TRACING THE CRIMINAL Part seven: Anaerobic criminals Institute for microbiology shows Anaerobes Survey of topics Pathogens with complicated diagnostics Before we start… n …it is something to think about. Until now (P1 to P6) we spoke mostly about bacteria, that do not need special approach. (Although, some bacteria from P06 did not match: gonococcus, brucella, legionella etc.) n Clinical doctor simply sends a specimen „for bacteriological culture“, and something would grow out of it. n Now, it is the END! Now, we will have bacteria that do not match to this system. And so: n 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. n In other agents (e. g. mycoplasms or chlamydia) it is often necessary to take serum and to perform indirect diagnostics n Remember especially this for your practice! Clostridia – clinical characteristics Story one n 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. n The GP used local treatment for the wound, and then recommended re-vaccination agaist one serious disease n If she would get the disease, it would be very dangerous, including spasms of her body Neurotoxic clostridia n The criminal that threatened Mr. 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. n 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. Tetanus A tetanic man Once more tetanus Trismus (spasm of chewing muscles) Botulism Typical tongue appearance in case of botulism Botox: use of Clostridium botulinum toxin to became younger Story two n 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 mood, came deeply into Mustafa‘s tigh. n 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. The criminal is now n Clostridium perfringens, one of agents of gas gangrene (with C. novyi, C. septicum, etc.) n Gas gangrene is a typical war disease. It is nevertheless possible to get it even during peace, e. g. in case of catastrophes n Gas gangrene clostridia – or their enterotoxins – are intestinal pathogens, too Gas gangrene formation Necrotizing enterocolitis – this, too, may be caused by C. perfringens Story three n 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 Clostridium difficile and its action I Toxins of Clostridium difficile Pseudomembranous colitis Clostridia – survey Spore non forming anaerobes – clinical characteristics Story four n Mrs. Cancer was hospitalized because of intensive abdominal pain n Description methods found an abscessus of pelvic region. It showed, though, a tumor cervicis – later described as a carcinoma n 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. The disease is formed by Anaerobes in the body Anaerobic infection from oral cavity Newborn anaerobic pneumonia Gingivostomatitis: Prevotella gingivalis Common characteristics of spore-non-forming anerobes Relation of bacteria to oxygen (repeating) Remember, what condition enable bacterial growth What we know until now Now, we add four more groups Diagnostics of anaerobic bacteria, how to obtain anaerobiosis How to search for the anaerobic bacteria – I n Microscopy: More important than in aerobic bacteria, because of morphological diversity n 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 n Biochemical identification: catalase and oxidase usually negative, mutual differenciation possible biochemically, and chromatographical gas analysis (they are biochemically active) n Antigen analysis and indirect diagnostics are rarelly used in diagnostics Microscopy of anaerobic bacteria n We perform normal Gram staining. We differenciate bacteria according to shape and cell wall type into cocci and bacilli, G+ and G–. n Anaerobic rods vary in shape very much – one preparation contains various formations from filamentous to nearly coccal ones Note to microscopy of anaerobes: various shapes of anaerobes n 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). n 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) Culture of anaerobic bacteria n Anaerobic bacteria grow often in tiny, irregular colonies, that may have tails on margins. It smells typically. n 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 gen anaerobiose n Mechanically – VL broth is covered by parafin oil n Physical – in the anaerobic box, air is replaced by a mixture of anaerobic gases from a bomb n Chemically – in the anaerobic jar [n ]organic acids à H[2] and CO[2] n 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 Anaerobic box Anaerobic box (detailed description) Anaerobic jar (principle) Anaerobic jar (detailed description) Another anaerobic jar Biochemical differentiation n Different tests are used, in Czech conditions mostly ANAEROtest 23 Lachema. Anaerotest – example of reading Other sets for diagnostics of anaerobes Antibiotic susceptibility tests n Antibiotic susceptibility in anaerobic bacteria is tested on media enabling their growth, so not MH agar, but usually VL blood agar n 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 Ilustration photo How to get out facultative anaerobes n Many anaerobes are resistant to vancomycin and/or amikacin. (In the same time they are susceptible to „not so strong“ antibiotics, as penicillin or metronidazol; see the picture) Detection of toxin I: lecitinase Detection of toxin II: animal experiment for tetanic/botulinic toxin n Animal experiment is used in tetanus and botulism. In tetanus mouse is spastic, in botulism we can see pareses. Toxin detection using animal experiment n Look at the picture of tetanic mouse Appearance of an experimental animal is observed also in other situations, e. g. botulism. n In botulism, we can se pareses, not spasms Practical search for anaerobes (example in vaginal microbes) n 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. n 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) n Our imperfect anaerobiosis enables growth of microaerofilic bacteria, as you can see. Pictures of anaerobic bacteria Clostridium tetani C. botulinum Clostridium perfringens Clostridium septicum Clostridium difficile Bacteroides sp. Porphyromonas gingivalis Prevotella melaninogenica (black pigmentation) Peptostreptococcus magnus Peptococcus constellatus Veillonella sp. The End