The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria

Review

The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria Elizabeth M H Wellington, Alistair B A Boxall, Paul Cross, Edward J Feil, William H Gaze, Peter M Hawkey, Ashley S Johnson-Rollings, Davey L Jones, Nicholas M Lee, Wilfred Otten, Christopher M Thomas, A Prysor Williams

During the past 10 years, multidrug-resistant Gram-negative Enterobacteriaceae have become a substantial challenge to infection control. It has been suggested by clinicians that the effectiveness of antibiotics is in such rapid decline that, depending on the pathogen concerned, their future utility can be measured in decades or even years. Unless the rise in antibiotic resistance can be reversed, we can expect to see a substantial rise in incurable infection and fatality in both developed and developing regions. Antibiotic resistance develops through complex interactions, with resistance arising by de-novo mutation under clinical antibiotic selection or frequently by acquisition of mobile genes that have evolved over time in bacteria in the environment. The reservoir of resistance genes in the environment is due to a mix of naturally occurring resistance and those present in animal and human waste and the selective effects of pollutants, which can co-select for mobile genetic elements carrying multiple resistant genes. Less attention has been given to how anthropogenic activity might be causing evolution of antibiotic resistance in the environment. Although the economics of the pharmaceutical industry continue to restrict investment in novel biomedical responses, action must be taken to avoid the conjunction of factors that promote evolution and spread of antibiotic resistance.

Introduction

The pandemic of CTX-M ESBLs

Resistance to antibiotics used to treat serious bacterial infections results in substantially increased mortality.1 The upsurge in multidrug-resistant strains of Enterobacteriaceae and Pseudomonas aeruginosa Gramnegative bacilli over the past decade is threatening the successful treatment of infections caused by these bacteria.2 The most substantial reservoir of multidrugresistant Gram-negative bacilli is the gut of man and animals, particularly in those who are receiving antibiotics. The contamination of water, food, and the environment with multidrug-resistant Gram-negative bacilli is an important route for its spread, whether from man or animals, and is therefore a crucial area for control. The introduction of semi-synthetic penicillins (eg, ampicillin and carbenicillin) in the 1960s and their subsequent combination with beta-lactamase inhibitors (eg, amoxicillin plus clavulanic acid) enabled the successful treatment of infections caused by Enterobacteriaceae. Over the next 10 years, plasmid-encoded beta-lactamases (particularly TEM) substantially undermined this therapeutic advantage, resulting in the increased use of aminoglycosides (eg, gentamicin and amikacin), thirdgeneration cephalosporins (eg, cefotaxime and ceftazidime), and quinolones (eg, ciprofloxacin). However, again the bacteria developed resistance. In the late 1970s, the emergence of plasmid-mediated aminoglycoside resistance resulted in substantial use of third-generation cephalosporins and quinolones. The large increase in extended-spectrum beta-lactamases (ESBLs) after the early 2000s in Europe has been one of the most dramatic phenomena in antimicrobial resistance.2 ESBLs are generally acquired by horizontal gene transfer and confer resistance to oxyimino-cephalosporins; some are mutants of established plasmid-borne beta-lactamases (eg, TEM or SHV) or are mobilised from environmental bacteria (eg, CTX-M from Kluyvera spp).

During the 1990s, most reported ESBLs were TEM or SHV types and occurred at a low frequency, with the exception of CTX-M-2 from South America. In the past 10  years, and particularly since 2005, the incidence of ESBLs of the CTX-M type have increased dramatically.2 Four major groups of CTX-M enzymes, 1, 2, 8, and 9, have been identified and correspond to genes transferred from different Kluyvera species.3 Particular CTX-M types are also associated with geographical regions.2 In China, CTX-M-14 (a group 9 genotype) is the dominant type and has spread to become the second most reported worldwide after CTX-M-15 (group 1).4 CTX-M-15 is the most widely distributed and most commonly recorded type in the world, having reached endemic prevalence in much of Asia, southern Europe, and South America.4,5 In Europe, findings from the SENTRY study showed a significant increase in ESBL rates for both Escherichia coli (11·6%) and Klebsiella spp (17·6%) in 2008 compared with 2004–06 (<10% in both cases).6 Understanding the role of faecal carriage might be the key to understanding the high levels of ESBLs that occur in India and China, where the transmission is linked to poor sanitation. Carriage of CTX-M in China and India is poorly studied, but available data suggest a rate of 22% in India7 and 7% in elderly Chinese people.8

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Effect of the environment on the global spread of antibiotic resistance

Lancet Infect Dis 2013; 13: 155–65 School of Life Sciences (Prof E M H Wellington PhD, A S Johnson-Rollings PhD) and Institute of Education (N M Lee PhD), University of Warwick, Coventry, UK; Environment Department, University of York, Heslington, York, UK (Prof A B A Boxall PhD); School of Environment, Natural Resources and Geography, Bangor University, Bangor, UK (P Cross PhD, D L Jones PhD, A P Williams PhD); Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, UK (E J Feil PhD); European Centre for Environment and Human Health, Exeter University Medical School, Knowledge Spa, Royal Cornwall Hospital, Truro, UK (W H Gaze PhD); Health Protection Agency, West Midlands Public Health Laboratory, Heart of England NHS Foundation Trust, Bordesley Green East, Birmingham, UK (Prof P M Hawkey MD); School of Immunity and Infection (Prof P M Hawkey) and School of Biosciences (Prof C M Thomas PhD), University of Birmingham, Edgbaston, Birmingham, UK; and The SIMBIOS Centre, University of Abertay Dundee, Dundee, UK (Prof W Otten PhD) Correspondence to: Prof Elizabeth M H Wellington, School of Life Sciences, University of Warwick, Gibbet Hill Site, Gibbet Hill Road, Coventry CV4 7AL, UK e.m.h.wellington@warwick. ac.uk

The widespread and increasing prevalence of CTX-Ms is causing a shift in prescribing away from third-generation cephalosporins and quinolones to carbapenems such as imipenem and meropenem. Of grave concern at present is the rise in carbapenemase genes typically associated with Klebsiella spp, particularly in Greece, India, and China. This rise was because of the high incidence of CTX-M ESBLs, which resulted in heavy use of carbapenem 155

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antibiotics to treat patients infected with these bacteria. The resistance genes emerging in India include NDM-1, which is linked with medical tourism and from waste water seepage and tap water.9 In China, NDM-1 has now been found in Acinetobacter baumannii,10 but most clinically significant carbapenem-resistant isolates across the country carry KPC-2 or IMP-4.11 IMP-4 was originally described in Citrobacter youngae in China in 1998.12 KPC-2 carbapenemase is endemic in the USA, Israel, Greece, and parts of South America.2 However, these carbapenemases are now spreading to other parts of the world.

The mobile resistome Resistance genes are commonly associated with mobile genetic elements (the mobilome) and can be transferred between distantly related bacteria corresponding to different phyla. Mobile elements tend to be variably present or absent within a population; hence they do not carry genes essential for cell function. Equipped with the appropriate transfer machinery, resistance genes can in principle be acquired from any source (figure 1). However, in practice gene flow is probably structured by ecology, with species that share similar niches drawing from similar gene pools. For this Review, we will focus on the mobilome that connects Enterobacteriaceae in human beings, animals, and terrestrial and aquatic environments. Resistance genes tend to be associated with mobile genetic elements including transposons and integrons. Time

R–

Species A, strain 1

R+

Species A, strain 1

Mobile element

α Genome

α Plasmid

R+

Species A, strain 2

R–

Species A, strain 1

R+

Release of antibiotics into the environment and development of reservoirs of antibioticresistant bacteria

α α

β

Plasmid Conjugation pilus

Species A, strain 2

R+

Species B, strain 1

β

β

R+

R–

α Genome

Figure 1: The mobilome The gene pool on shared mobile elements. Resistance genes (red) evolve on the chromosome and move by transposition to the plasmid. Narrow host range plasmids (α) allow spread between strains while broad host range plasmids (β) allow transfer to distantly related bacteria. R–=sensitive phenotype. R+=resistant phenotype.

156

Although there are many different transposable elements, the same elements tend to be implicated repeatedly in recent independent acquisition events. For example, the genes for CTX-M enzymes are associated with copies of ISEcp113 and NDM-1 is associated with IS26.14 Sequence comparisons of several IncW plasmids show the acquisition of genes both by transposable elements and integrons. Although the much studied plasmid R388 carries no transposable elements but has a number of genes associated with the integron In0, another IncW plasmid, R7K, has followed a parallel history of resistance gene acquisition involving transposable elements.15 Transposable elements and integrons that have acquired resistance genes can in turn become linked with a conjugative transfer system, which will further increase the mobility of the gene. Although the best known conjugative elements are plasmids, integrative and conjugative elements are increasingly being shown to also play an important part.16 For many integrative and conjugative elements, mobilisation is associated with the stress response, which can be activated by exposure to antibiotics and pollutants in the environment. Some plasmids are limited in their host range, but others, such as the IncW plasmids, have a broad host range and can thus promote exchange with environmental bacteria. In Enterobacteriaceae, IncFI/FII, IncI, IncL/M, IncA/C, and IncK plasmids are associated with genes for CTX-M enzymes (eg, the IncK plasmids responsible for the spread of CTX-M-14 in Spain).17 The limited range of plasmids implicated in the spread of resistance in Enterobacteriaceae might be expected since those plasmids selected by the previous generation of antibiotics will be very common, hence most likely to acquire new resistance genes. A more subtle explanation is that transposition events tend to insert DNA into locations where previous insertions have taken place.18,19 Thus, plasmids might confer some protective properties on the host, because invading transposons might preferentially integrate into the plasmid rather than the host chromosome.

After use in human beings, antibiotics and any metabolites will be emitted to the sewerage system20 and, depending on their polarity, water solubility, and persistence, the compounds might be degraded, associate with sewage sludge, or released to rivers. Sludge-associated drugs will enter agricultural systems when the sludge is used as a fertiliser.21 Antibiotics for use in human beings can also reach agricultural soils directly through irrigation with wastewaters and surface waters.21 Veterinary pharmaceuticals and their metabolites can be released into the environment either directly, from use in aquaculture and the treatment of animals on pasture, or indirectly during the application of manure and slurry from intensive livestock facilities www.thelancet.com/infection Vol 13 February 2013

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to land.22 Compounds that are released to the soil system can subsequently be transported to surface water or groundwater23,24 and be cycled and re-cycled within the environment (figure 2). Antibiotic-producing bacteria occur naturally throughout the environment,25 colonising plants, soil, and detritus in aquatic environments, aquatic plants, and animals. The large-scale mixing of these environmental bacteria with exogenous bacteria from anthropogenic sources such as farm drainage and waste processing provides the ideal selective and ecological conditions for new resistant strains to arise; thus, soil, water, and other nutrient-enriched habitats can act as hotspots for horizontal gene transfer.26 Human beings can be exposed to antibiotics, antibiotic resistance genes (ARGs), or antibiotic-resistant bacteria in the environment by several routes: (1) crops that have been exposed to contaminated sludge, manure, and slurry; (2) livestock that have accumulated veterinary drugs and resistant flora through the food chain; (3) fish exposed to pharmaceuticals released to surface waters either intentionally (aquaculture treatments) or unintentionally; (4) abstracted groundwater and surface water containing residues of pharmaceuticals that is then used for drinking water; and (5) coastal waters used for recreation or shellfish production (figure 2). Several antibiotics have been detected in drinking water, including clofibrate and tylosin.27,28 Antibiotics have also been detected in fish in effluent-dominated or effluentinfluenced water bodies,29 and food crops also take up antibiotics.30–32 Exposure can also occur via the inhalation of dust emitted from facilities where livestock are intensively reared.33 After ingestion, most bacteria harmlessly pass through the gut without causing disease; however, there are ample opportunities for horizontal gene transfer within the human host, allowing ARGs to become part of the gut microflora. Wastewater can contain complex mixtures of pharmaceuticals, detergents, and bacteria of human and animal origin. The primary role of wastewater treatment is to eliminate organic substances to avoid eutrophication in receiving waters, which is achieved by removing solids (primary treatment), degrading organics (secondary treatment), and disinfecting effluent in sensitive areas to protect bathing waters or shellfish production (tertiary treatment).34 Over 1 million tonnes of dry solids are generated each year in the UK, most of which is disposed of in landfill sites. However, under the Safe Sludge Matrix35 there are tight controls on the type of sludge disposed to different categories of soils.

Persistence of antibiotics in the environment The Dangerous Substances Directive 76/464/EEC36 lists 129 substances that are regarded as so toxic, persistent, or bioaccumulative that efforts to control their release and prevent pollution should be given the highest priority. However, because antibiotics are not listed and are www.thelancet.com/infection Vol 13 February 2013

Livestock

Meat

Human beings

Manure and slurry

Other livestock species

Crops and soil

Faeces

Discharge: raw or from septic tank

Sewage system

Surface and ground waters

Marine water

Wild birds and other vectors

Shellfish

Figure 2: Environmental reservoirs of resistance genes The associations between potential sources of antibiotic-resistant bacteria.

therefore not routinely tested for, their high prevalence in the environment has received little attention.37 Many antibiotics are not inherently biodegradable and some synthetic antibiotics can persist in soils for long periods of time at high concentrations.38,39 A range of antibiotics have been detected in soils, surface water, sediments, and groundwater,40,41 including fluoroquinolones, sulphonamides, tetracyclines, and macrolides (table 1). Although the reported concentrations of antibiotics are generally low (eg, <1 mg/L in surface waters), the substances have been recorded throughout the year across various hydrological, climatic, and land-use settings. Some substances (eg, the tetracyclines and fluoroquinolones) also persist in the environment for months to years.30,42,43 Antibiotics can also enter the environment during the manufacturing process. This situation is particularly problematic in India and China, where antibiotic manufacturing occurs on a substantial scale but regulations tend to be somewhat lax. In one monitoring study in India,44 ciprofloxacin concentrations as high as 2·5 mg/L were reported in river water downstream of a wastewater treatment plant receiving wastewater from 90 bulk drug manufacturers. Other antibiotics were also detected in the river, including enoxacin, enrofloxacin, lomefloxacin, ofloxacin, and trimethoprim.

Effects of sewage disposal on resistance gene prevalence Substantial evidence has accumulated in recent years linking the high prevalence of ARGs in the environment with anthropogenic sources. A Brazilian study of a hospital sewage treatment works showed that ESBL-producing Klebsiella pneumoniae were present at all stages of sewage treatment.45 β-lactam and aminoglycoside resistance genes have been isolated by exogenous isolation from activated sludge (biologically treated sewage) in Germany, showing 157

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General behaviour

Sewage sludge

River water

Ground water

Drinking water

Chloramphenicol

Not persistent and mobile

–

3

×

–

2,4-diaminopyridines

Persistent and immobile

3

3

×

×

Fluoroquinolones

Persistent and immobile

3

3

×

×

β-lactams

Not persistent and mobile

–

×

×

Macrolides

Slightly persistent and slightly mobile

3

3

Sulphonamides

Persistent and mobile

3

Tetracyclines

Persistent and immobile

–

Fish

Soil

Crops

Examples of monitored drugs

–

–

–

–

–

3

3

Trimethoprim

–

3

–

Ciprofloxacin, norfloxacin, ofloxacin

×

–

–

–

Amoxicillin, cloxacillin, dicloxacillin, meticillin, nafcillin, oxacillin, penicillin G, penicillin V

×

–

–

–

–

Azithromycin, clarithromycin, lincomycin, roxithromycin, spiramycin, tylosin

3

3

×

–

3

3

Sulfamethoxazole, sulfadiazine, sulfamerazine, sulfamethazine, sulfapyridine

3

×

×

3

3

3

Chlortetracycline, doxycycline, oxytetracycline, tetracycline

Persistence describes how long the compound will stay in the natural environment; mobility describes the potential of a substance to move from soils to surface waters. 3=detected. ×=not detected. –=no monitoring done. Data from Boxall and colleagues22 and Monteiro and Boxall.42

Table 1: Occurrence of antibiotics in the natural environment, fish, crops, and drinking water from published studies

that final-stage sludge can be a source of ARGs.46 In Portugal, β-lactamases, including TEM, IMP, and OXA-2 derivatives, have been identified in aquatic systems and ESBL resistance genes in sewage sludge.47 ESBL-producing Enterobacteriaceae were also detected in five samples of sewage from human beings in Spain.48 ESBLproducing E coli survived the wastewater treatment process of a modern secondary treatment facility in Ireland,49 including CTX-M groups 1 and 9. Gaze and colleagues50 reported that treated liquid sewage sludge contained 10⁷ bacteria per gram that were carrying class 1 integrons, revealing a potentially huge reservoir of antibiotic-resistant bacteria, many with detergent efflux pumps (qac; figure 3). DNA extracted from dewatered and limed sludge spread to arable land, which would be predicted to contain much lower numbers of bacteria, contained similar numbers of integrons. The qac genes borne by integrons give resistance to quaternary ammonium compounds, conferring protection against detergents and biocides, raising the possibility of co-selection by detergents for antibiotic resistance.50,51 qacEΔ1 is a marker of clinical class 1 integrons because it is always flanked by sul1, which confers sulphonamide resistance;52 this integron type is thought to have evolved during the use of antibiotics by man and these integrons are abundant in natural environments affected by human activity (figure 3). Insertion sequences such as ISCR1 are also associated with class 1 integrons, facilitating mobilisation and expression of resistance genes from the metagenome through rolling circle transposition (figure 4).53 Exposure to selective pressures, including some antibiotics, upregulates IntI1 expression, therefore increasing cassette gene recombination rates; the SOS response induces LexA deactivation, which derepresses IntI1, increasing the rate of cassette gene integration and excision. Crucially, although sewage sludge has been reported to contain ARGs and pathogenic bacteria, the extent of this problem and the potential for transfer of resistance to soil bacteria and ultimately its effect on human-associated bacteria is poorly studied. A study by Golet and 158

colleagues54 suggested that sewage sludge is the main reservoir of fluoroquinolone residues from wastewater and outlined the importance of sludge management strategies to assess whether most of the human-excreted fluoroquinolones enter the environment. Findings from field experiments of sludge application to agricultural land confirmed the long-term persistence of trace amounts of fluoroquinolones in sludge-treated soils and suggested a limited mobility of fluoroquinolones into the subsoil. Persistence of fluoroquinolones is particularly relevant because they seem to co-select for class 1 integrons and integron-borne ESBL genes because quinolone resistance genes (qnr) are situated in class 1 integron structures, which also carry ESBL resistance.55

Resistance gene dissemination in manure In the UK, about 350–400 tonnes of antibiotics were used per year in food-producing animals in 2006–11 (data from National Office of Animal Health, Enfield, UK). An estimated 70 million tonnes of animal manure waste are spread onto agricultural land per year in the UK.56 Continued antibiotic selection in the soil environment can facilitate recruitment of novel genes by exotic bacteria from naturally occurring soil bacteria, as has occurred for Kluyvera spp. and the dissemination of CTX-M progenitors.57 As discussed earlier, these genes are now widespread in human-associated bacteria and there is increasing evidence of their distribution in farm animals and ecosystem compartments. Third-generation cephalosporins are used in veterinary medicine, including ceftiofur, which is licensed for use in cattle and pigs.58 E coli strains producing CTX-M-2 have been isolated from cattle faeces in Japan.59 β-lactamase and ESBLs have also been detected in E coli isolates from healthy chickens, food, and sick animals in Spain.60–62 ESBLs have been reported from E coli in pig slurry from Spain, including SHV-12, CTX-M-1, CTX-M-9, and CTX-M-14.63 Multi-antibiotic-resistant bacteria, including potential Gram-negative opportunistic pathogens, were detected in subsurface flow several months after pig slurry was applied to agricultural soils, www.thelancet.com/infection Vol 13 February 2013

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Wildlife as reservoirs of ARGs Although there have been many studies designed to quantify and qualify ARGs in soil and water, the scientific community remains largely ignorant of the complex transmission dynamics of resistance genes in environmental settings.67–69 However, the potential publichealth risks associated with the colonisation of wildlife by pathogens has been acknowledged for decades. For example, reports that the colonisation of gulls by human pathogens might pose a public health risk via the contamination of water supplies date back to the early 1980s.70 The colonisation of wildlife by antibiotic-resistant bacteria through contact with sewage or animal manure might be important in the global dissemination of resistance genes, with grave implications for public health, ecosystem function, and animal disease.71,72 The persistence of bacteria harbouring exogenous resistance genes within wildlife populations, even in the absence of direct selection from antibiotics or continual pressure from anthropogenic perturbation, is still largely unknown. This question has attracted increased attention over the past 2 or 3 years, with a particular focus on wild birds (particularly waterfowl and birds of prey) and small woodland mammals.73,74 The present evidence is somewhat confusing, which is not surprising in view of the complexities of the ecological and anthropogenic factors. Findings from an influential study in the late-1990s showed a high prevalence of acquired ARGs within faecal bacteria from woodland rodents in the Wirral, UK.75 Because there was no direct antibiotic use on this land, the investigators argued that this prevalence was not the direct result of anthropogenic disturbance and that restrictions on antibiotic use would have a minimal effect on wildlife reservoirs. By contrast, a near absence of resistance in bacteria recovered from the faeces of rodents and ungulates was found in remote areas of Finland.76 Thus, there are degrees to which wildlife are really wild, and there is good evidence that proximity to human populations, rather than direct antibiotic use on the land, is sufficient to substantially affect the gut flora of local wildlife.77 Such a notion is supported by a study comparing levels of resistance in E coli recovered from animals with varying amounts of contact with people, from wild Antarctic animals to pet dogs.78 The situation seems to be somewhat different for wild birds, where ecological factors such as migratory behaviour and high population densities increase the www.thelancet.com/infection Vol 13 February 2013

intl1 qacEΔ1 qacE

1·40

1·20

1·00

Prevalence (%)

showing their persistence and dissemination to water catchments.64,65 Investigation of the effects of pig manure and sulfadiazine on bacterial communities in soil microcosms using two soil types showed that in both soils manure and sulfadiazine positively affected the quotients of total and sulfadiazine-resistant culturable bacteria.66 In many river catchments, the bulk of faecal coliforms are believed to be of agricultural origin because of direct excretion from animals and from subsurface and overland flow after waste application.

0·80

0·60

0·40

0·20

0

Reed bed sediment remediation of textile mill effluent

Fully digested sewage Pig slurry from sludge containing tylosin-fed pigs amended detergents and antibiotic with oxytetracycline residues and sulfachloropyridizine

Fallowed Cotswold soil with no history of sludge or slurry amendment

Sample

Figure 3: Prevalence of integrons in polluted environments Molecular prevalence of class 1 integrons (intI1) and integron-associated qac genes, qacEΔ1 and qacE.

Integrase upregulation

Increased promoter activity

Novel antibiotic and quaternary ammonium compound

Acquisition and expression of chromosomal genes

Exposure to antibiotic residues

SOS response

Environmental cassette gene metagenome

Insertion sequences

Environmental metagenome

LexA intl1

cassette

qacEΔ1

sul1

ISCR1

Mobilised DNA

(variable number)

Figure 4: Resistance gene acquisition by integrons Stress response via LexA inactivation leads to increased IntI1 expression, modulation of resistance gene expression via insertion sequence transposition, integration of antibiotic resistance genes (blue circles) and quaternary ammonium compound resistance genes (red circles), and transposition and expression of chromosomal resistance genes from the metagenome.

likelihood of the presence of clinically relevant resistance genes carried by birds even in areas of low anthropogenic effect.79 For example, antibiotic-resistant E coli were detected in 8% of a sample of 97 Arctic birds from Siberia, Alaska, and northern Greenland,80 which contrasts with the low frequency of resistance noted in mammalian populations from similarly pristine environments. Furthermore, although declining in coastal areas, gull populations have at least doubled in many cities in the UK 159

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over the past decade, and this urbanisation will increase the chance of transmission between people and birds.81 The possibility that resistance genes are transmitted between wildlife populations, livestock, and the clinic is supported by an emerging picture that essentially the same types of elements occur in all three settings. The first report of an ESBL-producing E coli isolated from wildlife populations was that by Costa and colleagues82 in 2006, who noted CTX-M-14, CTX-M-1, SHV-12, and TEM-15 in various bird and mammal hosts in Portugal. Since then, more than 30 wild animal species, particularly birds, have been found to harbour ESBLproducing E coli (table 2). The most commonly reported genes tend to be CTX-M, which shows the dramatic increase in frequency of these genes in clinical and agricultural settings. For example, populations of blackheaded gulls in Sweden, a country with a low frequency of nosocomial resistance, harbour ESBL-producing E coli strains with the same CTX-M types as are dominant among human isolates.85 Similarly, ESBLproducing E coli from gulls on beaches in Portugal frequently carried CTX-M-15,84 which is the most prevalent gene in local hospitals. Thus, there are accumulating data that support the importance of wildlife as a reservoir and route for transmission of clinically relevant resistance genes.

Social issues driving antibiotic resistance Social interventions are essential to reduce antibiotic misuse within the health-care industry and the home. Studies have tended to focus on health professionals in developed countries, with several campaigns aiming to raise awareness in clinicians and pharmacists to improve antibiotic prescription.96,97 Although such campaigns have led to some notable reductions in the total administration of antibiotics,96,97 there is widespread Region

CTX-M

SHV

TEM

OXA

Gulls Poeta et al83

Portugal

1, 14a, and 32

··

52

··

Simoes et al84

Portugal

1, 9, 15, and 32

··

··

··

Bonnedahl et al85

Sweden

··

··

··

Dolejska et al86

Czech Republic

1 and 15

2 and 12

··

··

Bonnedahl et al87

France

1 and 15

··

1

··

Hernandez et al88

Russia

14 and 15

··

··

··

Costa et al82

Portugal

14 and 1

Pinto et al89

Portugal

Radhouani et al90

Portugal

32 and 1

Silva et al91

Azores

14

Literak et al92

Poland

Garmyn et al93

Belgium

Tausova et al94

Central Europe

15 and 27

Guenther et al95

Germany

15

14 and 15

Other birds 1

1, 9, and 15 ··

Table 2: Reports of extended-spectrum β-lactamases in birds

160

12

52 and 1

··

5

1 and 20

··

··

1

··

12

··

··

··

1b

1

12

52

··

··

··

··

··

··

··

evidence of inappropriate antibiotic use in health-care environments.98 For instance, time-pressured physicians might knowingly administer inappropriate antibiotics to retain a patient’s loyalty,99 in part because of the commercial promotion of new antibiotics.100,101 Various social factors can impede large-scale reductions in antibiotic prescription, such as an increasing capacity to afford health care, rising health-care expectations, the number of vulnerable individuals who experience repeated infections, and poor professional attitudes.102,103 The rapid increase in internet access has resulted in a corresponding increase in the unregulated purchasing of antibiotics, accompanied by low-quality patient care and increased risk of environmental contamination through unregulated disposal.104 Public use or misuse of antibiotics is caused by several social factors, including increased incidence of selfmedication, ethnic origin, country of residence, income, and education level.99,101,105,106 An individual’s awareness (or absence of awareness) of the potential consequences of antibiotic misuse influences their behaviour.105,107 In a study comprising 11 countries of varying socioeconomic status, Grigoryan and colleagues105 found low awareness of antibiotic resistance in countries with high levels of antimicrobial resistance and antibiotic misuse. Efforts to promote the adoption of improved public antibiotic use have tended to focus on high-income countries.96,108,109 The methods used seem to affect the success rate,108 and not all campaigns have proved successful.109 Antibiotic use varies substantially throughout the agricultural world because of differing behaviours of farmers in different cultural and economic contexts and varying levels of effective legislation and guidance. For example, the practice of feeding antibiotics to promote livestock growth is banned in the EU but widespread in livestock systems elsewhere in the world.110,111 Educating farmers to reduce antibiotic use might prove more remedially effective than legislative enforcement,110 although there have been calls for a ban on the prominent marketing of antibiotics to livestock farmers under various trade names because of its link with agricultural overuse.112 Finally, the way society values the natural environment must change because unanticipated effects on the ecosystem and human health can result from disposal of domestic and industrial wastes—for example, dissemination of and selection for antibiotic resistance. Many proposed interventions (eg, compulsory screening of medical tourists for resistant organisms on return to their home country113 or capital investment in sewage treatment facilities in developing countries) are as yet untested and predicting their capacity to reduce levels of antibiotic resistance is problematic. Furthermore, not all interventions will necessarily be implementable or applicable because of the socioeconomic, legislative, or practical barriers that exist between countries. Lastly, emerging antimicrobial resistance presents substantial implications for future www.thelancet.com/infection Vol 13 February 2013

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health-care costs.114 Assessment of the effectiveness and practicality (particularly in terms of cost) of mitigation strategies seems to be increasingly urgent (panel).

Modelling the problem Despite the increasing awareness of public health boards of the increase in ARGs in the environment and the serious clinical consequences that result, substantial problems persist in the gathering of quantitative information on the magnitude and complexity of the problem.115 Mathematical modelling can play a crucial part in connecting disciplines towards a better understanding of complex biological, physical, social, and environmental interactions of ecological systems, and has proved particularly powerful in predicting the emergence of epidemics in the context of shifting host demographics. Furthermore, modelling of antibiotic-resistant bacteria and infectious diseases in hospitals has facilitated the creation of novel management strategies.116–118 Recent data on selection at low antibiotic concentrations119 shows that minimum selective concentrations of some antibiotics are in the same order of magnitude as environmental residues. Based on the selection pressure at a particular concentration, the relative growth rate of resistant and susceptible strains and the mutation or gene transfer rate that confers resistance, the time for a resistant organism to become fixed within a population can be predicted. Modelling processes within more complex environmental reservoirs has been attempted, where antibiotic concentration, partitioning between sediment and water column, bioavailability, dissolved organic matter, particulate organic matter, and growth of susceptible and resistant strains were used as variables.120 The investigators showed that their model reproduced reported trends of tetracycline resistance in the Poudre River, CO, USA and suggested that densities of bacteria could not be explained by inputs alone, but relied on growth or gene transfer within the aquatic environment. To analyse the potential effect of agricultural use of antibiotics on the emergence of antibiotic resistance in human populations, Smith and colleagues121 made the simple assumption that introducing a new antibioticresistant strain affects prevalence regardless of whether the whole organism or only genetic material was transferred. Although focused on human epidemiology, this model did not take into consideration the complex ecology and feedbacks between human populations and the environment, which others have proposed as an important factor of change.122–124 Salisbury and colleagues123 proposed an analysis framework for the management of antibiotic resistance in food-producing animals located around three interrelated hazards: antibiotic use, the antibiotic-resistant bacterium, and the ARG. The purpose of the framework was to guide data collection to inform decision makers on ways to reduce risk. Singer and colleagues67 posed the question “can landscape ecology untangle the complexity of antibiotic resistance” in the www.thelancet.com/infection Vol 13 February 2013

Panel: Mitigation strategies Wastewater treatment • Reduced microbial pollution, the present best practice in wastewater treatment (UV treatment). Only undertaken in so-called sensitive areas. • Dedicated hospital wastewater treatment. • Adoption of new technologies to remove pharmaceuticals from wastewater such as ozonation and membrane technology. • Further investment in wastewater system to reduce combined storm overflow discharges of raw sewage. Farming • Observe best practice in reducing livestock access to water courses. Implementation of buffer zones to reduce runoff. • Treatment of animal wastes to reduce microbial pollution. • Reduction in antibiotic use in agriculture—prophylactic use supporting unsustainable farming practice. Use alternative therapies where possible (probiotics). Medical • Ensure best practice in prescription of antibiotics, use alternative therapies such as bacteriophage or probiotics where possible. • Green drug choice: use of degradable pharmaceuticals rather than environmentally persistent compounds. • Ethical procurement: purchase from sources not polluting the environment with pharmaceuticals. Overall, reduced reliance on pharmaceuticals and personal care products is needed, as is reduction of unnecessary use of bioactive products that are ultimately discharged to the environment where unanticipated consequences can occur (eg, co-selection for antibiotic resistance by biocides or surfactants). Further research into the effects of pharmaceuticals and personal care products on microbial populations in the natural environment is necessary.

farm environment, accounting for spatial and temporal heterogeneity of the environment and its effect on evolution, dissemination, and persistence of resistance. However, so far models remain simple and have not attempted to account for the complexity of natural systems, and perhaps, as suggested by Smith and colleagues,125 “complexity of bacterial population biology and genetics makes it practically impossible to trace bacteria (or resistance factors) from the farm to the hospital, or to directly attribute some fraction of new infections to agricultural antibiotic use”. An important challenge in developing informative models is that ecological and co-evolutionary processes occur at many different spatial scales and different phenomena operate at different levels of importance at these different scales.122 For example, isolated populations tend to be dominated by genetic stochasticity, whereby factors that affect colonisation and extinction dynamics tend to dominate at the metapopulation scale, whereas 161

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Search strategy and selection criteria We identified published work through searches of PubMed, Medline, Google Scholar, and Web of Science, without date restrictions, and from references from relevant articles; the date of the final search was August, 2012. Search terms were “antibiotic resistance genes”, “drug resistance”, “CTX-M beta-lactamase”, “integrons”, and “mobilome”. Only papers published in English were included.

phylogenetic patterns and historical events dominate at larger geographical scales.126 Without bridging these scales, specific risk factors might not be immediately apparent and might be overlooked by biomedical and health agencies.127,128 Ultimately, the way forward is to balance ecological realism with mathematical tractability and to develop multi-level ecosystem approaches, combined with ecological and epidemiological theories and, importantly, data for specific systems.

Conclusion The potential threat posed by the continued evolution of ARGs seems sufficiently grave and imminent that reliance upon stakeholder behavioural change should be considered a high-risk strategy. The absence of full environmental fate and effect data of antibiotics inhibits an effective assessment of the potential risk through environmental pathways. Modelling such aspects should help to improve our understanding of the relative risks posed by contamination of water with ARGs. The future development of more effective biodegradable antibiotics might facilitate their rapid degradation in the environment; in view of the varying rates of low mineralisation and degradation of present-day antibiotics the importance of tackling the social drivers of their misuse is clear. There is now sufficient evidence to support the hypothesis that one of the most important emerging public health threats is that of large-scale dissemination of multi-resistant pathogens in the hospital environment, the community, and the wider environment. Rapid demographic, environmental, and agricultural changes are all contributing to a global antibiotic resistance crisis, which, if not stopped, will emerge as one of the major causes of death in the coming decades. Mitigation strategies are possible and a combined approach based on environmental, agricultural, and medical aspects is needed to tackle this problem. Contributors All authors contributed equally to the manuscript. Conflicts of interest PMH has received research funding and consultancy payments from Pfizer, Novartis, Novacta, Merck, Novolytics, and Wyeth; ABAB has received research funding from GSK and AstraZeneca and consultancy payments from Huvepharma; none of these were made in relation to the manuscript. EMHW, PC, EJF, WHG, DLJ, NML, WO, ASJ-R, CMT, and APW declare that they have no conflicts of interests.

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