Ratri Ariani K.

Ida Parwati

 Mikrobiologi

okuler terus berkembang. Perkembangan ilmu pencegahan dan eradikasi infeksi mata. Infeksi mata berat jmlnya Infeksi mata berhub dengan obat imunosupresif, transplantasi organ , penggunaan lensa kontak, pasca operasi katarak, implantasi lensa mata.

Infeksi mata: penyebab kebutaan. Anamnesis Pemeriksaan fisik Pemeriksaan mikrobiologi

Penanganan infeksi mata

Rencana terapi

Identifikasi kuman
Mencegah komplikasi

Tinjauan pustaka: Anatomi mata Patogenesis infeksi mata Berbagai infeksi mata Pemeriksaan mikrobiologi

Berbentuk seperti pir, saraf optikus sbg tangkai. Volume: 30 cc, 1/5 bag ruangannya Bag terbesar: lemak, otot Diameter: 24,5 mm Arteri utama: A. oftalmika, cabang A. karotis interna. Vena: V. oftalmika superior, inferior.

Faktor risiko infeksi mata: - Usia - Jenis kelamin - Status imun - Kondisi sosial ekonomi

S. epidermidis, S. aureus, Corynebacterium sp., Propionibacterium acnes. Bertambah usia bakteri Gram (-) Antibiotik jangka panjang perubahan mikroorganisme tumbuh jamur & bakteri resisten
Lahir:

Air mata: - Tdd protein, elektrolit, produk antimikroba (lisozim, laktoferin, beta lisin, komplemen, Ig) - Substansi seluler: Limfosit, PMN - Lensa kontak perubahan flora normal diganti mikroorganisme patogen

Mikroorganisme Bakteri Aerob Coccus Gram Positif Coccus Gram Negatif Batang Gram Positif Batang Gram Negatif

Spesies Staphylococcus epidermidis Staphylococcus aureus Micrococcus sp. Streptococcus pyogenes Streptococcus pneumoniae Streptococcus viridians Moraxella catarrhalis Corynebacterium sp. Bacillus sp. Proteus sp. Klebsiella sp. Eschericia coli Pseudomonas aeruginosa Moraxella sp.

Persentase 30-80% 3-25% 1-28% 0-3% 0-3% 0-1% 2-5% 5-83% 0,7-4.2% 0,4-1% 0-0,5% 0-1% 0-2% 0-2%

Mikroorganisme Bakteri Anaerob Jamur

Spesies Propionibacterium sp. Peptostreptococcus Bacteroides sp. Lactobacillus sp. Clostridium sp. Candida sp. Alternaria sp. Cladosporium sp. Aspergillus sp. Penicillium sp. Helminthosporium sp. Cephalosporium sp. Geotrichum sp. Rhodotorula sp. Fusarium

Persentase 0-33% 0-2% 0-1% 0-2% 0-2% 0,27-8.9% 0,99-17,1% 0,99-14,2% 0,36-9,3% 0,63-4,1% 0,31-7,7% 0,31-4,1% 0,27-4,1% 0,27-2,2% 1,6-2,0%

Blefaritis

Jenis infeksi Bakteri Virus Jamur

Jenis infeksi Endoftalmitis

Bakteri

Virus

Jamur

S. aureus Virus Herpes S. epidermidis Simpleks S. pneumoniae Virus VaricellaStreptococcus Zoster sp.lain Cytomegalovirus P. aeruginosa Measles virus Bakteri Gram negatif lain Actinomyces Propionibacterium propionicum S. aureus S. pyogenes S. pneumoniae H. influenzae S. aureus S. pneumoniae

Candida sp. Aspergillus sp. Volutella sp. Acremonium sp.

Kanalikulitis

Dakriosistitis

Dakrioadenitis

Candida albicans Aspergillus sp.

Blefaritis - BP: apus tepi kelopak mata (aplikator kapas / kalsium alginat) dibasahi HBSS / normal saline. - Inokulasi: agar darah, agar coklat, agar MacConkey, Sabouraud dextrose agar - BP pus: inokulasi pada BA, CA, MA, dan (Brain heart insusion broth) BHIB. - Pewarnaan

Konjungtivitis: - Penyebab tersering pada anak-anak adalah H. influenzae, S. pneumoniae, S. aureus. - Epidemi konjungtivitis:S. pneumoniae & H. influenzae.

- Kanalikulitis: infeksi pada kanalis lakrimalis

(Actinomyces israelii , Propionibacterium propionicum) Dakriosistitis: infeksi pada sakus lakrimalis dapat disebabkan bakteri & jamur Dakrioadenitisbakteri patogenik (S. aureus, Streptococcus sp)

Selulitis Orbita: - Definisi: infeksi pada jaringan di sekitar bola mata, infeksi akut, paling sering disebabkan bakteri. - Berpotensi serius menjalar ke posterior komplikasi pada SSP & sinus paranasal.

Penyebab selulitis orbita: - Bakteri: S. aureus, S. pyogenes , S. pneumoniae. H. influenzae . - Jamur: Mucormycosis.

Teknik pembedahan: implantasi lensa meningkatkan kejadian infeksi iatrogenik. (Propionibacterium acnes)

Hal yang perlu diperhatikan pd pengambilan BP: Dari lokasi infeksi yang tepat, dihindari dari kontaminasi. Saat pengambilan harus tepat. Jumlah BP harus cukup Alat-alat dan tempat pengambilan BP harus steril. Pengambilan BP: sebelum diberi antimikroba.

BP kerokan (scraping) untuk biakan organisme intraseluler diperiksa dalam 24-48 jam, (bakteri) dan 3-7 hari (virus). Harus dibiakan pada media yang sesuai sebelum pengambilan spesimen, kulit di sekitar mata harus dibersihkan terlebih dahulu. bedside culture

Pus konjungtiva diambil menggunakan aplikator kapas steril. BP dari kedua mata dibiakan secara terpisah. Pengambilan BP kerokan untuk deteksi C. trachomatis menggunakan aplikator kalsium alginat. Isolasi tidak segera dilakukanmedia Carry Blair, media transpor Stuart (dikirim ke lab. <4 jam,suhu kamar).

Menggunakan BP yang sangat sedikit. BPmengandung mikroorganisme yang mudah mati dibutuhkan media cair. Sudah memakai antibiotik topikal dibutuhkan media pengkaya Inokulasi bedside culture.

 Alat-alat

: bunsen, spatula Kimura, pisau bedah steril, kaca obyek, anesetsi topikal, media cair, media padat, aplikator steril kalsium alginat (untuk biakan bakteri), aplikator poliester atau kapas (untuk biakan virus)

- Inokulasi pada agar darah, agar coklat, Brucella Blood Agar. - Pus inokulasi pada agar darah, agar coklat, agar MacConkey, dan BHIB; & dibuat 3 buah apusan.

1. 2. 3. 4. 5.

Spatula Kimura dibakar dahulu. Meneteskan anestesi topikal. Digunakan bagian tumpul pisau bedah steril BP diambil dari kelopak mata atas & bawah. Diinokulasi pada agar darah, agar coklat, BBA, C. trachomatis sucrose phosphate broth , virus Hanks balanced salt solution. 6. Dibuat 3 atau 4 buah apusan (pewarnaan Gram, Giemsa, pewarnaan imunofluoresen (Chlamydiae).

1. Aplikator dibasahi HBSS.

2. BP diambil dari lower conjunctival fornix (dari medial ke lateral). 3. Aplikator dimasukkan ke dalam 1 ml HBSS. 4. Inokulasi agar darah dan agar coklat dengan 100 L HBSS. 5. Agar darah diinkubasi 1 malam (aerob, 37oC), agar coklat diinkubasi 10%CO2, 37oC. 6. Dilakukan hitung kuman, identifikasi bakteri, pemeriksaan sensitivitas antibiotik. 7. Pewarnaan Gram

1-10 CFU/100 L

10-50 CFU/100 L

50-100 CFU/100 >100 CFU/100 L L

S. pyogenes S. pneumoniae S. aureus Citrobacter sp. Enterobacter sp. E. coli Klebsiella sp. Proteus sp. Morganella sp. S. mercescens N. gonorrheae Neisseria sp. lain Moraxella sp. Acinetobacter sp. Achromobacter sp. Haemophilus sp. P. aeruginosa Pseudomonas sp. lain

Streptococcus GrupS. epidermidis Corynebacterium B ( hemolitik atauCONS sp. nonhemolitik). Micrococcus sp. Streptococcus GrupBacillus sp. C ( hemolitik, hemolitik atau nonhemolitik). Streptococcus sp. lain. Moraxella catarhalis

Media biakan

Suhu

Kondisi

Agar darah Brucella Blood Agar Agar coklat SDA Agar MacConkey BHIB Thioglycolate broth Lowenstein Jensen

35oC 35oC 35oC 25oC 35oC 25oC 35oC 35oC

Aerob Anaerob 5-10% CO2 Aerob Aerob Aerob Aerob Aerob

Cepat, sangat sensitif. Teknik pemeriksaan in vitro untuk replikasi atau ampifikasi DNA hanya dalam beberapa jam. 3 tahap: denaturasi, annealing, dan ekstensi. (satu siklus). PCR digunakan secara rutin untuk mendeteksi virus Herpes Simpleks, virus Varicella Zoster , adenovirus, C. trachomatis, dan M. tuberculosis

Media padat: agar darah, Brucella Blood Agar, agar MacConkey, SDA, agar coklat. Media cair: Brain Heart Infusion, BrothThioglycolate broth. Jika tidak ditemukan koloni bakteri setelah 48 jam 7 hari (25oC) untuk melihat infeksi jamur. Biakan bakteri anaerob s/d 2 minggu. Jika ditemukan koloni bakteri identifikasi kuman, sensitivitas terhadap antibiotik

Pewarnaan untuk apusan cairan vitreus dan aqueus: pewarnaan Gram, Giemsa, KOH, pewarnaan Gomaris Methanamine Silver , Calcoflour White. Sebelum diwarnai, apusan difiksasi dahulu dengan metanol 95% selama 5 menit.

TERIMA KASIH

Micrococcaceae Kingdom: Bacteria Phylum: Actinobacteria Class: Actinobacteria Subclass: Actinobacteridae Order: Actinomycetales Suborder: Micrococcineae Family: Micrococcaceae Genera Acaricomes Arthrobacter Citricoccus Kocuria Micrococcus Nesterenkonia Renibacterium Rothia Sinomonas Zhihengliuella

Streptococcaceae Scientific classification Kingdom: Division: Class: Order: Family: Bacteria Firmicutes Bacilli Lactobacillales Streptococcaceae Genera Lactococcus Lactovum Streptococcus

Deteksi antigen - BP: swab endoserviks, urine - + 30 mnt

-

The Chlamydia Rapid Test (Swab/Urine) is a rapid chromatographic immunoassay for the qualitative detection of Chlamydia trachomatis in female cervical swab, male urethral swab and male urine specimens to aid in the diagnosis of Chlamydia infection.

Female Swab Male Swab Sensitivity 88.5% Specificity 96.7% 78.4% 92.9%

Male Urine 90.9% >99.0%

Malnutrition Recurrent infections Immunosuppressin agents for organ transplant recipients Chemotherapy for cancer Genetic predisposition Skin damage Antibiotic treatment Medical procedures Pregnancy AIDS

Pneumocystis carinii Candida albicans Staphylococcus aureus Streptococcus pyogenes Pseudomonas aeruginosa Polyomavirus JC polyomavirus Acinetobacter baumanni Toxoplasma gondii Cytomegalovirus Aspergillus sp Kaposi's Sarcoma caused by Human herpesvirus 8 (HHV8) Cryptosporidium Cryptococcus neoformans Histoplasma capsulatum Clostridium difficile

Immature dendritic cells (iDC) are activated and matured by commensal bacteria, for example, lactic acid bacteria (LAB). These LAB-activated mature dendritic cells (DC) produce cytokines able to activate NK cell cytotoxicity and induce their proliferation. Activated NK cells can in turn, via the release of relevant cytokines, recruit (GM-CSF) and activate iDC (TNF- and IFN-). Alternatively, activated NK cells can exert an editing of DC by killing some of the iDC. At the same time, the early release of IFN- by NK cells interacting with LAB-activated DC, most likely in secondary lymphoid organs such as the mesenteric lymph nodes, is critical for shaping the following adaptive immune response toward a type 1 T cell response. Remarkably, some LAB display opposite outcomes and could hamper T cell type 1 polarization.

The classical mechanistic explanantion is illustrated above. Delayed type hypersensitivity results when an antigen presenting cell, typically a tissue dendritic cell which has picked up antigen, processed it and displayed appropriate peptide fragments bound to class II MHC is contacted by an antigen specific TH1 cell patrolling the tissue. The resulting activation of the T cell produces cytokines such as chemokines for macrophages, other T cells and, to a lesser extent, neutrophils as well as TNFbeta and IFNgamma. The consequences are a cellular infiltrate in which mononuclear cells (T cells and macrophages) tend to predominate. It is usually maximal in 48-72 hours.

Drug inactivation or modification: for example, enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of -lactamases. Alteration of target site: for example, alteration of PBPthe binding target site of penicillinsin MRSA and other penicillin-resistant bacteria. Alteration of metabolic pathway: for example, some sulfonamideresistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid. Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface.

The four most important antibiotic resistance mechanisms are alteration of the target site of the antibiotic, enzyme inactivation of the antibiotic, active transport of the antibiotic out of the bacterial cell, and decreased permeability of the bacterial cell wall to the antibiotic

Baird-Parker agar is a type of agar used for the selective isolation of gram-positive Staphylococci species. It contains lithium chloride and tellurite to inhibit the growth of alternative microbial flora, while the included pyruvate and glycine promote the growth of Staphylococci. Staphylococcus colonies show up black in colour with clear zones produced around them.

commonly used for specimen collection in pediatrics, public health microbiology, ophthalmology, food pathology Calcium alginate swabs contain no fatty acids that may be inhibitory to many strains of gonorrhea.

A type of blood agar plate in which the blood cells have been lysed by heating the cells to 56C. Chocolate agar is used for growing fastidious (fussy) respiratory bacteria, such as Haemophilus influenzae.

Brucella Blood Agar (BRU) is an enriched non-selective agar medium.Brucella Blood Agar is intended for the isolation, quantitation and partial identification of obligate anaerobic bacteria from clinical specimens.

Composition: Peptone - 17 g Proteose peptone - 3 g Lactose - 10 g Bile salts - 1.5 g Sodium chloride - 5 g Neutral red - 0.03 g Agar - 13.5 g Water - add to make 1 litre; adjust pH to 7.1 +/- 0.2

MacConkey agar (MAC)

-

The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective. Lactose and neutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies

Sabouraud agar is used to culture fungi and has a low pH that inhibits the growth of most bacteria; it also contains the antibiotic gentamicin to specifically inhibit the growth of Gram-negative bacteria.

Fungal media Sabouraud agar Sabouraud agar is used to culture fungi and has a low pH that inhibits the growth of most bacteria; it also contains the antibiotic gentamicin to specifically inhibit the growth of Gram-negative bacteria. Hay infusion agar Specific for the culturing of slime moulds (which are not fungi). Potato dextrose agar PDA is used to culture certain types of fungi. Malt extract agar Malt extract agar has a high content of peptone and is acidic. It is essentially used in the isolation of fungal microorganisms.

CFU/ mL = CFU/plate x dilution factor On the plate shown, milk was diluted 1 to 100 (10 2), 1.0 mL of the dilution was plated and 40 colonies formed. Therefore the count per mL in the milk was: 40 colonies x 102 x 1/1 = 4 x 103/mL

The glycogen, mucin, and fungi will be stained purple and the nuclei will be stained blue. Fixation: 10% formalin. Solutions and Reagents: 0.5% Periodic Acid Solution: Periodic acid ---------------------- 0.5 g Distilled water -------------------- 100 ml

Schiff Reagent: Test for Schiff reagent: Pour 10 ml of 37% formalin into a watch glass. To this add a few drops of the Schiff reagent to be tested. A good Schiff reagent will rapidly turn a redpurple color. A deteriorating schiff reagent will give a delayed reaction and the color produced will be a deep blue-purple. Mayers Hematoxylin Solution:

Procedure: 1. 2. 3. 4. 5. 6. 7. 8. Deparaffinize and hydrate to water. Oxidize in 0.5% periodic acid solution for 5 minutes. Rinse in distilled water. Place in Schiff reagent for 15 minutes (Sections become light pink color during this step). Wash in lukewarm tap water for 5 minutes (Immediately sections turn dark pink color). Counterstain in Mayer's hematoxylin for 1 minute. Wash in tap water for 5 minutes. Dehydrate and coverslip using a synthetic mounting medium.

Results: Glycogen, mucin and some basement membranes --- red/purple Fungi ------------------------ red/purple Background ------------------- blue

Silver staining is a common special staining technique used in medical laboratories. Gomori's Methenamine Silver (GMS) stain is used for fungi and bacteria. The fungi and bacteria are turned black, while everything else is stained green with Light green SF solution.

Shell vial cultures Following inoculation of the specimen, the shell vial was centrifuged at 700 g at room temperature followed by incubation at 36C for 1 h for adsorption. The inoculum was discarded and 1 ml of maintenance medium (MEM with 1 % foetal bovine serum) was added. The vial was incubated for 24 hrs at 36C in a CO2 incubator. The coverslip was removed, fixed in cold acetone for 30 minutes at -70C and stained by an indirect immunofluorescence assay (IFA) using a polyclonal antibody to HSV-1

Tube cultures Following inoculation of the specimen into a tube culture, the culture was incubated for 1 h at 36C for adsorption, the inoculum was removed and 1.5 ml of maintenance medium (MEM with 1% fetal bovine serum) was added. Cultures were incubated at 36C in a CO2 incubator for five days and observed for the presence of cytopathic effect (CPE) everyday. Cultures were terminated on the fifth day or as soon as CPE was observed (Fig. 2), whichever was earlier. Cells were scraped from the tube, washed in PBS, pH 7.2 and spotted onto a sterile glass slide. Smears were air dried, fixed in cold acetone for 30 minutes at -70C and stained by an indirect immunofluorescence assay (IFA) using a polyclonal antibody to HSV-1