Investigating The Efficacy of Nisin When Combined With First Line Antibiotics in The Treatment of MRSA 252

UNIVERSITY OF NOTTINGHAM
Investigating interactions of nisin with first-line antibiotics in MRSA 252 and S. aureus NCTC
6571
Biochemistry Research project

Joseph Clonan
1/1/2013

Project Supervisor: Dr. Boyan Bonev
Word Count: 8,425

Joseph Clonan Biochemistry Research Project - 4099822
1

Contents
Abstract................................................................................................................................. 3
Background: ...................................................................................................................... 3
Method: ............................................................................................................................. 3
Results: ............................................................................................................................. 3
Conclusion: ....................................................................................................................... 3
Introduction ........................................................................................................................... 4
The onset of bacterial resistance to antibiotics .................................................................. 4
Mechanisms underlying acquired bacterial resistance.................................................... 4
Staphylococcus aureus: a highly promiscuous genome .. Error! Bookmark not defined.
Methicillin resistant Staphylococcus aureus ................................................................... 6
First line treatments for MRSA ....................................................................................... 7
Further approaches to treating MRSA ............................................................................ 8
Antibiotics ........................................................................................................................ 11
Cell-wall synthesis inhibitors ........................................................................................ 11
Protein Synthesis inhibitors .......................................................................................... 14
Macrolides ................................................................................................................... 17
Tetracyclines ................................................................................................................... 18
Metabolic inhibitors ...................................................................................................... 19
Polymixins .................................................................................................................... 20
Hypotheses of interactions between nisin and antibiotics ............................................. 20
Methods .............................................................................................................................. 22
Nisin Purification .............................................................................................................. 22
2

Extraction of Nisin from Nisaplin (Dannisco Dupont, UK) ............................................. 22
Extracting nisin fractions with Toyobutyl column .......................................................... 22
Reverse phase high pressure liquid chromatography of 10ml HCl wash using C18
column ......................................................................................................................... 22
MALDI-TOF-Mass Spectrometry using the LaserToF................................................... 23
Testing the activity of nisin ............................................................................................... 23
Establishing Minimum Inhibitory Concentrations .............................................................. 24
Sensitivity testing for NCTC 6571 and MRSA 252 ........................................................ 24
Calculating MICs of the antibiotics to make ISA at MIC concentrations ....................... 26
Combining nisin and antibiotic in the same well to observe interactions determining
change in zone size ..................................................................................................... 26
Results ................................................................................................................................ 29
Testing the activity of nisin ............................................................................................... 30
Calculating MICs for antibiotics ....................................................................................... 31
Evaluating interactions of nisin and antibiotics combined in the same well ...................... 34
Discussion .......................................................................................................................... 39
Nisin activity and efficacy................................................................................................. 39
Limitations of methodological approach and confounders................................................ 40
Suggestions for future research ....................................................................................... 41
Concluding statements ........................................................................................................ 42
Acknowledgements ............................................................................................................. 42
Glossary of terms ................................................................................................................ 43
REFERENCES ................................................................................................................... 44
3

Abstract

Background: As a result of the widespread use of antibiotics, bacteria have both cause
and opportunity to acquire resistance as a result of chromosomal changes or the exchange
of genetic material through conjugative plasmids or transposons. L antibiotics are potent
antimicrobial peptides originating from bacteria, which have low propensity for generating
resistant bacteria.
Objective: To investigates whether the lantibiotic nisin, may enhance the activity of first-line
antibiotics.
Method: Nisin is combined with antibiotics observe inhibition zone sizes in MRSA, and S.
aureus NCTC 6571.Changes in the MIC of nisin were observed when nisin was in the
presence of antibiotic incorporated into agar. Zone diameters were measured around wells
containing either antibiotic, or antibiotic with nisin to observe interactions.
Results: Nisin enhanced the activity of vancomycin and trimethoprim, and facilitated activity
of colistin which was inactive when used alone. However, nisin was found to be antagonistic
to the activity of tetracycline (p<0.025).
Conclusion: Observations of synergy between nisin and colistin against MRSA offer a
novel approach to combining lantibiotics with antibiotics. Further investigation is required to
progress this approach toward clinical application in tackling MRSA or other multi-drug
resistant bacteria.

4

Introduction
The onset of bacterial resistance to antibiotics
The World Health Organisation defines antimicrobial resistance as resistance of a
microorganism to an antimicrobial medicine to which is was previously sensitive.
The first recorded case of resistance to antimicrobial chemotherapy was in 1907 when Paul
Ehrlich found protozoan trypanosomes resistant to arsenic, but drug-resistant bacteria were
first found in hospitals and treatment centres where antibiotics were being used.[1] Strains of
Streptoccoccus pyogenes resistant to sulphonamides were observed in hospitals as early as
1930[2]. Penicillin was introduced as a therapeutic agent in 1940, just two years before the
first recorded incidence of penicillin resistant staphylococci in 1942.[3] By 1944 several
penicillin resistant strains of Staphylococcus aureus had been documented, and theory
established describing how resistant had developed.[4] Clearly then, bacteria have high
capacity to evolve and resist antimicrobial chemotherapy through direct contact of bacteria
and antibiotics (assuming the antibiotic does not kill the entire population), use of resistance
genes, horizontal transmission of these genes, or serendipitous mutations conferring
resistance.[5]
Mechanisms underlying acquired bacterial resistance
Bacteria can undergo transformation by integrating naked DNA from other cells into a
homologous region on their own genome. This naked DNA is typically only small lengths of
fragmented DNA. Altered penicillin binding proteins (PBPs) in penicillin-resistant bacteria are
an example of genetic transformation and recombination. PBPs are variable and can be
bound by many different antibiotic -lactam rings, so penicillin-resistance indicates
modification of multiple genes coding for PBPs, demonstrating the extent of shared genes in
bacterial species, known as mosaic genes.[6]
Transposons and conjugal plasmids are mobile genetic elements which may integrate into
plasmids or recipient DNA regardless of sequence homology. This method of conjugating
5

DNA into the chromosome is capable of transferring long fragments of DNA i.e. many genes,
and can even occur between bacteria and eukaryotic cells via direct cell to cell contact. [7]
Transduction occurs when bacteriophages integrate their own DNA into host chromosome
and replicate through the lysogenic cycle until triggered to enter the lytic cycle, whereupon
they use the host cells replication machinery to replicate and lyse the cell; in doing so it is
possible resistance genes are inserted from the host cell chromosome into the phage DNA,
which the phage will then integrate into other host cells genomes, facilitating the spread of
resistance genes. [8]
Some resistance genes contained within bacterial chromosomes are regulated by operons
which only activate when the relevant antibiotic is present. -lactamase inhibitors can act as
inducers for -lactamase production.[9]
Genetic islands refer to the integration of large continuous fragments of DNA into the
bacterial chromosome. This may happen via inheritance of plasmids which may have
recombined into the bacterial chromosome or remain in the cytosol as autonomous
replicons; or by integration of lysogenic phage material into the bacterial chromosome; or via
linear DNA fragments being inserted into the chromosome via recombination next to
homologous sequences or transposition. This method of acquiring multiple genes at once
facilitates gain of function changes in the chromosome i.e. the inheritance of complex
phenotypic traits, in a single step.[10]
Staphylococcus aureus: a highly promiscuous genome
S.aureus is an opportunistic gram positive bacterium, and a major contributor to community
and hospital acquired infections. S.aureus is not a recent problem typical symptoms are
described in Exodus chapter 9 as shhin in the sixth plague of Egypt, translated as
relentless festering skin boils which were difficult to heal. S.aureus typically occupies the
nasal cavities of carriers and is not problematic whilst colonising a host, but if opportunistic
6

infection occurs S.aureus may release toxins such as alpha haemolysin into the
bloodstream, or cause septicaemia, and other more localised infections such as skin lesions.
The risk of S.aureus infection increases if a patient is colonised with S. aureus, or if they are
admitted to a hospital unit which has dealt with staphylococci infections before such as the
intensive care unit, or exposure to another patient who has MRSA.[11] [12]
The S.aureus genome is highly variable, with the capability, propensity and motivation to
acquire resistance genes, which led to the emergence of virulent and drug resistant strains
[13], creating the continuously evolving task of treating infection for clinicians. Due to the
genetic plasticity of S.aureus, there are many well-defined resistance genes present across
strains.
Methicillin resistant Staphylococcus aureus
Methicillin was developed by Beecham in 1959 to combat penicillinase-producing bacteria,
and was the first-line antibiotic for staphylococcal infections at the time. Two years later,
MRSA was first documented, and has since become an endemic problem in hospitals and
communities as indicated in Table 1.
Drug
Year Drug
Introduced
Years to report
of resistance
Years until 25%
resistance in
hospitals
Years until 25%
resistance in
the community
Penicillin 1941 1-2 6 15-20
Vancomycin 1956 40 - -
Methicillin 1961 <1 25-30 50
Table 1: Time taken for resistant S. aureus strains to arise and become established in
communities and hospitals.[14]
Resistance to methicillin is conferred via the mecA gene on the SSSmec operon (part of the
Staphylococcal cassette chromosome mec or SSCmec a mobile genetic element within
Staphylococci). The SSCmec recombinase enzymes ccrA and ccrB, facilitate site-specific
integration and excision of SSCmec into/from the S. aureus chromosome, enabling lateral
transfer of resistance genes.
mecA is controlled by the regulatory genes mecI and mecR1. mecI binds and represses the
mecA; when a -lactam antibiotic is present, mecR1 cleaves mecI and initiates mecA
transcription. mecA codes for an altered penicillin-binding protein PBP2a [15] diminishes
7

the binding affinity for -lactams[16] In Methicillin-sensitive S. aureus, unmodified PBPs are
bound by the -lactam ring, causing inhibition of cell wall synthesis, and cell death.[17]
-lactamases hydrolyse the -lactam ring in penicillin antibiotics, and remove the trailing
carboxyl group to inactivate the antibiotic. The BlaZ gene codes for -lactamase in S.
aureus.[18] BlaR1 cleaves BlaI to initiate BlaZ transcription and produce -lactamase. BlaI
and mecI bind identical DNA sequences and are thus able to co-repress BlaZ and mecA.
MRSA 252 is a -lactamase and is resistant to -lactam antibiotics such as ampicillin.
S. aureus
resistanc
e genes
Susceptible
antibiotics
Mechanism Propagator
NCBI
ID
MRS
A 252
NCTC
6571
mecA penicillins
Altered penicillin
binding proteins
Mosaic Gene NCBI [2]
erma macrolide
Methylation of
adenine 2058 of
23S rRNA
rRNA adenine N-6-
methyltransferase
142332
tet38 tetracycline
Tetracycline
efflux pump
Major facilitator
superfamily
transporter
129794
Table 2: Known antibiotic resistance genes found in the MRSA 252 chromosome
Table 2 shows some of the defined resistance genes found in the MRSA 252 genome for the
antibiotics covered in this project. S. aureus NCTC 6571 is susceptible to all antibiotics used
in this project, aide from colistin, making it a suitable control strain to test alongside MRSA
252. [19]
First line treatments for MRSA
Many first-line antibiotics are becoming less useful and inevitably redundant as populations
of bacteria surface with partial or total resistance. [20] The current first line treatment for
MRSA is the glycopeptide vancomycin, administered intravenously. The restricted use of
vancomycin in hospitals has limited its contact with MRSA, reducing the incidence of MRSA
evolving with resistance to vancomycin. Despite this, vancomycin-resistant MRSA has been
documented and necessitated the development of new MRSA treatments such as
8

daptomycin a membrane destabilising lipopeptide, linezolid an oxazolidinone protein
synthesis inhibitor, and tigecycline a glycylcycline protein synthesis inhibitor. [21]
Vancomycin also has poor tissue distribution, making it difficult for the drug to reach all sites
of infection, further necessitating the need for new therapeutic agents capable of both
eradicating the pathogenic bacterial populations and penetrating to required sites of action
easily.[22]
In the UK, all hospital in-patients are tested for presence of MRSA, and where necessary
decolonised with antimicrobial skin creams and nasal cavity sterilising ointments, often
containing mupirocin or polyhexanide biguanide.[23]
Further approaches to treating MRSA
Antimicrobials such as nisin are now being investigated as potential alternatives to
antibiotics in modern contemporary medicine. Lantibiotics such as nisin have long been used
in the food industry as an antimicrobial preservative - nisin was approved for by the Joint
FAO/WHO Expert Committee as a food additive in 1969. Despite this longevity of use,
lantibiotics retain their antimicrobial efficacy and few cases of resistance have been
described[10]. Lantibiotics are easy to derive from natural sources, have wide spectrum of
antimicrobial activity and are not prone to provoking bacterial resistance. [5]
The aim of this project is to investigate the interactions between nisin and first-line antibiotics
from each major class penicillins, aminoglycosides, macrolides, tetracyclines,
sulphonamides, polymixins and the glycopeptide vancomycin.
Nisin
Nisin, or European E number 234, is a lantibiotic commonly used as a preservative in food
and drink products such as beer and processed cheese. It effectively prevents contamination
by gram-positive bacteria including Listeria monocytogenes and Clostridium botulinum [24]
at nanomolar concentrations as low as 1.25mg/L. [25]
9

Nisin permeabilizes the bacterial membrane making it non-selectively permeable to amino
acids, ATP, and ions and results in depletion of membrane potential[26] [27]. It also targets
undecaprenyl pyrophosphate (PP on Figure 2) disrupting cell wall synthesis[28], and
accelerates the cell division process in bacteria and disrupts membrane regulation.[29]
The Joint Expert Committee on Food Additives (JECFA) indicates that nisin is a safe and
suitable food antimicrobial with an non-toxic daily intake of 0-33,000 units/kg body weight
(the equivalent of 0.33g/kg bodyweight per day), as well as having FDA Generally
Recognised as Safe status since 20/04/2001[30]. Purified nisin is a water-soluble micronized
powder, available as 2.5% commercial preparations such as Nisaplin and Novasin
(Danisco/Dupont).
Recombinant nisin is produced by Lactococcus Lactis NZ9840, which self-protects from nisin
with the ABC transporter NisFEG which transports nisin away from the cytoplasmic
membrane and lipoprotein NisI which prevents membrane aggregation.[31]
Nisin is a 34 residue post-translationally modified polycyclic peptide with only primary
structure shown in Figure 1. It contains the rare amino acids dehydrobutyrine (Dhb) and
dehydroalanine (Dha), which form thio-ether linkages with alanines, called lanthionines.[32]
Dha and Dhb are susceptible to attack by nucleophiles present at higher pH, resulting in a
decreased solubility as solvent pH increases. [33]. Nisin belongs to the type-A class of
lantibiotics, which are generally elongated and helical shaped peptides with a net positive
charge. [34]
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Figure 1: Structure of nisin; whilst not having any secondary structure, the primary
sequence of nisin is held together through sulphur bridges between alanine and dehydrated
residues, and hydrophobic interactions between residues. Nisin maintains a three
dimensional conformation due to hydrophobic interactions of residues in its primary structure
and five lanthionine rings A-E.
The cation amphipathic nature of nisin causes it to associate with anionic moieties on the
phospholipid surface of the cell membrane, which disrupts the dynamics of the polar
phospholipid head group in extracellular fluid. [35].
When nisin aggregates at the membrane, the N-terminal backbone amides of nisin bind the
pyrophosphate moiety of lipid II via hydrophobic interactions to configure nisin and lipid II
into a pyrophosphate cage complex consisting of 4 lipid II molecules and 8 nisin molecules
[36, 37]; this is the nonspecific hydrophilic pore through which ions, amino acids, and ATP
leave or enter the cell. The C-terminus of nisin traverses the cell membrane to propagate
and anchor the cage complex into the membrane.[31]
Lipid II is a composite of N-acetylglucosamine (NAG) and N-acetylmumaric acid (NAM) and
a pentapeptide such as L-Ala-D--Glu-L-Lys-D-Ala-D-Ala, attached to the carboxyl group of
NAM. It is estimated that nisin-lipid II complexes form pores of diameter 2-2.5nm[38]
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Antibiotics
Cell-wall synthesis inhibitors
Many antibiotics including penicillin, inhibit bacterial division or kill bacteria by disrupting the
synthesis of the bacterial cell wall.[39] Bacterial cell walls consist of cross-linked
peptidoglycan chains, and synthesis is tightly regulated by autolysin degradation and
synthesis of new peptidoglycan.
In the final step of bacterial cell wall synthesis, glycopeptide monomers are added to
peptidoglycan on the extracellular surface of the bacterial membrane. [40] The cell wall is
vital for resisting the high osmotic pressure to prevent the cell from bursting. It also helps
regulate membrane traffic, and prevents access of antibiotics such as colistin.
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Figure 2: Biosynthesis of bacterial cell wall component peptidoglycan
Glycopeptides
Vancomycin binds with high specificity to the C-terminal D-Ala-D-Ala moieties on lipid II, by
forming hydrophobic interactions with NAM and NAG, shown as M and G on Figure 2
respectively. This binding has three direct impacts on cell wall synthesis; lipid II cannot be
incorporated into the peptidoglycan component of the cell wall; binding to NAM and NAG
diminishes synthesis of peptidoglycan polymers, and prevents cross-linking of existing
polymers.[41]
In vancomycin resistant MRSA, an epitope substitution of D-Ala-D-Lac in place of D-Ala-D-
Ala on the end of cell wall peptide chains prevents vancomycin binding, thus cross-linking of
peptide chains can still occur and bacterial cell wall synthesis is unaffected. Nisin has been
13

shown to remain fully active against vancomycin-resistant MRSA, indicating the mechanism
of lipid II binding is different between nisin and vancomycin. [42]
Since both nisin and vancomycin target lipid II, it is possible that the concentration of lipid II
would be reduced sufficiently to hinder formation of pores with nisin. Even so, the
multifaceted cell wall synthesis inhibition offered by combining the two should yield at least
modest additive inhibition.
Ampicillin
Ampicillin is a broad-spectrum penicillin derivative, released by Beecham in 1961 as a
counter-measure against gram-negative bacteria; an amino-group is added to penicillin,
allowing it to penetrate the outer membrane only found on gram negative bacteria. The -
lactam ring at the core of ampicillin is similar to the terminal D-Ala-D-Ala motif on cell wall
precursors, and binds serine 405 on transpeptidase PBP in the final step of cell wall
synthesis. The cell cannot cross-link peptidoglycan properly resulting in interruption of the
cell wall, leakage of cytosolic material, and osmotic swelling of the cell resulting in lysis
causing cell death. Immobilisation of PBPs also leads to lack of peptidoglycan for cell wall
formation at the septum during mitotic binary fission.

Figure 3: The structure of ampicillin and target of B-lactamase enzymes
14

Resistance to ampicillin may arise through altered PBPs which have very low binding affinity
for the -lactam ring, and also through production of -lactamase. MRSA 252 is a -
lactamase producer[43] and thus less susceptible to ampicillin than NCTC 6571. MRSA 252
is not known to have altered PBPs; therefore resistance to ampicillin is surmountable with
concentrations of antibiotic high enough to overwhelm -lactamase. Nisin prevents cell wall
synthesis before it forms pores in the cytoplasmic membrane, by binding and sequestering
lipid II, at concentrations lower than that required to porate the cytoplasmic membrane.[44]
At low concentrations, nisin and ampicillin both inhibit cell wall synthesis at different targets
and should be synergistic. If nisin permeabilises cell membrane, this synergy may be less
important but nonetheless noticeable.
Protein Synthesis inhibitors
Protein synthesis is a tightly regulated process essential to producing metabolic, regulatory,
and structural proteins within a cell. This makes it an excellent target for antibiotics such as
aminoglycosides, macrolides and tetracyclines, which bind the bacterial ribosome at unique
points shown in Figure 4, to interfere with protein synthesis. This disrupts normal cell
regulation, depletes key metabolic factors such as respiratory enzymes, blocks mitotic cell
division, and leads to inevitable cell death.
The interaction of a protein synthesis inhibitor of nisin depends on its mechanism of entry
into the cell; macrolides diffuse through the peptidoglycan and cytoplasmic membrane with
ease, whereas aminoglycosides and tetracyclines rely upon charged moieties and co-
transporters to enter the cell.
15

Figure 4: Targets of protein synthesis inhibitors on the 30S and 50S subunits of the bacterial
ribosome
Aminoglycosides
Aminoglycosides are rapidly attracted to the cytoplasmic membrane by negatively charged
membrane components such as LPS, and then enter the cytoplasm in modest quantities via
a hydrophilic pathway and in more significant quantities via an energy-dependent transport
system. [45] Once in the cytosol, aminoglycosides target ribosomes and bind the 30S
subunit blocking translocation of peptidyl tRNA from the A-site to the P-site which inhibits
initiation of translation, peptide bond formation, and peptide elongation causing the release
of incomplete and sometimes toxic proteins which halts protein synthesis.
16

Figure 5: The structure of gentamycin and the targets of some of the mechanisms of
gentamycin resistance
Resistance to aminoglycosides may be conferred by enzymatic inactivation as shown in
Figure 5. MRSA 252 is not thought to produce aminoglycoside-modifying enzymes, but
these resistance genes are relatively easily acquired through autonomous plasmid
replicons.[46] Bacteria may also resist aminoglycosides via reduced membrane permeability,
or modified ribosomes with low binding affinity.
Both nisin and gentamicin have a higher affinity for bacterial membranes when more
negatively charged lipids are present, but gentamicin does not target cell wall synthesis or
interact with lipid II therefore should not influence nisin activity. At high concentration, nisin
porates the cell wall, causing depletion of proton-motive gradient, and disrupts osmotic and
nutrient balance; inhibition of protein synthesis may potentiate this by blocking cellular
upregulation of proteins involved in cell wall synthesis. Depletion of the proton-motive
gradient may diminish the energy-dependent transport of gentamicin, however it may still
17

gain access through nisin-mediates pores and the hydrophilic pathway , accelerating and
prolonging protein synthesis inhibition, thus nisin and gentamicin could have a synergistic
interaction.
Macrolides
Macrolides such as erythromycin are lipophilic molecules which easily cross the
peptidoglycan layer of the cell wall and the plasma membrane of gram-positive bacteria with
ease. Macrolides bind the 50S subunit of the bacterial ribosome, inhibiting translocation of
peptidyl tRNA bound at the A-site to the P-site of the rRNA complex. The A-site remains
occupied preventing the addition of new tRNA to the nascent polypeptide chain. This also
interferes with initiation of translation and release of peptides, causing incomplete nonsense
or toxic proteins to be released.

Figure 6: The structure of erythromycin and targets of some mechanisms of erythromycin
resistance [47]
Resistance to macrolides typically involves enzymatic inactivation, or macrolide efflux pumps
indicated in Figure 6. As shown in Table 2 MRSA 252 produces 23S methyl-transferase,
18

found on the ermA gene, which diminishes the affinity binding affinity of erythromycin to the
ribosome. It is unlikely then, that nisin will potentiate the effects of erythromycin in MRSA
252.
Erythromycin freely diffuses across the cytoplasmic membrane nisin mediated poration of
the membrane may accelerate erythromycins entry into the cell. Nisin could potentiate the
efficacy of erythromycin in the same way as gentamicin, thus is it possible nisin and
erythromycin could behave synergistically in NCTC 6571, which is sensitive to
erythromycin.[48]
Tetracyclines
Tetracyclines bind the 16S component of the 30S ribosomal subunit, blocking tRNA binding
the A-site, preventing amino acids being added to the nascent peptide chain, inhibiting
protein synthesis. The uptake of tetracycline is driven by proton-motive force, exploiting
ATP-dependent membrane transporters and chelating divalent cations on the cytoplasmic
membrane to enter the cell.[49]
Once across the cytoplasmic membrane, tetracycline acquires positive charge by forming
complexes with divalent cations and diffuses freely through the cytosol to the ribosome.
Tetracycline is poly-charged when not in the cell, thus allowing a higher concentration of
tetracycline to enter the cell before equilibrium is reached.
Tetracycline resistance has been identified in S. aureus through tetracycline efflux via
antiport proteins, which pump the tetracycline-divalent-metal-cation complex outside of the
cell in exchange for a proton. Enzymatic modification leading to inactivation of tetracycline
may occur in the cytosol. [50].
The MRSA 252 genome is known to contain tet38, the gene coding for an ATP-dependent
tetracycline efflux pump which removes tetracycline from the cell.[51] Therefore MRSA 252
should be less susceptible to tetracycline than NCTC 6571.
19

Since uptake of tetracycline is dependent upon proton-motive force and membrane potential,
nisin pore-mediated depletion of these factors will diminish tetracycline presence in the cell,
thus antagonising its action. The net effect of combined nisin and tetracycline should be
equal to the net effect of nisin alone, as tetracycline will not have access to ribosomes to
inhibit protein synthesis.
Metabolic inhibitors
Trimethoprim was historically used with sulfamethoxazole in the treatment of MRSA in the
UK, but this strategy has become less popular due to side effects of sulfamethoxazole.[52]
Trimethoprim has thousand-fold greater affinity for bacterial DHFR than human DHFR[53],
making this an excellent therapeutic target.
Trimethoprim acts in the cytosol to inhibit reduction of dihydropteroic acid by dihydrofolic
acid reductase. This prevents production of tetrahydrofolic acid as (shown in Figure 7) which
is an essential precursor to thymidines, purines and methionine. This prevents DNA and
RNA synthesis which halts the replication of bacteria, starves bacterial cells of thymidine
triphosphate, leading to thymineless death [54].

Figure 7: Trimethoprim inhibits dihydrofolate reductase (DHFR) to block tetrahydrofolic acid
synthesis.
The inhibition of DHFR may be circumvented by bacteria if they are able to acquired
thymidine by other means. Bacterial infection sometimes causes the release of polymerised
DNA from infected tissues; this polymerised DNA may be processed by thermonuclease
enzymes [55], to release thymidine from the DNA, which would allow bacteria to continue
DNA replication despite the blockade of folate synthesis.
20

There are other resistance mechanisms such as membrane impermeability and specific
efflux pumps, natural and regulational changes resulting in unsusceptible DHFR, as well as
acquired mutational and recombinant changes in target enzymes. [53]
Poration of the cytoplasmic membrane will allow trimethoprim to enter the cell more easily,
thus diminishing DNA/RNA synthesis and may prevent transcription of genes responsible for
upregulation of proteins involved in cell wall synthesis. In cells where nisin has not porated
the cytoplasmic membrane, there could still be a synergistic effect when nisin and
trimethoprim are combined.
Polymixins
Colistin is a membrane destabilising polymixin which behaves similarly to detergents due to
its hydrophilic and lipophilic moieties. By displacing ions of same charge, in particular the
divalent cations Ca
2+
and Mg
2+
, the poly-cationic regions of colistin disrupt the bacterial outer
membrane, whilst hydrophilic and lipophilic regions engage with the cytoplasmic membrane,
effectively solubilising the membrane in an aqueous environment. Cytosolic molecules are
then free to leak from the cell, the proton EMF is diminished, and ionic balance disrupted.
[56] Gram positive bacteria are resistant to polymixins as the cell wall blocks access to the
inner membrane.[57]
Nisin may allow colistin to access the cytoplasmic membrane by destabilising the
peptidoglycan layer, thus colistin could act on the membrane with nisin to cause multiple
sites of disruption, causing multifaceted leakage of intracellular components and cell death.
If this hypothesis is correct, nisin and colistin may potentiate each other and demonstrate
significant synergistic inhibition.
Hypotheses of interactions between nisin and antibiotics
The uptake of antibiotics by bacterial cells is often based on theory and not well defined, so it
is difficult to predict the exact interactions of an agent like nisin with antibiotics. Combining
nisin with first-line antibiotics may exaggerate their efficacy and produce a synergistic effect
21

greater than the sum of effects of both agents. There may be no synergy, but still a greater
effect than either antimicrobial alone i.e. an additive effect; or nisin may reduce the effect of
antibiotics, such as in the prediction of tetracycline i.e. an antagonistic effect. Table 3
summarises the predictions based on current knowledge of antibiotic uptake and mechanism
of action.
Antibiotic Expectation
GENTAMICIN Synergy
VANCOMYCIN Synergy
TETRACYCLINE Antagonism
ERYTHROMYCIN Synergy
AMPICILLIN Synergy
COLISTIN Synergy
TRIMETHOPRIM Additive effect
Table 3: Predicted interactions of antibiotics with nisin.
In order to investigate the interactions of nisin with first-line antibiotics, nisin must be purified
and tested for activity, as commercial nisin preparations typically contain only 2-2.5%
nisin.[25]

22

Methods
Nisin Purification
Extraction of Nisin from Nisaplin (Dannisco Dupont, UK)
1 litre of 10mM HCl was made from 37% HCl (Sigma Aldrich, UK) and Milli-Q water
(Millipore, UK). 4g Nisaplin was equilibrated to room temperature then suspended in 400ml
10mM HCl (Sigma Aldrich, UK) and mixed by swirling. 0.4M (52.852g) ammonium sulphate
crystals slowly added to the HCl-nisin suspension and mixed for 30mins at 300rpm using a
magnetic flea. This suspension was centrifuged at 10,000 RPM for 20minutes to pellet
precipitate impurities. The addition of ammonium sulphate raises the salt concentration of
the solution increasing the propensity of nisin to bind the Toyobutyl column through
hydrophilic interactions. The solution was adjusted to pH4 by addition of NaOH using pH
electrode then left at 4C overnight to allow nisin to precipitate.
Extracting nisin fractions with Toyobutyl column
400ml nisin loading fraction was run through Toyobutyl-650M column (Toyopearl, UK) at
2ml/min. Five column volumes of ammonium sulphate washed through (column volume is
20ml, but 120ml run through to ensure proper washing), followed by two 50ml washes of
deionised water until absorbance at 220nm <0.5. Nisin precipitate was run through the
column and 10ml of elution collected at peaks of absorbance at 254nm (an effective way of
identifying the presence of dissolved organics). 10ml nisin extract was eluted from the
Toyobutyl column with HCl wash and stored at 4C for later use in high pressure liquid
chromatography (HPLC).
Reverse phase high pressure liquid chromatography of 10ml HCl wash using
C18 column
0.1% (v/v) TFA (Sigma Aldrich, UK) in Milli-Q water and 0.1% (v/v) TFA in acetonitrile
(Fisher Scientific, UK) were used as solvents to elute over a linear gradient from 5% to 100%
23

over 40 minutes. The HPLC apparatus was switched on for 30 minutes to allow the lamp to
warm up for absorbance readings at 220nm, and the solvents inducted into the apparatus for
10 minutes to prime the tubes. A blank run was carried out first (deionised water in the
siphon) to remove air from the system and ensure proper flow through the tubes. 2ml
aliquots of the HCl wash were loaded into the siphon for actual runs, and fractions labelled
F1 were eluted when absorbance peaked at 25minutes F1 is expected to contain the full
nisin peptide (34 residues). F1 was analysed mass spectrometry to confirm the identity of
the organic solutes, and purity of the elutions. F1 was placed in a spherical flask for rotary
evaporation, via partial submersion in 40C water bath for 8 hours, to remove the acetonitrile
before the freeze drying process. F1 was freeze dried overnight yielding 8.26mg of pure
nisin.
MALDI-TOF-Mass Spectrometry using the LaserToF
Reversed-phase purified peptides or solutions of synthetic peptides in 0.1% (v/v)
trifluoroacetic acid (TFA) were co-spotted onto stainless steel targets with a saturated
solution of the matrix -cyano-4-hydroxy-cinnamic acid (-CHC). Spectra were acquired and
summed in positive ion reflectron modes over the required m/z range using a LaserToF-TT
(SAI Ltd), with MALDIMainFrame Vn 1.04.21 software, and with the analyser calibrated
against protonated molecular ions of synthetic peptides. Spectra were manually scrutinised
and sample ion profiles compared with negative control sample data.
Testing the activity of nisin
Nisin was diluted to 0.2mg/ml solution in sterilised water for sensitivity testing on
Lactococcus lactis MG1614 as it is sensitive to most antibiotics (MG 1614 cultured in M17
broth, from University Of Nottingham Culture Collection). M17 agar was prepared according
to manufacturers instructions. 10ml 10% (v/v) lactose solution added to 50C molten agar
through a sterile 2m macrofilter on a pipette; 2ml Bromocresol purple (Sigma Aldrich, UK)
was also filtered through a sterile 2m macrofilter and then added to the molten agar.
24

Bromocresol purple turns yellow in the presence of lactic acid, indicating L. lactis is thriving
and metabolising lactose.
20ml plates were poured on a level surface in a sterile air hood. Nisin was diluted over a
range from 0.2mg/ml to 5g/ml (Table 7), and a control solution of deionised water was
included. 8 wells were punched in the agar using sawn-off pipette tips and 10l of each nisin
solution deposited into each well; the plates were then left overnight at 4C to allow nisin to
diffuse properly, and then inoculated with 50l L. lactis MG 1614, using a sterile glass
spreader.
These plates were left to incubate overnight in a 5% CO
2
incubator at 37C, and removed
the next day to measure inhibition zones according to BSAC Susceptibility Testing Version
11, to confirm activity of nisin.
Establishing Minimum Inhibitory Concentrations
Accurate MICs needed to be established for each antibiotic with respect to MRSA 252 and
NCTC 6571, to incorporate antibiotic into ISA at MIC. Once MIC agar was produced,
nisin could be added to wells in the agar at various concentrations (2mg/ml to 0.05mg/ml),
then the plates inoculated with MRSA 252 or NCTC 6571, and inhibition zones measured
around wells. Zone diameters were input at agardiffusion.com to determine the MIC of nisin
in the presence of sub-lethal antibiotic, compared to the MIC of nisin in ISA. All plates were
repeated three times.
Sensitivity testing for NCTC 6571 and MRSA 252
Preliminary tests were carried out to establish the sensitivity of the S. aureus strains. Stock
MRSA 252 and NCTC 6571 from University of Nottingham Culture Collection were grown on
horse blood agar and kept at 4C. These stocks were subcultured overnight in Iso-sensitest
broth (ISB, made as per manufacturers instructions) in a 37C orbital incubator each time
inoculum was required. Inocula were diluted to an optical density of 0.5 each time, using
25

reading from a spectrophotometer ((Amersham Biosciences GeneQuant Pro) (Macfarlane
standard, BSAC Susceptibility Testing Version 11, 3.1.4)
Stock solutions of antibiotics were made up at concentrations shown in Table 4, diluting with
sterile water and ethanol as appropriate.
Antibiotic
Stock
Concentration
(mg/ml)
GENTAMICIN 10
VANCOMYCIN 5
TETRACYCLINE 5
ERYTHROMYCIN 10
AMPICILLIN 5
COLISTIN 2
TRIMETHOPRIM 5
Table 4: Initial stock and dilutions used for sensitivity testing: suggested stock
concentrations were taken from www.openwetware.org, as well as preparation protocol.
Iso-Sensitest agar (ISA) was made as per manufacturers instructions (Oxoid, Hampshire,
UK) and autoclaved at 121C for 15minutes.
20ml ISA plates were poured on a level surface with an electronic pipette (Pipetboy, Integra
Biosciences, Switzerland), and 6 wells punched in each plate with a sawn-off pipet tip
(1.5cm removed from end of tip).
10l nisin was pipetted into wells at 100%, 50%, 20%, 10%, 5% and 2.5% of stock
concentration (2mg/ml), and left to diffuse overnight at 4C. The following day, the remaining
antibiotics were pipetted into wells at respective concentrations, and then plates inoculated
with 50l MRSA 252 or NCTC 6571 subculture. These plates were then put in a 5% CO
2

incubator at 37C and left overnight. The following day zone diameters were measured to
determine sensitivity of strains to antibiotics, and establish an approximate MIC for each
antibiotic.
26

Calculating MICs of the antibiotics to make ISA at MIC concentrations
MICs were calculated at www.agardiffusion.com which uses a dissipative diffusion model
[58] (DDM). The DDM plots the squared radius of inhibition zones (y-axis) against natural
logarithm of antibiotic concentration (x-axis) which produces a straight line through points
leaving an x-axis intercept log(c) which is the natural log of the minimum inhibitory
concentration. The model is integrated to which produces the graph and the MICs calculated
by linear diffusion model (LDM) and DDM methods.
ISA plates were made, and 6 wells were punched in each plate and 10l of each antibiotic
was pipetted into each well, from 100% stock concentration to a 2.5% dilution. Plates were
inoculated with 50l MRSA 252 and NCTC 6571 subcultures then left overnight in a 5% CO
2

incubator at 37C. The following day zone sizes were measured to calculate antibiotic (and
nisin as control) MICs using DDM.
Antibiotics were added to 50ml of molten ISA in a falcon tube at half MIC concentrations
calculated with DDM, to make ISA plates with MIC antibiotic incorporated into the agar. 6
wells were punched in each plate and 10l of each antibiotic was pipetted into each well,
from 100% stock concentration to a 2.5% dilution. Plates were inoculated with 50l MRSA
252 and NCTC 6571 subcultures then left overnight in a 5% CO2 incubator at 37C. The
following day zone sizes were measured to calculate the MIC of nisin in the presence of
each antibiotic, and alone, to determine interactions.
Combining nisin and antibiotic in the same well to observe interactions
determining change in zone size
Using MICs calculated to produce MIC antibiotic-incorporated agar inhibited growth on
plates for most of the antibiotics, resulting in no inhibition zones present to determine an MIC
for nisin in the presence of antibiotics.
An alternative approach to defining interactions between antibiotics and nisin was required;
nisin and antibiotics were pipetted into the same wells, and then directly compared to wells
27

containing antibiotic alone. Plates were also made with wells containing nisin alone as a
control, but only wells containing nisin and antibiotic (combination treatment) and antibiotic
alone were statistically compared.
Antibiotic
NCTC
6571
MRSA
GENTAMICIN 0.5 0.5
VANCOMYCIN 0.25 0.25
TETRACYCLINE 0.0125 0.0125
ERYTHROMYCIN 0.5 -
AMPICILLIN 0.3125 12.5
COLISTIN - -
TRIMETHOPRIM 1 1
NISIN 1 1
Table 6: Concentrations of antibiotics used in wells (mg/ml).
Concentrations which produced inhibition zones of diameter 150.5mm shown in Table 7,
were used were used to produce consistent inhibition zones which could be easily measured
for variance in diameter should nisin have an additive, synergistic or antagonistic effect.
Colistin produced no zones as both strains are unsusceptible, and erythromycin produced no
zones in MRSA 252 as it is resistant.
ISA plates were made, and nisin was added to three of the wells at 10l each, as indicated
in Figure 7. The plates were left to diffuse overnight at 4C, and the following day 10l
antibiotic added at concentrations indicated in Table 6, to all wells. Plates were then
inoculated with 50l NCTC 6571 or MRSA 252 subcultures. Plates were then left overnight
in a 5% CO
2
incubator at 37C, and zone sizes measured the next day.
Shapiro-Wilk tests were used to compare the antibiotic zone sizes with the antibiotic and
nisin zone sizes, which indicated the data was non-parametric. Mann Whitney tests were
used to compare the two data sets to evaluate any statistically significant difference between
the two sets, CI=95%, N=12.
28

Figure 7: Arrangement of wells on combination plates.

29

Results
The nisin fraction in RPHPLC was analysed for purity via MALDI-TOF mass spectroscopy.
As the molecular weight of nisin is 3354g/mol a large peak expected at 3354M/Z (mass:
charge ratio). Figure 8 shows the large peak at 3354 with small peaks either side, which are
likely to be truncated or protonated nisin molecules. The dominance of the large peak
indicates a high percentage of pure nisin in the RPHPLC sample.

Figure 8: Mass spectrum analysis of nisin elution obtained by HPLC. Y-axis is intensity; X-
axis is atomic mass.

30

Testing the activity of nisin
After the purity of the sample was confirmed, it was freeze dried to leave 8.16mg 0.2%
yield from nisaplin. The activity of nisin was tested on L. lactis MG1614 producing inhibition
zones shown in Table 7, which were used to calculate an MIC of 2.7g/ml.
Nisin
Concentration
(g/ml)
Plate 1
zone
diameters
(mm)
Plate 2
zone
diameters
(mm)
100 17.5 17
50 15 15
40 12.5 13
30 11 11
20 8.5 8.75
10 6 8
5 0 0
0 0 0
Table 7: Nisin inhibition zone diameters on MG1614 plates

31

Calculating MICs for antibiotics
After the activity of nisin was confirmed, the sensitivity of MRSA 252 and NCTC 6571 to the
antibiotics was tested, and MICs calculated with the diffusion model; these are compared to
literary MICs (Table 8). The MIC for colistin in both strains was not determined as it
produced no measurable inhibition around wells at the concentrations used, so stock
solution of colistin was used (0.2mg/ml). The MIC of erythromycin for MRSA 252 could not
be calculated as MRSA 252 is resistant to erythromycin so the MIC of erythromycin for
NCTC 6571 was used for MRSA 252.
Antibiotic
NCTC 6571 MIC
from
literature(mg/ml)
NCTC 6571
MIC from
DDM
(mg/ml)
MRSA 252
MIC from
literature
(mg/ml)
MRSA 252
MIC from
DDM (mg/ml)
GENTAMICIN 0.001[59] 0.0155 0.0026[60] 0.0125
VANCOMYCIN 0.002[61] 0.0115 0.004[50] 0.0115
TETRACYCLINE 0.25[62] 0.004 0.256[63] 0.0235
ERYTHROMYCIN 0.5[64] 0.0905 0.5 ND
AMPICILLIN 0.03[65] 0.1 0.128[9] 0.1645
COLISTIN 0.016 ND 0.025 ND
TRIMETHOPRIM 0.5[66] 0.0885 0.5[67] 0.083
Table 8: Half MICs for MRSA 252 and NCTC 6571 calculated with diffusion model,
compared to values cited from literature.

32

The model used at agardiffusion.com produced MICs for each antibiotic, and an R
2
value
stating goodness-of-fit of the regression line plotted. An R
2
value tending towards unity
indicates a better fit. Figure 9 shows the regression plot for erythromycin, where the x-axis
intercept is the natural log of the MIC. The linear diffusion model values are also shown, but
the R
2
value indicates the DDM gives a better fit and is therefore more accurate MIC. Figure
10 shows decreasing zone diameter with decreasing antibiotic concentration which were
both input at agardiffusion.com.

Figure 9: Square zone diameter VS Natural log antibiotic concentration graph, and MIC
values for gentamicin on MRSA 252
33

Figure 10: ISA plate testing the sensitivity of MRSA 252 to trimethoprim; concentrations
ranging from 5mg/ml at the top well decreasing to 0.025mg/ml in a counter-clockwise order.
Once MICs had been calculated for each antibiotic, antibiotic-incorporated agar plates
were made, and inhibition zones measured around wells of nisin to determine changes in the
MIC of nisin.
All plates inoculated with MRSA 252 inhibited bacterial growth, except those containing
colistin and erythromycin, to which MRSA 252 is resistant. The MIC for nisin in the presence
of colistin (Table 9) was not determined as there was only one inhibition zone, and the
regression model requires at least two points. Erythromycin decreased the MIC of nisin in
MRSA 252 from 0.715mg/ml to 0.376mg/ml.
All plates inoculated with NCTC 6571 inhibited bacterial growth, except those containing
colistin, so inhibition zones could not be measured around nisin wells to produce an MIC for
nisin. Colistin decreased the MIC of nisin from 0.336mg/ml to 0.145mg/ml. Table 9 also
shows the MIC of nisin is substantially higher for MRSA 252 than NCTC 6571.

34

Antibiotic
Nisin MIC for
MRSA 252
(mg/ml)
Nisin MIC for
NCTC 6571
(mg/ml)
ERYTHROMYCIN 0.376 ND
COLISTIN ND 0.145
NISIN 0.715 0.336
Table 9: Inhibition zone diameters around wells of nisin (2mg/ml to 0.5mg/ml) in antibiotic-
incorporated ISA; agar antibiotic concentrations shown in Table 8; ND = not determined.
Using this method of antibiotic agar-incorporation to calculate nisin MICs in the presence of
antibiotics only allowed the comparison of nisin MICs for erythromycin and colistin. To
evaluate interactions between nisin and the other antibiotics chosen, it was decided to put
antibiotics in the same well as nisin, and compare zone diameters to those of wells
containing only antibiotic.
Evaluating interactions of nisin and antibiotics combined in the same well
For MRSA 252 Figure 11 shows increase in zone diameter for vancomycin, colistin and
trimethoprim with the addition of nisin. The increased zone sizes for trimethoprim and colistin
are easily visible in Figure 13 and 14, respectively. Adding nisin to tetracycline was shown to
decrease zone diameter. All of these results were confirmed to be statistically significant by
Mann Whitney tests (Table 10). Gentamicin and ampicillin also show a modest increase in
zone diameter, but overlapping error bars and Mann Whitney tests indicate this increase is
not statistically significant. Erythromycin did show increased zone diameter when nisin was
added, but had zone diameters of zero on its own (as MRSA 252 is resistant). Therefore the
significance values for erythromycin in MRSA 252 in table 11, compared erythromycin with
nisin to nisin alone, and show that nisin zone diameters were not significantly increased with
the addition of MRSA 252.
For NCTC 6571, all antibiotics increased zone diameters, and again tetracycline which was
clearly antagonised by nisin as shown by decreased zone diameters. Mann Whitney test
results shown in Table 10 which indicate all antibiotics exhibit significant changes in zone
35

diameters, and error bars (Figure 12) do not overlap, indicating firm changes in zone
diameters.

Figure 11: Mean zone diameters of zones with antibiotic (white) and antibiotic with nisin
(grey) for MRSA 252, with error bars indicating 2 Standard Errors

36

.
Figure 12: Mean zone diameters of zones with antibiotic (white) and antibiotic with nisin
(grey) for NCTC 6571, with error bars indicating 2 Standard Errors
37

Figure 13: ISA plate inoculated with MRSA 252; trimethoprim alone and in combination with
nisin; N indicates combination of nisin and antibiotic in the well; unmarked wells contain
antibiotic only.

Figure 14: ISA plate inoculated with MRSA 252; colistin alone and in combination with nisin;
N indicates combination of nisin and antibiotic in the well; unmarked wells contain antibiotic
only.

38

NCTC 6571 VALUES MRSA 252 VALUES
Antibiotic
Z-
value
P-
value
Significance Z-value P-value Significance
Gentamicin -2.918 0.004 Yes -1.260 0.208 No
Vancomycin -2.287 0.005 Yes -2.923 0.003 Yes
Tetracycline -2.908 0.004 Yes -2.961 0.003 Yes
Erythromycin -2.966 0.003 Yes -0.848 0.396 No
Ampicillin -2.929 0.003 Yes -1.642 0.101 No
Colistin -2.908 0.004 Yes -2.918 0.004 Yes
Trimethoprim -2.923 0.003 Yes -2.913 0.004 Yes
Table 10: MRSA 252 and NCTC 6571 Mann Whitney test values for the differences in mean
zone diameters for antibiotic only and antibiotic with nisin zones N=12, CI=95%
Low P-values in Table 10 indicate a high level of statistical significance. As CI=95%, the two-
tailed p-value must be below 0.025 to indicate significance. Z- Values of larger magnitude
indicate strong significance.

NCTC
6571
MRSA252

Antibiotic
Antibiotic
alone
Combination
Antibiotic
alone
Combination
GENTAMYCIN 13.75 15.67 13.92 14.33
VANCOMYCIN 14.08 15.92 15.50 18.42
TETRACYCLINE 15.75 9.50 15.42 10.00
ERYTHROMYCIN 10.42 12.17 0.00 7.50
AMPICILLIN 18.25 20.92 10.92 11.75
COLISTIN 3.50 13.83 3.67 12.17
TRIMETHOPRIM 21.33 26.75 21.67 27.67
NISIN 7.79 - 7.78 -
Table 11: Average zones diameters for antibiotic alone, and in combination with nisin (mm),
and for nisin alone.

39

Discussion
Nisin activity and efficacy
8.16mg of nisin was purified from 4g nisaplin, giving a 0.204% yield. Antimicrobial activity of
this nisin was confirmed on L. lactis, where it demonstrated good inhibition of growth down to
a minimum concentration of 10g/ml. An MIC of 2.7g/ml was calculated with the dissipative
diffusion model, but in the sensitivity assay, zero inhibition was observed at 5g/ml. The data
used to calculate the MIC was suitable for the diffusion model, demonstrating the difficulty of
applying theoretical MICs calculated via agar diffusion to real agar plates. Nisin will only form
pores at high concentrations, so a sudden drop in activity at concentrations higher than the
MIC may be observed, depending on inoculum size.[34]
The MIC of nisin for MRSA 252 was markedly higher than NCTC 6571; 0.715mg/ml
compared to 0.336mg/ml. It is difficult to explain this increase as MRSA 252 is not thought to
have modified Lipid II so binding of nisin to undecaprenyl pyrophosphate should theoretically
have been indifferent between strains.
MRSA 252 and NCTC 6571 seemed equally sensitive to most antibiotics. MRSA 252 was
not sensitive to erythromycin due to the presence of 23S rRNA methylase, and less sensitive
to ampicillin than NCTC 6571 due to it producing -lactamase. MRSA 252 was reported to
have a tetracycline efflux pump, but there was no statistical difference in sensitivity of NCTC
6571 (p=0.282).
The presence of erythromycin reduced the nisin MIC for MRSA 252 from 0.721mg/ml to
0.375mg/ml, suggesting synergy between nisin and erythromycin. This is unlikely as MRSA
252 is resistant to erythromycin through modification of erythromycins target in the ribosome,
which should theoretically prevent any interaction between erythromycin and the ribosome.
The lack of growth on plates with other antibiotics indicates the concentration of the antibiotic
in agar was above MIC, killing the bacteria.
40

The presence of colistin reduced the MIC of nisin for NCTC 6571 from 0.336mg/ml MIC to
0.145mg/ml in the presence of colistin, suggesting nisin facilitates colistin as predicted. Nisin
may enable colistin to access and destabilise the cytoplasmic membrane, possibly through
the inconsistency in the peptidoglycan layer conferred by nisin.
The combination of nisin and antibiotics in wells increased zone diameters in both NCTC
6571 and MRSA 252 for vancomycin, colistin, and trimethoprim. Nisin was also shown to
facilitate colistin in the diffusion assays, as predicted.
Vancomycin was predicted to have modest synergy with nisin as they both bind lipid II on
different targets, reducing the availability of lipid II for pore formation with nisin. Modest
synergy was suggested by small increase in zone diameter seen when nisin was added to
vancomycin.
The synergy observed with trimethoprim and nisin may be due to nisin pores facilitating the
entry of trimethoprim into the cell.
Tetracycline was shown to be antagonised by nisin in both strains. This was expected as
nisin pores can deplete the electromotive force, and membrane potential, which tetracycline
depends on to enter the cell and inhibit protein synthesis.
Limitations of methodological approach and confounders
When preparing inocula to spread on plates, colonies from a single plate of either MRSA 252
or NCTC 6571 were subcultured in ISB overnight. Since the bacterial assays were carried
out over a protracted period (six weeks), it is possible the plates become contaminated with
other bacteria, or the bacteria may have settled on the plates and become less able to
thrive[8]. This may have led to variation in MICs between methods.
The stock solutions of antibiotics also were also made fresh when required. Old stock
solutions may have become contaminated, or the antibiotics may have degraded in solution
over time, explaining why fresh stocks are more efficacious.
41

The dissipative diffusion model relies on plotting a straight line through points generating an
x-axis intercept, which includes outlying values which can change the intercept and distort
the MIC. It would be prudent to gather as many data as possible to offset these effects, and
exclude outlying values stringently. Data collection in this project was highly retentive and no
attempt was made to exclude outlying values.
When comparing sensitivity of bacteria to various antibiotics, diffusion dynamics through the
solid agar must be considered with caution, as high MICs may cause deceptively large
zones of inhibition zones in antibiotics which diffuse fast. [58]
Suggestions for future research
ISA plates were made by adding antibiotic to the molten agar, and not nisin, because of the
constrained availability of nisin; it would be useful to incorporate nisin into the agar at MIC,
and add antibiotics to wells to evaluate the potentiation of antibiotics by sub-MIC nisin.
The most significant synergy was observed with nisin and colistin. Colistin is less used
because of toxicity issues caused by the high therapeutic dose[56] , so it would be relevant
to investigate if nisin can potentiate colistin to lower the therapeutic dose to a non-toxic level.
Colistin and nisin could be investigated as a facilitator combination to evaluate if they could
act synergistically to increase the efficacy of other antibiotics against gram-negative bacteria,
as colistin solubilises the outer membrane, allowing nisin to porate the cytoplasmic
membrane and form pores to facilitate entry of antibiotics into the cell. This needs further
investigation to develop an approach to using this combination clinically.
When eluting nisin fractions in RPHPLC, a second, smaller peak was observed immediately
after the nisin peak, which is believed to be a truncated 31-residue nisin mutant. This could
be purified and tested for activity, as current data on nisin mutants suggests modifying native
nisin can increase its efficacy [68].
42

Concluding statements
The aim of the project was to determine if nisin and first line-antibiotics interact to produce a
synergistic or antagonist effect, or do not interact at all and produce an additive effect. It was
not possible to determine the MIC for nisin in the presence of most as the antibiotic-
incorporated agar was at concentrations too high to allow bacterial grow zones to form, no
zones were measured. It was then decided to analyse zone diameters around wells
containing both antibiotic and nisin, and it was determined by statistical analysis that
vancomycin, trimethoprim and colistin are all potentiated by nisin, and tetracycline was
antagonised.
Acknowledgements
I would like to extend personal thanks to Boyan Bonev and Alan Cockayne for providing help
and support throughout the project. I would also like to thank to David Griffin for his excellent
technical support.
43

Glossary of terms
PBP Penicillin binding protein
MIC Minimum inhibitory concentration
ISA Iso-Sensitest agar
ISB Iso-Sensitest broth
LDM Linear diffusion model
DDM Dissipative diffusion model
OD Optical density
NAM N-acetylmuramic acid
NAG N-acetylglucosamine
RPHPLC - Reverse phase high pressure lipid chromatography
MALDI-TOF Matrix assisted laser desorption/ionisation time of flight (spectroscopy)
44

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