Ecology and Evolution of Antimicrobial Resistance in Bacterial Communities

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Article: Bottery, Michael J. orcid.org/0000-0001-5790-1756, Pitchford, Jonathan W. orcid.org/0000- 0002-8756-0902 and Friman, Ville Petri orcid.org/0000-0002-1592-157X (2020) Ecology and evolution of antimicrobial resistance in bacterial communities. ISME Journal. ISSN 1751-7362 https://doi.org/10.1038/s41396-020-00832-7

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PERSPECTIVE

Ecology and evolution of antimicrobial resistance in bacterial communities

1 1,2 1 Michael J. Bottery ● Jonathan W. Pitchford ● Ville-Petri Friman

Received: 17 April 2020 / Revised: 29 October 2020 / Accepted: 3 November 2020 © The Author(s) 2020. This article is published with open access

Abstract Accumulating evidence suggests that the response of bacteria to antibiotics is significantly affected by the presence of other interacting microbes. These interactions are not typically accounted for when determining pathogen sensitivity to antibiotics. In this perspective, we argue that resistance and evolutionary responses to antibiotic treatments should not be considered only a trait of an individual bacteria species but also an emergent property of the microbial community in which pathogens are embedded. We outline how interspecies interactions can affect the responses of individual species and communities to antibiotic treatment, and how these responses could affect the strength of selection, potentially changing the trajectory of resistance evolution. Finally, we identify key areas of future research which will allow for a more complete understanding of

1234567890();,: 1234567890();,: antibiotic resistance in bacterial communities. We emphasise that acknowledging the ecological context, i.e. the interactions that occur between pathogens and within communities, could help the development of more efficient and effective antibiotic treatments.

Introduction result, clinical susceptibility to antibiotics might not translate well to the successful treatment of polymicrobial infections The global use and misuse of antibiotics has led to the where pathogens are typically embedded in complex mul- evolution and spread of bacterial resistance to all routinely tispecies microbial communities. Interspecies interactions used antibiotics. In order to effectively tackle the spread of are not typically accounted for when assessing antibiotic antimicrobial resistance (AMR) through antimicrobial sensitivity and resistance evolution in the context of anti- stewardship or development of novel antimicrobial com- biotic treatments. In this perspective, we focus on the role pounds, understanding how bacteria adapt and evolve to of interspecies bacterial interactions on the selection of pre- survive antibiotic treatments is crucial. Here we argue that to existing resistance, and on how interactions within bacterial fully understand the evolution of AMR we must study the communities could change the evolutionary trajectory of emergence of resistance within the context of microbial de novo resistance, i.e., the evolutionary dynamics and communities. While the composition, structure and interac- outcomes of antibiotic exposure, particularly within a clin- tions within microbial populations are known to affect the ical context. horizontal gene transfer (HGT) of antibiotic resistance [1–3], The ability of a bacterial strain to survive antibiotic it is becoming evident that intra- and interspecies interac- exposure is commonly assessed in monoculture measure- tions also influence how species respond and evolve under ments of growth, through broth microdilution or agar antibiotic exposure within microbial communities. As a dilution assays, to quantify a minimum inhibitory con- centration (MIC) [4]. These in vitro methods test the sus- ceptibility of individual bacterial strains determining MIC breakpoints and providing informed choices of anti- * Michael J. Bottery microbial interventions. However, our understanding and [email protected] interpretation of MIC measurements has improved in recent 1 Department of Biology, University of York, Wentworth Way, years. Higher initial population densities can result in lower York YO10 5DD, UK susceptibility to some antibiotics, a phenomenon called the fi 2 Department of Mathematics, University of York, Heslington, inoculum effect [5, 6]. In addition, bio lm growth can York YO10 5DD, UK dramatically increase the effective MIC of a population and M. J. Bottery et al. alter the rates of resistance evolution when compared to evolvable trait, as has been demonstrated in the evolution of planktonic cells [7, 8]. Finally, it is increasingly evident that tolerance through the optimisation of lag-times to match the sub-MIC antibiotic concentrations can positively select for intervals of antibiotic exposure [21]. Whereas resistance and resistance mutations, increase HGT of antimicrobial resis- tolerance are considered properties of a population, persis- tance genes (ARGs), and elevate mutation rates; all of tence describes the ability of a small sub-population of a which will increase the likelihood of resistance evolution clonal bacterial population to enter a state of dormancy, [9–12]. As such the minimal selective concentration (MSC) allowing it to survive high concentrations of antibiotic while and predicted no effect concentration have become impor- the rest of the population is rapidly killed [22, 23]. Both tant metrics for understanding and controlling the evolution tolerance and persistence allows surviving bacteria to and spread of resistance [13]. resume normal growth after the depletion of the antibiotic. The effect of ecological context, i.e., the type and dynamics of microbial interactions that occur between pathogens and the surrounding community, is often over- Interspecies interactions can alter responses looked when considering antibiotic efficacy and the evolu- to antibiotics tion of resistance. We currently predominantly consider antibiotic sensitivity as an independent trait of an individual Interspecies interactions can have a profound effect upon strain. However, it is clear that inter- and intra-species the outcome of antibiotic treatment. We define three main interactions within both clinical and environmental com- ways in which bacterial communities, or subpopulations of munities, as well as the physical properties of the associated communities, can survive exposure to antimicrobials due to microbial community, can alter the physiological state and interspecies interactions: (1) Collective resistance, interac- antibiotic susceptibility of bacteria [14]. Interspecies inter- tions within a community that elevate the ability of its actions are especially common in polymicrobial infections members to resist the action of an antibiotic and continue to where multiple species contribute to disease [15]. These grow in the presence of antibiotics thus increasing the MIC interactions not only affect the severity of infections but of the community. (2) Collective tolerance, interactions could also alter the exposure and physiological responses to within a community that alter cell state, such as slowing antibiotics. We thus argue that bacterial ability to withstand down metabolism, and thus slow down the rate of cell death antibiotic treatments, and the consequent evolution of during transient exposure to antibiotics without an increase resistance to those treatments, should be viewed as an in MIC. (3) Exposure protection, interactions within a emergent property of a microbial community rather than community that protect its sensitive members during anti- simply a trait of a particular species. biotic treatment by reducing the effective concentration of To discuss the effects of antimicrobial exposure on antibiotic [24, 25]. These definitions are not mutually microbial communities we must first distinguish the dif- exclusive; interactions could have multiple different effects, ferent ways in which bacteria survive in the presence of and multiple different mechanisms may act simultaneously antibiotics. Resistance is often defined as the inheritable within a community. ability of a cell to grow at high concentrations of an anti- Many of the known mechanisms that provide collective biotic, irrespective of the duration of exposure [16]. Its resistance, tolerance or exposure protection are density- mechanisms are many and varied [17]. Resistance can be dependent, providing protection to large, dense populations intrinsic, whereby the physiological properties of all mem- [25] through the inactivation of antibiotics, biofilm forma- bers of a species allow it to resist the action of certain tion and quorum sensing-activated responses [26–28]. antibiotics; or acquired, either through de novo mutation or While such interactions are often considered to take place via the acquisition of ARGs via HGT. However, bacterial among cells of the same (or closely related) genotypes, the populations can also survive antibiotic treatment without same mechanisms can also occur between different geno- encoding specific resistance mechanisms. Tolerance types or species in diverse multi-species communities. For describes the ability of a bacterial population to survive example, an analysis of the multi-species networks of transient exposure to a high concentration of an antibiotic urinary tract infections showed that clinical isolates from the without a change in their MIC [16]. This is often mediated same patient often protect each other from antibiotics, with by a change in the physiological state of the cells within a the protective effects correlating with interspecies interac- population due to their environmental context, such as tions that foster growth benefits [29]. However, the majority slowing down essential cellular processes following a lack of interactions within multispecies communities are argued of resources [18], interactions with a host’s immune system to be competitive, with most pairwise interactions resulting [19] or interactions with other bacterial species [20], in a net reduction in community productivity [29, 30]. resulting in a reduced death rate in the presence of anti- Disruption of the complex network of interactions within biotic. Alternatively, tolerance can be a heritable and communities, whether cooperative or competitive, through Ecology and evolution of antimicrobial resistance in bacterial communities exposure to antimicrobials will thereby alter community The benefit of antibiotic inactivation is not limited to peri- structure and change the growth and survival of its con- plasmic or secreted enzymes; the intracellular inactivation stituent members. of chloramphenicol by Staphylococcus aureus or Strepto- Resistance mechanisms that inactivate antibiotics are coccus pneumoniae, via a plasmid borne acetyltransferase, widespread, providing both acquired and intrinsic resistance allows the survival of sensitive S. pneumoniae through the to some of the most widely used antibiotics including detoxification of the local environment in liquid culture, on aminoglycosides, chloramphenicol and β-lactams. Such semi-solid surfaces and in in vivo mouse models [26]. resistance mechanisms can be considered as cooperative Computational modelling suggests that the benefits of such traits; the benefit of antibiotic inactiaving enzyme produc- cooperative antibiotic inactivation is greatest when com- tion is not confined to the producer, rather it is shared munity interactions are mutualistic, as mutualism drives the across the community [31, 32]. Cooperative enzymatic spatial mixing of the communities and hence the sharing of inactivation provides exposure protection through reducing the benefit of antibiotic inactivation [37]. As a result, anti- the environmental concentration of an antibiotic (Fig. 1a), biotic inactivation could provide protection to a microbial which can be exploited by sensitive members of the same, community allowing sensitive members of the community and different, species within a community [26, 33–35]. For to survive otherwise lethal antibiotic treatments. example, the enzymatic inactivation of β-lactam antibiotics Collective resistance, collective tolerance and exposure via β-lactamase producing Escherichia coli can protect protection to antibiotics can also be provided through bio- Salmonella enterica serovar Typhimurium from con- film formation (Fig. 1b). Bacterial biofilms comprise of centrations of antibiotic that would usually kill them [33]. cells growing as aggregates surrounded by a self-secreted The presence of β-lactamases has been reported in a wide polymer matrix of exopolysaccharides, DNA and proteins. range of clinical samples taken from polymicrobial infec- Biofilms provide exposure protection to their members tions (reviewed in [36]) suggesting that they likely con- through limiting the diffusion of antibiotics into the popu- tribute to the resilience of infections to β-lactam antibiotics. lation [38] and increasing the protection provided by

Fig. 1 Community interactions, as well as resistance genotype, of secreted signalling molecules, such as indole and DSF, can trigger affect the response to antibiotic exposure. In all panels, cell growth antibiotic resistance states in otherwise susceptible community mem- state is represented by either hatched (unable to grow) or solid fill (able bers through increasing the expression of resistance genes. d Reliance to grow). a Resistant bacteria that inactivate antibiotics reduce the upon cross-feeding networks can be detrimental for a resistant species local antibiotic concentration, providing exposure protection to sur- if its growth depends on cross-feeding on secretions of susceptible rounding sensitive species. This benefit is dependent upon the density community members. In this scenario, tolerance to antibiotics is and spatial structure of the population, as well as the diffusion rate and lowered to the level of the most susceptible community member as rate of inactivation of the antibiotic. b Some species are unable to form cross-feeding interactions are lost due to antibiotic killing (dashed biofilms in isolation but are able to gain improved antibiotic tolerance white arrows) thus the resistant species is unable to grow due to the by participating in established biofilms of other species. c The receipt loss of essential recourses. M. J. Bottery et al. antibiotic inactivation [34]. In addition, biofilms can induce polymicrobial pulmonary infection, alters P. aeruginosa tolerant cell states due to nutrient and oxygen gradients that biofilm structure and stimulates the synthesis of proteins lead to a reduction in metabolic activity in the centre of the that provide resistance to cationic antimicrobial peptides biofilm [27], increasing the proportion of persister cells such as polymyxin [55]. In addition to cellular cross talk, within the population [23]. Biofilms can also increase levels exotoxins — presumably produced to inhibit the growth of of resistance by altering the expression of pre-existing competitors — can promote the transition of competing ARGs [39]. Compared to single-species biofilms, inter- bacterial species into an antibiotic tolerant physiological species interactions within multi-species biofilms can lead state. For example, P. aeruginosa induces highly tolerant to further increases in survival during antibiotic treatment small colony variants of S. aureus through the secretion of through altering spatial structure of the biofilm, increasing 4-hydroxy-2-heptylquinoline-N-oxide, which inhibits the the expression of resistance mechanisms, and allowing for electron transport chain, dramatically slowing down the individually expressed antimicrobial defences to protect the growth rate of S. aureus [20, 56]. Given the vast diversity of whole community [40–42]. However, competition between bacterial secreted products, and the complexity of multi- genotypes via the secretion of toxins [43–45] and the species communities, it is likely that we have barely scrat- seizing of space via the use of adhesins [46, 47] and matrix ched the surface of understanding how these secretions alter production [48] may limit social mixing of different geno- antibiotic resistance and tolerance. types within a biofilm and eradicate competing species [49]. Competitive interactions, via interference or exploitation, Thus, competition may help to privatise public goods within are prevalent within mixed bacterial communities due to multi-species biofilms, limit between-genotype cooperation overlaps in metabolic requirements and limitation of space and break down interactions that increase the ability of the [29, 30, 57]. Disruption or alteration in the intensity of community to survive antibiotic treatment. interspecies competition, or cooperation, through exposure Much work on biofilms has focused on the interspecies to antibiotics can alter community structure by making interactions within the lungs of Cystic Fibrosis (CF) patients, otherwise inaccessible resources available for exploitation where multi-species biofilm formation is common. Within by other members of the community. These changes in mixed biofilms Pseudomonas aeruginosa can cause a meta- community structure may alter the ability of component bolic shift in S. aureus, reducing its growth and providing S. members to survive antibiotic exposure. Antibiotic treat- aureus with protection to vancomycin [50]. Reciprocally, S. ment of brewery multi-species biofilms, which were domi- aureus can enhance tobramycin tolerance of P. aeruginosa by nated by competitive interactions, resulted in reduced levels increasing aggregation and altering biofilm architecture in CF of competition due to the inhibition of highly competitive model systems [51]. However, the activity of antibiotics species within the population. This led to increased growth against multi-species biofilms appears to depend on both of species that were otherwise suppressed within the community composition and the antibiotic treatment, with population [58] and in turn elevated the antibiotic tolerance some combinations of bacterial species decreasing rates of of these species, likely due to the density-dependent nature killing by antibiotics, while others had no effect [52]. Whether of collective tolerance mechanisms. Similar results were interactions within biofilms facilitate or limit antibiotic resis- observed within experimental bacterial communities [59], tance or tolerance [53] might depend on the mode of the where antibiotic treatment benefited members of the com- interaction, i.e., direct cell contact versus diffusible molecules, munity that had marginally elevated levels of tolerance. In as the physical properties of the community alter the prob- contrast, the mutualistic interaction of cross-feeding can ability of cell-cell contact and diffusion rates. lead to inter-dependence between multiple species within a Bacteria secrete a vast variety of compounds ranging community; thus lowering the antibiotic sensitivity of all from signalling molecules involved in quorum sensing to members of the cross-feeding network to that of the weakest exotoxins involved in virulence and competition. Secreted member of the consortium (Fig. 1d) [37, 60]. Taken toge- products within communities have been shown to directly ther, these findings suggest that how individuals and com- alter gene expression, metabolic processes and the growth munities respond to antibiotic exposure depends on a dynamics of co-residing bacterial species, altering their complex network of interactions including social exploita- ability to survive antibiotic exposure (Fig. 1c). For example, tion, cooperation, competition and communication. interspecies signalling via the secretion of indole by E. coli activates the expression of an indole-dependent multi-drug efflux pump in Pseudomonas putida, which cannot produce Ecological context can influence the the indole itself, leading to elevated levels of resistance in selection for antimicrobial resistance P. putida [54]. Likewise, the secretion of diffusible signal factor by Stenotrophomonas maltophilia, a Gram-negative How does a multi-species community alter the selection for bacterium that often co-occurs with P. aeruginosa during AMR within its members? The answer to this question Ecology and evolution of antimicrobial resistance in bacterial communities depends upon the members of the community in question relatively higher concentrations of antibiotic [66]. However, and the specific interactions that occur between them. the selection dynamics of resistance would typically depend Multiple interactions — including competition, exploita- upon the nature of protection provided by the community. tion, commensalism and mutualism — take place in com- When protection is provided by a single resistant species, munities and each of them has the potential to alter the for example through exposure protection via antibiotic strength of selection acting upon resistance mechanisms of inactivation, selection for this resistant genotype would be its members. Consequently, AMR evolution within com- expected to follow negative frequency dependence. When munities may occur at different rates, result in different the resistant strain is rare exposure protection will be pre- magnitudes of resistance, or be associated with different ferentially directed towards the resistant genotype. As the levels of cost compared to single species populations. resistant strain becomes more common, exposure protection The presence of interacting species could further affect will be shared with a larger proportion of sensitive members the evolutionary trajectory of de novo resistance by chan- of the community and thus the relative fitness of the resis- ging which mutations and molecular mechanisms are tant genotype will decrease. In addition, if protection is selected for. provided by a single keystone species, protection might be Previous studies of the evolutionary dynamics of ARGs more vulnerable to stochastic events and density fluctua- have mostly focused on resistant and sensitive strains within tions of the protective species. In contrast, if protection is single-species populations [9, 32, 61]. These studies indi- provided by multiple species present in the community, or cate that the concentration of antibiotic required to posi- by the physical properties of the community (e.g., biofilm tively select for resistant bacteria, MSC, is far below the matrix), collective resistance or tolerance would likely be MIC of their sensitive counterparts [9, 61]. Low antibiotic more stable due to relatively higher functional redundancy concentrations at the MSC may be non-lethal to the sensi- of “protection” within the community. Ecological stability tive strains, but they reduce the growth rate of sensitive cells of collective resistance and collective tolerance could thus sufficiently to outweigh the inherent costs associated with alter the length of antibiotic exposure, and therefore alter the harbouring resistance [62–64]. Low levels of antibiotics timescales in which selection for resistance can act [67, 68]. could thus already select for an increase in the abundance of Inactivation of antibiotics may result in transient and short- antibiotic resistant cells within a population (Fig. 2a/b). lived antibiotic exposure leading to changes at ecological There are few experimental studies comparing how anti- rather than evolutionary timescales, that is, changes in the biotic resistant and sensitive strains respond to antibiotic composition and frequencies of different species within the selection in the absence and presence of other competing community similar to the effect of ecological disturbances. bacteria. Klümper et al. [14] recently demonstrated that In contrast, interspecies interactions that increase antibiotic complex multi-species anaerobic communities derived from tolerance may prolong antibiotic exposure at sub-inhibitory pig faeces can increase the concentration of antibiotics concentrations, allowing the accumulation of de novo required to select for resistant E. coli strains embedded mutations upon which selection can act [69] (in contrast to within the community. Mechanistically, it was shown that >MIC selection which typically acts upon the standing this increase in MSC was caused either by protection of the genetic variation within the population [Fig. 2e] [11]). Thus, sensitive E. coli strain by the community (Fig. 2d) (as communities could alter the trajectory of resistance evolu- previously shown with cooperative antibiotic inactivation in tion, allowing the stepwise generation of low effect, low single species populations [31, 32]) or through competition cost resistance mutations that together provide high levels elevating the magnitude of costs associated with the resis- of resistance (Fig. 2f). Similar results have been observed tance (Fig. 2c) with no resulting change in the MIC of the during sub-MIC selection for resistance in S. enterica, sensitive strain [14]. More generally, these results are in line which resulted in changes in multiple different resistance with previous studies showing that competition can con- mechanisms at the molecular level, including alteration of strain bacterial adaptation [65] supporting the idea that the ribosomal RNA target, reduction in antibiotic uptake antagonistic bacteria-bacteria interactions are likely to limit and induction of antibiotic-modifying enzymes [11]. the evolution of antibiotic resistance in communities. Interspecies competition inherent to complex microbial Interactions within a community that increase the prob- communities can also elevate the magnitude of cost of ability of the survival of its sensitive members during resistance [14, 70], which may favour the selection for antibiotic exposure will also alter the selection dynamics of low cost antibiotic resistance mutations, or alternatively, ARGs within the community [31, 32]. When a resistant increase the likelihood of compensatory mutations that strain provides protection to an otherwise sensitive strain overcome the associated costs of resistance. Similar trade- within a community, the MSC of resistance would be offs between cost and resistance have been observed when expected to increase (Fig. 2d). This is because sensitive comparing the evolution of resistance between planktonic strains would be able to outcompete resistant strains at and biofilm lifestyles [71], with members of biofilms M. J. Bottery et al.

Fig. 2 Fitness and selection consequences of differential effects of may arise by community protection (d). e, f The emergence of de novo antibiotic concentration on growth rate. a Theoretical max growth resistance mutations can be altered by community protection. e In the rates in pure culture of ‘isogenic’ strains differing only in resistance or absence of a protective community antibiotic exposure above the MIC sensitivity to antibiotic. Shaded area represents the range of antibiotic acts upon standing genetic variation, often selecting for a single high concentrations in subsequent panels (b–d) exploring competition resistance, high cost mutation. e Communities that provide exposure outcomes and minimal selective concentration (MSC) based on these protection may reduce the realised antibiotic exposure to sub-MIC relative growth rates. b The MSC is defined as the antibiotic con- levels, allowing for the sequential accumulation of low cost, low centration at which growth rate of the resistant strain exceeds that of resistance mutations that together provide high levels of resistance for the sensitive strain (relative fitness >1). c, d In a community, the MSC example via epistasis. Letters inside panels (S, R, A, AB, etc.) can be increased by two basic mechanisms. One is increased costs of represent the accumulation of different mutations during selective resistance, which may arise by increased competition for nutrients (c) sweeps. and the other is reduced antibiotic effect upon sensitive strains, which acquiring low cost mutations that provided overall lower might affect antibiotic resistance evolution in microbial levels of resistance compared to resistance mutations communities. acquired during planktonic growth. Moreover, it has recently been shown that bacterial resistance evolution to phage can be changed in the presence of competitors from Significance of interspecies interactions for more costly surface receptor-mediated resistance to less the success of antibiotic treatments costly CRISPR-based resistance [70]. While antibiotic and phage resistances are not directly comparable, both Even though interspecies interactions during polymicrobial are mediated by multiple different mechanisms with infections — as well as between focal pathogens and resi- varying magnitudes of cost, and their evolutionary tra- dent microbiota of the body, such as those found in the oral jectories could thus be similarly shaped by the presence of cavity or gastrointestinal tract [72, 73] — are known to alter competitors. More experimental studies are needed to the outcome of antibiotic treatments [74, 75], these inter- better understand the multiple ways interacting species actions are often overlooked. For example, relying on Ecology and evolution of antimicrobial resistance in bacterial communities testing the antibiotic susceptibility of pathogens in mono- evolution of resistance within focal pathogen strains. In cultures could confound our ability to select the most addition, these interventions may be effective against appropriate antibiotics for clinical interventions because cooperative antibiotic inactivation mechanisms of other these tests might not reliably reflect community-scale members of multi-species communities that are protecting responses. Indeed, in vitro antibiotic susceptibility does focal pathogens from treatment. However, it is not clear not always translate to successful treatment of poly- how communities affect the evolution of resistance towards microbial infections, where disease is caused by multiple enzyme inhibitors [86]. Other cooperative virulence traits interacting pathogens [76]. In fact, the response of indivi- such as EPS production are also vulnerable to perturbation; dual species to antibiotic treatment within a community can inhibition of EPS production in S. enterica combined with be opposite to that in isolation, with sensitive species being antibiotic treatment proved to be an evolutionarily robust able to grow and tolerant species being inhibited in the strategy to decrease collective tolerance and reduce the presence of antibiotic [77]. Given that many chronic probability of resistance evolution in vitro [87]. The infections that require long term, repeat antibiotic treat- potency of such interventions targeting cooperative traits ments — such as infections of the CF lung, urinary tract within polymicrobial biofilms is yet to be investigated. and diabetic wounds — arecausedbypolymicrobial More combinatorial approaches targeting the disruption of communities, it is important to start considering how the interactions that elevate tolerance may be discovered as we composition of bacterial communities modulates not only gain a greater understanding of the intricacies of interaction disease severity but also the success of antibiotic treat- networks shaping collective tolerance within bacterial ment outcomes [15, 78]. communities. Culture-independent methods, such as 16S rRNA sequencing and shotgun metagenomics, are providing an increasingly detailed understanding of the composition of Conclusion microbiota during infections. These methods have allowed for the tracking of community structure and species richness Bacteria typically coexist in complex multi-species com- within individual CF patients through time, across infection munities and the interactions within these communities can types and during antibiotic treatment [79, 80], provided dictate how bacteria respond to antibiotic exposure. This detailed understanding of the response of host microbiota has important clinical, ecological and environmental con- during Clostridium difficile infection [81] and directly sequences, altering levels of tolerance, the selection of identified resistance genes during polymicrobial urinary resistance and the trajectory of resistance evolution. Con- tract [82] and bone and joint infections [83]. As our ability ventionally, conclusions about the antibiotic susceptibility to identify and track resistance determinants within micro- of a pathogen are drawn from pure culture measurements of bial communities improves, the prospect of using clinical cells in a generally homogenous state. Such information metagenomics to design personalised drug regimens may be adequate for the treatment of infections caused by a becomes more appealing [84]. Despite these advances, we single strain. However, it may not be informative of the still face a major challenge in identifying which species are antibiotic susceptibility of a focal pathogen during poly- interacting within microbial communities and how these microbial infection or embedded within a commensal may alter both disease progression and the outcomes of microbial community. We propose that to effectively design antimicrobial treatment. In most cases the specific interac- antimicrobial stewardship for pathogens residing in com- tions during polymicrobial infection, or between focal munities we need to view antibiotic resistance as an emer- pathogens and the resident commensal communities, remain gent property that arises as a result of combined effects unknown. To incorporate multi-species interactions into of antibiotic exposure and microbial interactions within future treatment design requires identification of not only communities. the species present, but also the interactions between them. While understanding AMR in a community context is How can improved knowledge on microbial ecology be challenging given the diversity and complexity of microbial translated into more effective treatment of polymicrobial interaction networks, it can be achieved through the careful infections? A detailed and personalised understanding of the combination of complementary approaches: (1) antibiotic microbial taxa present during polymicrobial infections, susceptibility testing, where appropriate, should be conducted along with the interactions between them, may facilitate upon communities in addition to single-cell cultures because new approaches to antibiotic resistance [74]. Resistance resistance is determined by the interactions taking place mechanisms that inactivate antibiotics have been the target within that specific community; (2) This should be combined of inhibitory compounds, such as the co-treatment of β- with analyses exploring the consequences of antibiotic lactams with β-lactamase inhibitors [85] and such treat- treatments on community structure and functioning, which ments are commonly used in an attempt to overcome the could further change community susceptibility to antibiotics M. J. Bottery et al. during long term or repeated treatments which are common 5. 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