Introduction

In this chapter you will learn about how bacteria are classified and how they can be identified in the laboratory. Antimicrobial susceptibility testing will also be discussed.

Bacteria are the smallest self-replicating microorganisms or microbes. The smallest of these are mycoplasmas – bacteria without outer cell membranes. Individual cells cannot be seen with the naked eye, we have to examine for them using compound light microscopy. Some of the longest bacteria are the branching filamentous ones like Nocardia. Bacteria are usually observed using either from x400 to x1000 magnification.

Below is an example of bacterial being cultured artificially from a rectal swab and a Gram's stain  of bacteria from a single black colony showing that they are the same as they are derived from one ancestor i.e. clones.

Bacteria replicate by binary fission, first replicating their genome, plasmid and then creating a cross wall and pinching off to form daughter cells. The genetic material in the daughter cells is exactly the same as the parent cell. Thus, natural clones. The picture on the right above shows size variation in bacterial cells which is due to the daughter cells that have just formed are small, but will elongate as they grow. Hence the unevenness in length and the presence of diplobacilli or even long chains of bacilli. Under adverse conditions some bacteria that usually form short rods will elongate into very long filaments without dividing. The beta-lactam antibiotics that prevent peptidoglycan cross wall formation in the bacterial outer membrane may encourage this.

See this YouTube animation on how bacteria divide. Another, more humorous, take is this YouTube video about multiplying bacteria.

bacterial outer membrane

The outer membrane of bacteria are unique and confer on them shape and resistance to desiccation. It allows them to traffic substances in and out of the cell either by selective permeability or by active transport. Bacteria have surface structures that assist in adhesion, motility and the exchange of genetic material.  

The Gram’s stain makes use of the different structures of the gram-positive and gram-negative bacteria. Essentially, the gram-positive bacterium has a thick peptidoglycan layer that makes them more impermeable and resistant to drying out than the thin-walled lipid-rich gram-negative bacteria. When using the Gram’s stain, acetone-alcohol decolouriser leaches the purple crystal violet stain out of gram-negative bacteria, whereas gram-positive bacteria, will retain the stain. The weaker pink (safranin) counter-stain will then only be able to colour gram-negative bacteria where the crystal violet stain had been leached by the acetone-alcohol. Thus, purple gram-positive and pink gram-negative.

The Gram’s stain is useful to distinguish bacteria in a mixed sample and can assist you in clinical practice in the selection of empirical antibiotics.

Some bacteria, like mycoplasmas,  don’t have an outer cell membrane, they just have a very flexible plasma membrane that is rich in sterols. This means that they have to live a parasitic lifestyle as they don’t survive well in the environment. When animals become infected with mycoplasmas they are difficult to treat since they lack the outer membrane. The immune system also struggles to recognise them as foreign. Lacking that membrane, they will stain gram-negative. However, due to a lack of a rigid structure, they are difficult to observe using the Gram’s stain. Fluorescent stains are much better at detecting them cytologically.

bacterial spores

Bacteria don’t only survive by having a thick outer cell membrane, but also by the production of resistant structures known as spores. Some of the gram-positive Bacillota produce tough intracellular structures known as endospores. Only a few bacteria will produce exospores. The bacteria endospore consists of a dense genetic material core surrounded by a triple layer outer membrane that is completely impermeable to molecules. Spores are metabolically inactive and can survive drying out and some UV light penetration.

The endospore can survive a long time in an animal host without stimulating an immune response. An example is the endospores of Clostridium chauvoei, the agent of blackquarter/blackleg in cattle, will survive and be inactive in muscles for a long time.

bacterial glycocalyx – capsules and slime layers

A glycocalyx, consisting of capsules (organised structure) or slime layers (non-organised structures), are an outer polysaccharide (sugar) coating produced by many pathogenic bacteria. Some will have proteins in them.

They have several functions:

1. Promote bacterial adhesion to cell surfaces
2. Resists/delays phagocytosis by neutrophils and macrophages by hiding their receptors and some Complement that binds to the bacteria
3. Provide nutrients and moisture
4. Are a permeability barrier
5. Contribute to biofilms (see Bacterial pathogenesis)
6. Can be a barrier to some bacteriophages. This is a two-way street as some bacteriophages are adapted to attack a particular capsular type and won’t do so if the capsule is not produced. Unencapsulated bacteria will exchange genetic material by conjugation easier.

An introduction to Bacterial Taxonomy

Bacteria are the most diverse Domain with a rich abundance of species and numbers. There are more bacteria in your intestines than cells in your body. There are more than one hundred trillion microbes in your intestines with more than 1 100 species. This does not include microbes found on the skin and mucosae. The human body has about 30 trillion cells. You will have the opportunity in the first practical to observe the growth of bacteria from various samples.

Importance:

Bacteria in the same taxonomic group often stain the same and have similar morphology and laboratory culture conditions. They also tend to cause similar diseases and have a similar antimicrobial susceptibility range.

For example. Mannheimia haemolytica is a gammaproteobacteria (gram-negative) belonging to the Family Pasteurellaceae. This Family are usually commensals in the upper oro-respiratory and urogenital tracts and cause respiratory and urogenital tract disease. This Family shows susceptibility to beta-lactam drugs and fluoroquinolones (enrofloxacin) but is often resistant to the aminoglycosides (gentamicin). This is very different to the coliforms which although gammaproteobacteria belong to the Enterobacteriales. The coliforms are often susceptible to aminoglycosides and less susceptible to the beta-lactams.

Bacteria belong to a Domain on their own as they contain both RNA and DNA, have no nucleus or organelles and have unique structures and processes.

How are bacteria classified?

When bacteria were first observed under the microscope they were classified on their staining characteristics: Gram-positive or gram-negative; and morphology: cocci, bacilli, spiral, filamentous etc. The next step in bacterial classification included their biochemical characteristics i.e. what sugars did they use. Whilst these characteristics are still very useful in the identification of bacteria, they are no longer being used to classify the bacteria. Nowadays, the genetic characteristics of bacteria, especially sequencing of conserved genes, are used to classify bacteria. All bacteria have a gene that encodes for 16sRNA. 16sRNA is a structural component of the 30S small ribosome unique to bacteria. As this gene has a slow evolution, it can be used to identify bacteria to genus and even species level. A difference of 97% or more in

nucleotide sequences between 2 bacteria indicates that they are a different species. This gene does not distinguish highly related species. Thus, other genes have to be used with the most suitable being the recA gene.  It encodes for a protein that is essential for DNA recombination and repair.

The official bacterial nomenclature is published in the International Code of Nomenclature of Prokaryotes (ICNP) – it is currently being updated. All new bacterial species have to be published in the International Journal of Systematic and Evolutionary Microbiology.

Below is a tree of Bacteria showing the bacterial phyla. The gram-negative phyla are on the left and the gram-positive phyla are on the right. The size shows the diversity of bacterial species within each phylum.

An example of two common bacteria and their official designations are shown below: You will note that there is no Kingdom. Sometimes other sub-divisions are used. Examples are Division (i.e. Terrabacteria) and Sub-order. Whilst these assist in grouping similar bacteria together, they are not currently in wide use.

Below are classifications of the different classes of bacteria that only include the Genus names of bacteria you will encounter as pathogens of animals. Note that pathogenic bacteria are only about 10% of all the known species of bacteria and you will learn about half of these.

Some pictures below showing their morphology using the Gram’s stain. They are divided into gram-positive and gram-negative bacteria. The manufacture and control of the outer membrane of bacteria requires a lot of genes. Thus, bacteria with similar staining characteristics are also genetically related.

Below are examples of the different morphological types of gram-negative bacteria

Other ways of classifying bacteria

The taxonomic positions of bacteria are nowadays based on genotyping methods. In the past it was dependent on morphological appearance and their ability to use different substrates. However, for diagnostic and disease management purposes, they can be characterised in different ways. Below is a ranking order of how bacteria can be classified. It includes genotypic (genus, species and clade/genotype), phenotypic (biovar), serological (serovar), toxin type, antibiotype and bacteriophage typing methods. Below is an example of Salmonella, a member of the Enterobacteriales and the cause of gastrointestinal and systemic disease in animals and people.

Observe how each type of characterisation is written. You should also be using this in written assignments/examinations.

Characterisation of disease can also be done based on disease/lesions. For example, the toxic Clostridium species are divided into:

1. Enterotoxic – intestinal pathogens (Clostridium perfringens)
2. Histiotoxic – Tissue toxins (Clostridium septicum)
3. Neurotoxic- affecting the central and peripheral nervous system (Clostridium botulinum)

identification of bacteria in practice and the laboratory

Bacteria are microscopic and thus cannot be observed without assistance. However, their presence can be suspected by the type of disease that they cause.

Indicators that a bacterial infection may be present:

• Purulent inflammation. The exudate from affected tissue is often cloudy, has blood in it and discoloured. That is, a gold coloured exudate could have Staphylococcus aureus in it
• Many infections caused by obligate anaerobes and the coliforms can exude an unpleasant odour. Pseudomonas aeruginosa has a fruity smell.
• Some tissues are more prone to bacterial infections i.e. the skin, external ear canal, urinary and respiratory tracts.

For example. A female dog is urinating frequently and appears to be in pain. The urine is cloudy, red-stained and has an unpleasant odour.  Escherichia coli is the most likely cause of this infection.

Below is a picture showing the relative size of bacteria in comparison to other organisms. (Don’t learn)

Bacteria being  microscopic, can be identified in several ways:

1. Cytologically – under the microscope

They are the smallest microorganisms that can be observed as individuals using a light compound microscope. Generally, this can be done on shape and Gram staining reaction. Note that large bacteria can be observed using 100x; many at 400x magnification, but most will only be evident at 1000x magnification.

2. By culture, followed by other identification methods

3. Genotyping

16sRNA genotyping is used to identify bacteria to genus and even species level. If a particular bacterium is suspected, just a portion of a unique sequence can be amplified in a polymerase chain reaction (PCR) cycler until a point when the product is measurable. For conventional PCRs, the product is measured by allowing it to move under an electric current through an agarose gel until it reaches a point based on its size where it will accumulate forming a band. This band is stained and compared to known DNA sizes (DNA ladder) that were run at the same time as the amplicon. This process is simplified in real-time PCR (qPCR) where the build-up of a fluorescent dye is measured with a spectrophotometer. One of the dyes known as SYBR intercalates in double stranded DNA. Otherwise different coloured fluorescent dyes can be added to DNA probes that will build up in the amplification process if the target DNA is present.

Methods that cleave the genes at different sites create DNA strings of different lengths can be used to provide a unique fingerprint and identify the bacterium to species and strain level. These methods include pulsed-field gel electrophoresis and Tandem repeat sequences. Whole genome sequencing can also be done.

For the diagnosis of disease, a qPCR provides the fastest and most affordable approach.

Why we like the qPCR

1. Are sensitive so can detect small amounts of target DNA/RNA
2. Are as specific as you desire. i.e. If you want to identify a particular bacterium to Genus level i.e. Salmonella. Primers that initiate where amplification occurs on a DNA strand can ensure that only genus specific amplicons are formed. You can, based on the primer sequences selected identify species, subspecies and genotype level.
3. Multiplex qPCR. Can detect a number of different target oligonucleotide strands in a single PCR. For example,  infectious foal diarrhoea a single multiplex qPCR will detect Rotavirus; Salmonella, Clostridium perfringens and Clostridioides difficile.
4. Very rapid – most cases same day.
5. DNA but not RNA is very robust. Can be submitted on dry swabs or filter paper
6. Easily automated and can be used as an in-practice test
7. Sample preservation is easy as DNA will survive drying out

Why PCRs are not always the answer

1. High level of sensitivity means that you can detect low numbers of the organism, even when it is not the cause of disease
2. Some samples have DNA/RNA inhibitors/destroyers in them i.e. faeces – requires special processing. Bigger problem with wet samples any DNAses and RNAses will be active in a liquid phase.
3. The cause has to be suspected or be excluded as a cause. Primers select site for amplification target specific oligonucleotides on a nucleotide strand.
4. Cannot process large sample sizes, so the suspected cause must be in the sample used. One can use means to increase the amount of target DNA/RNA in a sample i.e. by concentrating a fluid sample and using the sediment; by using the buffy layer (white blood cell layer after centrifugation) in whole blood for blood-borne viruses and bacteria
5. Not as versatile in detecting antibiotic resistance
6. Can be more expensive and genomic sequencing can take longer as samples often have to be sent to specialist laboratories

4. MALDITOF MS

Cultured bacterial colonies can be rapidly identified to species level using a spectrophotometric method known as Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDITOF MS). In this method, macromolecules such as proteins are ionised with little fragmentation of their structure. The mass of the ionized macromolecules is measured by accelerating them, whilst they are in the gaseous phase, across a high voltage in a vacuum tube. The smaller particles will reach a detector at the end of the vacuum tube first. Thus, a molecule size pattern will be generated and can be compared to a database containing the molecule size patterns of known bacteria. The pictures below describe the process in more detail. An excellent You-tube video on the method is found at this URL: https://youtu.be/8R1Oyqx5KfE

Why is it rapidly becoming the bacterial and fungal identification method for diagnostic laboratories?

1. Fastest method, just requires a single colony and can even be done directly on certain samples such as blood.
2. Cheaper and more accurate than biochemical arrays; Cheaper with equivalent identification accuracy to 16sRNA or other gene sequencing.
3. Can be done in a diagnostic laboratory – does not have to be sent away as cultures/samples for gene sequencing have to be done
4. Can detect some types of resistant bacteria i.e. methicillin-resistant Staphylococcus aureus

Why can’t it completely take over from traditional culturing and identification methods or genotyping?

1. Performance in samples that have mixed bacterial populations is poor. Thus has to be done on pure cultures/samples.
2. Identification spectral pattern database is growing but is still relatively small for veterinary pathogens
3. Not validated for all sample types
4. Not as versatile as antibiotic susceptibility testing in detecting antibiotic resistance

traditional growing and identification of bacteria

During the practicals you will have the opportunity to grow and identify some bacterial pathogens. Use this information together with the Laboratory Manual.

Bacterial Culturing

Purpose of culturing:

1. To identify isolated bacterial colonies based on appearance
2. Permits selection and purification of different species from a mixed culture
3. Viable bacteria can be amplified to high numbers that can be used for further tests such as identification of them,antimicrobial susceptibility testing and any further research on them. They can also be used for disease control i.e. the production of vaccines, provision of virulent bacteriophages etc.

Principle:

When a single bacterial cell is deposited on the surface of a nutritive medium, it begins to divide after thousands of cells are formed, a visible mass appears = a colony (clones of the original bacterium)

Each species of bacteria will exhibit its own characteristic colony.

Identification methods used in cultures:

1. Selective and differential medias
2. Gram’s stain and other special stains
3. Biochemical identification usually via preliminary tests and then commercial identification biochemical array tests
4. Antigen detection tests i.e. latex agglutination test for Salmonela enterica.
5. Genotyping. Refer to the following section 3. The qPCR that is provided as a picture example is used to speciate Cryptococcus, a pathogenic yeast either from a culture or directly from the sample.
6. MALDITOF. Refer to Section 4.

Media types include:

1. Enrichment media – media that encourage the growth of bacteria in samples where they are in low numbers or are weakened i.e. by antibiotics. One of the most well-known media is  tryptose broth medium that has been adapted to grow bacteria in blood samples. They are often used for samples collected from normally sterile sites such as joints where infection is suspected. Bacteria in these sites might be inhibited but not killed by antibiotics or the inflammatory response.

 

1. Non-selective media – media that encourage the growth of most bacteria. Blood agar is such a medium as it is rich in proteins and iron.
2. Differential media – media that can distinguish between different types of bacteria. The general bacterial media used is an enriched nutrient agar medium containing blood. This allows most of the bacterial pathogens encountered in veterinary science to be cultured. Bacterial colonies (a clone from an individual bacterium) can be distinguished by their morphology and haemolytic pattern. Haemolysis can be: beta-haemolytic where the blood in the agar is completed haemolysed by bacterial enzymes leading to a clearing around the colony; alpha-haemolytic where bacterial enzymes only partially break down the blood leading to a greenish discoloration in the agar around the colony and: gamma-haemolysis where there is no evidence of blood breakdown around the colonies. Chromogenic agars are differential agars where specific bacterial species will show up as a different coloured colony.
3. Selective media – This is media that preferentially encourages the growth of a particular bacterial genus or species. They often contain inhibitors such as antibiotics, bile salts or have a high salt content. Most selective medias also are differential. For example, MacConkey agar with crystal violet and bile salts preferentially encourages the growth of Enterobacteriaceae (coliforms). MacConkey agar also has a sugar lactose that when fermented will cause the media around the bacteria to become acidic and thus change the neutral red indicator in the agar from pale pink to a bright pink/purple and even stain the colonies. Cetrimide agar contains the antiseptic cetrimide, quaternary ammonium compounds, that is inhibitory to most bacteria but does allow the highly drug-resistant bacterium Pseudomonas aeruginosa to grow. This media is also differential as it encourages Pseudomonas aeruginosa to produce a characteristic apple-green pigment known as pyoverdin.

Incubation conditions – temperature and atmospheric conditions

Once you have decided which media best suits the group of bacteria you wish to grow. you have to decide on the incubation conditions.

Temperature

As a rule of thumb, use the body temperature of the host animal for bacterial pathogens. So for mammals you would use 35 – 37°C; most birds 42°C and reptiles 28°C. The growth conditions for fish bacteria vary. If they originate from tropical waters then it is 28-30°C. If they originate from cold waters such as the Antarctic it will be a lot lower. Bacteria that grow at very high temperatures like the volcanic bacterium Geobacillus stearothermophilus that grow at 65°C are known as thermophiles. Most pathogenic bacteria of mammals and birds such as Escherichia coli are mesophiles since they grow at temperatures above 25°C but below 45°C. Those bacteria that will only grow at lower temperatures, like Aeromonas salmonicida a fish pathogen, are psychrophiles.

Gaseous conditions

Bacteria can be classified based on their preferred gaseous conditions of growth.

1. Aerobic. These bacteria grow best in normal air with about 21% oxygen. Pathogenic bacteria like Pseudomonas aeruginosa that are aerobic are usually environmental in origin. However, they also have the ability to tolerate low oxygen conditions that would be found in animals.
2. Facultative anaerobes. These bacteria are versatile, growing under any atmospheric conditions. Most animal pathogens fall in this category. Facultative aerobic pathogens are grown in air, but are supplied with additional (5%) carbon dioxide to enhance their growth.
3. Obligate anaerobes. These bacteria can only grow in the absence of oxygen. Oxygen is toxic to many of them, killing them within minutes. To grow these bacteria not only does the air have to be free of oxygen, but the media must be reduced or contain reducing agents to mop up any oxygen radicals. These bacteria are uniquely susceptible to the antibiotic metronidazole. A good example of an obligate anaerobe is Fusobacterium necrophorum the cause of necrotic laryngitis in livestock.
4. Microaerophilic. These bacteria grow in reduced oxygen (6%) and increased carbon dioxide (10%). Campylobacter species, such as Campylobacter fetus subsp. venerealis the cause of bovine genital campylobacteriosis (vibriosis) is an example.
5. Capnophilic. These bacteria have to have additional carbon dioxide, usually at 5 to 10% levels, to grow. Brucella ovis, the cause of contagious ram epididymitis is an example.

Duration of incubation

Most samples will grow colonies on a suitable growth media after 24 hours of incubation at a suitable temperature and under the correct atmospheric conditions.

Exceptions include:
1. Slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, the agent of Johne’s disease in ruminants, which can take up to 3 months to grow.

2. Obligate intracellular bacteria. These bacteria require cell cultures to grow. An example is Coxiella burnetii the agent of Q-fever.

3. When the appropriate media has not been selected or is not known. Most bacteria will grow on artificial agar. However, we only know the growth conditions of the known pathogenic bacteria. Bacteria present in low numbers in a mixed bacterial population may not be easy to detect on non-selective agar as they will be overgrown by other faster and more numerous bacteria. This is why selective media should sometimes be used.

Purification of mixed cultures

Identification of the individual bacterial species requires the presence of high numbers of colonies (bacteria) originating from a single bacterium. Thus a single represented colony is collected and streak diluted onto fresh blood agar and incubated for another day. Below are pictures showing the differences between mixed bacterial species growth (left picture) and on the right a picture of one of the bacterial colony types that were purified and incubated for 24 hours to get growth.

Once the colonies are purified they can be identified using some rapid preliminary tests and then slower biochemical test arrays. They can also be subjected to antibiotic susceptibility testing.

To identify the bacterium fully further identification tests can be carried out

1. Primary identification tests

Bacterial colony growth patterns on differential and selective agars

Examine and describe the growth of the purified colony growth on the enrichment/non-selective, differential and selective agars.  This will help you identify the cultured bacteria. The most common agar used is nutrient agar supplemented with 7% blood i.e. horse blood agar. This agar allows most bacteria to grow and shows the differences in colony morphology very clearly. Haemolytic patters are also seen.

• Beta-haemolysis is full breakdown of the red blood cells in the agar by bacterial enzymes (haemolysins) which causes clearing of the agar. The zone of beta-haemolysis can be very narrow or wide. Bacteria that produce a large quantity of haemolysins will produce a wide zone and those that produce less, a more narrow zone. A lot of pathogenic bacteria will produce a zone of beta-haemolysis.
• Alpha-haemolysis is when there is partial destruction of red blood cells in the agar that results in a green discolouration of the agar around the bacterial colony. This is less common and is most often associated with streptococci (gram-positive cocci) originating from the oral cavity.
• Gamma or no haemolysis.

 Gram’s stain

The Gram’s stain on each colony type allows one to select further identification tests and to decide on which antibiotics to test for. i.e. Penicillin which has a predominant gram-positive spectrum to gentamicin which has a predominant gram-negative spectrum.

Catalase- oxidase- and spot indole tests

These are very rapid tests that allow one to better select other tests for identification of the bacterial species isolated.

2. Rapid antigen detection tests

These tests can be used to rapidly identify common pathogens. They can make use of different antigen binding tests such as the agglutination, immunofluorescence tests and ELISA. For example, veterinary diagnostic laboratories commonly use the following rapid antigen identification tests:

• Rapid latex agglutination test for Staphylococcus aureus
• Rapid latex agglutination test for streptococcal wall antigens
• Rapid agglutination test for Salmonella enterica

2. Biochemical test arrays.

A number of tests are set up with each test containing a single substrate that the bacterium will use. In fermentation tests, the bacterium that ferments the sugar will change the colour of the pH indicator. In tests that test for use of a  substrate there will either be growth or non growth of that bacteria. Growth is often indicated by cloudiness or a change in colour. A lot of tests will generate a pattern based on positives. This positive pattern can be queried against a database where the closest identification match will be found.

4. Describe the principles of antibiotic susceptibility testing of bacteria.

During the practicals, you will have the opportunity to carry out, read and interpret some of these tests

Introduction

Testing for in vitro antibiotic susceptibilities is where the real strength of culture lies. This test is an expression of all forms of phenotypic antibiotic resistance, even those where the genetic resistance determinant is not known. Thus, they are used to drive good antibiotic stewardship in veterinary practice.

A word of warning. Do not use this test to decide what antibiotic to use, but rather what antibiotics to exclude because the bacteria tested are resistant to them. The tests do not take into account how the antibiotic behaves in the body. Know the antibiotic pharmacokinetics, pharmacodynamics and mode of action.

For example:

Potential sulphonamides are a combination of antibiotics that prevent bacteria from manufacturing tetrahydrofolic acid which is required for DNA and RNA synthesis. However, pus (containing degranulated neutrophils and necrotic cells) is a rich source of tetrahydrofolic acid, purines and pyrimidines. Bacteria can actively take them up without the need to utilize their folic acid pathway. This means that although potentiated sulphamides will block the folic acid pathway in the bacteria, the bacteria are still able to manufacture DNA and RNA and consequently protein. Bottom line: In vitro susceptibility of the pathogenic bacteria to potential sulphonamides, however, no effect on bacteria in pus.

There are basically two test types available:

1. Disk diffusion test
2. Minimum inhibitory concentration test

Disk diffusion test

Principle

Filter paper disks containing antibiotics are placed on a bacterial lawn and the bacteria incubated overnight. The antibioitic in the disk diffuses into the agar across a concentration gradient. Bacteria will not grow within inhibitory antibiotic concentrations. A circular zone of inhibition of growth (ZOI) is observed. The diameter of this ZOI is measured in millimeters and compared to a reference, known as the clinical breakpoint. ZOI diameters greater than the breakpoint is susceptible and less than the breakpoint resistant. Some antibiotics will have a range between the susceptible and resistant breakpoint that is known as intermediate.

The disk diffusion test is still used widely in veterinary laboratories for the following reasons:

1. Easy and cheap test to perform
2. Can customise the antibiotics to test

However, it is not the preferred test method for the following reasons:

1. Not validated for all bacteria i.e. obligate anaerobes, slow growers such as mycobacteria and mycoplasmas
2. Greater test variability – it should always be run with a control. It means that if not run properly there will be variable results
3. Overestimate resistance. Mainly because modern test interpretative standards used in the test interpretations are no longer taking the disk diffusion test into account
4. It is a qualitative test and cannot be used to customise antibiotic dosing regimens when treating patients with renal and liver disease

 

Minimum inhibitory concentration tests

Principle

These tests consist of serial 2-fold dilutions of antibiotic. The lowest concentration of antibiotic inhibits bacterial growth is considered the minimum inhibitory concentration (MIC). This value is compared to reference clinical breakpoint values for the antibiotic and in many cases the specific bacterial species. The results are then interpreted as susceptible if the value is less than the breakpoint value and resistant if it is more than that value. For some antibiotics there is a middle concentration range between susceptible and resistant. MICs in this range are considered intermediate.

Why are they popular?

1. Easy to standardise and less subject to user-associated variation, thus highly reliable
2. Commercial tests and automated systems are available
3. Can be used for most groups of bacteria and fungi
4. Modern breakpoint references only use MIC values
5. Can be used to modify antibiotic dosing in critical care patients i.e. those with hepatic or renal disease

Why are they less used in veterinary diagnostic laboratories?

1. Commercial tests – Cannot select the antibiotics or the dilution ranges which may be inadequate for some bacterial species
2. Home-made tests are labour-intensive and require reference controls
3. Expensive

There are several formats of these tests:

1. Microtitre broth MIC test – most popular. Easy to set up and commercial tests are available. Also easy to automate.
2. Agar MIC test – Most reliable. However, very labour-intensive and no commercial tests available. Use as a research tool.
3. E-test – Used for single antibiotics. Easy to set up. Commercial strips available. Less versatile than the microtitre broth MIC test as it can only test a few antibiotics. Used when a veterinarian wants to  use specific MIC of an antibiotic that is not registered for animals to tailor the antibiotic doses for a specific animal patient.

Interpretation of antimicrobial susceptibility tests

Susceptible: Use the antibiotic according to the manufacturer’s recommendations

Intermediate: Use the antibiotic at the highest non-toxic dose or decrease the dosing intervals i.e. from twice a day to 3 times a day. If possible avoid the use of antibiotics to which the target bacterium is intermediately susceptible as there is a risk that a portion of the bacterial population will not be killed and develop true resistance

Resistant: The antibiotic cannot be used at therapeutic recommended concentrations.

Note that this principle works well when the antibiotics are not used topically i.e. for oral and injectable use. They are less applicable for topical antibiotics. The main reason for this is that topical antibiotics can be administered at high concentrations without toxicity developing.

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