Lecture 4

Identifying and Classifying Microorganisms (Chapter 10)

• Phenotypic characteristics can be used to identify microorganisms
• These methods do not require sophisticated equipment
• Microscopic morphology is used to determine size, shape and staining characteristics of a microbe
• Microscopy can provide enough information to make a presumptive identification, e.g., Trichomonas (protozoa) & yeast in vaginal secretion.
• Round worms' eggs in stool can be identified by their shape and size.
• Gram staining is a rapid differential method, but it is generally not sensitive or specific enough to diagnose the cause of infections
• Gram staining can be useful in narrowing the possible identities of a microbe
• Gram staining can provide enough information to start appropriate antimicrobial therapy, e.g. Streptococcus pneumoniae (Gram +ve encapsulated diplococci) or Neisseria gonorrhoeae (Gram −ve diplococci in a urethral secretion)
• Some microbes have unique characteristics that can be detected with special staining; e.g., M. tuberculosis (acid-fast stain)

Culture Characteristics

• Colony morphology gives initial ideas to identify certain microbes
• Colonies of streptococci are generally small compared to staphylococci
• Colonies of Serratia marcescens are red when incubated at 22°C (pigment production) and white at 37°C
• Pseudomonas aeruginosa produces a soluble greenish pigment that discolors the growth media and has a distinct fruity odor.
• Blood agar media is a type of differential media
• MacConkey agar is both selective and differential media

Metabolic Capabilities

• Culture characteristics can give initial ideas of the identity of bacteria
• Biochemical tests are necessary for a more conclusive diagnosis
• Some biochemical tests are rapid, detecting the presence of enzymes; however, most tests generally require at least 18 hours of incubation
• Catalase: most bacteria that can survive in the presence of oxygen are catalase +ve and break down H₂O₂ to release O₂, which causes bubbling
• Streptococcus is an important exception, being lactic acid bacteria
• If a throat culture has beta-hemolytic colonies on blood agar and is catalase positive, Streptococcus pyogenes can be ruled out.

Biochemical Tests

• Most biochemical tests are based on a pH indicator or chemical reaction that results in a color change when a compound is degraded, e.g., no color change indicates sugar has not yet been used
• Fermentation of sugar often produces acid, which lowers pH causing a color change (pink to yellow); gas production can also occur
• Urea degradation by urease creates CO₂ & ammonia, leading to a bright pink medium in alkaline conditions

Biochemical Tests cont'd

• Strategy: use of a dichotomous key, for identifying bacteria
• A flow chart of tests gives either a positive or negative result
• Biochemical tests are usually initiated simultaneously to help speed up the identification process

Biochemical Tests cont'd

• In some biochemical tests, the organism can be tested without culturing it e.g., breath test for Helicobacter pylori
• A urea-containing drink labelled with a carbon isotope is ingested. H. pylori presence causes the release of labelled carbon dioxide. The levels are measured in exhaled air. It is less invasive than biopsy and is faster and cheaper.
• Modifications to traditional biochemical tests include the use of rapid test strips, biolog microtiter plates and Vitek 2 automated system, which use miniature cards.

Serological Testing

• Proteins & polysaccharides present on the bacterium surface can be used as identifying markers
• The most useful markers are the molecules that are part of surface structures such as cell wall, capsule, flagella, and pili
• Antibodies to surface proteins and polysaccharides are frequently used for identifying various bacteria
• Methods that use antibodies to detect antigens are called serology
• Some serological tests are specific, simple, and rapid e.g. to identify Streptococcus pyogenes (cell wall components)

Protein Profile

• A relatively new technology for determining organism's protein profile is MALDI-TOF (matrix assisted laser desorption ionization-time of flight mass spectrometry)
• The technology is important in clinical laboratories to identify bacterial isolates quickly for patient care because of its speed (<15 minutes)

Identification Methods Based on Genotype

• Used in the identification of microbes that are difficult to cultivate
• Nucleic acid probes and nucleic acid amplification tests are used to detect specific nucleotide sequences, which can characterize particular microbial species
• DNA probes bind to single-stranded DNA from an unknown organism, if a match is found, it is the species being tested

Nucleic Acid Probe Method

• Fluorescence in situ hybridization (FISH)
• Nucleic acid probes are used to identify intact microbes in environmental & clinical samples
• FISH uses 16S rRNA-specific probes

Nucleic Acid Amplification Tests (NAATS)

• Amplify specific nucleotide sequences of microbes from samples such as body fluids, soil, food, and water
• Used to detect microbes that are present in extremely low numbers and those that cannot be cultured
• Polymerase chain reaction (PCR) is one of the most common NAATS
• After ~30 PCR cycles, the amplified DNA fragment is visualized on an ethidium bromide-stained agarose gel
• Alternatively, a DNA probe can be used to detect the amplified DNA

Sequencing Ribosomal RNA Genes

• Ribosomal RNA (rRNA) genes are highly conserved and stable for identifying and classifying microbes
• The three main types of rRNA are 5S, 16S, and 23S rRNA
• Some regions of 16S rRNA are virtually the same in all prokaryotes, whereas others have quite variable sequences
• For some organisms, the 16S rDNA variable region is used to identify organisms, particularly useful for microbes that are difficult or impossible to culture

Whole Genome Sequencing (WGS)

• WGS is a feasible method for identifying isolates
• The data from WGS is useful to predict antibiotic resistance and study disease outbreaks

Characterizing Strain Differences

• Sometimes it is desirable to distinguish among different strains of bacteria when only certain strains cause disease
• Examples include E. coli and Salmonella strains that cause intestinal disease.
• Strain determination is useful in tracing the source of outbreaks (foodborne or healthcare-associated diseases)
• It's also used in bioterrorism and biocrimes investigations and in diagnosis of many diseases
• Methods used to distinguish strains include: biochemical typing, serological typing, whole genome sequencing, phage typing and antibiograms

Biochemical Typing

• Biochemical tests are used in identifying different species and also to distinguish between various strains
• A biovar or biotype is a strain that has a characteristic biochemical pattern, e.g., a biochemical variant of Vibrio cholerae called El Tor caused a worldwide epidemic

Serological Typing

• Proteins & carbohydrates that differ among various strains can be used as markers to differentiate strains
• E.g., E. coli vary in the O antigen of LPS (lipopolysaccharide) and H antigen of flagella (0157:H7 refers to the structure of LPS and its flagella).
• A group of strains with cell surface antigens different to other strains are called a serovar or serotype

Whole Genome Sequencing (WGS)

• WGS can be used to detect subtle differences among phenotypically identical strains
• Molecular typing can be done by comparing the restriction fragment length polymorphisms (RFLPs) produced by restriction enzymes used in digesting genomic DNA

Phage Typing

• A bacteriophage/phage is a virus, which infects and multiplies within bacteria and causes lysis of the infected cell

• Different strains of bacteria have different susceptibilities to various types of phages

• The clearing patterns around the bacteriophage spots indicate the susceptibility of the test organism to different phages

• Different patterns can be used to determine the strain differences

• Phage typing is largely replaced by molecular methods

Antibiograms

• Antibiotics susceptibility patterns or antibiograms are used to distinguish different strains
• Different strains will have different patterns of clearing around antibiotic disks
• This method has largely been replaced by other molecular methods, including WGS

The Eukaryotic Members of the Microbial World (Chapter 12)

• Only selected topics will be discussed from Chapter 12 that are relevant to the course
• Fungi, protozoa, algae, arthropods, and multicellular parasites will be included
• Many eukaryotic members are microscopic
• Techniques used for bacteria and Achaea are similar
• Many of these organisms cause disease in plants, humans, and animals
• The terms fungi and protozoa describe non-photosynthetic members; algae describes the photosynthetic members of eukaryotes

Fungi

• A study of fungi is known as mycology
• Fungi includes yeast, mold, and mushroom
• The terms yeast, mold, and mushroom describe the morphological forms (not necessarily the classification)
  • Yeast are single-celled fungi
  • Molds are multicellular filamentous fungi
  • Mushrooms are simply the reproductive structures of certain fungi, some of which are edible
• Fungi can be macroscopic or microscopic
• All fungi have chitin in their cell walls; some also have glucan in their cell walls
• Glucan synthesis is a target for anti-fungal medications
• Fungal membranes contain ergosterol, while animal cell membranes contain cholesterol. Ergosterol is also a target for anti-fungal medication
• Fungi prefer organic compounds for energy and carbon source, often dead organisms
• Most fungi are aerobic; some yeasts are facultatively anaerobic and perform alcoholic fermentation, producing ethanol.
• Fungi are found in virtually every habitat with organic matter; terrestrial habitats are common locations
• Some fungi species can tolerate high concentrations of salt, sugar, or acids to the point that these conditions kill most bacteria
• Fungi are a common reason for food spoilage.
• Some fungi are resistant to pasteurization and to UV rays
• Fungi prefer moist environments with a relative humidity of 70% or more
• Mildew & molds grow in damp environments and cause allergies and other health problems.
• Various fungal species can grow in a range of temperatures (6-50°C), with an optimum range of 20-35°C.
• Most fungi thrive in acidic environments with a lower pH, often in fruits and vegetables.

Reproduction in Fungi

• The reproductive cells of fungi come in a variety of sizes and shapes
• The term "spore" refers to reproductive cells that can either be formed sexually or asexually
  • The asexual reproductive cells of most molds are called conidia
• Some yeasts reproduce by binary fission (mitosis). Others reproduce by budding, where a small outgrowth produces a new cell
• Cell eventually die after producing a number of buds
• Molds have hyphae (filaments), which grow into a mycelium (network of filaments) in one place
• Reproductive spores are typically a single cell and about 3-30 µm in diameter, depending on the species
• Spore germination in fungi begins when the spore grows hyphae in the direction of a food source
  • Accumulation of hyphae form the mycelium

Fungi cont'd

• Dimorphic fungi can exist in two forms, such as yeast-like cells and mycelia, based upon environmental conditions
• Many fungi cause disease in plants
• Only a few species cause disease in humans; examples include athlete's foot and jock itch.
• However, immunocompromised hosts are most vulnerable to fungal diseases
• Dimorphic fungi such as Coccidioides immitis and Histoplasma capsulatum can grow as molds in soil and as yeast in the lungs

Medically Important Fungi

• (A table showing different groups of fungi and associated diseases)

Fungi cont'd

• If disease is caused directly by fungi, then it is called a mycosis
• Diseases caused by fungi can result from intoxication (e.g., ingestion of fungal toxins, such as aflatoxins from Aspergillus species), allergies, or hypersensitivity to fungal components (e.g., spores are a common cause of asthma and hay fever).

Economic Importance of Fungi

• Yeast (Saccharomyces) is used in the production of wine, beer, and bread, and in cheese-making
• Saccharomyces cerevisiae is used in genetic and biochemical studies
• Penicillin, griseofulvin, and other antimicrobial agents are synthesized from fungi
• Yeast are genetically engineered to produce human insulin, human growth hormone, somatostatin, and a hepatitis B vaccine
• Fungi commonly spoilage food products, leading to starvation

Protozoa

• Protozoa are microscopic, unicellular organisms that lack photosynthetic capability
• Protozoa are typically motile at some stage of their life cycle
• Protozoa most commonly reproduce asexually by fission
• Protozoa live in various environments such as marine, freshwater, or terrestrial environments
• Protozoa require large amounts of moisture
• Protozoa lack the rigid cell wall found in algae or fungi
• Protozoa have a specific shape determined by the rigidity or flexibility of the material beneath the plasma membrane
• Protozoa may have flagella, cilia, or pseudopodia for movement
• Protozoa use pinocytosis or phagocytosis to obtain food and water
• Protozoa may be polymorphic depending on the stage of life cycle (either a trophozoite-feeding form or a cyst-resting form).
• Cysts allow for the dispersal and survival of protozoa under harsh conditions, much like bacterial endospores
• Protozoan cysts are not as resistant as bacterial endospores

Medically Important Protozoa

• (A table listing various groups of protozoa and the diseases they cause)

Algae

• Algae are simple photosynthetic eukaryotes
• Algae use light energy to convert carbon dioxide and water into carbohydrates
• Algae are primarily aquatic, and can be either macroscopic or microscopic, unicellular or multicellular
• Algae have rigid cell walls primarily composed of cellulose.
• Algae usually do not cause disease directly in humans, but some algal toxins can be poisonous to humans when consumed (e.g., Paralytic Shellfish Poisoning)
• Some algal toxins are not harmful to the shellfish but can accumulate in the shellfish and then cause illness in humans when consumed

Next Lecture

• Read chapter 13 (Viruses, Viroids, and Prions)

Test your knowledge on microbial identification methods with this quiz. Explore various phenotypic methods, microscopic morphology, and biochemical tests essential for identifying bacterial species. Perfect for microbiology students or enthusiasts looking to deepen their understanding.