Potential interactions between radio contrast media and
antibiotics and their effect on discography - an in vitro study
Poster No.:
C-0027
Congress:
ECR 2014
Type:
Scientific Exhibit
Authors:
R. D. Langer , K. F. W. Neidl , G. Petroianu , D. E. Lorke ; Al Ain
1
2
2
2 1
2
- Abu Dhabi/AE, Al Ain/AE
Keywords:
Contrast agents, Neuroradiology spine, Interventional nonvascular, CT, Experimental, Myelography, Infection
DOI:
10.1594/ecr2014/C-0027
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Page 1 of 15
Aims and objectives
Lindblom (1) described discography -an invasive diagnostic procedure- firstly in 1948.
Iodine containing radio contrast medium (RCM) is injected into the nucleus pulposus of
an intervertebral disc. Discography indications have significantly changed over time, due
to further development of non-invasive cross-sectional imaging modalities, namely MRI.
However, especially in the United States of America (USA), and in Australia provocative
discography is again increasingly been used in patients with equivocal findings at MRI/
CT, suffering from recurrent post-operative severe lower back, pre-operatively before
spinal fusion, or before intradiscal (ID) injection of cortisone or anesthetics (2-10).
The most serious, but rare complication is iatrogenic bacterial discitis. Several studies
have shown that prophylactic intravenous (IV) injection of antibiotics can significantly
reduce the incidence of bacterial discitis. Therefore in 1989 Fraser and collaborators
(11, 12) conducted animal experiments, administering intravenous cefazolin, exactly 30
minutes before inoculation of bacteria into animals' discs, in order to prevent bacterial
discitis. Further animal studies demonstrated that after pre-interventional systemic
antibiotics administration the applied antimicrobials could be detected in the intervertebral
discs (with higher concentrations in the annulus fibrosus than in the nucleus pulposus).
In all animal studies it was emphasized that timing of systemic antibiotic prophylaxis was
critical, and that the time frame was limited (13-17). Post-interventional systemic injection
of antibiotics was never deemed beneficial. The most serious disadvantage of antibiotic
prophylaxis is the increase of bacterial resistance.
An attractive alternative is intradiscal (ID) injection of an antibiotic during discography.
One preliminary study on the efficacy of antibiotics, combined with one RCM, i. e. iohexol,
was conducted by Klessig in 2003 (18). To our knowledge, data of potential efficacy of
different RCM alone on bacterial strains have not been published.
ID injection of an antibiotic provides high local concentration in the disc, whereas bacterial
resistance is highly unlikely because of sole local - and not systemic application.
Therefore the aim of the present in vitro study was to test the efficacy of three
commonly used antibiotics on laboratory strains of five different bacteria. Furthermore
the antimicrobial effect of two commercially available RCM, in use for discography, and
one new RCM, on those five bacteria was evaluated in order to detect potential cross
reactions between RCM and antibiotics.
Methods and materials
Page 2 of 15
Bacterial strains
In our study the following bacterial strains were used: Staphylococcus (S) aureus (ATCC
29213), Staphylococcus (S) epidermidis (ATCC 12228), Klebsiella (K) pneumoniae
(ATCC 700324), Escherichia (E) coli (ATCC 25922), and Pseudomonas (P) aeruginosa
(ATCC 27853) (American Type Culture Collection, Manassas, VA, USA).
Culture methods
0
All bacterial strains were plated on blood agar for overnight growth at 37 C (well plates
from Nunc Comp., Roskilde, Denmark). Freshly grown isolated colonies from blood agar
plates were picked up, and inoculated overnight in Trypton Soy Broth (TSB, OXOID ltd,
0
Basingstoke, Hampshire, UK) at 37 C for all assays.
Bacterial dose
On the next day the bacterial concentrations were measured for optical density (OD) at
7
600 nm. Bacterial concentrations were diluted to 1x10 / mL, from which 50 µl each were
dispensed per well.
Radio contrast media and antibiotic
Three nonionic iodinated radio contrast media were used, i.e. iohexol and iodixanol,
both in use for discography, and one new dimeric nonionic substance, iosimenol. The
iodine (I) concentrations of those compounds are as follows: iohexol (Amersham Health,
Carringtonhill, Ireland) 652 mg compound/ mL stock solution, iodixanol (Amersham
Health, Carringtonhill, Ireland) 648 mg compound/ mL stock solution, and iosimenol
(Koehler Chemie, Alsbach-Hahnlein, Germany) 660 mg compound/ mL stock solution.
Eight dilutions of each compound were prepared (table 1).
Table 1
Concentration of iodinated nonionic RCM tested
Dilution
Iohexol
Iosimenol
Iodixanol
1
324 mg/ml
330 mg/ml
326 mg/ml
2
295 mg/ml
264 mg/ml
260 mg/ml
3
194 mg/ml
198 mg/ml
195 mg/ml
4
162 mg/ml
165 mg/ml
163 mg/ml
Page 3 of 15
5
81 mg/ml
82.5 mg/ml
81.5 mg/ml
6
40.5 mg/ml
41.25 mg/ml
40.7 mg/ml
7
20.25 mg/ml
20.6 mg/ml
20.35 mg/ml
8
10.12 mg/ml
10.3 mg/ml
10.17 mg/ml
Six serial dilutions of the three antibiotics ampicillin (Asia Pharmaceutical Ind., Syria),
gentamicin (Sandoz, Cairo, Egypt), and ceftriaxone (Gulf Pharmaceutical Industries,
RAK, UAE) were prepared (table 2), and dispensed on the wells inoculated with the five
above listed bacterial strains.
Table 2
Concentration of antibiotics tested
Dilution
Ampicillin
Gentamicin
Ceftriaxone
1
256 µg/ml
256 µg/ml
256 µg/ml
2
128 µg/ml
128 µg/ml
128 µg/ml
3
64 µg/ml
64 µg/ml
64 µg/ml
4
32 µg/ml
32 µg/ml
32 µg/ml
5
16 µg/ml
16 µg/ml
16 µg/ml
6
8 µg/ml
8 µg/ml
8 µg/ml
Assay procedure
50 µl of each RCM alone, 50 µl of each antibiotic alone, and 50 µl of combinations (of
each RCM with each antibiotic) were dispensed in the wells, starting with the highest
concentrations of the respective RCM and antibiotics according to table 1. 50 µl of TSB
and 50 µl of RCM, antibiotics, and RCM and antibiotics in combination were added
and mixed. Then serial dilutions were obtained, and from the last dilution 50 µl were
discarded leaving 50 µl in each well. Subsequently 50 µl of bacterial culture from freshly
7
grown bacterial culture (1 x 10 /mL) were added. Positive and negative control wells were
0
maintained without RCM or antibiotics. Plates were incubated at 37 C overnight, and
read by ELISA (Elisa Reader Magellan from Tecan Austria GmbH, 5082 Grodig, Austria)
at 450 nm. All experiments were repeated ten times for the RCM alone, and five times,
respectively, for the experiments using the three antibiotics, and the three antibiotics in
combination with the three different RCM. All results were recorded for calculation and
statistical analysis.
Page 4 of 15
Calculation and statistical analysis
Bacterial growth in all treated groups was calculated as percentage of control growth,
applying the following formula:
(Experimental OD - sterility OD / growth control OD - sterility OD) x 100
To test statistical significance the Mann-Whitney test was performed. SPSS 15.0 (SPSS
Inc. Chicago, Illinois) software package was used for all statistical evaluations, and
Microsoft Excel 2007 (Microsoft Coop, Redmond, WA, USA) was used for graphical
presentation. P#0.05 was set for statistical significance.
Results
As ubiquitous microbes S aureus, S epidermidis und E coli were chosen; K pneumoniae
and P aeruginosa were used as organisms responsible for hospital born infections.
For each bacterium tested the inhibition of growth was identical for each antibiotic within
one dilution for all five repetitions. The best overall inhibition of bacterial growth was
recorded for gentamicin (fig. 1) for all five bacterial strains tested. Inhibition of bacterial
growth was significant for all bacterial strains tested, and for all dilutions compared with
the controls. All three antibiotics inhibited the growth of S aureus best. Ampicillin also
significantly inhibited the growth of all bacterial strains (fig. 1). Growth of S aureus, S
epidermidis and E coli was inhibited at all dilutions; however, significant growth inhibition
of K pneumoniae and P aeruginosa was only observed at high concentrations (low
dilutions) (K pneumoniae at dilutions 1-4, P aeruginosa at dilutions 1-2). Inhibition of
bacterial growth by ceftriaxone, compared with control groups, was also significant for
all bacterial strains (fig. 1). Significant growth inhibition of S aureus, S epidermidis, K
pneumoniae and E coli by ceftriaxone was detected at all dilutions, whereas growth of P
aeruginosa pneumoniae was only significantly inhibited at high concentrations (dilutions
1-3).
When applied together with the three antibiotics, iohexol, iodixanol, or iosimenol did
not change the inhibition of bacterial growth of the particular antibiotic. Neither a
statistically significant inhibition, nor an enhancement of the antibiotic activity was
observed. Moreover we could not detect a synergism between any antibiotic combined
with any RCM with regard to inhibition of growth of all bacterial strains. Fig. 2, 3, and 4
demonstrate the inhibition of bacterial growth by gentamicin (fig. 2), ampicillin (fig 3), and
ceftriaxone (fig. 4) alone, and when combined with the three RCM.
Page 5 of 15
The concentrations for each antibiotic in combination with iohexol, iodixanol, or the new
compound iosimenol, sufficient to inhibit all five bacteria were as follows: gentamicin
64 µg/ mL and 128 µg/ mL for E coli, respectively, ampicillin 256 µg/ mL (undiluted),
ceftriaxone 128 µg/ mL, with some limitations for inhibition of growth of P aeruginosa.
Consequently each antibiotic tested maintained its efficacy in the presence of the three
RCM against all five organisms. There was no obvious enhancement or antagonism
between the three antibiotics and the three RCM tested.
When investigated alone iohexol, iodixanol, and iosimenol showed evidence of mild
antibiotic activity as well, but only at high concentrations (fig. 5). Statistically significant
inhibition of bacterial growth for all laboratory strains was only observed at dilutions 1
and 2 for the three RCM.
Images for this section:
Fig. 1: Bacterial growth after treatment with six dilutions of three antibiotics in percent
of positive controls
Page 6 of 15
Fig. 2: Bacterial growth after treatment with six dilutions of gentamicin +/- three contrast
media in percent of positive controls
Page 7 of 15
Fig. 3: Bacterial growth after treatment with six dilutions of ampicillin +/- three contrast
media in percent of positive controls
Page 8 of 15
Fig. 4: Bacterial growth after treatment with dilutions of ceftriaxone +/- contrast media in
percent of positive controls
Page 9 of 15
Fig. 5: Bacterial growth after treatment with eight dilutions of three contrast media in
percent of positive controls
Page 10 of 15
Conclusion
•
The antibiotic which inhibited best bacterial growth of all five laboratory
strains was gentamicin.
•
Inhibition of growth of K pneumoniae and P aeruginosa was limited after
administration of ampicillin and ceftriaxone at high dilutions (i.e. low
concentrations).
•
All three antibiotics, ampicillin, gentamicin, and ceftriaxone, retained
their efficacy in the presence of the RCM iohexol, iodixanol, and the new
substance iosimenol.
•
Iohexol, iodixanol, and iosimenol alone showed only minor antibiotic effects
at high concentrations (low dilutions).
•
Intradiscal injection of the three antibiotics at discography may offer an
adequate antibiotic prophylaxis against post-interventional bacterial discitis.
•
Intradiscal injection of antibiotics could replace systemic injection before
discography, with its inherent risk of generating antibacterial resistance, and
its limited effect caused by the short time frame of peak intradiscal antibiotic
concentration.
Acknowledgements
The authors express their sincere gratitude to the UAEU Research Affairs Office for
funding this Individual Research Project under Grant Number 01-15-8-11/08, and for
granting our research group the 'Research Project Award' at the 10
Research Conference, 2009.
th
Annual UAEU
The authors honestly thank Ms. Naila M Mohamed, UAEU student, for dedicated
assistance during the in-vitro experiments.
The authors express their high appreciation to Prof. U Speck, Charité, Berlin, for his
courtesy of providing us with the radio contrast media.
Page 11 of 15
Personal information
Ruth D Langer MD PhD
Present position:
Professor and Chair of Radiology
College of Medicine and Health Sciences (CMHS) - UAE University
Senior Consultant at Tawam Hospital, Al Ain UAE, in Affiliation with Johns Hopkins
Medical International (JHMI) (CMHS Teaching Hospital)
Tel/ Fax: +971 3 7137 124/ 211 / +971 3 767 2067
Mobile: 050 5834867
Email: [email protected]
Member of the CMHS Priority Research Groups 'Genetics' and 'Oncology'
Previous positions:
1994-1997 Full Professor and Chair of the Dept. of Diagnostic Radiology at the University
of Essen / Germany
1995-1997 Medical Director of the Center of Radiology at the University of Essen /
Germany
2005-2010 Associate Dean for Clinical Affairs CMHS - UAEU
2010-2013 Vice-Dean CMHS - UAEU
Images for this section:
Page 12 of 15
Fig. 6: Prof. Dr. Ruth D Langer
Page 13 of 15
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