defining the differences between hospital and community...
TRANSCRIPT
Defining the differences between hospital
and community-associated MRSA with
macrophage interactions.
Research Project
Master in Biomedical Sciences
Marco Giulio Loreti
S2934930
Supervisor: Prof. Dr. Jan Maarten van Dijl
Molecular Bacteriology – Department of Medical Microbiology
1
Contents
Abstract.................................................................................................................................................4
1 Introduction.......................................................................................................................................5
1.1 Diseases Epidemiology............................................................................................................ .5
Bacteremia................................................................................................................................................................5
Infective Endocarditis...............................................................................................................................................6
Skin and Soft Tissue Infections................................................................................................................................6
Pleuropulmonary Infections......................................................................................................................................6
Osteoarticular Infections...........................................................................................................................................6
Cardiovascular Device Infections.............................................................................................................................6
1.2 History of Resistance.............................................................................................................. .6
1.3 The USA300 ........................................................................................................................... .8
1.4 Ability to Survive Inside Cells................................................................................................. .9
1.5 NF-κB and Macrophages........................................................................................................ .9
1.6 Hydrogen Peroxide Resistance................................................................................................ .9
1.7 Aims of the study.................................................................................................................... 11
2 Material and Methods..............................................................................................................................................12
2.1 Bacterial Strains and Culture Conditions................................................................................ 12
2.2 Cell Culture and Growth Conditions...................................................................................... 13
2.3 MRSA Survival Assay................................................................................................................................... 13
MRSA Survival Assay – CFU Counting...............................................................................................................13
MRSA Survival Assay – FITC Staining ..............................................................................................................14
2.4 SDS-Page............................................................................................................................... 14
2.5 Western Blot Analysis .................................................................................................................................. 15
Procedure................................................................................................................................................................15
2.6 Protein Quantification............................................................................................................. 16
2.7 Relative Protein Expression ................................................................................................... 16
2.8 Hydrogen Peroxide Survival Assay....................................................................................... 16
Procedure ...............................................................................................................................................................17
3 Results.............................................................................................................................................18
3.1 Growth Curve of Isolates........................................................................................................ 18
3.2 MRSA Survival Assay – CFU Counting................................................................................ 18
3.3 MRSA Survival Assay – Fluorescein /FITC Staining .......................................................... 21
3.4 Comparison of NF-κB Subunit p65 ...................................................................................... 22
3.5 Hydrogen Peroxide Survival Assay........................................................................................ 25
4 Discussion....................................................................................................................................................................26
2
5 Bibliography....................................................................................................................................30
6 Supplementary.................................................................................................................................35
3
Abstract
S. aureus has always been a dangerous pathogen in human history. For example, before the discovery of
antibiotics, the mortality rate of this bacterium was higher than 80% when it was present in the bloodstream.
Even after the widespread introduction of penicillin and other antibiotics, S. aureus has retained a prominent
role in human diseases by developing resistance. The USA300 lineage is one of the rapidly spreading
meticillin resistant (MRSA) lineages that cause both community associated (CA) and hospital associated
(HA) diseases. Moreover, in relatively recent years, the rise of community-associated MRSA (CA-MRSA)
have infected many outside the hospital settings.
The goal of this study was to explore differences in the behavior of the classic hospital-associated MRSA
(HA-MRSA) with this new group (CA-MRSA) in features of survival inside white blood cells, effects on the
NF-κB pathway and resistance to hydrogen peroxide.
Six HA-MRSAs and six CA-MRSAs strains isolated from Denmark patients were employed in our study.
Infection assays of both groups inside THP-1 derived macrophages were first optimized regarding
multiplicity of infection and time of incubation. In addition, fluorescence microscopy confirmed the presence
of the bacteria inside the macrophages.
Blood agar plating and flow cytometry were both tested to verify which system was more reliable to check
the survivability of the bacteria inside macrophages. Flow cytometry was considered more promising and
showed a trend of higher internal survivability for the community-associated strains.
Interestingly, western blot analysis of the total p65 protein, one of the five members of NF-κB transcription
factor, revealed a statistically significantly higher level among the HA isolates than the CA isolates. In mice
NF-κB pathway was shown to be involved in phagocytosis of S. aureus by macrophages.
Subsequently, the effect of different concentrations of hydrogen peroxide was studied for the two groups.
HA-MRSA isolates exhibited a stronger resistance. This result concurs with a previous transcriptome
analysis of stress response genes which yielded a higher expression for hospital-associated.
Our work contributes to the studies that clarify the differences of such groups and will provide a better
understanding of the topic, in order to improve diagnosis and treatment of the associated infections.
4
1 Introduction
Staphylococcus aureus is a Gram-positive, coagulase-positive, motile, facultative anaerobic bacterium that
belongs to the genus Staphylococcus of the Staphylococcaceae family. Alexander Ogston, in 1880, described
S. aureus as a grape-like cluster of circular bodies [1]. In addition, Friedrich Julius Rosenbach observed a
peculiar golden color pigmentation in S. aureus strains, giving it the name “aureus”.[2]Staphylococci lack an
outer membrane as additional protection layer from potential harms. [3]
These bacteria can be coagulase positive or negative, meaning that they can or cannot have the power to clot
blood by converting fibrinogen to fibrin.
Traditionally, it was thought that the coagulase test was a clear way to distinguish S. aureus from the other
more abundant and less virulent coaugulase negative Staphylococci (CNS), but it is now clear that some
strains of S. aureus lack the ability to clot[4]. The test still retains useful screening capability since it is often
the case for S. aureus to be coagulase positive.
Staphylococci and S. aureus are found in a conspicuous number of mammals including human [5-9] and in
chickens and other birds as well [10]. It is not uncommon for these organisms to travel from one host of one
species to another of a different one. Both the zoonosis (from animal to humans) and the reverse zoonosis
(from humans to animals) are well-known in literature [5-9].
.
In humans S. aureus is resident on the skin and mucosa of many healthy individuals. It is estimated, by
several longitudinal surveys, that approximately 20% of individuals are persistent S. aureus nasal carriers,
around 30% are intermittent carriers and, roughly 50% are non-carriers [11-14].
1.1 Diseases Epidemiology
Unfortunately, this bacterium is not only very common in the population but it is also highly pathogenic and
responsible for many diseases and conditions both among humans, and livestock. Bacteremia, infective
endocarditis as well as skin and soft tissue, pleuropulmonary, osteoarticular, implants-related infections and,
rarely, meningitis are few of the many diseases caused by S. aureus [15, 16].
Bacteremia
The incidence of S. aureus bacteremia (SAB) in the developed countries ranges from 10 to 30 per 100.000
person per year[17].
SAB is extensively correlated with age, showing a high incidence in the first year which decreases during
adulthood and gradually rises again in the advancing ages. In particular, the incidence of SAB is more than
100 in 100.000 individual per year among subjects 70+ years of age [17] but is only 4,7 in 100.000 younger
persons per year among U.S. military employees [18]. Severe SAB sometimes lead to conditions such as
osteomyelitis, infective endocarditis, and pneumonia.
5
Infective Endocarditis In the industrialized world S. aureus has become the major cause of infective endocarditis according to the
International Collaboration on Endocarditis Prospective-Cohort Study from 1999 to 2003, with more than
25% of cases, especially in patients health-cared exposed. [19, 20]
Skin and Soft Tissue Infections For many years, S. aureus has been the leading cause of several skin and soft tissues infections (SSTIs) like
impetigo, boils (which are not very dangerous for health), carbuncle, cellulitis, and Staphylococcal scalded
skin syndrome which can become life threatening [21-23].
Pleuropulmonary Infections
Many pleuropulmonary infections are caused by S. aureus and, consequently, many pneumonia cases. Its
involvement in community-acquired pneumonia has been attested since the first half of the 19 th century
[24,25]. Moreover, during the 1918 pandemic, caused by H1N1 Influenza A virus, S. aureus was responsible
for dangerous respiratory complications [26]. Nowadays in hospital related pneumonia its involvement is
predominant for each type: hospital-acquired pneumonia, ventilator-associated pneumonia, and health care-
associated pneumonia. Additionally, thanks to its debilitating condition, cystic fibrosis is often accompanied
by infections: S. aureus and H. influenzae are the most common infecting pathogens in 10 years old or
younger children with cystic fibrosis [27].
Osteoarticular Infections
In chronic osteomyelitis cases S. aureus is the most found pathogen with a percentage around 32% [28]. The
same pattern can be found in adult native septic arthritis where it is the main cause with around 44,6% of
cases [29] and in prosthetic hip and knee joint infection (53%) [30]. All these three are the major classes of
osteoarticular infections and S. aureus is the main pathogen in all of them.
Cardiovascular Device Infections
Following a cardiac surgery, a sternal wound infections (SWI) can occur with serious health risk and,
according to European and U.S. studies, S. aureus is the primary cause of SWI [31]. This has been seen for
example in a dutch study of 2003[32], while concerning the cardiac device itself, its role in infections is up to
46%[15]. Furthermore, concerning vascular catheter infections a U.S. study found [33] that S. aureus is the
most common, after coagulase-negative staphylococci.
1.2 History of Resistance
Since its high capability of causing diseases it is no surprise that a treatment to manage S. aureus infections
has always been highly desirable.
At the time of Ogston and Rosenbach discovery, and for several years later, in fact, the presence of S. aureus
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bacteremia carried a heavy prognosis, with mortality rate >80% [34]. With the discovery of Penicillin in
1928 and its usage for treating infections the survival of patients improved considerably (Figure 1A).
Penicillin-like drugs are β-lactam antibiotics that work by inhibiting the function of bacterial PBPs
(penicillin binding protein), proteins that are paramount to the creation of the bacterial wall. Normally, PBPs
catalyze the formation of cross-links in the peptidoglycan layer of the wall by removing a D-alanine residue
from N-acetylmuramic acid peptide chains. The β-lactam ring has a structural similarity to D-alanyl-D-alanine
residues and binds irreversibly to PBP (Figure 1B), stopping the creation of a stable bacterial wall and
causing the death of the microorganism.
Figure 1: (A) Chronological representation of antibiotic-resistant S. aureus and pandemic in U.S.A from the first half of
XX century to the first half of XXI century.(B) Transpeptidase reaction of PBP, and penicillin G structural similarity to
the substrate of the bacterial enzyme for the building of the bacterial wall.
After few years, though, penicillin-resistant strains of S. aureus with β-lactamase enzymes emerged [35],
forcing the development of new drugs. In 1959 meticillin was created as an antibiotic impervious to β-
lactamase but its discovery was, sadly, immediately followed by the discovery of meticillin resistant strains
(MRSA)[36]. These MRSAs use to survive PBP2a, which is encoded by the gene mecA contained in the
Staphylococcal cassette chromosome mec. PBP2a has a low affinity for the β-lactam ring making the
antibiotic useless.
Worryingly, new strains resistant to other antibiotics, like vancomycin and daptomycin, are being discovered
continuously [37]. This should not be a cause of surprise because such speed in developing resistance is
common in bacteria since they reproduce very quickly and through the push of natural selection they can
evolve rapidly. MRSA has not used this fast adaptability only to resist antibiotics, but also to expand its
7
arsenal of virulence factors. These includes genes for adherence to the host cells [38] or for fighting and
evading the immune system [39-41] etc. It was its strong ability to modify itself and evolve that set up the
rise of community-associated MRSA (CA-MRSA).
Around two decades ago, in fact, MRSA infections used to be mostly in the hospitalized,
immunocompromised patients, in the very young and very old.
During the 90's, though, a new group of S. aureus emerged, not anymore related to the previous categories
but affecting also slices of the population with a healthy immune system: community-associated MRSA.
These CA-MRSA infections tend to be more SSTIs [42] and on a molecular level they differ considerably
from the previous HA-MRSA (hospital-associated). For example, the size and types of the mobile genetic
element SCCmec are different between the two groups. HA-MRSA exhibit bigger SCCmec of type I, II or III
while CA-MRSA has smaller SCCmec usually belonging to type IV, V or VII. It has been hypothesized that
this smaller size should grant CA-MRSA a higher chance of transmitting resistance compared to HA-MRSA
[43,44]. As a matter of fact, of the eleven known SCCmec, type III is the biggest and VI is the smallest.
Nevertheless, since the higher exposure of HA-MRSA to antibiotics in hospitals, and the fact that SCCmec
can carry resistance to many antimicrobial drugs [45], it is no surprise that HA-MRSA are generally more
resistant to non β-lactam antibiotics when compared to CA-MRSA [42]. Additionally, the more common
presence of Panton-Valentine leukocidin (PVL) gene in CA-MRSA, compared to HA-MRSA, which encode
for a cytotoxin (of bacteriophage origin) that destroys leukocytes [42] is another notable difference.
As it is the higher production of Phenol-soluble modulins (PSM) toxins by CA-MRSA as well implicated in
virulence and white blood cells destruction [46] PSM is a α-helical peptide toxin and its gene can be both
present in the core genome as well in the SCCmec MGE, in combination with the meticillin resistance,
highly increasing the virulence [47].
Taken together, this data clarify that the virulence factors can dramatically change between one strain and
another and also between the two main group HA and CA; especially now that they start to infect even
outside of their normal settings, with CA strains infections in healthcare facilities.
1.3 The USA300
The USA300 lineage is one of the rapidly spreading MRSA and it was first identified in the United States of
America in 2001. The USA300 naming is based on the pulsed-field gel electrophoresis (PFGE) pattern of the
isolates. Meaning that the genomic DNA was digested by smaI restriction enzyme, and it showed a distinct
pattern on a gel. It widely spread both in the community and in hospitals all over the world in a decade [42].
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1.4 Ability to Survive Inside Cells
Contrary to what was thought, S. aureus is not only an extracellular pathogen but it is also able to colonize
and survive inside host cells which include osteoblasts, fibroblasts, endothelial and epithelial cells and even
white blood cells like macrophages, polymorphonuclear neutrophils (PMN), T- lymphocytes, etc [39-41,48-
54].
The bacteria makes possible the engulfment of itself via bacterial expression of fibronectin-binding proteins
that enables it to form a connection with the extracellular matrix (ECM) and then with the cell integrin α5β1
which also bind fibronectin. The bridge derived by these connections allows the bacteria to be internalized
[55].
Comparing the survivability of different HA and CA-MRSA strains inside epithelial cells has showed that
the CA-MRSA strains tend to better manage the intracellular milieu compared to the HA-MRSA. This
difference in ability to survive seems linked to the bacterial proteomes. [Mekonnen et al, unpublished data]
Even more surprisingly, studies show that S. aureus is able to survive in mature phagolysosomes, the
specialized organelle for microbial destruction inside phagocytes[39,53,55,56].
1.5 NF-κB and Macrophages
The nuclear factor kB (NF-κB) protein is a transcription factor found in many animals, humans included
[57]. The NF-κB heterodimer p50-p65 resides in the cytoplasm in an inactive form in complex with I kappa
B alpha (IκBα) in resting cells. However, stress stimuli such as radiations, bacterial LPS, and pathogen
infections lead to phosphorylation of IκBα by I kappa B kinase (IKK) leading to its ubiquitin-dependent
proteasomal degradation. As a consequence, NF-κB translocates to the nucleus where it activates many (pro)
inflammatory genes. Usually, macrophages recognize typical pathogen-associated molecular patterns
(PAMPS) via Toll-like receptors to activate NF-κB, together with other pathways [58,59].
A 2014 study showed how this transcription factor is activated or, alternatively, becomes more expressed by
S. aureus infection and how it is vital for its correct phagocytosis operated by mouse macrophages RAW
264.7. NF-κB inhibition clearly reduced the capability of RAW 264.7 cells to engulf the bacteria as well as
the cascade of genes in the inflammation process [60].
1.6 Hydrogen Peroxide Resistance
S. aureus is known to be catalase positive, meaning that it has the ability to convert hydrogen peroxide into
water and oxygen, preserving itself from harmful reactive oxygen damages [4]. To avoid ROS damage,
recent studies showed that catalase works together with other enzymes like superoxide dismutase (SOD) [61]
and staphyloxanthin [62] to improve survival of the bacteria. This is of particular interest because of the way
phagocytes, like neutrophils and macrophages, use ROS generated by nicotinamide adenine dinucleotide
9
phosphate (NADPH) oxidase to kill bacteria [63]. Once internalization of the pathogen in the phagosome is
complete, the latter matures into a phagolysosome with the addiction of proteases, lysozymes and also ROS
to the compartment [63]. It is therefore clear why S. aureus has been evolutionarily pushed in the production
of antioxidant enzymes and stress response genes.
It is possible to spot a difference between CA and HA-MRSA, since a higher transcription of some stress
response genes has been seen in the latter ones [Mekonnen et al., unpublished data].
Figure 2: Bar graph representing the fold of induction of mRNA transcriptome of CA-MRSA strains (blue line) and the HA-MRSAs on average. See chart for the name of stress response genes.
Table 1. Chart showing the stress response genes more transcripted on average in HA-MRSA strains.
10
Name M1 aureus label Fold of induction Genes name Product
SR1 BN843_17420 2,3 General stress proteinDUF948 domain containing
protein
SR2 BN843_16270 2,4 SigmaB-controlled gene product CsbD family protein
SR3 BN843_24220 2,4 General stress protein 26 General stress protein
SR4 BN843_8030 2,9 Organic hydroperoxide resistance proteinOrganic hydroperoxide resistance protein-like
SR5 BN843_21760 3,5Non-specific DNA-binding protein Dps / Iron-binding
ferritin-like antioxidant protein / Ferroxidase
Starvation/stationary phase protection protein
Stress response genes
Fold
of i
nd
uctio
nm
RN
A
SR-1SR-2
SR-3SR-4
SR-50
1
2
3
4
1.7 Aims of the study- Seeing how MRSA is capable of survive in many cell types, and how this can correlated with the protein
expression for the infection of epithelial cells, we wonder if the same situation also applies for white blood
cells, like for example macrophages. Finding a similar correlation could help delineate the protein expression
differences between HA and CA-MRSA infections and create the basis for future studies about pathways
related to the invasion of both the two groups in macrophages.
-A clear pathway of reaction to the infection in macrophages has been found in murine macrophages [60],
with the involvement of NF-κB for phagocytosis. Consequently, the question is if an analogous scenario is
present in human macrophages as well and if a divergence is present between HA and CA-MRSA groups.
-The innate immune system employs many strategies to resist infections by recruiting cells, activating the
complement system, engulfing pathogens, and lysing them. Since neutrophils and macrophages [63], make
use of ROS molecules to destroy internalized microorganisms and since S. aureus shows differences between
CA and HA-MRSA to resist stress, it would be interesting to perform a study to verify if a higher level of
expression of stress response genes relates indeed with a higher ability to survive ROS molecules like
hydrogen peroxide. This would allow future investigations to justify, in part, different patterns of
survivability between HA and CA-MRSA after internalization by leukocytes.
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2 Material and Methods 2.1 Bacterial Strains and Culture Conditions
We used 12 clinical USA300 isolates, of which six belong to HA, and the remaining 6 belong to the CA
group. These isolates were collected from Denmark patients. The HA-MRSA exhibits S. aureus-specific
staphylococcal protein A (spa type) t024, while CA-MRSA shows spa type of t008. The isolates were grown
overnight (O/N) in trypticase soy broth (TSB) medium at 37°C and shaking at 116 rpm in a water bath. The
following day, isolates were diluted down to an optical density (OD600) of 0,05 in Roswell Park Memorial
Institute 1640 medium (RPMI 1640 Gibco®) supplemented with 2mM L-Glutamine (PAA – GE Healthcare)
and grown with the same condition up to 0,5 OD600. The main culture was started by diluting this pre-culture
again to OD600 0,05 with the same medium and conditions, and grew until exponential phase or stationary
phase accordingly to the experiment.
Later, an optimization of the liquid growth procedure was done. The procedure consisted in combining the
pre-culture and the main culture and in using an OD600 of 0,1 as starting point and up to 90 minutes in
stationary phase.
On solid medium, the plating was performed on BA plates either by mixing a gobbet from a -80 °C glycerol
stock with 50 μL MQ water, then spreading the solution with glass beads or by spreading directly 100 μL
from the main culture.
Table 2. Characteristics of isolates used in this study.
Name of isolate
Year of isolation
Age of carrier
OriginEpidemiological behavior
spa-type
SCCmec PVL ACME
D3 2003 48 Denmark HA t024 IV - -
D15 2004 39 Denmark CA t008 IV + +
D17 2004 82 Denmark HA t024 IV - +
D22 2004 79 Denmark HA t024 IV - +
D29 2004 40 Denmark CA t008 IV + +
D30 2004 88 Denmark HA t024 IV - +
D32 2005 61 Denmark CA t008 IV + +
D37 2005 24 Denmark CA t008 IV + +
D53 2005 65 Denmark HA t024 IVa - +
D61 2005 5 Denmark CA t008 IV + +
D66 2005 61 Denmark HA t024 IVa - +
D69 2005 26 Denmark CA t008 IV + +
12
Newman / / / Lab strain / / / /
2.2 Cell Culture and Growth Conditions
Monocytic cell line THP-1 was grown and maintained in 75 cm2 flask (TPP) containing 15 mL of RPMI
1640 Gibco® supplemented with Phenol red (Promega), 2 mM L-Glutamine and 10% FBS (Silantes) and
was incubated at 37°C in 5% CO2. Once 70-80 % confluence was reached, the cells were pelleted (1000 rpm
for 5 minutes at RT), and enumerated by using hemocytometer chamber with trypan blue as exclusion
method. Later, cells were seeded in 24-wells plates or 6-wells plates with numbers from 3x10^5 to 5,2x10^6
cells/mL depending on the experiment. The THP-1 cells were differentiated to macrophages via incubation
with 5 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich) for 2 days [50] at 37°C in 5% CO2.
2.3 MRSA Survival Assay in Macrophages
Two approaches were tried to assess different patterns in the intracellular survivability of CA and HA-MRSA : CFU counting and FITC staining.
2.3.1 MRSA Survival Assay – CFU Counting
The invasion of THP-1 derived macrophages by S. aureus was performed with a methodology similar to a
previous 2011 study of S. aureus infection but with some alterations convenient to our case [65].
As first pilot, we used a multiplicity of infection (MOI) of 50 with 2 hours incubation of undisturbed
phagocytosis at 37°C followed by an addiction of gentamicin (Sigma Aldrich) at a final concentration of 400
μg /mL (total volume of 500 μL). At time point 12, 24 and 48 hours the plates were washed three times with
PBS and the cells were detached with trypsin-EDTA (100 μL) for 5 minutes. Afterwards, 200 μL of
RPMI1640/FBS10%/2mM L-glutamine was added and the cells were resuspended in 500μL saponin 1% to
lyse them. The content was plated on BA at 37°C O/N with 104, 105, 106 dilutions. The following day we
were able to count the colonies and estimate the survivability of the strains at different time periods by ratio
between the number of CFU from bacteria-only plates and bacteria from lysed macrophages.
The use of gentamicin was necessary to kill all the uningested bacteria by the THP-1 macrophages as it was
proven to be the antibiotic of choice to avoid killing intracellular bacteria [65] while still getting rid of the
extracellular ones [66]. For the good result of the experiment, some passages were seen to be instrumental,
like the washing with PBS prior the collection of cells from the plate to remove the remaining antibiotic or
before the addiction of trypsin-EDTA to avoid inhibition by FBS via α1-antitrypsin and an excess of
proteins.
Later on, several MOI were used depending on the test and 50 to 1 MOI was found to yield an optimal
invasion rate, like already seen in other studies [67,68]. Once infected, the plates were incubated at 37°C and
5% CO2 for 1 hour then the plates were added with 25, 50 or 100 μg /mL gentamicin and at different times:
30, 60, 120 and O/N the macrophages were washed with PBS, detached with 200 μL tryspin-EDTA for 10
13
minutes. This was followed by an addiction of 300 μL RPMI1640/FBS10%/2mM L-glutamine and the
addiction of 500 μL saponin 1% for 5 minutes was used to lyses the cells like before.
2.3.2 MRSA Survival Assay – FITC Staining
From the main culture, volumes of bacterial suspension (1,8 mL to 14mL) were collected and centrifuged
(5000 g for 8 minutes), washed with warm (37°C) phosphate-buffered saline (PBS) and resuspended in 1 mL
0,5M NaHCO3 buffer (pH 8) freshly prepared. From the suspension, 990 μL were added to 10 μL of
fluorescein isothiocyanate (FITC) (stock 10 mg/mL) to get a final concentration of 100 µg/mL. The
isothiocyanate group bounds the fluorescein to the amine and sulphydryl groups of the bacterial protein
allowing visualization. Subsequently, the samples were incubated for 30 minutes at room temperature in the
dark (aluminum foil around tubes) on end-over-end rotation (or shaker) before a centrifugation at 7000g for
5 minutes. Three washes were performed in 500 μL PBS to remove unbound FITC (5000 rcf/g for 2 minutes)
with a last resuspension in 1 mL PBS. When necessary, for comparison, 100 μL of this stained bacteria
suspension were plated on BA plates (37°C O/N). Prior the incubation with macrophages the OD600 of the
stained bacteria was measured (100 μL of stained bacteria + 900 μL PBS) and Mekonnen's standard was used
to estimate the number of CFUs and to adjust the volumes for MOI of 50. The incubation lasted 1 hour at 37
ᵒC, (5% CO) for phagocytosis/infection before the medium was removed, 200 μL of trypsin-EDTA (37°C for
10 minutes) were added followed by 300 μL RPMI to stop trypsin action then a centrifugation (5000 rpm for
5 minutes). The pellets were resuspended in 500 μL trypan blue 0,2% to quench signals of not internalized
bacteria followed by centrifugation (5000 rpm for 5 minutes). Final steps were three washes in 500 μL PBS
(5000 rcf/g for 2 minutes) and a resuspension in 500 μL formhaldehyde 4%. Samples were stored at 4°C and
a FACS was run the following day. Before running the flow cytometry, the pellet was resuspended by
pipetting and vortexing.
The phagocytic index was calculated as the percentage of FITC positive cells divided by 100 times the mean
fluorescence, which is actually equivalent to the formula below:
Phagocytic Index = [Percentage of macrophages containing at least one bacteria] X [Mean Bacterial count
per macrophage containing organism(s)] /100
The equivalence derives from the fact that a FITC positive cell has to contain at least one bacteria and the
mean fluorescence is to the average number of ingested bacteria [71].∝
2.4 SDS-Page
For separating proteins, PAGE technique was performed assembling pre-casted gels in the electrophoresis
apparatus and using Nupage™ MES SDS Running buffer 1X to allow current to flow. The gels employed
were NuPage™ Novex™ Bis-tris midi protein gels (20 wells) and NuPage™ Novex™ Bis-tris gel (12
wells). Samples were mixed with Nupage™ 4x LDS loading sample and Nupage™ 10x sample reducing
14
agent to reach a correct 1x working dilution. 3 μL of Precision Plus Protein™ All Blue Prestained Protein
Standards (Bio-Rad) were used for measuring the size of the proteins as ladder. The PAGE was run at 200
Volts for 45 minutes.
2.5 Western Blot Analysis
The extraction of proteins was performed from THP-1 derived macrophages cultivated in 6-wells plates after
48 hours of differentiation (see section 2.2 Cell Culture and Growth Conditions). The wells were washed
with 500 μL PBS before infection, which lasted 1 hour with a MOI of 50. Afterwards a second wash with
500 μL PBS was performed, then 400 or 500 μL NP-40 lysis buffer was added depending on the number of
cells previously counted. NP-40 lysing buffer was supplemented with a tablet of protease inhibitor (Roche
cOmplete™, Mini) every 5 mL of buffer to avoid degradation of proteins.
Lysis buffer NP-40 was prepared following the Cold Spring Harb Protocol [69]. For 100 mL necessary
volumes are stated in the below chart:
Table 3. Chart of components of NP-40 lysis buffer
Content Volume
5 M NaCl 3 mL
10% NP-40 10 mL
1 M Tris (pH 8) 5 mL
Demineralized H2O 82 mL
Procedure
Western blot was performed for proteins detection with an electroblotting device stacking nitrocellulose
membrane, soaked in buffer K, on top of Whatmann paper soaked in Methanol buffer A1 (lower part) and
A2 (upper part). The gel was soaked in buffer K, positioned on the membrane and topped with more
Whatmann paper buffer K soaked as well. The stack was positioned between two conduction plates and run
at 200mA (big gel) and 100 mA (small gel) for 75 minutes. Afterward the membrane was stained with
Ponceau S (10mL for 5 minutes shaking), washed with demiwater (5 minutes shaking) then cut along the 50
kB to separate the portion with p65 (65 kDa) from the one with β-Actin (42 kDa) or GAPDH (38kDa) and
blocked in 5% skimmed milk in TBS-T ON at 4°C to reduce the possibility of Ab binding not specifically.
The next day the membrane was rinsed once with TBS-T before adding the primary antibody (1:1000
dilution) in TBS-T and hybridized for 1 hour, shaking at RT, and afterwards rinsed again 4 times with TBS-
T. Subsequently, the membrane was incubated in secondary antibody diluted 1:10000 in PBS-T for 60 or 80
minutes shaking at RT, in the dark in PBS-T and then washed away with PBS-T for 30, 20 and 10 minutes
or 3 times for 5 minutes. Two final washings with PBS were performed for 10 minutes or for 30 and 20
minutes to remove the Tween before scanning the membrane with an Odyssey machine.
15
Table 4. List of materials employed for WB
Protein gels
Whatmann paper 13 cm by 7.5 cm for big gels (20-26 wells)
8 cm by 6.5 cm for small gels (12 wells)
Nitrocellulose membrane
Methanol buffers A1, A2 and K
Skimmed milk 5% in PBS
PBS
PBS - T
TBS – T (pH 7,5) 6,05 g Tris + 8,76 g NaCl adjust with + 1 mL of Tween 20 in demiwater (up to 1 liter)
Table 5. List of antibodies employed for Western Blot assays
Primary Antibody Secondary AntibodyRabbit anti-NF-κB p65 (mAb #8242) Goat anti-rabbit (IRDye 800CW LI-COR 800nm channel)
Rabbit anti-β-Actin (mAb #4970) Goat anti-rabbit (IRDye 800CW LI-COR 800nm channel)
Mouse anti-GAPDH (mAb #10R-G109a) Goat anti-mouse (IRDye 800CW LI-COR 800nm channel)
2.6 Protein Quantification
To quantify the protein content inside samples we followed the Pierce TM BCA protein assay kit protocol (as
specified by the manufacturer), which is based on the Biuret reaction of proteins which reduce Cu2+ to Cu+.
This cuprous ion reacts with bicinchoninic acid to give a purple color. This kit allows an estimation of
protein content from 25 up to 2000 μg/mL. From the OD562 of the ladder's samples, which have known
amount of proteins, one is able to obtain a graph and a function and therefore a linear regression to calculate
the amount of protein for the test's samples.
2.7 Relative Protein Expression
The software ImageJ was used to acquire values of relative protein expression compared to a housekeeping
protein, like β-actin or GAPDH, from bands pictures of a membrane. The software converts the intensity of
the bands selected into areas that are possible to analyze.
2.8 Hydrogen Peroxide Survival Assay
To assess the ability of CA- and HA-MRSA isolates resistance to stress, six different concentrations of H2O2
in RPMI 1640/2mM L-glutamine were prepared and employed: 1, 5, 10, 50, 100, 200 mM, see chart for volumes.
16
Table 6. Volumes necessary for the creation of 50 mL solutions of RPMI 1640/2mM L-glutamine with different concentrations of H2O2.
Desired concentration (mM) Stock ( μL) RPMI1640 ( μL)
1 5,7 49994,3
5 28,3 49971,7
10 56,7 49943,3
50 283,4 49716,6
100 566,8 49433,2
200 1133,7 48866,3
Procedure
D3, D15, D17, D29, D61, D66 isolates were grown until 90 minutes into stationary phase for the pre/main
culture in 60 mL of RPMI 1640/2mM L-glutamine (usual conditions of growth) then ODs were measured to
calculate via the usual standard to have 5x107 CFU for each strain. Volumes of bacterial culture were added
to the volumes of different concentrations of H2O2 in 10 mL tubes for a total volume of 2 mL in each tube.
The mixture incubation lasted 1 hour in shaking incubator. A plating of 100 uL was performed on BA agar
plates, with different dilutions depending on the experiment but all in a range from undiluted to 10 5 dilutions
for the H2O2 exposed samples and up to 106 for the control bacteria only. The CFU were counted the
following day.
17
3 Results
3.1 Growth Curve of Isolates
As specified in the materials and methods section 2.1, MRSA isolates were first grown in TSB O/N at 37°C
in water bath at 116 rpm. The next morning, culture was diluted to an OD600 of 0.05 in RPMI 1640 medium
supplemented with 2 mM L-glutamine, and grown until an OD600 of 0.5 at 37°C in water bath at 116 rpm as a
pre-culture. Later the same day, culture was diluted to an OD600 of 0.05 in RPMI 1640, and grown till 90
minutes into the stationary growth phase with usual temperature and rpm. The two groups of isolates, namely
the CA- and HA-MRSA isolates showed similar growth curve (Figure 3).
Figure 3: Growth curve of representative isolates during pre- and main- culture. Two representative CA-MRSA isolates
D15 and D32, and two representative HA-MRSA isolates D53 and D66 were used for the growth curve. All cultures
were inoculated in 15 mL TSB in 250 mL flasks and kept ON in a waterbath at 37°C, 116 rpm. Cultures were then
moved the next day to 20 mL of RPMI 1640 supplemented with 2 mM L-glutamine in 250 mL flask in water bath at
37°C, 116 rpm for the preculture and 30 mL for the mainculture. The growth pattern of the strains showed no particular
difference between CA and HA-MRSA strains for the total time of growth of around 24 h.
3.2 MRSA Survival Assay – CFU Counting
We questioned if there is a difference in the survival of the CA- and HA- MRSA isolates inside macrophages
that, in turn, could explain differences in the pathogenesis of the two groups of isolates. The verification
under the inverted light microscopy of the first pilot experiment yielded the complete lack of human cells
after the first washing step done in order to remove the medium prior the infection. This could probably be
due to an incomplete differentiation/attachment of the cells to the bottom of the wells and to an excessive
pull from the suction device used during the washing steps. To avoid this issue, the suction procedure was
modified with the use of two pipette tips to slow down the vacuum pull.
The next experiments display a correct presence of surrogate THP-1 macrophages allowing us to proceed in
the assay.
18
Among the ones that exhibit growth we listed the most interesting case. The count of the colonies, and the
estimated survivability of the strains, is calculated for different time periods after the lysis of the THP-1
derived cells and the plate of the content. To obtain the baseline of survival some plates were created from
the original bacterial culture from each strain after series of dilutions. The maximum dilution plated, 10 5, was
the one used for every strains for reference (from main culture) although this exhibit numbers of CFU from
500 to 600 which is not ideal in the ideal range for counting [70].
Regarding the CFU from the macrophages infection, we counted the plates with 104 dilution for 30 minutes
and 1 hour and 103 for 2 hours, except the strain D53 for which we counted 10 3 for 1 hour and 102 for 2
hours. By doing so, the CFU numbers were all included within the standard 30 to 300 interval [70].
After 30 minutes of incubation the CA strains showed more colonies with 43,17% survival for D15 and
35,97% for D32, while HA ones displayed 35,70% for D53 and 8,03% for D66. A laboratory type (Newman)
showed a 7,18% percentage of survival (Figure 4A).
After a total time of 60 minutes the two HA strains decreased to 22,97% (D15) and 9,12% (D32) while the
CAs revealed a strange pattern with a 1,36% survival for D53 and a surprising 31,49% survival for D66, at
the same time the NM strain was at 1,20% (Figure 4B).
Subsequently, at 120 minutes D15 (CA) had around the same percentage of survival with D66 (HA),
respectively with 1,06% and 1,05% while D32 (CA) with 0,70%, D53 (HA) with 0,49% and NM with 0,66%
showed lower levels (Figure 4C).
Finally, in the O/N plates we assisted at the lowest percentage of survival with 0,09% for D15, 0,04% for
D32 and D53, 0,03% for D66 and 0,01% for NM (Figure 4D). To summarize, all strains percentages
diminished over time with one particular exception of D66 from 30 to 60 minutes (from 8,03% to 31,49%).
19
Figure 4. Bar graph showing the survival of the MRSA strains after (A) 30 minutes, (B) 60 minutes (C) 120 minutes and (D) overnight incubation inside THP-1 macrophages. It is visible a general decreasing pattern with the exception of D66 (B) at 60 minutes.
20
3.3 MRSA Survival Assay – Fluorescein /FITC Staining
Since the CFU approach was not definitive, we used flow cytometry based assay to assess the survival of
MRSA isolates inside macrophages. The activation and seeding of Thp-1 cells were similar to the CFU
approach. Isolates were grown like explained in section 2.1. Bacterial count was roughly correlated to an OD
measurement (OD600) of 0.5, which is approximately 127,000,000 cells (Mekonnen's standard).
Firstly we verified that the bacteria were indeed engulfed by the macrophages under fluorescent microscope
(Figure 5A) and secondly we assessed the phagocytosis rate of the two groups of isolates.
Figure 5. (A) Fluorescence microscopy showing FITC-stained MRSA isolate. (B) Representative image of the gate and the spike FITC positive isolate. (C) Bar graphs showing phagocytic index for HA-MRSA D17 and CA-MRSA D37.
21
D37 shows a higher trend of being internalized compared to D37 for both 50 MOI and 100 MOI.
Each of the two MOI considered, 50 and 100, had two samples for each strain, D17 (HA) and D37 (CA),
plus two negative controls of only macrophages were added and, as expected, gave no positive signal for
FITC, but only autofluorescence signals. Both the two MOI showed a slightly higher phagocytic index for
the CA strain D37 compared to the D17, with an average PI for D17 MOI 50 of ≈ 38000 and ≈ 45000 for
D37, while for MOI 100 ≈ 37000 and ≈ 44000 respectively. although this was not statistically significant by
Student’s t-test.
Afterward the flow cytometry machine generated strange values during the analysis. High number of
elements were counted even in simple washing solutions in combination with problems in the flow of the
liquid probably due to clogging material inside. This technical issue prevented us to continue the survival
assay
3.4 Comparison of NF-κB Subunit p65
We speculated that the level of p65 protein among CA- and HA- MRSA isolates differs.
After the estimation of the total quantity of proteins (Pierce TM BCA protein assay kit) it was possible to load
for every sample the same mass of protein in the LDS-PAGE. After a WB and an image analysis with
ImageJ, two major feature were revealed: the complete lack of a band for the NF-κB subunit p65 and a
difference in intensity in the control protein β-Actin between CAs and HAs.
Of the five clinical HA-MRSA from Denmark loaded, in fact, we observed the disappearance of the D3 band
for p65 (65 kDa) and at the same time a lower intensity of all the HA bands for β-Actin (42 kDa) compared
to CA bands of around 1/3 (Figure 6A).
22
Figure 6 (A) The WB contains the two bands for p65 and β-Actin in each sample excluding D3, from a LDS-PAGE loaded with 10,07 μg per sample. β-Actin works as a control of intensity of expression for the NF-κB p65 but its expression seems to be less intense in the HAs strains (from D3 to D66). β-Actin size is around 42 kDa while p65/RELA is 65 kDa. (B): Bars graph showing the average intensity of β-Actin bands in CA strains compared to HAs. The HAs group shows a reduction of intensity of around 1/3 compared to the CAs with arbitrary unit (Au) 63 against 42. This difference was statistically significant according to the Student's t-test (P ≤ 0.05).
Although the decreased level of β-Actin is interesting, to our purpose of measuring the expression of p65 this
constitutes a problem. We decided, then, to strip the membrane of the β-Actin Ab via 2% LDS + 7μL β-
Mercapto-ethanol in PBS for 30 minutes and then wash 3 times in PBS for 10 minutes and finally we added
GAPDH Ab (mouse) O/N as per usual protocol. GAPDH proved to be more uniform for all the strains than
β-Actin so were able to quantify p65 more accurately.
To have a baseline for the expression of p65 in pathogen-free conditions we added controls, which consisted
of a lysate of macrophages not previously infected with MRSAs. These samples displayed bands with an
intensity comparable to the other infected samples.
23
To investigate the lack of the D3 band we performed more tests with the former protocol and we saw the
reappearance of the band. The figure below (Figure 7) shows a WB executed with the same conditions of the
initial one excluding the fact that we loaded 6,34 μg of proteins for each sample instead of 10,07 μg.
Nevertheless, we were still able to visualize all the bands, including D3, and match to GAPDH ones to infer
protein expression.
Finally, the analysis of variance (ANOVA), performed with the software GraphPad Prism, showed that there
is no statistical significance between the 3 group (HAs, CAs and CTRL), but a Student's t-test did show that
the difference between the MRSA groups is significant for a higher expression for HAs.
Figure 7. (A): WB of a gel loaded with 6,34 μg of proteins per sample run with the protocol of previous ones. To visualize the bands the upper part of the membrane was photographed with the Odyssey machine with a linear manual of 7 while the lower part with one of 4, this was necessary since the bands above were sensibly less visible.(B). Average of relative protein expression for p65 in the control was 50, while for the CA-MRSA was 35 and for the HA-MRSA 54 AU (arbitrary unit). A statistical correlation was visible between the two MRSA groups under Student's t-test.
24
A
B
3.5 Hydrogen Peroxide Survival Assay
A pilot test was performed with the strains D3, D17 (HAs) and D15, D29 (CAs) with dilutions for the H 2O2
exposed samples of 104 and 105 and from 104 to 106 for the control ones. The following day very few colonies
(all under 30 CFU) were visible in the BA, except in the 104 and 105 CTRL ones, due probably to a
concentration of H2O2 too high.
The following experiments were done with the strains D3, D66 (HAs) and D61 (CA) with the same
conditions except for different dilutions of 103, 104 and 105 for the control and undiluted, 101 and 102 for the
exposed ones. It was possible to visualize more CFU for all the strains, especially for the CA-MRSAs
strains, D3 and D66. It was also visible that D66 possessed the most CFUs between the three strains for
every concentration of H2O2.
A final count and quantification for a more exact survival trend was performed.
Indeed, HA-MRSA D61 appeared to be the strain with the least amount of colonies compared to the CA-
MRSA in every dilution. On the other hand, D66 was the one with the highest number of CFUs.
In the figure 8 below it is visible that less colonies were present as the concentrations of H2O2 increased,
with almost no CFUs in 50 mM for all strains and none whatsoever in 100 mM. A particularly noticeable
decrease in the numbers of colonies was detected for D66 from 10mM to 50mM (see figure 8 and
Supplementary).
Figure 8. Line graph showing the trend of survival for the two HA-MRSA strains D66 and D3 and for CA-MRSA strain D61. The latter one for the same concentrations show less survival compared to the other two strain.
25
4 Discussion
A discernible deviation between community associated MRSA and hospital associated MRSA strains in
features like survivability under immune system attack, chemical attack, and what type of immunoresponse
the different strains of S. aureus induce, could prove to be a very useful tool for future diagnosis, prognosis
and even treatment in patients infected with such strains. Our work aims to add to studies that for almost two
decades have looked for and discovered relevant differences. These previous works proved for example a
higher prevalence of CA-MRSA in skin and soft tissue infection (SSTIs) [42] hence a recent view of their
higher viability inside epithelial cells should come as no surprise [Mekonnen et al, unpublished data], but
considering also their higher tendency of lysing leukocytes [46] it is also logical to wonder how CA-MRSA
behave compared to HA-MRSA inside macrophages. This prompted us to perform a first series of tests using
chemically differentiated THP-1 cells as macrophages.
It is known from literature, since 1982 [72], that THP-1 cells display differentiation into macrophages via the
anchorage to the inferior side of the flask in which they are contained after the exposition to PMA (phorbol
12-myristate 13-acetate) [59,72]. We devised an appropriate protocol with sign of adequate differentiation
into macrophages. The concentration of PMA used to induce maturation without undesirable gene up-
regulation [64] was 5 ng/mL and we opted for a weaker pulling force from the suctions device by using two
pipette tips on the opening of the aspirator.
Following tests showed indeed the wanted layer of cells attached to the bottom of the well, suggesting the
efficacy of the protocol.
Concerning survivability of MRSA in these white blood cells, an illustrative test showed the survival rate of
4 clinical strains , two CAs (D15 and D32) and two HAs (D53 and D66) plus a laboratory strain after half an
hour, one and two hours and overnight infection of THP-1 macrophages. All the strains kept steadily
decreasing in numbers at different rates excluding HA strain D66 which unexpectedly showed a recovering
high survival at 31,49% in the one hour infection plate, though it was only 8,03% at 30 minutes and in the 2
hours and O/N ones showed a similar pattern to the other strains with 1,06% and 0,03% respectively.
This singular divergence, which was not present in the previous optimizing tests, could be of course due to
the different MOI (compared to the expected 50) due to a procedural mistake or inaccuracy of the counting
method or to a wrong dilution in the last phase prior to the plating. In the first case, the mistake could have
taken place during the seeding of macrophages or the addition of bacteria resulting in an altered ratio either
with more macrophages ,which could have shielded the bacteria from the antibiotic, or with simply more
bacteria added and therefore capable of increased levels of infection. In the other hypothesis, a 10 1 dilution
could have been skipped since a 3,15% value instead of 31,49% would be closer to the other HA-MRSA
D53 (1,36%).
Other possibilities could involve a different pattern of macrophages maturation between the different wells,
since not properly specialized macrophages would show less capability of neutralize internalized MRSAs, or
the lack of gentamicin which would lead to counting also extracellular bacteria.
Finally, assuming all the conditions were met like intended, the unusual survival rise could have been caused
26
by a sudden multiplication of D66, which started after 30 minutes and quickly decreased after 2 hours. This
hypothesis is supported by the fact that after 2 hours D66 was, together with D15, the best surviving strain
(respectively 1,05 and 1,06).
Concerning the other strains, CA one D15 displayed the best survivability compared to any other strain
except at 1 hour for D66 incubation, while the second CA D32 exhibited better percentages compared to the
HA D53 ones, especially after 1 hour (9,12% vs 1,36%) and 2 hours (0,70% vs 0,49%) but less so for the
other times. The lab strain Newman showed the worst survival rate at every time studied. Before drawing
conclusions a premise is to be considered. The ability to escape from the phagosomes, mature
phagolysosomes and ultimately macrophages by lysis is a known ability for some MRSAs [56], an ability
that normally would improve its virulence. In our experiment, though, such evasion during the incubation
would yield a low percentage of survival due to the addition of gentamicin in the wells. This set up,
therefore, should be considered interesting mainly for studying an higher ability to survive and reside inside
macrophages between CAs and HAs, a residency that S. aureus is even able to prolong by activating anti-
apoptotic pathways inside the phagocytes [67]. In this context, excluding D66, CA-MRSAs seem to perform
better compared to HA-MRSAs and in general MRSAs perform better than the lab strain. However,
considering D66 such conclusion cannot be drawn and therefore the necessity of investing more in the matter
becomes unavoidable.
To resolve the previous issues of slow counting and possible procedural errors, we then decided to utilize a
more advance technique that involves FITC staining of the bacteria to study the phagocytosis of MRSAs
inside macrophages and that has shown promising results in other works both in neutrophils and
macrophages [73,74].
The FITC staining protocol proved to be efficient, as visualized by the fluorescence microscope, in staining
the MRSA strains before the incubation and allowing visualization once internalized but posed some
limitations as well. For example, the phagocytic index allows only to estimate a comparison between the
survival of strains that infected the human cells but cannot give an exact number of the starting CFUs. In
fact, to assess the number of CFUs for the incubation only a standard of 127M of CFU per 1 mL at 0,5 OD600
was used, without a reliable quantification for every sample. An easy solution is to plate after the staining on
BA to be able to estimate the following day by counting. Another issue is the fact that, once again, the
procedure does not consider the bacteria that manage to escape since their signals is quenched by adding
trypan blue.
Prior to being halted by technical issues, this system gave us an hint of higher internalization rate and
internal survival for CA-MRSAs D37, although statistically not very significant.
Subsequently, to see how a macrophage react to S. aureus infection, specifically in the NF-κB pathway, we
collected lysates of infected cells to use for Western blot analysis. The first notable result was a difference in
β-actin expression level, which was supposed to be a loading control for NF-κB sub-unit expression levels
p65. CA-MRSAs showed a stronger signal for β-actin.
β-actin is involved in phagocytosis [75] and therefore one of the possible explanations for its different
27
expression between HA and CA could be that the latter ones are in higher number phagocytized, as the flow
cytometer survival assay seems to suggest. Moreover, the initial lack of D3 band in the first experiment is
intriguing. The same strain showed, in fact, both β-actin and GAPDH excluding the possibility of missing
macrophages and even in case of missing bacteria should be possible to see a p65 band since the controls
(only macrophages) showed bands for p65 expression as well. While this could seem contradictory, since
these samples were not infected/incubated with MRSA, a 2000 study actually established a physiological
production of NF-kB for normal survival in macrophages [76]. In addition, another study proved how during
differentiation NF-κB functional protein is accumulated in the cytoplasm of PMA-induced macrophages and
how this is necessary for priming for its future translocation to the nucleus once bacterial LPS are detected
later on [69]. Therefore, p65 (from NF-kB) bands should appear even in the control deprived of bacterial
infection therefore it’s surprising to observe the complete lack in the D3 sample.
It is also quite interesting the similar strength in the signal of p65 between controls and infected ones with no
statistical significant deviation. However, a deviation was present between the two MRSA groups and,
considering the larger number of elements in those groups (compared to the controls), this is sign of a trend
of higher NF-κB for HA-MRSA compared to CA-MRSA. Should this trend be confirmed in the future
together with the role of NF-kB for phagocytosis in human THP-1 (like it has been seen in murine
macrophages RAW264.7 cells [60]), it would be proof that HA-MRSA are indeed more capable of activating
NF-kB inflammation pathway and also more internalized.
However, in view of the fact that we observed in a test more β-actin activation in CAs, the fact that β-Actin
is linked to phagocytosis as well and that the FITC-stained CA-MRSA showed higher phagocyting index in
another test we can’t for sure confirm this double trend to be the case in humans.
Concerning the H2O2 resistance test, the very high dilution of CFUs and high H2O2 concentration appeared to
be responsible for the lack of CFU in the pilot test, since colonies were seen in the CTRL H 2O2 free and in
the following experiments with less dilutions. In these ones, D61 was the only CA-MRSA strain used, while
D3 and D66 were the HA-MRSA that showed a clear pattern of higher resistance to H 2O2. These results
agree with the higher transcription of stress response genes seen by Mekonnen et al., especially for SR4 and
SR5 (Staphylococcus aureus M1 complete genome: labels BN843_8030 and BN843_21760) [77], which are
both responsible for ROS resistance and transcripted from 2,9 to 3,5 higher than CA-MRSA.
SR4 encode for an organic hydroperoxide resistance protein-like part of the Ohr and OsmC family and
specifically in the Ohr subfamily, while SR5 produce non-specific DNA-binding protein Dps that protects
the DNA by reducing the numbers of chromosome single and double breaks [78] in many prokaryotes. How
HA-MRSAs possess this higher resistance could be related to a gained ability after CA separation or the lost
of resistance by CAs, maybe thanks to a CA-improved ability to evade phagocytosis by lysing macrophages
like it has been seen in PMN [41], freeing the bacteria from the ROS stress very quickly.
Further studies should evaluate these hypotheses firstly by using more MRSA strains, both from community
and hospital, to confirm this HA's H2O2 resistance.
28
To summarize, HA-MRSAs seem to activate more the NF-kB pathway, related to phagocytosis in mice, and
to resist better to ROS stress. It is still unclear if they are also indeed more phagocytized in light of some
discordant results. Future studies should focus on clarifying if CAs or HAs are more internalized and, using
more strains, on confirming the higher resistance to ROS of HAs as well as their higher NF-kB inducing
capability. Such studies, together with our results, will definitely prove to be useful tools for discriminating
the two groups which would help both the academic knowledge and the clinical practice.
29
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6 Supplementary
Figure 9. BA plates of HA-MRSA strains on the side (D3,D66) and CA-MRSA in the middle (D61). As the concentration of H2O2 rises it is possible to see a fewer numbers of colonies in the plates, especially for [50] mM, 102
dilution.
35
D3 D66D61
CTRL 10^4 dil
50mM, 10^2
10mM, 10^2
5mM, 10^2
1mM, 10^2