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Microbiology: the first two lectures

Starting a new subject with a brand new teacher, and for once, I think the teacher is more nervous than the students. I hope he calms down enough to finish his sentences, and wait for that flash of awareness or recognition before he moves on to the next slide. I was left behind for most of his lecture, which was scattered and incomplete. The notes, furthermore, were incomplete (lacking images) and wildly disorganized (repetitive but without logic to the order). Welcome to NCNM.

But this study of microbes is very important, so I need to keep up with it. I have never taken microbio before. The closest I've come is learning about all the causes of food poisoning as part of becoming a Coconino County (Arizona) approved Food Manager (certified to supervise Food Handlers). So I will begin the process of transcribing my notes and looking up everything that I didn't get on the first run through.

First, a quiz:
A. What was the leading cause of blindness prior to 1800?
B. When was the first report of Helicobacter pylori, the bacterium now known to cause stomach ulcers?
C. What was the first commercial antibiotic, what was it for, and when was it produced?
D. What is the ultimate microbial symbiosis (from the human perspective)?
E. What specific state of infection contradicts Koch’s 3rd postulate?
F. What specific person has historically been used to contradict Koch’s Postulate?

Answers:
A. gonorrhea
B. 1875
C. Salvarsan, syphillis, 1909
D. mitochondria
E. the asymptomatic carrier state of infection
F. Mary Mallon, aka Typhiod Mary

LECTURE 1

According to the World Health Organization (WHO), infectious Diseases account for 29 of the 96 major causes of Human morbidity and mortality

Immunologically naive population = large group of folks who have not been exposed to a particular antigen for many generations, and so have no immune recognition of it. Smallpox in the 1500's killed thousands in Mexico, Central and South America because one slave of an invading army had it. In the 1600's smallpox killed 90% of the Massachusetts Bay Indians. In the 1700's the Brits/French in N America distributed blankets infested wtih smallpox causing the death of some 30% of the population.

Global warming affects microbe distribution by changing vector ranges: Malaria incidence in Papua New Guinea in 2000 was 638, in 2005 was 4986. Mosquitoes are expanding their ranges! West Nile Virus in US: first detected in 1999 and now in 48 continental states, 7 Canadian provinces and throughout Mexico.

HISTORICAL THEORIES OF DISEASE

Miasmatic Theory of Disease (middle ages to mid 1800's): cholera and Black Deather were thought to be caused by MIASMA, bad air, ie the stench of rotting bodies, the smell of death, the stink of sickness, etc. Florence Nightingale was a proponent of this theory of disease. Now miasma is defined as unhealthy vapors rising from the ground or other sources, or an unwholesome atmosphere.

Germ Theory, lots of people contributed, dispelled the idea that disease was spontaneously generated, or a punishment from a god/the gods. Leewenhoek created a simple microscope and was the first to observe CELLS. Jenner (1749-1923) was the father of VACCINATION, injected milkmaids "cow pox" pus into healthy subjects who then recovered from smallpox, also studied cholera. Semmelweis (1840's) is credited with our penchant for handwashing. Koch had 4 postulates to establish that a certain microbe caused a certain disease. Pasteur figured out that diseases come from something, and made vaccines for rabies and anthrax. Lister is "father of modern antiseptics" incl: listerine. Genus listeria named for him. Iwanowski found viruses but didn't know what they were. Fleming accidentally discovers penicillin while trying to cure typhoid, gets Nobel Peace price in 1945 with some other folks help in the lab. Fleming often warned of the potential problem of "acquired resistance" of microbes to antibiotics--and he was RIGHT.

KOCH'S POSTULATES
1. microorg must be found in all orgs that have the disease and not in health orgs
2. microorg must be isolated and grown in a pure culture
3. cultured microorg must cause disease when instroduced into health org
4. microorg must be re-isolated from innoculated, diseased holst and identified as identical to original

PROBLEMS with KOCH'S POSTULATES
1. not all orgs that have the disease are sick: carriers
2. not all microorgs can be isolated and grown in a pure culture
3. microorg may mutate and cease to cause disease or cause some other disease
4. same as 3

LECTURE 2

Bacteria are prokaryotes, have no organelles, identified by gram stains, DNA AT/GC content, morphology and biochemical characteristics. Bacterial morphologies: coccus (Staphylococcus aurea, Streptococcus pyrogenes), bacillus (Bacillus anthracis, Salmonella typhimurium, Clostribium tetani, Clostribium botulinum), spirochete (syphillis & Lyme disease).

Bacteria are either gram positive or gram negative.

GRAM POSITIVE cell wall has thick peptidoglycan layer and teichoic acids, which are not found in gram negative cells.

GRAM NEGATIVE cell wall
--thin peptidoglycan layer outside cytoplasmic membrane, containing a network of NAM and NAG with NAM's crosslinking the chains. NAM = n-acetylglutamic acid, and NAG = n-acetyle muramic acid. Enzyme known as transpeptidase does the cross linking and penicillin interferes with it so cell bursts.
--There is a pleriplasmic space only in gram negative.
--Gram-negative bacteria are those bacteria that do not retain crystal violet dye in the Gram staining protocol.
--Many species of Gram-negative bacteria are pathogenic, meaning they can cause disease in a host organism. This pathogenic capability is usually associated with certain components of Gram-negative cell walls, in particular the lipopolysaccharide (also known as LPS or endotoxin) layer. In humans, LPS triggers an innate immune response characterized by cytokine production and immune system activation. Inflammation is a common result of cytokine production, which can also produce host toxicity.
--The proteobacteria are a major group of Gram-negative bacteria, including Escherichia coli, Salmonella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella and alpha-proteobacteria as Wolbachia and many others. Other notable groups of Gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria. Crenarchaeota: Unique because most bacteria have gram-positive molecules in their capsules, it has gram-negative.
--Medically relevant Gram-negative cocci include three organisms, which cause a sexually transmitted disease (Neisseria gonorrhoeae), a meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis).
--Medically relevant Gram-negative bacilli include a multitude of species. Some of them primarily cause respiratory problems (Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary problems (Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens), and primarily gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis, Salmonella typhi).
--Gram negative bacteria associated with nosocomial infections include Acinetobacter baumanii, which cause bacteremia, secondary meningitis, and ventilator-associated pneumonia in intensive care units of hospital establishments.

CAPSULE of bacterial cell is a thick mucous-like layer of monosaccharides linked to form a polysaccharide with a molecular weight of 100-1000 KDa. It acts as a cloak for the microbe, limiting host immune response, helping resist phagocytosis by host macrophages, prevent drying, assist in formation of biofilm, and gram positive capsule can be seen using india ink stain. EXCEPTION: bacillis anthrasis capsule has a different makeup, contains d-glutinate instead.
--good for making vaccine

india ink - nigrosin = a "negative" stain - polysaccharide capsule exludes the stain and appears as a clear halo surrounding the microbe against a dark background

QUELLING REACTION = to identify Streptococcus pneumoniae. If the rxn is positive the capsule becomes opaque and appears to enlarge (swelling).

Viruses are noncellular

Hemolysis was mentioned in class as a halo around the dot of a microbial colony on a nutrient agar plate....looking it up I find lots of other definitions:

hemolysis = Alteration, dissolution, or destruction of red blood cells in such a manner that hemoglobin is liberated into the medium in which the cells are suspended, e.g., by specific complement-fixing antibodies, toxins, various chemical agents, tonicity, alteration of temperature. Syn: hematolysis, erythrolysis, erythrocytolysis .

Also Haemolysis may be caused by bacterial haemolysins, by antibodies that cause complement dependent lysis, by placing red cells in a hypotonic solution or by defects in the red cell membrane..

Also: hemolysis (he·mol·y·sis) (he-mol´ə-sis) [hemo- + -lysis] disruption of the integrity of the erythrocyte membrane causing release of hemoglobin; it may be caused by bacterial hemolysins, by antibodies that cause complement-dependent lysis, by placing erythrocytes in a hypotonic solution, or by defects in the cell membrane. Called also erythrocytolysis, erythrolysis, and hematolysis.

STRAINS of microorgs, aka "isolates" share the vast majority of their observable characteristics

BIOTYPING uses biochemical tests to differentiate, such as: ability to ferment certain sugars, make H2S, grow in high salt, grow in high urea, obligate aerobes/anaerobes, facultative anaerobes, microaerophilic, obvlicgate intracellular microbes, etc. Problem with this method: it's slow.

SEROTYPING is fast, highly specific and allows identification of microbes that won't grow in culture. Identify the antibodies of the host (sero-conversion) by matrix with microbial antigen. Color = positive result.

PHAGE TYPING = infect bacteria wtih virus. Problem: technically cumbersome and spendy but doesn't yield a lot of info.

ANTIBIOTIC SENSITIVITY PATTERNS = Cover agar with colonies, drop on circles with different antibiotics, see where colony growth is inhibited, know what antibiotic works.

PCR = polymerase chain reaction. I wrote this up in a prior biochem post. Very fast, sensitive and highly specific, use to amplify sections of DNA.

The NAME of a microbe contains genus, species, strain and mutation info. For example:
Escherichia coli, 0157:H7 toxA- . If it says toxA- then it doesn't have the toxA mutation.

LIPOPOLYSACCHARIDE = LPS = ENDOTOXIN....?? O-specific side chains are variagle in length, shorter ones give "rough" mutants....short have capsule, long/smooth have no capsule, lipid A is the toxic component of endotoxin. The prototypical examples of endotoxin are lipopolysaccharide (LPS) or lipo-oligo-saccharide (LOS) found in the outer membrane of various Gram-negative bacteria.
From wikipedia:
--LPS is a major component of the outer membrane of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack. LPS is an endotoxin, and induces a strong response from normal animal immune systems. The only Gram-positive bacteria that possesses LPS is Listeria monocytogenes, the common infective agent in unpasteurized milk.
--LPS acts as the prototypical endotoxin, because it binds the CD14/TLR4/MD2 receptor complex, which promotes the secretion of pro-inflammatory cytokines in many cell types, but especially in macrophages. An "LPS challenge" in immunology is the exposing of the subject to an LPS which may act as a toxin.
--LPS also increases the negative charge of the cell membrane and helps stabilize the overall membrane structure.

LIPID A is a lipid component of an endotoxin held responsible for toxicity of Gram-negative bacteria. It is the innermost of the three regions of the lipopolysaccharide (LPS, also called endotoxin) molecule, and its hydrophobic nature allows it to anchor the LPS to the outer membrane. While its toxic effects can be damaging, the sensing of lipid A by the human immune system may also be critical for the onset of immune responses to Gram-negative infection, and for the subsequent successful fight against the infection.

NOSOCOMIAL infections = result from treatment in a hospital or a healthcare service unit, secondary to the patient's original condition. Infections are considered nosocomial if they first appear 48 hours or more after hospital admission or within 30 days after discharge. Nosocomial comes from the Greek word nosokomeion (νοσοκομείον) meaning hospital (nosos = disease, komeo = to take care of ). This type of infection is also known as a hospital-acquired infection. The most common nosocomial infections are of the urinary tract, and various pneumonias.

EXCEPTIONS TO GRAM NEGATIVE/POSITIVE DIVISION:
1. mycobacteria (has hard "waxy" surface, slow growing, hard to kill, use acid fast stain)
2. spirochetes (a phylum of gram negative bacteria but dark field microscopy used) (eg: Treponema pallidum, Borrelia burgdorferi)
3. mycoplasma (a genus of bacteria that has no cell wall only membrane, difficult to treat with antibiotics, eg: M. pneumoniae)

ACID FAST:
--exception #1 to gram neg/pos categories
--eg: Mycobacterium tuberculosis (consumption), mycobacterium leprae
--use Ziehl Neelsen Stain
--Acid-fastness is a physical property of some bacteria referring to their resistance to decolorization by acids during staining procedures.
--Acid-fast organisms are difficult to characterize using standard microbiological techniques (e.g. Gram staining), though they can be stained using concentrated dyes, particularly when the staining process is combined with heat. Once stained, these organisms resist the dilute acid and/or ethanol-based de-colorization procedures common in many staining protocols—hence the name acid-fast.
--The high mycolic acid content of certain bacterial cell walls, like those of Mycobacterium, is responsible for the staining pattern of poor absorption followed by high retention. The most common staining technique used to identify acid-fast bacteria is the Ziehl-Neelsen stain, in which the bacteria are stained bright red and stand out clearly against a blue background.
--Acid-fast bacteria can also be visualized by fluorescence microscopy using specific fluorescent dyes (auramine-rhodamine stain, for example). Some bacteria may also be partially acid-fast.

DARK FIELD MICROSCOPY: Dark field microscopy (Dark Ground Microscopy) is an optical microscopy illumination technique used to enhance the contrast in unstained samples. It works on the principle of illuminating the sample with light that will not be collected by the objective lens, so not form part of the image. This produces the classic appearance of a dark, almost black, background with bright objects on it.

MYCOPLASMA
-has no cell wall only membrane
-difficult to treat with antibiotics
-eg: M. Pneumoniae
--Mycoplasma is a genus of bacteria that lack a cell wall. Because they lack a cell wall, they are unaffected by some antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. They can be parasitic or saprophytic. Several species are pathogenic in humans, including M. pneumoniae, which is an important cause of atypical pneumonia and other respiratory disorders, and M. genitalium, which is believed to be involved in pelvic inflammatory diseases. They may cause or contribute to some cancers.
--The genus Mycoplasma is one of several genera within the class Mollicutes. Mollicutes are bacteria which have small genomes, lack a cell wall and have low a GC-content (18-40 mol%). --There are over 100 recognized species of the genus Mycoplasma. Their genome size ranges from 0.58 - 1.38 megabase-pairs. Mollicutes are parasites or commensals of humans, animals (including insects), and plants; the genus Mycoplasma is by definition restricted to vertebrate hosts. Cholesterol is required for the growth of species of the genus Mycoplasma as well as certain other genera of mollicutes. Their optimum growth temperature is often the temperature of their host if warmbodied (e.g. 37 degrees Celsius in humans) or ambient temperature if the host is unable to regulate its own internal temperature. Analysis of 16S ribosomal RNA sequences as well as gene content strongly suggest that the mollicutes, including the mycoplasmas, are closely related to either the Lactobacillus or the Clostridium branch of the phylogenetic tree (Firmicutes sensu stricto).
--Mycoplasmas are often found in research laboratories as contaminants in cell culture. Mycoplasmal cell culture contamination occurs due to careless working or contaminated cell culture medium ingredients. The Mycoplasma cell is usually smaller than 1 µm and they are therefore difficult to detect with a conventional microscope. Their presence in cell cultures or media can skew experimental results.

SPIROCHETES
--Spirochaetes is a phylum of distinctive Gram-negative bacteria, which have long, helically coiled cells. Spirochetes are chemoheterotrophic in nature, with lengths between 5 and 250 µm and diameters around 0.1-0.6 µm.[citation needed]
--Spirochaetes are distinguished from other bacterial phyla by the location of their flagella, sometimes called axial filaments, which run lengthwise between the cell membrane and outer membrane. These cause a twisting motion which allows the spirochaete to move about.
--The spirochaetes are divided into three families (Brachyspiraceae, Leptospiraceae, and Spirochaetaceae), all placed within a single order (Spirochaetales). Disease-causing members of this phylum include the following:
Leptospira species, which causes leptospirosis
Borrelia burgdorferi, which causes Lyme disease
Borrelia recurrentis, which causes relapsing fever
Treponema pallidum, which causes syphilis
Most spirochaetes are free-living and anaerobic, but there are numerous exceptions, including the above.

LYSOZYME is in mucosal surfaces and can digest peptidoglycans which are thinner in gram negative bacteria.

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