Veterinary Microbiology 121 (2007) 116–124 www.elsevier.com/locate/vetmic
Susceptibility of bacteria isolated from pigs to tiamulin and enrofloxacin metabolites Anne Kruse Lykkeberg a,*, Bent Halling-Sørensen a, Lars Bogø Jensen b a
Department of Pharmaceutics and Analytical Chemistry, The Danish University of Pharmaceutical Science, Universitetsparken 2, DK-2100 Copenhagen, Denmark b Danish Institute for Food and Veterinary Research, Bu¨lowsvej 27, DK-1790 Copenhagen V, Denmark Received 19 May 2006; received in revised form 21 November 2006; accepted 22 November 2006
Abstract Susceptibilities to metabolites of tiamulin (TIA) and enrofloxacin (ENR) were tested using selected bacteria with previously defined minimal inhibitory concentrations (MIC). The TIA metabolites tested were: N-deethyl-tiamulin (DTIA), 2b-hydroxy-tiamulin (2b-HTIA) and 8a-hydroxy-tiamulin (8a-HTIA), and the ENR metabolites were: ciprofloxacin (CIP) and enrofloxacin N-oxide (ENR-N). Bacteria, all of porcine origin, were selected as representatives of bacterial infections (Staphylococcus hyicus and Actinobacillus pleuropneumoniae), zoonotic bacteria (Campylobacter coli) and indicator bacteria (Escherichia coli and enterococci). Furthermore the effects of these compounds were tested on the microbial community of active sludge to test any negative effect on colony forming units (CFU). DTIA had a potency of 12.5–50% of the potency of TIA. 2b-HTIA and 8a-HTIA had potencies less than 1% of the potency of TIA. ENR-N had a potency of 0.75–1.5% of the potency of ENR, while CIP and ENR had similar potencies. Results obtained here indicate that CIP and DTIA could contribute to the selective pressure for upholding antimicrobial resistant bacteria in animals under ENR or TIA treatment. The most potent metabolites CIP and DTIA showed considerable potencies against activated sludge bacteria compared to the parent compounds. EC50 (mg/ml) for ENR, CIP, TIA and DTIA were 0.018 [95% CI: 0.028–0.149], 0.064 [95% CI: 0.007– 0.046], 6.0 [95% CI: 3.6–9.8], and 9.7 [95% CI: 5.8–16.3], respectively. This indicates that the compounds can change the bacterial population in the sludge, and hereby alter the properties of the sludge. # 2006 Elsevier B.V. All rights reserved. Keywords: Enrofloxacin; Tiamulin; Metabolites; Resistance; Bacteria; Actinobacillus pleuropneumoniae; Campylobacter coli
1. Introduction
* Corresponding author. Tel.: +45 35 30 60 00/62 68; fax: +45 35 30 60 10. E-mail address:
[email protected] (A.K. Lykkeberg).
The aim of this study was to investigate the potencies of metabolites of antimicrobials used in animal production and to study these metabolites ability to select for antimicrobial resistant bacteria.
0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.11.020
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Fig. 1. Chemical structures of TIA and the three metabolites tested.
Furthermore the most potent metabolites were tested on activated sludge bacteria to find the potencies of the compounds to the environmental bacteria. As model compounds tiamulin (TIA) and enrofloxacin (ENR) were used. TIA and ENR are widely used antimicrobials in the treatment of infections in pigs (Emborg et al., 2005). In this work three TIA metabolites (phase I), produced in swine liver microsomes, have been tested on bacterial isolates from pigs. These metabolites have been isolated and identified in a previous work (Lykkeberg et al., 2006). The structures of the metabolites are N-deethyl-tiamulin (DTIA), 8ahydroxy-tiamulin (8a-HTIA) and 2b-hydroxy-tiamulin (2b-HTIA) (Fig. 1). The activities of the metabolites have not been published before, but according to several articles the metabolites of TIA are generally less bioactive than the parent compound (Berner et al., 1983, pp. 1317–1321; Committee for Veterinary Medicinal Products, 1999; Dreyfuss et al., 1979, pp. 496–503). ENR is extensively metabolised in many animal species to the metabolite ciprofloxacin (CIP) (Fig. 2), which is an antimicrobial used for human treatment. In pigs the formation of CIP is limited, however (Nielsen and Gyrd-Hansen, 1997, pp. 246–250). In a previous work Lykkeberg (2006) identified the new ENR
metabolite enrofloxacin N-oxide (ENR-N) (Fig. 2) produced in liver microsomes of pigs. This metabolite has not previously been found produced in any mammal. CIP and ENR have been shown to have similar potencies when tested against selected bacteria. The potency of ENR-N has not previously been investigated. To test the ability to uphold antimicrobial resistance by the isolated metabolites we tested the susceptibility of selected bacteria with known susceptibility to TIA and the fluoroquinolone CIP isolated from the continuous surveillance program for antimicrobial resistance in Denmark-DANMAP (Emborg et al., 2005).
2. Materials and methods 2.1. Study compounds Tiamulin hydrogen fumarate was purchased from Apodan (Løgstør, Denmark), ENR was purchased from Aldrich (Steinheim, Germany) and CIP was purchased from Bayer (Aprath, Germany). The TIA metabolites were isolated from swine liver microsomes in a previous work (Lykkeberg et al., 2006) and the ENR metabolite ENR-N was synthesized from
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Fig. 2. Chemical structures of ENR and the two metabolites tested.
ENR and m-chloroperbenzoic acid using a method previously described by Craig and Purushothaman (1969, pp. 1721–1722). 2.2. Antimicrobial susceptibility testing of TIA and metabolites Ten different Actinobacillus pleuropneumoniae (8 susceptible and 2 resistant to tiamulin hydrogen fumarate) isolates and 11 different Staphylococcus hyicus (7 susceptible, 2 intermediate and 2 resistant to tiamulin hydrogen fumarate) isolates were selected. The isolates were all of porcine origin isolated in 2004 (one A. pleuropneumoniae from 2002). Susceptibilities to the compounds were determined by broth dilution susceptibility tests following CLSI (former NCCLS) guidelines (NCCLS, 2003, 2004). The tests were performed in 96 wells microtitration plates with round bottom wells (Nunc, Roskilde, Denmark). A. pleuropneumonia ATCC 27090 was included in the A. pleuropneumonia experiments and S. aureus ATCC 29213 was included in the S. hyicus experiments as controls. The compounds were tested in two-fold dilutions in the following concentration range: TIA from 0.25 to 32 mg/ml and the metabolites from 0.25 to 128 mg/ml. All MIC-values (mg/ml) of the A. pleuropneumoniae and S. hyicus isolates were tested twice. The minimal inhibitory concentration (MIC) was read as the lowest concentration without
visible growth. Strains of S. hyicus and A. pleuropneumoniae were considered resistant if a MIC-value of 32 mg/ml or more were obtained (Emborg et al., 2005). It should be mentioned that the isolates were selected from DANMAP based on MIC-values of tiamulin hydrogen fumarate. In this work we have corrected for the fumarate salt, and fumarate is therefore not included in the mass of TIA in the MIC-values. The consequence of this is that the MIC-values found in this work were a little lower, and hereby redefining some isolates as sensitive (MIC = 16 < 32), which previously had been defined resistant (MIC 32) based on the break point. 2.3. Antimicrobial susceptibility testing of ENR and metabolites Thirty E. faecium (10 susceptible, 10 intermediate and 10 resistant to CIP), 19 E. faecalis (13 susceptible and 6 intermediate to CIP) and 26 Escherichia coli (14 susceptible and 12 resistant to CIP) were selected from isolates of porcine origin obtained in 2004. Twenty-six Campylobacter coli (23 susceptible and 3 resistant to CIP) all from 2003 were included in this study. All isolates were tested for susceptibility to ENR, CIP and ENR-N. Susceptibilities to the compounds were determined by agar dilution tests following CLSI (former NCCLS) guidelines (NCCLS, 2003, 2004). E. coli ATCC 25922, E. faecalis ATCC 29212, S. aureus
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ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 were included in each experiment as controls. The compounds were tested in two-fold dilutions; ENR and CIP from 0.03 to 4 mg/ml and ENR-N from 0.03 to 32 mg/ml. All MIC-values (mg/ml) were tested twice. Strains of E. faecium, E. faecalis and C. coli are considered resistant if a MIC-value of 4 mg/ml or more were obtained and for E. coli the break point of resistance were 0.125 mg/ml (NCCLS, 2003). 2.4. Growth inhibition testing on sludge bacteria ENR and TIA and the two metabolites CIP and DTIA, which showed largest potencies against the bacteria in the first assays, were tested for growth inhibition of sludge bacterial community. The activated sludge bacteria used in this investigation were obtained from the primary aeration tank of a pilot scale activated sludge waste water treatment plant located at the Institute of Environmental Science and Technology, The Technical University of Denmark, Lyngby, Denmark, receiving municipal wastewater from the Lyngby area. Within 1 h after collection, the sludge was pre-conditioned for 20–24 h at 20 8C. Viable plate counting (pour plate method) of aerobic sludge bacteria was used in all the growth inhibition toxicity tests. The method was applied and validated exactly as described in detail by HallingSørensen (2001) and in ISO 15522 (1999). The
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EC50 value (mg/l) is defined as the concentration of the antimicrobial, which inhibit the growth of the half of the plate colonies. The EC50 values were calculated by nonlinear regression analysis using the GradPad Prism 3.00 software.
3. Results The MIC-values of TIA against the two reference bacteria strains A. pleuropneumoniae ATCC 97090 and S. aureus ATCC 29213 were kept within the acceptable limits (Odland et al., 2000, pp. 453–455). The MIC-values of TIA and the metabolites are shown in Table 1. Furthermore MIC-values of CIP against the four reference bacteria strains E. coli ATCC 25922, E. faecalis ATCC 29212, S. aureus ATCC 29213 and Ps. aeruginosa ATCC 27853 were kept within the acceptable limits defined by CLSI. The MIC-values of ENR and the metabolites are shown in Table 2. 3.1. Susceptibility of S. hyicus and A. pleuropneumoniae to TIA and metabolites The selected S. hyicus and A. pleuropneumoniae isolates were screened for susceptibility to TIA metabolites. No susceptibility to 8a-HTIA was detected in the concentration range investigated (see Table 3). The susceptibility of the TIA sensitive S.
Table 1 MIC-values (mg/ml) of TIA and the metabolites against two reference bacteria strains Strain A. pleuropneumoniae, ATCC 97090 S. aureus, ATCC 29213 a b c d
Acceptable limits of TIAa 8–32 0.5–2d
c
TIAb
DTIA
2b-HTIA
8a-HTIA
8 0.5
32 4
>128 128
>128 >128
Tiamulin hydrogen fumarate. Tiamulin. Pfaller et al. (2001, pp. 67–70). Odland et al. (2000, pp. 453–455).
Table 2 MIC-values (mg/ml) of ENR and metabolites against four reference bacteria strains Strain
Acceptable limits of CIPa
CIP
ENR
ENR-N
E. coli, ATCC 25922 E. faecalis, ATCC 29212 S. aureus, ATCC 29213 Ps. aeruginosa, ATCC 27853
<0.03 0.25–2 0.125–2 0.25–1
<0.03 0.5 0.5 0.5
<0.03 0.5 0.125 2
2 >16 >16 >16
a
NCCLS (2003).
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Table 3 The MIC-values of the S. hyicus and A. pleuropneumoniae isolates against TIA (grey cells) and the three metabolites DTIA (italicized), 2b-HTIA (underlined) and 8a-HTIA (bold)
hyicus to 2b-HTIA was strongly reduced with a factor of 128 compared to TIA and the selected A. pleuropneumoniae isolates did not show any susceptibility to 2b-HTIA at all in the tested range. The potency of 2b-HTIA was therefore estimated to be less than 1% of TIA. For DTIA the isolates showed a reduction of the susceptibility by a factor between 2 and 8 compared to TIA, hereby making the potency of DTIA between 12.5 and 50% of TIA (Table 3). 3.2. Susceptibility of E. coli, E. faecium, E. faecalis and C. coli to ENR and metabolites The MIC-values of the tested bacteria-strains against ENR and the metabolites are shown in Table 4. Half of the tested E. faecium isolates showed susceptibilities to CIP, which were increased by a factor of two compared to ENR. In contrast ENR-N had no effect on neither the tested E. faecium nor the tested E. faecalis in the concentration range tested. Only small differences were detected in the susceptibilities of E. faecalis to ENR and CIP. E. coli and C. coli were in general more susceptible to ENR and CIP than the enterococci. They showed a highly reduced susceptibility to ENR-N in the concentration range tested compared to ENR. The susceptibilities of E. coli and C. coli to ENR-N were reduced by a factor of 64–128 compared to ENR. This estimates the potency of ENR-N to 0.75–1.5% of
ENR. No difference in susceptibility to CIP and ENR was detected for the tested E. coli isolates. In contrast the selected C. coli isolates showed susceptibilities to CIP, which were reduced by a factor of 2–4 compared to ENR, estimating the potency of CIP to be 25–50% of ENR. 3.3. Growth inhibition of sludge bacteria Table 5 shows the EC50 values (mg/l) for the growth inhibition assay used for the aerobic activated sludge bacteria of ENR, CIP, TIA, DTIA and the reference compound 3,5-dichlorophenol. The potency of the reference compound 3,5dichlorophenol was found in accordance with previously obtained results with this testing method (Halling-Sørensen, 2001, pp. 451–460).
4. Discussion TIA and the three metabolites formed in an in vitro swine liver microsome assay were tested on pathogenic bacteria isolates of S. hyicus and A. pleuropneumoniae. S. hyicus cause skin diseases in pigs and A. pleuropneumoniae cause swine pneumoniae. TIA is used in the treatment and prophylaxis of both diseases. Very low potencies, less than 1% of TIA, were determined for 8a-HTIA and 2b-HTIAwhen compared
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Table 4 The MIC-values of the E. faecium, E. faecalis, E. coli and C. coli isolates against ENR and the two metabolites CIP (italicized) and ENR-N (bold). The grey cells illustrate the MIC-values of ENR
to TIA. When TIA interacts with the ribosomes the tricyclic ring system of TIA will be positioned in a tight pocket in the 50S subunit, through a network of hydrophobic interactions (Schlu¨nzen et al., 2004, pp. 1287–1294). Hydroxylations in the ring system prevent these hydrophobic interactions resulting in a considerable reduced binding to the active site. This is consistent with the activities found for the metabolites. The bacteria tested were more susceptible to DTIA, which had a potency of 12.5–50% of the potency of TIA. Both the bacteria susceptible and intermediate to TIA had the same reduced
susceptibility to DTIA compared to TIA indicating a conserved action mechanism. The nitrogen in TIA has an effect on the water solubility and gives a better penetration into the tissues (Egger and Renshagen, 1976, pp. 923–927). A deethylation does not change the charges of the antimicrobial considerable, and it is therefore not expected to have much effect on the binding to the active site. This is in accordance with the activities of DTIA found in this work. The three TIA metabolites tested have only been identified in an in vitro metabolism study
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Table 5 EC50 values against sludge bacteria Compound
EC50 (mg/ml)
95% confidence limit
TIA DTIA ENR CIP 3,5-Dichlorophenol
6.0 9.7 0.018 0.064 6.2
3.6–9.8 5.8–16.3 0.007–0.046 0.028–0.149 5.8–6.7
(Lykkeberg et al., 2006), and it is not known in what extend the metabolites are formed in vivo. In this study DTIA showed a considerably potency, and could therefore be part of the selective pressure for development of TIA resistance if present in the animal. The two hydroxylated metabolites had negligible activities and do most likely not contribute to the development of TIA resistance. The concentrations of the tested metabolites and their distribution in blood samples await further investigation. ENR and the two metabolites CIP and ENR-N were tested on the zoonotic C. coli and on Gram-negative (E. coli) and Gram-positive (enterococci) indicator bacteria to investigate the contribution to antimicrobial resistance of the metabolites. The bacteria are all found in the intestines of animals. CIP is the primary metabolite of ENR and it is produced in vivo in large amounts in several animal species such as: 20–35% in horses (Kaartinen et al., 1997, pp. 378–381) and 50% in cows (Kaartinen et al., 1997, pp. 378–381; Varma et al., 2003, pp. 303–305) but not in high concentrations in pigs (Nielsen and Gyrd-Hansen, 1997, pp. 246–250). CIP and ENR were found to have comparable potencies in this study, but some variation between the bacterial species was detected. CIP was most potent to E. faecium, and ENR was most potent to C. coli. This variation was however within experimental variations in the used set-up. The selected bacteria tested showed a large reduction in susceptibility to ENR-N compared to ENR in the concentration range tested, indicating the potency of ENR-N to be 0.75–1.5% of the potency of ENR. The nitrogen in ENR is responsible for the lipid solubility and enhances the ability to penetrate tissues (Schentag and Domagala, 1985, pp. 9–13). The oxidation of the nitrogen cause considerable changes in the charges and will therefore have an effect on the activity. The deethylation in CIP
has only minor effects on the pKa-levels of the nitrogen and the binding in active site is therefore not effected considerable. These conclusions are in accordance with the activities found for the metabolites in this work. ENR-N has until now only been found formed in an in vitro study in liver microsomes of pigs (Lykkeberg, 2006), and it has never to our knowledge been identified in vivo. It is therefore not known in what extend it is formed in vivo, and if it is excreted in the faeces or in the urine. ENR-N showed a considerably reduced potency to the bacteria tested compared to ENR, and does probably not contribute to the development of resistance. ENR and TIA and the most potent metabolites CIP and DTIA were tested for growth inhibition of sludge bacteria (Table 5). The sludge bacteria are a heterogenic bacterial community (mixed bacterial culture), which contain both Gram-negative and Gram-positive bacteria. Presence of antimicrobials and their metabolites may reduce the capacity to degrade other xenobiotics or to nitrify ammonia of the bacterial community in the sludge. This more general investigation shows that ENR, CIP, TIA and DTIA can reduce the CFU in active sludge. The fluoroquinolones are as expected much more potent than TIA and DTIA. Table 5 shows further more that the EC50 (mg/ml) obtained for TIA and DTIA are equally potent to sludge bacteria as no statistical significant differences is found between the matched test results ( p > 0.05 and 95% CI overlapping for matched inhibition tests). Similar results were found for ENR and the metabolite CIP as matched tests, and they show equal potency and no statistical significant difference ( p > 0.05 and overlapping 95% CI for matched inhibition tests). TIA, DTIA, ENR and CIP all thus have an effect on the environmental bacterial community, which may change the bacterial population in the sludge.
5. Conclusion The susceptibility of S. hyicus and A. pleuropneumoniae were tested on TIA and three metabolites. Similar results were obtained for these two bacterial species. 8a-HTIA and 2b-HTIA showed very low potencies to the bacteria and their contribution to the
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selection of TIA resistant bacteria would therefore be neglectable. DTIA had a potency of 12.5–25% of the potency of TIA, and would be expected to select for TIA resistance if present. The susceptibilities of ENR and two metabolites were tested using indicator bacteria and the zoonotic C. coli. The metabolite ENR-N had a potency of 0.75– 1.5% of the potency of ENR, while the metabolite CIP had a potency almost like ENR. DTIA and CIP showed considerable potencies to activated sludge bacteria compared to the parent compounds TIA and ENR. This experimental set-up tested the effect of the presence of antimicrobials and metabolites of these on the microbial community. Results indicate a general reduction in CFU that could have an effect on the capacity of the active sludge to degrade other xenobiotics, but the experiment is not designed to quantify the effect on single members of the society. Results obtained here indicate that some of the metabolites of antimicrobials used for animal treatment can select for antimicrobial resistance. Presence of selective compound will even in very small concentration give resistant bacteria a growth advantage to sensitive tipping the population towards higher prevalence of resistance. Therefore in vivo studies of the metabolism of the antimicrobials and presence of their metabolites in animals and manure are needed to estimate their influence on the overall selection for antimicrobial resistant bacteria. It is possible that the metabolites can remain in the animal long after excretion and destruction of the original administered antimicrobial, and hereby uphold the antimicrobial resistance. This awaits investigation.
Acknowledgements The excellent technical assistance by Susanne Hermansen at The Danish University of Pharmaceutical Sciences is highly acknowledged as well as assistance and introduction to the microbiology by Vibeke Hansen and Anette Nielsen at the Danish Institute for Food and Veterinary Research (DFVF). The investigations at DFVF were financed from the Danish Directorate for Food, Fishery and Agri Business (project no. 3401-65-03-45).
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