TRACING THE CULPRIT Part seven: Anaerobic culprits Institute for microbiology shows 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… •…it is something to think about. Until now (P01 to P05, partially even P06) we spoke mostly about bacteria, that do not need special approach. (but in P06 it already did not match for some bacteria: 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 are going to deal with bacteria that do not match to this system. [USEMAP] 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 clotted blood ( serum is examined) for indirect diagnostics •Remember this not just for clinical microbiology examination, but also – and even more – for your future practice! 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 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 culprit that threatened Mrs. Cabbage 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. Tetanus Author: Sir Charles Bell – http://www.anatomyacts.co.uk/exhibition/object.asp?objectnum=62, Public domain, https://commons.wikimedia.org/w/index.php?curid=968572 Opisthotonus_in_a_patient_suffering_from_tetanus_-_Painting_by_Sir_Charles_Bell_-_1809.jpg Story two •Mustafa, 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 Mustafa‘s thigh. •Several days later, Mustafa came to one of field hospitals. His thigh was inflated and at knocking it was possible to hear breaking bubbles. Mustafa was operated immediately. The culprit 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 Gas gangrene formation Plynatá sněť Story three •Mr. Bones was third week in the hospital because of bacterial inflammation of bone marrow. The inflammation was treated by clindamycine (lincosamidic antibiotic). Suddenly, Mr. Bones started to have heavy diarrhoea. The department did not have metronidazol, and so they used the old method: Mr. Bones had to drink an ampoule of vancomycin – an antibiotic, that is normally administered only administratively. The culprit was •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 over-multiplies. •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 chemoterapeutic metronidazole now. There exist another method, so called „stool transplantation“. • [USEMAP] Clostridium difficile infection 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 over-multiplication, in cases of coming to places that are not normal for them, appearance of a strain, producing big amounts of a toxin etc. [USEMAP] Spore non forming anaerobes (and lactobacilli) – clinical characteristics Story four •Mrs. Cancer 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. Cancer a surgical treatment of the abscess and than also a cancer was possible, although hysterectomy was necessary. Fortunately, no metastases was found. The disease is formed by nA mixture of strictly anaerobic, but also facultative anaerobic bacteria nIt is likely, that the mixture was previously present in Mrs. Cancer‘s vagina, without making any problems nThe cancer broke the anatomical barrier, and so microbes came to other places, causing the abscess nSpore-not-forming anaerobic bacteria have limited ways of transmission because of their characteristics nMajority of infections are endogenous • Common characteristics of spore-not-forming anaerobes •They are present as a part of common microflora: –in the large bowel they form 99 % of the total amount of microorganisms, up to 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 over-multiplication, it is a dysmicrobia, requiring treatment •In inflammation usually there is no single pathogen, but rather a mixture, „Veillon microflora“ Spore not-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 Clark 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… [USEMAP] 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 anaerostats enable them to grow better. 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 the practices P1 to P6 we made acquaintance with four groups of microbes growing at aerobic conditions – some of them strictly aerobic as e. g. pseudomonades, 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 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 biochemically, and chromatographical gas analysis (they are biochemically active) •Antigen analysis and indirect diagnostics are rarely used in diagnostics Sampling and material transport 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. during surgery. *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 coccoid 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 an endospore (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 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 facultative 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 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 consumed Covering of VL-broths by paraffin oil Anae2 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 staff Anaerobic jar (principle) Anae3 Palladium catalyser (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 catalyser (beneath the lid) construction for placing of Petri dishes Anaerobiose generator (packet with chemicals) screw closer Compressive valve www.medmicro.info, photo O. Z. 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 www.medmicro.info 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 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 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 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 used to be performed by means of diffusion disc test (not on MH agar, but VL blood agar). Now it is mostly replaced by E-test (the MIC value is counted at crossing of the zone and the strip 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. n Lecitináza n„Negative I“ no lecithinase production. „Negative II“ a lecithinase is produced, but not the tested one Positive Negative I Negative II Half with antitoxin 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. 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. Background counterstained using not malachite 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 Rabies 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 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 •At a test positive for both toxin(s) and antigen we can see two lines and dots. One line and dots means positivity of antigen, but not toxin. „Dots only“ mean a 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. The End [USEMAP] Photo : Inst. of microbiology H:\Monika-práce\atlas\IMG_3837aPrevotella.jpg