TRACING THE CRIMINAL Part seven: Anaerobic criminals Institute for microbiology presents L [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… •…there 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. 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. mycoplasmas or chlamydias) it is often necessary to take serum and to perform indirect diagnostics. •Remember especially this for your practice! No regarding the examination – this should be clear for you even after 20 years. [USEMAP] Clostridia – clinical characteristics Story one •Mrs. C. 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 practitioner. •The GP used local treatment for the wound, and then recommended re-vaccination against 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. C. was Clostridium tetani, causative agent of tetanus. The disease is typical by a small, local inflammation, 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). 519944432_858d235bef_m http://sarasbioblog.blogspot.cz/2010/10/clostridium-tetani.html 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 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 •M., Kosovo Albanian, decided to visit 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 M‘s thigh. •Several days later, M. came to one of field hospitals. His thigh was inflated and at knocking it was possible to hear breaking bubbles. M. was operated immediately. http://pharmacie.univ-lille2.fr The criminal is… •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 •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, sometimes they even cause necrotizing colitis http://www.cdc.gov/foodborneburden/images/clos1.png http://www.cdc.gov/foodsafety/clostridium-perfingens.html Gas gangrene Gas gangrene formation 64 gangaz http://pharmacie.univ-lille2.fr Story three •Mr. B. was third week in the hospital because of bacterial inflammation of bone marrow. The inflammation was treated by clindamycin (lincosamidic antibiotic). •Suddenly, Mr. B. started to have heavy diarrhoea. The department did not have metronidazol, and so they used the old method: Mr. B. had to drink an ampoule of vancomycin – an antibiotic, that is normally administered only administratively. The agent is… •Clostridium difficile, or its toxin. •The 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. •Destroying of concurrence is mostly due to treatment by some antibiotics, formerly mostly lincosamids, but unfortunately, now also aminopenicillins and other drugs. Lincosamids are effective against majority of strictly anaerobic bacteria, but not C. difficile. •Treatment is performed mostly using antibacterial chemotherapeutic metronidazol now. There exist another method – faecal bacteriotherapy („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 http://www.csgh.info/obr/0904_180_b.jpg http://www.csgh.info/arch_detail.php?stat=397 Clostridia – survey •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. C. tetani Causes tetanus C. botulinum Produces botulotoxin Clostridium perfringens, C. septicum, C. welchii a aj. Gas gangrene clostridia (+ intestinal pathogenicity) C. difficile Enteropathogenous [USEMAP] Spore non forming anaerobes (and lactobacilli) – clinical characteristics Story four •Mrs. C. was hospitalized because of intensive abdominal pain •Description methods found an abscess of pelvic region. It showed, though, a tumour cervicis – later described as a carcinoma •In Mrs. C. a surgical treatment of the abscess and than also a cancer was possible, although hysterectomy was necessary. Fortunately, no metastases was found. http://www.vetbook.org/wiki/dog/images/b/b0/Prevotella.jpg http://www.vetbook.org/wiki/dog/index.php/Prevotella_spp The disease is formed by •A mixture of strictly anaerobic, but also facultative anaerobic bacteria. •It is likely, that the mixture was previously present in Mrs. C‘s vagina, without making any problems. •The cancer broke the anatomical barrier, and so microbes came to other places, causing the abscess. •Non-sporulating anaerobic bacteria have limited ways of transmission because of their characteristics. •Majority of infections are endogenous. 47 Anaerob obecný Common characteristics of spore-non-forming anerobes •They are present as a part of common microflora: •in the large bowel they form 99 % of the total amount of microorganisms, about one kilogram of them •in oral cavity they live thanks to biofilm – they are inside and so they have no access to the air that would be harmful for them •in 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 •In inflammation 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 •Bacteroides fragilis pneumonia in newborn (B. fragilis isolated from the placenta and blood culture from the newborn). Anaerobic cultures were obtained because of a faecal odours in the amniotic fluid • 82 Anaerobní pneumonie novorozence aapredbook.aappublications.org Gingivostomatitis: Prevotella gingivalis 77 Porphyromonas gingivalis stomatitida www.mamagums.com 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 Story five •Miss C. had chronic problems 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 gynaecologist 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 (mostly 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 anaerobic boxes 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 •In 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 •Each 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 paraffin 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 differentiation possible by biochemical tests, and chromatographic gas analysis (they are biochemically active). •Antigen analysis and indirect diagnostics are rarely used in diagnostics. Sampling and transportation 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 differentiate 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 differentiate between those with rounded ends (Bacteroides, Prevotella, Porphyromonas) and those with pointed ends, often spindle-shaped (Fusobacterium, Leptotrichia). 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 http://upload.wikimedia.org/wikipedia/commons/thumb/a/a4/Bakterien_Sporen.png/220px-Bakterien_Spore n.png http://cs.wikipedia.org/wiki/Spora_%28bakterie%29 39 tetanus http://www.geocities.com Clostridium tetani Terminal endospore http://img.docstoccdn.com/thumb/orig/123452532.png http://www.docstoc.com/docs/123452532/clostridium-tetni Clostridium botulinum 01 Cbotulinum www2.bc.cc.ca.us http://www.extension.org/sites/default/files/w/b/b4/Clostridium_botulinum_small.jpg http://www.extension.org/pages/13215/clostridium-botulinum 36 CLPE 37 CLPE http://www.geocities.com Clostridium perfringens Endospores are not always visible inside the vegetative cells! Clostridium difficile 11 Clostridium_difficile_spores http://medecinepharmacie.univ-fcomte.fr http://t0.gstatic.com/images?q=tbn:ANd9GcTlgN0rI6zOJkZhypk4xY9gZB1FCIj89FUfy45lbX_sYRACQ7d1zQ http://depts.washington.edu/molmicdx/mdx/tests/cdiff.shtml 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“ Inst. for microbiology, 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 anaerobic 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 paraffin 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 catalyser hydrogen reacts with oxygen, and water is formed, so oxygen is exhausted Covering of VL-broths by paraffin oil Anae2 Inst. for microbiology, photo O. Z. Anae1 Inst. for microbiology, photo O. Z. source of anaerobic gases space for entering culture plates entrances for hands of personel Anaerobic jar (principle) •Palladium catalyser (beneath the lid) necessary for the second phase •Generator of anaerobiose (packet with chemicals) necessary for the whole reaction Anae3 Inst. for microbiology, photo O. Z. Anaerobic jar Anae3 air-proof lid palladium catalyser (beneath the lid) construction for placing of Petri dishes Anaerobiose generator (packet with chemicals) screw closer pressureventile Inst. for microbiology, 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 to have quite large, irregular, badly smelling colonies. •Other anaerobic bacteria have rather small colonies. •Some anaerobic bacteria (Prevotella melaninogenica) have pigmented colonies. cloperf Inst. for microbiology 60 Clostridium-septicum http://pharmacie.univ-lille2.fr Clostridium septicum Prevotella melaninogenica (black pigmentation) 46 PrevMEL 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. •We write results of the strains („+“ or „-“) and count the octal code •We assess the result according 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 • strain A strain B 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 •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) •Antibiotic susceptibility in anaerobic bacteria was tested using diffusion disc test (not on MH agar, but rather VL agar); recently, this test is not used and testing, if performed, is done using an E-test (MIC = the value evaluated in crossing of the margin of the zone and the strip) Detection of toxin I: lecithinase •Lecithinase 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. Tetanic mouse microvet.arizona.edu http://www.microbeworld.org/index.php?option=com_jlibrary&view=article&task=download&id=245 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. 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, similarly as ELISA or immunofluorescence. •The most typical example is pregnancy test. •The principle was explained in J09 practical session. It is mostly used for Clostridium difficile toxin producing strains. • In positive case, both test and control strip is usually visible; in negative case, control strip is visible only. 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 The End http://pharmacie.univ-lille2.fr [USEMAP]