Mycobacterium & Bioterrorism PDF

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Summary

Mycobacterium- Slender, curved, rod shaped and non-motile. - Has a cell-wall containing a high amount of lipids that resists staining. - Acid-fast bacilli that resist decolourizers and disinfectants. - They are BSL 3 organisms with aerosol modes of transmission. They are strict aerobes, slow growing...

Description

MLSC-3131U, Clinical Microbiology II Mycobacterium -

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Slender, curved, rod shaped and non-motile. Has a cell-wall containing a high amount of lipids that resists staining. Acid-fast bacilli that resist decolourizers and disinfectants. They are BSL 3 organisms with aerosol modes of transmission. They are strict aerobes, slow growing and require complex media. Mycobacterium tuberculosis, causes the TB infection. When infected with TB, people may be either reactive or non-reactive. Immune response depends on the amount of exposure and strain virulence. Raised, dry, non-pigmented, buff coloured. LowensteinJensen media at 35 degrees. Mycobacterium leprae, causes Hansen disease (leprosy) which is an infection of the skin, mucous membrane and peripheral nerves. It is not highly contagious. Worldwide incidence of leprosy is decreasing and is spread by person to person contact or by inhalation of aerosols. A person must be genetically predisposed to getting leprosy to contract the infection. It cannot be grown in the lab, but it can be stained with an acid-fast stain. Diagnosis done through staining, microscopy and clinical symptoms (loss of sensation, thickening of peripheral nerves). The best sample for mycobacterium is sputum. Sputum contains an abundance of nonmycobacterial organisms which overgrow slow growing mycobacterium. However, the sample is processed before plating to remedy this. Processing is done with NaOH to decontaminate the sample, liquefy the mucous, and kill non-mycobacterial organisms.

Staining -

The lipid rich cell walls of mycobacterium cause them to be stain resistant to basic dyes. Phenol is used in special stains for mycobacterium because it works on their cell walls. Once stained with phenol stain, it can be acid-fast stained. This is a rapid, presumptive, and inexpensive ID tool. The sputum is placed on a slide, dried and heat fixed for staining.

Ziel-Neelson Stain -

Primary stain with carbolfuchsin ZH-Heat, and K-Cold. Decolourized with acid-alcohol-HCl 3% and uses methylene blue for a counterstain. Only acid-fast bacteria retain the first stain (red) all the rest appear the colour of the counterstain.

Fluorescent Stain -

Primary stain with Auramine O that does not give it a colour. Instead, it makes it fluorescent. Decolourized with 0.5M HCl for 1 minute.

MLSC-3131U, Clinical Microbiology II -

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Counterstained with a potassium permanganate quencher for 1 minute. Quenching serves to quench the fluorescent reaction so that non-specific fluorescence is omitted. Over quenching will quench the acid-fast bacilli fluorescence. Acid-fast bacteria will fluoresce yellow. The smear can be examined at a lower magnification for less time. Uses a fluorescent microscope.

Trouble Shooting Staining Methods -

Insufficient washing of Auramine stain will result in a lot of non-specific fluorescence. Insufficient decolourization from thick sputum slides or mistiming can result in all pink organisms. Insufficient quenching causes non-specific fluorescence (false positive) and over quenching results in mycobacterium losing fluorescence (false negatives). Slides should be read all over to properly find TB.

Cultivation of Mycobacteria -

Egg Based Media, Lowenstein-Jensen malachite green is a media that will use malachite green to supress gram positive organisms. Agar-based Media, includes Middlebrook which is useful in that it is clear compared to the coloured Egg-Based media and has more defined ingredients.

ID Approach to Mycobacterium -

Chromatography, allows for species-specific mycolic acid profiles. Nucleic Acid Hybridization Probes, available for M. tuberculosis. PCR-Based Assays, very specific. Sequencing of 16s rRNA Genes

Bioterrorism -

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The use of biological agents to harm humans, animals, or crops. In 2001, anthrax-contaminated letters were sent in the US. 22 cases of anthrax were identified with 11 being from inhalation and 11 being cutaneous. 6 of the inhalation infected people died. The anthrax incident caused healthcare workers to revaluate the history and use of bioterrorism to prevent future tragedies. Healthcare professionals should be able to recognize and report possible biological weapon effects to the appropriate authorities to quickly divert a large-scale disaster.

MLSC-3131U, Clinical Microbiology II Biosafety Containment Levels -

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CL1, do not normally cause disease (Bacillus subtilis). CL2, known to cause disease (Hep B, Salmonella) CL3, transmitted by respiratory route and serious disease (Bacillus Anthrax) CL4, transmitted by respiratory route, cause serious disease, and have no available treatment (Ebola). The most likely bioterrorism organisms are CL3 or higher. Include Bacillus anthracis, Francisella tularemia, Yersinia pestis and Brucella. The microbiology department may be involved in a bioterrorism case for: o Specimen collection for suspected biological agents. o Specimen processing and presumptive ID of possible biological agents. o Shipping suspected agents to a public health lab of CL3 or higher. Lab technologists must be able to recognized CL3 organisms so that they can stop working on them as soon as possible and ship them to a CL3 laboratory for further analysis. All organisms suspected of being Burkholderia pseudomallei, Bacillus anthracis, Francisella tularemia, Yersinia pestis or Brucella should be worked on in the BSC. As soon as you suspect a CL3 organism it is to be shipped out to a qualified facility for further work. These organisms are usually suspected by the time a gram stain is done. The ministry of health must be notified immediately of any suspected case of high-risk pathogens. Prior to sending any specimen out a Medical Microbiologist should be notified. Specimens suspected of being a CL4 agent are forwarded to NML in Winnipeg.

Bacillus anthracis -

Infection happens through inhalation or spore ingestion. Aerobic GPB and may have central/subterminal spores. Non-hemolytic, catalase positive and non-motile. Must be sent to the public health lab.

Yersinia pestis -

Bipolar staining GNB. Slow growing, non-hemolytic, tiny and have a fried-egg look on BAP. Tiny NLFs of MAC agar and the organism is biochemically inert. Caused the Bubonic plague.

Francisella tularensis -

Very short GNBS that are pleomorphic and poorly staining. NG on MAC agar and hunters, rabbits and ticks are its most common reservoir. Catalase positive (weak) and oxidase negative.

MLSC-3131U, Clinical Microbiology II Brucella -

GNB that is small, glistening, non-hemolytic and non-pigmented after 2-3 days of incubation. Shows poor growth on MAC agar and is oxidase positive....