criminal is now •Clostridium perfringens, one of agents of gas gangrene (with C. novyi, C. septicum, etc.) •Gas gangrene is a typical war disease. It is nevertheless possible to get it even during peace, e. g. in case of catastrophes •Gas gangrene clostridia – or their enterotoxins – are intestinal pathogens, too 38 CLPE http://www.geocities.com Gas gangrene Gas gangrene formation 64 gangaz http://pharmacie.univ-lille2.fr Necrotizing enterocolitis – this, too, may be caused by C. perfringens 15 clost1 enteritis necrotisans http://www.niah.affrc.go.jp Story three •Mr. Bones was third week in the hospital because of bacterial inflammation of bone marrow. The inflamation was treated by clindamycine (lincosamidic antibiotic). Suddenly, Mr. Bones started to have heavy diarrhoea. The departement did not have metronidazol, and so they used the old methode: Mr. Bones had to drink an ampule of vancomycine – an antibiotic, that is normally administered only administrativelly. The agent nClostridium difficile, or its toxin nThe microbe is present commonly in the intestine; a problem appears when the toxin starts to be produced, and mostly when its concurrence is destroyed and it overmultiplies. nDestroying of concurrence is mostly due to treatment by some antibiotics, formerly mostly lincosamids, but unfortunatelly, now also aminopenicillins and other drugs. Lincosamids are effective against majority of strictly anaerobic bacteria, but not C. difficile. nTreatment is performed mostly using antibacterial chemoterapeutic metronidazol now. There exist another method, so called „stool transplantation“. Clostridium difficile and its action I 90 cDiffLarge www.cdiff-support.co.uk 90 cDiffLarge Clostridium difficile and its action II www.cdiff-support.co.uk Toxins of Clostridium difficile 95 toxiny cldi www.zuova.cz INTESTINAL MUCOSA DISRUPTION B toxins Polymorphonuclears are bound to A toxins A toxins B toxins kill enterocytes Pseudomembrane Fibrin, mucin, dead polymorphonuclears Toxins of Clostridium difficile INFLAMMATION OF INTESTINAL MUCOSA Pseudomembranous colitis 92 pseudomembranous_plaques sitemaker.umich.edu 96 povrch střeva cldi www.zuova.cz Clostridia – survey C. tetani Causes tetanus C. botulinum Produces botulotoxin Clostridium perfringens, C. septicum, C. welchii a aj. Gas gangrene clostridia (+ intestinal pathogenicity) C. difficile Enteropathogenous It is necessary to know that even clostridia take normally part on common intestinal microflora. Problems start in overmultiplication, in cases of coming to places that are not normal for them, appearance of a strain, producing big amounts of a toxin etc. 09 clostridium_tetani_2s [USEMAP] www.biotox.cz Spore non forming anaerobes (and lactobacilli) – clinical characteristics Story four •Mrs. Cancer was hospitalized because of intensive abdominal pain •Description methods found an abscessus of pelvic region. It showed, though, a tumor cervicis – later described as a carcinoma •In Mrs. Cancer a surgical treatment of the abscessus and than also a cancer was possible, although hysterectomy was necessary. Fortunatelly, no metastases was found. 53 Porphyromonas http://pharmacie.univ-lille2.fr The disease is formed by nA mixture of strictly anaerobic, but also facultative anaerobic bacteria nIt is likely, that the mixture was previously preent in Mrs. Cancer‘s vagina, without making any problems nThe cancer broke the anatomical barrier, and so microbes came to other places, causing the abscessus nNon-sporulating anaerobic bacteria have limited ways of transmission because of their characteristics nMajority of infectons are endogenous 47 Anaerob obecný Common characteristics of spore-non-forming anerobes nThey are present as a part of common microflora: nin the large bowel they form 99 % of the total amount of microorganisms, about one kilogram of them nin oral cavity they live thanks to biofilm – they are inside and so they have no acces to the air that would be harmful for them nin vagina they are not present in all females, but about 70 % of women have some anaerobes in vagina; in case of overmultiplication, it is a dysmicrobia, requiring treatment nIn inflamation usually there is no single pathogen, but rather a mixture, „Veillon microflora“ www.microbes-edu.org Anaerobes in the body 72 kde jsou anaeroby gsbs.utmb.edu Anaerobic infection from oral cavity 80 infekce tváře aapredbook.aappublications.org Newborn anaerobic pneumonia 82 Anaerobní pneumonie novorozence aapredbook.aappublications.org Bacteroides fragilis pneumonia in newborn (B. fragilis isolated from the placenta and blood culture from the newborn). Anaerobic cultures were obtained because of a fecal odor in the amniotic fluid Gingivostomatitis: Prevotella gingivalis 77 Porphyromonas gingivalis stomatitida www.mamagums.com 45 Propionibacterium_acnes_Hemoc Spore non-forming anaerobes (most common species in humans) Cocci Bacilli G+ Peptococcus Peptostreptococcus Propionibacterium*** Eubacterium G- Veillonella Fusobacterium, Leptotrichia* Bacteroides, Prevotella, Porphyromonas** *pointed ends of the rod **round ends of the rod ***it is not a full anaerobe 27 Veillonella http://www.geocities.com http://www.geocities.com Story five •Miss Clark had chronical probles with her vaginal infections •Topic antibiotics in form of vaginal globules of cream with applicator gave her only partial help, pathogens often came back again •Finally, her gynecologist advised to use a probiotic drug with some „good“ bacteria, that would bring back the original vaginal microflora and not allow the pathogens to multiply again •The main „good bacterium“ was… Lactobacillus acidophilus, „Döderlein‘s bacillus“ •Lactobacilli are quite robust Gram-positive rods. They are called lactobacilli, because they ferment various substrates (moslty glucose and lactose) to lactate •Lactobacilli are the most important part of normal vaginal microflora, and also important part of intestinal microflora •Lactobacilli are not anaerobic bacteria. Nevertheless, as they are often microaerophilic, they are usually not able to grow at the normal atmosphere. On the contrary, the imperfect anaerobiose of our common anaerobic jars and anaerostats enable them to grow better. [USEMAP] Relation of bacteria to oxygen (repeating) Remember, what condition enable bacterial growth Conditions Normal ¯ O2 CO2 No O2 Strict aerobes yes yes yes no* Facultative anaerobes yes yes yes yes Aerotolorant bact. Microaerofilic bact. no yes (yes) no* Capnofilic bacteria no (yes) yes no* Strict anaerobes no no no yes** *In practice often growing – common anaerobiose is not ideal **In practice, sometimes not growing – common anaerobiose is not ideal. Such bacteria (EOS – Extremely oxygen sensitive) are not commonly culturable What we know until now nIn practicals P1 to P6 we made acquaintance with four groups of microbes growing at aerobic conditions – some of them strictly aerobic as e. g. pseudomonads, some facultative anaerobic as e. g. Escherichia coli. Aerobes cocci cocci rods rods Now, we add four more groups nEach of the four groups have anaerobic „brothers“. Their characteristics differ considerably from aerobic bacteria and have some common characteristics. Only genus Clostridium, spore forming, is different Aerobes a anaerobes [USEMAP] cocci rods cocci rods Diagnostics of anaerobic bacteria, how to obtain anaerobiosis How to search for the anaerobic bacteria – I •Microscopy: More important than in aerobic bacteria, because of morphological diversity •Culture: It is necessary to get anerobiosis using anaerobic jars or boxes. In liquid media it is sufficient to pour parafin oil over the medium. VL (viande levure) broth, VL blood agar and various special media are used •Biochemical identification: catalase and oxidase usually negative, mutual differenciation possible biochemically, and chromatographical gas analysis (they are biochemically active) •Antigen analysis and indirect diagnostics are rarelly used in diagnostics Sampling and transporation in anaerobic cultivation •Priority has liquid specimen, e. g. pus, the best is to send it in syringe with a cap after elimination of oxygen* •When a swab is sent, it is necessary to send it in a transport medium; on the other hand, common, e. g. Amies medium, is sufficient •It is also possible to talk with laboratory and to inoculate the specimen directly to media, e. g. peroperationally. *for safety reasons, unlike in older recommendations, it is no more recommended to use a syringe with needle Microscopy of anaerobic bacteria •We perform normal Gram staining. We differenciate bacteria according to shape and cell wall type into cocci and bacilli, G+ and G–. •Anaerobic rods vary in shape very much – one preparation contains various formations from filamentous to nearly coccal ones •In Gram-negative rods, it is useful to differenciate between those with rounded ends (Bacteroides, Prevotella, Porphyromonas) and those with pointed ends, often spindle-shaped (Fusobacterium, Leptotrichia) bacthodin Note to microscopy of anaerobes: various shapes of anaerobes •Students sometimes confuse a spore (unstained formation, only its margins are visible) and enlargements of rods (visible in some non spore-forming and Gram negative rods). •In real spore-forming microbes it is useful to follow position of the spore. In Clostridium tetani the spore is terminal (at the end of the cell) Clostridia True endospore Enlargement 39 tetanus http://www.geocities.com Clostridium tetani Terminal endospore C. botulinum 01 Cbotulinum www2.bc.cc.ca.us 85 Clostridium%20botulinum,%201000x,%20oil acpcommunity.acp.edu/ 36 CLPE 37 CLPE http://www.geocities.com Clostridium perfringens Endospores are not allways visible inside the vegetative cells! Clostridium difficile 11 Clostridium_difficile_spores http://medecinepharmacie.univ-fcomte.fr 94 C_diff_spores www.straightfromthedoc.com Clostridium difficile 23 BAFR Bacteroides fragilis http://www.geocities.com Bacterloides fragilis Bacteroides sp. bacthodin Sooner these objects were usually called „Sphaerophorus necrophorus“ = „globe and death bearing bacterium“ www.medmicro.info, photo O. Z. 24 Fusobact http://www.geocities.com Fusobacterium sp. 28 Peptostreptococcus http://www.geocities.com Peptostreptococcus sp. Peptostreptococci are anaerobic G+ cocci in chains, while peptococci are anaerobic G+ cocci in clusters. Veillonella sp. 76 veilonella http://www.primer.ru Veillonella is a very small anaerobic G– coccus Culture of anaerobic bacteria •Anaerobic bacteria grow often in tiny, irregular colonies, that may have tails on margins. It smells typically. •Aerobic culture on blood agar enables only growth of strictly aerobic and facultatively anaerobic bacteria. So, if a bacterium does not grow here, but does grow in anaerobic conditions, it is a strictly aerobic bacterium. To culture anaerobes, we use VL blood agar (in practice we say simply „VL agar“). To anaerobic culture: How to get the anaerobiose •Mechanically – VL broth is covered by parafin oil •Physically – in the anaerobic box, air is replaced by a mixture of anaerobic gases from a bomb •Chemically – in the anaerobic jar –organic acids à H2 and CO2 –in the second phase on palladium catalysator hydrogen reacts with oxygen, and water is formed, so oxygen is consumpted Covering of VL-broths by parafin oil Anae2 www.medmicro.info, photo O. Z. Anae1 www.medmicro.info, photo O. Z. Anae1 www.medmicro.info, photo O. Z. source of anaerobic gases space for entering culture plates entrances for hands of personel Anaerobic jar (principle) Anae3 Palladium calalysator (beneath the lid) necessary for the second phase Generator of anaerobiose (packet with chemicals) necessary for the whole reaction www.medmicro.info, photo O. Z. Anaerobic jar (detailed description) Anae3 air-proof lid palladium calalyser (beneath the lid) construction for placing of Petri dishes Anaerobiose generator (packet with chemicals) screw closer pressureventile www.medmicro.info, photo O. Z. Another anaerobic jar 20 anaerobicjar1_50 51 Fuso_spp Fusobacterium sp. http://pharmacie.univ-lille2.fr gold.aecom.yu.edu Morphology of colonies of anaerobic bacteria •Clostridia use tu have quite large, iregullar, badly smelling colonies •Other anaerobic bacteria have rather small colonies •Some anaerobic bacteria (Prevotella melaninogenica) have pigmented colonies cloperf www.medmicro.info 60 Clostridium-septicum http://pharmacie.univ-lille2.fr Clostridium septicum Prevotella melaninogenica (black pigmentation) 46 PrevMEL http://pharmacie.univ-lille2.fr 52 leptotrichiabuccaliscolonies Leptotrichia buccalis http://pharmacie.univ-lille2.fr Peptostreptococcus magnus 74 Peptostreptococcus magnus Peptostreptococcus anaerobius 75 Peptostreptococcus anaerobius www.zuova.cz www.szu.cz Biochemical differentiation •Different tests are used, in Czech conditions mostly ANAEROtest 23 Lachema. strain A strain B We write results of the strains („+“ or „-“) and count the octal code We assess the result acording to the codebook ATTENTION – the codebook is divided into several parts according to morphology of anaerobic bacteria. It is necessary to search in the proper part of the codebook ANAEROtest 23 – example + 1 - 1 + 1 + 1 - 1 - 1 - + - 2 + 2 + 2 - 2 + 2 - 2 - - + 4 - 4 + 4 + 4 + 4 - 4 + NEC 5 2 7 5 6 0 NEC = negative control (that is why it is ANAEROtest 23 and not 24 Other sets for diagnostics of anaerobes 69 biochemie B_fragilis 48 Identifikace anaerobů http://pharmacie.univ-lille2.fr www.microbes-edu.org Antibiotic susceptibility tests •Antibiotic susceptibility in anaerobic bacteria is tested on media enabling their growth, so not MH agar, but usually VL blood agar •The most classical therapy is usually classic penicillin. But Bacteroides genus is resistant (unlike genera Prevotella and Porphyromonas, formerly also part of old genus Bacteroides, that are susceptible) Susceptibility zones – a set that can be used for anaerobes Antibiotic Abbrev. Reference zone Penicilin (basic penicilin) P 20 mm Amoxicilin + klavulanate (protected aminopenicilin) AMC 20 mm Chloramfenikol C 21 mm Klindamycin (lincosamid) DA 21 mm Imipenem (karbapenem) IPM 16 mm Metronidazol (imidazol) MTZ 16 mm 67 B_frag_ATB Ilustration photo http://pharmacie.univ-lille2.fr Detection of toxin I: lecithinase nLecithinase production is detected as strain precipitation on the yolk agar. Nevertheless, there are many lecithinases, and one only, that of Clostridium perfringens is interesting for us, we have to test, whether the lecithinase may be inhibited by a specific antitoxin. „Negative I“ no lecithinase production. „Negative II“ a lecithinase is produced, but not the tested one Lecitinase Detection of toxin II: animal experiment for tetanic/botulinic toxin •Animal experiment is used in tetanus and botulism. In tetanus mouse is spastic, in botulism we can see pareses. 70 myš tetanus jen malá Tetanic mouse microvet.arizona.edu Toxin detection using animal experiment •Look at the picture of tetanic mouse Opistotonus is typical both for mice and humans 10 tetanus www.biotox.cz mysh0001 bar Drawing by Petr Ondrovčík (1959–2007) Graphically adapted. Backgound counterstained using not malachit green, but „Paint“ programme by Microsoft Tetanic mouse Appearance of an experimental animal is observed also in other situations, e. g. botulism. •In botulism, we can se pareses, not spasms mysh0002 bar mysh0003 bar mysh0004 bar mysh0005 bar Botulic mouse Rage mouse Septic mouse Dead mouse Detection of toxins III: Immunochromatographic tests •Immunochromatographic tests are based on binding of individual components, simillarly as ELISA or immunofluorescence •The most typical example is pregnancy test •The principle was explained in J08 practical session. •In case of Clostridium difficile toxin producing strains, unlikely other cases, we make a parallel testing of both antigen and A and B toxins •A test positive for both toxin(s) and antigen we can see two lines and dots. One line and dots meast positivity of antigen, but not toxin. Only dots mean completely negative, but valid, test. Principle (only for illustration) + – Test area Control area Practical search for anaerobes (example in vaginal microbes) •For vaginal swabs where anaerobic culture is requested we use VL agar with disks of vancomycin and amikacin. Usually, anaerobic bacteria grow between these two disks. •Besides eventually present anaerobic flora, we can see a lot of vaginal lactobacilli, microaerofilic bacteria commonly found in vaginal swabs (and rather rarely present in normal aerobic culture) •Our imperfect anaerobiosis enables growth of microaerofilic bacteria, as you can see. 56 P_intermedia13-03-2003 [USEMAP] The End http://pharmacie.univ-lille2.fr