Microbial Uses in Biotechnology

The Structure of Microbes Prokaryotes Archaebacteria Includes halophiles, thermophiles, “extremophiles” Eubacteria On skin, pathogens, soil, water Generally smaller than Eukaryotes (1-5μm vs μm) What are some other characteristics of prokaryotes? (cell wall (gram stain), no nucleus, binary fission, 20 min growth rate…) Do you know how to isolate single colonies?

Microorganisms as Tools Microbial Enzymes Taq (DNA polymerase), cellulases, proteases, amylases Bacterial Transformation The ability of bacteria to take in DNA from their surrounding environment Cells must be made competent (to take up DNA)

Electroporation A mixture of bacteria and plasmid are briefly electrically shocked

Cloning and Expression Techniques Fusion Proteins Use recombinant DNA methods to insert the gene for a protein of interest into a plasmid containing a gene for a well-known protein that serves as a “tag” The tag allows for isolation and purification Ex. GFP – Green Fluorescent Protein Used to follow certain molecules through cell processes

Microbial Proteins as Reporters Examples: the lux gene which produces luciferase Used to develop a fluorescent bioassay to test for TB (the lux gene is in a virus that only infects M. tuberculosis). If the bacteria is present, the virus infects the cells and the bacterial cells glow!

Using Microbes for a Variety of Everyday Applications Food Products Rennin used to make curds (solid) and whey in production of cheese Recombinant rennin is known as chymosin (first recombinant food product approved by FDA)

Therapeutic proteins Recombinant insulin in bacteria What is Type I diabetes (insulin-dependent diabetes mellitus) Inadequate production of insulin by beta cells in the pancreas

Field Applications of Recombinant Microorganisms Ice-minus bacteria (remove ice protein producing genes from P. syringae) P. fluorescens containing the gene that codes for the bacterial toxin from Bacillus thuringiensis (kills insects) Bt toxin!

Using Microbes Against Other Microbes Antibiotics Penicillin was the first Act in a few key ways Prevent replication Kill directly Damage cell wall or prevent its synthesis How do antibiotic resistant strains arise? How can studying bacterial pathogens lead to new drugs?

Microbial Genomes Microbial Genome Program (MGP) –the goal is to sequence the entire genomes of microorganisms that have potential applications in environmental, biology, research, industry, and health Sequencing Strategies 

Microbial Diagnostics Using Molecular Techniques to Identify Bacteria RFLP - restriction fragment length polymorphism PCR – Polymerase Chain Reaction PulseNet (Contaminated food) - a network run by the Centers for Disease Control and Prevention (CDC) which brings together public health and food regulatory agency laboratories around the United States

Combating Bioterrorism The use of biological materials as weapons to harm humans or animals and plants we depend on for food Examples in History Throwing plague infected dead bodies over the walls of their enemies Using Biotech Against Bioweapons Postal service x-raying packages Antibody tests in the field PCR tests in the field

RISE OF THE SUPERB UGS! An intro to bacteria, infectious diseases, and antibiotic resistance

RISE OF THE SUPER BUGS! An intro to bacteria, infectious diseases, and antibiotic resistance

How abundant are bacteria? Bacterial Abundance Total bacteria on Earth5 x Number of stars in the universe7 x Age of the universe in seconds4.4 x Bacteria in the human gut1 x Global gross product ($/year)7 x Cells in the human body1 x Texts sent in x People on Earth6.9 x X

How many types of bacteria are there? Bacterial Distribution Insect Species1-10 million Bacterial species in the soil4 million Bacterial species in the air4 million Bacterial species in the ocean2 million Bird Species10,000 Bacterial species in the human mouth600 Bacterial Species in the human gut500 Pathogenic Species55

Infectious Diseases Pathogen: a biological agent that causes disease Pathogen: a biological agent that causes disease Can be a virus, bacteria, fungi, protozoa, or parasite Can be a virus, bacteria, fungi, protozoa, or parasite Infectious diseases are communicable or transmissible from one person to another Infectious diseases are communicable or transmissible from one person to another

Virus: Influenza (H1N1) Fungi: Plant pathogen (Rigidoporus laetus) Protist: Malaria (Plasmodium)

Mycobacterium tuberculosisSalmonella typhimurium Staph Infection: Staphylococcus aureus

The Plague First recorded over 5,000 years ago First recorded over 5,000 years ago The teeth of individuals from Russia, Eurasia, and Estonia contained the plasmids of the bacterium Yersinia pestis (Y. pestis) The teeth of individuals from Russia, Eurasia, and Estonia contained the plasmids of the bacterium Yersinia pestis (Y. pestis) Amulet to ward off plague inscribed with a quotation from the Akkadian Erra Epic BCE

The Plague The Three Pandemics The Three Pandemics Plague of Justinian 541–542 CE Mostly bubonic; est. 100 million deaths Mostly bubonic; est. 100 million deaths The Black Death present somewhere in Europe in every year between 1346 and 1671 CE Again mostly bubonic Poor census records disallow for accurate death toll

The Plague The Three Pandemics The Three Pandemics The Black Death present somewhere in Europe in every year between 1346 and 1671 CE Again mostly bubonic Poor census records do not allow for accurate death toll

The Plague The Three Pandemics The Three Pandemics began in China's Yunnan province in 1855 Circumvented the globe though mainly found in China and Russia finally eradicated in 1959 in Maui and Hawaii

The Plague Plague as a Weapon ancient China and medieval Europe detail the use of infected animal carcasses, such as cows or horses to contaminate enemy water supplies Infected human carcasses were catapulted over walls during sieges

The Plague Unit 731 Unit 731 a covert biological and chemical warfare research and development unit of the Imperial Japanese Army that undertook lethal human experimentation during the Second Sino-Japanese War (1937–1945) of World War II

The Plague Unit 731 Unit 731 Japanese researchers performed tests on prisoners with Bubonic plague, cholera, smallpox, botulism led to the development of the flea bomb used to spread bubonic plague Japan planned to use plague as a biological weapon against San Diego, California. on September 22, 1945

The Plague

What causes symptoms? What causes symptoms? Bacterium Yersinia pestis Bacterium Yersinia pestis Gram-negative, rod-shaped coccobacillus

The Plague Found in fleas Found in fleas Which are found on Which are found onrodents

The Plague Symptoms Symptoms

The Plague Symptoms – Symptoms –Bubonic

The Plague Symptoms – Pneumonic Symptoms – Pneumonic Healthy Lung Plague Lung

The Plague Symptoms - Septicemic Symptoms - Septicemic

Antibiotics Compounds that kill bacteria or inhibit their growth Compounds that kill bacteria or inhibit their growth Naturally produced by microorganisms to kill others Naturally produced by microorganisms to kill others Now many are made synthetically Now many are made synthetically

Why is resistance bad? Because you can’t treat the disease! Because you can’t treat the disease! The patient will remain sick  The patient will remain sick 

How do you get resistance? Usually from mutation in DNA Usually from mutation in DNA Genetic mutations can be passed to offspring… Genetic mutations can be passed to offspring… Leads to evolution! Leads to evolution!

Genetic Mutation NATURAL SELECTION!

Susceptible + Antibiotic Antibiotic Resistant

What are MRSA and VRE?

MRSA MRSA stands for Methicillin-Resistant Staphylococcus aureus MRSA are gram-positive Staph aureus bacteria that have become resistant to this antibiotic.

MRSA In the United States approximately 60% of ICU staphylococcal infections are now caused by MRSA hospital-acquired MRSA (HA-MRSA) typically spread from patient to patient, frequently using healthcare personnel as intermediaries Community-acquired MRSA (CA-MRSA) is also increasing

And now, let’s look at what MRSA really looks like…

Vancomycin-Resistant Enterococci (VRE) Enteroccocci bacteria normally in human intestines, female genital tract, and the environment can sometimes cause infections Vancomycin is an antibiotic which in some instances, enterococci have become resistant to Most VRE infections occur in hospitals.

And now, let’s look at what VRE really looks like…

NDM-1 New Delhi Metallo-beta-lactamase-1 (NDM-1) an enzyme that makes bacteria resistant to a broad range of beta-lactam antibiotics first detected in a Klebsiella pneumoniae isolate from a Swedish patient of Indian origin in 2008 Later detected in India, Pakistan, the United Kingdom, the United States, Canada, and Japan

Usually found in: Escherichia coli and Klebsiella pneumoniae, but the gene for NDM-1 can spread from one strain of bacteria to another by horizontal gene transfer!

KPCs Klebsiella pneumoniae carbapenemases (KPCs) 2001, North Carolina - first KPC-producing K. pneumoniae isolate reported mechanism of resistance for a range of Gram- negative bacteria often not detected by routine susceptibility screening present serious treatment challenges, due to limited antibiotic options

Usually found in: Klebsiella pneumoniae, no longer limited to K. pneumoniae Enterobacteriaceae-producing KPCs have also been reported in Brazil, China, Colombia, Norway, United Kingdom, India, Sweden, and more recently, Italy and Finland

Acknowledgements The Infectious Disease Control Unit of the Department of State Health Services thanks Ginger Shields, RN Emergency Preparedness Specialist Texarkana-Bowie County Health Department for the creation and use of this presentation.