Microbiology 1

2D Electrophoresis separates proteins of microbial cells by pH and protein size.

2 D Electrophoresis separates proteins of a sample in two steps. First, proteins get separated by application of an electric current to a gel that has a pH gradient, which separates the proteins according to their isoelectric point. Then this gel is loaded onto an SDS PAGE gel, and current again is applied. This then separates the proteins also according to size, and the individual proteins can be visualized using various stains as shown on the right.

A-B toxins are common toxins produced by pathogens.

A common type of toxin produced by bacteria are the so-called A-B toxins. These consist of two proteins that are linked together and work together to cause toxic effects, usually with one protein binding the cell membrane, and the second protein causing some intracellular effect as shown here in these examples. This could be blockage of ribosomal translation as in the case of the toxin produced by Clostridium diphteriae bacteria, or to produce increased intracellular cAMP levels and opening of ion channels as caused by the toxin of Vibro cholera bacteria. Similar action happens with the neurotoxins of clostridium tetani or botulinum

A phase contrast microscope makes use of the fact that light gets transmitted and refracted differently through microbes than water, which magnifies their shape and structures within the microbes.

A phase contrast microscope makes use of the fact that light gets refracted around cell walls and organelles of microbes differently than across water or liquid cell content. This allows better identification of structures within microbes compared to brightfield microscopy, and it produces this glow-like appearance of microbes.

Bacterial replication happens at an "ori"gin on circular DNA.

All bacterial DNA is circular, and its replication starts at a place called "ori", or origin of replication. From there, bacterial polymerases replicate DNA in two replication forks as shown here, producing a leading and lagging strand where DNA replication is slower as it has to happen in a piecemeal fashion. Once the replication forks moved away far enough, new replication can start again at the origin, thus allow continuous replication of multiple DNA circular DNA genomes before each ring is complete. One caveat to mention here is that bacterial DNA is not found in a neat circle as shown here, but actually is highly coiled up more akin to a badly tangled extension cord. To untangle this mess and allow replications, bacteria use an enzyme called topoisomerase that breaks and unwinds the DNA for replication, and of course this can be used for antibiotic targeting.

Several related methods exist to isolate and identify proteins of microorganisms.

Although genetic methods offer powerful ways to identify bacteria, there are times when researchers want to study proteins created by microbes, or if a certain microbe produces a toxin or other protein of interest. Besides cloning genes and making recombinant proteins, researchers will isolate microbial proteins and separate them using various electrophoretic and chromatographic methods. The most common is SDS-PAGE, which separates proteins by size. If transferred to a membrane that binds proteins, antibody probes against the protein of interest can be used to determine if a microbe produces a specific protein, for example leukotoxin in some strains of oral bacteria. At other times, researchers may be interested in the totality of all proteins produced by a microorganism. Here, proteins get separated by a process called 2D electrophoresis, and each individual area of interest can be analyzed with mass spectroscopy that allows identification of known proteins.

Bacteria living in liquid culture media always show exponential growth until the nutrients run out.

As bacteria want nothing more than to replicate, bacteria will do that once they find a new medium containing nutrients. What happens is that once bacteria find a new food source or medium, the bacteria adapt to it by turning on metabolic machinery to utilize the new food source. During this time, bacteria don't grow, and this short time period is called lag phase. Once the bacteria are ready, nothing is in the way of replication and the bacteria show exponential growth. This continues until either space or nutrients start to run out, and toxic waste products accumulate. As a consequence, the bacteria adapt and focus on producing proteins that allow them to compete with other bacteria. At this time, the bacterial number stabilizes and this is called stationary phase. Most likely, most bacteria in nature or inside the human body are always in stationary phase. Eventually, bacteria that don't reproduce accumulate cellular damage to genes and proteins, and eventually they cannot replicate. Instead, bacteria structures degrade with bacteria beginning to break open, thus causing bacterial numbers to dwindle in the decline phase. Some bacteria will undergo sporulation at this time, where bacteria will produce a tough, reduced form of themselves that safeguards the genetic material and allows them to wait for better times when new nutrients become available.

Bacterial proliferation happens through cell fission.

As each circular DNA is made, the bacteria cell wall and membrane needs to grow. The chromosomes are distributed to each pole of the expanding cell, and the cell membrane and wall partition the cell, leading to two new bacterial cells, which already have started replicating their new genome. As a result, bacteria can grow rapidly, with some bacteria only needing about 30 minutes for each replication and division cycle. This is the reason why some bacterial infections can progress so quickly, and why food left out at room temperature can spoil within hours.

Why does this matter?

At the core of this question is a philosophical question. When is something alive? Is Covid19 a life form? Or just a collection of RNA and proteins? What about prions? What about other polymerizing organic molecules?

In bright field microscopy, light shines through the glass slide to highlight the shape of STAINED bacteria.

Brightfield microscopy is the simplest method, as bacteria are placed on a glass slide with light shining through the glass into the microscope. Naturally, as microbes are small and translucent, the microbes need to be stained in order to become identifiable.

What's in a name?

Example of name changes for bacteria that are associated with periodontitis

Fluorescence in Situ Hybridization uses fluorescent DNA probes to identify microbes in the context of tissues or biofilms.

Fluorescence in situ hybridization is a powerful method to identify microbes in biofilms. Here, micoscopic specimen are treated to expose their DNA, and a fluorescent DNA probe specific for each microbe of interest is allowed to hybridize with the specimen DNA. Wherever it binds, it can be seen under a fluorescence microscope, where each identified microbe will light up in a different color as seen here.

The 16S rRNA encodes part of the bacterial ribosome.

For both prokaryotes and eukaryotes, the units are similar, but the eukaryotic version is slightly larger and has slightly larger subunits. For prokaryotes, the 30s unit is made of a number of proteins and a piece of RNA called 16S ribosomal RNA.

In dark field microscopy, light shines at bacteria from the side, allowing view of live, motile bacteria.

For darkfield microscopy, the view is illuminated from the side, causing bacteria to reflect light and become visible in front of a dark background. This allows viewing of live, motile microorganisms, or microbe shapes such as this cork-screw like spirochete bacterium

Culturing Methods can characterize microorganisms like this:

For example, organisms such as bacteria can be categorized if they require oxygen for growth, as in obligate aerobic, or if oxygen completely prevents growth, such as in obligate anaerobic bacteria. There are also graduations in oxygen requirements, where a small amount of oxygen can be tolerated in facultative anaerobes, of if oxygen is not a requirement for growth, as in facultative aerobes. Temperature can influence growth patterns and morphology development such as in candida species, and high temperature can be a requirement for some bacteria such as campylobacter species. Parasites, Viruses and bacteria such as chlamydia that live inside cells may require culturing on a layer of feeder cells. Other bacteria such as neisseria, bordetella and many oral bacteria have very complex nutritional needs requiring special media for these fastidious bacteria. All of these characteristics often allow identification of clinically relevant bacteria, and there are microbial test kits based on culturing methods that allow clinicians to identify bacteria such as caries-associated Streptococcus mutans. Microbes can also be characterized and possibly identified through their growth characteristics. Bacteria can often be identified by their slow or fast growth, the colony morphology such as rough, smooth, flat, domed, star-shaped, mold appearance; color of colony, as for example, staphylococcus produces yellow colonizes, some oral bacteria black and smelly colonies. Some bacteria break down the culture medium causing discoloration such as beta-hemolytic streptococci, or infiltrate it if they are mobile. Some bacteria produce gas that can be measured and have unique metabolic byproducts. Probably of most clinical interest is that by culturing one can determine how much a bacterium is resistant against an antibiotic, depending on much antibiotic has been added to the medium prior to culture.

Sanger Sequencing allows reading the genetic sequence.

If PCR is performed with addition of chain-terminating nucleotides, PCR will randomly produce DNA fragments of up to about 90 base pairs, stopping at the given chain-terminating nucleotide. If this is done with all four bases adenine, guanine, cytosine, thymine, and the fragments separated using chromatography, a DNA sequence can be derived from these measurements. If automated, and repeated for every 70-90 basepairs, sequences of entire genes and genomes can be determined, thus allowing genome and transcriptome analyses. Genetic sequences then can be compared against known genetic sequences in large databases, thus identifying microbes, or at least provide the names of related microbes to the unknown microbes in the sample.

These are very common media for growth or identification of bacteria:

In order to grow microbes and especially bacteria, various culture media need to be used depending on growth requirements of each organism. These are called non-selective media as they allow growth of many bacteria, and usually contain a rich source of animal protein such as blood. So called selective media can be used to favor growth of certain bacteria and allows identification of these. These media usually contain antibiotics or antimicrobials, or lack certain nutrients so that only few desired bacteria can grow as shown on the right.

LPS can cause a large variety of serious effects.

LPS can cause a variety of severe effects especially if LPS is released into the blood stream in sufficient amounts as it occurs during sepsis. LPS will trigger blood coagulation causing thrombosis and disseminated intravascular coagulation of the entire blood. LPS activates complement leading to widespread blood vessel damage. Likewise, LPS will trigger endothelial cells to increase vascular permeability as well, leading to blood volume loss, shock and possibly death. LPS also triggers release of all kinds of cytokines that activated various immune cells, create fever and hypoglycemia, which deprives the brain of glucose and may also lead to coma or death.

PCR amplification success indicates presence or absence of a genetic sequence.

Let's look at these techniques in more detail. If a specific 16S rRNA sequence is known, complimentary primers can be constructed that match the ends of the corresponding DNA sequence derived from the ribosomal RNA using reverse transcriptase enzyme. Using a PCR amplifier that separates the strand to allow binding of the primers, and copying of the now single stranded DNA pieces with thermophilic DNA polymerase, it is possible to make many copies of the DNA sequence flanked by the primers as long as the primers can find complementary matching DNA. The resulting amplified DNA can be separated using an agarose gel and visualized with DNA stains, producing an image as shown above. If 16sRNA genetic material is present, it will amplify, producing bright bands of heavy molecular weight as shown here. If there is no matching sequence, nothing happens and only a large amount of low-molecular weight primer will be seen.

Lipoteichoic acid presents a unique chemical structure of gram-positive bacteria.

Let's talk about unique chemical structures encountered in bacteria since your immune system uses these structures to detect bacteria. Some bacteria also use these same structures to cause inflammation on purpose, so that inflammation damages surrounding tissue, which releases nutrients that these bacteria need for growth. Some of these unique chemical structures are also targets of antibiotic agents. Lipoteichoic acid is a polymer unique to gram-positive bacteria that consist of a glycerol - phosphate pattern with attached sugars. Although this vaguely resembles human lipids with this glycerol-phosphate pattern, the addition of sugars makes this unlike anything that exists in the human body and it is a prime signal for gram-positive bacterial invasion that toll-like receptor 2 on human immune cells can recognize.

Life on Mars?

Most of life as we know of exists as microbes, and the number of microbial organisms outnumbers by far any other life forms we know of on planet earth. Now, what gets some people excited is the possibility of extraterrestrial life. The chance of encountering movie-like aliens is almost zero, but there is a chance that microbes may exist elsewhere in the universe, especially given circumstantial evidence of microbe-shaped rock features found on a mars meteorite found several years ago, as shown here.

Mycobacteria are neither gram-positive or negative: Their complex cell walls contain long-chain mycolic acids that allow acid fast staining.

Mycobacteria are weakly gram-staining bacteria that require a special stain, the acid-fast staining, for identification. This is because mycobacteria have a specialized, lipid rich cell wall made of complex carbohydrates and mycolic acids that are long-chain, branched fatty acids. This specialized cell wall also allows these bacteria to be very resistant against disinfectants, sterilization, heat or chemicals. Some mycobacteria produce yellow or orange pigments when exposed to light, and usually require a special medium for growth, which also tends to be very slow.

MALDI-TOF Mass spectroscopy can identify proteins by determining characteristic protein fragment spectra.

Now with the proteins separated, how can researchers identify them? The tool for that is mass spectroscopy such as the matrix-assisted laser desorption time of flight technique, abbreviated MALDI-TOF. Here, a high intensity laser vaporizes and ionizes the protein obtained from a 2 D electrophoresis gel. The ionized protein and molecule fragments get drawn into a charged mass analyzer, and depending how heavy the fragment is, different fragment sizes will reach the ion detector at different times. This then produces a mass spectrum as shown on the lower right, which then can be compared to existing databases of spectra from known protein, thus identifying the protein if the spectrum matches. By extension, microbes can also be identified with MALDI-TOF as each organism has its own unique protein composition.

Ribosomal RNA can be used to identify bacteria.

Now, culturing microbes is cumbersome and takes days or weeks to complete, maybe too late for some clinical applications. Moreover, the majority of microbes is not yet cultivable because of their complex growth requirements. There is a genetic solution to this problem and it involves study of 16 subunit ribosomal RNA. What is this? As you remember, bacteria, archaea and eukaryotes use ribosomes to translate genetic information from messenger RNA into amino acid chains and proteins. Ribosomes contain two parts, a larger subunit that is involved in amino acid assembly, and a smaller subunit that reads the messenger RNA.

Bacterial infections are common in the U.S.

Of course caries and periodontal disease are common, with prevalence rates of plaque-mediated gingivitis and caries upwards of 70%. However, since caries and periodontal disease rarely grab attention as much as infections that cause hospital stays and deaths, here are some stats to ponder about. Based on data that the Centers for Disease Control collect, here is a list of the most common bacteria infections that happened in 2012. Although one should not get to focused on the exact numbers since a physician for each case had to diagnose it and report it properly, these are large numbers, considering that for example, new HIV infections are about 35,000 a year. These are big numbers, if you compare it to leukemias where only several thousand people get these cancers a year, or consider that this is as many people as live in Claremont. So, the number of people getting HIV every year is the same as if everyone in a suburb of Los Angeles got HIV every year.

Electron Microscopy is the method of choice for identifying structures within cells, or for studying viruses.

One of the disadvantages of conventional light microscopy is that resolution is limited by the wavelength of visible light, and generally it is impossible to see structures that are smaller than about 0.5 micrometers in size, which is a little less than the size of typical bacteria. In order to see a detailed view of microbial structures, electron microscopy is needed, and electrons instead of photons are used to illuminate the specimen. Electron microscopy requires a more elaborate technical setup, but the principle is the same in that there is a source of electrons that illuminates the sample, and through a series of electric coils the electrons are focused on a fluorescent screen which creates a visible image that can be recorded or viewed.

General bacteriology

One role where bacteriophages may be of significance may be in oral diseases since common oral diseases such as periodontitis and caries are caused by bacterial biofilms where bacteriophages are major factors in driving bacterial diversity and genetic exchange. And it is not only oral disease, but also systemic diseases like atherosclerosis and gastric ulcers that can be influenced by bacteria, or caused by bacteria such as staph infections.

Quantitative PCR allows estimates on the number of specific microorganism.

Quantitative PCR also uses the polymerase chain reaction, but uses a so-called Taqman probe in addition to the primers that can bind to the genetic material of interest. This probe contains a fluorescent dye and an inhibitor called quencher that prevents fluorescence as they are in close contact on this Taqman probe. Initially, the genetic material is made to react with this probe first, which will bind to all matching areas. When this prepared genetic material is then amplified using the polymerase chain reaction, the polymerase will not only fill in the complementary DNA strand, but also dislocate the probe and break it apart. As the probe gets broken apart by the polymerase, fluorescent dye and quencher become separated, and fluorescence can be detected by the q-PCR machine. The more DNA of interest was present in the sample, the more probe will have bound initially, and the earlier will a fluorescence signal be detected by the machine. This allows then an estimate of how much DNA of interest, or how much of a certain species was present in a sample.

Antibody-staining can identify specific organisms in a histological context.

Since antibody production against specific microbes and proteins is a well-established method, countless methods that use antibodies to identify bacteria or proteins have been developed, all of which involve antibodies that can bind a particular protein or microbe. These antibodies can be used to stain and identify specific proteins on specific microbes in a histology slide of culture liquid, biofilm or tissue.

These are common stains to identify microorganisms:

Since bacteria and small fungi are not readily visible in brightfield microscopy, various staining methods have been developed to allow better visualization of these microbes. A strong potassium hydroxide solution is used to show fungal elements sometimes as it dissolves other elements. India ink allows identification of cryptococcus species that cause brain infections. The most common differentiating stain for bacteria is the gram staining method, which stains gram-positive bacteria that have thick cell walls purple, and cells that lack a thick wall stain red. Wright Giemsa stain is used in to stain blood cells, and it is also used to identify blood-borne parasites. Mycobacterium species such as tuberculosis are identified using Acid-fast stains, which resist removal of the stain once bound to bacteria.

Here are genetic methods that identify bacteria:

Since 16s ribosomal RNA can be used to identify bacteria, a variety of genetic methods can be used to answer several questions about microbes in a sample. For example, reverse transcription copies of RNA isolates gained from a clinical sample can be amplified using polymerase chain reaction to identify within hours if a sample contains specific microbes. With quantitative PCR, it is possible to gauge the amount of RNA attributable to a specific bacterium, and if normalized to the amount of bacteria, it can tell you the proportion of microbial species in a sample. In order to know the sequence of 16S ribosomal RNA, someone had to sequence it first using the Sanger Method, and this is still being done for newly described microbes. While sequencing was expensive and very time consuming at one time, it is now possible to sequence thousands of genetic sequences at once with next generation sequencing methods. This allows sequencing of the entire genetic content of a sample, producing what is called a "genome" for DNA and "transcriptome" for all RNA content. If this is done for a microbial sample, it allows identification of all known and unknown or uncultivable species in a sample, and thus characterization of the entire "microbiome". If sequences are known for all microbes of interest, a quick method of identification of hundreds of species can be performed using microarrays that contain genetic probes for all the species of interest. The microarray technique is related to the older blot and checkerboard assays, where genetic probes identify specific genes - the Southern blot- or transcripts - the Northern blot in a sample. The checkerboard assay uses the same techniques, but allows simultaneous study of multiple clinical specimen or microbes. Genetic probes are also used in fluorescence in situ hybridization assays, where a fluorescent dye combined with a genetic probe binds to specific microbes, thus highlighting its position in relationship to other cells or microbes on a microscopy slide.

LPS is a highly inflammatory molecule, and the reason many sterile medical devices should not be reused since it CANNOT be destroyed by conventional sterilization methods.

Since it is so unique compared to molecular patterns found in human cells, LPS always elicits a high level of inflammation because it is readily detected by immune cells, and anything contaminated with LPS such as root surfaces or medical devices will produce a lasting immune response and delayed wound healing. Since LPS cannot be destroyed by sterilization, implanted medical devices such as IV infusion needles, anesthesia needles, drains, healing caps and dental implants are manufactured in sterile environments from new materials and never re-used. Although LPS can be recognized by the immune system and is the target of antibodies, LPS does not readily elicit an antibody response by itself and it is not possible to create a vaccine against a specific LPS.

Superantigens cause excessive immune activation

Some bacteria produce proteins that interfere with normal antigen-presenting cell - CD4+ t-cell binding. These are called super antigens, and bind MHC and T-cell receptor regardless of the fit of the receptors or antigen present. This results in excessive immune cell activation, and an exaggerated immune response including shock.

Peptidoglycans are assembled near the cell wall from monomers created intracellularly.

The main significance of peptidoglycans is however that they are found uniquely in all bacteria, and thus present a good target for antibiotics. Peptidoglycan synthesis is a complex process that requires many enzymes (as shown here in the boxes such as MurA, MurB, MurC, and so on), and begins by linking of muramic acid and glucosamine to Uracyldiphosphate, and the various enzymes adding N-acetyl residues and amino acids to the sugars. Then, these monomers get attached to a lipid called undecaprenyl at the cell membrane which allows eventually transfer of the peptide to the bacterial cell surface. This also allows additional processing of the monomers that includes addition of amino acids such as glycines or alanines to the existing amino acid chain and synthesis of N-acetylmuramic acid and N-acetyl glycosamine dimers. Once transported to the bacterial surface, the dimers get further polymerized and cross-linked by a transpeptidase which is also called penicillin binding protein. It is this enzyme that mistakenly binds antibiotics with a beta-lactam ring such as penicillin as it appears chemically similar to its usual substrate

Microbial Cultures use grow characteristics to identify bacteria, and determine resistance against antimicrobial agents.

The next oldest method of microbiology after microscopy is the study of growing, cultured bacteria. Microbial cultures are used to isolate and propagate microbes from clinical samples. This method also allows testing if microbes are alive, and to what extent they are resistant against antimicrobial agents. Most significant, especially before genetic tests existed, it allowed identification of microbes based on their growth characteristics.

Transmission Electron Microscopy "shines" electrons through microorganisms "stained" with heavy metal salts to highlight intracellular structures.

The principle I described is that of bright field microscopy, and with electron microscopy it allows a close up of microbial structures as shown here. Transmission electron microscopy of specially prepared samples usually can show cell membranes (as seen here), genetic material, cell organelles and various surface projections of cells and bacteria. It also shows bacterial morphology in great detail as with these rod-shaped bacteria.

Bacteria are characterized by morphology.

The second method to describe bacteria is by their morphology. There are the round cocci, the rod-shaped bacilli, the comma-shaped vibrio, spiral-shaped spirillium and spirochetes, and elongated rounded coccobacillus and fusiform bacteria.

Bacteria are broadly divided into two classes: gram+ and gram-.

The vast majority of bacteria can be divided into two groups with a simple stain test as shown here, and as described before. In short, crystal violet will stain irreversibly bacteria with thick cell wall, whereas gram-negative bacteria loose that stain with some type of decolorizer, and need to be stained again with a different stain, safranin red, to make them visible. The gram test divides bacteria into those that have thick cell walls, the gram-positives, and those that don't, the gram-negatives. That is important, because the presence or absence of this cell wall allows different growth characteristics, and different immune responses against these bacteria. Now there is a small exception to this, the acid-fast bacteria, that don't stain well with gram stains and they will be discussed separately, but for the most part the bacterial world divided into gram-positive and gram-negative bacteria, and this is one of the two general methods to classify and describe bacteria.

Bacterial gene expression follows usually an operon/repressor pattern.

Typically, in bacteria several genes that encode proteins that perform related functions are clustered together. For example, the lac operon contains enzymes that encodes proteins needed to metabolize lactose such as galatosidase to split the sugar into monomers, and a permease and acetylase to transport the enzyme to the extracellular environment. Now, if lactose is not present, making these proteins would waste precious energy and amino acids, and the mechanism to prevent production of these enzymes is a repressor. So, what happens here is that in the bacterial haploid genome, all these genes for proteins, also called cistrons, are clustered together on the genome along with their regulatory DNA sequences. If these genes produce proteins responsible for disease or antibiotic resistance, these are also called pathogenicity islands. Continually, transcription proteins bind to the promoter of the repressor region, and transcribe a long mRNA sequence of the entire lac operon. As this mRNA gets transcribed, the repressor protein is made first. The repressor protein can then bind galactose, the end product of the enzymatic split of lactose catalyzed by galactosidase enzyme which is encoded by this lac operon. If lactose is not present, the repressor protein can also bind to the remaining lac operon mRNA and prevent further translation of this mRNA, thus preventing further production of lac operon proteins and saving energy for other processes.

Unlike the O-antigen, the core and lipid A component of the LPS are less variable since they are essential to LPS integration into the bacterial membrane.

While to O antigen is highly variable, the core polysaccharide is much less variable and contains a sequence of five sugars terminating in ketodeoxyoctulonate (KDO) which links the sugars to the lipid A portion of LPS. The lipid A portion is a dimer of two sugars that are linked to a variety of unbranched and branched fatty acids. The structure of hydrophilic sugars and fatty acid tails resembles that of cell membrane lipids, and it allows formation of a second lipid membrane around the bacterial cell. Unlike cell membrane phospholipids, the chemical structure of lipid A is much different to molecular patterns found in human cells, which usually produce only straight, even-numbered fatty acids and use glycerol as a backbone for lipids. Therefore, lipid A is a good marker for immune cells to detect gram-negative bacteria and it is usually detected by toll like receptor 4 of immune cells. As usual in microbiology, there are exceptions to the rule, and some bacteria including oral bacteria associated with gum disease have evolved a different type of lipid A that does not trigger this receptor in an effort to undermine immune defense. from: Medical Microbiology, 7th edition. P Murray, K Rosenthal, M Pfaller Since it is so unique compared to molecular patterns found in human cells, LPS always elicits a high level of inflammation because it is readily detected by immune cells, and anything contaminated with LPS such as root surfaces or medical devices will produce a lasting immune response and delayed wound healing. Since LPS cannot be destroyed by sterilization, implanted medical devices such as IV infusion needles, anesthesia needles, drains, healing caps and dental implants are manufactured in sterile environments from new materials and never re-used. Although LPS can be recognized by the immune system and is the target of antibodies, LPS does not readily elicit an antibody response by itself and it is not possible to create a vaccine against a specific LPS.

Tetracycline highlights

§Bind 30s ribosome §Resistance: tetracycline pump, breakdown §Calcium supplement inhibits tetracycline §Broad spectrum, mostly bacteriastatic §Treat: Acne, pneumonia, rickettsia, Lyme disease, §Accumulates in cells, bone, teeth -> discoloration of teeth §Phototoxity, tinnitus; nephrotoxicity Not shelf stable; causes liver failure if expired

Antibiotics aim to target molecular mechanisms unique to bacteria.

**FLAG Antibiotics aim to control bacteria through targeting of molecular mechanisms unique to bacteria. For example, antibiotics such as beta lactams that include penicillins and cephalosporins target enzymes involved in cell wall synthesis. As bacteria that cannot grow, cannot reproduce, it stops bacterial growth. In addition, loss of the cell wall makes it more likely for the bacterium to loose viability as osmotic changes could rupture the bacterium. Detergents target bacteria by disrupting the cell membrane, and this works well with gram-negative bacteria, as well as enveloped viruses. Although it does not work well with gram-positive bacteria, it still can reduce general bacterial load as it makes it more likely that bacteria get dislodged from any surface through surfactant action, thus reducing overall bacterial counts. Other antibiotics target DNA synthesis such as the quinolones that inhibit gyrase and prevent DNA replication. Other antibiotics target the unique ribosomes of bacteria such as clindamycin and tetracycline used in dentistry. Lastly, there are few antibiotics that are antimetabolites and interfere with bacterial metabolism. There is a question you may ask, and that is where did antibiotics come from? It happens that bacteria and fungi often compete for the same nutrients in the same environment, and therefore, fungi and even some bacteria have developed antibiotic substances that they use to kill off competing bacteria in their environment.

Gram-positive bacteria have cell walls containing lipoteichoic acid and peptidoglycans.

**FLAG Going back to the gram positive and negative division, gram-positive bacteria are surrounded by a dense, thick wall of peptidoglycans and a unique molecule, called lipoteichoic acid that links that wall with with the cytoplasmic membrane. This cell wall however is not impermeable to nutrients, and gram-positive bacteria have surface proteins to grab nutrients, perform enzymatic reactions and maintain the shape of the cell wall and bacterium. Since this cell wall is important to gram-positive bacteria, and unique to them, eukaryotic organisms such as humans have developed enzymes such as lysozyme that attack this cell wall, and antibiotics such as penicillin are much more effective against gram-positive bacteria. On the plus side, some gram-positive bacteria use the same mechanism to encapsulate themselves into spores than can resist very harsh environments and disinfectants.

Gram-negative bacteria have an outer membranes and lipopolysacharides shells.

**FLAG In contrast to the wall of gram-positive bacteria, gram-negative bacteria are surrounded by a simple outer membrane that anchors a fluid, flexible shell of lipopolysaccharides based on a thin peptidoglycan film. To allow nutrients to cross this water-insoluble shell, gram-negative bacteria insert pore proteins into this outer membrane that allows nutrients to reach into the periplasmic space, where nutrient binding proteins grab those nutrients, ready for carrier proteins to transport these into the bacterial cytoplasm.

Bacteria store nutrients, proteins and metabolites in inclusion bodies.

Although gram-negative and gram-positive bacteria have different cell walls, the intracellular structure is similar with the bacterial chromosome and ribosomes. Bacteria need to store materials such as nutrients, proteins and other metabolites in inclusion bodies inside the cells. Gram-negative bacteria also have the option of storing material in the periplasmic space underneath the outer membrane. One thing that distinguishes bacteria is that they can produce various attachment fibers such as fimbriae or pili and also can produce slime capsules that also have adhesive properties.

Fluorescence Microscopy identifies individual organisms by specific proteins or genes.

Although the previously listed stains have been a main-stay for many decades, the greatest breakthrough in microscopy of microbes was possible with fluorescent dyes that can be linked to any probe specific to a certain protein or gene sequence. This allows direct visualization of specific bacteria as seen on the left, or identification of dead or alive bacteria in a sample as seen on the right. Regardless of fluorescence staining, a specialized microscope needs to be used that contains lasers at defined wavelengths which cause the fluorescent dyes to glow.

Peptidoglycan are another molecular pattern unique to bacteria.

Another molecular pattern that is unique to bacteria are peptidoglycans, which are also usually detected by Toll-like receptors, usually TLR2, which are the prime detectors of gram-positive bacteria. Peptidoglycan are the predominant component of cell walls of gram-positive bacteria, but are also found in small amounts in gram-negative bacteria as well. Peptidglycans are made of long polymers of N-acetylglucosamine and N-acetylmuramic acid carbohydrates, which then are arranged in crystalline sheets forming a capsule around bacteria. These sugars don't exist really in human cells, which is the reason why they are readily recognized by the immune system. Like LPS, peptidoglycan fragments will elicit a strong immune reaction, but by themselves do not elicit a strong antibody response. Instead, peptidoglycans and LPS can serve as so-called adjuvants in vaccines, where they can boost antibody response to a particular protein if they are added to the vaccine since the strong immune response will favor antibody production against foreign proteins.

Light microscopy provides general morphology of bacteria.

As mentioned, light microscopy is the oldest method for studying microbes, and light microscopy uses a set of biconvex lenses to create a magnified virtual image of very small objects in front of the objective lens. Light microscopy is useful for a general view of microbes against a backdrop of tissue, or to study the general morphology of bacteria and relationship with other bacteria for identification purposes. With some types of microscopy and specialized stains, it is possible to identify individual bacteria.

All microorganisms are classified:

As there are millions of life forms, study of these life forms requires a classification system. The classification system used in biology is derived from the taxonomy system created by 18th century Swedish botanist Carl Linnaeus. Nowadays, life forms are categorized based on shared genetic characteristics and morphology, with species describing similar individuals capable of interbreeding, and ever-wider categories describing broader similar characteristics up to the domains of life. This certainly applies to microbiology, as shown here with the example of E.coli. Here, each category describes an ever narrower group of bacteria with ever closer similarities such as metabolic capacity, cell structure and genetic similarity.

Most bacteria use biofilms as growth strategy to avoid hostile environments and improve survival.

As you see from the different metabolic requirements during the different stages of bacterial growth, it is important for bacteria to find out how many of bacteria exist in the environment. Moreover, bacteria often can coordinate their cellular behavior depending on how many like-minded bacteria of the same species are around. A very common system is the so called LuxI/R type quorum sensing system of gram-negative bacteria. Here, the LuxI gene is continually active and codes for a protein that leads to production of acyl-homoserine-lactone which freely diffuses out of the cell. If enough of acyl-homoserine-lactone is present, presumably because enough other bacteria produce it, some of this leaks back into the cell, and it will bind to the LuxR protein that then can turn on transcription of genes for proteins useful in crowded conditions.

Microarrays can be used to detect thousands of genes and measure relative expression of genes.

If a 16s rRNA gene or any other gene of interest is known, and a large number of samples need to be analyzed, microarrays allow identification of it in hundreds or throusands of samples. For example, a glass slide can be printed with DNA probes specific for hundreds of different 16s RNAs of various bacteria. In the meanwhile, a sample is amplified using a generic 16S ribosmal RNA primer set that will amplify any known 16s ribosomal RNA genetic material. This then is conjugated with fluorescent dyes, and the amplified, labeled material is allowed to bind the probes on the glass slab. If it binds, it will produce a signal for each 16S RNA present in the sample. An alternative approach prints the samples on the glass slab, and two fluorescent DNA probes for two different genes are allowed to react with the glass slab. This produces an image as shown here on the lower right, where presence or absence of each gene shows up in red or green, or yellow if both genes are present.

The key metabolite for bacteria is pyruvic acid, which is fermented to various end products.

In order for bacteria to create cell walls and various lipids, they have to create energy and simple carbon-containing precursor molecules. In order to accomplish this, bacteria release all types of enzymes that break down complex molecules in the external environment such as proteins and polycarbohydrates into simple carbohydrates and amino acids, which can be taken up across the cell membrane into the bacterial cytosol. While this is similar to eukaryotic cells, bacterial cells are more specialized in catalyzing fewer biochemical reactions and quite often do not engage in respiration, and for some bacteria, oxygen is very toxic to them. Instead, they break down simple molecules typically to pyruvic acid. For example, glucose is most commonly broken down by using the Embden-Meyerhof-Parnas glycolytic pathway into pyruvic acid, producing two NADH molecules and 2 pyruvic acid molecules from one glucose molecule. To recycle NADH and gain energy, pyruvic acid is fermented to various end products depending on the bacterial species as shown here.

Scanning Electron Microscope "shines" electrons from the side on metal-plated microorganisms to reveal surface structures. This can only be done in hard & dry specimen.

In principle somewhat similar to dark field microscopy, in scanning electron microscopy, an electron beam illuminates an metal-coated specimen from the side, and the microscope picks up the scatter from electrons bouncing off the sample. This creates a stunning views of microbial life as if you had microscopic vision and watched a lamp shine on a collection of microbes as seen here. Scanning electron microscopy is a great method to visualize surface details of microbes and anything else such as insects, microchips, teeth and other hard structures. The downside of scanning electron microscopy is that it requires a relatively hard and dry specimen as the preparation process usually involves vacuum and a plasma spray of metal.

Plasmids are circular pieces of DNA that can independently replicate themselves.

One step up from transposons are plasmids. Unlike transposons, plasmids are circular pieces of DNA that contain their own replication origin, that allows for independent replication of the plasmid. Usually, the plasmids also contain genes coding for proteins that cause mobilization of the plasmid and allow transfer of it to other cells. In a way, plasmids can be seen as intracellular parasites almost like a virus, but without a capsule.

Plasmids can transfer from one bacteria to another in a process called conjugation using pili.

One step up from transposons are plasmids. Unlike transposons, plasmids are circular pieces of DNA that contain their own replication origin, that allows for independent replication of the plasmid. Usually, the plasmids also contain genes coding for proteins that cause mobilization of the plasmid and allow transfer of it to other cells. In a way, plasmids can be seen as intracellular parasites almost like a virus, but without a capsule.

The CRISPR-Cas system allows bacteria to neutralize phage attacks.

Since bacteriophage attack is often lethal for bacteria, bacteria have developed a system to counter phage attacks. This is the CRISPR-Cas system, where Cas is a group of enzymes that process incoming phage DNA, and insert small bits of it into the bacterial DNA for memory, and reuse these small bits (the CRISPR - clustered regularly interspaced short palindromic repeats) to specifically recognize incoming phage DNA. This then targets this incoming phage DNA for destruction by an enzyme called Cas9, and prevents the phage DNA from taking over the bacterium.

Bacteria have a much simpler cell structure than eukaryotes

So what are bacteria. Bacteria are small single-celled organisms that have generally a smaller size than eukaryotic cells and much simpler cell structure including a much smaller genome than eukaryotic cells. Unlike eukaryotes, bacteria don't have a nuclear membrane; chromosomes; strands of a DNA diploid genome; Golgi bodies; Lysosomes; Mitochondria or Endoplasmic reticulum; a cell membrane containing glycoproteins, proteoglycans, hundreds of different receptor and attachment proteins, various lipids and lipoproteins; and if bacteria move, they have simple-structured flagella. Instead, bacteria have a single, circular DNA haploid genome floating in the cytoplasm. They have smaller 70S ribosomes (50S+30S) instead of 80S (60S+40S). The cytoplasmic membrane does not contain sterols, but may contain a cell wall. Reproduction is simpler bacteria, not going through complex sexual behaviors, using different sexes or haploid and diploid lifecycles. Instead, bacteria undergo simple binary fission. Respiration simply takes place simply within the bacterial cell membrane. For attachment and environmental sensing, bacteria use a few proteins that can perform a variety of functions with the same protein.

Some bacteria can be identified through unique metabolic products.

Some microbes produce typical metabolites and enzymes that can be used to identify them. For example, the enzyme catalase that breaks down hydrogen peroxide is produced by straphylococci, but not streptococci, and can be used to differentiate between these bacteria. Another example is the proteolytic substrate BAPNA, which is cleavable by enzymes produced by periodontal bacteria, and thus can be used to identify bacteria associated with periodontitis. Anaerobes and aerobes can be distinguised with either oxidase test or fluid thioglycollate tubes. Many medically relevant enteric bacteria can be distinguished by their metabolic abilities, which can be tested with glucose fermentation tubes, urea broth and the IMViC tests.

EIA/ELISA can be used to measure the amount of produced protein/ or number of bacteria.

Specific antibodies can also be used in methods called either enzyme-immunobased essay (EIA) or enzyme linked immunosorbent assay (ELISA). Many different ELISA setups exist that fit specific measuring purposes. A common form is the Sandwhich ELISA. Here a dish containing small wells is prepared so that the surface of the well is covered with an antibody that can bind a protein of interest. These wells are then exposed to either test solutions containing an unknown amount of target protein, or reference solution containing known concentrations of the target protein. After removing these solutions, and rinsing the wells with buffered non-binding protein solution, antibody is added that can bind to the target protein which now will have been captured on the plate. This antibody then can be detected with another antibody that recognizes bound antibodies and carries an enzyme that can make a dye which can be measured with a spectrometer. The amount of dye produces is proportional to the amount of bound target protein, and therefore it can measure how much target protein was present in the samples. This method can also be used in a paper format where a test strip with antibodies printed on it is first bathed with a spit or urine sample and then bathed with different solution containing antibody-linked enzymes and coloring agents. This is used for commercial kits that allow you to identify different bacteria, such as a strep throat kit.

Summary - How are these tests used?

Summary - How are these tests used?

16S rRNA can identify bacteria since it has areas that can accumulate species-specific mutations.

The ribosomal RNA is key for the translation process, and can assume a highly complex structure with many loops and double-stranded regions that supports the function of ribosomes. As protein translation is key for cell survival, ribosomal RNA contains critical RNA sequences that cannot be changed without endangering cell survival, and less critical areas where nucleic acid sequence is not too important for ribosomal function. Therefore, there are ribosomal RNA sequences that are conserved through all organisms since any mutation would prevent survival, and variable and hypervariable regions where mutations are permissible over the course of generations. These 9 variable regions labeled "V1" to "V9" therefore accumulate mutations over the course of generations, and differences in genetic sequence therefore mark relatedness of different lineages. Since ribosomal function is common to all prokaryotes and eukaryotes, and the ribosomal RNA ancient in origin, every species likely has an unique ribosomal RNA sequence that can be used to identify each species. Also, assuming that the mutation rate stayed constant throughout time and across species, one can also compare ribosomal RNA and build family trees based on similarity of the ribosomal RNA, leading to the dendrogam showing the genetic relationships between millions of living organisms on a previous slide.

Bacteria regulate translation also through complex mRNA folding.

The tryptophan operon contains promoter and repressors like the lac operon as shown on the left, but also shows the ability of bacteria to fine tune their expression of protein through manipulating the way mRNA can fold, leading to more stable or less stable mRNA, and subtler changes in protein expression. Here, if tryptophan is present in high amounts, mRNA translation proceeds quickly and the mRNA is quickly fed through the ribosomes. However, because it is quickly fed through the ribosomes, the initial leader mRNA is kept from folding up to sequence 3 and 4, which allows the complimentary last segments 3 and 4 to bind to each other, forming a terminator RNA hair loop that ends further translation. If on the other hand, only a small amount of tryptophan is present, translation takes longer, and the ribosomes cannot prevent segments 2 and 3 to bind to each other, creating an RNA configuration that is more stable and allows continued translation of ezymes that make tryptophan. If there is virtually no tryptophan, the bacterial cell needs to conserve tryptophan for more essential tasks and can shut down production of tryptophan-producing enzymes as all the RNA segments bind, folding the RNA and preventing translation at all.

Transposons are simple genetic elements than can "jump" in and out of genetic material.

These can be simple transposons, which are a gene or a few genes that are flanked by repetitive DNA sequences. As such, these repetitive sequences can bind to each other and pinch off the genes within the sequence from the parent bacterial chromosome. It can also bind to the a genetic sequence somewhere else on the bacterial genome that has the same repetitive sequence, and thus insert itself into that area. Transposons usually are very small and may not even encode a gene - they may just disrupt a gene or alter the resulting protein sequence so that the gene produces a different protein. The key, though, for transposons to be viable is that it does not affect survival of the organism it changes. If you want to see what transposons can do, look for Indian corn. Here the random different colors of corn kernels are caused by transposons encoding various corn pigments.

DNA fragments and plasmids can also get into bacteria through "transformation".

Under certain conditions, bacteria can randomly take up genetic fragments of other bacteria with similar genetic structure and incorporate those fragments into their own genome. This is called transformation, and this can also work for uptake of plasmids. Some bacteria also contain plasmids that encode a plasmid mobilizes, it induces formation of a tube called sex pilus that forms a channel between two bacteria. This channel allows transfer of the plasmid, or by mistake, a bit of the bacterial genome. The fourth possibility of gene material transfer is also accidental. Here, some bacteria have the ability to take up bits of loose DNA from the outside of environment that got released when another bacterium got broken up. This is called transformation. All these processes happen in the environment, but also can be used in a microbiological laboratory to create genetically modified bacteria.

Bacteriophages are usually viruses preying on bacteria, but occasionally may inadvertently carry bacterial genes and transfer these to other bacteria in a process called transduction.

Very much a virus, but affecting bacteria are bacteriophage, which you have seen before. These also typically contain circular pieces of DNA that contain their own replication origin, replication and capsule enzymes, and are intracellular parasites. Now, these bacteriophage not only contain their own genetic material, but can integrate themselves into the bacterial DNA if it is useful to their survival using CRISPR sequences as mentioned before. Now, as they integrate themselves, they have to excise themselves at some point, and this is errorprone. Occasionally, a bit of the bacterial genome gets excised as well and packaged into the phage DNA, and just as random, this bacterial DNA gets eventually deposited inanother bacterial cell the next time the phage excises itself.

LPS is the key, unique feature of gram-negative bacteria, and characteristic of bacterial species.

While lipoteichoic acid is the prime marker for gram-positive bacteria, lipopolysaccharide is the prime marker for gram-negative bacteria. Lipopolysaccharides contain three major molecular regions: the lipid A portion, the core poly saccharide and the O-antigen. The O-antigen is a carbohydrate polymer made of various sugar motifs that repeat itself, like the example given here mannose-rhamnose-galactose, for up to 40 times. Since it is made of carbohydrates, it is a highly hydrophilic and protective surface layer that protects the underlying cell membrane from attack by immune cell proteins. The interesting thing about the sugar sequence is that it is highly variable between species and even bacterial strains, and it is a major target for strain-specific antibodies in the immune defense against gram-negative bacteria. Therefore it can also be used to identify specific bacteria through a process called serotyping in a laboratory process, allowing clinicians to detect dangerous bacteria such as Escherichia coli O157 strains.

Why all this genetic transfer ability?Because it facilitates spread of useful genes for bacteria.

Why do genetic transfers including phage attacks happen at all, and why would bacterial evolution allow it.? It allows it, because very often genes usesful for bacterial survival are carried by it such as antibiotic resistance genes. This contributes to what is called horizontal gene transfer, or the spread of genes between bacterial species, in contrast to vertical transfer, which is the passage of genetic information down to bacterial offspring from the parent bacterium through cell division. Horizontal gene transfer is a problem because it leads to the spread of bacterial resistance, which is a big public health problem. Now, bacteria don't necessarily want to participate uncontrollably in these transfers, and actually have developed some mechanisms that control and limit these processes. For example, bacteria contain restriction enzymes, which cut specific palindromic DNA sequences typical of invading plasmids, and thus cause these plasmids to become degraded, thus preventing unlimited plasmid infection. Likewise, bacteria produce cas9 enzyme that is a restriction enzyme specific to bacteriophage. Bacteria can also have mutations of the direct repeat sequences that prevent transposon insertion. Therefore, most bacteria have evolved to allow some degree of gene transfer, that is enough to provide occasional uptake of genetic material beneficial to bacteria while limiting excessive susceptibility to mobile elements.

Beta-lactam antibiotics work by mimicking the transpeptidase substrate and inactivating the enzyme.

Why is that so? The beta lactam ring looks like the usual glycine/alanine peptide chain the enzyme recognizes. Since the 4-sided beta-lactam ring is highly reactive, it binds covalently to the enzyme and permanently inactivates it. If enough enzyme is inactivated by the presence of a sufficient and sustained concentration of antibiotic, it prevents the bacterial cell to make more cell wall and preventing growth of new bacteria. Moreover, the bacterial cell eventually dies as the existing peptidoglycans degrade over time and the cell wall breaks down, causing the bacterial cell membrane to rip as well and spilling the cell content.

Immune Systems evolved to protect multicellular organisms from bacterial invasion using...

§... §Coagulation §Complement §Adaptive immunity (i.e. antibodies, cytotoxic T-cells) Blood coagulation can also be seen as a method to contain microorganisms as it is a quick molecular way to wall off microorganisms and prevent spread of the infection through the rest of the organism. A famous example is the blood of horseshoe or Limulus crabs that coagulates when it encounters endotoxin, and this is used in the Limulus assay to check for endotoxin contamination of medical products. In humans, a vast range of bacterial products can trigger coagulation including most cell wall products of bacteria. The idea of coating bacteria to control their spread is also a function of complement systems, which can bind to various bacterial cell wall components and recruit enzymes that perforate bacteria covered in complement. Probably the most recent major innovation in controlling microorganisms is the use of adaptive immunity, which probably dates back to origin of jawless fish about 550 million years ago, which developed immune cells that can recognize foreign proteins, and somewhat later fish developed the ability to secrete primitive antibodies directed against foreign proteins. In mammals such as humans, there is a large family of specialized immunoglobulins such as IgA, IgM, IgG, IgD and IgE antibodies that recognize foreign material and recruit other proteins and cells to destroy anything covered in antibodies. Moreover, these immune systems also developed cells that can recognize specific molecular signatures of intracellular parasites and destroy infected cells using cytotoxic T-cells.

Immune Systems evolved to protect multicellular organisms from bacterial invasion using...

§Antimicrobial substances, peptides & enzymes §Physical barriers §Phagocytic cells As bacteria can replicate faster and are quicker to utilize nutrients than eukaryotic cells, eukaryotic cells have evolved mechanisms to stall bacterial growth and kill bacteria. For single-celled eukaryotes and even other bacteria, this usually involves creation of antimicrobial chemicals such as antibiotics, or even chemicals such as hydrogen peroxide or oxygen that kill bacteria. For multicellular organisms, this is even truer as they depend on cell interactions and protein matrixes that hold the multicellular organism together, which can be destroyed by bacteria. Therefore, every multicellular organism, even as simple as sea sponges produces a mix of antimicrobial substances that limit the growth of microorganisms. In humans, examples of these include cathelicidin and the defensins, which contain peptide sequences that disrupt bacterial cell membranes; hydrogen peroxide and hypochlorite, which produce oxidative damage to microorganisms, and lysozyme, which is an enzyme that digests cell-walls of gram-positive bacteria. Another very ancient mechanism of preventing bacterial infection is the use of water-tight physical barriers such as skin tissue and intestinal lining that keeps most bacteria out. Not surprisingly, one of the main functions of epithelia is to produce substances that limit microbial growth. The next ancient mechanism of controlling bacteria is to the use of phagocytic cells that can detect, ingest and kill bacteria, which is a mechanism of infection control shared by organisms ranging from nematodes to insects and any vertebrate organism from fish to humans. Humans have a whole family of phagocytic cells including neutrophils, eosinophils, basophils, macrophages, microglia, synovial type A cells and dendritic cells that perform specific functions.

Clindamycin Highlights

§Bind 50s ribosomes & inhibit transpeptidation §Bactericidal (at high dose- 300 mg), Bacteriostatic (at low dowse - 150 mg) §Treats anaerobic gram-negative bacteria §Side: effects: diarrhea, nausea, vomiting; pseudomembraneous colitis (Clostridium difficile) §Alternative to Penicillin in penicillin-allergic patient

NASA's definition of life

§Complexity §Consumption §Homeostasis §Responsiveness §Reproduction

Bacteria cause disease through

§Disturbance of normal microflora §Nutrient competition §Inflammation §Proteolytic tissue damage §Bacterial toxins Bacteria can cause disease through a variety of mechanism. Sometimes, the underlying problem of disease may be very subtle, as there is a change in the normal microflora which normally inhabits a site in the human body like the intestine or oral cavity. This can change how these organs function, producing symptoms of disease such as in inflammatory bowel syndrome, caries or periodontal disease. Bacteria also compete for nutrients and a disturbed balance of bacteria may cause nutritional deficiency. For more acute bacterial infections, general signs and symptoms of infection such as fever and pain are caused by the inflammatory response to bacteria as they get recognized by the immune response. Inflammation also typically results in local tissue damage as immune cells release proteolytic enzymes to remove diseased tissue and bacteria. Bacteria also can produce proteolytic enzymes that damage tissue, and they can also produce proteins and noxious substances that are toxic themselves.

Bacteria evade the immune system by ...

§Encapsulation §Hemagglutination §Intracellular lifestyle §Antigen switching §Proteolytic digestion of immune proteins §Toxins directed against immune cells The immune system evolved to fight off bacterial infections, and bacteria have evolved to evade getting killed by immune cells. A lot of bacteria have evolved to produce slime capsules that prevent attack by either immune cells or enzymes. Other bacteria use a similar strategy by attaching themselves and surrounding themselves with red blood cells using hemagluttinating proteins. A number of bacteria such as mycobacteria and listeria evade the immune system by hiding within human cells out of sight of the immune system, and sometimes even within the immune cells such as listeria in macrophages. As antibodies target proteins and other molecules made by bacteria, a number of bacteria have evolved genetic mechanisms that allow rapid changes of surface molecular patterns that make antibodies targeting old patterns useless. Some bacteria produce proteases that destroy antibodies or complement, or produce pore-forming toxins such as leukotoxins that kill immune cells.

What is Taxonomy?

§Hierarchical grouping §Similarity §Carl Linnaeus (1707-78) §[Genus] [species] §Escherichia coli §Homo sapiens

What are "...~omics"?

§How does bacterial behavior change in different environments? §Transcriptomics: Gene expression changes §Proteomics: Protein production changes §Phenomics: Changes in bacterial characteristics §Microbiomics: Changes of the entire microbial community

Metronidazole Highlights

§Inhibits DNA synthesis by inhibiting ferredoxin, which normally protects DNA in gram negative bacteria & protozoans from DNA damage §Broad spectrum & treats protozoans; treats c. difficile infection §Few side effects §Side effects: decreased appetite, nausea, metallic taste; Disulfiram-like reaction

Dental Use (FYI)

§Oral abscess: §Penicillin VK 500 mg; 1 capsule every 6 hours; 28 capsules §Amoxicillin 500 mg; 1 capsule every 8 hours; 21 capsules §Alternatives: §Keflex 500 mg: 1 capsule every 6 hours; 28 capsules §Clindamycin 150 mg; 2 capsule every 6 hours; 56 capsule

Penicillin-class highlights

§Penicillin binding proteins mistake beta-lactam antibiotic for cell wall precursors §Kill bacteria (bacteriocidal) §Augmentin: Amoxicillin + beta-lactam §Methicillin: overcomes beta-lactamase §Penicillin VK: oral infections; strep throat; neisseria; syphillis §Wider spectrum: Amoxicillin -> periodontal infections; shigella, salmonella; listeria, haemophilus influenza §Low toxicity: low pregnancy risk §Side effects: GI upset, diarrhea; Allergy; Yeast infections

Important Antibiotics in Dentistry

§Penicillin-class antibiotics §Lincosamide and Macrolide antibiotics §Clindamycin §Erythromycin, Arithromycin §Tetracyclines §Metronidazole

Antibiotic resistance mechanisms involve ...

§Pumps §Inactivating enzymes & Decoy proteins §Mutations of target sites §Alternate metabolic pathways Obviously, bacteria want to survive, and have evolved antibiotic resistance mechanisms to counteract those molecules. Many antibiotic mechanisms that are transferrable between bacteria through transposons, plasmids and bacteriophages are molecular pumps that bind and eject those antibiotic substances from the cellular environments. An example of this are tetracycline pumps that eliminate tetracycline from the inside of the cells and allow bacteria to continue protein synthesis. Another type of protein that can inhibit antibiotics are decoy proteins and inactivating enzymes that bind and destroy antibiotics. This includes beta-lactamase which is an enzyme that destroys penicillins and cephalosporins. A simpler method of achieving bacterial resistance is by simply by mutation of the affected enzyme. If the binding site of the antibiotic is altered through mutation, it likely will not bind and the antibiotic becomes ineffective. Last, bacteria may circumvent the effect of some antibiotics, especially antimetabolites, if they can use alternative pathways that can by pass the inhibition. Needless to say, all these provide evolutionary advantages to bacteria possessing them, and if an antibiotic is present in the environment, it will select those bacteria and allow spread of antibiotic resistance.

Bacteria have mobile genetic elements.

§Transposons §Plasmids §Bacteriophage Unlike mammalian cells which usually don't share genetic materials, prokaryotic cells typically contain genetic elements that can be exchanged with other cells quite readily. Commonly there are three mechanisms: Transposons, Plasmids and Bacteriophages.

Why does gene transfer matter?

§Vertical gene transfer: §parent bacteria à offspring bacteria §Horizontal gene transfer §Bacteria à other nearby bacteria §Why does this matter? §Transfer of antibiotic resistance (i.e. MRSA) §Transfer of pathogenic molecules (i.e. Shiga toxin between Escherichia and Shigella bacteria)