Application of Microbiological Screening Tests in Assessment of Environmental Exposure to Antibiotics: Preliminary Studies

Abstract

Contact of aquatic microbiocenoses with antibiotics present in the environment can cause the former to develop resistance to antimicrobial drugs. Therefore, the search for methods to detect antibiotics and drug-resistant microorganisms in the environment is important. The presented paper proposes a simple procedure to assess environmental exposure to antibiotics and the presence of non-susceptible microorganisms. Medium solutions with selected antibiotics and a microbial growth indicator were applied to test plates, and were inoculated with water samples from various ecosystems. After incubation, the susceptibility of the microorganisms to antibiotics was determined and presented in chronic microbial toxic concentration (MTC) values. It was confirmed that the presented procedure enables the assessment of the antibiotic susceptibility and adaptation potential of unselected microorganisms from different aquatic ecosystems. However, the MTC values depend on the inoculum volume, the density and seasonal activity of the microorganisms, the method of inoculum preparation, and the incubation time of the test plate. The described procedure may be practically applied as a screening test to identify the presence of drug-resistant microorganisms. Additionally, it may also be suitable as a method to assess environmental exposure to antibiotics. However, prior standardisation is required before implementing this procedure in quantitative studies.

Keywords: antibiotics; bioassay; environmental pollution; microbial drug resistance; microbial toxic concentration.

Figure 1

Scans of test plates containing DOX (C_max 10 mg/L) inoculated with different volumes of inoculum from Przemsza River (a) or Brynica River (b). Incubation duration: 24 h; temperature: 303 K.

Figure 2

MTC values determined for DOX using inoculum from Brynica River and Przemsza River. (a) Effect of inoculum volume—incubation duration: 24 h; collection date: 29 September. (b) Effect of incubation duration—inoculum volume: 30 µL; collection date: 17 April. (c) Effect caused by peptone addition (50 mg/L) to inoculum and incubation for 24 h before inoculation—inoculum volume: 30 µL; incubation duration 24 h, collection date: 17 April. (d) Effect caused by differences in inoculum collection date—inoculum volume: 30 µL; incubation duration: 24 h.

Figure 3

MTC values determined for TYL using inoculum from Brynica River and Przemsza River. (a) Effect of inoculum volume—incubation duration: 24 h; collection date: 29 September (b) Effect of incubation duration—inoculum volume: 30 µL; collection date: 29 September (c) Effect caused by peptone addition (50 mg/L) to inoculum and incubation for 24 h before inoculation—inoculum volume: 30 µL; incubation duration 24 h; collection date: 29 September (d) Effect caused by differences in inoculum collection date—inoculum volume: 30 µL; incubation duration: 24 h.

Figure 4

Relative MTC values (a) and absolute MTC values (b) determined after 24 and 48 h of incubation for test plates containing a gradient of antibiotic concentrations and inoculated with 30 µL of the studied environmental samples.

Figure 5

Procedure for the preparation of the test plate.