Fermentative Proteobacteria and Biofilms and Quorum Sensing

Diversity of Bacteria

• The lecture will focus on bacteria regarding their phylogenetic classifications.
• There will be a summary of the major traits of the different classes and selected species of Gammaproteobacteria.

Learning Objectives

• Compare and contrast the physiology and importance of members of the Enterobacteriales and the Vibrionales.
• Summarize mixed acid and butanediol fermentation.
• Describe the use of metabolic assays to distinguish between enteric bacteria.
• Describe how bacteria use regulatory mechanisms to control complex processes and quorum sensing.

Diversity of Bacteria

• Bacteria are phylogenetically diverse.
• More than 90% of characterized genera and species come from four phyla.
• The four Phylas include:
  • Proteobacteria
  • Actinobacteria
  • Firmicutes
  • Bacteroidetes

Proteobacteria

• Consist of five clusters containing several genera.
• Each cluster has a Greek letter designation.
• Alphaproteobacteria, such as Rhizobium, can survive at very low nutrient levels and have unusual morphologies like stalks and buds.
• Betaproteobacteria, such as Bordetella pertussis, use nutrients diffusing from anaerobic decomposition areas, including hydrogen gas, ammonia, and methane.
  • This includes chemoautotrophs.
• Gammaproteobacteria is the largest group, including Acinetobacter, Pseudomonas, Escherichia coli, Salmonella, and Serratia marcescens.
• Deltaproteobacteria, such as Desulfovibrio, are predators on other bacteria and important contributors to the sulfur cycle.
• Epsilonproteobacteria, such as Helicobacter, are slender, helical or curved Gram-negative rods, motile by flagella, and microaerophilic.
• Proteobacteria constitute a major bacterial lineage (phylum).
  • The name comes from the Greek god Proteus, known for assuming many shapes.
• This group encompasses many commonly encountered bacteria, divided into five classes: Alpha, Beta, Delta, Gamma, and Epsilon.
• Proteobacteria are the most metabolically diverse bacteria, utilizing chemolithotrophy, chemoorganotrophy, and phototrophy.
• These bacteria are also morphologically diverse.
• A majority of known bacteria that are of medical, industrial and agricultural importance
• It represents a major branch on the bacterial phylogenetic tree.
• Defined primarily by rRNA sequences.
• Genome sequencing has shown about half of the bacterial genomes in NCBI are proteobacteria and are well studied because they are easily cultured.
• All proteobacteria are Gram-negative and can be found in almost all environments.
• Fermentation is a diverse array of physiological traits including aerobic respiration, nitrification, nitrogen fixation, denitrification, sulfate reduction, phototrophy, metal and methane oxidation, and sulfur and sulfide oxidation.
  • Many pathogens are proteobacteria

Gammaproteobacteria

• Key genera includes: Enterobacter, Escherichia, Klebsiella, Proteus, Salmonella, Serratia, and Shigella
• It is the largest and most diverse class of Proteobacteria with diverse metabolic and ecological characteristics

Fermentative Proteobacteria

• Enteric Bacteria are are all within the Gamma Proteobacteria.
• ("enter" is Greek for "gut"), and their habitat is within the intestines of certain animals.
• It represents a large group of facultative aerobic rods of medical and molecular biological significance.
• Enteric bacteria are Gram-negative and facultative aerobes.
  • They can be motile or nonmotile, are nonsporulating rods, and use peritrichous flagella.
• These bacteria possess simple nutritional requirements.
• Oxidase (negative) and catalase (positive) tests are used to distinguish enteric from other bacteria.
• They ferment sugars to produce a variety of end products and are medically and scientifically important, containing many pathogens and industrially important bacteria.

Famous Genera

• Salmonella: S. typhi causes typhoid fever.
• Escherichia (E. coli): It is a commensal and pathogen in gastrointestinal infections.
• Shigella: S. dysenteriae is closely related to E. coli.
• Yersinia: Y. pestis causes plague.
• Klebsiella: It fixes N₂ and can cause pneumonia.
• Serratia: It produces the red pigment prodigiosin.
• Erwinia: It is a common plant pathogen.
• Vibrio: The name comes from the Latin "vib" meaning 'whip mark'.
• Photobacterium: bioluminescent.
• Aeromonas: Contains fish pathogens
• Enteric bacteria can be separated into two broad groups based on the byproduct's generated during anaerobic fermentation of glucose.
  • Mixed-acid fermenters.
  • 2,3-butanediol fermenters.

Fermentation

• Two broad fermentation patterns include mixed-acid fermentation seen in E.coli, Salmonella, Shigella, Citrobacter, Proteus, and Yersinia.
  • Three acids formed are acetic acid, lactic acid and succinic acid.
  • Also generated are ethanol, CO2, and H2_in equal amounts via formate hydrogenlyase but not butanediol.
• The other is 2,3-butanediol fermentation seen in Enterobacter aerogenes, Klebsiella, Erwinia, Serratia.
  • The main fermentation products are butanediol, ethanol, CO2, and H2 with undetected levels of acids.

Examples of Mixed-Acid Fermenters

• Escherichia are universal inhabitants of the intestinal tract of humans and warm-blooded animals.
  • Synthesize vitamins for the host and some strains are pathogenic, causing diarrhea, urinary tract infections in women, gastrointestinal infections, and foodborne disease.
• Salmonella and Shigella are closely related to Escherichia, and usually are pathogenic and characterized immunologically by surface antigens.
  • Salmonella causes typhoid fever and gastroenteritis.
  • Shigella causes bacillary dysentery.
• Proteus, the genus has rapidly motile cells capable of swarming motility.
  • Colonies form concentric rings.
  • It is a frequent cause of urinary tract infections in humans and produce urease.

Butanediol Fermenters

• Enterobacter aerogenes or Enterobacter cloacae
  • Closely related group of organisms found in water, sewage, and the intestinal tract of warm-blooded animals
  • It may cause urinary tract infection
• Klebsiella
  • Found in soil and water
  • Most strains fix nitrogen
  • Klebsiella pneumoniae occasionally causes pneumonia
• Serratia
  • Found in water, soil, and the intestinal tract of insects, vertebrate animals, and humans.
  • May cause hospital-acquired (nosocomial) infections and forms red pyrrole-containing pigments called prodigiosins
• Cronobacter
  • Discovered in 2007
  • C. sakazakii causes meningitis and is found in a variety of environments and foods.
  • Coliforms are useful indicators of water contamination, and they proliferate in the intestines of humans and animals

Coliforms

• Coliforms are facultatively aerobic, Gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose within 48 hours at 36°C.
• They are an operational grouping that include taxonomically unrelated microorganisms, but many coliforms are enteric bacteria.
• The presence of total coliforms, especially E. coli, in water indicates contamination and inadequate treatment to remove faecal contaminants
• Escherichia are found in the GI tract of humans and warm-blooded animals and synthesizes vitamins (Vit K) in the gut.
  • Escherichia is facultatively aerobic, consuming oxygen in the gut, resulting in anoxic conditions
  • Pathogenic strains can cause diarrhea in infants, urinary tract infections in women, and infections through contaminated foods (meat, milk) or water.
• Certain strains form the K antigen (capsular polysaccharide) that permits attachment and intestinal colonization, along with enterotoxin.
• These cause the primary symptoms of diarrhea.
• Strains, such as Enterohaemorrhagic E. coli eg. EHEC 0157:H7, can cause sporadic outbreaks of severe foodborne disease through the consumption of contaminated foods, such as raw or undercooked ground beef, unpasteurized milk, or contaminated ground water.
• Salmonella is usually pathogenic and causes typhoid fever and gastroenteritis, with over 2500 distinct serovars.
• Classifications are based on three cell surface antigens which are: - O - cell wall (somatic) antigen - H - flagellar antigen - V - outer polysaccharide antigen - typhoid fever
• Salmonella typhi is serovar of Salmonella that exhibits both systemic infections and typhoid fever.
  • This exists primarily in humans as it's primary reservoir
• It is also genetically identical by about 98%.
• Salmonella adapts and grows under aerobic and anaerobic conditions

Shigella

• Primarily pathogenic and causes of severe gastroenteritis called bacillary dysentery (bloody diarrhea)
  • It produces both endotoxin (enterotoxin) and neurotoxin which result in gastroenteritis.

Identifying Enteric Bacteria

• Diagnostic tests and differential media are used to separate the various genera into two broad groups.
• Molecular methods are in use and under development.
  • Genus and species-specific probes for individual genes using PCR; Tests for genes involved in pathogenicity; Partial or complete 16S rRNA sequencing and ribotyping (RFLP of rRNA genes)
• Use of Enterotube Multitest System and Analytical Profile Index (API)
• Methyl Red (MR) test: Determines whether the microbe performs mixed acids fermentation when supplied with glucose.
• E. coli produces a positive MR test by turning the Methyl Red indicator to show an acid change.
• Voges-Proskauer test which detects acetoin in a bacterial broth culture.
• It is performed by adding α-naphthol and KOH to the Voges-Proskauer (VP) broth which has been inoculated with bacteria.
• A cherry red color is a postive result while yellow-brown color is negative.
• Enterobacter and Klebsiella produce positive results here
• Triple Sugar Iron (TSI) test is for microorganism to ferment sugars and to produce H2S.
  • Used to identify enteric bacteria especially Salmonella & Shigella.
  • It tests the fermentation of sugars (lactose, sucrose, glucose).
  • Sodium thiosulphate tests for Sulphur source and ferrous sulphate for Iron source.
  • Acids produced change phenol red from red to yellow.
• Urease Test identifies organisms capable of hydrolyzing urea to produce NH3 and CO2.
  • Used to distinguish urease-positive Proteus from other Enterobacteriaceae.
  • Phenol red indicator turns yellow in acidic conditions and red/magenta in basic conditions.
• Indole Test: Determines the ability of the organism to cleave indole to tryptophan using tryptophanase.
  • Aeromonas, Klebsiella oxytoca Enterococcus Vibrio, Escherichia coli, and Edwardsiella can do this
• Motility Test: Determines the differentiation of Enterobacteriaceae and many other groups.
  • Motility Test Agar uses a semi-solid concentration is reduced to 0.4% (compared to 2%) allows movement of motile bacteria
• Motility is detectable as diffuse as growth radiating from the central stab line for Proteus mirabilis, Salmonella, and Escherichia (some).
• Gas Production from Glucose: The Durham tube shows change from red to yellow during glucose production by Escherichia coli.
• Citrate Test determines utilization (sodium citrate as the main carbon source):
  • Escherichia coli (negative)
  • Enterobacter aerogenes, Salmonella, Proteus (positive)
  • Use of a pH indicator bromothymol blue

Oxidase Test

• Key test distinguishing Enterics from other bacteria of similar morphology.
• Enterics are ALWAYS negative.
• Used to determine if the organism produces certain cytochrome c oxidases using disks impregnated redox indicators N,N,N',N'-tetramethyl-p-phenylenediamine vs N,N-dimethyl-p-phenylenediamine
• Non-enterics reduce nitrate to nitrogen gas which results in gas detecting.

Grammaproteobacteria

• Key genera: Alivibrio, Pseudomonas, Vibrio, Photobacterium.
• Pseudomonadales are Gram-negative, anaerobic or aerobic, chemoorganotrophs that use a wide variety of organic compounds and are metabolically diverse.
• Many Pseudomonads show naturally are drug resistant to antibiotics due to transmembrane pumps to remove antimicrobial drugs and R plasmids and has a tendency to forming biofilms.
  • Opportunistic pathogen -P. aeruginosa: respiratory (cystic fibrosis), wound (burns) and urinary tract infections; P. syringae and P. marginalis are plant pathogens.

Biofilms

• Most microbes grow attached to surfaces (sessile) rather than free floating (planktonic) via complex, slime-enclosed communities that are ubiquitous in nature in water, and can be formed on any conditioned surface.
• Mature biofilms are dynamic complexes that exhibit heterogeneity.
  • This is characterized through differences in metabolic activity between individuals due to interactions, or localized nutrient concentrations.
• Biofilm effects: protection from harmful agents, illness when found on medical devices, and releases contamination via the water systems.

Quorum Sensing

• Archeal and Bacterial cells in biofilms communicate in a population density-dependent via quorum sensing.
• Small proteins are stimulated to convert a microbe to competent to uptake DNA and releases of bacteriocins. -This ensures sufficient species cells are present before initiating a response that requires a certain cell density (e.g., toxin production).
• N-acylhomoserine lactone (AHL) autoinducer (Al) molecules are produced at low levels by many Gram-negative organisms.
• Once inside the cell, it induces expression of target regulator genes.
• Each bacterial species produces a specific autoinducer signalling molecule which diffuses freely across the cell envelope.
• AHL reaches high concentrations only with multiple nearby individuals.
• Once high concentrations, the molecules bind to and induce activators or kinases on neighboring cells.

Processes Regulated by Quorum Sensing Involve Host-Microbe Interactions

• These interactions include Vibrio fischeri's symbiosis with the bobtail squid through bioluminescence.
• Pathogenicity includes increased virulence factor production
• Rhizobium interactions with plants.
• The virulence factors of Escherichia coli 0157:H7 produces a cytotoxin of high virulence.
• Quorum Sensing can also be regulated by AI-3 with epinephrine-like signaling.
• Quorum-sensing disruptors are being researched as biofilm dispersing medicines as this stops bacterial interactions and the resulting virulence.

Bioluminescence

• Several bacteria emit light called bioluminescence.
  • Photobacterium, Alivibrio, and Vibrio isolates are facultative anaerobes but are only bioluminescent when O2 is present.
• Autoinduction exhibits luciferase and bacterial proteins dependent on population presence.
• The lux operon (luxCDABE) encodes bioluminescence and requires regulatory proteins for induction.
• Requires high levels of activator and the production of many proteins together.
• Luminescence in bacteria is catalyzed by the enzyme:luciferase which uses O2 and NADH to make the molecules involved in bioluminescence.
• Expression of luminescence requires high population density.