Table Of ContentCHAPTER 1
Antibiotic Resistance Profiling
Stephen J. Hammonds
1. Introduction
Antibiotic resistance is not an absolute property. Even within a single
species, strains can exhibit different degrees of antibiotic resistance.
When a bacterium is tested for resistance, it should be compared, quan-
titatively with a standard.T his standardm ay be the resistanceo f a wild-type
strain, a typical strain, or a “type” strain, appropriate to the test strain.
Most commonly in these studies, large numbers of test isolates are
screened for their resistance on one or more antibiotics. The test strains
may be mutant or recombinant derivatives of a known parental strain, or
they may be fresh isolates from an independent source. The degree of
resistance is quantified by incorporating known concentrations of an
antibiotic into solid growth medium. The bacterial test strains are then
inoculated onto the medium. After a period of growth, the plates are
examined for growth and the lowest antibiotic concentration that inhibits
the growth of each bacterium is determined. This measurei s the minimum
inhibitory concentration (MIC). This is then compared with the observed
MIC of a standard strain or with a reference threshold value (I).
This is an accurate quantitative technique that uses well-established
culture and test methods. In addition, the expected MIC values for many
different species of bacteria are readily available in the literature (2). The
method described here is suitable for bacterial species commonly used in
genetic studies and genetic engineering, such as Escherichia coli. The
technique is readily adaptable to other species by adjusting the media
and culture conditions appropriately. However, the effect of such changes
on the activity of the antibiotic should be consideredc arefully (seeN ote 1).
From: Methods In Molecular Biology, Vol 46: Dagnostlc Bacteriology Protocols
Edited by J Howard and D M. Whltcombe Humana Press Inc , Totowa, NJ
1
2 Hammonds
The technique described is based on the standard method used in the
United Kingdom for determination of the sensitivity of the pathogenic
bacteria to antibiotics. It forms the basis of the well-known “breakpoint”
method for sensitivity testing, widely employed in diagnostic bacteriol-
ogy laboratories. The antibiotics that are most appropriate for testing
against the various species of pathogenic bacteria may be found in
Lambert and O’Grady (2).
The technique may also be used to determine the sensitivity of bacte-
ria to a range of antibiotics to obtain a pattern of antibiotic sensitivities
known as an antibiogram. These antibiograms may be used in bacterial
taxonomy.
2. Materials
1. Phosphate-buffered saline (PBS): 20 mi!4 sodium phosphate, pH 7.4,
0.85% (w/v) NaCl. Sterihze by autoclaving at 121°C for 15 min. Store at
4°C indefinitely, but warm to room temperature before use as a culture dil-
uent, because the viabthty of some strains can be affected by cold diluent.
2. Phosphate buffer: 20 mM sodium phosphate, pH 7.0. Sterilize by auto-
claving at 121°C for 15 min and store indefimtely at 4°C.
3. Antibiotics: 1 mg/mL in sterile 20 mM phosphate, pH 7.0. Sterilize by
filtration through a 0.22~pm filter and store for up to 24 h at 4°C. Any
antibiotics may be used, but solubihty in water 1sc rucial. This may require
the use of antibiotrc salts (see Note 2). Antibiotic preparations supplied for
clinical use are often unsuitable, because they are sometimes inactive in
vitro. Furthermore, they often contam additional, nonantibiotic substances
that could interfere with the assay. Details of the stability and storage
conditions can be obtained from the suppliers. In general, though, the
undissolved powders should be kept desiccated, m the dark and at low
temperature.
4. Liquid medium: Any rich medmm suitable for the organism under study.
For many strains, tryptone soya broth (TSB), also known as tryptrcase soy
broth, is suitable.
5. Test agar: Mueller-Hmton agar and dragnostic sensitivity agar have been
developed specifically for antibiotic testing purposes and are commercially
available. Dtssolve the agar mix according to the manufacturer’s mstruc-
tions and heat to 100°C for 5-10 min to dissolve the solids. Adjust the pH
to exactly 7.4 and autoclave at 121°C for 15 mm. Allow the agar to cool
and keep it, molten, at 45°C until required. Prepare the molten agar on the
day of use. Other media can be used but be sure that the components of
such media have no interfering effects on antibiotic activity (see Note 1).
Antibiotic Resistance Profiling 3
3. Method
1. Inoculate 10 mL of TSB with the test strains. Always mclude at least one
control or reference strain (often the parental strain from which the test
isolates were derived).
2. Grow the cultures at then optimal temperature to early stationary phase
(usually 18-24 h). Vary the mcubation temperature and time as appropri-
ate to the strain. The anticipated cell density is 10’ viable cells/ml.
3. Dilute the cultures 1 in 100 in PBS (see Note 3).
4. Immediately before use, prepare a series of five lo-fold dilutions of the
antibiotic in 20 mM phosphate. Always use a fresh tip for each dilution to
avoid carryover of concentrated antibiotic solution to dilute.
5. Dispense 2 mL of each dilution mto premarked Petri dishes. Also, pipet
2 mL of neat antibiotic solution and 2 mL of phosphate buffer only into
two more dishes.
6. Dispense 18 mL of molten agar mto each dish and thoroughly mix in the
antibiotic by swirling the plate on the bench.
7. Allow the agar to set at room temperature and chill the plates at 4°C to
harden the media.
8. Dry the plates m a plate-drying oven at 55°C for 20 min.
9. Spot each of the diluted cultures onto each of the antibiotic dilution plates,
using sterile loops or applicator sticks (see Notes 1 and 3). Be sure to mark
the onentatton of the plates to allow later identification of the spots. It may
be helpful to use a template where many spots are to be tested.
10. Invert the plates and incubate them under the appropriate conditions
(typically, 37°C for 18-24 h).
11. Examine the surface of the plates and determine the lowest concentration
of antibiotic that inhibits growth of each strain (MIC). Ensure that the
control and/or reference strains have produced the anticipated results.
12. Determine the MIC more accurately by using a twofold dilution series of
antibiotrc, starting with the dilution above the MIC.
4. Notes
1. The activity of many antibiotics 1s significantly affected by the physical
and chemical conditions in the bacterial culture medium (3):
a. Acid conditions, which arise by fermentation of carbohydrate m the
medium, may reduce the activity of lincomycin, erythromycin,
cephaloridine, and the aminoglycosides;
b. Alkaline conditions reduce the activity of methicillin, cloxacillin,
fucidin, and the tetracyclmes;
c. Anaerobic conditions reduce the activity of ammoglycosides;
Hammonds
d. Aerobic conditions reduce the activity of metronidazole;
e. Divalent cations such as magnesium and calcmm reduce the activity of
aminoglycosides and tetracyclines against Pseudomonas aeruginosa; and
f. Pyrtmidines and related compounds reduce the acttvtty of sulfonamtdes
and trimethoprtm.
2. Some antibrotics are insoluble in water in the form of the base. However, tt
is often the case that soluble salts exist for these antibiotics (e.g., erythro-
mycin lactobionate, trimethoprim lactate, and polymyxin sulfate). It is
sometimes also possible to dissolve the base m a minimal volume of 0. 1M
NaOH (e.g., nitrofurantoin, nalidixtc acid, and the sulfonamides), or O.lM
HCl (e.g., rifampicm), or ethanol (e.g., chloramphenicol, erythromycin)
before dilution to the required volume with 20 mM phosphate buffer.
Details of the solubibty and other properties of antibiotics may be found m
The Merck Index (4).
3. The concentration of the moculum is less important in the agar incorpora-
tion technique than in other methods. However, too high a concentration
of bacteria does cause a sigmftcant reduction in the activity of
sulfonamides. Consequently, the correct dilution of bactertal suspension
must be used.
References
1 Phillips, I. (1991) A guide to sensitivity testing. Report of the working party on
antibiotic sensitivity testing of the British Society for antimicrobial chemotherapy
J. Antimicrob Chemother 27(Suppl. D), l-50.
2 Lambert, H. P. and O’Grady, F (1992) Antibiotic and Chemotherapy, 6th ed
Churchill Livingstone, Edinburgh, UK.
3. Houang, E T., Hince, C , and Howard, A. J. (1983) The effect of composition of
culture media on MIC values of antibiotics, in Antibiotics: Assessment of
Antimicrobial Activity and Resistance (Russell, A. D. and Quesnel, L B , eds ),
Academic, London, pp. 3 l-48.
4 Budavari, S. (1989) The Merck Index. Merck, Rahway, NJ.
CHAPTER2
Bacteriocin Typing
l)rone L. Pitt and Michael A. Gaston
1. Introduction
Bacteriocins are proteins produced by bacteria which are lethal for other
members of the same species and, occasionally, for other species. In gen-
eral, bacteriocins are active in very low concentrations against specific
strains. This property has been widely utilized for the identification of
types of strains within several bacterial species and most methods are
based on the inability of a strain (indicator) to grow on an agar surface that
has previously supported the growth of the bacteriocin producer strain.
Bacteriocin typing is a simple, but powerful, technique to distinguish
between strains of bacterial species but is often overlooked in favor of
more technically sophisticated methods. At its simplest level no stored
reagents or even reference strains are required, as a collection of isolates
can be tested against each other for bacteriocin production/sensitivity.
The information gained in this way is likely to be less valuable than that
obtained with an established reference typing method but may be suffi-
cient for the identification of unique strains in an outbreak situation.
1.1. Nomenclature
The naming of bacteriocins has been problematic. Some workers prefer
to use the term bacteriocin of, for example, Klebsidu or Pseudomonas,
whereas others refer to klebocin or pyocin, respectively. This trivial form
of nomenclature is widely used but the discovery of new bacteriocins may
lead to taxonomic confusion.
From’ Methods m Molecular B/ology, Vol. 46. D!agnost/c Bacteriology Protocols
E&ted by J Howard and D. M Whltcombe Humana Press Inc , Totowa, NJ
5
Pitt and Gaston
1.2. Characterization
Bacteriocins are a large heterogeneous group of antibiotic substances
that differ greatly in their molecular size and their resistance to heat,
chloroform, and proteolytic enzymes. Three basic groups have been
defined:
1. Cohcin-like agents ranging 111m olecular size from 3 x lo6 to 1 x lo8 Daltons;
2. Bacteriophage-like particulate bacteriocins that resemble phage tails, e.g.,
R (retractile) and F (flexuous) pyocins of Pseudomonas aeruginosa; and
3. Low molecular weight bactenocms, also called mlcrocins, which are bac-
tericidal peptldes (I-3)
1.3. Activity Spectra
The bacteriocins of Gram-negative bacteria often have a narrow spec-
trum of activity. In contrast, the bacteriocins produced by Gram-positive
bacteria have a broad host range and are sometimes active on taxonomi-
tally unrelated genera (4).
1.4. Mode of Action
The modes of action of some bacteriocins have been established. For
example, colicins cause specific inhibition of protein synthesis or induce
a single endonucleolytic break in RNA, whereas others inhibit DNA syn-
thesis (5). Some bacteriocins inhibit macromolecular synthesis by
uncoupling electron transport systems or cause disruption of the perme-
ability barrier of the cell wall by enzymatic action, as is the case with the
megacins of Bacillus megaterium. Specific receptors are required for
the adsorption of bacteriocins and resistant strains often lack, or have
occluded, surface receptors. Receptors that have been identified include
glycoproteins, outer membrane proteins, and lipopolysaccharides.
1.5. Resistance and Tolerance
A bacteriocin is inactive against strains possessing the same
bacteriocinogenic factor. These factors may be plasmid borne or chro-
mosomal. Strains may produce more than one type of bacteriocin, but
the immunity of producer strains to bacteriocins is often extremely
specific and they may be sensitive to a closely related bacteriocin. Some
bacteria produce a specific inhibitor protein that complexes with the bac-
teriocin and protects the producer strain from its action. Alternatively,
immunity may be associated with the inability of the producing bacterium
to adsorb homologous bacteriocin (6). A distinction should be made
Bacteriocin Typing 7
between resistant and tolerant strains; the former do not adsorb bacterio-
tin, as do tolerant strains, but the lytic action is blocked subsequently.
1.6. Practical Considerations
The inhibition of strains by bacteriocins can be confused with the
action of bacteriophages and vice versa. In some strains this can be fur-
ther complicated by the simultaneous production of both bacteriocin and
bacteriophage. A simple way to distinguish between the two groups is to
prepare a logarithmic dilution series of a suspension containing the
inhibitor and spot a sample of each dilution onto the indicator strain.
Inhibition owing to bacteriocins will generally only be visible to a
dilution of lo3 or lo4 and will “fade out,” whereas bacteriophages will
usually be active to at least a dilution of lo7 and will show individual
plaques at high dilutions.
1.7. Typing Schemes
Bacteriocin typing schemes can be classified as active or passive
methods. In the first, the bacteriocins produced by the test (field) strains
are detected by their activity on a panel of standard indicator strains,
whereas in the second method, bacteriocins from a set of producer strains
are tested for activity on the field strains. The discrimination achieved by
either method should be determined by experiment as well as the sta-
bility (reproducibility) of the patterns of inhibition. Schemes with
high reproducibility will allow patterns to be designated as types, but
this is impractical for those schemes that exhibit excessive variation on
repeated testing.
Three different methodological approaches have been used for routine
typing of isolates. The original method of “scrape and streak” (7),
described in Section 3.3., is not commonly used today because it is too
labor-intensive; it takes 3 d to complete and reactions can be equivocal.
Essentially, the bacteriocin-producing strain is grown in a streak across
the diameter of an agar plate. Following incubation, this growth is
removed and the indicator strain is inoculated at a right angle over the
original streak. Bacteriocin activity is evidenced by inhibition of the
indicator strain.
In the lysate method (B), described in Section 3.2., bacteriocins of a
producer strain are induced by mitomycin C and cell free lysates are
spotted onto lawns of the indicator strain. This method is not widely used,
perhaps because of the need to prepare lysates that not only require stan-
8 Pitt and Gaston
dardization of bacteriocin concentration but also deteriorate on storage.
The influence of bacteriophages that are also induced by mitomycin C
can be reduced by UV irradiation of the lysate, but this is a time-consum-
ing and variable procedure.
The principle of the overlay method (9) (see Section 3.1.) is that spots
of a dense suspension of bacteria containing preformed bacteriocin are
allowed to grow for a short period in which more bacteriocin is formed.
The thin agar layer serves to concentrate the bacteriocins around and
under the spot of growth. Bacteriocin activity is visualized by inhibition
of the growth of the indicator strain contained in the overlay; diffusion is
enhanced by the use of soft agar. This method also allows the differen-
tiation of low molecular weight diffusible bacteriocins from the particu-
late high molecular weight structures by their inhibition zone diameters.
The major advantages of this procedure over others is the rapidity and
the clarity of reactions that can be obtained.
1.8. Prior Considerations
The choice of methodology for bacteriocin typing is greatly influ-
enced by the growth characteristics of the species to be examined and
the equipment available. We recommend that the following factors
are determined by experiment in order to optimize conditions for a par-
ticular species.
1.8.1. Media
Some media constituents are inhibitory to bacteriocin production.
For example, bile acids are antagonistic to Proteus mirabilis bacterio-
tins (10) and, therefore, McConkey agar is unsuitable for bacteriocin
typing of this species. Other compounds that have been reported to affect
bacteriocin synthesis include casein, amino acids, manganese, and sug-
ars, particularly glucose (4). The addition of enzyme inhibitors such as
iodoacetic acid has been advocated to improve the clarity of inhibition
reactions (11).
1.8.2. Temperature
Bacteriocins are often produced in higher yield if the strain is
incubated at a temperature slightly below the growth optimum. This
serves two purposes; first, it reduces the amount of cell growth and, sec-
ond, it delays the production of bacteriocin, inactivating substances such
as proteases.
Bacteriocin Typing 9
1.8.3. Induction
Most bacteriocins may be recovered in higher yield if an inducing
agent is used. Mitomycin C is the agent most commonly used. This
compound damages DNA and, as a result, a complex cellular mechanism,
the SOS regulatory system, is activated. DNA repair occurs in the absence
of template instruction resulting in the creation of many nonsense or
deletion mutations. Lysis of these mutants occurs at a rapid rate with a
release of cellular contents that include bacteriocins and lysogenic phages.
Most workers have used mitomycin C to induce bacteriocin production
in liquid media but care should be taken to rule out the contribution of
phage in the lysate to the inhibition zone formed on agar lawns of suscep-
tible bacteria. Anaerobiosis can also induce bacteriocin synthesis and nitrate
can be incorporated into a growth medium to encourage anaerobic growth
of Pseudomonas (nitrate acts as an alternative electron acceptor when
cleaved by nitrate reductase to produce molecular oxygen). Bacteriocin-
inactivating substances may also be suppressed by anaerobiosis and recep-
tors may not be synthesized in the absence of oxygen.
2. Materials
1. Nutrrent broth and solid media appropriate to the growth requirements of
the species (see Note 1). In the agar overlay method, add 0.5 g of bacterro-
logrcal grade agar to 100 rnL of 1% (w/v) peptone, boil for 15 mm to
dissolve the agar, and sterilize at 121°C for 15 min. Drspense into sterile
bijoux bottles in 2.5-mL volumes.
2. Mitomycin C: 100 ug/mL in sterile water, store m lOO-200~yL aliquots in
foil-wrapped bottles at -70°C. Discard the stock after 6 mo and do not
refreeze. This 1s a hazardous agent and appropriate safety precautions
should be taken.
3. Saline: 0.2M in distilled water. Sterilize by autoclavmg and store at room
temperature.
4. Chloroform (see Note 2).
5. Multipomt inoculator (see Note 3).
6. Membrane filters: 0.45 urn pore size, sterilizing grade.
3. Methods
3.1. Agar-Overlay Method (Fig. 1)
1. Pipet 10 mL of molten agar into g-cm diameter Petri dishes. Allow the
agar to set and dry the surface at 37°C for 15 min. Store plates m a sealed
plastic bag at 4°C overnight.
10 Pitt and Gaston
Bacteriocin productlon Bacteriocin detection
Fig. 1. Bacteriocm typmg by the method of Fyfe et al. (9).
2. At the same time, grow the bacteriocin-producing strams overnight on
nutrient agar plates.
3. Disperse 5-10 colonies with a cotton swab m 2 mL of sterile distilled water
to give a smooth suspension of approximately lOlo cfu/mL.
4. Dispense the suspensions into wells of a sterile reservoir block and apply
spots of 0.3-0.5 pL with a multipomt moculator onto the surface of the
lo-mL agar plates.
5. Incubate plates at a suitable temperature (see Note 4).
6. Remove plates from incubator and, m a fume hood, invert the agar surface
for 15 min over filter paper pads (30 x 30 mm) that have been impregnated
with 0.5 mL of chloroform (see Note 5).
7. Place the plates, open, in a 37OC incubator for 30 min to dry the surface
and dispel all chloroform vapor.
8. Add 0.05 mL of a log-phase broth culture of an indicator strain to 2.5 mL
of molten overlay agar at 50°C.
9. Mtx gently and pour the total volume rapidly over the entire surface of
the plate.
10. Incubate the plate overnight at the optimal temperature for growth and
record zones of inhibition.
3.2. Lysate Method
1, Grow the producer strains m broth overnight.
