Antibiotic resistance and the evolution of group-beneficial traits. II: a metapopulation model

Inspired by the evolution of antibiotic resistance in bacteria, we have developed a model that examines the evolution of "producers" (who secrete a substance that breaks down antibiotics) and non-producers. In a previous study, we found that frequency-dependent selection could favor an intermediate frequency of producers in a single, large population. Here we develop a metapopulation model that examines the evolution of producers and non-producers. Our results indicate that in a metapopulation with many groups, each of size N, the equilibrial frequency of producers decreases with group size. Even when N is high (e.g. 150 individuals/group), however, a significant frequency of producers is still predicted. We also found that the equilibrial frequency of producers increases as the minimum numbers of producers necessary to provide protection to non-producers increases. Lastly, increasing the benefit/cost ratio (b/c) for producers increases their equilibrial frequency.     

Antibiotic production in a spatially structured environment

Ecological factors influencing the effects of antibiotic production were explored experimentally and theoretically. A spatially structured model was used to model the dynamics of antibiotic-producing and nonproducing bacteria in which growth of the nonproducers was reduced by neighbouring antibiotic producers. Various factors affecting spatial interactions between the bacteria were examined for their impact on antibiotic producers. Spatial clustering had a positive impact on the effect of antibiotic production, as measured by the decline in growth of the nonproducing strain, while increasing the initial density of the nonproducing strain had a negative impact. Experiments examined the growth of antibiotic-producing Streptomyces species and a nonproducing, antibiotic-sensitive strain of Bacillus subtilis that were coinoculated on surface media. There was an effect of the Streptomyces on Bacillus growth in some experiments but not in others. In light of the predictions from the model, unintentional clustering of cells is a more likely explanation for this finding than different initial Bacillus densities. The importance of spatial structure seen in this study is consistent with a terrestrial rather than an aquatic distribution of antibiotic-producing bacteria, and may have implications in the search for novel antibiotics.     

Cheating and resistance to cheating in natural populations of the bacterium Pseudomonas fluorescens

Bacteria perform cooperative behaviors that are exploitable by noncooperative cheats, and cheats frequently arise and coexist with cooperators in laboratory microcosms. However, evidence of competitive dynamics between cooperators and cheats in nature remains limited. Using the production of pyoverdine, an iron‐scavenging molecule, and natural soil populations of Pseudomonas fluorescens, we found that (1) nonproducers are present in the population; (2) they co‐occur (<1cm³) with pyoverdine producers; (3) they retain functional pyoverdine receptors; and (4) they can use the pyoverdine of on average 52% of producers. This suggests nonproducers can potentially act as social cheats in soil: utilizing the pyoverdine of others while producing little or none themselves. However, we found considerable variation in the extent to which nonproducers can exploit producers, as some isolates appear to produce exclusive forms of pyoverdine or kill nonproducers with toxins. We examined the consequences of this variation using theoretical modeling. We found variance in exploitability leads to some cheats gaining increased fitness benefits and others decreased benefits. However, the absolute gain in fitness from high exploitation is lower than the drop in fitness from low exploitation, decreasing the mean fitness of cheats and subsequently lowering the proportion of cheats maintained in the population. Our results suggest that although cooperator‐cheat dynamics can occur in soil, a range of mechanisms can prevent nonproducers from exploiting producers.     

Mechanism of bacitracin resistance in gram-negative bacteria that synthesize exopolysaccharides.

Four representative species from three genera of gram-negative bacteria that secrete exopolysaccharides acquired resistance to the antibiotic bacitracin by stopping synthesis of the exopolysaccharide. Xanthomonas campestris, Sphingomonas strains S-88 and NW11, and Escherichia coli K-12 secrete xanthan gum, sphingans S-88 and NW11, and colanic acid, respectively. The gumD gene in X. campestris is required to attach glucose-P to C55-isoprenyl phosphate, the first step in the assembly of xanthan. A recombinant plasmid carrying the gumD gene of X. campestris restored polysaccharide synthesis to bacitracin-resistant exopolysaccharide-negative mutants of X. campestris and Sphingomonas strains. Similarly, a newly cloned gene (spsB) from strain S-88 restored xanthan synthesis to the same X. campestris mutants. However, the intergeneric complementation did not extend to mutants of E. coli that were both resistant to bacitracin and nonproducers of colanic acid. The genetic results also suggest mechanisms for assembling the sphingans which have commercial potential as gelling and viscosifying agents.     

Growth Interactions during Bacterial Colonization of Seedling Rootlets

Rootlet elongation and bacterial growth on rootlets were determined after inoculation of cucumber and spinach seedlings with Pseudomonas strains differing in production of siderophores and HCN. Siderophore producers grew more profusely than nonproducers on both species and promoted rootlet elongation on cucumber. Coinoculation of siderophore producers and nonproducers resulted in restricted growth of the latter. The total populations of nonproducers of HCN in the presence of HCN producers were not decreased, but the tenacity of their association with the rootlet surface was altered.     

Growth interactions during bacterial colonization of seedling rootlets

Rootlet elongation and bacterial growth on rootlets were determined after inoculation of cucumber and spinach seedlings with Pseudomonas strains differing in production of siderophores and HCN. Siderophore producers grew more profusely than nonproducers on both species and promoted rootlet elongation on cucumber. Coinoculation of siderophore producers and nonproducers resulted in restricted growth of the latter. The total populations of nonproducers of HCN in the presence of HCN producers were not decreased, but the tenacity of their association with the rootlet surface was altered.     

Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."     

COEVOLUTION BETWEEN COOPERATORS AND CHEATS IN A MICROBIAL SYSTEM

In many circumstances organisms invest in cooperative activities to increase their mutual fitness but are susceptible to cheats that obtain the benefits of cooperation without investment. Natural selection may favor cooperators that resist cheats, and cheats that avoid such resistance; in theory the coevolutionary interaction may be sustained and dynamic. Here, we report evidence of antagonistic coevolution between cooperators and cheats involved in biofilm formation by Pseudomonas fluorescens bacteria. Two distinct phenotypes occur in static culture tubes: one that can form a biofilm at the air–broth interface and thus obtain improved access to oxygen, and one that colonizes the broth phase but which can also invade, and weaken, the biofilm produced by the other type. Over serial passage, biofilm producers (considered here as cooperators) evolve to become better at resisting invasion, and biofilm nonproducers (cheats) evolve to be more efficient invaders. Each type has higher performance (resistance in the case of cooperators and biofilm invasion for cheats) in competition with isolates of the other type from their past compared to that from their future, indicating a dynamic coevolutionary interaction. Such coevolution may have important consequences for the maintenance of cooperation.     

Selection of resistant bacteria at very low antibiotic concentrations

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.     

Distribution and Quantification of Antibiotic Resistant Genes and Bacteria across Agricultural and Non-Agricultural Metagenomes

There is concern that antibiotic resistance can potentially be transferred from animals to humans through the food chain. The relationship between specific antibiotic resistant bacteria and the genes they carry remains to be described. Few details are known about the ecology of antibiotic resistant genes and bacteria in food production systems, or how antibiotic resistance genes in food animals compare to antibiotic resistance genes in other ecosystems. Here we report the distribution of antibiotic resistant genes in publicly available agricultural and non-agricultural metagenomic samples and identify which bacteria are likely to be carrying those genes. Antibiotic resistance, as coded for in the genes used in this study, is a process that was associated with all natural, agricultural, and human-impacted ecosystems examined, with between 0.7 to 4.4% of all classified genes in each habitat coding for resistance to antibiotic and toxic compounds (RATC). Agricultural, human, and coastal-marine metagenomes have characteristic distributions of antibiotic resistance genes, and different bacteria that carry the genes. There is a larger percentage of the total genome associated with antibiotic resistance in gastrointestinal-associated and agricultural metagenomes compared to marine and Antarctic samples. Since antibiotic resistance genes are a natural part of both human-impacted and pristine habitats, presence of these resistance genes in any specific habitat is therefore not sufficient to indicate or determine impact of anthropogenic antibiotic use. We recommend that baseline studies and control samples be taken in order to determine natural background levels of antibiotic resistant bacteria and/or antibiotic resistance genes when investigating the impacts of veterinary use of antibiotics on human health. We raise questions regarding whether the underlying biology of each type of bacteria contributes to the likelihood of transfer via the food chain.     

Persistence of antibiotic resistance in bacterial populations

Unfortunately for mankind, it is very likely that the antibiotic resistance problem we have generated during the last 60 years due to the extensive use and misuse of antibiotics is here to stay for the foreseeable future. This view is based on theoretical arguments, mathematical modeling, experiments and clinical interventions, suggesting that even if we could reduce antibiotic use, resistant clones would remain persistent and only slowly (if at all) be outcompeted by their susceptible relatives. In this review, we discuss the multitude of mechanisms and processes that are involved in causing the persistence of chromosomal and plasmid-borne resistance determinants and how we might use them to our advantage to increase the likelihood of reversing the problem. Of particular interest is the recent demonstration that a very low antibiotic concentration can be enriching for resistant bacteria and the implication that antibiotic release into the environment could contribute to the selection for resistance. Several mechanisms are contributing to the stability of antibiotic resistance in bacterial populations and even if antibiotic use is reduced it is likely that most resistance mechanisms will persist for considerable times.     

Carriage of antibiotic-resistant bacteria in urban versus rural wild boars

The Western European population of wild boar (Sus scrofa) has increased its distribution over the past several decades, and some populations have colonized areas strongly influenced by human activity. Wild boars are known carriers of antibiotic-resistant bacteria acquired from the environment, and urban populations of wild boars may be more exposed than their rural counterparts. In this work, we compared the frequency of antibiotic resistance in indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium) isolated from urban wild boars with that from rural wild boars in NE Spain. We further assessed whether bacterial isolates from the urban wild boars had a higher probability of showing antibiotic resistance when their host was highly associated to urban features. Seventy-two and 100 bacterial isolates from urban and rural habitat, respectively, were screened for antibiotic resistance against a set of antibiotics (13 per bacterial species). We found a significantly higher frequency of E. faecium showing resistance to tetracycline (70.0% vs 36.4%) and high-level resistance to streptomycin (30.0% vs 4.5%) in urban wild boars compared to rural wild boars (p?<?0.05). E. faecalis was more frequently resistant to trimethoprim in urban than rural wild boars (33.3% vs 0.0%, p?<?0.05). In isolates from urban origin, 55.6% of the likelihood of detecting antibiotic resistance depended only on the bacterial species, being more likely in the enterococci than in E. coli. These results suggest that urban wild boars may be more exposed to certain antibiotic-resistant bacteria or antibiotic resistance genes that they may acquire from the urban environment, although implications are uncertain.     

Non-inherited antibiotic resistance

In addition to their impressive, well-publicized and well-researched propensity to evolve and acquire genetically determined mechanisms for resistance to antibiotics, bacteria that are inherently susceptible to these drugs can also be phenotypically refractory to their action. This phenomenon of 'non-inherited resistance' to antibiotics has been known since the beginning of the antibiotic era but, relative to inherited resistance, it has been given little attention. Here, we review the in vitro and in vivo evidence for the different forms of non-inherited resistance and the mechanisms responsible. With the aid of a simple mathematical model and computer simulations, we show how non-inherited resistance could extend the duration of antibiotic treatment, cause treatment failure and promote the generation and ascent of inherited resistance in treated patients.     

Bioengineering nisin to overcome the nisin resistance protein

The emergence and dissemination of antibiotic resistant bacteria is a major medical challenge. Lantibiotics are highly modified bacterially produced antimicrobial peptides that have attracted considerable interest as alternatives or adjuncts to existing antibiotics. Nisin, the most widely studied and commercially exploited lantibiotic, exhibits high efficacy against many pathogens. However, some clinically relevant bacteria express highly specific membrane‐associated nisin resistance proteins. One notable example is the nisin resistance protein that acts by cleaving the peptide bond between ring E and the adjacent serine 29, resulting in a truncated peptide with significantly less activity. We utilised a complete bank of bioengineered nisin (nisin A) producers in which the serine 29 residue has been replaced with every alternative amino acid. The nisin A S29P derivative was found to be as active as nisin A against a variety of bacterial targets but, crucially, exhibited a 20‐fold increase in specific activity against a strain expressing the nisin resistance protein. Another derivative, nisin PV, exhibited similar properties but was much less prone to oxidation. This version of nisin with enhanced resistance to specific resistance mechanisms could prove useful in the fight against antibiotic resistant pathogens.     

Pyoverdin cheats fail to invade bacterial populations in stationary phase

Microbes engage in cooperative behaviours by producing and secreting public goods, the benefits of which are shared among cells, and are therefore susceptible to exploitation by nonproducing cheats. In nature, bacteria are not typically colonizing sterile, rich environments in contrast to laboratory experiments, which involve inoculating sterile culture with few bacterial cells that then race to fill the available niche. Here, we study the potential implications of this difference, using the production of pyoverdin, an iron‐scavenging siderophore that acts as a public good in the bacteria Pseudomonas aeruginosa. We show that (1) nonproducers are able to invade cultures of producers when added at the start of growth or during early exponential growth phase, but not during late exponential or stationary phase; (2) the producer strain does not produce pyoverdin in the late exponential and stationary phases and so is not paying the cost of cooperating during those phases. These results suggest that whether a nonproducing mutant can invade will depend upon when the mutation arises, as well as the population structure, and raise a potential difficulty with the use of antimicrobial treatment strategies that propose to exploit the invasive abilities of cheats.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic producers, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.     

Antibody‐mediated crosslinking of gut bacteria hinders the spread of antibiotic resistance

The body is home to a diverse microbiota, mainly in the gut. Resistant bacteria are selected by antibiotic treatments, and once resistance becomes widespread in a population of hosts, antibiotics become useless. Here, we develop a multiscale model of the interaction between antibiotic use and resistance spread in a host population, focusing on an important aspect of within‐host immunity. Antibodies secreted in the gut enchain bacteria upon division, yielding clonal clusters of bacteria. We demonstrate that immunity‐driven bacteria clustering can hinder the spread of a novel resistant bacterial strain in a host population. We quantify this effect both in the case where resistance preexists and in the case where acquiring a new resistance mutation is necessary for the bacteria to spread. We further show that the reduction of spread by clustering can be countered when immune hosts are silent carriers, and are less likely to get treated, and/or have more contacts. We demonstrate the robustness of our findings to including stochastic within‐host bacterial growth, a fitness cost of resistance, and its compensation. Our results highlight the importance of interactions between immunity and the spread of antibiotic resistance, and argue in the favor of vaccine‐based strategies to combat antibiotic resistance.     

Tracking Acquired Antibiotic Resistance in Commensal Bacteria of Galápagos Land Iguanas: No Man,No Resistance

Background

Antibiotic resistance, evolving and spreading among bacterial pathogens, poses a serious threat to human health. Antibiotic use for clinical, veterinary and agricultural practices provides the major selective pressure for emergence and persistence of acquired resistance determinants. However, resistance has also been found in the absence of antibiotic exposure, such as in bacteria from wildlife, raising a question about the mechanisms of emergence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. Since previous studies yielded some contrasting results, possibly due to differences in the ecological landscapes of the studied wildlife, we further investigated this issue in wildlife from a remote setting of the Galapagos archipelago.

Methodology/Principal Findings

Screening for acquired antibiotic resistance was carried out in commensal enterobacteria from Conolophus pallidus, the terrestrial iguana of Isla Santa Fe, where: i) the abiotic conditions ensure to microbes good survival possibilities in the environment; ii) the animal density and their habits favour microbial circulation between individuals; and iii) there is no history of antibiotic exposure and the impact of humans and introduced animal species is minimal except for restricted areas. Results revealed that acquired antibiotic resistance traits were exceedingly rare among bacteria, occurring only as non-dominant strains from an area of minor human impact.

Conclusions/Significance

Where both the exposure to antibiotics and the anthropic pressure are minimal, acquired antibiotic resistance traits are not normally found in bacteria from wildlife, even if the ecological landscape is highly favourable to bacterial circulation among animals. Monitoring antibiotic resistance in wildlife from remote areas could also be a useful tool to evaluate the impact of anthropic pressure.     

Bacterial evolution and the cost of antibiotic resistance.

Bacteria clearly benefit from the possession of an antibiotic resistance gene when the corresponding antibiotic is present. But do resistant bacteria suffer a cost of resistance (i.e., a reduction in fitness) when the antibiotic is absent? If so, then one strategy to control the spread of resistance would be to suspend the use of a particular antibiotic until resistant genotypes declined to low frequency. Numerous studies have indeed shown that resistant genotypes are less fit than their sensitive counterparts in the absence of antibiotic, indicating a cost of resistance. But there is an important caveat: these studies have put resistance genes into naive bacteria, which have no evolutionary history of association with the resistance genes. An important question, therefore, is whether bacteria can overcome the cost of resistance by evolving adaptations that counteract the harmful side-effects of resistance genes. In fact, several experiments (in vitro and in vivo) show that the cost of antibiotic resistance can be substantially diminished, even eliminated, by evolutionary changes in bacteria over rather short periods of time. As a consequence, it becomes increasingly difficult to eliminate resistant genotypes simply by suspending the use of antibiotics.