MYCOBACTERIUM TUBERCULOSISCORYNEBACTERIUM DIPHTHERIAEDISAMPAIKAN OLEH:PROF. DR.dr. NOORHAMDANI AS, DMM, SpMK (K)KaLab/KaSMF MIKROBIOLOGI KLINIK FKUB/RSUD DR SAIFUL ANWAR (RSSA) MALANGKetua Tim Pencegahan dan Pengendalian Infeksi RSSAKetua Tim Program Pengendalian Resistensi Antimikroba RSSA

Types of MicroorganismsPrinciples of InfectionTransmissionHost resistanceVirulence and pathogenicityControl of transmission and infectionDevelopment of InfectionOnset and courseClinical signs and symptomsDiagnostic testsAntimicrobial Drugs

BacteriaVirusesChlamydiae, Rickettsiae, MycoplasmasFungiProtozoaParasites (not microorganisms)Helminths

CocciDiplocciStreptococciStaphylococciStaphylococcus aureusBacilliBacillus anthracis, Clostridium tetaniSpiralsBorrelia sp.Treponema pallidum

Respiratory tractGastrointestinal TractGenitourinary tractUnnatural routes opened up by breaks in mucous membranes or skinDifferent levels of host defense mechanisms are enlisted depending on the number of organisms entering and their virulence.

Extracellular BacteriaHumoral immune responseHumoral antibodies produced by plasma cells in regional lymph nodes and submucosa of respiratory and gastrointestinal tractsThe antibodies remove the bacteria and inactivate bacterial toxins to protect the host cell from invading organisms.

Extracellular Bacteria

Intracellular Bacteria

Antibody neutralizes bacterial toxinsComplement activationAntibody and complement split product C3b bind to bacteria, serving as opsonins to increase phagocytosis.C3a and C5a induce local mast cell degranulation Other complement split products are chemotactic for neutrophils and macrophages.

Intracellular BacteriaCell-mediated immune response (Delayed-type hypersensitivity) Activate Natural Killer (NK) cells provide early defense against bacteria.

In delayed type hypersensitivity, cytokines secreted by CD4+ T cells, such as IFN gamma, activate macrophages to kill ingested pathogens more effectively.

Four Steps in Bacterial InfectionAttachment to host cellsProliferationInvasion of host tissueToxin-induced damage to host cellMany bacteria have developed ways to overcome some of these host defense mechanisms

Disease can also be caused by the immune response to the pathogen.

Pathogen-stimulated overproduction of cytokines can lead to symptoms of bacterial septic shock, food poisoning, and toxic shock syndrome.

Bacteria that can survive intracellularly within infected cells can result in chronic antigenic activation of CD4+ T-cells, leading to tissue destruction by a cell-mediated response with characteristics of a delayed type hypersensitivity reactionCytokines secreted by CD4+ cells can accumulate, leading to the formation of granulomas. The concentrations of lysosomal enzymes in the granulomas can cause tissue necrosis.

Gram positive, rod-like organismBacterial disease caused by a secreted exotoxin.Spread via airborne respiratory dropletsExotoxin destroys underlying tissue, forming a tough, fibrous membrane compose of fibrin, white blood cells and dead respiratory cellsAlso responsible for systemic manifestations.

C.diphtheriae(Neisser stain)Loefflers serum agar slant> metachromatic granule> Club Shaped

Damage to different organs such as the heart, liver, kidneys and nervous system.Choking layer of bacteria and dead cells in the respiratory system, accompanied by an unworldly stenchDifficulty swallowing and breathingPus and blood discharge through nostrils following death from asphyxiation

The exotoxin is encoded by the tox gene carried by phage B (beta)Some strains can exist in the state of lysogeny.Exotoxin has two disulfide linked chains, a binding chain and a toxin chain. The binding chain interacts with ganglioside receptors on susceptible cells, facilitating internalization of the exotoxin. Inhibitory effect of toxin chain on protein synthesis leads to toxicity.Removal of the binding chain prevents exotoxin from entering the cell.

Elek test

Toxoid prepared by treating diphtheria toxin with formaldehyde.Reaction with formaldehyde cross-links the toxin, resulting in loss of toxicity and enhancement in its antigenicity.Usually administered with tetanus toxoid and inactivated Bordetelal pertussis in a combined vaccine that is given to children 6-8 weeks of age. Immunization with toxoid induces production of antibodies which bind to the toxin and neutralize its activity.

Intracellular PathogenLifelong InfectionPulmonary InfectionArrives at AlveoliPhagocytized by Alveolar Macrophages M. tuberculosis Blocks Phagosome From Fusing With lysosome (not nutrient containing vesicles, though)Other Phagocytes Are AttractedForms Multinucleated Giant Cells (Langhans Cells)

Bacilli shaped organismPulmonary infection by inhalation of small droplets of respiratory secretions containing a few bacilliInhaled bacilli are ingested by alveolar macrophages and multiply intracellulary by inhibiting formation of phagolysomes.Macrophages lyse and large numbers of bacilli are released. Cell mediated response by CD4+ T cells may be responsible for much of the tissue damage of the disease. Most common infection of tuberculosis is pulmonary tuberculosis.

X-ray : Pulmonary TB

Direct Smear : > Sputum > Ziehl-Neelsen Staining > Acid Fast Bacilli (AFB)

Cause tuberculosis, very important human pathogenThe WHO estimate there are 8 million new cases and 3 million deaths directly attributable to disease/year tuberculosis is the leading cause of death in many poor and developing countries.Factors:Drug abuseHIV AIDSMalnutritionPoor socio-economic Dirty environmentIndustrial area:air pollutionover populatedRobert Koch (1882): establish the cause of tuberculosis

M. tuberculosis; in tissue: thin straight rods, in artificial media: coccoid, filamentous.Size: 0.2 to 0.6 by 1.0 to 4.0mNon sporeformingNon motile, capsule (-)

The cell wall are complex, contain large amount of lipid that are composed of fraction A, B, C, and D.

Mycobacteria are difficult to stain (the cell wall has a very high lipid content)However after absorbing the dye, these organisms resist decolorization by acid alcoholAcid Fast Bacilli (AFB)

Staining Methodes:

1. Z.N. (Ziehl Neelsen) Basic stain: carbol fuchsin Counter stain: methylene blue red bacilli with blue background2. Kinyoun carbol fuchsin3. TTH (Tan Thiam Hok/ H.O.K.Tanzil)4. Auramine rhodamine (fluorescense dyes)5. Gram staining : Gram positive

ResistancePhysical effect :M.tuberculosis are highly resistant to dryingWhen exposed to direct sunlight, organisms from culture are killed within 2 hours, in sputum require an exposure for 20 30 hours. In dry sputum protected from direct sunlight: 6 8 months In dust (as droplets) remain infectious for 8 10 days

Resistance against chemical agents and antibiotic:Mycobacterium tuberculosis tend to be more resistant to chemical agent because of the hydrophobic nature of the cell surface (malachite green, penicilin incorporated into media without inhibiting the growth M.tuberculosis)Sensitive to :INHPyrazinamidStreptomycinRifampinResistance rapidly to PAS (para-aminosalicylic acid)

CultureFor growth, Mycobacterium need:Fatty acidAmino acidNitrogen compoundGlycerol as carbon sourceOptimum growth temperature: 35 370C.Aerobic atmosphere (M. tuberculosis obligate aerobe), grow better in atmosphere of 5 10 % CO2Incubation time : 1014 days , up to 4-8 weeks.For rapid growers: 7 days.

Media for primary culture: I.Solid mediaLowenstein-Jensen (L-J) medium-inspissated egg media agarKudoh agarPetragnani agarMiddlebrook 7H10 agar, 7H11-semisynthetic agar media

II.Liquid mediaDubos brothMiddlebrook 7H9 Middlebrook 7H12 Liquid media contain Tween 80 and albumin

Morphology of colonies on LJ mediumHuman strain:Non pigmented, roughNodular surface with irregular thin peripheryDry, creamy color

Bovine strain:Pyramid-shaped coloniesSmoothColorless

Can take 2 to 4 weeks to visualize coloniesLowenstein-Jensens media

Antigenic structureThe component of bacterial cell wall which have antigenic properties :PolysaccharidesPeptidesCell wall of Mycobacterium contain much lipid/wax hydrophobic properties

Difficult to be stained/ Impermeable to staining substances.Resistant to acid and alkali.Resist phagocytosis and intracellular killing mechanisms.Slow growth/long generation time

Lipid Fractions:Wax AWax BWax C (serpentine cord virulence)Wax D (delayed type hypersensitivity)

According to Seibert :M. tuberculosis composed of 5 fractions:4 fractions of protein A, B, C & D1 fraction of polysaccharides

Immunity:Cellular immunityDead M. tuberculosis gives low grade immunity Live attenuated M. tuberculosis (bovis) BCG vaccine gives better immunity

Virulence factors(Determinants of Pathogenicity)Produce serpentine cord correlated with the presence of glycolipid : trehalose-6,6- dimycolate.Produce enzyme catalase.Neutral red test (+).Mycobacterium do not produce toxin

CLINICAL MANIFESTATIONSPrimary TuberculosisInhaled bacilli alveoli and multiplies in peripheral lung tissue (tubercles, necrosis) break spread via blood flow and lymph miliary tuberculosisIngestion (food & beverage) mouth & tonsil enlarged of cervical lymph nodes cervical adenitis/scrofula intestines (mesenterial adenitis) peritonitisSkin direct contact ulceration adenitis of regional lymph nodes

*Pulmonary TB occurs in the lungs85% of all TB cases are pulmonary Extrapulmonary TB occurs in places other than the lungs, including the:LarynxLymph nodesBrain and spineKidneys Bones and jointsMiliary TB occurs when tubercle bacilli enter the bloodstream and are carried to all parts of the body

Reactivation Tuberculosis :Spreading focus - caseous necrosis from primary infectionUsually occurs in lung Reactivation type is characterized by: Chronic tissue lession Formation of tuberclesCaseationFibrosis

*Persons more likely to progress from LTBI to TB disease includeHIV infected personsThose with history of prior, untreated TBUnderweight or malnourished personsInjection drug useThose receiving TNF- antagonists for treatment of rheumatoid arthritis or Crohns diseaseCertain medical conditions

Symptoms of TB depend on where in the body the TB bacteria are growing. TB bacteria usually grow in the lungs. TB in the lungs may cause a bad cough that lasts longer than 2 weeks pain in the chest coughing up blood or sputum (phlegm from deep inside the lungs) Other symptoms of TB disease are weakness or fatigue weight loss no appetite chills fever sweating at night

Cytokines produced by CD4+ T cells activate macrophages, which kill the bacilli or inhibits their growth.High levels of interleukin-2 (IL-2), produced by macrophages, stimulates Th 1-mediated responses. IL-2 may also contribute to resistance by inducing production of chemokines that attract macrophages to the site of infection. CD4+ T cell mediated response mounted by those exposed to M. tuberculosis controls the infection and protects against later infection.

Tuberculosis is treated with several drugs including isoniazid, rifampin, streptomycin, pyrazinamide, and ethambutol.

Drug therapy must continue for at least 9 months to get rid of the bacteria since the intracellular growth of M. tuberculosis makes it difficult for the drugs to reach the bacilli.

Vaccine : attenuated strain of M. bovis called BCG (Bacillus Calmette-Guerin) Most effective against extrapulmonary tuberculosis.

Biofilms are multicellular aggregates of bacteria and yeast that congregate on surfaces.Biofilm may form on any surface exposed to biofilm-forming bacteria and some amount of water. Biofilms are formed to protect the bacteria from host defenses, antibiotics, and from harsh environmental conditions.

Bacterial biofilms can form almost anywhere, even on your teeth if you don't brush for a day or two.

When they accumulate in hard to reach places such as the insides of food processing machines or medical catheters however, they become persistent sources of infection.

Bacterial biofilms are often a cause of infections associated with medical implants such as catheters and IV lines and other medical devices. Biofilms are found almost everywhere in nature, including rivers, lakes, soil, water pipes, and even inside the human body.A common type of bacterial biofilm-responsible for plaque.

Due to the morphology of biofilms, bacteria capable of forming them are highly resistant to antibiotics, making treatment very difficult. In the US alone, one million nosocomial (hospital acquired) infections each year are caused by bacterial biofilms, leading to longer hospitalization, surgery, and even death.

Biofilm fouling of fiber filterPlacque on teethUU

The Power of MathematicsThe Excitement of Biology

BIOFILM DETECTION TISSUE CULTURE PLATE (TCP) METHOD

TUBE METHODCONGO RED AGAR METHOD

THANK YOU FOR YOUR ATTENTIONTERIMA KASIH

***Appendix 12 - Fundamentals of TB Presentation*Appendix 12 - Fundamentals of TB Presentation*