Download S.AUREUS RESPONSE TO ACCELERATED ELECTRONS AND

S.AUREUS RESPONSE TO ACCELERATED ELECTRONS
AND LOW DOSE X-RAYS*
R. FOCEA 1, A. POIATA2, D. CREANGA1, T. LUCHIAN1
1
“Alexandru Ioan Cuza” University, Faculty of Physics, Blvd. Carol I, No.11, 700506 Iasi, Romania
E-mail: [email protected], [email protected], [email protected]
2
“Gr.T.Popa”University of Medicine and Pharmacy, Faculty of Pharmacy, Iasi, Romania
E-mail: [email protected]
Received September 5, 2011
The impact of low level irradiation on the cell density of S. aureus samples
(ATCC 25923) was studied as well as the irradiated germ resistance to antibiotics. Both
electron beam irradiation and X-ray exposure were performed with doses between 30
and 120 Gy The bacterial cell density exhibited a significant minimum in the case of
the X-ray exposed samples, while for accelerated electrons a curve with a slight
maximum was recorded. Certain tendency of increasing the resistance to antibiotics in
the X-ray exposed samples was recorded but no detectable changes could be noticed for
accelerated electrons.
Key words: low dose X- rays, electron beam exposure, cell density, tobramycin,
ofloxacin, ampicillin.
1. INTRODUCTION
Staphylococcus aureus (S.aureus) is the second most common etiological
agent of nosocomial bloodstream infections after coagulase-negative
Staphylococcus. S. aureus was found responsible for 20.2% of the 24,179 cases of
nosocomial bloodstream infections included in the SCOPE project, yielding an
incidence rate of 10.3 cases of bacteremia per each 10,000 admissions[1].
S. aureus bacteremia can become complicated and progress to metastatic
infection, recurrence, severe sepsis, septic shock, and death [2]. The mortality rate
associated with S. aureus bacteremia (11–43%) has not varied over the last few
decades. Six to 12% of cases present recurrence [3-5] while up to 31% of patients
present metastatic infection [6-8]. Staphylococcus aureus is also reported to be the
*
Paper presented at the 12th International Balkan Workshop on Applied Physics, July 6–8,
2011, Constanta, Romania.
Rom. Journ. Phys., Vol. 57, Nos. 7–8, P. 1167–1176, Bucharest, 2012
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second most common cause of bloodstream infection after coagulase-negative
Staphylococcus in patients with neoplastic disease [9]. The possible side effects of
radiation therapy in the case of microbial loading of irradiated organs was taken as
phenomenological basis of the experiment carried out on Staphylococcus aureus
(ATCC 25923 germ) exposed to X-rays and electron beams. The purpose of this
study was to highlight the influence of radiation doses comparable to those used in
radiotherapy (relatively low doses compared to those required for sterilization of
microorganisms) on S. aureus growth as well as on its resistance to various types
of antibiotics (ampicillin, tobramycin and ofloxacin).
Ofloxacin is a broad-spectrum antibiotic that is active against both Grampositive and Gram-negative bacteria. Ofloxacin acts on DNA gyrase and
toposiomerase IV, enzymes which, like human topoisomerase, prevents the
excessive supercoiling of DNA during replication or transcription. By inhibiting
their function, the drug thereby inhibits normal cell division [13].
Ampicillin has in vitro activity against Gram-positive and Gram-negative
aerobic and anaerobic bacteria. The bactericidal activity of ampicillin results from
the inhibition of cell wall synthesis and is mediated through ampicillin binding to
penicillin binding proteins (PBPs). Ampicillin is stable against hydrolysis by a
variety of sectreted beta-lactamases, including penicillinases, and cephalosporinases
and extended spectrum beta-lactamases. By binding to specific penicillin-binding
proteins (PBPs) located inside the bacterial cell wall, ampicillin inhibits the third
and last stage of bacterial cell wall synthesis. Cell lysis is then mediated by
bacterial cell wall autolytic enzymes such as autolysins; it is possible that
ampicillin interferes with an autolysin inhibitor [14].
Tobramycin, an aminoglycoside antibiotic obtained from cultures
of Streptomyces tenebrarius, is used in combination with other antibiotics to treat
urinary tract infections, gynecologic infections, peritonitis, endocarditis,
pneumonia, bacteremia and sepsis, respiratory infections including those associated
with cystic fibrosis, osteomyelitis, and diabetic foot and other soft-tissue infections.
It acts primarily by disrupting protein synthesis, leading to altered cell membrane
permeability, progressive disruption of the cell envelope, and eventual cell death.
Tobramycin has in vitro activity against a wide range of gram-negative organisms
including Pseudomonas aeruginosa [15]. Previous experiments allowed the
assessing of the (electro) magnetic fields effects on various bacteria resistance to
several antibiotics [10-11], the general evidenced tendency being that of bacteria
resistance diminution to some antibiotics. Cancer patients, because of their
immunocompromised state and antibiotic treatment, may be susceptible to
opportunistic infections and to organisms that usually cause minimal infection. On
the other hand, there is much evidence that the inappropriate use of antibiotics may
be harmful by encouraging the colonisation of resistant organisms [12].
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The main issue of this study is related to the compatibility of the usual
radiotherapy dose range and the human body tissues potential loading with
S. aureus – from the viewpoint of its resistance to antibiotic administration.
2. MATERIAL AND METHODS
The microbiological material was represented by bacterial strain samples
from standard collections of Staphylococcus aureus (ATCC 25923 germ). The
inoculum was prepared in liquid culture medium – nutrient broth from Oxoid, with
standard composition, 3 ml equal volumes (in sterile glass tubes) being irradiated
in hospital conditions. Ten irradiation variants were developed: five corresponding
to X-ray irradiation times between 25 and 100 minutes (with absorbed dose values
of 31Gy, 62Gy, 87Gy, 107Gy and 128Gy) and five corresponding to electron beam
exposure with the same absorbed dose values.
2.1. X-RAY EXPOSURE
The samples were irradiated using a X-ray tube (SRT100 characterized by
70 kV and 10 mA; the filtration system was based on 0.75 mm Al and HVL: 1.3 mm
Al.), a superficial radiotherapy device used in the ”St. Spiridon” Emergency County
Hospital, Iasi, Romania. The sterile glass tubes containing the liquid culture
medium were placed in a special holder designed to allow irradiation from beneath
being exposed with the dose rate of 227cGy/min at a source-dish distance of 25
cm. The time of exposure was calculated at 1.5 cm depth in the samples, using
classical computerized software of radiotherapy treatment planning system.
2.2. ELECTRON BEAM EXPOSURE
Electron beam irradiation was carried out with 6 MeV energy electron beam
produced by a linear particle accelerator type VARIAN CLINAC 2100SC. All
glass tubes that contained 3 ml inoculated culture medium were placed in an
adequate holder that allowed the lateral uniform irradiation (Fig.1). The samples
were exposed to a dose rate of 240cGy/min, with doses between 31 and 128 Gy the same dose array that in the case of X-ray irradiation carried out also in this
study. The time of exposure (in the range of 16 - 68 min. respectively) was
calculated at 1.5 cm depth in the samples, using a classical computerised software
of radiotherapy treatment planning system. In both situations four repetitions were
assured for every exposure time by using identical glass tube samples.
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Fig. 1 – Irradiation geometry for photon (left) and electron (right) special cones –applicators –
are used to collimate the electron beams) beam exposure.
Fig. 2 – Schematic geometry for dose calculation. Point P is situated on the beam central axis
at depth z in the sample; SSD is the source to surface distance; the field size A is defined
on the surface of the sample.
Exposure time (t) required do deliver a radiation dose (D) at point P (Fig. 2)
using a single electron field A is calculated from Ec. (1) [16]:
t = D/ [Ḋ (zmax,10,100,hν)×RDF(A, hν)×PDD(z,A,f, hν) ×0.005029]
(1)
were D is the prescribed dose in the sample, D (zmax,10,100,hν) is the dose rate, at
point P, at depth zmax of dose maximum on the central axis of the beam, RDF(A,
hν) is the relative dose factor and PDD(z,A,f, hν) is the procentage depth dose at z
depth, for a A field size.
The values for D , RDF and PDD for the given irradiation geometry (6 MV
photon beam and 6 MeV electron beam, respectively, 20×20 cm2 field size, 1.5 cm
depth in the sample) were obtained after beam calibration procedures (in
accordance with dosimetric standard IAEA TRS-398) using a 3D Blue Water
Phantom, a PTW Farmer cylindrical ionizing chamber (for the photon beam), a
PTW Frieburg Markus flat ionizing chamber (for the electron beam) and a PTW
Unidose Electrometer.
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2.3. SPECTRO-COLORIMETRIC ASSAY
Cell density in the liquid culture medium was assessed by spectrocolorimetric assay, measurements being accomplished at 560 nm and 420 nm –
using a Shimadzu Spectrophotometer UV type 1700 Pharmaspec with quartz cells
(with distilled water as reference).
2.4. ANTIMICROBIAL SUSCEPTIBILITY
For measuring antibiotic resistance or antimicrobial susceptibility, the KirbyBauer [17] method was used. The antibiotics resistance of the irradiated bacteria
was tested on agarized nutrient broth against three antibiotic molecules (ampicillin
(A), tobramycin (TOB) and ofloxacin (OF)). The circular inhibition zones revealed
on the agarized microbial samples were observed by direct visual inspection, their
diameters being measured with 1 mm precision usual ruler. Four repetitions for
every microbial sample were carried out for very irradiation type and exposure time.
2.5. STATISTICS
Average values were calculated for every experimental variant using the four
repetitions included in the experiment plan, being used for graphical plots. Three
samples were irradiated for each dose of radiation taken into study. Standard
deviation was taken as error bar in each case. Student t-test was applied to evidence
statistical significance according to the threshold of 0.05.
3. RESULTS AND DISCUSSION
Cell density variations (Fig. 3) in the directly irradiated tubes exhibit
remarkable diminution for the X-ray exposure time of around 50 minutes where the
damages caused by the absorption of the radiation energy probably affected
seriously the cell viability and consequently their proliferative activity – as resulted
from measurements of light absorbance. The cell density appeared to be reduced
more than twice - as resulted from the data provided by spectral measurements
both at 560 nm and at 420 nm.
In Figure 4 the data obtained following the irradiation with accelerated
electrons (6 MeV energy) are represented; slight stimulatory effect given by 16%
increasing in the cell density was evidenced for the lowest dose, of about 31 Gy
(exposure time equal to 16 min) while significant diminution of approximately
25% (p<0.05) for the highest dose of 128 Gy (exposure time equal to 68 min) was
also revealed; decreasing tendency was recorded in all the other situations though
the variations were comparable with the standard deviation – so missing the
statistical significance.
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Fig. 3 – The cell density – as given by light absorbance – for low doses of X-radiation administration
(standard deviation 8%); a. u. – arbitrary units.
Fig. 4 – The cell density as given by light absorbance - for 6 MeV electron beam exposure;
(standard deviation 8%); a. u. – arbitrary units.
The dose array applied in the case of electron irradiation was the same as for
X-ray exposure but the corresponding irradiation times were different. The tubes
glass wall influence on absorbed dose in the samples was of about 1% for
megavoltage energies, mainly caused by increased electron scattering from the
glass which increases the dose to the sample [18].
From Figures 3 and 4 it seems that, although the X-ray and accelerated
electrons are known to have similar LET (linear energy transfer) they are able to
interact with living matter by different action mechanisms that result in the
different responses of the irradiated cell density, most probably related to the
different nature of the two radiation types (photons with secondary ionizing effects
and respectively corpuscular charged radiation with direct ionizing action); as well
the penetration depth is different for the two types of radiation – being much lower
for accelerated electrons. Cell density variations may result mainly from cell
divisibility changes which indicates that cell DNA replication might be affected by
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radiation absorption – probably single or/and double strand damages that led to
incorrect duplication and consequently to failing in cell reproduction. It is possible
that secondary electrons produced by X-ray absorption are able to induce more
serious DNA lesions than the accelerated electrons directly delivered into the
samples (much more absorbed by the glass tubes and the culture medium than the
X-photons); so, only part of DNA injuries could be recovered by the specific action
of bacterial enzymatic equipment – as suggested by the cell density relative
increase in the case of accelerated electron irradiation for irradiation times longer
than one hour.
There are results from literature, according to which there aren’t significant
differences between cells incubated in irradiated culture medium and cells
incubated in unirradiated culture medium, even for doses 10-100 Gy [19] so we can
aproximate that culture medium iradiation has no significant influence on our results.
Fig. 5 – The resistance of X-ray irradiated S. aureus cells to tobramycin (standard deviation 6%).
Fig. 6 – The resistance of X-ray irradiated S. aureus cells to ofloxacin (standard deviation 6%).
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It is also presumable that accelerated electrons triggered certain cellular
mechanisms that resulted not only in the DNA lesion partial repair but also in the
stimulation of cell division in the case of the shortest exposure time – as suggested
by Figure 4.
Regarding the antimicrobial susceptibility, according to the average values
reported in Figure 5, the graph of the growth inhibition discs of S. aureus given by
tobramycin shaped a diminution tendency for X-ray exposure – the lowest value
corresponding to the longest irradiation time although an increased value was
recorded foe the shortest exposure time; (with statistical significance: p<0.05).
Fig. 7 – The resistance of X-ray irradiated S. aureus cells to ampicillin (standard deviation 7%).
As the diameter of the growth inhibition areas inversely depends on the
resistance to antibiotic one could assume that general tendency of increasing in S.
aureus resistance to antibiotic was suggested by the above discussed graph. In the
case of two other antibiotic drugs: ofloxacin and ampicillin, it is also the longest Xray irradiation time that appeared to have some significant effect on the resistance
to antibiotic of S. aureus bacteria (Figs. 6-7) – consisting in the significant
diminution of the inhibition growth area which is equivalent to the bacteria
resistance increase following the radiation action. In the case of accelerated
electrons impact on the bacterial cell cultures there was no detectable change in the
resistance to the three tested antibiotics following the measurement of the growth
inhibition areas (data not shown).
It seems that, in the case of the studied S. aureus, for the low X-ray doses and
chosen antibiotics, there is no visible correlation between the response evidenced
by cell density assessing and that recorded at the level of antimicrobial
susceptibility. S. aureus irradiation within the specified dose range was not able to
induce quite significant changes in the bacteria mechanisms of resistance to
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antibiotics – this being an indicator on the microbial cell radioresistance to low
dose radiation range. Further experiments are needed to get a deeper insight in the
interaction of low-dose radiation with S. aureus germ aiming to get valuable
answers regarding the possible microbial side effects of radiotherapy.
4. CONCLUSION
The S. aureus response to low X-ray doses was evidenced mainly by cell
density assessment in the liquid culture medium that was directly exposed to
X-rays. The resistance to various antibiotic drugs seems not to be a good indicator
of the radiation effect since few significant changes were noticed following the
measurement of growth inhibition areas exhibited at the surface of agarized
cultures.
From the medical viewpoint, however, the resistance to antibiotics is crucial
when the patient health is concerned, so, the main conclusion of this work is that
no significant influence of the patient’s exposure to low radiation doses (usual in
the radiation therapy) is expected for the efficacy of an additional antibiotic
treatment at the level of the microbial loaded organs.
Acknowledgements. The authors greatly acknowledge the support of the European Social Fund
POS DRU [grant POSDRU/88/1.5/S/47646] and also the Radiotherapy Department of ”St. Spiridon”
Emergency County Hospital, Iasi, Romania, for this study.
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