INTRODUCTION TO BACTERIAL TAXONOMY

Dr. El-Safey Mohamed El-Safey

Introduction

Since the days of Koch and Pasteur bacteria were mainly classified according to morphological, physiological and biochemical criteria. These properties are usually counted equally and bacteria are grouped together as to how many of these features they have in common. This classical approach is also known as numerical taxonomy and the most widely used textbook in bacterial classification Bergey's Manual of Determinative Bacteriology depends heavily on numerical taxonomy.

More recently however it has become apparent that many of these original criteria have no bearing at all in defining a particular taxonomic group; this includes such important properties such as cell shape (apart from Spirillae), photosynthesis and to a lesser extent Gram-stain. Due to these shortcomings and the paucity in diversity within alternative techniques scientists were for a long time interested in the development of more useful classification tools.

Recent developments in molecular genetics and gene cloning provided exactly these tools giving rise to the new era of molecular taxonomy where a wealth of data can readily be obtained from any microorganisms.

2 BACTERIAL STRUCTURE, CLASSIFICATION, AND PHYSIOLOGY

BACTERIA

• Prokaryotes - lack a nuclear membrane (unlike eukaryotes) • Single-celled • Reproduce by simple division, i.e. binary fission • Cells - small, ~1 µm (mycoplasmas as small as 0.2 µm; bacillus as large as 10 µm) • Chromosome - single, circular, double-stranded DNA (borrelia - linear); up to 1 mm in length; 600 to 4500 kb in size. Smaller = more dependent on host/environment; larger can synthesize more of own constituents. • Extrachromosomal DNA - plasmids may be present. In cytoplasm, replicate independent of chromosome. Usually circular, sometimes linear (Borrelia). Few to several hundred kb. • Most are free-living, a few (rickettsiae, chlamydiae) are obligate intracellular parasites

THE PLACE OF BACTERIA IN THE GRAND SCHEME OF THINGS

Eukaryotes - nuclear membrane (true nucleus) • Animals • Plants • Protists - simple eukaryotes Algae Fungi Protozoa Prokaryotes - no nuclear membrane (primitive nucleus) • Eubacteria - true bacteria. Includes most bacteria, all pathogens, most well-studied. • Archaebacteria - primitive bacteria. Evolutionarily separated from eubacteria. Differences in cell walls (lack PG), membranes (ether- rather than ester-linked lipids), ribosome components and metabolism. Share some features with euk (introns, histones, etc). Groups of archaebacteria: Methanogens - produce methane

3 Halophiles - grow in high salt Thermophiles - grow at high temp Based on evolutionary relationships (more later), 3 domains (kingdoms) suggested: Archaebacteria Eubacteria Eukaryotes

BACTERIAL STRUCTURE

1. nucleoid (nuclear or chromatin body) • DNA (60%; 2-3% dry wt of cell); RNA (30%); protein (10%) • Does not have a nuclear membrane • Haploid chromosome in cytoplasm • 1 to 4 nuclear bodies/cell, number depends on growth rate example: E. coli at 37 o C - doubling time = 20 min. - 4 copies; with dt > 60 min - 1 copy (more later) • No histones, but ~6 chromosome-associated basic proteins involved in determining chromosomal structure • Polyamines, e.g., spermidine and putrescine, neutralize negative charges on phosphates • Size of E. coli chromosome = 3 x 10 9 daltons = 4.7 x 10 6 bp • Roughly 4000 +/- 1000 genes • Circular and double stranded (borrelia - linear) • 1.6 mm - supercoiled (underwound) to fit (occupies 10% of cell volume) • Smaller bacteria like Rickettsia and Chlamydia have chromosomes less than one- third the size of that of E. coli. Smaller chromosome, more dependent on host/environment. 2. cytoplasmic membrane (CM) (inner membrane, IM in gram -) • lipid bilayer with embedded proteins, little carbohydrate. • osmotic barrier, active transport of ions and metabolites, electron transport, oxidative phosphorylation,and photosynthesis (in some bacteria) • 7.5 nm thick • antibacterials: detergents, polymyxins, ionophores disrupt membrane 3. mesosomes - cytoplasmic membrane-associated organelle (more easily seen in gram-positive bacteria); an irregular invagination in the cytoplasmic membrane.

4 • Note: bacteria don't have the membranous organelles of eukaryotes, e.g., endoplasmic reticulum, mitochondria, golgi, etc. • May be attached to the nucleoid • Possible role in replication and cell division (septal meso), maybe in protein export (lateral meso) 4. cell wall (CW) (murein) - shape, barrier (osmotic resistance - CW removed by enzymes that degrade, cells will lyse in water, serum. Protoplasts = wall-less, osmotically sensitive (gm +); spheroplasts = same with some envelope component retained = gm -) • Peptidoglycan (PG) - highly crosslinked layers of NAM-NAG joined peptides, crosslinking in peptide. . Certain antibiotics ( -lactams, penicillins) prevent crosslinks, lysozyme degrades. • DAP (diaminopimelic acid) in PG is unique to bacteria • Cell wall thickness is basis for gram stain: • property of cell wall to retain a basic dye (Gram's crystal violet/KI/acetone/wash/safranin) • those that don't retain crystal violet are counterstained with safranin (red) • thick cell walls retain (= gram +); thin cell walls don't (= gram -). Something else may be involved. • Exceptions to gram-positive/gram-negative staining: • mycoplasmas - no cell wall. Triple-layered membrane, gets sterols from host. • mycobacteria - lipid interferes with strain; use an acid-fast stain (retain carbol fuschin when decolorized with HCl in alcohol). • both are related to gram-positives based on genetic analyses (rRNA sequence). In gm + only: • Teichoic acids (Wall TA = WTA) - oligosaccharides (roughly 30 ribitol residues with phosphodiester links). Covalently linked to PG. 50% of dry weight of CW; 10% of dry weight of cell. • Lipoteichoic acids, LTA (polyglycerol phosphates) attached to the cytoplasmic membrane. May be same or different structure as teichoic. • Both, bind ions, important in membrane integrity, adherence 5. outer membrane (OM) - only in gram-negative bacteria • bilayer composed of phospholipids, proteins (4 - 5 major, 10 - 50 minor), and LPS

5 • LPS = Lipid A + core polysaccharides + O Ag = Endotoxin (Lipid A is responsible for toxic effects –endotoxic shock: fever, hypotension, impaired organ function, acidosis, dissem. intravascular coag., death). • contains lipoprotein (lipid/protein molecule covalently attached to the cell wall) • second permeability barrier, excluding molecules >600 daltons, e.g., many antibiotics • contains porins (trimeric protein pores; channels for low MW water soluble substances, phage receptors) (Note that in gram-positive bacteria, molecules as large as 10 5 daltons can pass through the cell wall.) Bayer's junctions (adhesion zones) - points of connection between IM and OM. • outside = phage attachment/injection site, complement-mediated lysis • inside = growth zones, sites for translocation of OM proteins, polysaccharides, LPS, pili, flagella 6. periplasmic space - space between IM and OM (or CW in gram-positive) • very small or nonexistent in gram-positive lactamase-ك contains hydrolytic enzymes like alkaline phosphatase and • • contains amino acid and sugar binding (transport) proteins • contains chemoreceptors • contents can be released by osmotic shock - rapid dilution of hypertonic (0.5 M sucrose) cell suspensions with H 2 0, after treatment with EDTA 7. "Optional" structures (Gm + and Gm -) capsule • varying thickness • polysaccharide or protein • antiphagocytosis, attachment • structurally, antigenically variable within a species • tightly associated with cells; if it can be washed off easily, it's called slime flagella (singular = flagellum) - motility, chemotaxis, virulence. FIGURE 5 • 3 parts: basal body and hook in cell envelope plus extended filament • filament: long (7-15 µm) and thin (20 nm diam.) (can't see with light microscope - need to stain) composed primarily of a single, self-aggregating protein called flagellin • basal body and hook: motor for energy-dependent rotation of the the filament • one to several hundred per cell • peritrichous - all around cell

6 • polar - at one end of the cell • bipolar - both ends pili (fimbriae) (singular = pilus) - hair-like structures composed of single, self- aggregating protein (pilin) • shorter, thinner, and straighter than flagella (several µ long, 7.5 -10 nm thick) • types: • common pili - peritrichous; attachment • F (sex) pilus - single; gene transfer (conjugation); gram-negative only granular inclusions or inclusion bodies - -storage polymers; often visualized by hydroxybutyrate-ك-staining. Glycogen, polyphosphate, poly endospores (spores) - dehydrated cells that are resistant to heat (difficult to kill); more later toxins - excreted (either actively or by cell lysis) by bacterium, act on host cells. Virulence. Examples: • cholera toxin (Vibrio cholerae) - B subunit (pentamer) binds host cells (GM1); A subunit has toxic activity (increases adenlyate cyclase activity, cAMP concentrates, electrolytes are secreted, Na and Cl absorption in small bowel impaired, fluid and electrolytes lost). • tetanus toxin - (Clostridium tetani) - reaches peripheral nervous system, blocks release of neurotransmitter, acetylcholine, from nerve terminals at neuromuscular junctions. ADP-ribosylating activity. Released at bacterial cell death. Bacteriophage- encoded. enzymes - excreted, act on extracellular polymers, host cells; hyaluronidases, proteases, DNases. BACTERIAL TAXONOMY Classification - arrangement into taxonomic groups based on similarities and relationships. Realize that there is no "official" classification. Classifications are for our benefit and are frequently arbitrary and historical in nature. Nomenclature - assignment of names by International rules. Unlike classification, it is official. Latinized, italicized. Yersinia pestis, Y. pestis Change name if re-classification warrants. Usually, maintain the species name for continuity, change the genus name. Identification - determining group to which new isolate belongs.

7 Despite the lack of an official classification, the closest and most accepted, though often debated, standard is Bergey's Manual of Systematic Bacteriology. Published since 1923. Most recent editions (9th and above) from 1984 to 1989. Bergey's current classification of prokaryotes Recognizes 4 divisions (based on cell wall properties) I. Gracilicutes - gram-negative bacteria (thin CW) II. Firmicutes - gram-positive bacteria (thick CW) III. Tenericutes - cell wall-less bacteria (i.e., Mycoplasma or Mollicutes) IV. Mendosicutes - the Archaebacteria Nomenclature of classification and some examples: [further down = more similar] official nomenclature Kingdom Prokaryotae Division Gracilicutes Class Scotobacteria Subclass Order Spirochaetales Family Spirochaetaceae Tribe Genus Borrelia Species Borrelia burgdorferi Subspecies No official standing; Infrasubspecies - based on common properties Practical utility Biovar (biotype) - biochemical or physiological Serovar (serotype) - antigenic Pathovar (pathotype) - pathogenic Phagovar (phagotype) - bacteriophage lysis Morphovar (morphotype) - morphologic species = basic taxonomic unit. Difficult to define in bacteria. Artificial, based on determinative keys, i.e. common properties. Even though classification is becoming more genetics based (genotypic, more later), still largely phenotypic (observable traits). strain - specific isolate of the species```````````` Means of classification (and identification) of bacteria 1. Structural traits

8 • Size - average 1 µm wide x 2 µm long (Escherichia coli 1 x 1 x 2 µm; 2 x10 -15 l volume) can be smaller (rickettsiae, chlamydiae less than 1/2) or larger (Bacillus megaterium ~10µm) Fig 2.1 from Sherris • Shape - determined microscopically spheres (cocci), rods (bacilli), spiral forms overhead cocci - diplococci = pairs streptococci = chains staphylococci = clusters sarcina = tetrads bacilli - short or long fusiform - tapered ends filamentous curved - vibrios • staining properties gram stain acid fast (mycobacteria) capsules, flagella have specific stains • colony morphology - shape (flat, domed, irregular borders), pigmented, size, spreading (motile) • motility • endospores 2. Biochemical, physiological traits - determined by ability to grow on various media under various conditions, e.g., different sugars, carbon sources, pH, temp, aeration, growth inhibitors. Growth indicators (color). 3. Genetic relatedness. a. ability to exchange DNA - later b. G+C content of chromosomal DNA . TABLE 3 • %GC + %AT =100% - refer to %GC • %GC has taxonomic implications for bacteria • very different GC - reflects substantial divergence, unrelated • very similar - may be related. Must also hybridize; share phenotypic traits • wide range w/in prokaryotic kingdom; narrow within a species. c. DNA homology. Fig. 14.7 Sherris DNA filter hybridization - FIGURE 6

9 • Figure shows one of the methods for determining the %DNA homology between two isolates (there are many variations) • DNA of unknown is labeled (growth in 3 H thymidine; nick translation, non- radioactive methods), denatured (heat), reacted with denatured test DNA bound to a nitrocellulose filter, washed, amount of radioactivity retained on filter reflects %homology. • The more closely related the strains are, the greater the homology. • DNA homology does not go much beyond the genus or family level. RNA (esp. rRNA) does ( ). d. DNA-RNA homology. • same principles as DNA homology but hybridization is between DNA and rRNA. • three types of RNA: mRNA, tRNA, rRNA • more sensitive - can pick homologies not detected by DNA-DNA hybridization because: • rRNA is highly conserved - critical for protein synthesis - must have 2 o structure (base pairs with self), must bind proteins, bind other RNA. Any change in critical areas - likely detrimental (changes would have to be balanced by changes in genes for r-proteins, in 2 o structure, or DNA encoding other proteins). • constitutes small % of total genetic material - therefore, examining specific conserved genes, not whole chromosome that is less conserved. e. rRNA sequence. most useful genetic taxonomic method. • the DNA sequence that encodes rRNA is highly conserved among bacteria of common ancestry. • sequence DNA encoding rRNA of different isolates, determine % identity, have a "molecular clock" –an accurate determination of phylogenetic distance. • Phylogenetic trees are now established on basis of rRNA sequence. Important Points: • Gm (+) = single phylogenetic group. Include the wall-less mycoplasmas which recently evolved from the clostridia. Also Mycobacteria. Neither of these can be gram stained. • Low GC group genetically similar - DNA exchange. • Gm (-) = >10 groups

10 BACTERIAL PHYSIOLOGY AND GROWTH NUTRITIONAL REQUIREMENTS

1. Water - 70 to 80% of a bacterial cell is water 2. Carbon and energy source (may be same or different) Carbon source - CO 2 or organic molecules autotrophs - (lithotrophs)- can use CO 2 as the sole carbon source for all cellular organic molecules heterotrophs - (organotrophs)- require an organic molecule as the carbon source monosaccharides - glucose, galactose, fructose, ribose, etc. disaccharides - sucrose (E. coli can't use), lactose (S. typhimurium can't use) organic acids - succinate, lactate, acetate amino acids - glutamate, arginine alcohols - glycerol, ribitol fatty acids Most compounds can be degraded by some bacterium; different bacteria use different compounds. Energy source - light (photosynthesize), inorganic, or organic compounds photoautotrophs - grow with light and CO 2 (e.g., cyanobacteria) chemoautotrophs - oxidation of inorganic compounds (e.g., H 2 , S, H 2 S, Fe, NH 3 ); carbon from CO 2 photoheterotrophs - grow with light and organic carbon source (e.g., purple bacteria) chemoheterotrophs - use organic molecules for both carbon and energy source Most bacteria and all human pathogens are chemoheterotrophs. 3. Nitrogen - ammonium ion (NH 4 + ) is end product of all nitrogen assimilation pathways. Pathway depends on organism. 10% dry weight is N (proteins, nucleic acids) Inorganic source a. ammonia: NH 3 (actually NH 4 + ) ––> glutamate and glutamine ––––––––> biochemical pathways b. nitrogen (N 2 ) fixation - Azotobacter, Klebsiella, Rhizobium nitrogenase N 2 —> NH 3 (NH 4 + ) ———> glutamate and glutamine c. nitrate (NO 3 - ) or nitritre (NO 2 - ) nitrate reductase nitrite reductase (i) nitrate reduction: NO 3 - ——–—> NO 2 - —––———> NH 3 (NH 4 + )

11 (ii) denitrification: NO 3 - —>—>—> N 2 (under anaerobic conditions; use NO 3 - as electron acceptor, give off N 2 ) Organic source e. g., amino acids (e.g., glutamate, glutamine, proline) 4. Inorganic ions Large amounts of: SO 4 2- (source of S: amino acids, coenzymes, proteins), PO 4 3- (NAD, ATP, nucleotides, etc.), K + (principal cation, cofactor for enzymes), Na + (some halophiles), Mg 2+ (counter ion for anionic, polymers, e.g., ribosomal RNA, and cofactor, for enzymes), Fe 2+ (heme, iron-sulfur proteins), Ca 2+ (gm + cell walls, lots in spores). Trace elements (micronutrients): Zn 2+ , Mn 2+ , Mo 2+ , Cu 2+ (cofactors for enzymes), Co 2+ (B 12 ), Se (in tRNAs and incorporated into proteins as selenocysteine via UGA codon) Siderophores - chelating agents that transport Fe (insoluble Fe-hydroxides) • high affinity constants for Fe • specific cell surface receptors • several different systems Probably all of the trace elements have specific transport systems. 5. Oxygen • aerobe (strict) - requires O 2 (cannot ferment, i.e., cannot transfer electrons and protons directly to organic receptor). Respire - transfer to oxygen. • anaerobe (strict) - killed by O 2 (lack superoxide dismutase, catalase; ferment). • facultative anaerobe - grows with or without O 2 (respire or ferment). Uses it if it's there. • aerotolerant anaerobe - grows with or without O 2 (ferment always) • microaerophilic - grows best with low O 2 , can grow without Oxygen and toxicity: a. O 2 inactivates some enzymes (e.g., nitrogenase), toxic products resulting from O 2 can damage DNA b. strict anaerobes lack superoxide dismutase and catalase, can't remove toxic products of respiration ferredoxins, flavoproteins (electron transfer) flavoproteins (electron transfer) O 2 —> O 2 - (superoxide)(most toxic) O 2 –––>H 2 O 2 (hydrogen peroxide) SOD

12 2 O 2 - + 2 H + —–––––—> O 2 + H 2 O 2 catalase 2 H 2 O 2 —––––—> 2 H 2 O + O 2 6. CO 2 Heterotrophs require CO 2 for some biosynthetic reactions: • pyrimidine and arginine biosynthesis - carbamyl phosphate synthetase • fatty acid biosynthesis • anaplerotic reactions: pyruvate or PEP ——> oxalacetate or malate 7. Other growth factors Many heterotrophs (particularly pathogens) require additional organic growth factors, e. g., vitamins, amino acids, purine or pyrimidine bases, heme, etc. Use rich media containing enzymatic digests of meat, yeast extract, milk protein hydrolysates, blood, etc. Some pathogens like Mycobacterium leprae (grow in armadillo liver), Rickettsia, (grow in egg yolks), etc. still can't be cultivated on artificial media.

PHYSICAL REQUIREMENTS 1. Temperature - Bacteria can grow from -5 to ~100°C: • thermophiles - >50 o C • psychrophiles - 4 o to 20 o C • mesophiles - 20 o to 45°C (pathogens) Ranges and optimum temperatures for growth. Optimum usually close to that of the natural habitat. 2. pH • range and optimum again - different species can grow between 2 and 10. • E. coli range is 4.5 to 8 (pathogens have a narrower pH range but some tolerate intracellular lysosomal fusion where pH is low); optimum is 7.2 to 7.6 • internal pH is roughly constant at approx. 7.4 3. Ionic strength or salt tolerance • bacteria don't tolerate well a very low ionic strength medium like water. • halophilic bacteria can tolerate and even require high salt, e.g., 30% NaCl 4. Oxidation-reduction potential (E h ) • strict anaerobes can't tolerate oxygen, require reducing agents in the medium.

13 • need an E h < -0.2 V; most media in contact with air have an E h = + 0.2 to 0.4 V. • important for bacterial growth in wounds. Initial growth by aerobic bacteria uses up the oxygen, which causes a progressive decrease in E h , and wound infection then becomes possible by anaerobic pathogens such as Clostridium perfringens (gas gangrene).

NUTRIENT UPTAKE: 1. Hydrolysis of nonpenetrating nutrients (CM is a semipermeable membrane) • large molecules hydrolyzed by extracellular enzymes (proteases, nucleases, lipases, etc.) • pathogens often use these enzymes to attack tissue constituents. 2. Membrane transport - protein-mediated. Few molecules cross the membrane by themselves (H 2 O, NH 4 , O 2 are some that can). a . facilitated diffusion - passive mediated transport • no energy required. Carrier protein facilitates rapid equilibration between inside and outside of cell • example is glycerol transport in E. coli: Glycerol is trapped and concentrated inside the cell by phosphorylation to glycerol 3- phosphate by the enzyme glycerol kinase. Phosphorylated compounds don't pass through membrane. b. active transport - group translocation • requires energy (from phosphoenolpyruvate [PEP], ATP, etc.) and carrier protein. • concentrates metabolites in cell; moving against a gradient • metabolite is chemically altered (trapped) once inside cell. • phosphotransferase system (PTS) - widespread in bacteria; glucose is transported by PTS in E. coli (PEP as energy source). Net reaction is phosphorylation of sugar (now trapped in cell) at expense of PEP. c. active transport - substrate translocation • requires energy (proton gradient or ATP) and carrier protein. • concentrates metabolites in cell; can move against concentration gradient. • substrate unchanged following transport. Retained in cell because transport system has a higher affinity for metabolite located outside the cell. • transport may require a periplasmic binding protein and an OM protein, e.g., sugar (galactose, maltose, ribose, arabinose, etc.) or amino acid (histidine) binding proteins.

14 Transport across cytoplasmic (IM) is coupled to ATP hydrolysis. Shock-sensitive because binding proteins released from periplasmic space by osmotic shock. • alternatively, only cytoplasmic membrane protein required. Transport is coupled to simultaneous passage of protons (H + ) through the membrane. Energy derived from proton gradient set up by electron transport within the energized cell membrane. Example = lactose transport in E. coli . Permease on inside has decreased affinity for substrate. Shock-insensitive because no periplasmic proteins are involved.

The Bacterial Cell Wall

The bacterial cell wall is a unique structure, which surrounds the cell membrane. Although not present in every bacterial species, the cell wall is very important as a cellular component. Structurally, the wall is necessary for:

Maintaining the cell's characteristic shape- the rigid wall compensates for the flexibility of the phospholipid membrane and keeps the cell from assuming a spherical shape

Countering the effects of osmotic pressure- the strength of the wall is responsible for keeping the cell from bursting when the intracellular osmolarity is much greater than the extracellular teichoic osmolarity

· Providing attachment sites for bacteriophages-teichoic acids attached to the outer surface of the wall are like landing pads for viruses that infect bacteria

· Providing a rigid platform for surface appendages- flagella, fimbriae, and pili all emanate from the wall and extend beyond it

The cell walls of all bacteria are not identical. In fact, cell wall composition is one of the most important factors in bacterial species analysis and differentiation. There are two major types of walls: Gram-positive and Gram-negative.

15 There are 2 basic cell walls, Gram positive and Gram negative.

Gram Positive: The cell wall of Gram-positive bacteria consists of many polymer layers of peptidoglycan connected by amino acid bridges. A schematic diagram provides the best explanation of the structure. The peptidoglycan polymer is composed of an alternating sequence of N-acetylglucosamine and N-acetyl-muraminic acid. It's a lot easier to just remember NAG and NAMA. Each peptidoglycan layer is connected, or crosslinked, to the other by a bridge made of amino acids and amino acid derivatives. The particular amino acids vary among different species, however. The crosslinked peptidoglycan molecules form a network, which covers the cell like a grid. Also, 90% of the Gram-positive cell wall is comprised of peptidoglycan.

Gram-positive cell wall structures

Gram Negative

The cell wall of Gram-negative bacteria is much thinner, being comprised of only 20% peptidoglycan. Gram-negative bacteria also have two unique regions which surround the outer plasma membrane: the periplasmic space and the lipopolysaccharide layer. The periplasmic space separates the outer plasma membrane from the peptidoglycan layer. It contains proteins, which destroy potentially dangerous foreign matter present in this space. The lipopolysaccharide layer is located adjacent to the exterior peptidoglycan layer. It is a phospholipid bilayer construction similar to that in the cell membrane and is attached to the peptidoglycan by

16 lipoproteins. The lipid portion of the LPS contains a toxic substance, called Lipid A, which is responsible for most of the pathogenic affects associated with harmful Gram- negative bacteria. Polysaccharides which extend out from the bilayer also contibute to the toxicity of the LPS. The LPS, lipoproteins, and the associated polysaccharides together form what is known as the outer membrane.

Keep in mind that the cell wall is not a regulatory structure like the cell membrane. Although it is porous, it is not selectively permeable and will let anything pass that can fit through its gaps.

Gram-negative cell wall structures

17 Selected some media and biochemical tests to identification bacteria

I. Media Bile Esculin Agar CAMPY-BAP Chocolate Agar Eosin Methylene Blue (EMB) Agar Hektoen Enteric (HE) Agar MacConkey Agar Mannitol Salts Agar Chopped Meat Glucose Medium Triple Sugar-Iron Agar Tryptic Soy Agar/Broth CDC anaerobic blood agar Blood agar plates Campylobacter agar Colistin Nanadixic Acid agar Lysine Iron Agar Thiosulfate Citrate Bile salts Sucrose agar Thioglycollate medium

BILE ESCULIN AGAR

Bile esculin agar is a medium used to identify group D streptococci. These groups of bacteria have the ability to grow in the presence of bile, an emulsifying agent produced in the liver. Group D streptococci also have the ability to hydrolyze esculin. This hydrolysis of esculin turns the medium black and denotes a positive test. Other bacteria capable of growing in the presence of bile do not turn the medium black. A variation of this medium uses sodium azide to inhibit the growth of all other Gram- positive bacteria and Gram-negative bacteria.

CAMPY-BAP

18 Campy-BAP is a blood agar that is highly selective for the growth of Campylobacter species. It contains several antibiotics, which inhibit the growth of Gram-positve bacteria as well as most Gram-negative organisms. After the plate is inoculated, it is placed in a microaerophilic environment such as a sealed bag containing high carbon dioxide concentrations and low oxygen concentrations.

CHOCOLATE AGAR

Chocolate agar is a nutrient medium which is used in culturing fastidious organisms such as Haemophilus species and Neisseria. It is comprised of sheep blood that provides the X and V factors necessary for Haemophilus growth. Chocolate agar, however, does not reveal hemolysis data, so species differentiation among the members of Haemophilus must be performed in another manner.

EOSIN METHYLENE BLUE (EMB) AGAR

EMB agar is a differential medium used in identification and isolation of Gram- negative enteric rods. EMB agar also inhibits the growth of Gram-positive organisms. The differential basis of this medium involves two indicator dyes, eosin and methylene blue, that distinguish between lactose fermenting and non-lactose fermenting organisms. Lactose fermenters form colonies with dark centers and clear borders while the non-lactose fermenters form completely coloroless colonies.

19

HEKTOEN ENTERIC (HE) AGAR

Hektoen enteric agar is a medium designed for the isolation and recovery of fecal specimens belonging to the Enterbacteriaceae family, especially Salmonella and Shigella. It can differentiate between bacteria that ferment lactose and those that don't. Acid produced from fermenting lactose imparts a yellow-orange color to the medium due to the presence of a pH indicator. Non-lactose fermenters do not significantly change the color of the medium. HE agar can also detect the production of hydrogen sulfide gas, which turns parts of the medium black.

20

MACCONKEY AGAR

MacConkey agar is probably the most popular solid differential medium in the world. It is mainly used in identification of lactose fermenting, Gram-negative enteric pathogens and for inhibiting growth of Gram-positive organisms. Bacterial colonies that can ferment lactose turn the medium red. This red color is due to the pH indicators response to the acidic environment created by fermenting lactose. Organisms that do not ferment lactose do not cause a color change.

MANNITOL SALTS AGAR

21 A common medium used for the isolation of pathogenic staphylococci is the Mannitol Salts Agar. The high salt concentration of this medium inhibits the growth of most other organisms. Pathogenic staphylococci not only grow on the medium, but they also produce acid from it. This acid production turns the pH indicator from red to yellow. Non-pathogenic staphylococci can grow on the medium but produce no acid from it.

CHOPPED MEAT GLUCOSE MEDIUM

Chopped meat broth is used for culturing anaerobic organisms.

TRIPLE SUGAR IRON (TSI) AGAR

Triple sugar iron (TSI) agar is a medium used in the identification of Gram-negative enteric rods. The medium measures a bacterium's ability to utilize three sugars, glucose, sucrose and lactose, the concentrations of which are 0.1%, 1.0%, and 1.0%, respectively. A pH indicator incuded in the medium can detect acid production from fermentation of these carbohydrates. A yellow color change indicates acid in the medium while no color change indicates an alkaline surrounding. Inoculation of the tube is a two step procedure. First, a loop of bacteria is spread across the surface of

22 the agar. Second, a needle of bacteria is inserted (stabbed) into the bottom (butt) of the tube. Carbohydrate uilization can be determined through analysis of the extent of acid production. Acid production limited to only the butt of the tube is indicative of glucose utilization. This is because the concentration of glucose is lower than that of other sugars, thus the acid production is not very extensive. Acid production in the slant and butt indicates sucrose or lactose fermentation because of the relatively high concentrations of these sugars, thus leading to extensive acid production. TSI agar can also detect reduction of sodium thiosulfate to hydrogen sulfide. Hydrogen sulfide production will turn parts of the agar black. Production of other gases is marked by cracks in the agar as well as an air gap at the bottom of the test tube.

INTERPRETATION OF TUBES ABOVE TUBE 1 TUBE 2 TUBE 3 TUBE 4 TUBE 5 (UNINOCULATED) SLANT - A A K K BUTT - A A A A HYDROGEN - - - + + SULFIDE GAS - - + - - A=Acidic K=Alkaline

TRYPTIC SOY BROTH/AGAR

Tryptic soy agar and broth is a basic medium used for culturing many kinds of microorganisms. Tryptic soy agar is mainly used as an initial growth medium for the following purposes:

23 · observe colony morphology · develop a pure culture · achieve sufficient growth for further biochemical testing

· culture storage

Tryptic soy broth is used mostly to generate a large supply of bacteria for certain biochemical tests. It can also be used in the determination of bacterial numbers.

CDC ANAEROBIC BLOOD AGAR

Is an enriched non-selective medium containing sheep's blood. It is used as a primary plating medium to culture and maintain all types of anaerobes. Optimal incubation for this medium is at a temperature of 37° C for a period of 48 hours without oxygen. Bacteroides melaninogenicus pigment and hemolysis around Clostridium perfringes are seen with ABAP due to the vitamin K, hemin, and cysteine that are included in the medium

BLOOD AGAR PLATES Contain an enriched non-selective medium with sheep's blood. It is used as a primary plating medium to culture and maintain most aerobes and facultative anaerobes. Optimal incubation for this medium is at a temperature of 37° C for an incubation period of 24 hours in a carbon dioxide incubator. Rabbits blood can be substituted for sheep's blood for the growth of fastidious organisms. Blood agar plate contains the antibiotic optocin are used to differentiate Streptococcus pneumoniae (which is sensitive to optocin) from other Streptococci species. When S. pneumoniae is grown on this plate, there will be a zone of no growth around the paper disk. Optimal incubation for this medium is at a temperature of 37° C for an incubation period of 24 hours in a carbon dioxide incubator. Blood agar plates contain the antibiotic bacitracin are used to differentiate group A beta-hemolytic Streptococci (which are sensitive to bacitracin) from other Streptococci species. When group A beta Strep are grown on this plate, there will be a zone of no growth around the paper disk. CAMPYLOBACTER AGAR Is a selective sheep's blood medium with several antibiotics added. It is used to isolate Campylobacter jejuni. It will inhibit the growth of normal flora enteric bacteria

24 while supporting C. jejuni. Optimal incubation for this medium is at a temperature of 42° C for an incubation period of at least 48 hours in microaerophilic conditions

COLISTIN NANADIXIX ACID AGAR Is a selective and differential sheep's blood medium that supports the growth of gram-positive organisms. Optimal incubation for this medium is at a temperature of 37° C and an incubation period of 24 hours in a carbon dioxide incubator. This medium inhibits the growth of gram negative organisms.

LYSINE IRON AGAR

Is a purple colored medium in a slant tube that is used to detect glucose fermentation, lysine decarboxylation, lysine deamination and hydrogen sulfide production. Glucose fermentation is indicated by a change to yellow in the lower half of the tube (K/A or R/A). Lysine decarboxylation is indicated by a completely purple tube(K/K). Lysine deamination is indicated by a red color in the top half of the tube (R/K or R/A). Hydrogen sulfide production is indicated by a black precipitate in the middle of the tube. Some fermentation will also produce gas that gives bubbles or cracks in the agar.

THIOSULFATE CITRATE BILE SALTS SUCROSE AGAR

is used to identify Vibrio sp., and is used to detect the utilization of sucrose. Vibrio species that utilize sucrose produce yellow colonies on the agar, while those that do not produce blue-green colonies. Optimal incubation for this medium is at a temperature of 37° C and an incubation period of 24 hours in ambient air.

THIOGLYCOLLATE MEDIUM

is a liquid medium used as a backup for the plating media especially when the presence of an anaerobe is a possibility. A special seal is not needed for the growth of the strictest anaerobes because of the low EH that is provided by this medium. The position of growth in the tube will indicate the oxygen usage of the organism. Strict aerobes will grow only at the top and strict anaerobes only at the bottom with other usages growing in between. Optimal incubation for this medium is at a temperature of 37° C and an incubation period of 24 hours in ambient air

25 II. Some biochemical tests

Catalase Test Citrate Test Coagulase Test Indole Test Optichin Test Oxidase Test Urease Test The string of pearls Test

CATALASE TEST

Some bacteria and macrophages can reduce diatomic oxygen to hydrogen peroxide or superoxide. Both of these molecules are toxic to bacteria. Some bacteria, however, possess a defense mechanism, which can minimize the harm done by the two compounds. These resistant bacteria use two enzymes to catalyze the conversion of hydrogen peroxide and superoxide back into diatomic oxygen and water. One of these enzymes is catalase and its presence can be detected by a simple test. The catalase test involves adding hydrogen peroxide to a culture sample or agar slant. If the bacteria in question produce catalase, they will convert the hydrogen peroxide and oxygen gas will be evolved. The evolution of gas causes bubbles to form and is indicative of a positive test.

CITRATE TEST

The citrate test is used to determine the ability of a bacterium to utilize citrate as its only source of carbon. Bacteria can break the conjugate base salt of citrate into organic acids and carbon dioxide. The carbon dioxide can combine with the sodium from the conjugate base salt to form a basic compound, sodium carbonate. A pH indicator in the medium detects the presence of this compound by turning blue (a positive test).

26

COAGULASE TEST

Like the mannitol salts agar, the coagulase test is another method for differienting between pathogenic and non-pathogenic strains of Staphylococcus. Bacteria that produce coagulase use it as a defense mechanism by clotting the areas of plasma around them, thereby enabling themselves to resist phagocytosis by the host's immune system. The sample in question is usually inoculated onto 0.5 ml of rabbit plasma and incubated at 37 degrees celsius for one to four hours. A positive test is denoted by a clot formation in the test tube after the allotted time.

INDOLE TEST

Indole is a component of the amino acid tryptophan. Some bacteria have the ability to break down tryptophan for nutritional needs using the enzyme tryptophanase. When tryptophan is broken down, the presence of indole can be detected through the use of Kovacs' reagent. Kovac's reagent, which is yellow, reacts with indole and produces a red color on the surface of the test tube.

27

OPTOCHIN TEST

The optochin test is a presumptive test that is used to identify strains of Streptococcus pneumoniae. Optochin (ethyl hydrocupreine) disks are placed on inoculated blood agar plates. Because S. pneumoniae is not optochin resistant, a zone of inhibition will develop around the disk where the bacteria have been lysed. This zone is typically 14mm from the disk or greater.

OXIDASE TEST

Cytochrome oxidase is an enzyme found in some bacteria that transfers electrons to oxygen, the final electron acceptor in some electron transport chains. Thus, the enzyme oxidizes reduced cytochrome c to make this transfer of energy. Presence of cytochrome oxidase can be detected through the use of an Oxidase Disk which acts as an electron donator to cytochrome oxidase. If the bacteria oxidize the disk (remove electrons) the disk will turn purple, indicating a positive test. No color change indicates a negative test.

28

UREASE TEST

Urease is an enzyme that breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water. Members of genus Proteus are known to produce urease. Urease can be detected by plating bacteria onto an amide containing medium, specifically urea. When urea is broken down, ammonia is released and the pH of the medium increases (becomes more basic). This pH change is detected by a pH indicator that turns pink in a basic environment. A pink medium indicates a positive test for urease.

THE STRING OF PEARLS TEST is used for the identification of Bacillus sp., specifically Bacillus anthracis. A sample is swabbed on one quadrant of a nutrient agar plate. A microscope slide cover- slip is placed on the swabbed area, with a ten-unit penicillin disk placed directly adjacent to it. The plate is incubated for 3 hours in ambient air at 37° C. The plate is placed on a microscope stage without a lid. A drop of immersion oil is placed on the cover-slip, and the 100x lens is used to look for chains of bacilli that show pronounced swelling close to the penicillin disk, with the swelling diminishing further from the disk.

29 The String Test is used to identify Vibrio spp. A colongy is placed on a microscope slide and mixed with 0.5% aqueous sodium deoxycholate. In a positve test, the bacterial cells will be lysed by the sodium deoxycholate, causing the suspension to lose turbidity. The DNA from the cells is left in a viscous suspension. When an inoculating loop is immersed in the suspension and drawn slowly away, a mucoid "string" is produced.

30 Bacterial classification and nomenclatures

Gram-positive

Aerobic Cocci Aerobic Bacilli

Gram-negative

Aerobic Cocci Enteric Bacteria Pleomorphic Bacteria Non-Fomenters

Anaerobes

Gram-positive Anaerobes Gram-negative Anaerobes

GRAM-POSITIVE COCCI

The Gram-positive cocci are grouped together based on their Gram-stain reaction, thick cell wall composition, and spherical shape. Most of the organisms in these groups are members of the Micrococcaceae family All of the organisms in these groups are non-endospore forming chemosynthetic heterotrophs. We will discuss only the clinically relevent bacteria from Micrococcus and Staphylococcus of the Micrococcaceae family. Streptococcus and Enterococcus(formerly a species of Streptococcus) are discussed as well because of the many diseases they inflict on humans. The chart below shows the paths to identification of the genera discussed.

31

MICROCOCCUS

Microcoocus is a Gram-positive, aerobic bacterium which is a member of the Micrococcaceae family. Micrococcus cells can be observed under the microscope as spherical cells forming pairs or clusters. If cultured in broth or on nutrient agar, the colonies may be red or yellow when observed unstained. Although these bacteria are a common human skin contaminant, they are relatively harmless to humans because they maintain a saprophytic lifestyle. They can also be found in freshwater environments or in soil. Three common species of Micrococcus are M. luteus, M. roseus, and M. varians.

LABORATORY INDICATIONS:

o Catalase + o Oxidative action on glucose o Growth on mannitol

32

Gram-stained Micrococcus under the microscope

STAPHYLOCOCCUS

Clinically, the most important genus of the Micrococcaceae family is Staphylococcus. The Staphylococcus genus is classified into two major groups: aureus and non-aureus. S. aureus is a leading cause of soft tissue infections, as well as toxic shock syndrome (TSS) and scalded skin syndrome. It can be distinguished from other species of Staph by a positive result in a coagulase test(all other species are negative).

The pathogenic effects of Staph are mainly asssociated with the toxins it produces. Most of these toxins are produced in the stationary phase of the bacterial growth curve. In fact, it is not uncommon for an infected site to contain no viable Staph cells. The S. aureus enterotoxin causes quick onset food poisoning which can lead to cramps and severe vomiting. Infection can be traced to contaminated meats which have not been fully cooked. These microbes also secrete leukocidin, a toxin which destroys white blood cells and leads to the formation of pus and acne. Particularly, S. aureus has been found to be the causative agent in such ailments as pneumonia, meningitis, boils, arthritis, and osteomyelitis (chronic bone infection). Most S. aureus are penicillin resistant, but vancomycin and nafcillin are known to be effective against most strains.

Of the non-aureus species, S. epidermis is the most clinically significant. This bacterium is an opportunistic pathogen which is a normal resident of human skin. Those susceptible to infection by the bacterium are IV drug users, newborns, elderly,

33 and those using catheters or other artificial appliances. Infection is easily treatable with vancomycin or rifampin.

LABORATORY INDICATIONS:

Anaerobic glucose fermentation with acid production

Catalase +

Nitrate +

Coagulase +

Mucoid Staphylococcus aureus under the microscope

STREPTOCOCCUS

The Streptococcus genus consists of Gram-positive, aerobic bacteria which appear as chains under microscopic observation. The organisms in this genus are characterized by a coccus appearance, a thick cell wall, and aerobic action on glucose. Four different classification systems exist for this important microorganism:

CLINICAL

Pyogenic Streptococci

Oral Streptococci

Enteric Streptococci,

34 HEMOLYSYS

alpha-hemolysis

beta-hemolysis

gamma-hemolysis

SEROLOGICAL-Lancefield (A-H), (K-U) BIOCHEMICAL(physiological)

GROUP A

The first group in the Lancefield classification system includes only one species of Streptococcus, S. pyogenes. This particular opportunistic pathogen is responsable for about 90% of all cases of pharyngitis. A common form of pharyngitis is "Strep throat" which is characterized by inflamation and swelling of the throat, as well as development of pus-filled regions on the tonsils. Penicillin is usually administered to patients as soon as possible to quell the possibility of the infection spreading from the upper respiratory system into the lungs. Once in the lungs, the infection could give rise to pneumonia. Some cases also develop into rheumatic fever if left untreated. Other diseases linked to S. pyogenes are skin infections such as impetigo, cellulitis, and erysipelas. LABORATORY INDICATIONS:

Catalase -

Beta-hemolysis

Bacitracin sensitive

GROUP B

The B classification of Lansefield also includes only one bacterium, S. agalactiae. For years this bacterium has been the causative agent in mastitis in cows. Currently, it has been found to be a cause of sexually transmitted urogenital infections in females. Although infection is easily treated with penicillin, proper diagnosis is necessary for

35 women nearing labor because the infection can easily spread to the child via the birth canal. LABORATORY INDICATIONS:

· CAMP + · Beta-hemolysis

GROUP D

Type D Streptococcus is the next clinically important bacterium because of the multitude of diseases it is known to cause. Although many are harmless, the pathogenic strains cause complications of the human digestive tract. This group has recently been reclassified into two divisions: Enterococcus and non-Enterococcus. The Enterococci include E. faecalis, a cause of urinary tract infections, and E. faecium, a bacterium resistant to many common antibiotics. Diseases such as septicemia, endocarrditis, and appendicitis have also been attributed to group D Strep. Fecal matter from infected individuals is a source for isolation and identification techniques. Once identified, Group D Strep can be treated with ampicillin alone or in combination with gentamicin. LABORATORY INDICATIONS:

Hydrolysis of bile esculin (dark brown medium) -this indicates the ability of the bacteria to tolerate bile from the liver

Growth in high salt conc.

OTHER IMPORTANT STREP

Streptococcus pneumoniae

Because its surface carbohydrate antigens do not correspond to a specific Lancefield group, S. pneumonia is discussed separately. Although not given a letter designation, S. pneumoniae can be considered a Pyogenic (pus-producing) strain of Strep. It can be

36 distinguished from other Pyogenic bacteria by its high sensitivity to Optochin (no growth zone of inhibition). This bacterium causes pneumonia (obviously!), meningitis, and otitis media. It also demonstrates alpha-hemolytic growth on blood agar.

Viridans Group

The Viridans Streptococci, consisting of S. mutans and S. mitis, are alpha- hemolytic bacteria. These bacteria inhabit the mouth. In fact, a large percentage of tooth decay can be attributed to S. mutans.

GRAM-POSITIVE RODS

The Gram-positive rods in this section will be divided into three distinct varieties based upon their ability to produce endospores and their morphological appearance:

ENDOSPORE-FORMING

BACILLUS

REGULAR, NON-ENDOSPORE-FORMING

LACTOBACILLUS

LISTERIA

ERYSIPELOTHRIX

IRREGULAR, NON-ENDOSPORE-FORMING

CORYNEBACTERIUM

These bacteria are ubiquitous in nature and most are aerobic or facultatively anaerobic.

BACILLUS

Bacillus represents a genus of Gram-positive bacteria which are ubiquitous in nature (soil, water, and airborne dust). Some species are natural flora in the human intestines.

37 When grown on blood agar, Bacillus produces large, spreading, gray-white colonies with irregular margins. A unique characteristic of this bacterium is its ability to produce endospores when environmental conditions are stressful. The only other known spore-producing bacterium is Clostridium. Although most species of Bacillus are harmless saprophytes, two species are considered medically significant: B.anthracis and B. cereus.

B. anthracis

B. anthracis is the bacterium which causes anthrax in cows, sheep, and sometimes humans. Anthrax is transmitted to humans via direct contact with animal products or inhalation of endospores. Under the microscope, B. anthracis cells appear to have square ends and seem to be attached by a joint to other cells. The spores are best observed when the bacterium is cultered on artificial media. Sources of infection are usually industrial or agricultural and the infection is classified as one of three types:

CUTANEOUS INFECTION (95% of human cases)

INHALATION ANTHRAX (rare)

GASTROINTESTINAL ANTHRAX (very rare)

LABORATORY INDICATIONS:

Nonhemolytic (sheep blood agar)

Non-motile Gel hydrolysis - Catalase +

B. cereus

Unlike B. anthracis, B.cereus is a motile bacterium which can cause toxin-mediated food poisoning. It is known to inhabit many kinds of food including stew, cereal, and milk. Most recently, however, it has been found in fried rice. The two toxins released by the bacterium lead to vomiting and diarrhea, symptoms similar to those of Staphylococcus food poisoning. Because toxin production usually takes place after the

38 infected foods are cooked, proper cold storage of food is recommended immediately after preparation. LABORATORY INDICATIONS:

Hemolytic (sheep blood agar)

Motile Gel hydrolysis +

Glucose, maltose, & salicin fermentative Catalase +

LISTERIA

Listeria is a Gram-positive rod which is not capable of forming endospores. Although several species of this bacterium exist, our discussion will focus only on the two species of human pathogenic significance: L. monocytogenes and L. ivanovii. In particular, L. monocytogenes has been implicated in several food poisoning epidemics. This normal inhabitant of the gastrointestinal tract and of animal feces led to a 1986 outbreak in Massachusetts hospital patients. Those infected suffered from vomiting, nausea, and diarrhea. Apparently, the hospital patients contracted the microbe from the infected hospital food and were at high risk of infection. Those at high risk include newborns, pregnant women and their fetuses, the elderly, and persons lacking a healthy immune system. The bacterium usually causes septicema and meningitis in patients with supressed immune function. It also causes listeriosis which is an inflammation of the brain. Antibiotics are recommended for treatment of infection because most strains of Listeria are sensitive to ampicillin and gentamicin.

LABORATORY INDICATIONS:

Catalase +

Motile at room temperature

Growth at 4 degrees Celsius

Bile esculin hydrolysis

Beta-hemolysis

39 LACTOBACILLUS

Most species of this non-spore-forming bacterium ferment glucose into lactose, hence the name Lactobacillus. The most common application of Lactobacillus is industrial, specifically for dairy production. This genus also contains several bacteria that make up part of the natural flora of the human vagina. Because of their ability to derive lactic acid from glucose, these bacteria create an acidic environment which inhibits growth of many bacterial species which can lead to urogenital infections. Lactobacillus is generally harmless to humans, rarely inciting harmful infections or diseases. Treatment of this vancomycin-resistant microbe usually consists of high doses of penicillin in combination with gentamicin.

LABORATORY INDICATIONS:

· Catalase - · Lactic acid production from glucose · Growth on tomato juice agar

ERYSIPELOTHRIX

E. rhusiopathiae, the only species of this genus, is better known as a veterinary pathogen than as a human pathogen. When cultured on blood agar or some other nutrient medium, Erysipelothrix forms notably large colonies. This ubiquitous microbe has been found in many farm animals such as pigs, horses, and turkeys. Occasionaly, though, it can infect a human host and cause an inflammatory skin disease, Erysipeloid. Treatment usually consists of penicillin G, ampicillin, or cephalothin. Most clinical strains have been found to be resistant to the super- antibiotic, vancomycin.

LABORATORY INDICATIONS:

· Catalase - Non-motile TSI : H2S +

40 CORYNEBACTERIA

The coryneform group of Gram-positive bacteria includes several genera of non- spore-forming rods which are ubiquitous in nature. We will consider only two of these genera in our discussion of clinically significant microorganisms: Actinomyces and Corynebacterium. The first genus, Actinomyces, will be presented later when we focus on anaerobic Gram-positive bacteria. The second genus, Corynebacterium, is comprised of facultatively anaerobic bacteria which are normally saprophitic and harmless to humans. An exception is the bacterium C. diphtheriae which produces the toxin that causes diphtheria, a disease of the upper respiratory system in humans. Under the microscope (best viewed using Loeffler's methylene blue dye), C. diphtheria can be seen forming colonies which clump up or stick together. This is a characteristic associated with many higher forms of bacteria. Although other species of Corynebacterium can inhabit the mucous membrane, C.diphtheria is unique in its exotoxin formation. Treatment for the disease usually consists of administration of an antitoxin with penicillin.

LABORATORY INDICATIONS:

· Catalase + Nitrate + · Glucose fermentation Non-motile

NEISSERIA

The Neisseria genus consists of aerobic, non-spore-forming Gram-negative coccobacilli which inhabit the mucous membranes of many animals (and humans). These non-motile microbes require a moist environment and warm temperatures (human body temperature range) to achieve optimum growth. An important means of identification of Neisseria species is the oxidase test, for which all members test positive. Additionally, Neisseria grow well on chocolate agar containing antibiotics that inhibit growth of Gram-negative bacteria, Gram-positive bacteria, and molds. The two most clinically significant members of the genus Neisseria are N. gonorrhoeae and N. meningitidis.

41

N. gonorrhoeae

Infection by the diplococcoid bacterium N. gonorrhoeae is referred to as a gonococcal infection. Gonorrhea is transmitted between humans through intimate contact of the mucous membrane. This sexually transmitted organism can be carried by men and women for many years without any sign or symptoms. In infected males, the disease is characterized by a urethral discharge of pus and can eventually result in other complications such as prostatitis and periurethral abscess. The incubation period of the bacterium can last from a day to a week. Females with gonorrhea exhibit vaginal discharge, abdominal pain, and abnormal non-menstrual bleeding. Ironically, the widespread use of birth control devices such as the pill has actually increased the number of gonococcal infections in the United States. Use of the pill can lower the glycogen concentration of the vaginal membrane. This environmental change inhibits the growth of acid-producing bacteria, such as Lactobacillus, which are the natural flora of the vagina. The vaginal pH soon becomes less acidic and a variety of organisms are able to grow there. As with most other sexually transmitted diseases, gonorrhea is prevalent in young adult and homosexual populations. This disease may sound really bad, but it is treatable. N. gonorrheae is sensitive to ultraviolet radiation, drying, and antibiotics. Because chlamydia infection is often associated with a gonococcal infection, a regimen of ceftriaxone and doxycycline is used to kill both organisms. LABORATORY INDICATIONS:

· Oxidase + Glucose fermentative

N. meningitidis

42 It doesn't take a genius to figure out that N. meningitidis causes meningitis, inflammation of the membranes covering the central nervous system. The different strains of N. meningitidis are classified by their capsular polysaccharides. This bacterium is the second leading cause of meningitis in the United States. Early symptoms may include headache, fever, and vomiting. Death can quickly follow due to endotoxin shock or focal cerebral involvement. Infection doesn't always lead to death, however. The organism can often assume a carrier status with very few carriers actually developing the disease. Infected patients can be treated with penicillin, while rifampin may be used prophylactically as a means of preventing the disease state in carriers

ENTEROBACTERIACEAE

Members of genera belonging to the Enterobacteriaceae family have earned a reputation placing them among the most pathogenic and most often encountered organisms in clinical microbiology. These large Gram- negative rods are usually associated with intestinal infections, but can be found in almost all natural habitats. They are the causative agents of such diseases as meningitis, bacillary dysentery, typhoid, and food poisoning. As well as being oxidase negative, all members of this family are glucose fermenters and nitrate reducers. In most cases, the pathogenicity of a particular enteric bacterium can be determined by its ability to metabolize lactose. Non-utilizers are usually pathogenic while the lactose utilizers are not. Because many different species in this family can cause similar symptoms, biochemical tests are crucial to the identification, diagnosis, and treatment of infection. We will discuss the twelve genera of the Enterobacteriaceae family which are most commonly encountered in the clinical laboratory:

· ESCHERICHIA COLI • SHIGELLA · EDWARDSIELLA • SALMONELLA · CITROBACTER • KLEBSIELLA · ENTEROBACTER • SERRATIA · PROTEUS • MORGANELLA · PROVIDENCIA • YERSINIA

ESCHERICHIA COLI

43 E. coli, the whipping boy of microbiology and genetics labs around he world, is the most encountered bacterium in the clinical laboratory. Besides being the number one cause of human urinary tract infections, E. coli has been linked to diseases in just about every other part of the body. Pneumonia, meningitis, and traveler's diarrhea are among the many illnesses that pathogenic strains of E. coli can cause. As part of the normal flora of the human intestinal tract, E. coli plays a crucial role in food digestion by producing vitamin K from undigested material in the large intestine. Pathogenic strains of E. coli, however, can cause severe cases of diarrhea in all age groups by producing a powerful endotoxin. Treating E. coli infections with antibiotics may actually place the patient in severe shock which could possibly lead to death. This is do to the fact that more of the bacterium's toxin is released when the cell dies. Below are two examples of E. coli growing on eosin methylene blue(EMB) agar.

LABORATORY INDICATIONS:

· Lysine + • Citrate - · Indol + • Acetate + Lactose +

SHIGELLA

Shigella is closely related to Escherichia and is considered, by some scientists, to be another strain of E. coli. However, because Shigella is anaerogenic (does not produce gas from carbohydrates) and lactose(-), it is usually discernable from E. coli. In many cases, a Shigella infection will lead to diarrhea accompanied by fever. Shigella is also an invasive pathogen which can be recovered from the bloody stool of an infected host. Invasive pathogens colonize the host's tissues as opposed to growing on tissue surfaces. The four species in this genus are sometimes referred to by a letter designation based on their serologocal antigen:

44 § Serotype A- S. dysenteriae § Serotype B- S. flexneri § Serotype C- S. boydii § Serotype D- S. sonnei

Strain D is the causative agent of most cases of Shigella-related diarrhea (shigellosis), while strain C species are rarely encountered in the laboratory. Shigella is easily spread from host to host, which should make it the primary suspect in outbreaks of diarrhea. LABORATORY INDICATIONS:

· Lysine - · non-motile · -/+ TSI reaction (no gas) · Acetate - · Lactose -

EDWARDSIELLA

Edwardsiella tarda is the only species in this genus of enteric bacteria which is important to our discussion. E. tarda is biochemically similar to E. coli with the exception that E. tarda produces hydrogen sulfide. This bacterium is usually found in aquatic animals and reptiles. However, it has been known to cause gastroenteritis and wound infections in humans.

LABORATORY INDICATIONS:

· Lysine + • Hydrogen sulfide + · -/+ TSI reaction (with gas) • Indole + · Citrate –

SALMONELLA

Salmonella bacteria are instigated in more han 50,000 cases of bacterial food poisoning in the United States every year. Transmission of this microbe is usually through uncooked meats and eggs. Chickens are a major reservoir of Salmonella, which explains its ubiquitous presence in poultry products. Ingesting foods contaminated with significant amounts of Salmonella can cause intestinal infection which can eventually lead to diarrhea, vomiting, chills, and a really big headache. The 2200 known species of Salmonella are classified according to their surface antigens.

45 The capsular properties of this Gram-negative rod can cause serious complications in immunosuppressed individuals such as HIV/AIDS patients. In the United States, S. typhimurium and S. enteritidis are the two leading causes of salmonellosis (inflammation of the intestine caused by Salmonella).

While animals carry most Salmonella, S. typhi is unique because humans only carry it. This intracellular parasite can cause typhoid fever (enteric fever) which is characterized by fever, diarrhea, and inflammation of the infected organs. Most Salmonella infections can be treated with ciprofloxacin or ceftriaxone.

LABORATORY INDICATIONS:

Lysine + • Hydrogen sulfide +

-/+ TSI reaction (with gas) • Indole +

Citrate + • ONPG -z

Malonate -

CITROBACTER

Citrobacter is not considered to be an enteric pathogen because it is normal gut flora. When plated, Citrobacter colonies bare a strong resemblance to E. coli colonies. This group of bacteria is of small clinical interest. C. freundii is suspected to cause diarrhea and possibly extraintestinal infections. C. diversus has been linked to a few cases of meningitis in newborns.

LABORATORY INDICATIONS:

Lysine - • Hydrogen sulfide + (C. freundii) • Citrate +

+/+ TSI reaction (with gas) • Slow urease

KLEBSIELLA

The most clinically important speciies of this genus is Klebsiella pneumoniae. This large, non-motile bacterium produces large sticky colonies when plated on nutrient media. Klebsiella's pathogenicity can be attributed to its production of a heat-stable

46 enterotoxin. K. pneumoniae infections are common in hospitals where they cause pneumonia (characterized by emission of bloody sputum) and urinary tract infections in catheterized patients. In fact, K. pneumoniae is second only to E. coli as a urinary tract pathogen. Klebsiella infections are encountered far more often now than in the past. This is probably due to the bacterium's antibiotic resistance properties. Klebsiella species may contain resistance plasmids (R-plasmids) which confer resistance to such antibiotics as ampicillin and carbenicillin. To make matters worse, the R-plasmids can be transferred to other enteric bacteria not necessarily of the same species.

LABORATORY INDICATIONS:

Lysine + • Citrate + • Indol - • +/+ TSI (with gas)

Non-motile • Ornithine -

ENTEROBACTER

Enterobacter includes eleven species of highly motile bacteria. The Enterobacter species is biochemically similar to Klebsiella, but unlike Klebsiella Enterobacter is ornithine positive. Although this bacterium is part of the normal flora of the human intestinal tract, several species cause opportunistic infections of the urinary tract as well as other parts of the body. E. aerogenes and E. cloacae are two such pathogens that do not cause diarrhea, but that are sometimes associated with urinary tract and respiratory tract infections.

LABORATORY INDICATIONS:

Lysine + (except E. cloacae)

Citrate + • Indol -

+/+ TSI (with gas) • Motile Ornithine +

SERRATIA

Members of the Serratia genus were once known as harmless organisms that produced a characteristic red pigment. Today, Serratia marcescens is considered a harmful human pathogen which has been known to cause urinary tract infections,

47 wound infections, and pneumonia. Serratia bacteria also have many antibiotic resistance properties which may become important if the incidence of Serratia infections dramatically increases. Serratia can be distinguished from other genera belonging to Enterobacteriaceae by its production of three special enzymes: DNase, lipase, and gelatinase.

LABORATORY INDICATIONS:

· Lysine + • Citrate + • Indol - • +/+ TSI (No gas) · DNase +

PROTEUS

Proteus, like almost every other bacterium in this family, can cause urinary tract infections and hospital-acquired infections. Proteus is unique, however, because it is highly motile and does not form regular colonies. Instead, Proteus forms what are known as "swarming colonies" when plated on non-inhibitory media. The most important member of this genus is considered to be P.mirabilis, a cause of wound and urinary tract infections. Fortunately, most strains of P. mirabilis are sensitive to ampicillin and cephalosporins. Unlike its relative, P. vulgaris is not sensitive to these antibiotics. However, this organism is isolated less often in the laboratory and usually only targets immunosuppressed individuals. P. mirabilis and P. vulgaris can be differentiated by an indole test for which only P. vulgaris tests positive. LABORATORY INDICATIONS:

· Lysine - • Hydrogen sulfide + · Motile • Urease +

48 MORGANELLA

Not much exciting going on here. Morganella morganii is the only important species of this genus. It can cause urinary tract and wound infections, as well as diarrhea. Chloramphenicol is a good choice for treating Morganella infections.

LABORATORY INDICATIONS:

· Indole + • Ornithine + • Citrate +

PROVIDENCIA

Although rare, Providencia species have been associated with nosocomial (hospital acquired) urinary tract infections. One species, P. alcalifaciens, has been associated with some cases of diarrhea in children. Since infection is so rare, other genera of the family Enterobacteriaceae should be considered before Providencia as the causative agent of disease.

LABORATORY INDICATIONS:

· Indole + · Hydrogen sulfide - · Citrate + · Lysine - · Lactose -

YERSINIA

Two important species are included in the Yersinia genus: Y. enterocolitica and Y. pestis. Y. enterocolitica is the most often encountered species of Yersinia in the lab. This bacterium is an invasive pathogen which can penetrate the gut lining and enter the lymphatic system and the blood. Infection, which is usually through ingestion of contaminated foods, can cause a severe intestinal inflammation called yersiniosis. Release of its enterotoxin can cause severe pain similar to that found in patients with appendicitis. Y. enterocolitica is easy to identify because it is able to grow in cold temperatures and is motile at room temperature. Antibiotic treatment can consist of aminoglycosides, chloramphenicol, or tetracycline.

49 Although not an enteric pathogen, Y. pestis is included hear because it causes the bubonic, pneumonic, and septicemic plagues. Human contraction of bubonic plague is usually through flea bites. Once inside the body, Y. pestis releases a toxin that inhibits electron transport chain function. Swelling of the lymph nodes, skin blotches, and dilerium are sometimes observed within a few days of infection. Untreated infections usually result in death within a week of initial infection. Although not common in the United States, the plague has historically been a deadly pathogen, inflicting Europeans in epidemic proportions during the fourteenth, fifteenth, and sixteenth centuries. A lack of sanitation allowed the plague to go unchecked killing tens of millions along the way. Fortunately, antibiotics such as streptomycin and gentamicin are effective in killing Y. pestis.

LABORATORY INDICATIONS: Y. entercolitica

· Urease + · Ornithine + · +/+ TSI reaction (No gas) · Motile at room temperature

LABORATORY INDICATIONS: Y. pestis

· Urease – · Ornithine - · Non-motile at room temperature

PLEOMORPHIC GRAM-NEGATIVE RODS

HAEMOPHILUS

BORDETELLA

BRUCELLA PASTEURELLA LEGIONELLA

HAEMOPHILUS

50 The Haemophilus genus represents a large group of Gram-negative rods that like to grow on blood agar. The blood provides two factors which many Haemophilus species require for growth: X factor and V factor. Sometimes Haemophilus is cultured using something called the "Staph streak" technique. In this procedure, both Staphylococcus and Haemophilus organisms are grown together on blood agar. Haemophilus colonies will usually form small colonies called "satellites" around Staphylococccus colonies because Staph can provide the necessary factors required for optimium Haemophilus growth. Morphologically, Haemophilus bacteria usually appear as tiny coccobacilli under the microscope, but they are included with the Pleomorphic bacteria because of the many shapes they assume. A methylene blue stain of a smear can also help with identification. Haemophilus species are classified by their capsular properties into six different serological groups, (a-f). Species that possess a type b capsule are clinically significant because of their virulent properties.

H. influenzae

Infection by H. influenzae is common in children and its name may lead you to conclude that it is the causative agent of the flu. Actually, this bacterium causes a secondary respiratory infection that usually inflicts those who already have the flu. This species may exist with or without a pathogenic polysaccharide capsule. Although both strains occur as normal flora of the nose and pharynx, they can confer severe illness in patients that are immunosuppressed or that have pre-existing respiratory ailments. Strains that lack the capsule usually cause mild localized infections (otitis media, sinusitis), as opposed to the type b encapsulated strain of H. influenzae that can cause several serious infections. Most of these infections occur in unvaccinated children less than five years of age because they have not yet formed antibodies against the bacterium. H. influenzae infection can lead to a variety of diseases:

51 Meningitis- H. influenzae is the most common cause of bacterial meningitis in children between the ages of five months and five years. The initial respiratory infection can spread to the blood stream and eventually the central nervous system. A stiff neck, lethargy, and the absence of the sucking reflex are common symptoms in infected babies. A vaccine is available, but is not always effective in very young children. Adult meningitis is much less common and usually only occurs in those predisposed to illness.

Epiglottitis- H. influenzae is the number one cause of this potentially fatal disease, which may cause airway obstruction in children between the ages of 2 and 4.

Haemophilus infection has also been associated with chronic bronchitis, pneumonia, bacteremia, conjuctivitis, and a host of other illnesses.

For serious infections, third generation cephalosporins are the drug of choice. Resistance may develop when ampicillin is used in treatment.

OTHER SPECIES

H. aegyptius

This bacterium is biochemically identical to H. influenzae. It is known to cause pinkeye (conjuctivitis) and is spread very easily, especially among children.

H. ducreyi

Chancroid, a sexually transmitted disease characterized by painful genital ulcers, is associated with this bacterium. H. ducreyi does not require the V factor for growth nor does it ferment glucose.

BORDETELLA

Bordetella organisms are small, Gram-negative coccobacilli which are strict aerobes. The three species of this genus vary in motility and certain biochemical characteristics. The most important species in this genus is B. pertussis, the organism which causes whooping cough. This highly contagious bacterium makes its way into

52 the respiratory tract via inhalation and subsequently binds to and destroys the ciliated epithelial cells of the trachea and bronchi. It does this through the use of several toxins:

Pertussis toxin- an exotoxin which enters target cells and activates their production of cAMP, a molecule that acts as a second messenger in cell protein synthesis regulation

Tracheal cytotoxin- causes ciliated epithelial cell destruction

Hemoagglutinin- a cell surface protein which helps the bacterium bind to the host cell surface

Under the microscope, Bordetella are often bipolar stained and appear singly or in pairs. The best method for isolation of Bordetella is on Bordet-Gengou agar or Regan- Lowe medium.

There are about 5000 cases of whooping cough per year in the United States, usually afflicting children less than a year old. A vaccine has reduced the incidence of this disease one hundred fold since its introduction! Despite this extraordinary rate of eradication, controversy exists regarding the safety of the vaccine. Antimicrobial therapy for whooping cough usually consists of erythromycin.

Two other species of Bordetella are also of clinical importance. B. parapertussis is a respiratory pathogen that can cause mild pharyngitis. This bacterium is similar to B. pertussis but lacks some of the toxins which make its sibling so nasty. Biochemical testing can easily differentiate the two species. Bordetella bronchiseptica is usually a cause of pneumonia, otitis media, and other respiratory infections in animals. It is

53 seldom known to be a human pathogen. Its motility makes it easy to distinguish from the other two organisms in this genus.

DIFFERENTIATION OF BORDETELLA SPECIES

Growth on Nitrate Urease Motility Citrate Blood Free Peptone Reduction

B. pertussis - - - - -

B. parapertussis + + - - +

B. bronchiseptica + + + + +

FRANCISELLA

The Francisella genus contains Gram-negative, non-motile, strict aerobes which can cause human infection. The most clinically important species, F. tularensis, causes tularemia which is disease that humans can catch from tick bites. This highly infectious disease is carried by rodents, deer, pets, and many other animals. Humans can acquire the organism in several different ways through lesions in skin. Many times their will be an ulcer at the site of penetration. Also, inhalation of the organism occurs in roughly 10% of all cases. A sudden onset of flu like symptoms (headache, fever, chills) is observed in infected individuals. Lab identification can be made using a medium called cysteine-blood agar. The is effective because F. tularensis requires cysteine (an amino acid) for growth. Most hospital labs shouldn't process this organism, however, because it is highly contageous. The preferred antibiotic treatment for treating patients consists of streptomycin.

54

PASTEURELLA

Organisms of the genus Pasteurella are Gram-negative, non-motile, facultatively anaerobic coccobacilli. Unlike most of the pleomorphic organisms we have spoken of, Pasteurella is not an intracellular parasite. P. multocida is the species which most commonly infects humans. Although most members infect animals, humans can acquire the organism from dog or cat bites. Patients tend to exhibit swelling, cellulitis, and some bloody drainage at the wound site. Infection may also move to nearby joints where it can cause swelling and arthritis (not to mention a lot of pain). Fortunately, P. multocida is susceptble to penicillin, tetracycline, and chloramphenicol. For identification, this organism can be cultured on chocolate agar and can produce a really foul odor.

LABORATORY INDICATIONS:

Oxidase +

Non-motile

Catalase +

Non-hemolytic (some are beta-hemolytic)

Indole +

Ornithine decarboxylase +

LEGIONELLA

The first discovery of bacteria from genus Legionella came in 1976 when an outbreak of pneumonia at an American Legion convention led to 29 deaths. The causative agent, what would come to be known as Legionella pneumophila, was isolated and given its own genus. The organisms classified in this genus are Gram-negative

55 bacteria that are considered intracellular parasites. They grow well on buffered charcoal yeast extract agar, but it takes about five days to get sufficient growth. When grown on this medium, Legionella colonies appear off-white in color and circular in shape. Laboratory identification can also include microscopic examination in conjunction with a direct flourescent antibody (DFA) test. Since the initial discovery many species have been added to the Legionella genus, but only two are within the scope of our discussion: L. pneumophila and L. micdadei.

LABORATORY INDICATIONS:

Motile • Urease -

Catalase +

Nitrate -

Gelatinase +

L. pneumophila

L. pneumophila is the bacterium associated with Legionnaires' disease and Pontiac fever. Respiratory transmission of this organism can lead to infection, which is usually characterized by a gradual onset of flu-like symptoms. Patients may experience fever, chills, and a dry cough as part of the early symptoms. Patients can develop severe pneumonia which is not responsive to penicillins or aminoglycosides. Legionnaires' disease also has the potential to spread into other organ-systems of the body such as the gastrointestinal tract and the central nervous system. Accordingly, patients with advanced infections may experience diarrhea, nausea, disorientation, and confusion. The 1200 or so cases of Legionnaires' disease per year in the United States usually involve middle-aged or immunosuppressed individuals. Pontiac fever is also caused by L. pneumophila but does not produce the severity of the symptoms found in Legionnaires' disease. The flu-like symptoms are still seen in Pontiac fever patients

56 but pneumonia does not develop and infection does not spread beyond the lungs. Most L. pneomophila infections are easily treated with erythromycin. LABORATORY INDICATIONS:

· Beta-lactamase + · Hippurate hydrolysis +

L. micdadei

L. micdadei is the second most commonly isolated member of Legionella. This bacterium can cause the same flu-like symptoms and pneomonia which characterize an L. pneumophila infection. Unlike its relative, L. micdadei is sensitive to the penicillins because it does not produce beta-lactamase. LABORATORY INDICATIONS:

· Beta-lactamase - · Hippurate hydrolysis - · Acid fast

MISCELLANEOUS GRAM-NEGATIVE RODS

57 VIBRIO

CAMPYLOBACTER HELICOBACTER

VIBRIO

The Vibrio genus contains motile, Gram-negative bacteria that are obligate aerobes. Vibrio have a recognizable curved shape and a single polar flagella. Although Vibrio species are non-invasive pathogens, they cause some of the most serious cases of diarrhea and thousands of people die from infection annually. These waterborne organisms are transmitted to humans via infected water or through fecal transmission. Thus, in countries with poor sewage or water treatment cholera is sometimes seen in epidemic proportions.

V. cholerae

Several species of Vibrio are known to be human pathogens, the most famous of which is V. cholerae (the causative agent of cholera). Cholera is characterized by severe diarrhea which has a rice-water color and consistancy. The diarrhea is so severe that about 60% percent of cholera deaths are due to dehydration. After cholera organisms are ingested, they descend to the intestinal tract where they bind to the epithelium and release their exotoxin, choleragen. This induces the epithelial cells to excrete salt. The cells then lose water which passively flows out of the cells. Fluid and electrolyte replacement is the key to treating cholera patients. Lab identification of V. cholerae is almost conclusive if the collected specimen is plated onto thiosulfate citrate bile sucrose (TCBS) agar. V. cholerae will form small yellow colonies on this medium. Simultaneous administration of doxycycline kills the organism.

LABORATORY INDICATIONS:

· Oxidase + • Catalase + • Indole + · Lysine decarboxylase + • Ornithine deaminase +

OTHER VIBRIO

58 Another species of Vibrio that causes diarrhea is V. parahaemolyticus. Raw seafood such as sushi and oysters is the source of human infection. Along with severe diarrhea, patients can also experience cramps, nausea, and fever. This disease is self- limiting and only lasts about three days. Therefore, no antiobiotic treatment is necessary in most cases.

V. vulnificus is also obtained from eating contaminated seafood. Unlike other Vibrio species, this one is invasive and is able to enter the blood stream through the epithelium of the gut. Fever, vomiting, and chills are the symptoms normaly associated with infection of this organism. Additionally, wound infection may also occur from contaminated sea water. Cellulitis or ulcer formations may result. Treatment is usually with tetracycline.

LABORATORY INDICATIONS:

· Halophilic · Lactose fermenting (V. vulnificus)

CAMPYLOBACTER

Organisms of the genus Campylobacter are Gram-negative, microaerophiles that cause 5-11% of all diarrhea cases in the United States. Cell motility is achieved through polar flagella which emanate from a curved rod-shaped cell. The unique shape of the cell and flagella are extremely useful in Gram-stain identification. Campylobacter are microaerophiles, which means that they can survive in a low oxygen environment. What is unusual about the organism, though, is that it also prefers a relatively high concentration of carbon dioxide in the environment. You can imagine the difficulty of producing an environment suitable for culturing this organism.

The most commonly isolated species of Campylobacter is C. jejuni, an organism that causes gastrointestinitis. Humans acquire the organisms by eating undercooked

59 chicken or drinking contaminated milk and water. Infection usually leads to fever, cramps, and bloody diarrhea. The bloody diarrhea indicates that Campylobacter is an invasive pathogen that infiltrates the lining of the small intestine. Along the way, the organism excretes toxins that destroy the gut mucosa. Erythromycin is the preferred antibiotic for treatment.

As previously mentioned, these organisms require special environmental conditions for optimal growth. Campy blood agar is a selective medium which can be used for isolating C. jejuni. A series of biochemical tests can then ditinguish C. jejuni from other species of Campylobacter.

LABORATORY INDICATIONS (C. jejuni):

· Motile Hippurate hydrolysis · Catalase + • Nitrate +

HELICOBACTER

The Helicobacter genus features Gram-negative, micraerophilic organisms that are very similar to organisms of the Campylobacter genus. Both species are motile, catalase-positive, and have curved cell bodies. When you think of Helicobacter, the first thing that should come to your mind is stomach ulcers. That is because this organism is the leading cause of peptic ulcers and chronic gastritis in America. The mechanisms for pathogenesis are not well understood at this time, but the circumstantial evidence points to Helicobacter pylori as the causative agent. Infected patients can be treated with an antacid as well as tetracycline to treat the ulcers and inhibit the growth of the organism. The microaerophilic nature of Helicobacter means that it requires a low oxygen concentration and a relatively high carbon dioxide concentration. Sufficient colony growth can be seen in three days when the organism is plated onto Brucella- sheep blood agar and placed in a microaerophilic environment. A Gram-stain will reaveal weakly stained rod with a unique curvature. Helicobacter pylori is also strongly urease-positive, which may help in identification.

60

LABORATORY INDICATIONS (H. pylori):

· Urease + • Caalase + • Motile • Microaerophile

NONFERMENTERS

The nonfermenters are classified as such because of the way they metabolize glucose and other carbohydtrates. Specifically, the bacteria discussed in this section are Gram- negative rods that either don't ferment glucose for energy or do not use glucose at all. This is a very diverse group of organisms in that they are not classified by DNA homology. These are also some of the most difficult bacteria to identify because they are negative for so many tests. Many nonfermenters do not grow on MacConkey agar. The two most common identification used for these organisms are the OF-glucose test and the triple sugar iron (TSI) test. We will discuss only the clinically significant organisms.

PSEUDOMONAS

ACINETOBACTER OTHERS

PSEUDOMONAS

Pseudomonads are motile, Gram-negative rods that utilize glucose oxidatively. Members of this genus are classified into five groups based on ribosomal RNA homology. These bacteria are clinically important because they are resistant to most

61 antibiotics and they are capable of surviving in conditions that few other organisms can tolerate. They also produce a slime layer that is resistant to phagocytosis. Pseudomonas is often encountered in hospital and clinical work because it is a major cause of hospital acquired (nosocomal) infections. Its main targets are immunocompromised individuals, burn victims, and individuals on respirators or with indwelling catheters. Additionally, these pathogens colonize the lungs of cystic fibrosis patients, increasing the mortality rate of individuals with the disease. Infection can occur at many sites and can lead to urinary tract infections, sepsis, pneumonia, pharyngitis, and a lot of other problems. Rarely will you find Pseudomonas as a cause of infection in healthy individuals. Its non-invasive nature limits its pathogenic capabilities.

P. aeruginosa

Pseudomonas aeruginosa is the most frequently isolated non-fermenter in the laboratory. It has several features that distinguish it from other species of Pseudomonas:

· It can grow at 42 degrees celsius

· Produces a bluish pigment (pyocyanin) and a greenish pigment · Characteristic fruity odor

The basis of this organisms pathogenicity involves several toxins and chemicals which the bacterium secretes upon infection. The lipopolysaccharide layer helps the cell adhere to host tissues and prevents leukocytes from ingesting and lysing the organism. Lipases and exotoxins then procede to destroy host cell tissue which then leads to the complications associated with infection. P. aeruginosa prefers to inhabit moist environments but it can survive in a medium as deficient as distilled water. It will also grow on just about any laboratory medium and is beta-hemolytic on blood agar. Treatment of Pseudomonas infection consists of a combination of two antibiotics: for example an anti-pseudomonal penicillin and an aminoglycoside. The best way to reduce the spread of P. aeruginosa in the hospital is to use good aseptic technique on hospital instruments and when in contact with patients. LABORATORY INDICATIONS:

62 · Oxidase + Beta-hemolytic Characteristic odor and color · Motile

OTHER SPECIES

Burkholderia (Pseudomonas) cepacia is an opportunistic pathogen of cystic fibrosis patients. It also shows substantially greater antibiotic resistance than its relative, P. aeruginosa. However, B. cepacia can be distinguished from Pseudomonas species because it is lysine positive.

Although not of the same genus, Stenotrophomonas maltophila (formerly known as Xanthomonas maltophila) is very similar to the Pseudomonads. This motile bacterium is a cause of nosocomal infections in immunocompromised patients. S. maltophila also harbors significant resistance to many antibiotics considered effective for treating Pseudomonas infections. However, most strains of the bacterium are susceptible to trimethoprim sulfamethoxazole. S. maltophila can also be distinguished from Pseudomonas species by the lysine and DNAse tests for which it is positive.

ACINETOBACTER

Acinetobacter species are oxidase-negative, non-motile bacteria which appear as Gram-negative coccobacilli in pairs under the microscope. Identifying the different species of this genus can be done through the use of FLN (Flourescence-Lactose- Denitrification medium) acid results which determines the amount of acid produced from metabolizing glucose. Also, most members of Acinetobacter show good growth on MacConkey agar with the exception of some A. lwoffii strains. Although many species of Acinetobacter can cause infection, A. baumannii is the most frequently encountered species in the clinical laboratory. Like Pseudomonas, A. baumannii can be linked to many hospital acquired infections including skin and wound infections, pneumonia, and meningitis. A. lwoffi, in particular, is responsible for most cases of meningitis caused by Acinetobacter. Because most species are resistant to penicillin and chloramphenicol, a combination of aminoglycoside and ticarcillin is usually recommended for treatment.

LABORATORY INDICATIONS:

63 · Oxidase - · Non-motile · Penicillin resistance (most strains)

OTHER NONFERMENTERS

Flavobacterium

Members of Flavobacterium are ubiquitous organisms that can cause infection in premature infants and immunocompromised individuals. They are not difficult to distinguish from other nonfermenters because most species produce indole when grown in tryptophan broth. While most species of Flavobacterium metabolize glucose oxidatively, some have been shown to be really slow fermenters. In any case, all species are motile and oxidase positive. The species most often recovered from humans is F. meningosepticum, a penicillin resistant bacterium that can cause neonatal meningitis. Vancomycin is usually effective in Flavobacterium infection.

Alcaligenes

All the species in this genus are oxidase positive, motile, and can grow on MacConkey agar. The most coommon species A. faecalis, an opportunistic pathogen which usually inhabits soil and water. This organism has a characteristic fruity odor which distinguishes it from other Alcaligenes species.

64 GRAM POSITIVE ANAEROBES

· GRAM-POSITIVE BACILLI (SPORE-FORMING) o CLOSTRIDIUM · GRAM-POSITIVE BACILLI (NON-SPORE-FORMING) o ACTINOMYCES o BIFIDOBACTERIUM o EUBACTERIUM o PROPIONIBACTERIUM o LACTOBACILLUS (discussed with Gram-positive rods) · GRAM-POSITIVE COCCI o PEPTOSTREPTOCOCCUS

CLOSTRIDIUM

Members of genus Clostridium are Gram-positive, spore-forming rods that are anaerobic. These motile bacteria are ubiquitous in nature and are especially fond of soil. Under the microscope, they appear as long drumsticks with a bulge located at their terminal ends. A Gram-stain is a good method for identifying Clostridium because the cell incorporates the dye while the spore remains unstained. Clostridium shows optimimum growth when plated on blood agar at human body temperatures. When the environment becomes stressed, however, the bacteria produce spores that tolerate the extreme conditions that the active bacteria cannot. In their active form, these bacteria secrete powerful exotoxins that are responsible for such diseases as tetanus, botulism, and gas gangrene. The four clinically important species of Clostridium will be discussed here: C. tetani, C. difficile, C. perfringens, and C. botulinum.

C. tetani

Clostridium tetani is the bacterium that causes tetanus (lockjaw) in humans. C. tetani spores can be acquired from any type of skin trauma involving an infected device. If an anaerobic environment is present, the spores will germinate and eventually form active C. tetani cells. At the tissue level, the bacterium then releases an exotoxin called tetanospasmin that causes certain nervous system irregularities by means of retrograde tramsmission through neurons to the brain. One of the toxin's effects includes constant skeletal muscle contraction due to a blockage of inhibitory interneurons that regulate muscle contraction. Prolonged infection eventually leads to respiratory failure, among other things. If not treated early, the mortality rates of this

65 disease are high. Immunization is the best way to prevent C. tetani infections in children and adults. The process is started early with the first four shots being administered within two years of birth. The initial shots are then followed up with periodic booster shots given every ten years.

LABORATORY INDICATIONS:

· Motile • Terminal spores • Non-aerotolerant

C. botulinum

Clostridium botulinum produces one of the most potent toxins in existence and the cause of the deadly botulism food poisoning. Because Clostridium spores can be airborne, they sometimes find their way into foods that will be placed in anaerobic storage such as cans or jars. Once the jars are sealed, the spores germinate and the bacteria release their potent toxin. Patients will experience muscular paralysis as well as blurred vision. Immmediate treatment with an anti-toxin must take place for the patient to have a chance at survival. Infantile botulism is acquired in a similar manner but is much milder than the adult version. Honey, however, is the most common source of the spores that germinate in the child's intestinal tract. Bacterial proliferation and subsequent toxin production cause symptoms which last a few days and then subside without the use of an antitoxin.

LABORATORY INDICATIONS:

· Motile · No terminal spores · Non-aerotolerant · Lipase +

C. perfringens

This non-motile bacterium is an invasive pathogen that can be contracted from dirt via large cuts are wounds. C. perfringens cells proliferate after spore germination occurs and they release their exotoxin. The toxin causes necrosis of the surrounding tissue (Clostridial myonecrosis destroys muscular tissus). The bacteria themselves produce gas which leads to a bubbly deformation of the infected tissues. C. perfringens is

66 capable of necrotizing intestinal tissues and can release an enterotoxin that may lead to severe diarrhea. Treatment of infection can consist of penicillin G (to kill the organism), hyperbaric oxygen (?), and administration of an antitoxin.

LABORATORY INDICATIONS:

· Non-motile · No terminal spores · Non-aerotolerant · Double zone hemolysis

C. difficile

Clostridium difficile is a motile bacterium that can be part of the natural intestinal flora. Infection can occur through the use of broad-spectrum antibiotics which lower the relative amount of other normal gut flora. When this situation occurs, C. difficile proliferates and infects the large intestine. The bacterium then releases two enterotoxins that destroy the intestinal lining and cause diarrhea. The preferred method of treatment is oral vancomycin.

LABORATORY INDICATIONS:

· Motile · No terminal spores · Non-aerotolerant

67 GRAM-POSITIVE BACILLI (NON-SPORE-FORMING)

ACTINOMYCES

Actinomycetes are fungus-like bacteria that form filamentous branches. These Gram- positive obligate anaerobes are known to reside in the mouth and in the intestinal tract. Pathogenic proliferation of the organisms, which is usually a result of trauma to the region of infection, can lead to actinomycosis. The patient will form abscesses and swelling at the site of infection. A diagnosis can be made upon microscopic examination of pus. The fluid will have a granular texture which is caused by sulfur granules. These sulfur granuules are actually composed of the bacterium and its waste. The species of Actinomyces which is most commonly associated with actinomycosis is A. israelii, but several other bacteria in this genus are capable of causing the disease as well. Actinomycosis can often be treated with penicillin.

LABORATORY INDICATIONS:

· Indole - Catalase - · Lipase - DNase -

BIFIDOBACTERIUM

Members of the genus Bifidobacterium are anaerobic, Gram-positive bacilli that is rarely associated with infection. The only pathogenic species of this genus is Bifidobacterium dentium, a normal inhabitant of the gut flora. Under the microscope, these bacteria appear to be bone shaped, which makes them easy to identify. As obligate anaerobes, they require a very low oxygen tension to survive and to achieve moderate growth.

LABORATORY INDICATIONS:

· Non-motile · Catalase - · Forms branching filaments

68

EUBACTERIUM

Eubacterium species are not of much clinical importance. However, because they are normal flora of the intestinal tract, they may cause opportunistic infections. E. lentum, the most often isolated species, has been linked to endocarditis and some wound infections. Biochemical testing can distinguished Eubacterium from the other Gram- positive, anaerobic rods. Because Eubacterium species are negative for many tests, results nmay be somewhat ambiguous. It is important to know, however, that these bacteria tend to form clumps under microscopic observation.

LABORATORY INDICATIONS:

· Indole - · Catalase - · Hydrogen sulfide -

PROPIONIBACTERIUM

Propionibacterium species are some of the most common Gram-positive anaerobes that are isolated in the laboratory. One particular species, P. acnes, is a usually harmless microbe that has pathogenic potential. It has been linked to certain cases of endocarditis, wound infections, and abscesses. Ironically, it can infect acne sites on the skin but it does not cause them. Under the microscope, Propionibacterium clump up and may show a slight tendency to branch. Also, they show uneven staining patterns following a Gram-stain procedure. Colonies grow best in an anaerobic or microaerophilic environment using blood agar.

LABORATORY INDICATIONS (P. acnes):

· Indole + • Catalase + · Glucose fermentation Gelatin hydrolysis

69

PEPTOSTREPTOCOCCUS

The genus Peptostreptococcus contains very small bacteria that grow in chains. These anaerobic counterparts of Streptococcus are usually not harmfull. They are known to be normal flora of the skin, urethra, and the urogenital tract. If given an opportunity, however, they can cause infections of bones, joints and soft tissue. Their increasing resistance to such antibiotics as penicillin G and clindamycin makes them especially important to clinical work. P. magnus is the species that is most often isolated from infected sites.

LABORATORY INDICATIONS:

· Esculin hydrolysis - · Hydrogen sulfide - · Catalase - • Lactose -

BACTEROIDES

The Bacteroides genus of anaerobic bacteria comprise the majority of microorganisms that inhabit the digestive tract. 50% of most fecal matter is actually Bacteroides fragilis cells! Bacteroides organisms are the anaerobic counterpart of E. coli except they are somewhat smaller. They grow well on blood agar, and under the microscope, they may contain large vacuoles that are similar in appearance to spores. Members of Bacteroides species are not spore-forming, but they do produce a very large capsule. Their pathogenicity is limited, however, because they possess no endotoxin in their cell membrane. Infection only occurs after severe trauma to the abdominal region. Infection could lead to abscess formation and possibly fever. Antibiotic treatment usually consists of metronidazole or clindamycin.

LABORATORY INDICATIONS (B. fragilis):

· Indole - Catalase + · Esculin hydrolysis + · Glucose fermentation Lactose +

70

FUSOBACTERIUM

Fusobacterium organisms are anaerobic, Gram-negative bacilli that bare a strong resemblance to certain Bacteroides species. Under the microscope, they are normally spindle-shaped cells with sharp ends. Both bacteria are normal gut inhabitants but are capable of causing serious infection. Fusobacterium species, the most common of which is F. nucleatum, are associated with pleuropulminary infections and disease. They are also capable of causing infection in the oral cavity (the mouth). F. nucleatum has been cited as one of the causes of gingivitis. Identification of these bacteria

LABORATORY INDICATIONS (F. nucleatum):

· Indole + · Hydrogen sulfide - · Catalase -

VEILLONELLA

Veillonella are Gram-negative cocci that are the anaerobic counterpart of Neisseria. These non-motile diplococci are part of the normal flora of the mouth. The reason this organism is important is not due to its pathogenicity. Instead, we include Veillonella because it can be and is often mistaken for the more serious gonococcal infection. The most common species isolated from humans is V. parvula. Veillonella species are negative for almost every biochemical test with the exception of an occasional strain being positive for catalase.

71 Background on bacterial genetics (introduction) Structure and Function of Genetic Material 1. Genetics is the study of what genes are how they carry information, how their information is expressed, and how they are replicated and passed to subsequent generations or other organisms. 2. DNA in cells exists as a double-stranded helix; the two strands are held together by hydrogen bonds between specific nitrogenous base pairs: A-T and C-G. 3. A gene is a segment of DNA, a sequence of nucleotides, that codes for a functional product, usually a protein. 4. When a gene is expressed, DNA is transcribed to produce RNA; mRNA is then translated into proteins. 5. The DNA in a cell is replicated before the cell divides, so each daughter cell receives the same genetic information. Genotype and Phenotype 1. Genotype is the genetic composition of an organism—its entire DNA. 2. Phenotype is the expression of the genes—the proteins of the cell and the properties they confer on the organism. DNA and Chromosomes 1. The DNA in a chromosome exists as one long double helix associated with various proteins that regulate genetic activity. 2. Bacterial DNA is circular; the chromosome of E. coli, for example, contains about 4 million base pairs and is approximately 1000 times longer than the cell. 3. Genomics is the molecular characterization of genomes. 4. Information contained in the DNA is transcribed into RNA and translated into proteins. DNA Replication 1. During DNA replication, the two strands of the double helix separate at the replication fork, and each strand is used as a template by DNA polymerases to synthesize two new strands of DNA according to the rules of nitrogenous base pairing. 2. The result of DNA replication is two new strands of DNA, each having a base sequence complementary to one of the original strands.

72 3. Because each double-stranded DNA molecule contains one original and one new strand, the replication process is called semiconservative. 4. DNA is synthesized in one chemical direction called 5' à 3' (5' is phosphate end; 3' is hydroxyl end of deoxyribose). At the replication fork, the leading strand is synthesized continuously and the lagging strand, discontinuously. 5. DNA polymerase proofreads new molecules of DNA and removes mismatched bases before continuing DNA synthesis. Errors only occur ~1 time for every 1010 bases added. 6. Each daughter bacterium receives a chromosome identical to the parent's. RNA and Protein Synthesis 1. During transcription, the enzyme RNA polymerase synthesizes a strand of RNA from one strand of double-stranded DNA, which serves as a template. 2. RNA is synthesized from nucleotides containing the bases A, C, G, and U, which pair with the bases of the DNA sense strand. 3. The starting point for transcription, where RNA polymerase binds to DNA, is the promoter site; the region of DNA that is the endpoint of transcription is the terminator site; RNA is synthesized in the 5' —> 3' direction. 4. Translation is the process in which the information in the nucleotide base sequence of mRNA is used to dictate the amino acid sequence of a protein. 5. The mRNA associates with ribosomes, which consist of rRNA and protein. 6. Three-base segments of mRNA that specify amino acids are called codons. 7. The genetic code refers to the relationship among the nucleotide base sequence of DNA, the corresponding codons of mRNA, and the amino acids for which the codons code. 8. The genetic code is degenerate; that is, most amino acids are coded for by more than one codon. 9. Of the 64 codons, 61 are sense codons (which code for amino acids), and 3 are nonsense codons (stop codons) which do not code for amino acids and are stop signals for translation. 10. The start codon, AUG, normally codes for methionine (formylmethionine at the beginning or a protein). 11. Specific amino acids are attached to molecules of tRNA. Another portion of the tRNA has a base triplet called an anticodon.

73 12. The base pairing of codon and anticodon at the ribosome results in specific amino acids being brought to the site of protein synthesis. 13. The ribosome moves along the mRNA strand as amino acids are joined to form a growing polypeptide; mRNA is read in the 5' —> 3' direction. 14. Translation ends when the ribosome reaches a stop codon on the mRNA. 15. In prokaryotes, translation can begin before transcription is complete. Regulation of Gene Expression in Bacteria 1. Regulating protein synthesis at the gene level is energy-efficient because proteins are synthesized only as they are needed. 2. Constitutive enzymes are always present in a cell. Examples are genes for most of the enzymes in glycolysis. 3. For these genetic regulatory mechanisms, the control is aimed at mRNA synthesis. Repression 1. Repression controls the synthesis of one or several (repressible) enzymes. 2. When cells are exposed to a particular end product, the synthesis of enzymes related to that product decreases. 3. An example of a repressible genetic system is the synthesis of the amino acid tryptophan in E. coli. Induction 1. In the presence of certain chemicals (inducers), cells synthesize more enzymes. This process is called induction. 2. An example of an inducible genetic system is the production of b -galactosidase by E. coli in the presence of lactose, so lactose can be metabolized. The Operon Model of Gene Expression 1. The formation of enzymes is determined by structural genes. 2. In bacteria, a group of coordinately regulated structural genes with related metabolic functions and the promoter and operator sites that control transcription are called an operon. 3. In the operon model for an inducible system,a regulatory gene codes for repressor protein. 4. When the inducer is absent, repressor binds to the operator and no mRNA is synthesized. 5. When the inducer is present, it binds to the repressor so that it cannot bind to the operator; thus, mRNA is made and enzyme synthesis is induced.

74 6. In repressible systems, the repressor requires a corepressor in order to bind to the operator site; corepressor controls enzyme synthesis. 7. Transcription of structural genes for catabolic enzymes (such as b -galactosidase) is induced by the absence of glucose. cAMP and CRP must bind to a promoter in the presence of an alternative carbohydrate (such as lactose). 8. The presence of glucose inhibits metabolism of alternative carbon sources by catabolic repression. Mutation: Change in the Genetic Material 1. A mutation is a change in the nitrogenous-base sequence of DNA; that change causes a change in the product coded for by the mutated gene. 2. Many mutations are neutral (silent), some are disadvantageous (or even lethal), and others are beneficial. Types of Mutations 1. A base substitution occurs when one base pair in DNA is replaced with a different base pair. 2. Alterations in DNA can result in missense mutations (which cause amino acid substitutions) or nonsense mutations (which create stop codons). 3. In a frameshift mutation, one or a few base pairs are deleted or added to DNA. 4. Mutagens are agents in the environment that cause permanent changes in DNA. 5. Spontaneous mutations occur without the presence of a mutagen. The spontaneous mutation rate varies from genome to genome. Mutagens 1. Chemical mutagens include base-pair mutagens (for example, nitrous acid), nucleoside analogs (for example, 2-aminopurine and 5-bromouracil), and frameshift mutagens (for example, benzpyrene). 2. Ionizing radiation causes the formation of ions and free radicals that react with DNA; base substitutions or breakage of the sugar-phosphate backbone result. 3. Ultraviolet radiation is nonionizing, causing bonding between adjacent thymines (thymine dimers). Repair of Mutated DNA 1. Nucleotide excision repair can enzymatically repair mutational damage to DNA by cutting out and replacing the damaged portion. 2. Photoreactivation enzymes (light-repair enzymes; photolyases) can repair thymine dimers caused by exposure to ultraviolet radiation in the presence of visible light.

75 Frequency of Mutation 1. Mutation rate is the probability that a gene will mutate when a cell divides; the rate is expressed as 10 to a negative power. 2. Mutations occur randomly along a chromosome. 3. A low rate of spontaneous mutation is beneficial, providing genetic diversity needed for evolution. Identifying Mutants 1. Mutants can be detected by selecting or testing for an altered phenotype. 2. Positive selection involves the selection of mutant cells and rejection of nonmutated cells. 3. Replica plating is used for negative selection—to detect, for example, auxotrophs that have nutritional requirements not possessed by the parent (nonmutated) cell. Identifying Chemical Carcinogens 1. The Ames test is an inexpensive and rapid test for identifying possible chemical carcinogens. 2. The test assumes that a mutant cell can revert to a normal cell in the presence of a mutagen and that many mutagens are carcinogens. 3. Histidine auxotrophs (Salmonella) are exposed to an enzymatically treated potential carcinogen; reversions to the nonmutant state are selected. Genetic Transfer and Recombination 1. Genetic recombination, the rearrangement of genes from separate groups of genes, usually involves DNA from different organisms; it contributes to genetic diversity. 2. In crossing over, genes from two chromosomes are recombined into one chromosome containing some genes from each original chromosome. 3. Vertical gene transfer occurs during reproduction when genes are passed from an organism to its offspring. 4. Horizontal gene transfer in bacteria involves a portion of the cell's DNA being transferred from donor to recipient. 5. When some of the donor's DNA has been integrated into the recipient's DNA, the resultant cell is called a recombinant. Transformation in Bacteria 1. During this process, genes are transferred from one bacterium to another as "naked" DNA in solution.

76 2. This process was first demonstrated in Streptococcus pneumoniae, and occurs naturally among a few genera of bacteria. Conjugation in Bacteria 1. This process requires contact between living cells ('bacterial sex'). 2. One type of genetic donor cell is an F+; recipient cells are F-. F+ cells contain plasmids called F factors; these plasmids are transferred to the F- cells during conjugation. 3. When the plasmid becomes incorporated into the chromosome, the cell is called an Hfr (high-frequency recombinant). 4. During conjugation, an Hfr can transfer chromosomal DNA to an F-. Usually, the Hfr chromosome breaks before it is fully transferred. Transduction in Bacteria 1. In this process, DNA is passed from one bacterium to another via a bacteriophage and is then incorporated into the recipient's DNA. 2. In generalized transduction, any bacterial genes can be transferred. 3. In specialized transduction, only genes adjacent to a prophage (lysogenic bacteriophage integrated into bacterial chromosome) can be transferred. Plasmids and Transposons 1. Plasmids are self-replicating, closed circular extrachromosomal molecules of DNA carrying genes that are not usually essential for survival of the cell. 2. There are several types of plasmids, including conjugative plasmids, dissimilation plasmids, plasmids carrying genes for toxins or bacteriocins, and resistance factors. 3. Transposons are small segments of DNA that can move from one region of a chromosome to another region of the same chromosome or to a different chromosome or a plasmid. 4. Transposons are located in the chromosomes of organisms, in plasmids, and in the genetic material of viruses. They vary from simple (insertion sequences) to complex. 5. Complex transposons can carry any type of gene, including antibiotic-resistance genes, and are thus a natural mechanism for moving genes from one chromosome to another. Genes and Evolution 1. Diversity is the precondition of evolution.

77 2. Genetic mutation and recombination provide a diversity of organisms, and the process of natural selection allows the growth of those best adapted for a given environment.

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