This document summarizes a study that developed and evaluated a quantitative PCR (qPCR) method for rapid detection of Pseudomonas aeruginosa from positive blood cultures. The qPCR method targeted the ecfX gene and was able to identify P. aeruginosa from blood cultures in less than 1.5 hours with 97% sensitivity and 100% specificity. The qPCR method demonstrated improved speed and equivalent performance compared to conventional identification methods and could help clinicians initiate appropriate antibiotic therapy more quickly.

1. PowerPoint TemplateJurnalDivisiPenyakitInfeksidanMikrobiologi IRapid detection of Pseudomonas aeruginosa from positive blood cultures by quantitative PCRAnnals of Clinical Microbiology and Antimicrobials 2010, 9:21Vincent Cattoir, Audrey Gilibert, Jeanne-Marie Le Glaunec, Nathalie Launay, Lilia Bait-Mérabet,Patrick LegrandYUNI SETYOWATININGSIH /ARYATIJumat, 1 April 2011

2. 2Pseudomonas aeruginosa6,8 % BakteremiakarenabakteribatangGram negatifPENDAHULUANPatogenoportunisutamainfeksinosokomial, terutamadi unit perawatanintensifDi Amerika patogenterbanyakke 7 darialirandarah,

3. 3Pseudomonas aeruginosaLanjutan (PENDAHULUAN)Bakteremiasecaraklinistidakdapatdibedakandariinfeksikarenabakteri Gram negatiflainnyaTerapiantibiotikaawal yang tidaktepatmengakibatkandampaknegatifhebat.

4. Metodeidentifikasifenotipmembutuhkanwaktu lama dankebanyakanpunyapembatasaninherenBeberapametodeberbasis PCR telahditelitiuntukmengidentifikasiP.aeruginosa, terutamapadasampel yang diambildarisistimrespiratoriuspasien fibrosis kistik.Teknikmolekulertelahditelitisebagaimetode yang cepatdandapatdiandalkanuntukidentifikasipatogenbakterial.Lanjutan (PENDAHULUAN)4

5. 5Lanjutan (PENDAHULUAN)Target MolekulerqPCRecfX16 S rRNAalgDgyrBPseudomonas aeruginosaoprItoxAoprLAdahasilpositifpalsuAdahasilnegatifpalsu

6. Kelemahanmetode PCR  masihharusditeliti target gen yang sesuaikarenatelahdilaporkansebelumnya target gen oprIdanoprLtidak 100% spesifikuntukP.aeruginosa.Metode PCR dari BC positifcepat, akuratsampai level spesiesdilanjutkanpengobatanempiris yang sesuai6Lanjutan (PENDAHULUAN)KelemahanteknikidentifikasiP.aeruginosakonvensionaldari BC positifbutuhwaktu minimal 24 jam

7. 7

8. Spesimen1IdentifikasiFenotip2Ekstraksi DNA3PCR kuantitatif4Batas deteksi5METODE8

9. 9Diambildari 100 hasil BC positifdari 100 pasienrawatinapMETODEWaktupenelitian : Agustus 2008 sampaiJuni 2009SPESIMEN DarahbotolBacT/ALERT aerobdananaerobDiinkubasidalamalatBacT/ALERTPengecatan Gram : Batang Gram Negatif87 aerob +13 anaerob +Konsentrasibakteri KulturKuantitatifIdentifikasifenotipdenganmorfologikoloni, tesoksidasedanhasil API 20 EDiinokulasisecaraaerobdananaerob, 37oC, 24 - 48 jam pada TSA, Drigalski Agar, Blood Agar, Chocolate AgarDiinokulasiSecaraAnaerobDiinokulasiSecaraAerob

10. Ekstraksi DNA denganRapid Boiling Procedure100,5 ml darahSentrifus 850 g 2’Supernatandiambil1% Triton X-1000,5% Tween 2010 mMTris-HCl1 mM EDTAInkubasipada 100oC10 ‘Hasil : Template DNAWaktu : 20 menitSentrifus 11500 g 5’ Pellet diambil,diresuspen200 µL buferpelisisSupernatandiambilSentrifus 850 g 2’

11. 11PCR KUANTITATIFCAMPURAN AMPLIFIKASI :2 µl 10x LightCyclerFastStart DNA Master Hybridization (Roche)2 mM MgCl20,5 µM masing-masing primer0,2 µM masing-masingprobe5 µl template DNA volume akhir 20 µl. PrimeroligonukleotidaAmplifikasiKontrolnegatif: SuspensiTris-EDTA KontrolPositif: Ekstrak DNA P. aeruginosaATCC 27853 Gen ecfXfragmen152 bpProbe berlabelfluoresenDeteksi

12. 12Tabel 1 PrimeroligonukleotidadanProbehibridisasiLightCycler yang digunakandalampemeriksaanPCRa [FAM], fluorescein; [LC705], LightCycler™-Red 705; Ph, 3’-phosphate. b Extracytoplasmic function gene(GenBank accession no. DQ996551).

13. 13Lanjutan (PENDAHULUAN)LOKASI ecfXecfX-HP2154-172ecfX-F46-62ecfX-HP1135-151ecfX-R179-197

14. Tahap1Tahap 2Tahap3PCR KUANTITATIFSatuSiklus PCR Denaturasiawalpadasuhu 95 °C selama 10 menitSEQUENCING/ELONGATION72 °C, 20”DENATURASI95 °C, 10”ANNEALING50 °C, 10”14

15. PCR KUANTITATIF15DNA teramplifikasidiukurdengandeteksifluoresensipada705 nmWaktuProsesqPCR : 1,5 jamPadasaat yang bersamaan, dilakukankontrolinhibisi PCR untuksemuasampeldenganmenggunakantabungreaksikedua yang berisi 100 ngekstrak DNA darikontrolpositif.

16. BATAS DETEKSILigasiecfXPlasmid pRT-ecfXMedia berisi 30 µg/mLKanamycinEscherichia ColiPlasmid DNA diekstraksi,dimurnikan, ditambahelution buffer dandi-sequencing untukmenentukanadanyaecfX16

17. BATAS DETEKSIKonsentrasi DNA  SpektrofotometerBahan Plasmid DNA rujukandiencerkan serial  plasmid genome equivalen10 sampai 1010AnalisisKurvaStandar.AnalisiskuantitatifdilakukandenganLightCycler software v 3.5 (Roche)Rasio signal diukurpada 705 nm/ signal pada 530 nm untukmenghitungnilaiCrossing Point17

18. KurvaamplifikasiqPCRdariplasmid DNA reference material dengan 11 konsentrasi DNA Eksternal(dari 1011sampai 10 copies/ml)18

19. KurvakalibrasiqPCRmenggunakanpengenceran serial dariplasmid DNA reference material19

20. HASIL2033 strain P. aeruginosa53 strain Enterobacteriaceae100 BC9 strain Stenotrophomonasmaltophilia2 spesies gram negatif lain3 BC adalahpolimikrobialDijumpaiinhibisi PCR padadua (2 %) preparasi DNA

21. Tabel 2 HasiltesqPCRecfX Pseudomonas aeruginosadari 98 botol BC positifKulturdarahPolimikrobial . Didapatkan P. aeruginosasebanyak 20 CFU/ml dan K. pneumoniae 108 CFU/ml darikulturkuantitatif21

22. Lanjutan HASIL22qPCRternyatatidakdapatmendeteksiadanyaP.aeruginosadalamkulturPolimikrobialPenelitimelakukantesqPCRlangsungdariisolatP.aeruginosa ecfXberhasildiamplifikasiKulturkuantitatifditemukanbahwakonsentrasiP.aeruginosadi BC inisangatrendah (20 CFU/ml).Batas deteksi PCR Kuantitatifpenelitianiniadalah102 CFU/ml.

23. PadaDari 98 BC yang dapat ditafsirkan (98 %), assay qPCR menunjukkan sensitivitas 97%, spesifisitas 100 %, PPV 98,5 % dan NPV 100%.23

24. Lanjutan HASILSensitivitas 97%Spesifisitas 100 %98 BCNPV 98.5 %24PPV 100 %

25. DISKUSIDenganteknikFISH (disetujuioleh FDA) yang secaralangsungdigunakanpada BC positif, metodeqPCRterbarumenunjukkanperforma yang serupanamunlebihcepat (1,5 jam vs 2,5 jam) Inhibitor teknikqPCRyang potensialdarispesimen BC positifadalahcharcoal dan hemoglobin25

26. Lanjutan DISKUSIInformasiakuratdantepatwaktu yang diberikanolehassayqPCRiniakanmembantudoktermengidentifikasibakteremiaP. aeruginosadanmemberiterapilebihcepat 18-24 jam.SulituntukmemprediksikepekaanantibiotikapadaP. aeruginosadenganteknikmolekuler.P. aeruginosaMultidrug resistance  terapiantibiotikadikombinasi26

27. 27PenelitimengembangkanteknikqPCR identifikasicepat (< 1,5 jam) dengansensitivitasdanspesifisitastinggiuntukidentifikasiPseudomonas aeruginosadari BC positif. PenelitianlanjutanmengevaluasidampakklinisdarimetodeqPCRbaruinidibandingkandenganmetodekonvensional.21KESIMPULAN

28. www.themegallery.comThank You !www.themegallery.com

29. KurvaamplifikasiqPCRdariplasmid DNA reference material dengan 11 konsentrasi DNA Eksternal(dari 1011sampai 10 copies/ml)29TresholdCTCTCT

30. KurvakalibrasiqPCRmenggunakanpengenceran serial dariplasmid DNA reference material30

31. Inhibitor PCR Inhibitor PCR: Darah, jaringan, fabrics, soilSumber lain : material danreagen yang kontakdengansampelselamaproses PCRmisal : KelebihanKCl, NaCl, Ion detergen (Sodium Deoxycholate), Isopropanolol, PhenolCara mengatasi : - inhibitor PCR daridarahdanjaringantidakbisadihindari - inhibitor PCR misal saliva dengan media transfer swab pemurnian DNA dengancaraekstraksi  memurnikanTemplate DNA31

32. 32

33. PCR CONTAMINATIONContamination is defined as the unwanted presence of DNA (or RNA) ampliconsAt present, no experimental means exist which can directly detect the ultra-trace amounts of this template (e.g.,picogram amounts or less). The best way to monitor is to use reagent-only blanks The presence of contamination is then signalled by the appearance of the test amplicon in the reagent-only blank tubes.33

34. Triton X-100 Triton X-100 (C14H22O(C2H4O)n) is a nonionicsurfactant which has a hydrophilicpolyethylene oxide group (on average it has 9.5 ethylene oxide units) and a hydrocarbonlipophilic or hydrophobic group. 34

35. Tween 20Polysorbate 20 (a common commercial brand name is Tween 20) is a polysorbatesurfactant whose stability and relative non-toxicity allows it to be used as a detergent and emulsifier in a number of domestic, scientific, and pharmacological applications. It is a polyoxyethylene derivative of sorbitanmonolaurate, and is distinguished from the other members in the polysorbate range by the length of the polyoxyethylene chain and the fatty acid ester moiety. 35

36. Tris-HClTE buffer is a commonly used buffer solution in molecular biology, especially in procedures involving DNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that chelatescations like Mg2+. The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.36

37. TBETBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA.In molecular biology, TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water. EDTA is a chelator of divalent cations, particularly of magnesium (Mg2+). As these ions are necessary co-factors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation. 37

38. KANAMYCINKanamycin is used in molecular biology as a selective agent most commonly to isolate bacteria (e.g., E. coli) which have taken up genes (e.g., of plasmids) coupled to a gene coding for kanamycin resistance (primarily Neomycin phosphotransferase II [NPT II/Neo]). Bacteria that have been transformed with a plasmid containing the kanamycin resistance gene are plated on kanamycin (50-100ug/ml) containing agar plates or are grown in media containing kanamycin (50-100ug/ml). Only the bacteria that have successfully taken up the kanamycin resistance gene become resistant and will grow under these conditions. As a powder kanamycin is white to off-white and is soluble in water (50mg/ml).38

39. DampaknegatifPasientidaksembuhWaktuopnamelebih lamaMemicuresistensiantibiotika lain39

40. Kelemahan PCRTidakbisamenunjukkanbakteriinihidupataumati. KulturtetapdipercayasebagaiGold Standard  karenabakteritumbuhdanhidupTetapidarisampeldarahbiasanyahanya 20-30% yang positif  menunjukkansulitnyamencapaiGold Standard  butuhmetodealternatifuntukidentifikasi.40

41. Antimicrobial Resistance in P. aeruginosaIntrinsic resistance to most antibiotics is attributed to:Efflux pumps: Chromosomally-encoded genes (e.g. mexAB-oprM, mexXY, etc) and Low permeability of the bacterial cellular envelopeAcquired resistance with development of multi-drug resistant strains by:Mutations in chromosomally-encoded genes, or Horizontal gene transfer of antibiotic resistance determinants 41

42. MetodefenotipMetodekonvensionaluntukidentifikasiPseudomonas aeruginosabutuhwaktu lamaTesgula-gula :TesoksidasepositifMamputumbuhpada 42oCTSI slant  tidakadareaksiMH agar : Warnahijaukebiruan, merahataucoklatProduksinitrat, Gas +42

43. MetodefenotipLanjutanTesGula-gula :Arginindehidrogenase +Gelatin Liquified : variabelLysine decarboxylase –Urea Hydrolysis : variabelMengoksidaseglukosa & Xylosa (Lactose -, mannitolvariabel)43

44. Pseudomonas aeruginosaScientific Classification :Kingdom : BacteriaPhylum : ProteobacteriaClass : Gamma ProteobacteriaOrder : PseudomonadalesFamily : PseudomonadaceaeGenus : PseudomonasSpecies : Pseudomonas aeruginosa44

45. SynonimsBacterium aeruginosumSchroeter 1872Bacterium aeruginosum Cohn 1872Micrococcus pyocyaneusZopf 1884Bacillus aeruginosus (Schroeter 1872) Trevisan 1885Bacillus pyocyaneus (Zopf 1884) Flügge 1886Pseudomonas pyocyanea (Zopf 1884) Migula 1895Bacterium pyocyaneum (Zopf 1884) Lehmann and Neumann 1896Pseudomonas polycolor Clara 1930Pseudomonas vendrellinomennudum 193845

46. Although classified as an aerobic organism, P. aeruginosa is considered by many as a facultative anaerobe, as it is well adapted to proliferate in conditions of partial or total oxygen depletion.

47. This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor, and, in its absence, it is also able to ferment arginine by substrate-level phosphorylation.46

48. 47

49. Biofilms and treatment resistanceBiofilms of Pseudomonas aeruginosa can cause chronic opportunistic infections. They often cannot be treated effectively with traditional antibiotic therapy. Biofilms seem to protect these bacteria from adverse environmental factors. Pseudomonas aeruginosa can cause nosocomial infections and is considered a model organism for the study of antibiotic-resistant bacteria. 48

50. DIAGNOSISFirst, a Gram stain is performed, which should show Gram-negative rods with no particular arrangement.Then, if the specimen is pure, the organism is grown on MacConkey agar plate to produce colorless colonies (as it does not ferment lactose)Cetrimide agarP. aeruginosa may express the exopigmentpyocyanin, which is blue-green in color, and the colonies will appear flat, large, and oval. It also has a characteristic fruity smell. P. aeruginosa is catalase+, oxidase+, nitrase+, and lipase+. When grown on TSI medium, it has a K/K profile, meaning that the medium will not change color. Finally, serology could help, which is based on H and Oantigens.49

51. TREATMENTaminoglycosides (gentamicin, amikacin, tobramycin)quinolones (ciprofloxacin, levofloxacin, and moxifloxacin)cephalosporins (ceftazidime, cefepime, cefoperazone, cefpirome, but notcefuroxime, ceftriaxone, cefotaxime)antipseudomonalpenicillins: ureidopenicillins and carboxypenicillins (piperacillin, ticarcillin: P. aeruginosa is intrinsically resistant to all other penicillins)carbapenems (meropenem, imipenem, doripenem, but notertapenem)polymyxins (polymyxin B and colistin)[31]monobactams (aztreonam)In the rare occasions where infection is superficial and limited (for example, ear infections or nail infections), topicalgentamicin or colistin may be used.50

52. 51

53. PENGGUNAAN INKUBATOR SESUAI KEBUTUHAN LINGKUNGAN UNTUK PERTUMBUHAN BAKTERI52

54. TOPLES LILINKomponenToplesterbuatdarikacaTutuptoplesLilinputih yang kering53

55. ANAEROBIC JAR DENGAN GAS PACKKomponenAlat:Toplesterbuatdarimika yang dilengkapikaretmelingkardiatasnyaTutuptoplesdengan 3 lubangsaluranuntuk gas CO2, O2 dan N2Penjepitantaratutupdantoples yang diperkuatsekrupdiatasnyaGas packIndikatoranaerobKatalis54

56. INKUBATOR CO255

57. The basicsAerobic, opportunistic pathogenGram-negative bacillusFlagella56

58. PathogenesisVirulence Factors (structural components)“Alginate”Adherence proteins and “pili”Lipopolysaccharide (LPS)Procyanin57

59. Structure58

60. Structural ComponentsAdherence to host cells mediated by pili and nonpilusadhesins. LPS (lipopolysaccharide) inhibiting antibiotic killing and suppress neutrophil and lymphocyte activityAlginate – mucoidexopolysaccharide that forms a shiny biofilm protecting from antibodies, complement, phagocytosis, and antibioticsProcyanin – impairs ciliary function, mediates tissue damage through production of oxgen radicals59

61. PathogenesisVirulence Factors (toxins and enzymes):Exotoxin AExoenzyme SEndotoxinsPhospholipase CElastase and Alkaline Protease60

62. Exotoxin ASimilar in structure to Diptheria toxin Inhibits protein synthesis by ADP-ribosylating EF-2 (G-protein)Causes Dermatonecrosis in burn wounds, corneal damage in ocular infections, and tissue damage in chronic pulmonary infections.Also this toxin is immunosuppressive61

63. Exoenzyme S & TADP-ribosylates G-proteins including p21 RAS interfering with host cell growth62

64. Phospholipase CHeat labile hemolysinBreaks down lipids and lecithin causing tissue destructionStimulates inflammatory response63

65. Elastase and Alkaline ProteaseDestruction of elastin-containing tissues (blood vessels, lung tissue, skin), collagen, immunoglobulins, and complement factorsCan produce hemorrhagic lesions (ecthyma gangrenosum) associated with disseminated infectionInactivation of interferon and TNF-Alpha64

66. EpidemiologyUbiquitous in moist environmental sites in the hospital as well as natureNo seasonal incidenceCan transiently colonize the respiratory and GI tract of hospitalized patientsMinimal nutritional requirements and can tolerate broad temperature spectrum65

67. Clinical DiseasePulmonary InfectionsBurn Wound Infections and other skin and soft tissue infections (life threatening)UTI’s (especially catheterized)External Otitis (malignant OE, swimmer’s ear)Eye Infections and corneal ulceration via contaminated contact lens cleaning fluidsPseudomonal Endocarditis66

68. Pulmonary InfectionsCan range from asymptomatic colonization to severe necrotizing bronchopneumoniaColonization is seen in patients with cystic fibrosis, chronic lung disease, and neutropeniaMucoid strains are commonly isolated from chronic pulmonary patients and are more difficult to eradicatePredisposing conditions include previous therapy with broad spectrum abx (disrupts normal protective bacteria population and use of respiratory therapy equipment (can introduce the organism to lower airways)Mortality rate can be as high as 70% for invasive bronchopneumonia67

69. Ecthyma GangrenosumEcthyma gangrenosum is a well recognized cutaneous manifestation of severe, invasive infection by Pseudomonas aeruginosa that is usually seen in immunocompromised, burn patients, and other critically ill patients 68

70. Antibiotic ResistanceInherently resistant to many abxCan mutate to more resistant strains during therapyPenetration of abx highly dependent on outer membrane pores which can be alteredProduction of B-lactamasesCombination of active abx generally required for successful therapy69

71. Drigalski AgarDrigalski agar is a selective isolation and differentiation medium used to identify Enterobacteriaceae and other Gram-negative bacteria.Microorganisms that ferment lactose form yellow or yellowish-green colonies; the others produce blue, green, or bluish-green colonies.The presence of sodium deoxycholate and crystal violet inhibits the growth of Gram-positive.70

72. Chocolate AgarA non-selective, enriched growth medium. A variant of the blood agar plate. Contains red blood cells, which have been lysed by heating very slowly to 56 °C. Chocolate agar is used for growing fastidious (fussy) respiratory bacteria, such as Haemophilusinfluenzae. These bacteria need growth factors, like NAD and hematin, which are inside red blood cells; thus, a prerequisite to growth is lysis of the red blood cells. 71

73. Trypticase Soy AgarThe medium contains enzymatic digests of casein and soybean meal which provides amino acids and other nitrogenous substances.Dextrose is the energy source. Sodium chloride maintains the osmotic equilibrium, while dipotassium phosphate acts as buffer to maintain pH.The medium may be supplemented with blood to facilitate the growth of more fastidious bacteria or antimicrobial agents to permit the selection of various microbial groups from pure flora.72

74. MicrobiologyFamily PseudomonadaceaeAerobic, non-spore forming Gram negative straight or slightly curved rod (1 to 3 um in length), polar flagellaNon-fermentersCatalase and oxidase positiveMorphologic characteristics on lab media:Production of pigments:Soluble blue-coloured phenazine pigment called pyocyanin)Some strains produce red or black colonies due to pigments termed pyorubin and pyomelanin, respectivelyP. aerugnosa produces pyoverdin (diffusible yellow-green to yellow-brown pigment) which, when produced with pyocyanin gives rise to green-blue colonies on solid mediaTerm ‘aeruginosa” stems from green-blue hue 73

75. MicrobiologyTerm Pseudo = “false”; monas = “single unit”Term ‘aeruginosa” stems from green-blue huePseudomonas are classified as strict aerobes but some exceptions:May use nitrateBiofilm formation74

76. MicrobiologyPseudomonads classified into five rRNA homology groups:Pseudomonas (sensu stricto)Burkholderia speciesComamonas, Acidovorax, and Hydrogenophaga generaBrevundimonas speciesStenotrophomonas and Xanthomonas generaGenus Pseudomonas contains over 160 species but only 12 are clinically relevant75

77. MicrobiologyP. aeruginosa is the type species and may have highly varied morphologyTypical colonies may appear to spread over the plate, lie flat with a metallic sheen and frequently produce a gelatinous or “slimy” appearanceMost strains produce characteristic ‘grapelike’ or ‘corn taco-like’ odor76

78. P. aeruginosa on blood agar77

79. Non-Lactose FermenterLactose FermenterMacConkey Agar78

80. P. aeruginosaMucoid P. aeruginosaMacConkey Agar79

81. Epidemiology and TransmissionNatural habitat:Temperature between 4 to 36oC (can survive up to 42oC)Found throughout nature in moist environment (hydrophilic) (e.g. sink drains, vegetables, river water, antiseptic solutions, mineral water, etc.)P. aeruginosa rarely colonizes healthy humansNormal skin does not support P. aeruginosa colonization (unlike burned skin)Acquisition is from the environment, but occasionally can be from patient-to-patient spread80

82. Range of clinical infections caused by P. aeruginosaP. aeruginosa is an opportunistic infection:Individuals with normal host defenses are not at risk for serious infection with P. aeruginosaThose at risk for serious infections include:Profoundly depressed circulating neutrophil count (e.g. cancer chemotherapy)Thermal burnsPatients on mechanical ventilationCystic fibrosis patients81

83. Range of clinical infections caused by P. aeruginosaImmunocompetent Host:Most common cause of osteochondritis of dorsum of foot following puncture wounds (running shoes)Hot tub folliculitisSwimmer’s earConjunctivitis in contact lens users (poor hygiene or if lenses are worn for extended periods)Other Hosts:Malignant otitis externa in diabeticsMeningitis post trauma or surgerySepsis and meningitis in newbornsEndocarditis or osteomyelitis in IVDUsCommunity-acquired pneumonia in pts with bronchiectasisUTI in patients with urinary tract abnormalitis82

84. PLASMIDA plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria.83

85. PLASMIDPlasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host.plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer.84

86. TesOksidaseDigunakanuntukmembantuidentifikasispesies yang memproduksienzimoksidasePrinsipSepotongkertassaringdibasahidenganbeberapatetesreagenoksidase. Jikaorganismememproduksioksidase, phenylenediaminepadareagenakanteroksidasimenjadiwarna deep purple.Reagen yang dibutuhkan:ReagenOksidase yang berisilarutan 10 g/l tetramethyl-p- phenylenediaminedihydrochlorideCtt: reageninimudahteroksidasi. Jikareagentelahberwarnabiru, jangangunakan.85

87. MetodeLetakkankertassaringpadapetri disk yang bersih, tambahkan 2-3 ttsreagenoksidase

88. Denganbatangkaca, pindahkankoloniorganismetesdanoleskanpadakertassaring

89. Lihatperubahanwarnamenjadi blue-purple dalambeberapadetikHasilWarna blue purple (dalam 10 detik) positifmemproduksienzimoksidaseWarnabukan blue purple (dlam 10 detik) negatifCtt:abaikanwarna blue purple yang terjadisetelah 10 detikKontrolpositif: Pseudomonas aeruginosaKontrolnegatif: Eschericia Coli86

90. 87

91. Pseudomonas aeruginosaCultural features blood agar: large, flat, haemolitic colonies. Strict aerob. Most strain produce pyocyanin (yellow green in medium)McConkey:non lactose fermenting colonies with yellow green in medium KIA: Pink-red slope and butt, gas (-), H2S (-)identifikasi: gram negatif, oksidasepositif, citrate positifMIU: Motility positif, indolnegatif, tesureasetergantung strain 88

92. Citrate utilization testUntukmembantuidentifikasienterobactericiaea. Tesiniberdasarkankemampuanorganismemenggunakansitratsebagaisumberkarbondan ammonia sebagaisumber nitrogenPrinsip:Organismedikulturpada media yang mengandung sodium citrate, garam ammonium, danindikatorbromothymol blue. Pertumbuhanpada media ditandaiolehkekeruhandanperubahanwarnaindikatordarihijauterangmenjadibiru, olehkarenareaksialkali,mengikutipemakaiancitrat89

93. Bahan yang dibutuhkan: Media Koser’s citrate (Simon citrate dapatdigunakantapilebihmahal)Dengansterile straight wire, inokulasiorganismedi media kultur broth ke 3-4 ml media Koser’s Citrate (harushati-hati agar tidakmengkontaminasi media denganpartikelkarbon, sepertikawat yang terbakarInkubasipada 35-37oC, hingga 4 hari, periksatiaphariadanyapertumbuhan90

94. HasilKekeruhandanwarnabiru, hasiltespositif,citrateterpakai

95. Takadapertumbuhan, tesnegatif, citrate tidakterpakai

96. Kontrolpositif : Klebsiellapneumoniae

97. Kontrolnegatif : Eschericia coli91

98. CITRATE UTILIZATION TEST92InkubasiPositifnegatif

99. 93

100. Urease Test Tesureasepentinguntukmembedakanenterobacteriaceaeberdasarkankemampuanmemproduksiurease.PrinsipOrganismetesdikulturpada media yang mengandung urea danindikator phenol warnamerah. Jika strain memproduksiurease, enziminiakanmemecah urea menghasilkan ammonia dankarbondioksida. Denganmenghasilkan ammonia, media menjadi alkali yang ditunjukkanperubahanwarnamenjadimerahmuda94

101. TesureaseBahan yang dibutuhkan Media Motility Indole urea (MIU)Metodemenggunakankawatsterillurus, inokulasi media pada media MIUTempatkanindole paper strip dilehertabung MIU diatas media. Tutuptabungdaninkubasipada 35-37oC semalamPeriksaproduksiureasedenganmelihatwarnamerahmudapada media95

102. Hasil Media merahmuda, positifmenghasilkanurease Media tidakmerahmudanegatifmenghasilkanureaseKontrolkontrolpositifurease : Proteus vulgariskontrolnegatifurease : Eschericia ColiMotilitasditunjukkandenganadanyapenyebarankekeruhandarigaristusukan96

103. 97

104. TesIndolTesproduksiindolpentinguntukidentifikasienterobacteriaceae. Kebanyakan strain dariE.Colimemecahasam amino tryptophan denganmenghasilkanindole.Organismetesdikulturpada media yang mengandung tryptophan. ProduksiindoldideteksiolehreagenKovac’satau Ehrlich yang berisi 4 (p)-dimethylaminobenzaldehyde. Bahaninibereaksidenganindolsehinggamemproduksiwarnamerah98

105. Bahan media MIUKovac’s reagent stripsHasil strip kemerahan, positifmenghasilkanindolbukanwarnamerah, negatifmenghasilkanindolJikabelumjelas, tambahkan 1 ml reagent Kovacdanlihatadanyawarnamerahdalam 10 menit99

106. 100

107. gyrBDNA gyrase subunit BLocation : sitoplasmaGenomic location : 4275 - 6695 (+) Function : DNA replication, recombination and repair101

108. gyrB102Dari gyrB dicari primer misalnya 20 bpdenganurutansbb :Diambildari www.pseudomonas.com

109. Exotoxin A103

110. algD104Gene name : algDGenomic Location : 3962825 - 3964135 (+) Function : Cell wall / LPS / capsule Adaptation, Protection Secreted Factors (toxins, enzymes, alginate)

111. oprIGene Name :oprILocation : 3206915 – 3207166PseudoCAP Function Class : Membrane ProteinProduct name : Outer membrane lipoprotein OprI precursor105

112. oprLGene Name :oprLLocation : 1057400 - 1057906PseudoCAP Function Class :Membrane proteins,Transport of small moleculesProduct name : Peptidoglycan associated lipoprotein OprL precursor106

113. toxAGene Name : toxALocation : 1242500 - 1240584PseudoCAP Function Class : Secreted Factors (toxins, enzymes, alginate)Product name : Exotoxin A precursor107

114. PCRPCR is a technique used to amplify segments of DNA in vitroPCR can generate millions of copies of DNA fragments108

115. 109

116. BacT/ALERTFungsinya sama dengan BACTEC sebagai media pemupuk kuman.Macam Media :110FAN ANAEROBPEDIATRIC FAN FAN AEROB SA SN FA FN PF MP Lytic

117. Media BacT/ALERT111

118. Prinsip dan Cara Kerja BacT/ALERTMenggunakan sensor kolorimetri dan sinar refleksi untuk memonitor CO2 yang larut pada botol media.CO2 dihasilkan oleh mikroorganisme  berdifusi melewati membran dan melepas ion H+perubahan pH ditangkap oleh sensor warna pada dasar botol BacT/ALERT perubahan warna dari abu-abu menjadi kuning.112

119. 113Perubahan warna botolPrinsip kerja kolorimetri

120. 114

121. 115

122. API 20 EAPI 20 E is an identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods which uses 23 standardized and miniaturized biochemical tests and a database. The API 20 E strip consists of 20 microtubes containing dehydrated substrates.These tests are inoculated with a bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents.Incubate at 35-37°C for 18-24 hours.116

123. Preparation of the stripPrepare an incubation box (tray and lid) and distribute about 5 ml of distilled water or demineralized water [or any water without additives or chemicals which may release gases (e.g., Cl2, CO2, etc.)] into the honeycombed wells of the tray to create a humid atmosphere.Record the strain reference on the elongated flap of the tray.Remove the strip from its packaging.Place the strip in the tray.Perform the oxidase test on a colony identical to the colony which will be tested.Refer to the Oxidase Test Kit Package Insert.This reaction should be recorded on the result sheet as it constitutes the 21st test.117

124. Preparation of the inoculumOpen an ampule of NaCl 0.85 % Medium (5 ml) or an ampule of Suspension Medium (5ml) as indicated in the paragraph "Warnings and Precautions" or use any tube containing 5 ml of sterile NaCl 0.85% Medium, pH 5.5 - 7.0, or sterile distilled water, without additives.With the aid of a pipette, remove a single well-isolated colony from an isolation plate.Carefully emulsify to achieve a homogeneous bacterial suspension.118

125. Inoculation of the stripWith the same pipette, fill both tube and cupule of test CIT , VP and GEL with the bacterial suspension.Fill only the tubes (and not the cupules) of the other tests.Create anaerobiosis in the tests _A_D_H_, _L_D_C_, _O_D_C_, _H_2_S_ and U__R_E_ by overlaying with mineral oil.Close the incubation box.Incubate at 35-37°C for 18-24 hours.119

126. PCR(Polymerase Chain Reaction)

127. PendahuluanPCR ditemukanolehKaryMulispadatahun 1985.

128. PCR  suatutekniksintesisdanamplifikasifragmen DN A secara in vitro

129. Proses PCR miripdenganprosesreplikasi DNA in vivo

130. PCR membutuhkan template untaiganda yang mengandung DNA target yang akandiamplifikasi, enzim DNA polimerase, nukleotidatrifosfat, dansepasang primer oligonukleotida.121

131. PRIMERPrimer oligonukleotidadisintesismenggunakanalatDNA SynthesizerUntukmerancangurutan primer, perludiketahuiurutannukleotidapadaawaldanakhir DNA target.Padakondisitertentu, kedua primer menempelpadauntai DNA komplemennya yang terletakpadaawaldanakhir DNA target. 122

132. Lanjutan (PRIMER)Primer haruspunyasuhuTm(Melting Temperature) yang samadengansuhuprosesanneling menghindarimishybridizedan agar tidakmenempelkesegmen DNA lainKedua primer menempelpadaawaldanakhir DNA target  berfungsimenyediakangugushidroksilbebaspadakarbon 3’.SetelahituEnzim DNA Polimerasemengkatalisisprosespemanjangankedua primer tersebutdenganmenambahkannukleotida yang sesuai123

133. Primer dipilihdaridaerah yang khususuntukmenghindarimishybridizationdari sequence yang sama. Method BLASTseringdigunakanuntukmencari primer disemua area tersebut. Basic Local Alignment Search Tool124

134. Komponen PCR125DNA TemplateEnzim DNA PolymerasePrimerdNTP (deoxynucleosidetriphosphate)BufferIon Logam

135. 16 S rRNA12616S ribosomal RNA (or 16SrRNA) is a component of the 30S subunit of prokaryoticribosomes. It is 1,542 nucleotides in length.Multiple sequences of 16S rRNA can exist within a single bacterium.[2]In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid, accurate alternative to phenotypic methods of bacterial identification[9].

136. 16 S rRNA127It has several functions:Like the large (23S) ribosomal RNA, it has a structural role, acting as a scaffold defining the positions of the ribosomal proteinThe 3' end contains the anti-Shine-Dalgarno sequence, which binds upstream to the AUG start codon on the mRNAInteracts with 23S, aiding in the binding of the two ribosomal subunits (50S+30S)Stabilizes correct codon-anticodon pairing in the A site, via a hydrogen bond formation between the N1 atom of Adenine (see image of Purine chemical structure) residues 1492 and 1493 and the 2'OH group of the mRNA backbone

137. 12816 S rRNA

138. Enzim DNA Polimerase129Enzim DNA Polimerasebersifattermostabil, tahansampaisuhu 95oCBakteriThermusAquaticus(enzimdisebutTaqPolymerase)Meningkatkanspesifisitas PCR karenasintesis DNA dilakukanpadasuhu 72oC

139. dNTP (deoxynucleosidetriphosphate)130Untuksintesis DNA :Deoksiadenosintrifosfat (dATP)Deoksitidintrifosfat (dCTP)Deoksiguadintrifosfat (dGTP)Deoksitimidintrifosfat (dTTP)dNTPinimenempelpadagugus 3’ hidroksilbebas primer membentukrantaiuntaibarudenganrantai DNA templat

140. Buffer dan Magnesium klorida131Buffer yang biasanyaterdiriatasbahan-bahankimiauntukmengkondisikanreaksi agar berjalan optimum danmenstabilkanenzim DNA polymerase.Ion Magnesium membentukkomplekslarutantaradNTP.Ion Magnesium menstimulasiaktivitaspolimerase, meningkatkan Tm DNA untaigandadaninteraksidengan template

141. Primer132Panjangoligonukleotida :15 – 30 bpTemperatur melting ™ : 55 – 65oCKomposisinukleotidaLokasipada DNA targetPanjang target yang diamplifikasi

142. Pradenaturasi133Dilakukanselama 1-10 menitdiawalreaksiuntukmemastikankesempurnaandenaturasidanmengaktifasi DNA Polymerase (jenis hot-start alias baruaktifkalaudipanaskanterlebihdahulu).

143. DNA134DNA adalahasamnukleat yang mengandungmaterigenetikdanberfungsiuntukmengaturperkembanganbiologisseluruhbentukkehidupansecaraseluler. DNA terdapatpadanukleus, mitokondriadankloroplas. Perbedaandiantaraketiganyaadalah: DNA nukleusberbentuk linear danberasosiasisangateratdengan protein histon, sedangkan DNA mitokondriadankloroplasberbentuksirkulardantidakberasosiasidengan protein histon. Struktur DNA prokariotberbedadenganstruktur DNA eukariot. DNA prokariottidakmemiliki protein histondanberbentuksirkular, sedangkan DNA eukariotberbentuk linear danmemiliki protein histon (Klug & Cummings 1994: 315--316; Raven & Johnson 2002: 94).

144. Bentuk DNA135ContohGambar Double helix

145. PenyusunUtama DNA136Sesuaidengannamanya, DNA, Deoxyribose Nucleic Acid. Penyusunutama DNA adalahgula ribose yang kehilangansatu atom oksigen (deoksiribose).Gambar 2. Perbedaan Ribose danDeoksiribosePerhatikangambardiatas, padadeoksiribose, satu atom oksigenpadasalahsatu atom C ribose hilang.

146. Nukleotida137Tiap pita/rantai double helix terbuatdari unit-unit berulang yang disebutnukleotida. Satunukleotidaterdiridaritigagugusfungsi; satugula ribose, triphosphate, dansatubasa nitrogen.

147. 138Satuhal yang perludiingatadalahposisitriphosphatedanbasa nitrogen yang terikatpadaribosa. Gugustriphosphatterikatpada atom C no 5′ dariribosa (Lihatgambardiatas). Gugustriphosphateinihanyadimilikiolehnukleotidabebas. Sedangkannukleotida yang terikatpadarantai DNA kehilanganduadarigugus phosphate ini, sehinggahanyasatu phosphate yang masihtertinggal.Ketikanukleotidabergabungmenjadi DNA, nukleotida-nukleotidatersebutdihubungkanolehikatanphosphodiester. Ikatankovalen yang terjadiantaragugus phosphate padasatunukleotida, dengangugus OH padanukleotidalainnya. Sehinggasetiaprantai DNA akanmempunyai ‘backbone’ phosphate-ribosa-phosphate-ribosa-phosphate. Dan seterusnya..

148. 139GambarStruktur DNA Sederhana

149. Basa Nitrogen Pada DNA140Padastruktur DNA, gularibosadangugus phosphate yang terikatadalahsama. Yang berbedahanyalahpadabasa nitrogen. Jadisebetulnyaperbedaandisebabkanolehvariasisusunandaribasa-basa nitrogen yang terdapatpadarantai DNA. Adaempatmacambasa nitrogen. Adenin, Cytosine, Guainne, dan Thymine.

150. 141Ketikabasa-basa nitrogen tersebutterikatdalamnukleotida, makapenamaan-pun berubah. Ingatkembalipenjelasandiawaltentangnukelotida. Nukleotidaterdiridarigugustriphosphatedansatubasa nitrogen yang terikatpadasatumolekul ribose. Nah.. basa-basa nitrogen iniapabilaterikatpada ribose membentuknukleotidamakapenamaannya-pun berubah.Adeninmenjadi 2′deoxyadenosinetriphosphate, cytosinmenjadi 2′deoxycytidinetriphosphate, guainnemenjadi 2′deoxyguanosinetriphosphate, dan Thymine menjadi 2′deoxythymidinetriphosphate. Disingkatmenjadi A, C, G, dan T.

151. 142Perhatikanbahwaadaduapasangbasa yang mirip. A dan G sama-samamempunyaiduacincinkarbon-nitrogen, disebutgolonganpurine. Sedangkan C dan T hanyamempunyaisatucincinkarbon-nitrogen, masukgolonganpirimidin.

152. PenyebabBentuk DNA Double Helix143GambarIkatanHidrogenAntaraBasa-Basa Nitrogen

153. 144Interaksiikatanhidrogenantaramasing-masingbasa nitrogen menyebabkanbentukdariduarantai DNA menjadisedemikianrupa, bentukinidisebut double helix. Interaksispesifikiniterjadiantarabasa A dengan T, dan C dengan G. Sehinggajika double helix dibayangkansebagaisebuahtangga spiral, makaikatanbasa-basainisebagaianaktangga-nya. Lebardari ‘anaktangga’ adalahsama, karenapasanganbasaselaluterdiridarisatuprimidindansatupurin.

154. 145GambarStruktur Double HeliLengkap

155. StrukturAsam amino146

156. Transkripsi147Prosespengkopian DNA menjadi RNA inidinamakantranskripsi.

157. Translasi148mRNA hasiltranskripsikemudiandikeluarkanmenujusitoplasmasehinggabisadiproseslebihlanjutolehsuatuorganelsel yang bernamaribosom. Ribosomakanmembacaurutanbasa RNA danmenterjemahkannya (translate ) menjadiurutanasam amino tertentusesuaidenganresep yang dibawa mRNA. Di sinilahasam-asam amino itudirakitsesuaiurutan yang diresepkan gen (DNA) dankemudianmelipatmembentukstrukturtigadimensi yang fungsional.