Bacteriology

• They only way to accurately classify an organism as a bacteria is through DNA sequence analysis, because some bacteria have nuclei and organelles.
• Bacterial shapes include cocci, rods, comma shaped, and spirochete. They are much smaller than eukaryotic cells which allows rapid metabolism, so they divide and grow exponentially.
• The main microbiological media are liquid culture, agar/solid culture, selective media, differential media, defined medium and complex medium.
• Liquid culture grows bacteria in flasks to see growth and replication rates or to observe bacterial physiology. Agar/solid culture is used to isolate pure cultures for identification and quantification.
• Selective media is used to isolate specific bacteria and allows growth of only one type of bacteria. Differential media allows growth of multiple bacterial colonies but allows them to be distinguished from each other.
• Defined medium consists of very pure chemicals whereas complex medium contains digests of microbial, plant and/or animal products.
• Microbial growth typically occurs around pH 7.4. Bacteria can be aerobic (use O2 for ATP production) or anaerobic (don't require O2 for ATP production). Growth is also dependent on carbon, nitrogen, sulphur, phosphorous, minerals and pH.
• Aerotolerant anaerobes can tolerate and grow in air whereas obligate anaerobes are killed by oxygen
• The bacterial growth curve consists of lag, exponential, stationary, and death phases. In the lag phase bacterial metabolism is starting and they are adapting to the new environment. In the exponential phase there is rapid growth and metabolism. The stationary phase occurs when growth slows due to waste accumulation and reduced nutrients. During the stationary phase bacteria prepare for survival by producing antibiotics to kill neighbouring cells. The death phase has a decline in cell number.
• Gram staining uses crystal violet and safarin to identify gram positive and gram negative bacteria. Gram positive bacteria have a thick peptidoglycan cell wall whereas gram negative bacteria have an outer membrane and thin peptidoglycan layer. Gram negative bacteria are resistant to many antibiotics as the outer membrane acts as a barrier.
• Clinical microbiology techniques include staining & microscopy, culture, immunoassays and DNA sequencing
• Advantages of staining & microscopy are that it is cheap and fast
• Advantages of culturing are that it is gold standard for diagnosis and can be used to determine specific biochemical traits of bacteria. Disadvantages of culturing are that not all bacteria can be grown easily, it takes time and isn’t always accurate
• Immunoassays use antigens and secondary antibodies to detect antibodies against a bacteria in serum. They are useful if bacteria can’t be cultured. Direct antigen detection and lateral flow tests are examples of immunoassays.
• In DNA sequencing bacterial analysis, PCR amplification and either Taqman assay or qPCR-SYBR green are used to visualise DNA sequences on agarose gel.
• Taqman assay allows bacterial visualisation as Taq cleaves it to release a fluorescent label. qPCR-SYBR Green binds to dsDNA to allow visualisation.
• Advantages of DNA sequencing for bacterial analysis is that it’s quick and useful when culture is hard or long. Disadvantages are that the primers used determine what is found.
• Microbes can be either commensal, obligate pathogens or opportunistic pathogens. Obligate pathogens are always pathogenic whereas opportunistic pathogens are only sometimes pathogenic.
• Some pathogens have evolved to cause latent infections (eg M TB), acquire extra DNA for virulence factors (eg E. coli), and cause disease if they access deep tissue.
• Infections associated with biomaterial implants are often caused by biofilms, commensals, or opportunistic pathogens
• Biofilms are a collection of microorganisms surrounded by a polymatrix of protein, DNA and polysaccharide produced by the microorganisms
• Stages of biofilm formation are: 1. attachment of bacteria to surface using virulence factors 2. multiplication and cell-cell adhesion formation 3. extracellular polymatrix becomes larger and more complex 4. biofilm maturation causes some bacteria to disperse and attach to other sites
• Biofilms are clinically significant as they can protect bacteria from the immune response & antibiotics (to allow colonisation), may be metabolically dormant, can transfer virulence genes to other microorganisms, can communicate w diff species via quorum sensing, and can create new ecological niches for other microbes to exploit
• Biofilms and infections can occur when medical devices are implanted, either during surgery (early infection) or post-surgery (delayed & late infections). These biofilms are hard to treat as they typically have high antibiotic resistance so the implant may need to be removed.
• Surgical infection can be due to the air, instruments, surgical team or patients own skin. Post-surgery infections can be due to skin wounds, catheters, oral infection, or haematogenous infection
• Staphylococci are commensals found on the skin that can become opportunistic pathogens by entering wounds, or growing in biofilms on biomaterials. They are resistant to many antibiotics, can inhibit neutrophil phagocytosis, and can grow well and survive in most conditions.
• Biofilms can form on the inside and outside of catheters, causing catheter associated UTIs (CAUTIs). For example, protium mirabilis commonly causes CAUTIs. Longer term catheter use increases infection risk.
• Biofilm bacterial antibiotic resistance can be via biofilm inhibiting penetration, antibiotic inactivation, bacterial resistance and bacterial dormancy.
• The consequences of implant infection can be implant replacement, bacterial seeding into blood, permanent loss of mobility or death.
• Koch’s postulates suggest pathogens must: be present in all cases of a disease, multiply when inoculated into healthy susceptible host, be recoverable from experimentally infected host, & be isolated from diseased host & grown in pure culture
• Normal flora can prevent infection through colonisation resistance. Colonisation resistance prevents pathogens colonising epithelial surfaces such as in the mouth, nose, skin and small intestine. Antibiotics can remove normal flora and cause superinfection by resistant microbes.
• To be successful, pathogens need to be able to bind to & enter host cells,  survive & avoid host responses, replicate & be transmitted to new hosts.
• Disease is caused by a combination of pathogen & host factors. Pathogens produce molecules that contribute to disease and host responses can often cause pathology.
• Exotoxins are bacterial proteins that cause features of bacterial infection when injected. Vaccination can use inactivated exotoxins to reduce spread of bacterial infection.
• The 8 Ss for successful infection are the ability to: swim, sense env, switch on & off virulence factors, stick, stealth, subvert, spread, and scatter
• Bacterial virulence is multifactorial and requires multiple virulence factors. Virulence factors are needed to colonise and damage tissues, and differentiates pathogens from commensals. Comparative genomics can be used to determine the function of bacterial virulence factors.
• Bacterial mechanisms for successful infection are ability to swim via motile flagella (eg H pylori); sense environmental changes (eg vibrio fischeri use bioluminescence to help host avoid predation); switch virulence factors on and off via transcriptional & post-transcriptional regulation; stick to hosts via adhesins; stealth to avoid immune system (e.g. via capsules, LPS, antigenic mimicry); subversion to manipulate host cells for own needs; spreading via blood, lymph, immune cells, and tropism; and scattering via vectors, aerosols etc to continue transmission.
• N Gonorrhoea are gram negative enterobacteria that cause STIs. They have lots of pili for motility, have Opa proteins to stick to urethral tract epithelial cells, are spread in neutrophils, can supress T cell proliferation, and secrete IgA1 protease which degrades IgA to prevent immune attack.
• Microorganisms incl. bacteria, fungi, microscopic parasites, archaea, viruses, bacteriophages & virophages
• Main sites of human microbiomes are oral cavity, respiratory tract, skin, GIT & urinary tract