TRACING THE CRIMINAL Part Five: Gram-Negative Criminals II Institute for microbiology shows: L Survey of medically important G– rods Story Endo Group P04 grows Enterobacteriaceae (GLC +, OXI –) P04 grows Vibrionaceae (GLC +, OXI +) P04 does not Campylobacter and Helicobacter 3. + 4. grows G- non-fermenters (GLC –) 1. + 2. does not Pasteurellaceae P06 does not Legionella, Bordetella, Brucella etc. 14 HEIN Haemophillus http://medinfo.ufl.edu Pseudomonas aeruginosa – a microbiological everGREEN J 06 P textbookofbacteriology.net [USEMAP] Survey of topics Clinical characteristics – Pasteurellaceae Clinical characteristics – G– glucose non-fermenters Diagnostics of Pasteurellaceae Diagnostics of G– glucose non-fermenters Clinical characteristictics – Pasteurellaceae 15 bild1001927121_Baby_Intensivstation_Nahaufnahme23b8e03a0130ce Story One nFour-years old 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 would like to keep him at home, but as they have to be at work, they sent him to a nursery. nAfter 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… http://www.kinderaerzte-im-netz.de Who did this to Jimmy? nCriminal: Haemophilus influenzae ser. b (Hib) nHaemophili are short Gram negative rods. nHaemophili belong to the family Pasteurellaceae, together with Pasteurella (see later) 28 Hib http://www.co.monroe.mi.us Classification of haemophili nHaemophilus influenzae ncapsullar type b (Hib) – preventable (vaccine) ncaspullar types a, c, d, e, f nnon-encapsulated strains nHaemophilus parainfluenzae (much more common, much less pathogenous) nHaemophilus aphrophilus and many other species nHaemophilus ducreyi, causative agent of a sexually transmitted disease ulcus molle Pathogenicity of haemophili nThe most severe diseases caused by haemophili are epiglottitis, meningitis and sepsis. This is mostly typical for Haemophilus influenzae, serotype b. nOther common diseases are otitis media and sinutisitis (after Streptococcus pneumoniae and together with Moraxella catarrhalis) nTheir 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. [USEMAP] A Haemophilus disease 10 Haemophilus-Type-b http://www.immune.org.nz 13 HIB_vaccine Ulcus molle nIt is a sexually transmitted disease found mostly in sub-tropical and tropical countries 20 180px-Chancroid_lesion_haemophilus_ducreyi_PHIL_3728_lores 21 Cancro2 22 Cancro 23 měkký vřed http://upload.wikimedia.org www.fmt.am.gov.br www.fmt.am.gov.br http://www.sexsygdom.dk Ulcus molle – chancroid – caused by Haemophilus ducreyi Ulcus durum – chancre – one of symptoms of syfilis, caused by Treponema pallidum Story Two nJoana did 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. nThe 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. And the criminal was… Pasteurella multocida nPasteurella multocida is common flora in dog chaps. nIn humans, it causes mainly pyogene wound inflamations after being bitten by a dog or another animal. nIt smells simillarly as Haemophillus (some people say „like old rag“, but unlike Haemophilus, it grows on blood agar (but not Endo agar). nThe morphology is something between Streptococcus and Enterococcus, but it is Vancomycin resistant and this gives a suspicion to the microbiologist (especially at parallel susceptibility to penicillin) Pasteurella multocida 19 pasteurella 18 encefalite_bovina_f4 [USEMAP] http://www.biologico.sp.gov.br http://library.thinkquest.org Clinical characteristictics – Gram– glucose non-fermenters Story Three nMr. 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). 09 Skin-Ecthyma PSAE http://faculty.plattsburgh.edu Who is guilty this time? nIt is Pseudomonas aeruginosa, the most common so named „Gram– non-fermenters“ (G– NF) nOn the other hand, the guilty one could be any of that group, e. g. Acinetobacter, Burkholderia cepacia or Stenotrophomonas maltophilia nThose bacteria are mostly strict aerobes, instead of fermentation sugars, they breakdown them by aerobic respiration, and their adaptation to outer environment is clear also of other properties – they use to have low temperature optimum and they are often pigmented, so they fight with sun in outer environment 5 Green pigmented strain of Pseudomonas aeruginosa on MH www.medmicro.info (web of the Institute), photo by prof. Skalka Another picture of Pseudomonas aeruginosa P.aeruginosa - kultivace - kmen napadený bakteriofágem. Klikni! www.medmicro.info (web of the Institute) photo by prof. Skalka 04 280px-Pseudomonas 07 P 08 pseudo 05 P Exceptional Pseudomonas strain with blue pigmentation http://de.wikipedia.org textbookofbacteriology.net textbookofbacteriology.net www.uiowa.edu Pathogenicity of G– NF nCommonly: they are bacteria from outer environment, often plant pathogens, „not-brave-bacteria“, which are not able to infect a healthy person. Their target are patients with burns, clients of emergency units, transplant centers, e. t. c. nIn hospitalized persons they often cause not only wound infections, but we find them also in respiratory ways, and even in the bloodstream. nSo they are important agents of nosocomial infections nSometimes it is difficult to differenciate between an infection and a colonisation – especially in superficial wounds it is often useless to use other than topical antibiotics at finding of some of those bacteria In disabled persons, they can cause even such problems as a nail inflamation 03 pseudomonas%20i%20nogl Island www.kvarts.is Dr. Zahradníček‘s autocauistics nFriday, 13th January 2006: fall into a not-covered canalisation hole in the city of Padang, West Sumatera, Indonesia. Quite large wound, reaching tibial periost nSome three weeks later, the wound started to smell like Pseudomonas, and really, this bacterium was sucsesfully cultured from it. nDr. Zahradníček decided for local treatment (mixture of gentamicin and polymyxine) nThe therapy was succesfull (but it is not „lege artis and cannot be recommended for routine use) 151-05(33) Padang Photo O. Z. Story Four nLinda was a poor girl: she suffered because of an inborne disease, cystic fibrosis. nHer lung surfactant was different from surfactant of healthy people. So, he was often infected. nLast 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 nCystic 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 nMost common causative agents are Pseudomonas aeruginosa, Burkolderia cepacia and Staphylococcus aureus. Strains often become polyresistant and many children with cytic fibrosis die very young. 42 cystic fibrosis 43 hdc_0001_0001_0_img0072 http://www.humanillnesses.com 44 4083-250px-cftreatmentvest2-cystic-fibrosis http://goldbamboo.com More Gram non-fermenters: Pseudomonas fluorescens 31 PSFL http://www.bact.wisc.edu Burkholderia cepacia Burkholderia cepacia is responsible for rotten onions (Allium cepa), so it is really a typical plant pathogen 32 onion_compend http://www.apsnet.org 34 Burkholderia%20cepacia%20fig18 http://www.microbelibrary.org Burkholderia pseudomallei Burkholderia pseudomallei is causative agent of mellioidosis. Related B. mallei is causative agent of malleus (a zoonosis) 33 bpseud http://www.asm.org Stenotrophomonas maltophilia Stenotrophomonas maltophilia is a long name, but it is possible to learn it easily: it is narrow-nutrition-unit maltose-loving, so it is a „bacterial panda“, chewing maltose instead of bamboo J. 35 STMA 36 StenoMalto-BAP-24-h 37 Stenotrophomonas%20maltophilia%20fig20 http://www.scielo.cl http://clinicalmicrobiology.stanford.edu http://www.microbelibrary.org 40 acinetc1259th Acinetobacter sp. 38 Acinetobacter_calcoaceticus_04-2Tag-Columbia 39 ACBA http://www.bakteriologieatlas.de http://www.microbelibrary.org http://www.buddycom.com Greek: a-kineto- = „non motile“ Bacterial metabolism and relation of bacteria to oxygen nWe know already that G– non-fermenters are bacteria that do not ferment sugars, but performs aerobic respiration. Let‘s compare now two bacteria: nEscherichia coli lives in the intestine. It has enough nutrients, but not enough oxygen (unlike other gases J) preferes glucose (and other substrates) fermentation. Escherichia coli is a facultative anaerobe. Some other intestinal bacteria are strict anaerobes. nOn the other hand, Pseudomonas has oxygen enough, but nutrients not enough. It uses aerobic respiration: enables better exploitation of nutrients. Pseudomonas is a strict aerobe. Pseudomonas as a strict aerobe (unlike other bacteria) nUnlike 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) Strain Broth VL-broth Result I growth clear Strictly aerobic bacterium II clear growth Strict anaerobe III growth growth Facultative anaerobe [USEMAP] Diagnostics of Pasteurellaceae Survey of methods in Pasteurellaceae diagnostics nDirect methods nMicroscopy – short G– rods nCulture – Pasteurellaceae do not grow on Endo agar, Haemophilus even does not grow on Blood agar (except being co-cultivated with another microbe) nBiochemical identification – it is possible to use it nAntigen analysis – used in haemophili (Hib) nNucleic acid detection – not used routinely nIndirect methods used rarely Differentiation of Pasteurellaceae (differential diagnostics) nGram staining: Gram– rods × other bacteria nEndo medium: as we now, among clinically important bacteria, only Enterobacteriaceae, Vibrionaceae and Gram– non-fermenters are able to grow. Pasteurellaceae do not grow. nPasteurellaceae are detected by typical smell, biochemical properties, growth on individual media, typical antibiotic susceptibility etc. 11 HaemophilusInfluenzae To Haemophilus and Pasteurella diagnostics nPasteurella is able to grow on on blood agar nHaemophili are not able to grow on blood agar, they are not able to „open the RBC“. So, they grow on chocolate agar or Levinthal agar (filtrated chocolate agar) nOn BA, they are able to grow, if a bacterium that „opens the RBC“ is present (satelite phenomenon). Such bacterium is e. g. golden Staphylococcus nThey grow in tiny colonies, so we use a disc to disable growth of other bacteria (bacitracin, but in higher concentration than in bacitracin test) http://www.uni-ulm.de Satelite phenomenon nAs we know already, haemophilli need factors from RBC, but they are not able to break an RBC themselves. They need the RBCs to be broken nby heating – chocolate agar nby presence of another microbe nSatelite 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. nPresence of satelite phenomenon is a confirmation, that our bacterium is really a Haemophilus Haemophili on chocolate agar (left) and as a satelite on blood agar Haemophilus sp. - kultivace na čokoládovém agaru . Klikni! ChA Haemophilus sp. - satelitový fenomén. Klikni! BA (satelite) www.medmicro.info (web of the Institute) Satelite once more 26 satelit http://phil.cdc.gov Detection of haemophili Klin9 Klin9a Haemophili are more resistant than the bacteria of the common flora, so they grow inside the zone, but only around staphylococcus line (satelite phenomenon) www.medmicro.info Growth factors of Hemophili (= determination of individual species) nHaemophilli need factors from blood, but the need of individual factors is species specific. nH. parainfluenzae needs factor V (= NAD) nH. aphrophilus needs factor X (= hemin) nH. influenzae needs both factors. nWe 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 One disk is with factor X, second with factor V, third a mixture HEAP HEIN HEPA H.influenzae - růst kolem disku s faktory X+V. Klikni! H.parainfluenzae - růst kolem disku s faktorem V a s faktory X+V. Klikni! H. influenzae (left), H. parainfluenzae (right) www.medmicro.info Haemophillus influenzae: antigen analysis (intra-species diagnostics nAntigen analysis in Hemophillus influenzae is performed like in other bacteria. The main goal is diferenciation of Hib. Today, we have comercionally available sets, containing e. g. latex particles. We try to assess the capsular type of H. influenzae (a, b, c, d, e, or f). When the strain does not agglutinate with any sera, it is probably an un-encapsulated strain nFormerly, 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 H. influenzae: an example of the result nThe agglutination results for haemophili are observed simillarly as other agglutination reactions 49 stafaurex předělaný na hemofily collage with use of: www.microbes-edu.org Detection of Pasteurella using typical susceptibility pattern nNo 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 nOn the other hand, very little bacteria are susceptible to penicillin, escepically among G– bacilli. nSo, susceptibility to penicillin and resistance to vancomycin is quite typical for Pasteurella. Tests of atb susceptibility nHaemophilli do not grow on MH agar nUsually Levinthal agar (fitrated chocolate agar), is used for diffusion disc test – for this purpose, this agar is better than classical chocolate agar nOur laboratory uses „Haemophilus agar“, similar to Levinthal agar nReading of the zones is the same as for any other bacteria Antibiotic susceptibility testing: An example of Pasteurellaceae antibiotic set Antibiotikum Abbrev. Reference zone Ampicilin (aminopeniciline) AMP 22 mm Co-amoxicilin (am.+inhib.) AMC 18 mm Chloramphenicol C 29 mm Doxycycline (tetracycline) DO 29 mm Co-trimoxazol (mixure) SXT 16 mm Azithromycin (macrolid) AZM 12 mm [USEMAP] Diagnostics of Gram–non-fermenters Survey of methods for G– non-fermenters nDirect methods nMicroscopy – mostly G– rods, but Acinetobacter is a G– coccus nCulture – non-fermenters grow on majority of media, including Endo agar. As glucose-non fermenters, they are mostly also lactose-non fermenters, but their colonies are sometimes quite dark, because of pigmenation nBiochemical identification – possible, but tests cheking aerobic respiration (not fermentation) should be used. We also use mostly decreased temperature and prolonged incubation nantigen analysis, nucleic acid detection – not used routinely nIndirect methods used rarely Differentiation of G– non-fermenters (differential diagnostics) nGram staining: Gram– rods × other bacteria nEndo agar: they grow. As glucose-non fermenters, they are mostly also lactose-non fermenters, but their colonies are sometimes quite dark, because of pigmenation nNon-fermenters are differenciated from enterobacteria/vibria by non-fermenting glucose (e. g. Hajna medium remains completelly red after culture, no colour change; but eventual light brown colour does not matter, it is due to presence of pigments) Further diagnostics of individual genera and species of G– NFs nPseudomonas is usually detected by: nPresence of typical odour (young cultures) nThey form pigments, mostly green, sometiomes blue or maroon. Best visible on MH, worse on BA and Endo agar nPositive oxidase nOther non-fermenters, or not-sure Pseudomonas, shlould be differenciated biochemically, e. g. by NEFERMtest 24 Pseudomonas on MH agar and other media nRemember, that MH agar itself is nearly colourless (or slightly yelowish). nAll green colour you see is product of Pseudomonas, or moce preciselly, of its pigment pyoverdin nOn 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 Oxidase test in non-fermenters nAmong the most common G– non-fermermenters, Pseudomonas is oxidase positive, Burkholderia usually too; on he contrary, Stenotrophomonas is usually negative and Acinetobacter too. 08 oxidase3 medic.med.uth.tmc.edu/path/oxidase.htm NEFERMtest 24 nFor precise biochemical identification of G– non-fermenters we use mostly NEFERMtest 24 (or a simillar test of other provenience). nIt is a triple-strip (not double as last week) nThere is a different way of code-formation than for (for example) ENTEROtest 16: nfirst number is 0 (oxidase –) or 1 (oxidase +) nnext 6 numbers come from collumns H to C ncollumns B and A are not counted (they are eventually used for more detailed determination) P1010002upr nOne frame is used for four triple-strips (for four strains). Each strain is detected using 24 reactions. nRequires 30 °C, 48 h NEFERMtest 24 www.medmicro.info (web of the Institute), photo O. Z. Antibiotics susceptibility of G– NF nG– non-fermenters may be tested on common media. nWe use strong antibiotics, that should not be used for other infections nWe use here n3rd generation cephalosporins* (but only some of them – „anti-pseudomonad“ ones, like ceftazidime) nAnti-pseudomonad penicillins, monobactams and carbapenems* (imipenem, piperacillin/tazobactam) naminoglycosides (gentamicin, amikacin) nfluoroquinolones (ciprofloxacin, ofloxacin) npolypeptides (colistine) n*or combinations with beta-lactamase inhibitors An example of NF atb set Antibiotic Abbrev. Reference zone Piperacillin + tazobactam* TZP 22 mm Gentamicin (aminoglykos.) CN 18 mm Imipenem (karbapenem) IMP/IMI 22 mm Ciprofloxacin (quin 3 gen) CIP 29 mm Ceftazidime (CS 3 gen) CAZ 16 mm Colistin (polypeptide) CT 12 mm *antipseudomon. peniciline + b-lactamase inhibitor P.aeruginosa - diskový test citlivosti na antibiotika. Klikni! www.medmicro.info On this picture, Pseudomonas aeruginosa is probably susceptible to all tested antibiotics, but it is possible only set only contained discs with special anti-pseudomonad drugs. Even so there exist poly-resitant strains that have secondary resistances even to such antibiotics. Especially producers of so called metalo-betalactamases (MBL) use to be only susceptible to amikacin and colistin. E-test - kapkový tvar inhibiční zóny. Klikni! It would be also possible to use E-test (here) or microdilution test www.medmicro.info The End 30 PSFL [USEMAP] http://www.scienceclarified.com Inflamation of external ear – otitis media (bonus) nCommon in children (short horizontal Eustach tube) nCaused by: Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis nIn chronical cases also some G– rods nIt is necessary to differenciate otitis externa: here Staphylococcus aureus is the main pathogen (as in other skin inflamations), local therapy, e. g. Framycoin drops [USEMAP] Otitis media 10 JAMA_Childrens_EarInfections_lev20_AcuteOtitsMedia_JPP_01 09 otitis media [USEMAP] http://www.medem.com/MedLB/article_detaillb.cfm?article_ID=ZZZPMV6D1AC&sub_cat=544 http://www.otol.uic.edu/research/microto/Microtoscopy/acute1.htm Examination and treatment of otitis media nTherapy is indicated in case of a real inflamation (pain, redness, fever) and it does not react to anti-inflamatory treatment nDrug of choice is amoxicilin (e. g. AMOCLEN), an alternative is co-trimoxazol nEar swab examination is meaningfull only after paracentesis nOtherwise it is also possible to examinate pyogene liquid taken during paracenthesis [USEMAP]