MicroPara MIDTERMS

MicroPara MIDTERMS

MICROBIOLOGY & PARASITOLOGY What is microbiology? MICROBIOLOGY mikros "small or slight" bios "life" logia "study of" is the scientific study of microorganisms THE SCIENTIFIC METHOD Observation Question Hypothesis Experiment Result Conclusion MICROBIOLOGY Microorganisms are agents of infectious diseases They are ubiquitous in nature and in/on human body Most microorganisms harmless for humans Microorganisms are divided into: Bacteria (Bacteriology) Fungi (Mycology) Viruses (Virology) Prions (neurodegenerative disease) Parasites (Parasitology) HISTORY OF MICROBIOLOGY Most organisms are harmless. 99% are good bacteria 1% are bad bacteria ERAS OF MICROBIOLOGY DISCOVERY ERA TRANSITION ERA GOLDEN ERA MODERN ERA DISCOVERY ERA: SPONTANEOUS GENERATION ARISTOTLE: believed that living organisms could develop from non-living things ROGER BACON: described that the disease caused by a minute "seed" or "germ" A.V. Leeuwenhoek: Father of Bacteriology and Protozoology; description of protozoa (yeast and algae); described bacteria and protozoa as "animalcules" Sedillot (1978): termed "microbe" TRANSITION ERA Francesco Redi (1626 - 1697): showed that maggots would not rise from decaying meat (when covered). John Needham (1713 - 1781): supports spontaneous generation theory; proposed that animalcules arose spontaneously on the mutton gravy (broth); covered the flasks with cork (inspired by Redi), still microbes appeared Lazzaro Spallanzani (1729 - 1799): demonstrated that air carried germs to the culture medium; showed that boiled broth would not give rise to microscopic forms of life GOLDEN ERA Louis Pasteur: Father of Medical Microbiology; the “swan neck” shaped tube; in 1987, Pasteur suggested, mild heating at 62°C (145°F) for 30 mins. was enough rather than boiling to destroy undesirable organisms without ruining the taste of the product is called – PASTEURIZATION. Contributions of Louis Pasteur: He invented the processes of pasteurization, fermentation and the development of effective vaccines (rabies and anthrax). Pasteur demonstrated diseases of silkworm was due to a protozoan parasite. He coined the term “microbiology”, aerobic, anaerobic. He disproved the theory of spontaneous germination. He demonstrated that anthrax was caused by bacteria and also produced the vaccine for the disease. He developed live attenuated vaccine for the disease. John Tyndall (1820 - 1893): He discovered highly resistant bacterial structure, later known as endospore. Prolonged boiling or intermittent heating was necessary to kill these spores, to make the infusion completely sterilized, a process known as Tyndallisation. Lord Joseph Lister (1827-1912): He is the father of antiseptic surgery. Lister concluded that wound infections too were due to microorganisms. He also devised a method to destroy microorganisms in the operation theatre by spraying a fine mist of carbolic acid into the air. Robert Koch (1893-1910): He demonstrated the role of bacteria in causing disease. He perfected the technique of isolating bacteria in pure culture. Robert Koch used gelatin to prepare solid media but it was not an ideal because (i) Since gelatin is a protein, it is digested by many bacteria capable of producing a proteolytic exoenzyme gelatinase that hydrolyses the protein to amino acids. (ii) It melts when the temperature rises above 25°C. Koch's postulates: Postulate 1: The same microorganisms are present in every case of the disease. Postulate 2: The microorganisms are isolated from the tissues of a dead animal, and a pure culture is prepared. Postulate 3: Microorganisms from the pure culture are inoculated into a healthy, susceptible animal. The disease is reproduced. Postulate 4: The identical micro-organisms are isolated and recultivated from the tissue specimens of the experimental animal. Fanne Eilshemius Hesse (1850 - 1934): One of Koch's assistant first proposed the use of agar in culture media. It was not attacked by most bacteria. Agar is better than gelatin because of its higher melting pointing (96°c) and solidifying (40 – 45°c) points. Richard Petri (1887): He developed the Petri dish (plate), a container used for solid culture media. Edward Jenner (1749-1823): First to prevent small pox. He discovered the technique of vaccination. Alexander Flemming: He discovered the penicillin from penicillium notatum that destroy several pathogenic bacteria. Paul Erlich (1920): He discovered the treatment of syphilis by using arsenic. He Studied toxins and antitoxins in quantitative terms & laid foundation of biological standardization. IMPORTANT DISCOVERIES Bacteria: Hansen (1874) – Leprosy bacillus Neisser (1879) – Gonococcus Ogston (1881) – Staphylococcus Loeffler (1884) – Diphtheria bacillus Roux and Yersin – Diphtheria toxin Viruses: Beijerinck (1898) - Coined the term Virus for filterable infectious agents. Pasteur developed Rabies vaccine. GoodPasteur - Cultivation of viruses on chick embryos. Charles Chamberland, one of Pasteur’s associates constructed a porcelain bacterial filter. Twort and d’Herelle - Bacteriophages. Edward Jenner - Vaccination for Smallpox. MODERN ERA: Nobel Laureates 1901: Von behring - Dipth antitox 1902: Ronald Ross - Malaria 1905: Robert koch - Tb 1908: Metchnikoff - Phagocytosis 1945: Flemming - Penicillin 1962: Watson, Crick - Structur DNA 1968: Holley, Khorana - Genetic code 1997: Pruisner - Prions 2002: Brenner, Hervitz - Genetic regulation of organ development & cell death Bacteria Morphology & Shape Morphological Classification I. True Bacteria Cocci – spherical or an oval in shape a. Monococci – single (e.g., Monococcus spp.) (Micrococcus); Bacteria: Micrococcus flavus; Common Disease: Bacteremia - bloodstream infection b. Diplococci – in pairs; Bacteria: Streptococcus pneumoniae; Common Disease: Pneumonia – inflammation and fluid in your lungs, Meningitis – infection in brain and spinal cord c. Staphylococci – grape like cluster; Bacteria: Staphylococcus aureus; Common Disease: Boils and Furuncles – pus-filled bumps d. Streptococci – in chains; Bacteria: Streptococci pyogenes; Common Disease: Strep throat (pharyngitis)– common sore throat infection e. Tetrad – in group of four; Bacteria: Micrococcus species; Common Disease: Endocarditis – inflammation of the inner lining of the heart chambers and valves f. Sarcina – in group of eight; Bacteria: Micrococcus luteus; Common Disease: Endocarditis, Bacteremia II. Bacilli – rod shaped bacteria a. Bacillus – Latin word “stick”; Bacteria: Bacillus; Common Disease: Tuberculosis, Pneumonia b. Coccobacilli – spherical and rod-shape; Bacteria: coccobacilli; Common Disease: Whooping cough, Pneumonia c. spore-former – survive harsh environmental conditions d. diplobacillus – do not separate after cell division; Bacteria: Coxiella burnetii; Common Disease: Q Fever - caused by infected animals (cow, sheep, goat) e. palisades – V-shaped (Chinese letter); Bacteria: Corynebacterium diphtheriae; Common Disease: Diphtheria – infection in mucous membranes (nose and throat) f. Streptobacillus – non-motile; Bacteria: Leptotrichiceae; Common Disease: Leptospirosis III. Spirochetes – relatively long, slender, non-branched microorganisms or spiral shaped having several coils. IV. Mycoplasma – lacks rigid wall and are highly pleomorphic and of indefinite shape – occur in round/oval bodies and on interlacing filaments V. Rickettsia & Chlamydia – very small, obligate parasites (were closely related to viruses but now regarded as bacteria) Individual bacteria can assume one of three basic shapes: Bacteria that do not separate from one another after cell division form characteristic CLUSTERS that are helpful in their identification. Examples: Cocci (diplococci) Streptococcus pneumoniae (pneumonia) Neisseria gonorrhoeae (gonorrhea) FUNGI Unicellular (yeasts) or multicellular (moulds) Reproduce asexually (conidia) and sexually (spores*) Ubiquitous in nature some are parts of human normal flora Most opportunistic pathogens Cause severe infections in immunocompromised host Fungi Candida albicans (yeast) Candida glabrata (yeast) Candida parapsilosis (yeast) Habitat Environment, mucosa Environment, mucosa Environment, mucosa Survival on dry surfaces 1-120 days 120-150 days 14 days Spread in HC Contact, endogenous Contact, endogenous Contact, endogenous HAIS Various Various Various Specimens Various Various Various Prevention Clean hands, equipment Clean hands, equipment Clean hands, equipment Fungi Aspergillus species (mould) Mucor (mould) Rhizopus (mould) Habitat Environment, air Environment Environment Survival on dry surfaces Conidia and spores are resistant Conidia and spores are resistant Conidia and spores are resistant Spread in HC Inhalation, (contact) Inhalation Inhalation HAIS Various Various Various Specimens Various Various Various Prevention Safe water, air, reverse/protective isolation Safe food, reverse/protective isolation Safe food, reverse/protective isolation Survival is better if conditions are humid for most organisms, if microorganism is in biological material (blood, faeces, wound exudate), if the temperature is lower, and if bacteria are in higher numbers. Whenever indirect contact is involved, it is most frequently by hands of healthcare workers. VIRUS I Smallest infectious agents Require living cell for reproduction bacterial, plant or animal Consist of either DNA or RNA and a protein coat Some have also an outside lipid envelope VIRUS II Entering the cell, virus makes the cell synthesise its nucleic acid and proteins The cell is severely damaged or destroyed and infectious disease develops How a Virus Works The virus enters the cell body releasing RNA. Virus RNA invades the cell nucleus and takes over. Viral RNA uses the host cell to create new RNA and assemble more viral particles. New viral particles are released, sometimes destroying the cell in the process. Virus Adenovirus Coronavirus, including SARS Coxackie B virus Cytomegalovirus Habitat / Survival on dry surfaces Water, fomites, environment; 7 days - 3 months Humans; 3 hours (SARS virus: 72-96 hours) Humans; >2 weeks Humans; 8 hours Spread in HC Contact Droplet Faecal-oral; contact Blood products, tissue and organs HAIS Eye, respiratory infections Respiratory infections Generalised disease of newborn Various Specimens Serum sample Serum sample Serum sample Serum sample Prevention Individual eye drops Isolation, clean hands, environment Clean hands, environment Safe blood products, tissues/organs for transplantation Virus Human immunodeficiency virus Influenza Norovirus Respiratory syncytial virus Habitat Humans Humans Humans Humans Survival on dry surfaces >7 days 1-2 days 8 hours - 7 days Up to 6 hours Spread in HC Blood, body fluids, tissue, organs for transplant Droplets, contact Faecal-oral, contact Droplets, contact HAIS Acquired immune deficiency syndrome Influenza Diarrhoea Acute respiratory infections Specimens Serum sample Serum sample Serum sample NP exudate Prevention Safe blood products and tissues/organs for transplant Isolation, vaccination Clean hands, environment, safe food Isolation, clean hands, environment Virus Rotavirus Rubula virus (mumps) Rubivirus (rubella) Habitat Humans Humans Humans Survival on dry surfaces 6-60 days Spread in HC Faecal-oral, contact Droplets Droplets HAIS Diarrhoea Mumps (parotitis) Rubella (German measles) Specimens Stool Serum sample Serum sample Prevention Clean hands, environment Isolation, vaccination Isolation, vaccination Virus Morbillivirus (measles) Varicella-zoster virus Habitat Humans Humans Spread in HC Droplets Droplets, close contact HAIS Measles Varicella Specimens Serum sample Serum sample Prevention Isolation, vaccination Isolation, vaccination Prions Prions are proteinaceous particles Do not contain any nucleic acid Connected to several severe neurologic diseases Highly resistant to usual disinfection and sterilisation methods Possibility of iatrogenic transmission Through transplantation Through instruments contaminated with brain tissue, (dura) or cerebrospinal fluid of infected person The most important disease is Creutzfeldt-Jakob disease (familial spongiform encephalopathy that is hereditary disease) and variant Creutzfeldt-Jakob disease (bovine spongiform encephalopathy that can be transmitted from cattle to humans) and some other diseases. Parasites Protozoa are a diverse group of unicellular eukaryotic organisms, many of which are motile. Arthropods that can cause infections and also HAIs: Sarcoptes scabies that causes scabies. Lice also can be spread among hospitalised patients. Humans can be infested with parasitic worms that derive all their nutrients from their hosts. Most of these worms live in the intestines for at least part of their life cycle. Worm infestations are long-term diseases that produce few symptoms in their early stages. Parasite Cryptosporidium (protozoa) Plasmodium species (protozoa) Trichomonas vaginalis (protozoa) Enterobius vermicularis (helminth) Habitat Liver, erythrocytes Vaginal mucosa Intestinal tract Survival on dry surfaces 2 hours on dry surface Several hours in humid environment; Eggs: at least 1 year Spread in HC Faecal-oral Mosquito-borne; infected blood Contaminated equipment in gynaecology HAIs Malaria Vaginal infection Enterobiasis Specimens Blood Vaginal discharge Perianal tape Prevention Clean environment, clean hands Safe blood products Disinfected/sterilised equipment in gynaecology INTRODUCTION: Role of microbiology laboratory The diagnosis of infections performed by the laboratory has two important functions: Clinical: Diagnosis of infection in an individual patient for everyday management of infections Epidemiological: Support for infection prevention and control in searching for source and route of transmission of HAI Clinical and epidemiological role of the microbiology laboratory are very much connected: knowledge of an infective microbe in a patient helps find its source and route of transmission and prevent the spread to other patients. Minimal requirements for microbiology services - 1 Set up inside the facility; If not possible, negotiate a contract for diagnostic microbiology with the nearest laboratory Available every day, including Sundays and holidays (ideally on a 24-hour basis) Able to examine blood, cerebrospinal fluid, urine, stool, wound exudate or swab, respiratory secretions, and perform basic serological tests (HIV, HBV, HCV) Minimal requirements for microbiology services - 2 4. Identify common bacteria and fungi to species level 5. Perform susceptibility testing using disc-diffusion methodology 6. Perform basic phenotyping * Serotyping Salmonellae, Shigellae, P. aeruginosa, N. meningitidis * Biotyping S. typhi Clinical role: Diagnosis of infection Diagnosis should be rapid and accurate to the species level wherever possible: Role in prevention and control of healthcare associated infections: Outbreak investigation Surveillance of HAIs Alert microorganisms' reports Designing antibiotic policy Additional test during an outbreak: Sometimes the IP&C Team requires additional data to clarify endemic or epidemic situations. Microbiological tests may be required for blood products, environmental surfaces, disinfectants and antiseptics, air, water, hands of personnel, and anterior nares of personnel. HAI surveillance: The microbiology laboratory should produce routine reports of bacterial isolates—allows the IPC& Team to make graphs for specific pathogens, wards, and groups of patients. A ‘baseline incidence’ can be established; any new isolate can then be compared with this incidence. If the laboratory is computerised, these data can be made readily available. Alert organism reports: Identify possible agreed ‘alert’ microorganisms: Antibiotic policy: Antibiotic stewardship: Role of Clinical Microbiologist/ID specialist Interpretation of Microbiology data: Microbiologists interpret microbiological data for IP&C staff Results of isolation, identification, susceptibility tests, typing Ideally should be medical doctor specialist; if not possible, a properly educated scientist is required Role in education: Key points 1: Microbes are infectious agents not visible to the naked eye Widespread in nature and some cause human disease Diagnosis of infection by the microbiology laboratory has two important functions: Clinical and Epidemiological The laboratory should determine the most frequent microbes causing infections, including HAI pathogens Key points 2: The laboratory should perform basic typing of microorganisms The laboratory should produce routine reports for IP&C personnel to make incidence graphs for specific pathogens, wards, and groups of patients Medical microbiologists interpret microbiological findings for IP&C personnel and act together with clinical and nursing colleagues in prevention of HAI METHODS OF CULTURING MICROORGANISMS COLLEGE OF NURSING SPECIMEN COLLECTION PROCEDURES Should include instructions for: FIVE BASIC TECHNIQUES INOCULATE INCUBATE ISOLATION INSPECTION IDENTIFICATION INOCULATION The implantation of microorganisms into or onto the culture media. Culture: Propagations of microorganisms with various media Media: Nutrient used to grow microorganisms outside its natural habitat. Tools: Inoculating Loops and Needles INCUBATION ISOLATION Based on the concept that if an individual cell is separated from other cells on a nutrient surface, it will form a colony COLONY: A macroscopic cluster of cells appearing on a solid medium arising from the multiplication of a single cell. Three Basic Methods of Isolating Bacteria: Streak Plate Loop Dilution Spread Plate INSPECTION Colony morphology is a method that scientists use to describe the characteristics of an individual colony of bacteria growing on agar in a Petri dish. It can be used to help identify them. Colonies differ in their shape, size, colour and texture. IDENTIFICATION Bacteria: Each distinct circular colony should represent an individual bacterial cell or group that has divided repeatedly. Being kept in one place, the resulting cells have accumulated to form a visible patch. Most bacterial colonies appear white, cream, or yellow in color, and fairly circular in shape. Interpreting Plates: Bacteria grow tremendously fast when supplied with an abundance of nutrients. Different types of bacteria will produce different-looking colonies. Colony Morphology: The characteristics of a colony (shape, size, pigmentation, etc.). Basic elements to identify: Form: The basic shape of the colony (e.g., circular, filamentous). Elevation: The cross-sectional shape of the colony. Margin: The magnified shape of the edge of the colony. Surface: How the surface appears (e.g., smooth, glistening, rough, dull, rugose/wrinkled). Opacity: (e.g., transparent, opaque, translucent, iridescent). Chromogenesis (pigmentation): (e.g., white, buff, red, purple). A single visible colony represents a pure culture or single type of bacterium isolated from a mixed culture. Yeasts: Yeast colonies generally look similar to bacterial colonies. Some species, such as Candida, can grow as white patches with a glossy surface. Molds: Fungi that often appear whitish grey with fuzzy edges. They usually turn into a different color from the center outwards. MEDIA Classified according to three properties: Physical state, Chemical composition, and Functional types. Physical State: Chemical Content: Synthetic media: Chemically defined media. Non-synthetic or Complex media: Contain ingredients that are not chemically defined or pure (e.g., animal extract). MICROSCOPY Fluorescent Microscopy: Uses fluorescent staining. In fresh cheek scrapings: Fuzzy cells are cheek cells; distinct rods and cocci are bacteria (Red=dead, Green=live). Confocal Microscope. Transmission Electron Microscopy (TEM). Scanning Electron Microscopy (SEM). PREPARATION TECHNIQUES Wet Mounts Smears Stains Comparison of positive and negative stains.