Effects of Antibiotics on Aquatic Microbes

375 Pages·2013·4.04 MB·English

Effects of Antibiotics on
Aquatic Microbes

Iain Andrew Davies

For the degree of Doctor of Philosophy

University of York

Environment Department

August, 2010
Abstract
Antibiotics are designed to inhibit the growth of or kill bacteria. Of the many classes of antibiotics
that have been synthesized two particular classes of antibiotic have been frequently detected in the
natural environment, namely the tetracyclines and sulfonamides. Following use, these compounds
can enter the environment via the application of animal manures or domestic seawage sludge to
land as a fertiliser or from wastewater treatment plant effluents. Once in the environment the
compounds can persist and, depending on their properties, are distributed around the different
environmental media (i.e. surface waters, groundwaters, soils and sediments). Several studies have
suggested that tetracyclines and sulfonamides may have significant impacts on microbial function
in the natural environment. However most of these studies have used existing standardised test
procedures and/or unrealistic exposure conditions. The aim of the current study therefore was to
develop a more environmentally realistic test system for assessing the effects of antibiotics on
aquatic microbial communities and to apply this to assess the potential impacts of a sulphonamide
and a tetracycline antibiotic on aquatic microbial communities. The developed system, which was
an aquatic microcosm composed of fresh river water, return line treated sewage and OECD
synthetic sewage, was initially used to assess the effects of 3, 5-dichlorophenol, an OECD
reference toxicant. Effects of DCP were seen within the concentration range that is considered
valid by other standard microbial toxicity tests, such as OECD method 209 (3.2-32 mg/L). Since
the developed system was able to quantify effects on aquatic microbial communities, it can be seen
as being more environmentally relevant than existing standard tests, which rely on testing single
microbial species or sludge communities. The test system was then used to test the effects of
chlortetracycline (CTC) and sulfamethoxazole (SMX) on multisubstrate utilization. Following
addition of the study antibiotics, effects were observed for both antibiotics on total substrate
utilization and on the utilization of specific, ecologically relevant end-points (such as nutrient
cycling and polymer degradation) also. Effects were also observed on the structure of microbial
communities and on the functional diversity of substrate utilization (especially in the case of SMX
exposure) and these effects generally persisted for up to 3 d after addition of the study compounds
at a lower concentration of 0.1 mg/L for both compounds. The results indicate that both antibiotics
were inhibiting various aspects of substrate utilization, most of which were related to ecologically
relevant processes that occur in the aquatic environment. The results also suggest that SMX and
CTC exposure result in changes in the community structure of ecologically relevant groups of
microbes (such as bacteria involved in the nitrogen and carbon cycle bacteria). Microbial
communities that were exposed to CTC and SMX showed a varying degree of recovery, although
more long term effects were seen in SMX exposures; functional effects were observed for the
duration of the exposure. It was hypothesized that the observed recovery may have been caused by
the selection of antibiotic resistance in the exposed communities. Culture based resistance studies
showed that there was a significant temporal rise in CTC resistance at all CTC concentrations, but
2
not SMX resistance, across the dose range. In addition, a greater number of distinct microbial
morphotypes could be isolated from combined microcosms on day 7 compared with day 1. An
increase in CTC and SMX resistance in control exposures was also observed. In addition, distinct
resistant morphotypes developed in both dosed and undosed microcosms on day 7 of the study.
Furthermore, the results of a series of different culture based experiments suggested that multidrug
resistance was present in morphotypes that were isolated from dosed and control microcosms.
These data therefore suggest that multidrug resistance was present at the start of the exposure and
may also have been co-selected by exposure to CTC and SMX. In general, CTC and SMX show
signs that they may pose a risk to the environment and possibly the wider health of humans and
animals.

3
Contents

1. Introduction ................................................................................................................................... 23
1.2. Antibiotic Usage ........................................................................................................................ 27
1.3. Exposure Routes of Antibiotics into the Environment .............................................................. 27
1.5. Environmental Fate of Antibiotics ............................................................................................. 38
1.5.1. Adsorption and Absorption ..................................................................................................... 38
1.5.3. Degradation ............................................................................................................................. 39
1.5.3.1. Abiotic Degradation ............................................................................................................. 39
1.5.3.2. Biodegradation ..................................................................................................................... 40
1.6. Microbial Function in the Environment ..................................................................................... 40
1.6.1. The Nitrogen Cycle ................................................................................................................. 41
1.6.2. The Carbon Cycle ................................................................................................................... 42
1.7. Reported Effects of Antibiotics on Environmental Microbes .................................................... 44
1.7.1. Effects of Antibiotics on Single Species ................................................................................. 44
1.7.2. Effects of Antibiotics on Microbial Respiration ..................................................................... 48
1.7.3. Effects of Antibiotics on Microbial Growth ........................................................................... 49
1.7.4. Effects of Antibiotics on Specific Cellular Function .............................................................. 50
1.7.5. Effects of Antibiotics on Xenobiotic Degradation .................................................................. 54
1.8. Antibiotic Resistance ................................................................................................................. 55
1.8.1. Mechanisms of Resistance ...................................................................................................... 55
1.9. Current Testing Strategy Aimed at Protecting Environmental Microbes .................................. 59
1.9.1. Testing Strategy for Human Antibiotics ................................................................................. 59
1.9.2. Testing Strategy for Veterinary Antibiotics ............................................................................ 60
1.9.3. Reported Shortcomings of Microbial Standard Tests and Testing Strategy ........................... 62
1.10. Gaps in Knowledge and Major Concerns of Antibiotics in the Aquatic Environment ............ 64
1.11. Choice of Test Compounds ...................................................................................................... 64
1.12. Aims and Objectives ................................................................................................................ 65
2. General Materials and Methods .................................................................................................... 67
2.1. Introduction ................................................................................................................................ 67
2.1.1. Environmental Microbial Function Assays ............................................................................. 67
2.1.2. Multisubstrate Utilisation: The Biolog GN2 Assay ........................................................ 67
2.1.3. Advantages and Disadvantages of the Biolog GN2 Assay ..................................................... 72
2.1.4. Validity Criteria of Toxicity Tests .......................................................................................... 72
2.1.5. Use of Microcosms in Ecological Studies .............................................................................. 74
2.1.6. Rationale for Assay Choice..................................................................................................... 75
4
2.1.7. Choice of Statistical Analyses for Project ............................................................................... 76
2.1.7.1 Biolog Kinetic Data .............................................................................................................. 76
2.1.7.2. Multivariate Analysis and Ordination of Biolog Data ......................................................... 76
2.1.7.3. Functional Analysis of Biolog Data ..................................................................................... 77
2.1.7.4. Data Transformation and Analysis of CFU (Colony Forming Unit) Data ........................... 77
2.2. Materials and Methods ............................................................................................................... 78
2.2.1. Chemicals and Equipment ...................................................................................................... 78
2.2.2. Surface Water .......................................................................................................................... 78
2.2.3. Activated Sludge and Preparation of Treated Sewage ............................................................ 78
2.2.4. Preparation of OECD Synthetic Sewage................................................................................. 78
2.2.5. Microcosm Development ........................................................................................................ 79
2.2.5.1. Inoculum Type and Density ................................................................................................. 79
2.2.5.1.1. Procedure .......................................................................................................................... 79
2.2.5.1.2. Initial Data Analysis ......................................................................................................... 79
2.2.6. Initial Microcosm Studies ....................................................................................................... 79
2.2.6.1. Microcosm Set-up ................................................................................................................ 79
2.2.7. Validation of the Microcosm-Biolog GN2 system ................................................................. 82
2.2.7.1. Experimental Procedure ....................................................................................................... 82
2.2.8. Data Analysis .......................................................................................................................... 82
2.2.8.1. Kinetic Analysis ................................................................................................................... 82
2.2.8.2. Principal Component Analysis (PCA) and Ordination of PCA Data ................................... 83
2.2.8.3. Analysis of the Functional Diversity of Substrate Utilisation ............................................. 83
2.3. Results ........................................................................................................................................ 84
2.3.1. Inoculum Type and Density .................................................................................................... 84
2.3.2. Validation of the Biolog GN2/Microcosm System with 3, 5-DCP ......................................... 87
2.4. Discussion .................................................................................................................................. 95
3. Effects of CTC on Microbial Function ......................................................................................... 99
3.1. Tetracyclines .............................................................................................................................. 99
3.1.2. Chemistry of Tetracyclines ................................................................................................... 101
3.1.3. Tetracycline Mode of Action ................................................................................................ 102
3.1.4. Tetracycline Usage ................................................................................................................ 104
3.1.5. Occurrence of Tetracyclines in the Environment .................................................................. 105
3.1.6. Fate and Behavior of Tetracyclines in the Environment ....................................................... 108
3.1.6.1. Fate of Tetracyclines in Soil .............................................................................................. 108
3.1.6.2. Fate of Tetracyclines in the Aquatic Environment ............................................................ 111
3.1.7. Reported Effects of Tetracyclines on Environmental Microbes ........................................... 112
3.2. Materials and Methods ............................................................................................................. 115
3.2.1. Chemicals .............................................................................................................................. 115
5
3.2.2. Surface water ........................................................................................................................ 115
3.2.3. Activated Sludge and Preparation of Treated Sewage .......................................................... 115
3.2.4. Preparation of OECD Synthetic Sewage............................................................................... 115
3.2.5. Preparation of R2A Plates ..................................................................................................... 115
3.2.6. CTC Exposure Experiments .................................................................................................. 115
3.2.7. CFU (Colony Forming Unit) Counts .................................................................................... 115
3.2.8. Data Analysis ........................................................................................................................ 116
3.3. Chapter 3 Results ..................................................................................................................... 117
3.3.1. Effects of CTC on Organic Multi-Substrate Utilisation ....................................................... 117
3.3.1.1. Kinetic Model Plots ........................................................................................................... 117
3.3.1.2. Lag Phase versus Exposure Time ...................................................................................... 117
3.3.1.3. Max Rate versus Exposure Time ....................................................................................... 117
3.3.1.4. PCA Analysis ..................................................................................................................... 118
3.3.2. Effect of CTC on Amine and Amide Utilisation .................................................................. 124
3.3.2.1. Kinetic Model Plots of Amine and Amide Utilisation ....................................................... 124
3.3.2.2. Lag Phase of Amine and Amide Utilisation versus Exposure Time .................................. 124
3.3.2.3. Max Rate of Amine and Amide Utilisation versus Exposure Time ................................... 125
3.3.2.4. PCA Analysis of Amine and Amide Utilisation ................................................................ 125
3.3.3. Effect of CTC on Amino Acid Utilisation ............................................................................ 131
3.3.3.1. Kinetic Model Plots of Amino Acid Utilisation ................................................................ 131
3.3.3.2. Lag Phase of Amino Acid Utilisation versus Exposure Time ........................................... 131
3.3.3.3. Max Rate of Amino Acid Utilisation versus Exposure Time ............................................ 132
3.3.3.4. PCA Analysis of Amino Acid Utilisation .......................................................................... 132
3.3.4. Effect of CTC on Carbohydrate Utilisation .......................................................................... 138
3.3.4.1. Kinetic Model Plots of carbohydrate Utilisation ............................................................... 138
3.3.4.2. Lag Phase of Carbohydrate Utilisation versus Exposure Time.......................................... 138
3.3.4.3. Max Rate of Carbohydrate Utilisation versus Exposure Time .......................................... 138
3.3.4.4. PCA Analysis of Carbohydrate Utilisation ........................................................................ 139
3.3.5.1. Kinetic Model Plots of Carboxylic Acid Utilisation .......................................................... 145
3.3.5.2. Lag Phase of Carboxylic Acid versus Exposure Time ....................................................... 145
3.3.5.3. Max Rate of Carboxylic Acid Utilisation versus Exposure Time ...................................... 146
3.3.5.4. PCA Analysis of Carboxylic Utilisation ............................................................................ 146
3.3.6. Effect of CTC on Polymer Utilisation .................................................................................. 153
3.3.6.1. Kinetic Plot Models of polymer Utilisation ....................................................................... 153
3.3.6.2. Lag Phase of Polymer Utilisation versus Exposure Time .................................................. 153
3.3.6.3. Max Rate of Polymer Utilisation versus Exposure Time ................................................... 154
3.3.6.4. PCA Analysis of Polymer Utilisation ................................................................................ 154
3.3.7. Effect of CTC on Total CFU Counts .................................................................................... 160
6
3.3.8. Effect of CTC on the Functional Diversity of Multisubstrate Utilisation ............................. 161
3.4. Discussion ................................................................................................................................ 163
3.4.1. Effects of CTC on Total Organic Substrate Utilisation ........................................................ 163
3.4.2. Effect of CTC on Amine and Amide Utilisation .................................................................. 172
3.4.3. Effects of CTC on Amino Acid Utilisation .......................................................................... 174
3.4.4. Effects of CTC on Carbohydrate Utilisation ......................................................................... 176
3.4.5. Effect of CTC on Carboxylic Acid Utilisation ..................................................................... 178
3.4.6. Effect of CTC on Polymer Utilisation .................................................................................. 180
3.4.7. Effect of CTC on CFU Counts .............................................................................................. 181
4. Effects of Sulfamethoxazole on Microbial Function .................................................................. 184
4.1. Sulfonamides............................................................................................................................ 184
4.1.2. Chemistry of Sulfonamides ................................................................................................... 185
4.1.3. Sulfonamide Mode of Action ................................................................................................ 185
4.1.4. Sulfonamide Usage ............................................................................................................... 186
4.1.5. Occurrence of Sulfonamide Antibiotic in the Environment.................................................. 186
4.1.6. Fate and Behaviour of Sulfonamides in the Environment .................................................... 190
4.1.6.1. Sorption .............................................................................................................................. 190
4.1.6.2. Transportation of Sulfonamides ......................................................................................... 190
4.1.6.3. Dissipation of Sulfonamide Antibiotics - The Terrestrial Environment ............................ 192
4.1.6.4. Dissipation of Sulfonamide Antibiotics - The Aquatic Environment ................................ 192
4.1.7. Reported Effects of Sulfonamide Antibiotics on Microbial Function in the Environment ... 193
4.1.8. Aims and Objectives of Chapter ........................................................................................... 195
4.2. Materials and Methods ............................................................................................................. 196
4.2.1. Study outline ......................................................................................................................... 196
4.2.2. Chemicals .............................................................................................................................. 196
4.2.3 Surface water ......................................................................................................................... 196
4.2.4. Activated Sludge and Preparation of Treated Sewage .......................................................... 196
4.2.5. Preparation of OECD Synthetic Sewage............................................................................... 196
4.2.6. Preparation of R2A Plates ..................................................................................................... 196
4.2.7. Sulfamethoxazole Exposure Experiments ............................................................................ 196
4.2.8. CFU (Colony Forming Unit) Counts .................................................................................... 197
4.2.9. Data Analysis ........................................................................................................................ 197
4.3. Results ...................................................................................................................................... 198
4.3.1.1. Effect of Sulfamethoxazole on Total Substrate Utilisation ................................................ 198
4.3.1.2. Kinetic Model Plots ........................................................................................................... 198
4.3.1.3. Lag Phase Duration versus Exposure Time ....................................................................... 198
4.3.1.4. Max Rate versus Exposure Time ....................................................................................... 198
4.3.1.5. PCA Analysis of Total Substrate Utilisation ..................................................................... 199
7
4.3.2. Effect of SMX on Amine and Amide Utilisation.................................................................. 205
4.3.2.1. Kinetic Model Plots of Amine and Amide Utilisation ....................................................... 205
4.3.2.2. Lag Phase Duration: Amines and Amides ......................................................................... 205
4.3.2.3. Max Rate of Amine and Amide Utilisation ....................................................................... 205
4.3.2.4. PCA Analysis of Amine and Amide Utilisation ................................................................ 206
4.3.3. Effect of SMX on Amino Acid Utilisation ........................................................................... 212
4.3.3.1. Kinetic Model Plots of Amino Acid Utilisation ................................................................ 212
4.3.3.2. Lag Phase Duration: Amino Acid Utilisation .................................................................... 212
4.3.3.3. Max Rate of Amino Acid Production ................................................................................ 212
4.3.3.4. PCA Analysis of Amino Acid Utilisation .......................................................................... 212
4.3.4. Effect of SMX on Carbohydrate Utilisation ......................................................................... 219
4.3.4.1. Kinetic Plots of Carbohydrate Utilisation .......................................................................... 219
4.3.4.2. Lag Phase Duration: Carbohydrate Utilisation .................................................................. 219
4.3.4.3. Max Rate of Carbohydrate Utilisation ............................................................................... 219
4.3.4.4. PCA Analysis of Carbohydrate Utilisation ........................................................................ 220
4.3.5. Effect of SMX on Carboxylic Acid Utilisation..................................................................... 226
4.3.5.1. Kinetic Model Plots of Carboxylic Acid Utilisation .......................................................... 226
4.3.5.2. Lag Phase Duration: Carboxylic Acid Utilisation ............................................................. 226
4.3.5.3. Max Rate of Carboxylic Acid Utilisation .......................................................................... 227
4.3.5.4. PCA Analysis of Carboxylic Acid Utilisation ................................................................... 227
4.3.6. Effect of SMX on Polymer Utilisation ................................................................................. 233
4.3.6.1. Kinetic Plots of Polymer Utilisation .................................................................................. 233
4.3.6.2. Lag Phase Duration: Polymer Utilisation .......................................................................... 233
4.3.6.3. Max Rate of Polymer Utilisation ....................................................................................... 234
4.3.6.4. PCA Analysis of Polymer Utilisation ................................................................................ 234
4.3.6. Effect of SMX on Total CFU Counts ................................................................................... 240
4.3.7. Effect of Sulfamethoxazole on the Functional Diversity of Multisubstrate Utilisation ........ 241
4.4. Discussion ................................................................................................................................ 243
4.4.1. Effects of Sulfamethoxazole on Total Organic Substrate Utilisation ................................... 243
4.4.2. Effect of Sulfamethoxazole on Substrate Guild Utilisation .................................................. 249
4.4.2.1. Amines and Amides Utilisation ......................................................................................... 250
4.4.2.2. Amino Acid Utilisation ...................................................................................................... 251
4.4.2.3. Carbohydrate Utilisation .................................................................................................... 252
4.4.2.4. Carboxylic Acid Utilisation ............................................................................................... 254
4.4.2.5. Polymer Utilisation ............................................................................................................ 255
4.4.3. CFU Counts .......................................................................................................................... 257
5. Presence and Development of Antimicrobial Resistance in Aquatic Microcosms ..................... 259
5.1. Specific Antibiotic Resistance ................................................................................................. 259
8
5.1.1. Tetracycline Resistance......................................................................................................... 259
5.1.2. Sulfonamide Resistance ........................................................................................................ 260
5.1.3. Environmental Monitoring of Tetracycline and Sulfonamide Resistance Genes ................. 260
5.1.4. Multidrug Resistance (MDR) in the Environment ................................................................ 266
5.1.5. MDR in the Aquatic Environment ........................................................................................ 266
5.1.6. MDR in the Marine and Estuarine Environment .................................................................. 267
5.1.7. MDR in the Soil Environment .............................................................................................. 268
5.1.8. Multidrug Efflux in the Environment ................................................................................... 268
5.1.9. Reported Incidences of MEPPs in the Environment ............................................................. 269
5.1.10. Transfer of Genetic Elements between Environmental Microbes ...................................... 270
5.1.11. Direct Experimental Evidence for Gene Transfer between Environmental Bacteria ......... 270
5.1.12. Reported Transfer of Single Drug Resistance Determinants in the Environment .............. 271
5.1.13. Reported Transfer of Multiple Drug Resistance Determinants in the Environment ........... 272
5.1.14. Development of Antibiotic Resistance in the Environment ................................................ 273
5.1.14.1. External Sources of Resistance Genes in the Environment ............................................. 273
5.1.14.2. Antibiotics as a Selective Pressure for the Formation of Antibiotic Resistance in the
Environment .................................................................................................................................... 275
5.1.14.3. Selection of Single–Drug Resistance ............................................................................... 275
5.1.14.4. Co–Selection of Antibiotic Resistance: Development of Multidrug Resistance ............. 277
5.1.15. Fears over Antibiotic Resistance in the Environment: The Broader Issues ........................ 277
5.1.16. The Biolog PM11C and PM12B Assays as a Phenotypic Reporter of Multidrug Resistance
........................................................................................................................................................ 279
5.1.17. Aims and Objectives ........................................................................................................... 282
5.2. Materials and Methods ............................................................................................................. 283
5.2.1. Reagents ................................................................................................................................ 283
5.2.2. Preparation of R2A/CTC and Sulfamethoxazole Agar Plates .............................................. 283
5.2.3. Preparation of Biolog BUG B Media .................................................................................... 283
5.2.4. Preparation of Biolog PM Media .......................................................................................... 283
5.2.5. CFU Counts of CTC and Sulfamethoxazle–Resistant Bacteria ............................................ 283
5.2.6. Preparation of R2A Slopes .................................................................................................... 284
5.2.7. Morphotyping and Storage of Antibiotic Resistant Bacteria ................................................ 284
5.2.8. Biolog PM11C and PM12B Array Procedure ....................................................................... 284
5.2.9. Resistant CFU Counts ....................................................................................................... 285
5.2.10. Data Analysis ...................................................................................................................... 285
5.2.10.1. Total Resistant CFU Counts over Time: Formation of Culturable Resistance ................ 285
5.2.10.2. Morphotype Data ............................................................................................................. 285
5.2.10.3. Biolog PM Data: MDR Profiling of SMX/CTC-Resistant Morphotypes ........................ 286
5.3: Results ...................................................................................................................................... 287
9
5.3.1: Total Resistant CFU Counts: Formation of Single Drug Resistance over Time .................. 287
5.3.2. Total CFU Counts of CTC and SMX-Resistant Isolates: Day 1 versus Day 7 ..................... 289
5.3.3. CFU Counts and Morphotyping of Resistant Bacteria in Control Microcosms on day 1 and 7
........................................................................................................................................................ 290
5.3.4. Analysis of Day 7 Isolates that could not be Cross-Referenced with a Day 1 Control
Morphotype ..................................................................................................................................... 295
5.3.5. Temporal Expression of MDR by the Total Microbial Community (Control and Exposed) 298
5.3.5.1. Frequency of MDR Phenotypes on Day and Day 7 of CTC and SMX Exposures ............ 298
5.3.5.2. Temporal Change of MDR Profile of the Total Microbial Community ............................ 301
5.3.6. Co-Selection of MDR in CTC and SMX-Resistance Microbes by CTC and SMX Exposure
........................................................................................................................................................ 303
5.4. Discussion ................................................................................................................................ 306
5.4.1. Culture of CTC / SMX - Resistant Morphotypes from Aquatic Microcosms....................... 306
5.4.2 Development of CTC and SMX Resistance over Time in Aquatic Microcosms ................... 307
5.4.3. Presence of Multidrug Resistance on Day 1 of Aquatic Microcosm Experiments ............... 309
5.4.4. Development of Multidrug Resistance in Aquatic Microcosms ........................................... 310
6. General Discussion ..................................................................................................................... 315
6.1. General Conclusions of Thesis ................................................................................................ 324
Appendix………………………………………………………………………………………….327
Appendix 1a………………………………………………………………………………………329
Appendix 1b..……………………………………………………………………………………..331
Appendix 1c………………………………………………………………………………………332
Appendix 1d……………………………………………………………………………….……...333
References………………………………………………………………………………………...334

10