Lecture 2.2 - Bacteriology PDF

Lecture 2 Bacteria Lecture 3 1  General appearance of bacteria Lecture 3 2  Characteristics of bacteria Microscopic organisms Prokaryotes Lack a true nucleus Mostly have one chromosome Exa...

Lecture 2 Bacteria Lecture 3 1  General appearance of bacteria Lecture 3 2  Characteristics of bacteria Microscopic organisms Prokaryotes Lack a true nucleus Mostly have one chromosome Examples with more than one exist but are the exception e.g., Borrelia burgdorferi DO NOT all look the same Lecture 3 3  Size of bacteria Bacterial colonies can be visualised on agar plates Different species of bacteria will take on different shapes and colours on the same growth medium. The next few slides have little relevance to the module but show how differences can be seen. Lecture 3 4 American Society for Microbiology 12 May 2016 Title of Piece: Alexander Flamingo under a Cherry Tree Cherry tree and flamingo are made out of four different enteric pathogens: Klebsiella pneumoniae (pink), Citrobacter freundii (magenta to black), Salmonella typhimurium (black), and Morganella morganii (brown). K. pneumoniae, C. freundii, and M. morganii are commonly associated with hospital-acquired infections. Serotypes of Salmonella enterica cause salmonellosis (food poisoning), and the infamous serotype, Salmonella enterica subsp. enterica Typhi causes life-threatening illness, Typhoid fever. Salmonella-Shigella agar is commonly used in medical microbiology labs to selectively grow enteric pathogens and differentiate based on the organisms’ metabolism. K. pneumoniae and C. freundii ferment lactose, reducing pH of agar and causing bile salt precipitation and color change of neutral red from yellow to red. C. freundii and S. typhimurium use sodium thiosulphide to produce hydrogen sulphide, which reacts with ferric citrate to turn black. M. morganii does not ferment lactose nor produce hydrogen sulphide. The metabolism of non-lactose fermenters increases the pH of agar, which changes the colour of neutral red from red to yellow. Lecture 3 5  American Society for Microbiology – Agar Art (posted on 19 April 2017) I am a hungry frog Who lives in a mysterious bog. Somebody drew this fly as my bait So, I can appear on this agar plate. And that's how I got a moment to shine And hypnotize you with my beautiful smile. Serratia marcescens made my tongue red To show to the flies it's a serious threat. The skin colour of florescent green Came from Pseudomonas pigment pyoverdin. And my bloated belly of yellow Was painted by Staphlococcus aureus fellow. Who also made the sun rise in the sky In order for me to catch a silly fly. But, if you like me on Facebook, you bet I will keep this fly as my favourite pet! Lecture 3 6  American Society for Microbiology 23 May 2017 Dancing Microbes The artist, Ana Tsitsishvili from Tbilisi, Georgia, won third place with this arrangement of bacteria and fungi on brain-heart infusion agar. The common skin microbe, Staphylococcus epidermidis, is responsible for the white color; Rhodotorula mucilaginosa, common in milk, soil, and air, makes pink; Micrococcus luteus, frequently found in soil, water, air, and skin, is responsible for the lady's luscious yellow locks; Xanthomonas axonopodis, a pathogenic plant microbe, makes green. Combinations of these various microbes make up everything in between. Lecture 3 7  We have also tried this at RGU! Lecture 3 8  Size of bacteria To visualise bacteria, other than in colony form it is necessary to use microscopy E. coli cells This can be done using a light microscope at x 1000 magnification (oil immersion) Alternatively use electron microscopy Salmonella cells Lecture 3 9  Viewing bacteria – use of dyes Dyes are often used to stain cells to make them easier to see. E. coli cells – phase contrast Different dyes exist, each with benefits and disadvantages Many rely on cationic organic compounds combining with the negatively charged cell envelope E. coli cells – bright field following Gram staining Lecture 3 10  Viewing bacteria – Gram-staining One of the most common methods to stain bacteria is called Gram- staining It is named after the person who Hans Christian invented it; H. C. Gram, a Danish Gram (1853 –1938) bacteriologist Lecture 3 11  Viewing bacteria – Gram-staining Not all bacteria stain equally with Gram-staining. The process involves a series of steps – see image and at the end of the staining process cells will generally be one of two colours Lecture 3 12  Viewing bacteria – Gram-staining The differences in staining patterns result from differences in the structure of the bacterial cell wall – next slide Normally there is a standard staining pattern for a particular species. A Gram stain of mixed bacterial culture. Note that in a few organisms the cell wall Staphylococcus aureus ATCC 25923 (Gram-positive cocci) stained purple and structure changes under different growth Escherichia coli ATCC 11775 (Gram-negative bacilli) conditions – leading to a difference in Gram- stained red/pink. staining These species are known as Gram-variable organisms Lecture 3 13  Structures within bacteria Lecture 3 14  Bacterial structure Bacteria are unicellular The have clearly identifiable sub- cellular features Lecture 3 15  Bacterial cell wall - composition There are a number of differences between Gram- positive and Gram-negative walls Differences include wall thickness Gram-positive ~ 20-80 nm thick Gram-negative ~ 7-8 nm thick Lecture 3 16  Gram-positive cell wall - composition The Gram-positive wall is ~ 90% peptidoglycan This includes teichoic acids For more information on teichoic acids see: Baddiley J. (1970) Structure, biosynthesis, and function of teichoic acids. Accounts in Chemical Research Volume 3, pp 98-105 http://pubs.acs.org/doi/pdf/10.1021/ ar50027a003 Lecture 3 17  Gram-negative cell wall - composition The Gram-negative wall is ~ 5-20% peptidoglycan They lack teichoic acids There is an additional membrane layer present. Lecture 3 18  Lipopolysaccharide Layer The outer layer in Gram- negative bacteria is known as the LPS (lipopolysaccharide layer) Exists as a lipid bilayer Surrounds the peptidoglycan and cytoplasmic membrane Lecture 3 19  Bacterial spores Lecture 3 20  Bacterial endospores Some bacterial species produce internal spores Examples include species from the genera Clostridium and Bacillus These endospores are highly resistant to heat and many antimicrobials Therefore, they can persist in an environment for long periods of time Lecture 3 21  Bacterial endospores Endospores were first reported by John Tyndall in the 19th Century Treatment with chemicals normally able to kill cells (e.g., alcohols, hydrogen peroxide) failed Heat treatment at 100ºC failed to kill John Tyndall (1820 – 1893) endospores – although autoclaving at 121ºC is generally successful Also, highly resistant to UV radiation, gamma radiation, etc. Lecture 3 22  Bacterial endospores Endospores have a highly complex structure There are multiple layers associated with them Stewart G. C. (2015) Microbiology and Molecular Biology Reviews Vol. 79: pp 437-457 http://mmbr.asm.org/content/ 79/4/437.full Lecture 3 23  Case study: Anthrax Lecture 3 24  Case study: Anthrax The persistence of spores for long periods of time allows sporulating bacteria to survive in niches with variable nutrient supplies Good examples of this are organisms which can survive in soil Note that some medically-important organisms are able to survive in soil for long periods of time (e.g., Bacillus anthracis) Lecture 3 25  Case study: Anthrax Anthrax once able to divide again (i.e., after germination) can infect animals The toxins they produce can cause severe illnesses and even death. Anyone working with anthrax Warning needs to wear protective clothing sign from as seen in the bottom photo. 1986 Lecture 3 26  Case study: Anthrax Gruinard Island was infected with anthrax in 1942 Part of a biological warfare test during World War II Tested on sheep – which started to die after 3 days https://www.pressreader.com/ usa/id-magazine/ 20211101/281891596340438 Lecture 3 27  Case study: Anthrax Gruinard Island is approximately 2 km long and 1 km wide (196 hectares) In 1986, 80 tonnes of formaldehyde diluted in sea water was sprayed on the island In 1990 it was declared fit for habitation Lecture 3 28  Bacterial Genetics 3 December 2 29 024  Bacterial Genetics Bacteria have relatively small genomes Very little redundant DNA Around a couple of thousand genes Seldom (if at all) use introns Lecture 4 30  Bacterial Genetics Able to respond quickly to environmental changes Gene expression levels change rapidly (both upregulation and downregulation) Lecture 4 31  Bacterial Genetic Components Bacterial cells (generally) have only one chromosome – but they can have other forms of DNA present Lecture 4 32  Bacterial Plasmid DNA chromosome DNA Bacterial Genetic Components Bacterium with single plasmid Most of the essential genes are located on the bacterial chromosome (single copy) There are some other genetic Bacterium with more than one copy of a single plasmid components (plasmids) which generally contain non-essential (luxury) genes – can be multiple copies Bacterium with more than one kind of plasmid Lecture 4 33  Bacterial Genetics Generalities Single chromosome Genes - structural components and metabolism “Housekeeping genes” Only single copies of genes on chromosomes Plasmids Genes - antibiotic resistance, virulence, conjugation “Luxury genes” Often several copies of plasmids Lecture 4 34  Bacteria can be under “attack” from different sources of DNA Transformation Conjugation Transduction Lecture 4 35  Free DNA can infect bacteria by a process known as transformation Lecture 4 36  Bacterial Genetics Evidence for transformation Griffith F. (1928) Journal of Hygiene 27: 113-159 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2167760/pdf/jhyg00267- 0003.pdf Work carried out with Streptococcus pneumoniae Lecture 4 37  Transformation Recipient cell acquires DNA – normally from lysed cells Can occur naturally in some recipient species / genera e.g., Bacillus; Streptococcus; Haemophilus; Neisseria Lecture 4 38  Transformation A cell which can be transformed is known as a competent cell For cells which are not naturally competent, it is often possible to artificially make them competent Used for E. coli to allow genetic engineering to occur. Lecture 4 39  Plasmids can be passed from one bacterium to another using a process called conjugation Lecture 4 40  Conjugation Not all bacterial cells contain plasmids Lecture 4 41  Conjugation Conjugation requires cell-cell contact (normally same species) Contact initiated by the pilus on the donor Transfer of DNA is unidirectional Lecture 4 42  Conjugation Pilus of donor attaches to recipient Pilus disassembles drawing recipient closer Cell wall / cytoplasmic membrane fuse forming conjugation bridge Lecture 4 43  Conjugation DNA can be moved through the bridge from the donor to the recipient Only one strand is transferred – the donor keeps the other. Lecture 4 44  Conjugation Recipient can now replicate the DNA to become double stranded Donated DNA is the same in both cells now – both potential donors Lecture 4 45  Conjugation Transfer is facilitated by plasmids Example of this F+ strain passing plasmid to F- strain, which becomes a new F+ strain Plasmid carries all genes needed for its own transmission Lecture 4 46  Viruses in bacteria These are also referred to a bacteriophages (phages) Literally this means something which eats bacteria Actually, results in production of plaques as they kill bacteria in a radiating manner Lecture 4 47  Properties of Viruses Lecture 4 48  Bacterial viruses or phages infect bacteria by a process known as transduction Lecture 4 49  Bacteriophage Therapy Bacteriophages are viruses which infect bacteria discovered in 1915 Their potential therapeutic uses were described in the 1920s Used against dysentery (shigellosis) and staphylococcal skin infections Still used as a treatment in some Eastern European countries Used during WWII by Soviet and German armies to treat dysentery Lecture 8 50  Bacteriology in the Medical Context 3 December 2 51 024  Notifiable organisms as of 3rd August 2023 https://www.gov.uk/guidance/notifiable-diseases-and-causative-organisms-how-to-report#list-of-notifiable- diseases Organism Bacillus anthracis Chlamydophila Giardia lamblia Leptospira Rabies virus Variola virus psittaci interrogans Bacillus cereus Clostridium Guanorito virus Listeria Rickettsia virus Verocytotocigenic (food poisoning) botulinum, monocytogenes Escherichia coli perfringens, tetani Bordetella pertussis Corynebacterium Haemophilus Machupo virus Rift Valley fever Vibrio cholerae diphteriae, ulcerans influenzae virus Borrelia spp Coxiella burnetii Hanta virus Marburg virus Rubella virus West Nile virus Brucella spp Crimean-Congo Hepatitis A,B,C, Measles, Sabia virus Yellow fever virus haemorrhagic fever delta, E virus Monkeypox, Mumps virus virus Burkholderia mallei Cryptosporidium spp Influenza virus Mycobacterium Salmonella spp Yersinia pestis tuberculosis Burkholderia Dengue virus Junin virus Neisseria SARS-CoV-2 pseudomallei meningitidis Campylobacter spp Ebola virus Kyasanur Forest Omsk haemorrhagic Shigella spp disease virus fever virus Carbapenemase- Entamoeba Lassa virus Plasmodium Streptococcus producing gram- histolytica falciparum, vivax, pneumoniae, negatives ovale, malariae, pyogenese knowlesi Lecture 2 52  Diagnostic Microbiology Often causative pathogen must be identified This is necessary to have full and effective treatment Lecture 9 53  Identification of organisms After reaching lab identify the organism Staining (e.g., Gram-stain – year 1 labs) Specific growth medium – already have some idea about identity Biochemical assays Lecture 9 54  Types of Media Assuming you can make a guess at the organism – based on type of infection – select conditions: Aerobic versus anaerobic Optimal temperature - or temperature range Optimal pH – e.g., stomach versus colon Length of time required for growth Salt tolerance Haemolysis Degradation of certain compounds, e.g., sugars Lecture 9 55  Selective and Differential Media It is unlikely that the sample collected will be pure There will probably be several different species in it Need to grow pathogen alone Lecture 9 56  Phenotypic Identification Phenotypic feature used in Proteus mirabilis characteristic concentric screening: ripple-pattern growth Colony morphology Gram staining Microscopic morphology Cell association / aggregation Grow on selective / differential media Biochemical / metabolic profile Lecture 9 57  Biochemical Phenotype Pure culture are grown Then perform biochemical analyses Figure shows catalase breaking down H2O2 Staphylococcus spp. (catalase positive) Streptococcus spp. (catalase negative) Lecture 9 58  Biochemical Phenotype Many other tests can be performed Forms the basis of the API strip test produced by Biomerieux http://www.biomerieux- usa.com/clinical/api Lecture 9 59  Molecular Diagnostics Analysis of DNA can also be used Requires some knowledge of DNA sequences from organism Generally, uses 16S rRNA 16S rRNA gene Lecture 9 60