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Distinguishable Epidemics of Multidrug-Resistant Salmonella Typhimurium DT104 in Different Hosts

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Science  27 Sep 2013:
Vol. 341, Issue 6153, pp. 1514-1517
DOI: 10.1126/science.1240578

Sourcing Antibiotic Resistance

It is widely assumed that antibiotic resistance in farm animals contributes in a major way to antibiotic resistance in humans. Mather et al. (p. 1514, published online 12 September; see the Perspective by Woolhouse and Ward) analyzed hundreds of genome sequences from Salmonella isolates collected from both livestock and patients in Scotland between 1990 and 2004. The relative contributions of animal-derived and human-derived sources of infection were quantified and the phylogenetic diversity of resistance profiles was matched with bacterial phylogenies. The results suggest that most human infections are caught from other humans rather than from livestock and that humans harbor a greater diversity of antibiotic resistance.

Abstract

The global epidemic of multidrug-resistant Salmonella Typhimurium DT104 provides an important example, both in terms of the agent and its resistance, of a widely disseminated zoonotic pathogen. Here, with an unprecedented national collection of isolates collected contemporaneously from humans and animals and including a sample of internationally derived isolates, we have used whole-genome sequencing to dissect the phylogenetic associations of the bacterium and its antimicrobial resistance genes through the course of an epidemic. Contrary to current tenets supporting a single homogeneous epidemic, we demonstrate that the bacterium and its resistance genes were largely maintained within animal and human populations separately and that there was limited transmission, in either direction. We also show considerable variation in the resistance profiles, in contrast to the largely stable bacterial core genome, which emphasizes the critical importance of integrated genotypic data sets in understanding the ecology of bacterial zoonoses and antimicrobial resistance.

Salmonella enterica subspecies enterica is one of the most common bacterial pathogens of humans and other animals (1, 2). The global burden of disease caused by Salmonella infections is substantial, with more than 90 million human cases of gastroenteritis alone occurring each year (3). The annual cost of these infections is estimated to be about €3 billion in the European Union (4) and about $2.7 billion in the United States (5). The public health impact is exacerbated by antimicrobial resistance (AMR), which leads to increased morbidity, mortality, and treatment costs (6, 7). In our study, the interrelated epidemiologies of Salmonella and AMR were examined at the level of the genome. Our results challenge the established view that the human and animal epidemics are synonymous (811) and show that the phylogenetic associations both within and between the resistance determinants and the host bacteria are different.

Throughout the 1990s, there was a global epidemic of multidrug-resistant S. Typhimurium definitive type 104 (DT104) in animals and humans (12, 13). The DT104 epidemic was important because of its widespread prevalence and perceived zoonotic nature, as well as the high frequency of resistance to a wide range of commonly used antimicrobials, particularly ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The primary source of human DT104 infections is thought to be the animal population, particularly local animals, through both direct contact and food-borne transmission (9, 10, 14). Using whole-genome sequencing, we have studied the course of the epidemic in Scotland, a restricted geographical location that has a well-characterized collection of DT104 bacteria isolated from both humans and other animals over a 22-year period. By interrogating isolates collected from sympatric and contemporaneous hosts and using isolates from geographically diverse hosts from other countries to put them in phylogenetic context, we have an unprecedented opportunity to profile the evolution and spread of a local epidemic of this globally important zoonotic pathogen.

To investigate the evolutionary changes that characterize the long-term epidemic and to assess the roles of animals and humans in the spread of both the bacterium and AMR, we sequenced 142 isolates from humans and 120 isolates from other animals in Scotland sampled from 1990 to 2011. To set the Scottish isolates within a broader international context, 111 isolates were included from other locations around the world (table S1). Sequence reads were mapped against our reference chromosome of DT104 and its virulence plasmid, and genomes were assembled de novo to identify unique regions (15). Maximum likelihood methods were used to produce a phylogenetic tree based on single-nucleotide polymorphisms (SNPs) in the core genome (15). In this tree, we identified a main clade of related DT104 isolates (29 to 123 SNPs compared with the reference core genome) and a separate subset of 14 atypical isolates (620 to 709 SNPs compared with the reference core genome) (fig. S1). Within the main clade, there were 2765 variable sites across the 359 isolates, with pairwise differences between isolates ranging from 0 to 167 SNPs (Fig. 1). Most SNPs were sporadically distributed in the core genome throughout the phylogenetic tree, with relatively few genes showing nonsynonymous SNPs in >5 isolates (fig. S2). These data indicate that the rate of change within the core genome over the 22-year epidemic period, 3.4 × 10−7 substitutions per site per year [95% highest posterior density (HPD) interval: 3.1 × 10−7 to 3.7 × 10−7], is in line with that seen in other S. Typhimurium (16). Although confirmed phenotypically as DT104 by standard phage typing, the 14 atypical isolates are more similar genetically to the non-DT104 S. Typhimurium reference strain LT2, which demonstrates the lack of resolution of traditional typing techniques. Isolates from all geographical locations can be found near the root of the tree, consistent with the rapid global spread of DT104 (12). However, many of these isolates, particularly those from England and Wales, can also be found interspersed with Scottish isolates throughout the rest of the tree. The most deeply rooted are primarily derived from humans and do not have Salmonella genomic island 1 (SGI1), a 43-kb genomic island with a 13-kb multidrug-resistance region (17) that is characteristic of the epidemic strain.

Fig. 1 Phylogeny of Scottish and global S. Typhimurium DT104, rooted on S. Typhimurium SL1344.

Colored rings indicate, from the center out, the host and country of origin, and the unique genotypic AMR profile of each isolate (table S6); the asterisk indicates the location of the reference isolate HF937208. The same tree with bootstrap values added is shown in fig. S5.

To evaluate interspecies transmission and how DT104 circulated in Scotland, Bayesian phylogenetic diffusion models (18, 19) were used to reconstruct the host population of each branch of the phylogenetic tree (Fig. 2A), using the 135 human and 113 animal Scottish DT104 isolates. The most recent common ancestor of DT104 in Scotland is predicted to date to 1968 (95% HPD interval: 1962 to 1976), consistent with reports of antimicrobial-susceptible DT104 strains isolated from human cases of infection since the 1960s in England and Wales (13), with the first report of multidrug-resistant DT104 in Scotland in the mid 1980s (13). What can be observed in Figs. 1 and 2A is that, although there are isolates from a single host population that cluster together, isolates from animals and humans are also interspersed throughout the tree. However, quantification of the association between phylogeny and host population shows that the clustering is not fully randomized (15). Furthermore, similar to the maximum likelihood tree (Fig. 1), the most deeply rooted branches are estimated to be human (Fig. 2A), but it is difficult to infer with certainty the direction of transmission from these observations alone. Overall, the reconstructed host populations of the branches within the tree suggest that there were relatively few animal-to-human or human-to-animal transitions (Fig. 2B). Using Markov jumps to estimate the number of unobserved human-to-animal and animal-to-human transitions along each branch (15), we show the overall numbers of animal-to-human and human-to-animal transitions were similar, which indicates that the majority of human infections in Scotland were unlikely to have been sourced from the local animal population. Markov rewards, from which inferences may be made with respect to which populations might act as sources and which might act as sinks, showed that, although the human isolates represented just 54% of the sample, 62% (666 years, 95% HPD: 545 to 771) of the total phylogenetic tree length was in the human state (15). Conversely, 38% (400 years, 95% HPD: 318 to 521) of the total tree length was spent in the animal state. Our conclusions are consistent when we perform these analyses on animal isolates by species and with only cattle isolates, the main animal reservoir (15, 20). These results support two novel hypotheses: (i) DT104 was predominantly circulating separately within each population in Scotland, with a low frequency of spillover in both directions, and/or (ii) that animals and humans were each sinks for a different and separate source of infection, also with a low level of spillover. Scotland is a net importer of food (21); for example, by value, 58% of all red meat and 38% of raw beef are of non-Scottish origin (22). However, given a lack of surveillance data on food, attribution to this potential source cannot be quantified. Given the likely importance of imported food to the disease burden in humans, the greater global mobility of the human population, and the recognition of this as a risk factor for bacterial infections (23, 24), we suggest that, although these two hypotheses are not mutually exclusive, the second scenario is the more likely.

Fig. 2 Bayesian maximum clade credibility phylogenetic tree and most probable ancestral state reconstruction of host population for S. Typhimurium DT104 in Scotland.

(A) Branches with a reconstructed state (host population) posterior probability of >0.75 are colored red for human, blue for animal; branches with a state probability of <0.75 are colored gray. The same tree is shown in fig. S6 with branch width scaled by the posterior probabilities. (B) Posterior density plot of the numbers of human branches ancestral to human branches, human branches ancestral to animal branches, animal branches ancestral to human branches, and animal branches ancestral to animal branches integrated over the subsample of 3600 phylogenetic trees with reconstructed host population states along branches obtained by using BEAST. These results suggest (i) circulation of DT104 predominantly within animals and humans separately, with only a low frequency of spillover in both directions, and/or (ii) animals and humans were each sinks for different and separate sources of infection, with only a low frequency of spillover in both directions.

Our results show that the ecological diversity of AMR, at the level of the genome, is greater in the isolates from the human population than in those from animals and is not tightly coupled to the bacterial evolutionary history. Examining the distribution of the phenotypic AMR profiles throughout the molecular phylogenetic tree (fig. S3) (15) allowed us to investigate the degree to which the evolutionary history of the organism is linked to that of the resistance determinants it carries, regardless of whether they are genes or SNPs known to confer resistance. We show that isolates demonstrating the same phenotypic resistance profile, even those putatively conferred by SGI1 (17) (fig. S3A), do not cluster together exclusively.

The diversity of AMR was assessed further by interrogating the presence or absence of resistance determinants and profiles in the 147 Scottish isolates selected to cover the range of phenotypic resistance profiles observed in the epidemic period (Fig. 3) (15). A greater number of both resistance determinants and genotypic-resistance profiles are found in the human isolates. As demonstrated by rarefaction analysis (Fig. 3D), these results cannot be accounted for by differential sampling intensity. Multiple, independent recombination events in the multidrug-resistance region occurred, as variants of SGI1 (25) are found throughout the tree (fig. S4). Similarly, although there are some clades with a single AMR gene profile, in many instances, profiles can be found in multiple clades and may be due to the gain or loss by horizontal gene transfer of accessory AMR determinants and their plasmid vectors (tables S2 to S5). Although the propensity for horizontal transmission is well recognized (26), these results provide both a unique insight into the genetic flux occurring across a defined 22-year epidemic and a baseline for the concomitant genetic flux in the DT104 core genome. In contrast to the relatively stable core genome with an expected substitution rate, the AMR features of these isolates varied appreciably throughout the time period.

Fig. 3 Venn diagrams demonstrating the degree of overlap in AMR between the human and animal populations of S. Typhimurium DT104.

(A) The numbers of shared and unique AMR determinants (acquired resistance genes or SNPs known to confer resistance) in the 147 Scottish human and animal DT104 isolates investigated for AMR diversity; (B) the numbers of shared and unique AMR profiles (unique combinations of AMR determinants) in the 147 isolates; (C) the numbers of shared and unique AMR phenotypic profiles (unique combinations of AMR phenotypes) in the original 5200 surveillance isolates of DT104, 1990–2004, Scotland (11). (D) Rarefaction curves, with 95% confidence intervals (vertical lines), of the number of genotypic AMR profiles in the 147 isolates investigated for AMR diversity, demonstrating similar sampling intensity.

Most transmission of DT104 has hitherto been considered to be from animals to humans (2729), but this view has been based primarily on outbreak investigations. However, on the basis of whole-genome sequence analysis, we provide a historical perspective on the epidemic that leads to a different inference. This is the largest study of its kind, utilizing tools with the finest resolution available to investigate the associations of Salmonella Typhimurium DT104 and AMR between and within colocated human and animal populations, and has enabled us to address two separate issues with this data set, those of the origins and dissemination of AMR and also of Salmonella. First, the greater diversity of AMR genes and profiles in the human DT104 isolates suggests that there were other sources contributing to the diversity of the human resistance burden than the local animals; it is noteworthy that this analysis of molecular data leads to identical conclusions to those based on phenotype (11). Second, we show that a large proportion of transmission of the bacterium appears to have occurred within each host population, and/or that there were separate sources of DT104 for the humans and local animals, with a small degree of spillover in both directions. If the majority of human infections were obtained from locally produced food or direct contact with animals, the human isolates should be a subset of the animal isolates, but we do not observe this.

With humans having a more diverse source of infections than the local animal population, it is probable that imported food, foreign travel, and environmental reservoirs are significant sources of Salmonella and AMR in humans in our study (23, 30), but surveillance data simply do not exist in any coherent form locally, nor consistently across the international community, to quantify the importance of these different sources. If we are to address this deficit, there is an urgent need to construct relevant, robust, and harmonized surveillance protocols to capture this information. Similarly, although the Scottish isolates are similar to those from other countries, this investigation needs to be conducted in other organisms and environments to determine the generality of our conclusions across different agricultural, political, and sociological conditions. This study challenges current views on the contribution of the local animal reservoir as a source of Salmonella and AMR in humans. It demonstrates the critical importance of acquiring targeted genotypic data sets integrated across the veterinary and public health sectors in order to understand the ecology of bacterial zoonoses, identify the sources of AMR, and formulate responsible evidence-based control strategies.

Supplementary Materials

www.sciencemag.org/content/341/6153/1514/suppl/DC1

Materials and Methods

Figs. S1 to S7

Tables S1 to S12

References (3170)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: A.E.M. was funded by the William Stewart Fellowship while at the University of Glasgow. A.E.M., S.R.H., M.C.F., T.R.C., J.P., and N.R.T. are supported by Wellcome Trust grant 098051. P.L. and M.A.S. acknowledge funding from the European Union Seventh Framework Programme (FP7/2007-2013) under European Research Council Grant agreement no. 260864. M.A.S. is supported in part by NIH R01 grants AI107034 and HG006139 and NSF grant DMS-1264153. The authors would like to thank J. Cotton (Wellcome Trust Sanger Institute) for helpful discussions and assistance. Genome sequences are deposited with the European Nucleotide Archive (study accessions ERP000244, ERP000270, ERP000994) and DNA Data Bank of Japan (accession DRA000942). The finished chromosome and plasmid for Salmonella Typhimurium DT104 are submitted to the European Molecular Biology Laboratory, accessions HF937208 and HF937209, respectively.
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