Bacteriology and Sensitivity

Prokaryote

Single cell microbe with a simple cell structure

Most bacteria are vital for animal life (commensal) with very few being ..

Parasite or pathogens that cause disease

Commensal bacteria can become ..

Pathogenic

Prokaryotic cells contain no nucleus, this allows these cells to .. 

Reproduce, very rapidly, by binary fission (cytokinesis) and require a suitable environment to live and reproduce 

General requirements for life include .. 

• Water

• Essential nutrients (species dependant) 

• Correct pH 

• Correct temperature 

Some bacteria can form endospore that are ..

Dormant and resistant to hostile environmental conditions (hot, cold, radiation, disinfection etc..) 

An example of an endospore producing bacteria ..

Bacillus anthracis (anthrax)

Bacillus anthracis (anthrax) - transmission

• This bacteria can lie dormant in the environment(soil) for many years 

• Transmitted via inhalation, entry via an open wound or ingestion 

• It targets the gastrointestinal tract, skin and/or lungs

• Particularly high risk to farm and wild animals 

Bacteria are grouped into three categories based on their response to gaseous exchange

• Obligate aerobes

• Obligate anaerobes

• Facultative anaerobes

Obligate aerobes

Requires oxygen

Obligate anaerobes

Does not require oxygen

Facultative anaerobes

Both (adaptable) (requires oxygen + doesn’t require oxygen)

Aerobic bacteria can only survive in the presence of oxygen, an example of this is ..

Leptospira spp. 

Anaerobic bacteria can only survive and reproduce in areas where oxygen is not present or available, for example in the gastrointestinal tract, for example .. 

Clostridium perfringens 

Facultative bacteria are adaptable and can survive in environments both with or without oxygen present, for example

MRSA

Culture

Growing bacteria in a controlled laboratory environment, often by providing a nutrient-rich medium to support growth

Sensitivity

The susceptibility of bacteria to a specific antibiotic

Why would a Veterinary Surgeon choose to conduct bacterial culture and sensitivity testing? 

• To enable the cultivation of bacteria from a sample collected from a patient (for example, from the urine, skin, faeces etc..) 

• To ensure the identification of the cause of infection and/or antibiotic sensitivity test, ensuring the most appropriate antibiotic is selected for treatment. Reducing the risk of antibiotic resistance 

Why would a Veterinary Surgeon choose to conduct bacterial culture and sensitivity testing - simple version 

• Disease diagnosis

• Treatment management

• Antimicrobial selection - what type of antibiotic to prescribe due to the bacteria

• Prevent antimicrobial resistance

• Environmental surveillance

• Postmortem

Sampling and collection - Fluids

• Blood 

• CSF

• Synovial fluid 

• Exudates 

• Semen 

• Sputum etc.. 

Sampling and collection - Swabs

• Ear

• Skin

• Wounds 

• Abscess 

• Mucosa 

• Tracts/stomas etc .. 

Sampling and collection - other

Infection control surveillance

Prepping for taking a swab sample

• Ensure appropriate PPE is used

• Considered zoonotic risk

• Maintain asepsis throughout sampling/collection

• Locate area of interest

• If required, clip the hair if it is preventing access to the area of interest

• Remove curst from scabbed lesions to exposure exudate, if present

• If a large amount of exudate is present, obtain a swab sample prior to and post cleaning with sterile saline or debridement

Stages of collecting a swab sample - collection

1. Carefully remove the swab from the package being sure not to contaminate the tip

2. Pre-wetting the swab with sterile saline can increase the harvest in the area of interest is dry

3. Roll the sterile swab (do not drag) using a consistent and controlled swabbing motion, back and forth or a zigzag pattern across the entire area of interest

4. Apply sufficient pressure to ensure that contact between the swab and surface is always made

5. Avoid necrotic as this will harbours large quantities of bacteria

Stages of collecting a swab sample - What to do once the sample has been collected

6. Place the swab into the sterile tube with appropriate transport media contained within the tip of the tube

7. Label the tube with the patient information (full name, date, area sample obtained (+/- L or R)

8. Correct storage of swab (process ASAP or refrigerate for no longer than 24 hours) 

Stages of collecting a swab sample - Transport media and preservation and storage 

• Maintain viability of bacteria 

• Prevent reproduction 

• Provide essential nutrients 

Transport media preservation and storage aims (swabs) 

• Amies media with charcoal (useful for aerobic and fastidious bacteria)

• Amies without charcoal

• Virus transport medium

• Anaerobic transport medium

Bacteria will not survive on ..

A dry swab

Charcoal eliminates ..

Metabolic products of bacterial growth - neutralises bacterial toxins

Sample Storage and Preservation 

• Urine 

• Blood 

• Faeces 

• CSF 

• Synovial fluid 

Sample Storage and Preservation - Urine

Boric acid preservative (refrigerate)

• Specific for urine bacteriology 

• White power inside allows the bacteria to survive prior to analysis 

Sample Storage and Preservation - Blood 

Blood culture broth 

• Specific liquid culture

• Inject blood into it (good for identification of sepsis)

Sample Storage and Preservation - Faeces 

Sterile tube (refrigerate) 

Sample Storage and Preservation - CSF

EDTA (do not refrigerate - very fragile)

Sample Storage and Preservation - Synovial fluid  

EDTA

Sample Storage and Preservation - further considerations

• Keep specimen cool to avoid bacterial growth (refrigerate at 4 ̊C) (not blood or CSF) 

• Follow specific guidelines set by external lab to ensure valid results achieved 

• Full clinical history and patient signalment should be provided to the lab 

• Ensure sample containers are sterile to prevent erroneous results 

Aims of bacterial culture 

• Maintain viability of bacteria 

• Encourage reproduction 

• Inhibit the growth of unwanted microflora

• Isolate individual bacteria colonies for species identification 

Bacteria culture - Liquid nutrient broths

Used for bacteria that grow within the liquid, e.g. blood

Bacteria culture - Solid (jelly) nutrient solutions

• Agar-based media prepared in a flat petri dush (can be used for the isolation of the individual species) 

• Agar alone provides no nutrition for bacterial growth. Specific nutrients and growth are impregnated into the agar base. These are designed to encourage growth of particular organisms. Isolation of individual species can only be achieved on solid media. Un-inoculated media plates should be stored within the regenerator and not frozen.

Bacterial culture - Media types

• Simple media (nutrient broth, nutrient agar) 

• Enrichment media (blood, egg etc) 

• Selective/differential media (Stabourad’s agar) 

• Transport media - Incubate 18-24 hours at 37°C

Bacterial culture - Simple media

Provides basic nutrition for growth of nutritionally undemanding species (for example E.coli)

• Examples include nutrient broth and nutrient agar

Bacterial culture - Enrichment media

Nutrient agar base with added ingredient to encourage growth of fastidious bacteria - for example, blood, egg etc 

Bacteria culture - Selective/differential media

Basic nutrient agar with added nutrient to inhibit the growth of some bacteria stains, while not affecting other. Examples include Sabouraud’s agar for fungal culture (ringworm) 

Bacterial culture - colonies 

• Colonies of bacteria will appear as round raised lumps along the streak lines 

• Colonies of different species may show different characteristics helpful for identification - e.g. colour, texture 

Sensitivity Testing

• Allows selection of the most appropriate antimicrobial 

• Allows the patient to be treated quickly effectively 

• Reduces the risks associated with antimicrobial resistance 

• Need a culture beforehand 

Sensitivity Testing - Disc agar infusion method 

• A bacterial colony from loop or swab or spread over an agar plate

• A manufactured disc continued a range of antibiotics is applied to the plate surface

• Incubate

• The resistance zone cut off is specific to each antibiotic

• Accurate measurement of the zone diameter and comparison to an interpretations chart is necessary to interpret this test

Sensitivity Testing - what is the Disc agar infusion method 

• Most used method that determines the susceptibility of a microorganisms to a specify antimicrobial agent (efficiency, cost and convenience) 

• Antimicrobial agent diffuses into the solid medium from the filleter paper resulting in the inhibition of reproduction of the microorganism on its surface 

• A zone of inhibition form around the disc, beyond this zone an unaffected area of normal microbial growth is present 

Sensitivity testing - results

• Susceptible

• Intermediate

• Resistant  

Sensitivity testing results - Susceptible

Bacterial strain is inhibited by a concentration of antibiotic that is associated with a high likelihood of threptic success

Sensitivity testing results - Intermediate

Bacterial strain inhibited by a concentration of an antibiotic that is associated with an uncertain therapeutic effect 

Sensitivity testing results - Resistant

Bacterial strain is inhibited by a concentration of an antibiotic that is associated with a high likelihood of therapeutic failure