Resistance in the environment

K. Kümmerer*

Applied Environmental Research Section, Institute of Environmental Medicine and Hospital Epidemiology, Freiburg University Hospital, Hugstetter Straße 55, D-79106 Freiburg, Germany


    Abstract
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
Antibiotics, disinfectants and bacteria resistant to them have been detected in environmental compartments such as waste water, surface water, ground water, sediments and soils. Antibiotics are released into the environment after their use in medicine, veterinary medicine and their employment as growth promoters in animal husbandry, fish farming and other fields. There is increasing concern about the growing resistance of pathogenic bacteria in the environment, and their ecotoxic effects. Increasingly, antibiotic resistance is seen as an ecological problem. This includes both the ecology of resistance genes and that of the resistant bacteria themselves. Little is known about the effects of subinhibitory concentrations of antibiotics and disinfectants on environmental bacteria, especially with respect to resistance. According to the present state of our knowledge, the impact on the frequency of resistance transfer by antibacterials present in the environment is questionable. The input of resistant bacteria into the environment seems to be an important source of resistance in the environment. The possible impact of resistant bacteria on the environment is not yet known. Further research into these issues is warranted.

Keywords: environmental microbiology , gene transfer , ecotoxicity , xenobiotics


    Introduction
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
Antibiotics are used extensively to prevent or to treat microbial infections in human and veterinary medicine. Apart from their use in aquaculture, they are also employed to promote more rapid growth of livestock.

Most of the compounds used in medicine are only partially metabolized by patients and are then discharged into the hospital sewage system or directly into municipal waste water if used at home. Along with excreta, they flow with municipal waste water to the sewage treatment plant (STP). They may pass through the sewage system and end up in the environment, mainly in the water compartment. Antibacterial substances used for livestock enter the environment when manure is applied to fields. These antibiotics may either end up in soil or sediment or in ground water.

Antimicrobial agents are also used to treat infections in intensive fish farming where they are added directly to the water, resulting in high local concentrations in the water compartment and adjoining sediments. Some antibiotics such as streptomycins are used in fruit growing, others in bee-keeping. Disinfectants are widely used in the food and glue industries, medicine and livestock rearing. In addition to antimicrobials and disinfectants, resistant bacteria themselves are excreted by humans and animals14 and are emitted into sewage or manure and other environmental compartments.

The unwanted effects of microbial growth have long been controlled through use of antimicrobials. It has also long been recognized that susceptibility to such chemicals varies markedly between different groups of organisms and within these groups. The different mechanisms of action and the methods used to evaluate susceptibility are crucial for the results of susceptibility testing and the evaluation of resistance.5 Resistance is a description of the relative insusceptibility of a microorganism to a particular treatment under a particular set of conditions. Therefore, care must be taken in interpreting the literature and the reader is advised to refer for details to the literature. For antibacterials, resistance is usually quantified as the minimum concentration required to assert a definable effect (e.g. growth inhibition) on a population of cells. Wherever there is a change in susceptibility that renders an agent ineffective against a certain organism, this organism is referred to as resistant. Many organisms have always been insensitive to and are thereby intrinsically resistant to a particular agent by nature of their physiology or biochemistry. Susceptible organisms can become insensitive by mutation or by incorporation of the genetic information which encodes the resistance.

This short paper summarizes findings on antibiotic resistance in different environmental compartments. It is structured as follows: first, a brief background is given on resistance and antimicrobials in the environment. Next, different sources for the input of resistant bacteria and antibiotics into the environment such as hospital effluent and municipal sewage water including sewage treatment plants are discussed. Findings on other compartments of the aquatic environment such as surface water, ground water, sea water, and sediments are briefly summarized. Issues in our understanding of the interaction of bacteria and antimicrobials in the environment are outlined before research needs are addressed.


    Antibiotics, bacteria, and resistance in the environment
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
Antibiotics in the environment

Although antibiotics have been applied in large quantities for some decades, until recently the existence of these substances in the environment was accorded very little attention. Studies conducted in various countries have detected a number of antibiotics in the low microgram per litre or the nanogram per litre (Table 1) range in different environmental compartments, i.e. hospital effluent, municipal waste water, effluent from sewage treatment plants, surface water and in some cases ground water.14,69 The compounds detected are from different important antibiotic classes such as macrolides, tetracyclines, sulphonamides, quinolones and others as far as analytical methods are available. ß-Lactams have not been detected yet despite the fact that ß-lactams are used in the highest amounts.10 Obviously, most of the atibiotics are not fully eliminated during the sewage purification process. The results of investigations using test systems indicate that a number of antibiotics and disinfectants are not biodegradable in the aquatic environment.11,12 In soil, tetracycline concentrations in the range of several hundred micrograms per kilogram have been detected some months after manure application.13,14


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Table 1. Concentrations (ng/L) of antibiotics and disinfectants measured in the aqueous environment (for references see text)

 
The main processes of substance elimination in the environment, especially in waste water, sediments, and soil are due to bacteria. The concentration of antibiotics may be much higher if the active compounds are persistent and accumulate, e.g. by sorption to solid surfaces in certain environmental compartments such as sewage sludge, sediments or soil. In these cases, the role of antimicrobial concentration could be different from that in water. It is not known how strongly the antibiotics are sorbed and under what circumstances they are still available and active after sorption.

Bacteria in the environment

Apart from the issue of resistance (see below), it has to be kept in mind that bacteria form one of the most important groups of organisms in soil and in other environmental compartments as well as in natural or technical sewage treatment. Without bacteria, water would not be clarified. Bacteria are essential for the closing of nutrient and geochemical cycles such as the carbon, nitrogen, sulphur and phosphorous cycle. Without bacteria, soil would not be fertile and organic matter such as straw or leaves would accumulate within a short time. In soil, naturally occurring antibiotics from bacteria and fungi amongst others control the dynamics of bacterial populations. In contrast to these, most of the compounds used nowadays are semi-synthetic or synthetic. They are often much more stable and are not biodegradable by bacteria. They may therefore persist in the environment. Furthermore, they often have a different, e.g. broader, activity spectrum.

Antimicrobial resistance in the environment

The most common response of the cell to antibiotics is to cease growing (bacteriostasis), but for certain classes of compounds such as ß-lactams, continued growth is permitted, with inhibition of the target in the organism leading indirectly to cell death. In the treatment of an infection, bacteriostasis is often effective because the killing and elimination of the pathogen are mediated through host immune defences. Such augmentation is typically absent in the environment. In this respect, there is little knowledge of environments such as waste water, sludge, surface water, and soil compared with the medical use and effectiveness of antibacterials. Furthermore, in waste water, surface water, sediments, sludge and soil a cocktail of different active compounds may be present in contrast to the medical and veterinary application of antibiotics and disinfectants. Concentrations are normally some orders of magnitude lower in the environment than for therapeutic use.14,610

There has been growing concern about antimicrobial resistance for some years now. In a report by the UK House of Lords, it is stated: ‘Resistance to antibiotics and other anti-infective agents constitutes a major threat to public health and ought to be recognized as such more widely than it is at present.’15 There is a vast amount of literature related to use of antimicrobials and resistance against them in medicine, veterinary medicine and animal husbandry.1625 Resistance genes as well as resistant bacteria in the environment are increasingly seen as an ecological problem.25,26 The most prominent medical examples are vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and multi-resistant pseudomonads. With respect to the causes of resistance, the focus is on the use of antimicrobials in hospitals, by medical practitioners and in animal husbandry.

The selection pressure of antibiotics present above a certain concentration against the microbial biocoenosis is an important factor in the selection and spread of resistant bacteria. The transfer of resistance genes as well as the already resistant bacteria themselves is favoured particularly by the presence of antibiotics over a long period and at subtherapeutic concentrations. They play a role in the stimulation of resistance and its transfer by genetic material in bacteria. Exposure of bacteria to such sub-therapeutic antimicrobial concentrations is thought to increase the speed with which resistant bacterial strains are selected, e.g. if antibiotics are used as growth promoters1618 or by improper use in veterinary medicine and medicine.19,23 In general, the emergence of resistance is a highly complex process, which is not yet fully understood with respect to the significance of the interaction of bacterial populations and antibiotics,2426 even in a medicinal environment.24,25

Smith and co-workers27 identified a number of limitations in the current understanding of antibacterial resistance and difficulties in interpreting resistance data from environmental samples such as sediments under fish ponds. Westen makes three main points:28 (i) the use of one antibacterial agent can increase levels of resistance not only to that specific drug but to many others, even those using very different modes of antibacterial action (cross resistance); (ii) antibacterial resistance does not always respond in a predictable fashion correlating with the amount of drugs used or with the concentrations of residues in the environment; (iii) often, existing data which are used for the environmental effects of antibacterials are not adequate to establish how long bacteria maintain antibacterial resistance in the absence of continued selective pressure for that resistance.

On the one hand, the knowledge of subinhibitory concentrations of antimicrobials and their effects on environmental bacteria is scarce and contradictory, especially with respect to resistance. On the other hand, there is a huge volume of evidence that antibiotic resistance is already present in natural environments and that it is exchanged between bacteria.26

According to Murray,29 the transfer as well as the emergence of new combinations of resistance genes will happen most frequently in compartments with high bacterial density, i.e. biofilms. Such biofilms are not only found in a medical context. Bacterial density is very high both in aerobic and anaerobic septic tanks of sewage treatment works, and in biofilms, e.g. in drinking water pipes, in sediments, and soils. Biofilms are no taxonomic barrier to this horizontal genetic transfer. Bacteria are either resistant by nature or they may have become resistant by the use of antibiotics as well as in the environment by uptake of genetic material encoding resistance (e.g. in hospital effluent or manure) before they reach a sewage treatment plant or the soil. It is under discussion whether they may also become resistant in the sewage treatment plant or soil itself. The transfer of resistant bacteria to humans may occur via water or food if plants are watered with surface water or sewage sludge, if manure is used as a fertilizer or if resistant bacteria are present in meat.19,22,23 A prerequisite for a direct transfer of resistance is that the bacteria are able to survive or at least the genetic material is stable enough for transfer to the new environment, e.g. from the human body to surface water which is colder and much poorer in nutrients or the transfer from plants into animals. Therefore, the question is whether the input of antibiotics into the environment is an important source for resistant bacteria in the environment, i.e. was the concentration of the antibiotic and the bacterial density high enough, was the exposure long enough to promote resistance or to select resistant bacteria? Or is the transfer of resistance from already resistant bacteria following improper use of antibiotics much more important than the input of antibiotic compounds themselves?

Identification of resistance and resistant bacteria in the environment

It has been known for quite some time that cultivable bacteria represent only a small portion of the vast number of bacteria present in the environment and that the number that can be enumerated is higher than can be cultivated. The known and cultivable environmental bacteria add up to only 5–10% of the total number assumed to be present in waste water and waste water treatment plants.30 For soil, only 1% of the bacteria present are cultivable and can be identified by classical microbiological methods.31 Enrichment, cultivation and differentiation of bacteria are the most important steps. Within the last few decades, techniques have been developed to improve the situation. Methods relying on biomarkers such as chemotaxonomy or genetically based methods are now widely employed. Most of the recent work dealing with resistance in the environment found in the literature uses both approaches. Frequently, bacteria from a sample are cultivated first. Nutrients typical for known (pathogenic) bacteria or groups of bacteria with similar nutrient requirements are used for this purpose. That is, bacteria which are able to grow on the nutrient used are selected and then isolated. Other bacteria present are not monitored. Enterococci, Enterobactaericeae, Streptomyces spp., coliform bacteria, Escherichia coli and Acinetobacter spp., are quite typical (Table 2). In a second step, these isolates are classified according to their susceptibility to a certain antibiotic or a mixture of antibiotics to detect resistant bacteria or multi-resistant ones. For this purpose, antibiotic concentrations up to the milligram per litre range are used. These concentrations are orders of magnitude higher than the environmental ones. The bacteria identified as resistant are investigated further by monitoring the genetic material encoding the resistance found. Resistance genes in samples can be detected if they are extractable from the environmental matrix and if they can be amplified. The link between the presence of antimicrobials and the favouring of resistant bacteria as well as the transfer of resistance at concentrations as low as found for the antimicrobials in the environment is not yet established. Preliminary results indicate that the transfer of resistance and the selection of resistant bacteria are not favoured at antibiotic concentrations found in the environment.32


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Table 2. Examples of resistant bacteria and genetic material associated with resistance found in different environmental compartments (references see text)

 

    Sources of antibiotics and resistance in the environment
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
Hospital effluent

Antibiotics used in medicine for the treatment of infections and prophylactically are mainly released non-metabolized into the aquatic environment via waste water.

This can only have an effect if an active compound is present. Unused therapeutic drugs are sometimes disposed of down drains. Amongst other active compounds used, antibiotics and disinfectants are present in the effluent of hospitals.33 Ciprofloxacin, for example, was found in concentrations of between 0.7 and 124.5 µg/L in hospital effluent and was assumed to be the main source of genotoxic effects measured with the umuC test in hospital effluent.34 Ampicillin was found in concentrations of between 20 and 80 µg/L in the effluent of a large German hospital.35 Concentrations as high as 5 mg/L were found for benzalkonium chloride, a quaternary ammonium compound, in the effluent of European hospitals.9 The IC50s for nitrifying bacteria have been found to be in the order of 1–2 mg/L.9 Antibiotic concentrations calculated and measured in hospital effluents are in the same order of magnitude as the MICs for sensitive pathogenic bacteria.35 Resistant bacteria may be selected or favoured by antibiotic substances in hospital effluent. For single compounds, concentrations calculated and measured in hospital effluents are below MIC50 values. They may reach this range or even exceed MIC50 values in hospital effluent if not only single compounds but groups of compounds acting via the same mechanism are considered.35 Schwartz et al.36 found bacteria carrying vanA genes in hospital effluent. The mecA gene encoding resistance against methicillin in staphylococci was only found in bacteria in hospital waste water, but not in bacteria in municipal waste water.37 Gentamicin resistance genes were found in Acinetobacter, Pseudomonas and Enterobacteriaceae in hospital sewage.37 An important source of the resistance in hospital effluents is also the input of bacteria already resistant because of the use of antibiotics in medical treatment. There have been reports that the widespread use of biocides such as triclosan and quaternary ammonium compounds used in hospitals and homes could select for antibiotic-resistant bacteria.38 Triclosan for example has been shown to select for low-level antibiotic resistance in E. coli39 and high-level ciprofloxacin resistance in triclosan-sensitive Pseudomonas aeruginosa mutants.40 Others have suggested that the introduction of cationic biocides into clinical practice has been associated with the selection of S. aureus strains containing qacA/qacB genes under clinical conditions.41 Results of clinical trials are contradictionary.5 At present, no data are available for hospital effluents.

Municipal sewage and activated sludge of STPs

Antibiotics and disinfectants have been detected in sewage water (Table 1) at concentrations of a few micrograms per litre. Resistant bacteria are present in municipal sewage as well as in aeration tanks and the anaerobic digestion process of STPs.36,37,4250 Reinthaler and co-workers investigated the resistance of E. coli in three Austrian sewage treatment plants against 24 antibiotics by classical means, i.e. isolation, cultivation and resistance testing. E. coli were resistant to several antibiotics such as penicillins (ampicillin, piperacillin), cephalosporins (cefalothin, cefuroxime), quinolones (nalidixic acid), tetracycline and sulfamethoxazole/trimethoprim. Resistance rates were highest for tetracycline (57%).51 Bacteria carrying the vanA gene have been detected in waste water in Europe as well as in the USA.26,48,49 Also the ß-lactamase gene encoding AmpC was amplified by PCR in waste water.36 Gentamicin resistance genes have been found in Acinetobacter, Pseudomonas, and Enterobacteriaceae and in phylogenetically distant bacteria, such as members of alpha and beta-proteobacteria in municipal sewage.37 Tetracycline-resistant bacteria have also been identified in waste water44,46 as well as resistant P. aeruginosa.50

Resistant and multi-resistant pathogenic bacteria such as Acinetobacter spp.37,42 have been detected in waste water and STPs as well as transfer of resistance.37,45,47 Exchange of genes encoding for resistance between pseudomonads and E. coli in sewage sludge36 has been reported.

Up to 99% of Campylobacter spp. were eliminated from sewage water by treatment in an STP.52 A similar elimination rate was found for imipenem-resistant P. aeruginosa, ciprofloxacin-resistant E. coli and VRE.53 Elimination rates were 95–99% for E. coli, Pseudomonas spp. and Enterococcus spp. For resistant bacteria, the elimination rate was 93.5–100%. There was no difference between resistant and non-resistant bacteria. These results apply in winter and spring time. Jones found a seasonal pattern in the elimination rate of Campylobacter in an STP.52 Thus, studies should be conducted over a period of at least 1 year and take into consideration the treatment applied.

The prevalence of bacteria with reduced susceptibility to benzalkonium chloride was elevated in the effluent of a municipal STP.54 A strong selecting effect of benzalkonium chloride was found in biodegradability testing.55,56 In such tests, the benzalkonium chloride concentration was at least 100-fold higher than in hospital effluents or municipal sewage. Benzalkonium chloride and other quaternary ammonium compounds are sorbed by sewage sludge whereas pyridinium salts are not.57

Input of resistant bacteria into municipal sewage

It is often assumed that hospitals are the most important source for the input of resistant bacteria into municipal waste water. The numbers of resistant bacteria found in the effluent of an intensive care unit (ICU) of a hospital with maximum medical service spectrum were in the same range as those found for the influent of municipal STPs.53 Taking into consideration that the dilution of hospital effluent by municipal sewage is normally more than 100-fold,35 and that in municipal sewage without hospital effluent resistant bacteria are also present53 because of the use of antibiotics at home, the conclusion is that it is probably the general community which is responsible for the main input of resistant bacteria into STPs. Another point is that in Germany for example only one-quarter of the total consumption of antibiotics can be attributed to hospitals.35

The input into and the elimination of resistant bacteria in three different sewage treatment plants (a municipal one and two located in the countryside) were monitored.53 The STPs were different with respect to hospitals and old peoples' homes passing their waste water into the sewage as well as the technology used for water treatment and size (7500–600 000 population equivalents). No correlation between input, i.e. size and number of hospitals passing waste water and the STPs was found. Taking into consideration that hospital effluents contribute less than 1% of the total amount of municipal sewage, it is plausible that hospitals are not the main source of resistant bacteria in municipal sewage. The situation might be different for multi-resistant bacteria.

It is assumed that multi-resistant bacteria are selected mainly in hospitals and passed into waste water.50 The number of multi-resistant bacteria in sewage correlated with the size and the number of hospitals connected to an STP. The numbers and types of resistant bacteria found in the effluent of the ICU of the hospital offering maximum medical service showed that the number in the ICU effluent and in the influent of the STP are in the same range.53 Reinthaler et al. found the highest resistance rates in an STP receiving municipal sewage which also contained hospital effluent.51 More detailed studies are necessary to assess the different sources of input of resistant bacteria into STPs. Separate treatment of hospital effluent to reduce the input of resistant bacteria into the aquatic environment is not recommendable from the present state of knowledge.

Surface water

Concentrations of antibiotics in surface waters, i.e. in rivers and lakes, are in the low microgram per litre range for most compounds (Table 1). In a study using ciprofloxacin and ceftazidime, it was concluded that the average concentrations of these compounds actually found in surface water will be clearly below concentrations able to change bacterial populations.58 This was monitored by classical microbiological methods such as Gram-staining, aminopeptidase and catalase test as well as metabolic fingerprints using the Biolog system. However, some restrictions imposed by the methodology have to be taken into account in this study.

Bacteria resistant to antibiotics are present in surface water.36,43,59 Goni-Urizza et al.60 found a correlation between resistant bacteria in rivers and urban water input. Schwartz et al.36 were able to amplify AmpC ß-lactamase gene sequences by PCR in surface water. Genetic transformations have for instance been reported for E. coli.61 Antimicrobial resistance was also found in marine bacteria and bacteria living in estuaries.62,63 Gentamicin resistance genes were found in Acinetobacter, Pseudomonas, Enterobacteriaceae, and in phylogenetically distant bacteria such as members of alpha and beta proteobacteria in coastal water polluted with sewage water.37

Ground water

Antibiotics are rarely found in ground water and if they do occur, they are far below the microgram per litre range (Table 1). Leaching from fields fertilized with animal slurry or passing through sediments into the ground water might be a source of antibiotics in ground water. However, the volume load of antibacterial agents in ground water in rural areas with high concentrations of livestock has proved to be small.8 Antibiotic-resistant E. coli have been found with a surprisingly high incidence in rural ground water.64 The authors do not speculate much on the origin of this resistance but manure runoff from farms or leakage from septic tanks are clear possibilities for the input of resistant bacteria into ground water as well as broken sewage pipes.

Drinking water

Antibiotic-resistant bacteria were detected in drinking water as early as the 1980s and later in the 1990s.65,66 These authors found that resistant bacteria identified using classical microbiological methods, i.e. standard plate counting, occurred within the distribution network of drinking water supply systems. They concluded that the treatment of raw water and its subsequent distribution selects for antibiotic-resistant bacteria. In agreement with these data, increased phenotypic resistance rates were also detected at the drinking water sampling points in the study by Schwartz et al.36

These authors also found vanA and ampC genes in heterotrophic bacteria in drinking water biofilms. Enterococci were not detected. The authors concluded that this is an indication of the possible resistance transfer to autochthonous bacteria.

Sediments

Resistant bacteria may be present because of the application of antibiotics in fish or because of selection by the antibiotics present in sediments. High antibiotic load in sediments in concentrations sufficiently potent to inhibit the growth of bacteria were reported for aquaculture. The fact that the exposure is highly locally concentrated has to be considered critical. The substances used in fish farming can enter sediments directly from the water without undergoing any kind of purification process. Some investigations have demonstrated the presence and persistence of antibiotics applied extensively in fish farming in sediments beneath fish farms.6772

Quinolones, sulphonamides and tetracyclines are sorbed by organic matter. Therefore, they can accumulate. It is not yet known to what degree and under what circumstances the compounds are effective after sorption or whether they are released and may contribute to resistance. Antimicrobials may have qualitative and quantitative effects upon the resident microbial community of the sediments.73 Bacteria resistant against these compounds have been detected in sediments. Anderson & Sandaa72 and Samuelson et al.,73 for example, isolated tetracycline-resistant Gram-negative bacteria from polluted and unpolluted marine sediments. An increased antibacterial resistance in sedimentary bacteria is often the most sensitive environmental indicator of past antibacterial use.

Soil

Antibiotics used for veterinary purposes or as growth promoters are excreted by the animals and end up in manure as well as in disinfectants used in livestock. The manure is applied to agricultural land as a fertilizer. If antibiotics are used in animal husbandry, they pass from manure into the soil. Tetracyclines have been detected in soil in concentrations up to 0.3 mg/kg.13 Tetracyclines, some sulphonamides, and quinolones are strongly sorbed by soils.13,14,7478 Sulfachloropyridazine is not sorbed very much.79 Compounds from different antibiotic groups such as virginiamycin, sarafloxacin, tetracycline, oxytetracycline, chlortetracycline and cyclosporine A were only slowly biodegraded in soils.13,14,74,76,78,80 Tylosin disappeared soon after the application of manure.14 Enrofloxacin was degraded in laboratory tests by a white-rot fungus81 which may be present in soils but not in sewage or sewage sludge. Thus, the antibiotics may accumulate and reach concentrations in the MIC range. If they are still effective after sorption, resistant bacteria may be selected by antibiotic substances due to their application in animals, their use as growth promoters and in soil.

Sengelov et al. found that some soil microbial populations are affected by applying manure containing antibiotics.82 The bacterial composition returns to the original one some weeks after application of the manure. It was not investigated whether the change was due to antibiotics or to other constituents of the manure. Pang et al.83 reported the acquisition of Gram-positive tetracycline resistance genes in Mycobacterium and Streptomyces species in a laboratory test. This is one of the few reports of possible gene transfer between soil bacteria and human intestinal bacteria. Lorenz & Wackernagel also reported the exchange of genetic material between soil bacteria.84 Amino acid variations in the GyrA subunit of bacteria are potentially associated with natural resistance to fluoroquinolone antibiotics.85 A high incidence of bacteria resistant to fluoroquinolones was found in soil isolates. The origin of this resistance is not clear as enrofloxacin is widely used in agriculture. Resistance could be a result of the input of already resistant bacteria into soils following the application of the antibiotic pressure. It has been suggested that the development of new antibiotics should take into account the pattern of resistance in naturally occurring isolates.85

The spread of resistant bacteria and resistance genes by manure and sewage sludge used as fertilizer in agriculture or for land amendment has not been sufficiently investigated so far. Furthermore, antibiotics occur naturally in soils. Resistance against these antibiotics plays an important role in the population dynamics in soils. Antibiotics are a natural mechanism used by microbes in their natural ecology. The dynamics of soil microbiology and antibiotics has played out for millions of years. The abundance of natural antibiotics seems to be low on average and to be restricted to the nearest surroundings, i.e. the micro-environment of the bacteria. Tetracycline, for example, is produced by bacteria occurring naturally in soils. To the author's best knowledge, there are no findings of tetracycline in soils which had not been fertilized with manure containing tetracycline. In soils used as control when studying the input and fate of tetracycline in soils, tetracycline concentration was always below the detection limit.13 This situation may be different in tropical soils as the bacteria producing tetracycline naturally occur in such soils in higher density.

Most of the compounds used nowadays are synthetic or at least semi-synthetic. The speed of their depletion will probably be lower than that of naturally occurring compounds, but to the best of the author's knowledge, no information is available. Summarizing, at present there is insufficient information available on the impact of antibiotics on the structural and functional changes of bacterial populations in the environment which would allow for the assessment of potential risks related, for instance, to soil fertility. Input of resistant bacteria as well as of antibiotics could disturb the established well-balanced and important interdependencies.


    Understanding the interaction of bacteria and antimicrobials in the environment
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
Antibiotics and resistant bacteria are present in the environment. Antibiotics could favour resistant bacteria. As in other environments, the significance of this process depends on the antibiotic concentration, its bioavailability and other constraints. This varies in water, sewage sludge, soils, and sediments, because the concentration of antibiotics, the physicochemical constraints and the mobility of bacteria as well as their resistance genes vary. The ability to take up DNA from the environment is widespread among natural isolates. To understand the interaction of antimicrobial compounds and bacteria in the environment and for a sound risk assessment, the use of test systems and field studies is crucial.

A broad range of unknown bacterial species is present in the environment.30,31 When checking the toxicity of the antibiotics and the size of the biomass in the test guidelines of the International Standards Organization (ISO) or the Organization of Economic Cooperation and Development (OECD), it often appears that the bacteria are unaffected in the presence of antibiotics. But what does ‘unaffected’ mean? All bacteria present? And if so, are all the organisms affected in the same way? Does it mean that resistant bacteria are selected or that others or the same ones produce enzymes to degrade a compound which is not toxic to them? In other words, one has to take account of microbial ecology.25,86 It is known that antibiotics at subinhibitory concentrations can have an impact on cell functions and change the expression of virulence factors or the transfer of antibiotic resistance.87,88 In in vitro experiments, it was shown that gentamicin at a concentration of 100 µg/L improved the transfer rate in staphylococci. Other such as macrolides, quinolones or vancomycin did not have such an impact.32

When a complex mixture of bacteria is used, in some cases increased activity can be observed.89,90 Do the results depend on the test system and the inocula used? What is the role of (natural) resistance against bacteria?86 In other words, what do we mean by resistance? Even from a medical standpoint, this question is not unimportant.2426,29,91 Accordingly, several definitions of resistance can be found in the literature. Is it a matter of the ability of bacteria to ignore the toxic properties of the antibiotic, or do they cope with it despite its adverse effects?

Regarding the MICs for pathogenic bacteria the individual susceptibility of every species is well known and documented.92 In the environment the vast majority of individual bacteria are unknown and so are their MICs. Furthermore, even when resistance is encoded in the genes, it is sometimes not expressed. Why does resistance occur in the environment and what is its ecological significance? Are the environmental bacteria resistant by nature or is the resistance achieved through exposure to antimicrobials or other xenobiotics such as heavy metals? The nature and significance of resistance and the impact of the use of antibacterials on natural habitats such as soil are not yet understood.

What is the role of the concentration and type of antibacterial compound?2426 In some instances, the substance concentration is reduced by sorption. It is unclear whether elimination by sorption is an irreversible process. Some of the compounds may be deactivated or reduced in their activity by sorption. Antibiotics present in soil and sediment can lose their antimicrobial activity as a result of binding to sediment particles or complex formation with ions, which and for a few substances, this has been demonstrated. However, contradictory results concerning the reduced antibacterial activity and bioavailability due to binding or complex formation have been reported for one and the same substance.66,67,93,94 The reason could be the differences in sediment composition, which seem to play a key role in the effects of substances upon the resident population.


    Future developments
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
The volume of waste water will drop in future as water-saving measures are introduced and the growing quantity of antibiotics used will probably increase antibiotic concentration in urban waste water. This will depend on demographic developments and changes in standards of living. According to the present level of our knowledge, concentrations necessary to favour the spread of resistance will not be reached. We have to understand this issue much better, not only with regard to resistance but also in relation to the functions and services which bacteria in the environment offer us. Frequently, it is unknown whether resistance is natural or acquired, because of the lack of identification methods regarding environmental bacteria and a lack of investigation of environmental samples from areas where no antibiotics are present due to human use.


    Conclusions and work to be done
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
There are enough gaps in the existing body of scientific evidence to make it a risky response to increasing public concern to deny that there is a problem with respect to resistance in the environment. On the basis of our present knowledge, an increased direct impact of antibiotics on bacteria in the aquatic environment and in soils is questionable. The input of bacteria already resistant following the use of antibiotics in human and veterinary medicine seems to be the more important source of resistant bacteria in STPs and the environment. What has been learned so far is that it is critical to prevent the selection of resistant strains in the first place both in human and veterinary medicine. The opportunities and routes whereby this may be achieved are different in both fields. According to present knowledge, the prudent use of antibiotics in all areas seems to be the key to coping successfully with resistance in the environment. Therefore, the proper use of antibiotics and disinfectants in human medicine and livestock farming will significantly reduce the risk for the general public and for the environment. This not only includes limiting the duration of the selective pressure by reducing the treatment period and the continuous use of subtherapeutic concentrations. It also includes controlling the dissemination of antibiotics being used.

The use of antibiotics as growth promoters in fattening animals will diminish in Europe because of the complete ban on use of these substances by 2005.

The use of antibiotics and disinfectants in veterinary medicine for prophylactic and therapeutic reasons must be monitored to make sure that there are no excessive compensatory effects. Using proper hygienic procedures and housing for animals can successfully compensate for the abandonment of growth promoters as shown by examples from Sweden and Denmark. Experience gained in Norway shows that the use of vaccines can significantly reduce the use of antimicrobials in fish farming. It is also important to use the possibilities to reduce properly the consumption in medicine.15,21 Among other things, this will result in reducing the input of both resistant bacteria and antibiotics into the environment. These measures will also reduce costs. Therefore, the issue of resistance as a whole should be incorporated into the curricula of doctors and pharmacists. Public awareness has to be raised.

Knowledge about how antibiotic resistance arises, how resistant strains and resistance genes spread in nature and the significance of this for humans and nature is far from complete. There are not enough data available to draw a final conclusion especially with respect to the input of already resistant bacteria into the environment. This topic needs further consideration and investigation. An assessment of the different pathways also has to take into consideration the ingestion of resistant bacteria with food (e.g. poultry, pork).

The importance of the different sources of resistance found in the environment, i.e. the presence of antibiotics in the environment and the importance of resistant bacteria resulting from the use of antibiotics in the various fields has to be measured. For this purpose, it is important to make a more detailed assessment of the significance of culture-dependent and laboratory-based methods in relation to conditions found in the environment. This also applies to the concentrations of antibiotics applied in the identification of resistant bacteria in laboratory testing compared to the concentrations of antibiotics in the environment. Thresholds favouring selection and transfer of resistance genes between different species under environmental conditions should be established. For this purpose, the significance of the availability and activity of the antibiotics in the environment, i.e. the extent and importance of their sorption to sludge, particles in surface water, sediment, and soil should be determined. The significance of the semi-synthetic and synthetic compounds used compared to naturally occurring compounds needs to be known for a final risk assessment. The conditions and time scales under which antibiotics and resistance are lost in the environment are also of importance in relation to the input of antibiotics and resistant bacteria.


    Acknowledgements
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
I thank Ali Al-Ahmad (Genescan Europe AG), Sebastian Lemmen (Technical University Aachen), and Jörg Unger (Institute of Environmental Medicine and Hospital Epidemiology, Freiburg University Hospital) for fruitful discussions and comments on draft versions of the manuscript. Thanks also to the unknown reviewers for their helpful comments.


    Footnotes
 
* Tel: +49-761-2705464; Fax: +49-761-2705445; Email: klaus.kuemmerer{at}uniklinik-freiburg.de


    References
 Top
 Abstract
 Introduction
 Antibiotics, bacteria, and...
 Sources of antibiotics and...
 Understanding the interaction of...
 Future developments
 Conclusions and work to...
 Acknowledgements
 References
 
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