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
35
Embed
Defining the differences between hospital and community ...fse.studenttheses.ub.rug.nl/14704/1/MasterLS_BMS_2016_MGLoreti.pdfInfective Endocarditis In the industrialized world S. aureus
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
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
Skin and Soft Tissue Infections................................................................................................................................6
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
2.5 Western Blot Analysis .................................................................................................................................. 15
(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
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.
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
5 Bibliography
1. Reviews Of Infectious Diseases. Vol. 6, No.1. January-February 1984
3. Radhey S. Gupta - Origin of diderm (Gram-negative) bacteria: antibiotic selection pressure rather than endosymbiosis likely led to the evolution of bacterial cells with two membranes Antonie Van Leeuwenhoek. 2011 Aug; 100(2): 171–182. Published online 2011 Jun 30. doi: 10.1007/s10482-011-9616-8
4. Timothy Foster Medical Microbiology 4th Chapter 12 Staphylococcus - editior Baron S. ; 1996.University of Texas Medical Branch at Galveston 5. J.S. Weese - An outbreak of meticillin-resistant Staphylococcus aureus skin infections resulting from horse to human transmission in a veterinary hospital - Veterinary Microbiology 114 (2006) 160–164. doi:10.1016/j.vetmic.2005.11.054
6.Keith E. Baptiste – Meticillin resistant Staphylococci in Companion Animals - Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 12, December 2005 DOI:10.3201/eid1112.050241 7. F.C. Leonard - Meticillin-resistant Staphylococcus aureus in animals: A review- The Veterinary Journal Volume 175, Issue 1, January 2008, Pages 27–36 doi:10.1016/j.tvjl.2006.11.008
8. J. Scott Weese - Staphylococcal control in the veterinary hospital - Vet Dermatol 2012; 23: 292–e58 DOI: 10.1111/j.1365-3164.2012.01048.x
9. N.P. Markham - Staphylococci in man and animals: Distribution and characteristics of strains - Journal of Comparative Pathology Volume 76, Issue 1, January 1966, Pages 49-56 doi:10.1016/0021-9975(66)90047-8
10. Morgan M - meticillin-resistant Staphylococcus aureus and animals: zoonosis or humanosis? - J Antimicrob Chemother. 2008 Dec;62(6):1181-7. doi: 10.1093/jac/dkn405. Epub 2008 Sep 26. 11. J Kluytmans, A van Belkum, H Verbrugh - Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks - Clin Microbiol Rev, 10 (1997), pp. 505–520
12. NH Eriksen, F Espersen, VT Rosdahl, K Jensen - Carriage of Staphylococcus aureus among 104 healthy persons during a 19-month period - Epidemiol Infect, 115 (1995), pp. 51–60 - doi:10.1017/S0950268800058118
13. JL Nouwen, A Ott, MF Kluytmans-Vandenbergh, et al. - Predicting the Staphylococcus aureus nasal carrier state: derivation and validation of a “culture rule” - Clin Infect Dis, 39 (2004), pp. 806–811 – doi:10.1086/423376
14. L Hu, A Umeda, S Kondo, K Amako - Typing of Staphylococcus aureus colonising human nasal carriers by pulsed-field gel electrophoresis - J Med Microbiol, 42 (1995), pp. 127–132 doi:10.1099/00222615-42-2-127 15. Steven Y. C. Tong - Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management -July 2015 Volume 28 Number 3 Clinical Microbiology Rev doi:10.1128/CMR.00134-14.
16. W.N. Chang, C.H. Lu, J.J. Wu - Staphylococcus aureus Meningitis in Adults:A Clinical Comparison of Infections Caused by meticillin-Resistantand meticillin-Sensitive Strains - Infection, 2001, Volume 29, Number 5, Page 245 , doi:10.1007/s15010-001-1092-z
30
17. K.B. Laupland - The changing epidemiology of Staphylococcus aureus bloodstream infection: a multinational population-based surveillance study - Clinical Microbiology and Infection Volume 19, Issue 5, May 2013, Pages 465–471 doi:10.1111/j.1469-0691.2012.03903.x
18. Michael L. Landrum - Epidemiology of Staphylococcus aureus Blood and Skin and Soft Tissue Infections in the US Military Health System, 2005-2010 - JAMA. 2012;308(1):50-59. doi:10.1001/jama.2012.7139
19. Vance G. Fowler - Staphylococcus aureus EndocarditisA Consequence of Medical Progress - JAMA. 2005;293(24):3012-3021. doi:10.1001/jama.293.24.3012
20. David R. Murdoch, MD, MSc; G. Ralph Corey, MD; Bruno Hoen, MD - Clinical Presentation, Etiology, and Outcome of Infective Endocarditis in the 21st CenturyThe International Collaboration on Endocarditis–Prospective Cohort Study - Arch Intern Med. 2009;169(5):463-473. doi:10.1001/archinternmed.2008.603
21. Kumar, Vinay; Abbas, Abul K.; Fausto, Nelson; & Mitchell, Richard N. (2007). Robbins Basic Pathology (8th ed.). Saunders Elsevier. pp. 843 ISBN 978-1-4160-2973-1
22. Wolf K; et al. (2005). "Section 22. Bacterial infections involving the skin". Fitzpatrick's Color Atlas & Synopsis of Clinical Dermatology (5th ed.). McGraw-Hill. 23. Linda F. McCaig, L. Clifford McDonald, Sanjay Mandal, and Daniel B. Jernigan - Staphylococcus aureus–associated Skin and Soft Tissue Infections in Ambulatory Care - Emerg Infect Dis. 2006 Nov; 12(11): 1715–1723. doi: 10.3201/eid1211.06019
24. Wallace HJ - Specimen from a case of staphylococcal pneumonia - Proc R Soc Med. 1937 May; 30(7): 885–886. PMCID: PMC2075878 25. Johnson A. 1944. Report on a case of staphylococcal pneumonia with staphylococcal septicaemia: treated with penicillin. Ulster Med J. 1944 Nov; 13(2): 122-3-122-4, 144-149. PMCID: PMC2479234
27. Rajan S - Pulmonary infections in patients with cystic fibrosis. - Department of Pediatrics, Division of Pediatric Infectious Diseases, Columbia University, New York, NY 10032, USA. Seminars in Respiratory Infections [2002, 17(1):47-56] DOI: 10.1053/srin.2002.31690 28. Sheehy SH, Atkins BA, Bejon P, Byren I, - The microbiology of chronic osteomyelitis: prevalence of resistance to common empirical anti-microbial regimens. - J Infect 60:338–343. DOI :10.1016/j.jinf.2010.03.006
29. O. Clerc, G. Prod'hom, G. Greub - Adult native septic arthritis: a review of 10 years of experience and lessons for empirical antibiotic therapy - J. Antimicrob. Chemother. (2011) 66 (5): 1168-1173. doi: 10.1093/jac/dkr047
30. T.N. Peel, A.C. Cheng, , P.F.M. Choong - Early onset prosthetic hip and knee joint infection: treatment and outcomes in Victoria, Australia - Journal of Hospital Infection Volume 82, Issue 4, December 2012, Pages 248–253 doi:10.1016/j.jhin.2012.09.005
31. B. Söderquist, (2007) - Surgical site infections in cardiac surgery: microbiology - APMIS, 115: 1008–1011. doi: 10.1111/j.1600-0463.2007.00833.x 32 D. Jonkersa, T. Elenbaasb, P. Terporten - Prevalence of 90-days postoperative wound infections after
33.H. Wisplinghoff, T. Bischoff, S. M. Tallent - Nosocomial Bloodstream Infections in US Hospitals: Analysis of 24,179 Cases from a Prospective Nationwide Surveillance Study - Clin Infect Dis. (2004) 39 (3): 309-317. doi: 10.1086/421946
34 . Skinner D, Keefer CS. 1941. Significance of bacteremia caused by Staphylococcus aureus: a study of one hundred and twenty-two cases and a review of the literature concerned with experimental infection in animals. - Arch Intern Med (Chic). 1941;68(5):851-875. doi:10.1001/archinte.1941.00200110003001
35. Bondi JA, Dietz CC. - Penicillin resistant staphylococci - Proc. Royal Soc. Exper. Biol. Med. 1945;60:55–58. PMID: 21004029 :
36. Jevons MP. “Celbenin” - resistant Staphylococci - British Medical Journal. 1961;1(5219):124-125.
37. A. Mangili, I. Bica, D. R. Snydman, and D. H. Hamer - Daptomycin-Resistant, meticillin-Resistant Staphylococcus aureus Bacteremia - Clin Infect Dis. (2005) 40 (7): 1058-1060. doi: 10.1086/428616
38 T.J. Fostera, M. Höökb - Surface protein adhesins of Staphylococcus aureus - Trends in Microbiology, Volume 6, Issue 12, 1 December 1998, Pages 484–488 doi:10.1016/S0966-842X(98)01400-0
39.M. Kubica , K.Guzik , J. Koziel - A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages - PLoS One. 2008 Jan 9;3(1):e1409. doi: 10.1371/journal.pone.0001409.
41. M. J. Voyich, K. R. Braughton, D. E. Sturdevant - Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils - doi: 10.4049/jimmunol.175.6.3907 The Journal of Immunology September 15, 2005 vol. 175 no. 6 3907-3919
42. Michael Z. David - Community-Associated meticillin-Resistant Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging Epidemic - Clin. Microbiol. Rev. July 2010 vol. 23 no. 3 616-687 doi: 10.1128/CMR.00081-09
43. S. M. Lee, M. Ender, R. Adhikari et al - Fitness Cost of Staphylococcal Cassette Chromosome mec in meticillin-Resistant Staphylococcus aureus by Way of Continuous Culture - Antimicrob Agents Chemother. 2007 Apr; 51(4): 1497–1499. doi: 10.1128/AAC.01239-06
44. Collins J1, Rudkin J, Recker M - Offsetting virulence and antibiotic resistance costs by MRSA - ISME J. 2010 Apr;4(4):577-84. doi: 10.1038/ismej.2009.151. Epub 2010 Jan 14.
45. S. S Chatterjee and M. Otto - Improved understanding of factors driving meticillin-resistant Staphylococcus aureus epidemic waves - Clin Epidemiol. 2013; 5: 205–217. doi: 10.2147/CLEP.S37071
46. Rong Wang , Kevin R Braughton , Dorothee Kretschmer - Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA - Nature Medicine 13, 1510 - 1514 (2007) doi:10.1038/nm1656
47. S. Y. Queck ,B. A. Khan, Rong Wangm - Mobile Genetic Element-Encoded Cytolysin ConnectsVirulence to meticillin Resistance in MRSA - PLoS Pathog 5(7): e1000533. doi:10.1371/journal.ppat.1000533
48. M.C. Hudson , W. K. Ramp, N.C. Nicholson - Internalization of Staphylococcus aureus by cultured osteoblasts - Microbial Pathogenesis Volume 19, Issue 6, December 1995, Pages 409-419 doi:10.1006/mpat.1995.0075
32
49. O. Krut, H. Sommer, M. Krönke - Antibiotic-induced persistence of cytotoxic Staphylococcus aureus in non-phagocytic cells - J. Antimicrob. Chemother. (2004) 53 (2): 167-173. doi: 10.1093/jac/dkh076 50. R. A. Almeida, K. R. Matthews, E. Cifrian - Staphylococcus aureus Invasion of Bovine Mammary Epithelial Cells - Journal of Dairy Science Volume 79, Issue 6, June 1996, Pages 1021-1026 doi:10.3168/jds.S0022-0302(96)76454-8
51. O.Vesga , M.C. Groeschel, M.F. Otten - Staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu. - J Infect Dis. (1996) 173 (3): 739-742. doi: 10.1093/infdis/173.3.739
52. J.M. Balwit ,P. van Langevelde , J.M. Vann - Gentamicin-resistant menadione and hemin auxotrophic Staphylococcus aureus persist within cultured endothelial cells. - J Infect Dis. (1994) 170 (4): 1033-1037. doi: 10.1093/infdis/170.4.1033
53. C. Garzoni , W.L. Kelley - Staphylococcus aureus: new evidence for intracellular persistence. - Trends Microbiol. 2009 Feb;17(2):59-65. doi: 10.1016/j.tim.2008.11.005. Epub 2009 Feb 7.
54. AJM Vriesema, H Beekhuizen, M Hamdi - Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells. - doi: 10.1128/IAI.68.4.1765-1772.2000 Infect. Immun. April 2000 vol. 68 no. 4 1765-1772 55. T. J. Foster - Immune evasion by staphylococci - Nature Reviews Microbiology 3, 948-958 (December 2005) | doi:10.1038/nrmicro1289
56. RS. Flannagan, B.Heit, E. D. Heinrichs - Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination - Cellular Microbiology Volume 18, Issue 4, pages 514–535, April 2016 DOI: 10.1111/cmi.12527
57. S Ghosh, MJ May, EB Kopp (1998).- NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses -. Annual Review of Immunology 16: 225–60 doi:10.1146/annurev.immunol.16.1.225.
58. Malgorzata Miller, Annette Dreisbach, Andreas Otto, Jan Maarten van Dijl - Mapping of Interactions between Human Macrophages and Staphylococcus aureus Reveals an Involvement of MAP KinaseSignaling in the Host Defense - dx.doi.org/10.1021/pr200224x |J. Proteome Res. 2011, 10, 4018–4032
59. S. Takashiba, T. E. V. Dyke, S. Amar - Differentiation of Monocytes to Macrophages Primes Cells for Lipopolysaccharide Stimulation via Accumulation of Cytoplasmic Nuclear Factor κB - Infect Immun. 1999 Nov; 67(11): 5573–5578. 60. F. Zhu, W. Yue, Y.Wang - The nuclear factor kappa B(NF-κB) activation is required for phagocytosis of staphylococcus aureus by RAW264.7cells - Experimental Cell Research Volume 327, Issue 2, 1 October 2014, Pages 256–263 doi:10.1016/j.yexcr.2014.04.018
61. M. W.Valderas, J.W. Gatson, N. Wreyford, M. E. Hart - The Superoxide Dismutase Gene sodM Is Unique to Staphylococcus aureus: Absence of sodM in Coagulase-Negative Staphylococci - J Bacteriol. 2002 May; 184(9): 2465–2472. doi: 10.1128/JB.184.9.2465-2472.2002
62. A. Clauditz, A. Resch, K.P. Wieland - Staphyloxanthin Plays a Role in the Fitness of Staphylococcus aureus and Its Ability To Cope with Oxidative Stress - INFECTION AND IMMUNITY, Aug. 2006, p. 4950–4953 Vol. 74, No. 8 0019-9567/06/$08.00 0 doi:10.1128/IAI.00204-06
63. J. M. Slauch - How does the oxidative burst of macrophages kill bacteria? Still an open question - Mol Microbiol. 2011 May; 80(3): 580–583. Published online 2011 Mar 14. doi: 10.1111/j.1365-2958.2011.07612.x
64. E. K. Park, H. S. Jung, H. I. Yang - Optimized THP-1 differentiation is required for the detection of responses to weak stimuli - Infl amm. Res. 56 (2007) 45–50 1023-3830/07/0100045–6 DOI 10.1007/s00011-
33
007-6115-5
65. Edwards, A. M., Massey, R. C. Invasion of Human Cells by a Bacterial Pathogen. J. Vis. Exp. (49), e2693, doi:10.3791/2693 (2011).
66. Sun Woo Kim, Dong-Gun Lee, Su-Mi Choi - Once-Daily Gentamicin Administration for Community-Associated meticillin Resistant Staphylococcus aureus in an in vitro Pharmacodynamic Model: Preliminary Reports for the Advantages for Optimizing Pharmacodynamic Index - Yonsei Med J. 2010 Sep 1; 51(5): 722–727. Published online 2010 Jul 16. doi: 10.3349/ymj.2010.51.5.722
67. J. Koziel, A. M. Gudowska, T. Mikolajczyk - Phagocytosis of Staphylococcus aureus by Macrophages Exerts Cytoprotective Effects Manifested by the Upregulation of Antiapoptotic Factors - PLoS ONE. 2009; 4(4): e5210. Published online 2009 Apr 21. doi: 10.1371/journal.pone.0005210
68. J.H. Melehani , D. B. A. James , A.L. DuMont - Staphylococcus aureus Leukocidin A/B (LukAB) Kills Human Monocytes via Host NLRP3 and ASC when Extracellular, but Not Intracellular – PLOS Pathogens Published: June 12, 2015 http://dx.doi.org/10.1371/journal.ppat.1004970 69. Cold Spring Harb Protoc 2006. doi:10.1101/pdb.rec10423
70. S. Sutton - Accuracy of Plate Counts- JOURNAL Of Validation Technology (SUMMER 2011)
71. S.B. Barbuddhea, S.V.S Malika, L. K Gupta - Effect of in vitro monocyte activation by Listeria Monocytogenes antigens on phagocytosis and production of reactive oxygen and nitrogen radicals in bovines - Veterinary Immunology and Immunopathology Volume 64, Issue 2, 8 July 1998, Pages 149–159 doi:10.1016/S0165-2427(98)00129-9
72. -S .Tsuchiya, Y. Kobayashi, Y. Goto, - Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. - Cancer Res. 1982 Apr;42(4):1530-6. 73. S. Busetto, E. Trevisan, P. Patriarca, R. Menegazzi - A single-step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane-bound and ingested Candida albicans in phagocytosing neutrophils. - Cytometry. A 58, 201–206 (2004) DOI: 10.1002/cyto.a.20014
74. T. Hamza, B. Li - Differential responses of osteoblasts and macrophages upon Staphylococcus aureus infection - BMC Microbiology201414:207 doi: 10.1186/s12866-014-0207-5
75. G. Gerisch - Actin switches in phagocytosis - Commun Integr Biol. 2011 May-Jun; 4(3): 344–345. Published online May-Jun 2011. doi: 10.4161/cib.4.3.15041
76. LJ Pagliari, H Perlman, H Liu, RM Pope - Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis. - Mol Cell Biol. 2000 Dec;20(23):8855-65.
78.A.Martinez, R. Kolter - Protection Of Dna During Oxidative Stress By The Nonspecific Dna-Binding Protein Dps - J. Bacteriol. August 1997 Vol. 179 No. 16 5188-5194
Figure 1A: Frank R. DeLeo, Henry F. Chambers - Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era - J Clin Invest. 2009;119(9):2464-2474. doi:10.1172/JCI38226. Figure 1B: M. Palmer, A. Chan, T. Dieckmann - Notes to Biochemical Pharmacology Chap.11- Wiley
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