Water Microbiology and Genetic Transfer Mechanisms

Water Microbiology and Drinking Water Safety

  • Overview

    • Public drinking water is tested and treated to ensure it is potable and free from contamination with microbes.

    • Drinking water supplies are crucial nonliving reservoirs for gastrointestinal disease-causing agents.

    • Possible pathogens in contaminated drinking water:

    • Parasites:

      • Cryptosporidium

      • Giardia

    • Viruses:

      • Norovirus

    • Bacteria:

      • Salmonella

      • Shigella

      • E. coli

      • Vibrio cholera

      • Campylobacter

      • Leptospira

Fecal Contamination

  • Source of Pathogens

    • Fecal contamination of water from humans and animals can introduce pathogens.

    • Many pathogens are resistant to chlorine, particularly parasites, causing gastrointestinal outbreaks in the U.S.

  • Impacts of Fecal Contamination

    • Fecal contamination is harmful to health.

Detection of Pathogens in Water

  • Challenges in Detection

    • Many pathogens entering the water supply via fecal contamination are:

    • Difficult to culture.

    • Difficult to detect.

    • Short-lived.

    • Present in low concentrations.

    • Water quality tests rely on the detection of coliform bacteria, a sentinel group of bacteria that is easy to culture and detect.

  • Definition of Coliform Bacteria

    • Coliform bacteria: Gram-negative bacilli (rod-shaped) that are facultative anaerobes, ferment lactose, producing acid and gas, and only live in the gastrointestinal tract of animals.

    • Coliforms include species of Escherichia, Klebsiella, and Enterobacter.

    • Their presence indicates fecal contamination of a water supply and potential gastrointestinal pathogens.

Selective-Differential Media

  • Overview

    • Water screening utilizes selective-differential media, which select for Gram-negative bacteria and inhibit Gram-positive bacteria.

    • Differentiate between lactose fermenters (coliforms) and non-lactose fermenters (non-coliforms).

  • Types of Selective-Differential Media

    1. Rapid'E. coli 2 Agar

    2. Eosin Methylene Blue (EMB)

    3. Hektoen Enteric Agar (HEA)

    4. MacConkey Agar (MCA)

Rapid'E. coli 2 Agar

  • Application

    • Selective chromogenic medium to detect E. coli and other coliforms.

  • Mechanism

    • Detects two enzymatic activities:

    • β-D-Galactosidase (GAL)

    • β-D-Glucuronidase (GLUC)

    • Two chromogenic substances:

    • Specific to GAL; produces blue colonies if present.

    • Specific to GLUC; produces pink colonies if present.

    • Coliforms (GAL+/GLUC-) form blue/green colonies.

    • E. coli (GAL+/GLUC+) form violet to pink colonies.

    • Detection of GLUC indicates high specificity, as E. coli is one of the few enterobacteria with this enzyme.

  • Note

    • Some strains of E. coli (O157) are GLUC negative.

    • Certain strains of Salmonella and Shigella can be GLUC positive.

Eosin Methylene Blue (EMB) Agar

  • Composition

    • Contains dyes eosin Y and methylene blue.

    • Methylene blue inhibits Gram-positive bacteria growth; eosin Y reacts with acid from lactose fermenters.

  • Findings

    • E. coli produces a metallic green sheen due to excessive acid production, and the media turns pink-dark purple.

    • Other coliforms produce less acid and turn the medium to pink-dark purple.

  • Interpretation of Results

    • If no lactose fermentation occurs, the agar remains red, indicating non-coliform bacteria.

Hektoen Enteric Agar (HEA)

  • Purpose

    • Differentiates Salmonella and Shigella from other enterics.

  • Mechanism

    • Bile salts inhibit Gram-positive bacteria.

    • Three sugars are fermented (lactose, sucrose, salicin); fermentation causes media to turn yellow-pinkish orange.

    • If no fermentation occurs, the media remains blue-green, indicating the presence of non-coliforms (i.e., Shigella).

    • Contains ferric ammonium citrate which reacts with H2S gas to form ferrous sulfide, producing black media (indicative of Salmonella).

MacConkey Agar (MCA)

  • Function

    • Used to differentiate Salmonella and Shigella from other enterics.

  • Components

    • Contains bile salts and crystal violet to inhibit Gram-positive growth.

    • Neutral red indicator dye turns pink during fermentation (indicating coliform presence).

  • Observation

    • If there is no fermentation, MCA remains colorless, indicating the absence of coliforms with non-coliform bacteria present.

Genetic Transfer Mechanisms

Conjugation

  • Definition

    • Conjugation is the process by which genetic information (in the form of a plasmid) is transferred from a donor bacterial cell to a recipient cell through a sex pilus.

Transformation

  • Definition

    • Transformation is a type of horizontal gene transfer where competent bacterial cells incorporate free DNA from the environment into their genome.

Transduction

  • Process Overview

    1. Phage injects DNA into host bacterial cell.

    2. Phage enzyme breaks down host DNA.

    3. New phages are created, including phage and host DNA.

    4. Recipient cell takes up the transducing phage.

    5. Donor DNA is inserted into the recipient's chromosome due to recombination.

  • Diagramming the Process

    • Bacteriophages transfer DNA segments between bacteria, initiating genetic changes.

    • Defective phages can result from errors during assembly, packaging host DNA instead of viral DNA.

Bacteriophages

Lytic Infections

  • Process Description

    • Bacteriophages infect and replicate within bacteria by identifying susceptible cells via tail fibers (spike proteins).

    • Phage DNA injected into the bacterial cell leads to the use of host cell machinery to produce viral proteins and DNA.

    • Resulting viruses are assembled and released by lysing the host cell.

Lysogenic Infections

  • Characteristics

    • The phage DNA may integrate into the bacterial chromosome, becoming a prophage.

    • Bacteria continue normal reproduction, replicating the phage DNA with their own.

    • Events triggering prophage excision can lead to a transition to the lytic cycle.

Lytic vs. Lysogenic Cycle

  • Lytic Cycle Steps

    1. Phage attaches to the host cell and injects DNA.

    2. Phage DNA circularizes and begins lytic cycle.

    3. The cell lyses, releasing phage virions.

  • Lysogenic Cycle Steps

    1. Phage DNA integrates into host chromosome, becoming a prophage.

    2. Lysogenic bacteria reproduce normally until induction leads to lytic cycle initiation.

Laboratory Techniques

  • Transduction Lab

    • Using T4 bacteriophage to transduce E. coli strain BE.

    • Phage solutions grown on E. coli containing plasmid with Kanamycin resistance.

    • Phage used to infect E. coli strain BE where some phages may carry plasmid DNA (conferring ampicillin resistance).

Lab Procedure

  • Experiment conducted in pairs.

  • Required preparations:

    • Label tubes and plates to avoid confusion.

    • Prepare 10-fold serial dilutions as per the protocol.

    • Plate the neat (undiluted) and three dilutions, including a control plate with no phage added (5 plates total).

  • Infection Process

    • Add 10 μl of phage dilution to tubes of bacteria, mix, and let sit for 20 minutes at room temperature.

    • Allow the mixture to absorb, then incubate.

Identification of Unknown Enteric Bacteria

  • Differential media used to observe enzymatic activities in bacteria.

  • Indicators detect pH changes through various biochemical reactions.

  • Media Types Used:

    • Simmon’s Citrate Agar (SCA)

    • Urea agar

    • Sulfide-Indole-Motility (SIM)

    • Triple Sugar Iron (TSI) agar

Simmon’s Citrate Agar (SCA)

  • Composition

    • Contains sodium citrate.

  • Mechanism

    • Bacteria with citrase enzyme utilize sodium citrate and nitrogen to produce ammonia (NH3), raising pH.

    • Indicator (bromothymol blue) changes from green to blue upon alkalization.

Urea Agar

  • Function

    • Urease enzyme hydrolyzes urea, releasing ammonia and CO2.

    • pH change leads to alkaline shift, causing phenol red indicator to turn pink.

Sulfide-Indole-Motility (SIM) Medium

  • Sulfur Reduction

    • Reduction of sulfur produces H2S, leading to black precipitate when ferrous sulfate is present in media.

  • Indole Production

    • Tryptophan metabolism through tryptophanase hydrolyzes tryptophan, producing indole (detected by Kovac’s reagent).

    • A cherry red compound indicates indole production.

  • Motility Observation

    • Motile bacteria exhibit cloudiness away from stab line; non-motile bacteria remain localized.

Triple Sugar Iron (TSI) Agar

  • Composition

    • Contains three carbohydrates, ferrous sulfate for detecting H2S production, and phenol red for pH monitoring.

  • Biochemical Reactions

    • Acidic conditions from sugar fermentation turn phenol red to yellow.

    • Results Interpretation:

    • Yellow slant and butt (A/A): complete fermentation of glucose and at least one other sugar.

    • Pink-red slant and yellow butt (K/A): glucose fermenters only, utilizing amino acids after glucose depletion.

    • Red slant and butt (K/K): no fermentation, only peptones utilized.

    • Black butt indicates sulfate reduction.

Results for Laboratory Transformations

  • Comparative observation of plates with or without ampicillin showing differential growth based on the presence of plasmid-borne antibiotic resistance genes.

Control Plates Observations

  • Growth should be noted on plates containing plasmid for confirming viability.

  • Control assessments of resistance and mutation possibilities while monitoring colony characteristics.

Summary of Current Lab Tasks

  • Inoculate HEA, MAC, EMB, and Rapid E. coli agar with assigned water samples.

  • Conduct transplantation experiments with careful documentation of growth observations.

  • Record biochemical reaction results to identify unknown microbes effectively.

  • Evaluate transformation and conjugation results for comparative analysis.