Antigen antibody reactions used in bacteriology
Agglutination tests
Agglutination takes place when antibodies bind to particulate antigens such as bacterial
surface antigens or antigen-coated carrier particles. As a result of antigen-antibody
binding, clumping of antigens occurs. Agglutination tests may be carried out in
qualitative or quantitative forms.
Agglutination of bacteria
- used for identification of bacteria or to determine the antigenic structure of a
bacterium (e.g in Enterobacteriaceae: determination of O somatic and H flagellar
antigens permits grouping of these bacteria in serogroups and serotypes)
- put on glass slide
o at one end one drop of saline – suspend a small quantity of bacterial
culture: a homogenous suspension should from; this serves as a negative
control to assure that the tested bacterium does not agglutinate
spontaneously;
o at the other end one drop of solution which contains specific, known
antibodies – suspend a small quantity of bacterial culture: if the bacterium
expresses the corresponding antigens to antibodies, agglutination will
occur (positive result); otherwise a homogenous suspension will form
(negative result)
Agglutination tests using inert particles
Inert particles such as latex, bentonite (clay), charcoal adsorb antigens or antibodies and
play the role of support for soluble antigen and antibody reactions in passive
agglutination reactions.
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These tests are widely used in microbiology for identification of bacteria and viruses and
also for the detection of antibodies. Their main advantages are that are easily and rapidly
performed, which do not need special equipment and are affordable for routine diagnostic
laboratories.
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Antibiotic susceptibility testing (AST)
= to determine the in vitro susceptibility of different bacteria to antibiotics;
Susceptibility testing is indicated for any bacteria that cause an infectious disease
warranting antimicrobial chemotherapy, if its susceptibility cannot be reliably predicted
from knowledge of the organism’s identity.
Antibiotics
• substances with antibacterial effect
Classification of antibiotics according to composition
1. β-lactam antibiotics
penicillins
penicillin
aminopenicillins (ampicillin, amoxicillin)
penicillinase-stable penicillins (oxacillin, cloxacillin)
antipseudomonal penicilins (piperacillin, ticarcillin)
cehalosporins (I-IV generation)
I generation – cefazolin, cephalothin.
II generation – cefuroxim
III generation – ceftazidime, ceftriaxone, cefotaxim,
IV generation – cefepime
cephamycin (cefoxitin, cefotetan)
monobactams (aztreonam)
carbapenems (ertapenem, imipenem, meropenem)
β-lactam/lactamase inhibitor combination (amoxicillin+clavulanic acid,
ampicillin+sulbactam, piperacillin+tazobactam, ticarcillin+clavulanic acid)
2. macrolides
erythromycin, clarithromycin, azithromycine
3. lincosamides
clindamycin
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4. fluoroquinolones
norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, etc.
5. tetracyclines
tetracycline, doxycycline, minocycline
6. glycylcycline
tigecycline
7. aminoglycosides
kanamycin, gentamicin, amikacin, netilmicin
8. glycopeptides
vancomycin, teicoplanin
9. oxazolidinones
linezolid
10. phenicols
chloramphenicol
11. folate pathway inhibitors
trimethoprim-sulfamethoxazol
12. lipopetides
daptomycin
13. nitroimidazoles
metronidazole
AST methods are performed and interpreted according to available standards. The most
widely accepted standard is the one elaborated by CLSI (Clinical Laboratory Standards
Institute), an international, interdisciplinary nonprofit, standards-developing and
educational organization that promotes the development and use of voluntary consensus
standard and guidelines within the health care community. Laboratories in most European
countries (including Romania) use CLSI standard.
As the CLSI standard was based mostly on resistance data gathered from the US, and in
Europe there were mainly national and regional standards, in last years a huge effort was
undertaken in order to develop a European guideline based on particularities of European
resistance phenomena. This new guideline was developed by EUCAST (European
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Committee on Antibiotic Susceptibility Testing), and was issued in December 2009.
Therefore in the near future we expect that most laboratories in Europe will adhere to this
new standard which will result in some changes in performing antibiotic susceptibility
testing and interpretation of results.
Antibiotic susceptibility testing is indicated:
- In case of all clinically significant bacteria, when the causative agent of an infection
belongs to a species capable of exhibiting resistance to commonly used antibiotics.
The need for testing is decided by the microbiologist according to
- specimen type
- bacterial species
When the clinical relevance of an isolate is doubtful, the microbiologist decides in
consultation with the clinician based on clinical data.
Methods:
• qualitative
o disk-diffusion method (Kirby-Bauer)
• quantitative
o determination of minimal inhibitory concentration (MIC)
A. Qualitative methods
Disk diffusion method (Kirby-Bauer)
- routinely used
- it does not permit to determine the level of resistance
- results are expressed as: susceptible, intermediate or resistant
Susceptible (S):
• the strain is inhibited by the usually achievable serum concentrations of the
antimicrobial agent when the recommended dosage is used for the site of
infection
• susceptible bacteria do not present detectable resistance mechanisms
Intermediate (I)
• the therapeutic response rate might be lower than for susceptible isolates,
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• this category includes a buffer zone, which should prevent small, uncontrolled,
technical problems from causing major discrepancies in interpretation
o intermediate category implies clinical efficacy in body sites where the
drugs are physiologically concentrated (quinolones and β-lactams in urine)
or when the antibiotic can be used in a higher than normal dose (lactams)
Resistant
• the isolates are not inhibited by the usually achievable concentrations of the
antimicrobial drug with normal dosage schedules
• the presence of specific resistance mechanisms is likely
Technique
1. Preparation of the inoculum:
- The inoculum is prepared from fresh, pure culture
- Suspend 2-3 isolated colonies in sterile saline ( in a test tube)
- Adjust the turbidity of the suspension to 0.5 McFarland (which correlates with
1,5x108 CFU/ml)
• McFarland turbidity standard: can be used to visually approximate the
concentration of cells in a suspension. The McFarland scale represents
specific concentrations of CFU/ml. McFarland standards are labeled 0.5
through 10 and are filled with suspensions of Barium salts or latex. The
bacterial inoculum is adjusted to the turbidity of a 0.5 McFarland tube.
Examples of McFarland scale and corresponding CFUs
McFarland scale CFU (x108 CFU/ml)
0.5 1.5
1 3
2, etc. 6 etc.
2. Inoculation:
- Choose an appropriate culture medium for the tested bacterium
• Mueller-Hinton for most non-fastidious organisms
• Mueller-Hinton supplemented with blood (e.g. streptococci)
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• Special culture media (for Haemophilus spp . Neisseria spp.)
- Dip a swab into the bacterial suspension
- Remove excess by pressing the swab against the tube wall
- Swab uniformly (3 times) the entire surface of the culture medium – the goal of
inoculation this time is to obtain a uniform, confluent growth
3. Antibiotic discs
- Choose the appropriate antibiotics to be tested according to the bacterial species and
localization of infection.
- Lay the antibiotics on the surface of inoculated culture medium at least at a 1,5 cm
distance from the edge of the plate and at 3 cm distance from each other.
- Leave the plates at room temperature for 15 minutes.
4. Incubation
- Choose the proper atmosphere according to bacterial species:
• Normal atmosphere
• 5-10% CO2 (streptococci, Neisseria spp., Haemophilus spp..)
- Temperature 35-37°C
- Duration: 16-18 hours (for fastidious organisms : 20-24 hours)
5. Reading
- After incubation a confluent culture appears on the plate. Circular inhibition zones
(lack of growth) may appear around the antibiotic discs.
- Read the diameter of inhibition zones.
- Compare the obtained diameter to standard diameters in function of bacterial species,
antibiotic content of the disc.
Table: Example of standard diameters according to CLSI (Clinical Laboratory
Standards Institute USA) and EUCAST.
Standard Organism Antibiotic Resistant Intermediate Susceptible
CLSI Staphylococcus nitrofurantoin 300 µg ≤ 14 mm 15-16 mm ≥ 17 mm
aureus
EUCAST Staphylococcus nitrofurantoin 100 µg ≤ 13 - > 13
aureus
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Special interactions may appear between antibiotics:
- synergism/antagonism – these phenomena reflect special resistance mechanisms
- The presence of these interactions must be specifically sought for, otherwise
resistance to certain antibiotics might be overlooked
Synergism Antagonism
- The presence of isolated colonies inside an inhibition zone might represent
- resistant subpopulations
- contamination (check purity)
6. Interpretation:
- Results are reported as: susceptible (S), intermediate (I) or resistant (R) to the tested
antibiotic
- The value of the diameter is not reported
- Each TSA result must be critically interpreted.
Variables that may interfere with the results:
- Composition of culture media (cathion, thymidine content)
- pH (7,2-7,4)
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- depth of the agar plate (standard: 4 mm)
- inoculum - 0,5 McFarland
- discs (content, conservation)
- duration, temperature of incubation, atmospheric conditions
- reading of diameters (refracted or reflected light, isolated colonies, etc)
Quality control
- Perform quality control of discs and culture media using reference strains
B. Quantitative methods
- Quantitative methods allow to determine the minimal inhibitory concentration (MIC)
of an antibiotic
- MIC: the smallest concentration of the antibiotic that inhibits bacterial growth
- Indications to perform MIC testing:
- Strains isolated from severe diseases (sepsis, endocarditis, meningitis, etc)
- Inaccurate results by disc diffusion
- Bacteria for which disc diffusion method is not applicable (anaerobes, viridans
group streptococci, etc.)
- Special situations (testing of vancomycin for Gram-positive cocci, penicillin for
pneumococci, etc.)
- The CMI value might be used to adjust antibiotic dosage to ensure higher blood
levels of the antibiotic than the CMI.
- Similarly to diameters in disc diffusion, CMI values may be reported to standard
breakpoints and clinically interpret the results as S, I or R.
µg/ml) for ampicillin:
Table: Breakpoints (µ
Organism Susceptible Intermediate Resistant
Staphylococcus spp. ≤ 0,25 - ≥ 0,5
Enterococcus spp. ≤8 - ≥16
Enterobacteriaceae ≤8 16 ≥32
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Dilution method (macro/microdilution)
- The tested strain is added to tubes or wells containing decreasing (serial dilution)
amounts of the antibiotic.
- After incubation at 35-37°C overnight, growth of bacteria is observed visually (the
content of the tube or well becomes turbid)
- MIC: the lowest concentration that inhibits the growth is the MIC (clear well/tube)
MIC=2µg/ml
Maximum
concentration Minimum Growth control
(256 µg/ml) concentration
(0,25 µg/ml)
Agar dilution method
- Serial dilution of the antibiotic in Mueller-Hinton agar
- Inoculate the tested strains in spots
- MIC: the lowest concentration of antibiotic which inhibits bacterial growth (totally or
max. 1 colony may appear)
E-test
- Apply an E-test (plastic band impregnated with increasing amount of antibiotic) on
the surface of an inoculated Mueller Hinton agar plate.
- Incubate
- An elliptic inhibition zone appears.
- MIC: the concentration read on the E test where the inhibition zone crosses the band
MIC
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