LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XL, 2007, TIMIŞOARA
INVESTIGATIONS ON THE SYNERGIC EFFECTS OF
AMINOGLYCOSIDES, POLYMYXINS AND FLUOROQUINOLONES
COMBINATIONS AGAINST PSEUDOMONAS AERUGINOSA
ISOLATED FROM DOG
ANCA MĂRCULESCU, GH. RĂPUNTEAN, N.A. OROS, M. CERNEA
University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca
Summary
Many times, the treatment of severe bacterial infections determined by
multiresistant microorganisms need a combination of antibiotics.
This paper present the activity of some antibiotics, used alone and in combination,
on Pseudomonas aeruginosa strains isolated from dogs having chronic otitis. In order to
demonstrate the efficacy of these antibiotics on the isolated strains, the dilution method was
used and minimum inhibitory concentration was determined for each antibiotic and for the
combinations. The results were interpreted by the National Committee for Clinical
Laboratory Standards.
Antibiotics from the groups of fluoroquinolones, aminogycosides and polymyxins,
and combination between enrofloxacin-gentamicin, enrofloxacin-amikacin, enrofloxacincolistin were tested on 7 bacterial strains. The fractional inhibitory concentration index was
calculated and showed that the phenomenon of synergism appeared for all strains when
enrofloxacin-gentamicin and enrofloxacin-amikacin combinations were used. The
enrofloxacin-colistin combination showed synergism only in 57.14 % of cases, in 42.86 % of
strains being observed the additive phenomenon.
Based on these results comes the recommendation of using combination of
antibiotics when necessary, because of the higher efficacy together, much more than alone,
in the therapy.
Pseudomonas aeruginosa is a very special species of bacteria; it is
resistant to almost every antibiotic (Răpuntean Gh. et al., 1999). Pseudomonas spp
is known world-wide to produce, together with E. coli, Proteus spp., Pasteurella
spp., Bacteroides spp., chronic otitis in dog. It is common for ear infection to be
recurrent. The infection will not be successfully treated and recurrent otitis will not
stop until the predisposing disease is identified and controlled (Paterson Sue,
2006).
Bacterial culture’s and antibiotic sensitivity profiles should be used (Anand
et al., 2001) to determine which organisms are present and for the selection of
appropriate antibiotic therapy.
Only few antibiotics are efficient against Pseudomonas. Therefore, in order
to obtain a broadened antibacterial therapeutic spectrum and a synergic or additive
effect, combining two antimicrobial agents is used for the treatment of serious
infections (Oie et al., 2003).
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Materials and methods
The Pseudomonas strains used in this study were isolated from chronic
recurrent otitis in dogs. Routine isolation and identification methods were applied.
Susceptibility testing was carried out using broth dilution method (Boldizsar
et al., 2002).
Antimicrobial agents from de groups of aminoglycosides (gentamicin,
amikacin), polymyxins (colistin) and fluoroquinolones (enrofloxacin) were tested.
The stock solutions were prepared in sterile distilled water. Serial dilution,
beginning with 5 µg in enrofloxacin (ENR), 10 µg in colistin (CT), 10 µg in
gentamicin (G) and 30 µg in amikacin (K) were performed in 15 tubes for each
antibiotic. Every tube contained 1 ml broth in a twofold dilution, comparing with the
proximate tubes. One more tube contained no antibiotic and served as a control.
In order to determine the synergism between antibiotics, for each
combination of two antibiotics were prepared 15 more tubes of dilutions, by using
½ of the amount of antibiotics, as following: enrofloxacin-gentamicin (the first tube
= 2.5 µg ENR + 5 µg G), enrofloxacin-amikacin (the first tube = 2.5 µg ENR + 15
µg AK), enrofloxacin-colistin (the first tube = 2.5 µg ENR + 5 µg CT).
The strain grew on broth at 37°C for 18-24 hours and the inoculum was
placed in each of the tubes. The minimal inhibitory concentration (MIC) was
determined after aerobic incubation at 37°C for 18-24 hours and was considered
the lowest antimicrobial concentration that produced no visible bacterial growth
(Anand et al., 2001).
Fractional inhibitory concentration indices (FIC) were calculated (Hallander
et al., 1982) for all isolates with all combinations.
Results and discussions
The present study concerned the in vitro effect of antibiotics from the groups
of aminoglycosides, polymyxins and fluoroquinolones, alone (Pantozzi et al., 2000)
and in combination with another antibiotic, on seven bacterial strains of
Pseudomonas aeruginosa isolated from chronic otitis in dogs (Seol and. col, 2002).
As Mayer and Nagy reported in 1999, Pseudomonas spp. presented
synergism when combinations of aminoglycosides and fluoroquinolones were used
in therapy. Oie et al. (2003) studied combinations of antipseudomonal agents and
found synergy between some antibiotics.
We tested combination of fluoroquinolones-aminoglycosides and
fluoroquinolones-polymyxins and compared with the activity of antibiotic alone.
The MIC’s of the tested antibiotics were determined and interpreted
according to The National Committee for Clinical Laboratory Standards (NCCLS,
1999) (table 1).
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LUCRĂRI ŞTIINłIFICE MEDICINĂ VETERINARĂ VOL. XL, 2007, TIMIŞOARA
Table 1
Control Limits for Monitoring MIC’s with veterinary pathogens Pseudomonas aeruginosa
Antibiotic
MIC (µg/ml)
Enrofloxacin
≤1–4
Amikacin
≤1–4
Gentamicin
≤ 0,5 – 2
Colistin
≤ 4 – 16
The bacterial growth was also examined for the combinations enrofloxacingentamicin, enrofloxacin-amikacin, enrofloxacin-colistin.
Synergy studies are interpreted by calculating the fractional inhibitory
concentration (FIC) which compares the activity of an agent in combination (agent
A + agent B) with the activity of the antibiotic alone (agent A or agent B) according
to CF Referral Center for Susceptibility & Synergy Studies (2003).
The interpretation of the FIC’s values are presented in the table 2:
Table 2
Interpretation of the FIC’s value
FIC value
Interpretation
≤ 0.5
Synergistic
> 0.5 – 1,0
Additive
> 1.0 - ≤ 4
Indifferent
> 4.0
Antagonistic or not clinically achievable
The strains investigated by us have presented the following results
concerning the combination of enrofloxacin-amikacin (table 3):
Table 3
FIC’s values for the combination enrofloxacin-amikacin
Strain no.
FIC
Interpretation
1
0.375
Synergistic
2
0.15625
Synergistic
3
0.3125
Synergistic
4
0.5
Synergistic
5
0.1875
Synergistic
6
0.375
Synergistic
7
0.09375
Synergistic
Enrofloxacin-amikacin combination had synergical effect for all isolates of
Pseudomonas in this study, notifying the fact that we calculated very low values for
three of the strains – 0.09375, 0.15625 and 0.1875 respectively. Only for one strain
FIC was at the borderline (0.5), this value also being interpreted as synergism.
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The values in the case of combination between enrofloxacin and gentamicin
are reported (table 4) as follows:
Table 4
FIC’s values for the combination enrofloxacin-gentamicin
Strain no.
FIC
Interpretation
1
0.3125
Synergistic
2
0.28125
Synergistic
3
0.375
Synergistic
4
0.375
Synergistic
5
0.25
Synergistic
6
0.28125
Synergistic
7
0.3125
Synergistic
Synergism in 100 % percentage was demonstrated by combining
enrofloxacin with gentamicin. The FIC’s values were lower than 0.5, which is
considered borderline of synergical effect (NCCLS, 1999).
The results regarding the enrofloxacin-colistin combination are presented in
the table 5:
Table 5
FIC’s values for the combination enrofloxacin-colistin
Strain no.
FIC
Interpretation
1
0.5
Synergistic
2
0.75
Additive
3
0.25
Synergistic
4
0.75
Additive
5
0.3125
Synergistic
6
0.75
Additive
7
0.5
Synergistic
Although colistin is considered to be efficient in the case of Pseudomonas
(Baba E., Fukata T., 1981), the combination with a quinolone demonstrated
synergical effects only in 57.14 % of strains, 42.86 % being considered additive,
according to the CF Referral Center for Susceptibility & Synergy Studies (CF Ref.,
2003), at Columbia University.
This study demonstrated that combination of enrofloxacin with gentamicin,
amikacin et alistin showed better activities than each antibiotic alone, these being
revealed as synergic or additive effect.
So, there is the recommendation that, in the case of multiresistant bacterial
strains, especially Pseudomonas aeruginosa, to use combinations of antibiotics
that are known to have synergic or additive effects, better than an antibiotic alone,
and by this method the therapy will be successfully.
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Conclusions
Antibiotics from the groups of fluoroquinolones, aminoglycosides and
polymyxins, singly and in combination of two antibiotics, on 7 bacterial
strains of Pseudomonas aeruginosa
Broth dilution method was carried out in this paper and MIC’s of the tested
antibiotics were determined and interpreted according to The National
Committee for Clinical Laboratory Standards
Fractional inhibitory concentration indices (FIC) were calculated for all
isolates with all combinations
Enrofloxacin-amikacin combination have synergic effect for all isolates of
Pseudomonas in this study
Synergism in 100 % percentage was demonstrated combining enrofloxacin
with gentamicin
Combination between colistin and a enrofloxacin demonstrated synergic
effects only in 57.14 % of strains, 42.86 % being considered additive
We recommend the combination between fluoroquinolones and
aminoglycosides and also polymyxins, in the case of chronic otitis
produced by Pseudomonas aeruginosa in dog, because of the synergic or
additive activities of these antibiotics, according our results
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