Advertisement

Environmental metabolites of fluoroquinolones: synthesis, fractionation and toxicological assessment of some biologically active metabolites of ciprofloxacin

Abstract

Background, aim, and scope

Biowastes produced by humans and animals are routinely disposed of on land, and concern is now growing that such practices provide a pathway for fluoroquinolone (FQs) antibacterial agents and their environmental metabolites (FQEMs) to contaminate the terrestrial environment. The focus of concern is that FQs and FQEMs may accumulate in amended soils to then adversely impact on the terrestrial environment. One postulated impact is the development of a selective environment in which FQ-resistant bacteria may grow. To find evidence in support of an accumulation of antibacterial-like activity, it was first necessary to establish whether any biologically active FQEMs could be synthesized by physicochemical factors that are normally present in the environment. However, many FQEMs are not commercially available to be used as standards in such studies. FQEMs were therefore synthesized using well-defined processes. They were subsequently analyzed using spectroscopy (UV-vis) and high performance liquid chromatography with mass spectral detection. The antibacterial-like activities of fractionated FQEMs were then assessed in novel bacterial growth inhibition bioassays, and results were compared to those obtained from instrumental analyses.

Materials and Methods

Parent FQs were either exposed to sunlight or were synthesized using defined aerobic microbial (Mycobacterium gilvum or a mixed culture derived from an agricultural soil) fermentation processes. Mixtures of FQEMs derived from photo- and (intracellular) microbial processes were isolated by preparative chromatography and centrifugation techniques, respectively. Mixtures were subsequently fractionated using analytical high-performance thin layer chromatography (HPTLC), and excised analytes were tested in bioautography assays for their antibacterial-like activities. Two bacteria, Escherichia coli (E. coli) and Azospirillum brasilense (A. brasilense) were used as reporter organisms in testing FQ standards and any subtle differences between biologically active FQEMs of ciprofloxacin (CF).

Results and discussion

FQEMs produced in the photo-synthetic process had UV-vis profiles that were indistinguishable from the parent FQs, and yet mass spectral data revealed the presence of N-formylciprofloxacin (FCF). In contrast, the UV-vis profiles of FQEMs synthesized by M. gilvum and a mixed culture of microorganisms had UV-vis profiles that were similar to one another and markedly different to the parent fluoroquinolones. Mass spectral studies confirmed the presence of FCF and N-acetylciprofloxacin in both microbial ferments. In addition, a photo-FQEM (Cp 6), three M. gilvum FQEMs (Cm 5, Cm 8, and Cm 10) and a mixed culture FQEM (Cs 6) of CF and many other FQEMs of CF, norfloxacin (NF), and enrofloxacin (EF) were fractionated using HPTLC, although their identities have yet to be confirmed. Differences between bioautography results were obtained when E. coli or A. brasilense were used as reporter organisms. Parent FQs (CF and EF) and the FQEMs of CF (Cp 6, Cm 8, and Cs 6) displayed antibacterial-like activity when using E. coli as the reporter organism. In contrast, A. brasilense was insensitive to parent CF and sensitive to EF and all tested FQEMs of CF. Results are consistent with photo- and microbial processes modifying CF in different ways, with the latter changing the UV-vis chromophores. It can be inferred that a lack of detection of analytes (especially photo-FQEMs) when using UV-vis does not necessarily indicate an absence of analyte. Additionally, similarities between the UV-vis profiles of FQEMs extracted from the (monoculture) M. gilvum and the mixed culture microbial aerobic ferments are consistent with similar processes operating in both ferments. Results of HPTLC and bioautography studies revealed that mixtures of (photo- and microbial) FQEMs could be fractionated into individual components.

Conclusions

Bioactive FQEMs of ciprofloxacin, as a representative FQ, can be synthesized by photo- and microbial processes, and their detection required the use of both instrumental and bioautography analytical techniques. It is likely that such FQEMs will also be present on agricultural land that has been repeatedly amended with FQ-contaminated biosolids.

Recommendations and perspectives

The use of instrumental analytical techniques alone and especially photometric detection techniques will underestimate antibacterial-like activities of FQEMs. Moreover, the extraction technique(s) and the selected toxicological endpoint(s) require careful consideration when assessing bioactivity. It is therefore recommended that instrumental analytical techniques and several bioautography assays be performed concurrently, and bioautography assays should use a variety of reporter organisms. Two types of bacterial growth bioassays are recommended in any assessment of antibacterial-like activity derived from CF (and possibly from other FQs). A standardized E. coli bioassay should be used as a general screening procedure to facilitate intra- and inter-laboratory exchange of data. Additionally, soil-specific (region-specific) growth inhibition bioassays should be undertaken using several species of endemic soil bacteria. It is likely that the two sets of data will be useful in future risk assessment processes.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Acea MJ, Alexander M (1988) Growth and survival of bacteria introduced into carbon-amended soil. Soil Biol Biochem 20:703–709

  2. Adjei MD, Heinze TM, Deck J, Freeman JP, Williams AJ, Sutherland JB (2006) Transformation of the antibacterial agent norfloxacin by environmental Mycobacteria. Appl Environ Microbiol 72:5790–5793

  3. Adjei MD, Heinze TM, Deck J, Freeman JP, Williams AJ, Sutherland JB (2007) Acetylation and nitrosation of ciprofloxacin by environmental strains of Mycobacteria. Can J Microbiol 53:144–147

  4. Amoo SO, Ndhlala AR, Finnie JF, Van Staden J (2009) Antibacterial, antifungal and anti-inflammatory properties of Burchellia bubalina. South African J Botany 75:60–63

  5. Anadón A, Martínez-Larrañaga MR, Díaz MJ, Bringas P, Martínez MA, Fernández-Cruz ML, Fernández MC, Fernández R (1995) Pharmacokinetics and residues of enrofloxacin in chickens. Am J Vet Res 56:501–506

  6. Anadón A, Martínez-Larrañaga MR, Díaz MJ, Fernández-Cruz ML, Martínez MA, Frejo MT, Martínez M, Iturbe J, Tafur M (1999) Pharmacokinetic variables and tissue residues of enrofloxacin and ciprofloxacin in healthy pigs. Am J Vet Res 60:1377–1382

  7. Anadón A, Martínez-Larrañaga MR, Iturbe J, Martínez MA, Diaz MJ, Frejo MT, Martínez M (2001) Pharmacokinetics and residues of ciprofloxacin and its metabolites in broiler chickens. Res Vet Sci 71:101–109

  8. Bailey RR (1992) Quinolones in the treatment of uncomplicated urinary tract infections. Int J Antimicrob Agents 2:19–28

  9. Beausse J (2004) Selected drugs in solid matrices: a review of environmental determination, occurrence and properties of principal substances. TrAC Trends in Analytical Chem 23:753–761

  10. Bêhal V (2006) Mode of action of microbial bioactive metabolites. Folia Microbiol 51:359–369

  11. Blau K (1993) Acylation. In: Blau K, Halket JM (eds) Handbook of derivatives for chromatography, 2nd edn. Wiley, Chichester, pp 31–50

  12. Burhenne J, Ludwig M, Nikoloudis P, Spiteller M (1997) Photolytic degradation of fluoroquinolone carboxylic acids in aqueous solution. Part I: primary photoproducts and half-lives. Environ Sci Pollut Res 4:10–15

  13. Campbell JIA, Jacobsen CS, Sorensen J (1995) Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils. FEMS Microbiol Ecol 18:51–62

  14. Cardoza LA, Knapp CW, Larive CK, Belden JB, Lydy M, Graham DW (2005) Factors affecting the fate of ciprofloxacin in aquatic field systems. Water Air Soil Pollut 161:383–398

  15. Cester CC, Toutain PL (1997) A comprehensive model for enrofloxacin to ciprofloxacin transformation and disposition in dog. J Pharm Sci 86:1148–1155

  16. Chien C, Kuo Y, Chen C, Hung C, Yeh C, Yeh W (2008) Microbial diversity of soil bacteria in agricultural field contaminated with heavy metals. J Environ Sci 20:359–363

  17. Cox CE (1991) Oral temafloxacin compared to norfloxacin for the treatment of complicated urinary tract infections. Am J Med 91:S129–S133

  18. de Mouy D, Fabre R, Cavallo JD (2007) Infections urinaires communautaires de la femme de 15 a 65 ans: sensibilite aux antibiotiques de E. coli en fonction des antecedents: etude AFORCOPI-BIO 2003. Med Mal Infect 37:594–598

  19. Edlund C, Lindqvist L, Nord CE (1988) Norfloxacin binds to human fecal material. Antimicrob Agents Chemother 32:1869–1874

  20. Gay JD, DeYoung DR, Roberts GD (1984) In vitro activities of norfloxacin and ciprofloxacin against Mycobacterium tuberculosis, M. avium complex, M. chelonei, M. fortuitum, and M. kansasii. Antimicrob Agents Chemother 26:94–96

  21. Golet EM, Strehler A, Alder AC, Giger W (2002) Determination of fluoroquinolone antibacterial agents in sewage sludge and sludge-treated soil using accelerated solvent extraction followed by solid-phase extraction. Anal Chem 74:5455–5462

  22. Golet EM, Xifra I, Siegrist H, Alder AC, Giger W (2003) Environmental exposure assessment of fluoroquinolone antibacterial agents from sewage to soil. Environ Sci Technol 37:3243–3249

  23. Grobbel M, Lubke-Becker A, Wieler LH, Froyman R, Friederichs S, Filios S (2007) Comparative quantification of the in vitro activity of veterinary fluoroquinolones. Vet Microbiol 124:73–81

  24. Iravani A (1991) Treatment of uncomplicated urinary tract infections with temafloxacin. Am J Med 91:S124–S128

  25. Isbell RF (1996) The Australian soil classification. CSIRO, Melbourne

  26. Jacobs MR (1991) Evaluation of the bactericidal activity of temafloxacin. Am J Med 91:S31–S34

  27. Jones RN, Hoban DJ (1994) North American (United States and Canada) comparative susceptibility of two fluoroquinolones: ofloxacin and ciprofloxacin: a 53-medical-center sample of spectra of activity. Diagn Microbiol Infect Dis 18:49–56

  28. Jones RN, Kehrberg EN, Erwin ME, Anderson SC (1994) Prevalence of important pathogens and antimicrobial activity of parenteral drugs at numerous medical centers in the United States I. Study on the threat of emerging resistances: real or perceived? Diagn Microbiol Infect Dis 19:203–215

  29. Jork H, Funk W, Fischer W, Wimmer H (1990) Thin-layer chromatography: reagents and detection methods, vol 1. Physical and chemical detection methods. VCH Verlagsgesellschaft GmbH, Weinheim

  30. Jung CM, Heinze TM, Strakosha R, Elkins CA, Sutherland JB (2009) Acetylation of fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated from a municipal wastewater treatment plant. J Appl Microbiol 106:564–571

  31. Karl W, Schneider J, Wetzstein H-G (2006) Outlines of an "exploding" network of metabolites generated from the fluoroquinolone enrofloxacin by brown rot fungus Gloeophyllum striatum. Appl Microbiol Biotechnol 71:101–113

  32. Kim Y-H, Engesser K-H, Cerniglia CE (2003) Two polycyclic aromatic hydrocarbon o-quinone reductases from a pyrene-degrading Mycobacterium. Arch Biochem Biophys 416:209–217

  33. Kumar K, Gupta SC, Chander Y, Singh AK (2005) Antibiotic use in agriculture and its impact on the terrestrial environment. In: Sparks DL (ed) Advances in agronomy. Academic Press, New York, pp 1–54.

  34. Kümmerer K (2003) Pharmaceuticals in the environment: sources, fate, effects and risks, 2nd edn. Springer, Berlin, 55

  35. Lewis G (2011) Thesis: fate and dynamics of fluoroquinolone antibiotics as pharmaceutically active compounds (PhACs) in the soil environment. http://arrow.unisa.edu.au:8081/1959.8/119064. Accessed 29th December 2011.

  36. Lindberg RH, Björklund K, Rendahl P, Johansson MI, Tysklind M, Andersson BAV (2007) Environmental risk assessment of antibiotics in the Swedish environment with emphasis on sewage treatment plants. Water Res 41:613–619

  37. Mahamat A, Lavigne J-P, Bouziges N, Daurès J-P, Sotto A (2006) Profils de résistance des souches urinaires de Proteus mirabilis de 1999 à 2005 au CHU de Nîmes. Antimicrobial susceptibility of Proteus mirabilis urinary tract isolates from 1999 to 2005 at Nîmes university hospital. Pathol Biol 54:456–461

  38. Matthews JNS, Altman DG, Campbell MJ, Royston P (1990) Analysis of serial measurements in medical research. British Medical Journal 300:230–235

  39. McLafferty W, Turecek F (1993) Interpretation of mass spectra, 4th edn. University Science Books, Mill Valley, pp. 72–76

  40. Mitani K, Kataoka H (2006) Determination of fluoroquinolones in environmental waters by in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry. Anal Chim Acta 562:16–22

  41. Ramirez-Ronda C, Colon M, Saavedra S, Jacobo S, Corrado ML (1987) Treatment of urinary tract infection with norfloxacin: analysis of cost. Am J Med 82:75–78

  42. Sasabe H, Tsuji A, Sugiyama Y (1998) Carrier-mediated mechanism for the biliary excretion of the quinolone antibiotic grepafloxacin and its glucuronide in rats. J Pharmacol Exp Ther 284:1033–1039

  43. Smith SR (1996) Agricultural recycling of sewage sludge and the environment. CAB International, Wallingford

  44. Strehler A (2001) Entwicklung einer Extraktionsmethode zur Bestimmung von Fluorochinolones in Klarschlamm. ETH, Zurich

  45. Sunderland J, Tobin CM, Hedges AJ, MacGowan AP, White LO (2001) Antimicrobial activity of fluoroquinolone photodegradation products determined by parallel-line bioassay and high performance liquid chromatography. J Antimicrob Chemother 47:271–275

  46. Tanaka M, Takahashi K, Saika T, Kobayashi I, Ueno T, Kumazawa J (1998) Development of fluoroquinolone resistance and mutations involving GyrA and ParC proteins among Neisseria gonorrhoeae isolates in Japan. J Urol 159:2215–2219

  47. US Census Bureau (2009) http://www.census.gov/. Accessed 18 October 2010

  48. USEPA (1999) Biosolids generation, use, and disposal in the United States. US Environmental Protection Agency Municipal and Industrial Solid Waste Division Office of Solid Waste, EPA530-R-99-009

  49. UWI (1993) News: from waste to want-united water biosolids enrich farmlands. United Water Industries. http://www.uwi.com.au/index.php?zoom_query=From+Waste+To+Want-United+Water+Biosolids+Enrich+Farmlands&zoom_per_page=10&zoom_and=0&PID=1&SF=&Go.x=27&Go.y=9. Accessed 26 May 2011

  50. Vieno N, Tuhkanen T, Kronberg L (2007) Elimination of pharmaceuticals in sewage treatment plants in Finland. Water Res 41:1001–1012

  51. Waites KB, Canupp KC, DeVivo MJ (1991) Efficacy and tolerance of norfloxacinin treatment of complicated urinary tract infection in outpatients with neurogenic bladder secondary to spinal cord injury. Urology 38:589–596

  52. Walters E, McClellan K, Halden RU (2010) Occurrence and loss over three years of 72 pharmaceuticals and personal care products from biosolids–soil mixtures in outdoor mesocosms. Water Res 44:6011–6020

  53. Waters B, Davies J (1997) Amino acid variation in the GyrA subunit of bacteria potentially associated with natural resistance to fluoroquinolone antibiotics. Antimicrob Agents Chemother 41:2766–2769

  54. Wetzstein H-G, Schmeer N, Karl W (1997) Degradation of the fluoroquinolone enrofloxacin by the brown rot fungus Gloeophyllum striatum: identification of metabolites. Appl Environ Microbiol 63:4272–4281

  55. Wetzstein H-G, Schneider J, Karl W (2006) Patterns of metabolites produced from the fluoroquinolone enrofloxacin by Basidiomycetes indigenous to agricultural sites. Appl Microbiol Biotechnol 71:90–100

  56. Wetzstein H-G, Schneider J, Karl W (2009) Comparative biotransformation of fluoroquinolone antibiotics in matrices of agricultural relevance. In: Henderson KL and Coats JR (eds) Veterinary pharmaceuticals in the environment, Chapter 6. American Chemical Society, Washington, pp. 67–91

Download references

Acknowledgments

This research was funded by the Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE). The authors would like to acknowledge the support of the Centre for Environmental Risk Assessment and Remediation (University of South Australia) for this research.

Author information

Correspondence to Gareth Lewis.

Additional information

Responsible editor: Philippe Garrigues

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 732 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lewis, G., Juhasz, A. & Smith, E. Environmental metabolites of fluoroquinolones: synthesis, fractionation and toxicological assessment of some biologically active metabolites of ciprofloxacin. Environ Sci Pollut Res 19, 2697–2707 (2012). https://doi.org/10.1007/s11356-012-0766-7

Download citation

Keywords

  • Fluoroquinolone
  • Metabolite
  • Transformation product
  • Biosolid
  • Ciprofloxacin
  • Enrofloxacin
  • Norfloxacin
  • Pharmaceutically active compounds
  • PhACs
  • Pharmaceuticals and personal care products
  • PPCPs
  • Resistance to antibiotics
  • Risk