TRACING THE CRIMINAL Part Five: Gram-Negative Criminals II Institute for microbiology shows: Survey of medically important G - rods Pseudomonas aeruginosa – a microbiological everGREEN J Survey of topics Clinical characteristictics – Pasteurellaceae Story One n Jimmy is a fine boy, but his parents are members of a strange religious society and so they do not wish let him get vaccinated. They woud like to keep him at home, but they sent him to a kindergarden. n After a month Jimmy started to have a cold, difficult breathing, gasping for air, and it become so hard that emergency had to be called. Emergency even thougt about coniotomia, but finally it was not necessary. It was epiglottitis – a disease not too common today… Who did this to Jimmy? n Criminal: Haemophilus influenzae ser. b (Hib) n Haemophili are short Gram negative rods. Unlike e. g. enterobacteria, they do not grow on Endo agar. And even more: they even do not grow on blood agar, as they are unable to get inside erythrocytes to get the nutrients. n Haemophili belong to the family Pasteurellaceae, together with Pasteurella (see later) Classification of haemophili n Haemophilus influenzae n capsullar type b (Hib) n caspullar types a, c, d, e, f n non-encapsulated strains n Haemophilus parainfluenzae (much more common, much less pathogenous) n Haemophilus aphrophilus and many other species n Haemophilus ducreyi, causative agent of a sexually transmitted disease ulcus molle Pathogenicity of haemophili n The most severe diseases caused by haemophili are epiglottitis, meningitis and sepsis. This is mostly typical for Haemophilus influenzae, serotype b. n Other common diseases are otitis media and sinutisitis (after Streptococcus pneumoniae and together with Moraxella catarrhalis) n Their presence in throat is very common and their pathogenic role is very query. Especially in case of Haemophilus parainfluenzae, we usually do not suppose them to be pathogens. A Haemophilus disease Ulcus molle n It is a sexually transmitted disease found mostly in sub-tropical and tropical countries Story Two n Joana went walking in gardens as usually. Unfortunatelly, one garden fence was too old and rotten and the dog inside too strong. The dog run out and just met Joana. So, Joana was bitten into her leg. n The owners of the dog had proven that the dog was vaccinated against rabies. Nevertheless, some pus was found soon in the wound. So the pus was sent to the laboratory. Ant the criminal was… Pasteurella multocida n Pasteurella multocida. n It is common flora in dog mouth. n In humans, it causes mainly pyogene wound inflamations after being bitten by a dog or another animal. n It smells simillarly as Haemophillus, but unlike Haemophilus, it grows on blood agar (but not Endo agar). n The morphology is something between Streptococcus and Enterococcus, but is is Vancomycin resistant and this gives a suspicion to the microbiologist Clinical characteristictics – Gram– glucose non-fermenters Story Three n Mr. Phosphoros is a pyroman. Several days ago, he burned himself. Now, his burn is inflamated. He is hospitalised on a specialized centre and feels very badly. Doctors knew that it has no sense to try antibiotics accidentaly, so they performed a swab. Thanks to this, a target therapy was found, and Mr. Phosphoros healed. Of course, only temporarily: sooner or later, he will probably play with his matches again (like some students of the practical). Who is guilty this time? In disabled persons, they can cause even such problems as a nail inflamation Dr. Zahradníček‘s autocauistics n Friday, 13th January 2006: fall into a not-covered canalisation hole in the city of Padang, West Sumatera, Indonesia. Quite large wound, reaching tibial periost n Some three weeks later, the wound started to smell like Pseudomonas, and really, this bacterium was sucsesfully cultured from it. n Dr. Zahradníček decided for local treatment (mixture of gentamicin and polymyxine) n The therapy was succesfull. Padang Story Four n Linda was a poor girl: she suffered because of an inborne disease, cystic fibrosis. n Her lung surfactant was different from surfactant of healthy people. So, he was often infected. n Last time it was Staphylococcus aureus. This time it was different: the causative agent was Burkholderia cepacia, one of G– non-fermenters. Non-fermenters and Cystic fibrosis n Cystic fibrosis is a severe, inborn lung disease, with failure of production of normal lung surfactant. This leads to changed characteristics of lungs, including many times increased risk of infection n Most common causative agents are Pseudomonas aeruginosa, Burkolderia cepacia and Staphylococcus aureus. Strains often become polyresistant and many children with cytic fibrosis die very young. A few words concerning bacterial metabolism n As you know, clinically important bacteria use mostly one of two metabolism types: fermentation and aerobic respiration. n Escherichia coli, having enoght nutrients, but not enough oxygen (unlike other gases J) preferes glucose (and other substrates) fermentation. n On the other hand, Pseudomonas has oxygen enough, but nutrients not enough. It uses aerobic respiration: enables better exploitation of nutrients. n Adaptation to outer environment is also demonstrated by pigment production in non-fermenters – see next. Pseudomonas as a strict aerobe (unlike other bacteria) n Unlike strain I (Escherichia coli) and strain II (Bacterioides fragilis, a strict anaerobe), Pseudomonas aeruginosa (strain III) is a strictly aerobic bacterium (Bacteroides fragilis, more in P07) Diagnostics of Pasteurellaceae Survey of methods used for diagnostics of these bacteria n Direct methods n Microscopy – if we need it for differenciation n Culture – non-fermenters grow on majority of media, Pasteurellaceae are more specific n Biochemical identification – in both groups; in non-fermenters it is based on aerobic respiration reactions, it requires ê temp. and 2 d culture n antigen analysis – mostly in haemophili (Hib) Indirect methods used rarely More to culture of these bacteria n Haemophilli need factors from RBC, but they are not able to break an RBC themselves. So they are not able to grow on blood agar alone. They need it to be broken, e. g. by heating (àchocolate agar) or by presence of another bacterium (Satelite phenomenon) n Pasteurellae grow on blood agar, but not Endo agar n G– non fermenters grow on all common media (blood agar, Endo agar, Müller-Hinton agar) Differentiation of Pasteurellaceae (differential diagnostics) n Gram staining: Gram– rods × other bacteria n Endo medium: as we now, among clinically important bacteria, only Enterobacteriaceae, Vibrionaceae and Gram – non-fermenters are able to grow. Pasteurellaceae do not grow. n Pasteurellaceae are detected by typical smell, biochemical properties, typical antibiotic susceptibility etc. To Haemophilus and Pasteurella diagnostics n Pasteurella grows on blood agar n Haemophili are not able to grow on blood agar, they are not able to „open the RBC“. So, they grow on chocolate agar n On BA, they are able to grow, if a bacterium that „opens the RBC“ is present (satelite phenomenon). Such bacterium is e. g. golden Staphylococcus n They grow in tiny colonies, so we use a disc to disable growth of other bacteria (bacitracin, but in higher concentration than in bacitracin test) Satelite phenomenon n As we know allready, haemophilli need factors from RBC, but they are not able to break an RBC themselves. They need the RBCs to be broken n by heating – chocolate agar n by presence of another microbe n Satelite phenomenon is an example of the second way how to make haemophilli be able to exploit blood factors. That means the growth of Haemophilus around Staphylococcus line only. n Colonies are very tiny, they require careful observing! Detection of haemophili Growth factors of Hemophili n Haemophilli need factors from blood, but the need of individual factors is species specific. n H. parainfluenzae needs factor V (= NAD) n H. aphrophilus needs factor X (= hemin) n H. influenzae needs both factors. n We use discs with these factors: one with X, another with V, and the third with a mixture of both of them. Growth factor test of Hemophili Haemophili: antigen analysis n Antigen analysis in Hemophilli is performed like in other bacteria. The main goal is diferenciation of Hib. Today, we have comercionally available sets, containing e. g. latex particles e. t. c. n Formerly, so named co-agglutination with Staphylococcus strain was used: agglutinate was more dense because of Staphylococcus binding the Fc-end of anti-haemophilus antibody Antigen analysis of haemophili: an example of the result n Write down the result, as you can see it on the following picture (it belongs to strain K): Detection of Pasteurella using typical susceptibility pattern n No Gram-negative bacterium is susceptible to vancomycin. Vancomycin can be used for Gram-positive bacteria only, but here it is very strong; all streptococi and majority of staphylococci and enterococci are susceptible n On the other hand, very little bacteria are susceptible to penicillin, escepically among G– bacilli. n So, susceptibility to penicillin and resistance to vancomycin is quite typical for Pasteurella. Diagnostics of Gram–non-fermenters Differentiation of G– non-fermenters (differential diagnostics) n Gram staining: Gram– rods × other bacteria n Endo medium: as we now, among clinically important bacteria, only Enterobacteriaceae, Vibrionaceae and Gram– non-fermenters are able to grow. So, G– non-fermenters do grow n Non-fermenters are differenciated from enterobacteria/vibria by non-fermenting glucose (e. g. Hajna medium remains completelly red after culture, no colour change) Tests of atb susceptibility n Haemophilli do not grow on MH agar n Usually Levinthal agar (fitrateed chocolate agar), is used – for this purpose, it is better than classical chocolate agar n Our laboratory use „Haemophilus agar“, similar to Levinthal agar An example of Pasteurellaceae antibiotic set To non-fermenters diagnostics n Pseudomonas is usually detected by: n Presence of typical odour (young cultures) n They form pigments, mostly green, sometiomes blue or maroon. Best visible on MH, worse on BA and Endo agar n Positive oxidase n Other non-fermenters, or not-sure Pseudomonas, shloud be differenciated biochemically, e. g. by NEFERMtest 24 Pseudomonas on MH agar and other media n Remember, that MH agar itself is nearly colourless (or slightly yelowish). n All green colour you see is product of Pseudomonas, or moce preciselly, of its pigment pyoverdin n On BA and Endo, pigment production is not so strong, but partially visible, too. Nevertheless, something more visible on these media is the typical pearl smooth surface of the colonies Hajna medium for non-fermenters determination n The strains growing on Endo agar could be G– non-fermenters, Enterobacteriaceae or Vibrionaceae. n Typical for G– non-fermenters is absence of any change of colour of Hajna medium (it remains red; eventual light brown colour does not matter, it is due to presence of pigments) Oxidase test in non-fermenters n Among the most common G– non-fermermenters, Pseudomonas is oxidase positive, Burkholderia usually too; on he contrary, Stenotrophomonas is usually negative and Acinetobacter too. NEFERMtest 24 n For precise biochemical identification of G– non-fermenters we use mostly Nefermtest 24 (or simillar test of other provenience). n It is a triple-strip (not double as last week) n There is a different way of code-formation n first number is 0 (oxidase –) or 1 (oxidase +) n next 6 numbers come from collumns H to C n collumns B and A are not counted (they are eventually used for more detailed determination) Tests of atb susceptibility n G– non-fermenters may be tested on common media. n We use very strong antibiotics, that would be too strong for use with more common bacteria. Majority of them should be kept only for real G– non fermenters infections (but not mere colonisations by such strains!) An example of NF atb set Somebody use E-test… Pictures of Pasteurellaceae and Gram–non-fermenters Photographs of criminal database Haemophili Satelite once more Pasteurella multocida Photographs of criminal database Pseudomonas aeruginosa More Gram non-fermenters: Pseudomonas fluorescens Burkholderia cepacia Burkholderia pseudomallei Stenotrophomonas maltophilia Acinetobacter sp. The End