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BackgroundCarbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms.MethodsTo overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years.ResultsOur CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment.ConclusionsCollectively, the framework we developed provides a clearer, high-resolution

Human-associated microbial communities comprise not only complex mixtures of bacterial species, but also mixtures of conspecific strains, the implications of which are mostly unknown since strain level dynamics are underexplored due to the difficulties of studying them. We introduce the Strain Genome Explorer (StrainGE) toolkit, which deconvolves strain mixtures and characterizes component strains at the nucleotide level from short-read metagenomic sequencing with higher sensitivity and resolution than other tools. StrainGE is able to identify strains at 0.1x coverage and detect variants for multiple conspecific strains within a sample from coverages as low as 0.5x., Pattern Recognition and Bioinformatics

Human-associated microbial communities comprise not only complex mixtures of bacterial species, but also mixtures of conspecific strains, the implications of which are mostly unknown since strain level dynamics are underexplored due to the difficulties of studying them. We introduce the Strain Genome Explorer (StrainGE) toolkit, which deconvolves strain mixtures and characterizes component strains at the nucleotide level from short-read metagenomic sequencing with higher sensitivity and resolution than other tools. StrainGE is able to identify strains at 0.1x coverage and detect variants for multiple conspecific strains within a sample from coverages as low as 0.5x.

BackgroundCarbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms.MethodsTo overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years.ResultsOur CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment.ConclusionsCollectively, the framework we developed provides a clearer, high-resolution

Human-associated microbial communities comprise not only complex mixtures of bacterial species, but also mixtures of conspecific strains, the implications of which are mostly unknown since strain level dynamics are underexplored due to the difficulties of studying them. We introduce the Strain Genome Explorer (StrainGE) toolkit, which deconvolves strain mixtures and characterizes component strains at the nucleotide level from short-read metagenomic sequencing with higher sensitivity and resolution than other tools. StrainGE is able to identify strains at 0.1x coverage and detect variants for multiple conspecific strains within a sample from coverages as low as 0.5x.

Healthy development of the gut microbiome provides long-term health benefits. Children raised in countries with high infectious disease burdens are frequently exposed to diarrhoeal pathogens and antibiotics, which perturb gut microbiome assembly. A recent cluster-randomized trial leveraging >4,000 child observations in Dhaka, Bangladesh, found that automated water chlorination of shared taps effectively reduced child diarrhoea and antibiotic use. In this substudy, we leveraged stool samples collected from 130 children 1 year after chlorine doser installation to examine differences between treatment and control children's gut microbiota. Water chlorination was associated with increased abundance of several bacterial genera previously linked to improved gut health; however, we observed no effects on the overall richness or diversity of taxa. Several clinically relevant antibiotic resistance genes were relatively more abundant in the gut microbiome of treatment children, possibly due to increases in Enterobacteriaceae. While further studies on the long-term health impacts of drinking chlorinated water would be valuable, we conclude that access to chlorinated water did not substantially impact child gut microbiome development in this setting, supporting the use of chlorination to increase global access to safe drinking water.

Human-associated microbial communities comprise not only complex mixtures of bacterial species, but also mixtures of conspecific strains, the implications of which are mostly unknown since strain level dynamics are underexplored due to the difficulties of studying them. We introduce the Strain Genome Explorer (StrainGE) toolkit, which deconvolves strain mixtures and characterizes component strains at the nucleotide level from short-read metagenomic sequencing with higher sensitivity and resolution than other tools. StrainGE is able to identify strains at 0.1x coverage and detect variants for multiple conspecific strains within a sample from coverages as low as 0.5x., Pattern Recognition and Bioinformatics

Recurrent urinary tract infections (rUTIs) are a major health burden worldwide, with history of infection being a significant risk factor. While the gut is a known reservoir for uropathogenic bacteria, the role of the microbiota in rUTI remains unclear. We conducted a year-long study of women with and without history of rUTIs, from whom we collected urine, blood and monthly fecal samples for multi-omic interrogation. The rUTI gut microbiome was significantly depleted in microbial richness and butyrate-producing bacteria compared to controls, reminiscent of other inflammatory conditions, though Escherichia coli gut and bladder dynamics were comparable between cohorts. Blood samples revealed signals of differential systemic immunity, leading us to hypothesize that rUTI susceptibility is in part mediated through a syndrome involving the gut-bladder axis, comprising gut dysbiosis and differential immune response to bacterial bladder colonization, manifesting in symptoms. This work highlights the potential for microbiome therapeutics to prevent and treat rUTIs.

As many malaria-endemic countries move towards elimination of Plasmodium falciparum, the most virulent human malaria parasite, effective tools for monitoring malaria epidemiology are urgent priorities. P. falciparum population genetic approaches offer promising tools for understanding transmission and spread of the disease, but a high prevalence of multi-clone or polygenomic infections can render estimation of even the most basic parameters, such as allele frequencies, challenging. A previous method, COIL, was developed to estimate complexity of infection (COI) from single nucleotide polymorphism (SNP) data, but relies on monogenomic infections to estimate allele frequencies or requires external allele frequency data which may not available. Estimates limited to monogenomic infections may not be representative, however, and when the average COI is high, they can be difficult or impossible to obtain. Therefore, we developed THE REAL McCOIL, Turning HEterozygous SNP data into Robust Estimates of ALelle frequency, via Markov chain Monte Carlo, and Complexity Of Infection using Likelihood, to incorporate polygenomic samples and simultaneously estimate allele frequency and COI. This approach was tested via simulations then applied to SNP data from cross-sectional surveys performed in three Ugandan sites with varying malaria transmission. We show that THE REAL McCOIL consistently outperforms COIL on simulated data, particularly when most infections are polygenomic. Using field data we show that, unlike with COIL, we can distinguish epidemiologically relevant differences in COI between and within these sites. Surprisingly, for example, we estimated high average COI in a peri-urban subregion with lower transmission intensity, suggesting that many of these cases were imported from surrounding regions with higher transmission intensity. THE REAL McCOIL therefore provides a robust tool for understanding the molecular epidemiology of malaria across transmission settings.

As many malaria-endemic countries move towards elimination of Plasmodium falciparum, the most virulent human malaria parasite, effective tools for monitoring malaria epidemiology are urgent priorities. P. falciparum population genetic approaches offer promising tools for understanding transmission and spread of the disease, but a high prevalence of multi-clone or polygenomic infections can render estimation of even the most basic parameters, such as allele frequencies, challenging. A previous method, COIL, was developed to estimate complexity of infection (COI) from single nucleotide polymorphism (SNP) data, but relies on monogenomic infections to estimate allele frequencies or requires external allele frequency data which may not available. Estimates limited to monogenomic infections may not be representative, however, and when the average COI is high, they can be difficult or impossible to obtain. Therefore, we developed THE REAL McCOIL, Turning HEterozygous SNP data into Robust Estimates of ALelle frequency, via Markov chain Monte Carlo, and Complexity Of Infection using Likelihood, to incorporate polygenomic samples and simultaneously estimate allele frequency and COI. This approach was tested via simulations then applied to SNP data from cross-sectional surveys performed in three Ugandan sites with varying malaria transmission. We show that THE REAL McCOIL consistently outperforms COIL on simulated data, particularly when most infections are polygenomic. Using field data we show that, unlike with COIL, we can distinguish epidemiologically relevant differences in COI between and within these sites. Surprisingly, for example, we estimated high average COI in a peri-urban subregion with lower transmission intensity, suggesting that many of these cases were imported from surrounding regions with higher transmission intensity. THE REAL McCOIL therefore provides a robust tool for understanding the molecular epidemiology of malaria across transmission settings.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is one of the most common healthcare-associated pathogens. To examine the role of inter-hospital patient sharing on MRSA transmission, a previous study collected 2,214 samples from 30 hospitals in Orange County, California and showed by spa typing that genetic differentiation decreased significantly with increased patient sharing. In the current study, we focused on the 986 samples with spa type t008 from the same population.MethodsWe used genome sequencing to determine the effect of patient sharing on genetic differentiation between hospitals. Genetic differentiation was measured by between-hospital genetic diversity, F ST , and the proportion of nearly identical isolates between hospitals.ResultsSurprisingly, we found very similar genetic diversity within and between hospitals, and no significant association between patient sharing and genetic differentiation measured by F ST . However, in contrast to F ST , there was a significant association between patient sharing and the proportion of nearly identical isolates between hospitals. We propose that the proportion of nearly identical isolates is more powerful at determining transmission dynamics than traditional estimators of genetic differentiation (F ST ) when gene flow between populations is high, since it is more responsive to recent transmission events. Our hypothesis was supported by the results from coalescent simulations.ConclusionsOur results suggested that there was a high level of gene flow between hospitals facilitated by patient sharing, and that the proportion of nearly identical isolates is more sensitive to population structure than F ST when gene flow is high.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is one of the most common healthcare-associated pathogens. To examine the role of inter-hospital patient sharing on MRSA transmission, a previous study collected 2,214 samples from 30 hospitals in Orange County, California and showed by spa typing that genetic differentiation decreased significantly with increased patient sharing. In the current study, we focused on the 986 samples with spa type t008 from the same population.MethodsWe used genome sequencing to determine the effect of patient sharing on genetic differentiation between hospitals. Genetic differentiation was measured by between-hospital genetic diversity, F ST , and the proportion of nearly identical isolates between hospitals.ResultsSurprisingly, we found very similar genetic diversity within and between hospitals, and no significant association between patient sharing and genetic differentiation measured by F ST . However, in contrast to F ST , there was a significant association between patient sharing and the proportion of nearly identical isolates between hospitals. We propose that the proportion of nearly identical isolates is more powerful at determining transmission dynamics than traditional estimators of genetic differentiation (F ST ) when gene flow between populations is high, since it is more responsive to recent transmission events. Our hypothesis was supported by the results from coalescent simulations.ConclusionsOur results suggested that there was a high level of gene flow between hospitals facilitated by patient sharing, and that the proportion of nearly identical isolates is more sensitive to population structure than F ST when gene flow is high.

Whole genome sequencing of pathogens from multiple hosts in an epidemic offers the potential to investigate who infected whom with unparalleled resolution, potentially yielding important insights into disease dynamics and the impact of control measures. We considered disease outbreaks in a setting with dense genomic sampling, and formulated stochastic epidemic models to investigate person-to-person transmission, based on observed genomic and epidemiological data. We constructed models in which the genetic distance between sampled genotypes depends on the epidemiological relationship between the hosts. A data augmented Markov chain Monte Carlo algorithm was used to sample over the transmission trees, providing a posterior probability for any given transmission route. We investigated the predictive performance of our methodology using simulated data, demonstrating high sensitivity and specificity, particularly for rapidly mutating pathogens with low transmissibility. We then analyzed data collected during an outbreak of methicillin-resistant Staphylococcus aureus in a hospital, identifying probable transmission routes and estimating epidemiological parameters. Our approach overcomes limitations of previous methods, providing a framework with the flexibility to allow for unobserved infection times, multiple independent introductions of the pathogen, and within-host genetic diversity, as well as allowing forward simulation.

Healthcare-associated infections (HCAIs) remain a problem worldwide, and can cause severe illness and death. The increasing level of antibiotic resistance among bacteria that cause HCAIs limits infection treatment options, and is a major concern. Statistical modelling is a vital tool in developing an understanding of HCAI transmission dynamics. In this thesis, stochastic epidemic models are developed and used with the aim of investigating methicillin-resistant Staphylococcus aureus (MRSA) transmission and intervention measures in hospital wards. A detailed analysis of MRSA transmission and the effectiveness of patient isolation was performed, using data collected from several general medical wards in London. A Markov chain Monte Carlo (MCMC) algorithm was used to derive parameter estimates, accounting for unobserved transmission dynamics. A clear reduction in transmission associated with the use of patient isolation was estimated. A Bayesian framework offers considerable benefits and flexibility when dealing with missing data; however, model comparison is difficult, and existing methods are far from universally accepted. Two commonly used Bayesian model selection tools, reversible jump MCMC and the deviance information criterion (DIC), were thoroughly investigated in a transmission model setting, using both simulated and real data. The collection of whole genome sequence (WGS) data is becoming easier, faster and cheaper than ever before. With WGS data likely to become abundant in the near future, the development of sophisticated analytical tools and models to exploit such genetic information is of great importance. New methods were developed to model MRSA transmission, using both genetic and epidemiological data, allowing for the reconstruction of transmission networks and simultaneous estimation of key transmission parameters. This approach was tested with simulated data and employed on WGS data collected from two Thai intensive care units. This work offers much sc

Whole genome sequencing of pathogens from multiple hosts in an epidemic offers the potential to investigate who infected whom with unparalleled resolution, potentially yielding important insights into disease dynamics and the impact of control measures. We considered disease outbreaks in a setting with dense genomic sampling, and formulated stochastic epidemic models to investigate person-to-person transmission, based on observed genomic and epidemiological data. We constructed models in which the genetic distance between sampled genotypes depends on the epidemiological relationship between the hosts. A data augmented Markov chain Monte Carlo algorithm was used to sample over the transmission trees, providing a posterior probability for any given transmission route. We investigated the predictive performance of our methodology using simulated data, demonstrating high sensitivity and specificity, particularly for rapidly mutating pathogens with low transmissibility. We then analyzed data collected during an outbreak of methicillin-resistant Staphylococcus aureus in a hospital, identifying probable transmission routes and estimating epidemiological parameters. Our approach overcomes limitations of previous methods, providing a framework with the flexibility to allow for unobserved infection times, multiple independent introductions of the pathogen, and within-host genetic diversity, as well as allowing forward simulation.

Healthcare-associated infections (HCAIs) remain a problem worldwide, and can cause severe illness and death. The increasing level of antibiotic resistance among bacteria that cause HCAIs limits infection treatment options, and is a major concern. Statistical modelling is a vital tool in developing an understanding of HCAI transmission dynamics. In this thesis, stochastic epidemic models are developed and used with the aim of investigating methicillin-resistant Staphylococcus aureus (MRSA) transmission and intervention measures in hospital wards. A detailed analysis of MRSA transmission and the effectiveness of patient isolation was performed, using data collected from several general medical wards in London. A Markov chain Monte Carlo (MCMC) algorithm was used to derive parameter estimates, accounting for unobserved transmission dynamics. A clear reduction in transmission associated with the use of patient isolation was estimated. A Bayesian framework offers considerable benefits and flexibility when dealing with missing data; however, model comparison is difficult, and existing methods are far from universally accepted. Two commonly used Bayesian model selection tools, reversible jump MCMC and the deviance information criterion (DIC), were thoroughly investigated in a transmission model setting, using both simulated and real data. The collection of whole genome sequence (WGS) data is becoming easier, faster and cheaper than ever before. With WGS data likely to become abundant in the near future, the development of sophisticated analytical tools and models to exploit such genetic information is of great importance. New methods were developed to model MRSA transmission, using both genetic and epidemiological data, allowing for the reconstruction of transmission networks and simultaneous estimation of key transmission parameters. This approach was tested with simulated data and employed on WGS data collected from two Thai intensive care units. This work offers much sc

Healthcare-associated infections (HCAIs) remain a problem worldwide, and can cause severe illness and death. The increasing level of antibiotic resistance among bacteria that cause HCAIs limits infection treatment options, and is a major concern. Statistical modelling is a vital tool in developing an understanding of HCAI transmission dynamics. In this thesis, stochastic epidemic models are developed and used with the aim of investigating methicillin-resistant Staphylococcus aureus (MRSA) transmission and intervention measures in hospital wards. A detailed analysis of MRSA transmission and the effectiveness of patient isolation was performed, using data collected from several general medical wards in London. A Markov chain Monte Carlo (MCMC) algorithm was used to derive parameter estimates, accounting for unobserved transmission dynamics. A clear reduction in transmission associated with the use of patient isolation was estimated. A Bayesian framework offers considerable benefits and flexibility when dealing with missing data; however, model comparison is difficult, and existing methods are far from universally accepted. Two commonly used Bayesian model selection tools, reversible jump MCMC and the deviance information criterion (DIC), were thoroughly investigated in a transmission model setting, using both simulated and real data. The collection of whole genome sequence (WGS) data is becoming easier, faster and cheaper than ever before. With WGS data likely to become abundant in the near future, the development of sophisticated analytical tools and models to exploit such genetic information is of great importance. New methods were developed to model MRSA transmission, using both genetic and epidemiological data, allowing for the reconstruction of transmission networks and simultaneous estimation of key transmission parameters. This approach was tested with simulated data and employed on WGS data collected from two Thai intensive care units. This work offers much sc

Healthcare-associated infections (HCAIs) remain a problem worldwide, and can cause severe illness and death. The increasing level of antibiotic resistance among bacteria that cause HCAIs limits infection treatment options, and is a major concern. Statistical modelling is a vital tool in developing an understanding of HCAI transmission dynamics. In this thesis, stochastic epidemic models are developed and used with the aim of investigating methicillin-resistant Staphylococcus aureus (MRSA) transmission and intervention measures in hospital wards. A detailed analysis of MRSA transmission and the effectiveness of patient isolation was performed, using data collected from several general medical wards in London. A Markov chain Monte Carlo (MCMC) algorithm was used to derive parameter estimates, accounting for unobserved transmission dynamics. A clear reduction in transmission associated with the use of patient isolation was estimated. A Bayesian framework offers considerable benefits and flexibility when dealing with missing data; however, model comparison is difficult, and existing methods are far from universally accepted. Two commonly used Bayesian model selection tools, reversible jump MCMC and the deviance information criterion (DIC), were thoroughly investigated in a transmission model setting, using both simulated and real data. The collection of whole genome sequence (WGS) data is becoming easier, faster and cheaper than ever before. With WGS data likely to become abundant in the near future, the development of sophisticated analytical tools and models to exploit such genetic information is of great importance. New methods were developed to model MRSA transmission, using both genetic and epidemiological data, allowing for the reconstruction of transmission networks and simultaneous estimation of key transmission parameters. This approach was tested with simulated data and employed on WGS data collected from two Thai intensive care units. This work offers much sc

Whole genome sequencing of pathogens from multiple hosts in an epidemic offers the potential to investigate who infected whom with unparalleled resolution, potentially yielding important insights into disease dynamics and the impact of control measures. We considered disease outbreaks in a setting with dense genomic sampling, and formulated stochastic epidemic models to investigate person-to-person transmission, based on observed genomic and epidemiological data. We constructed models in which the genetic distance between sampled genotypes depends on the epidemiological relationship between the hosts. A data augmented Markov chain Monte Carlo algorithm was used to sample over the transmission trees, providing a posterior probability for any given transmission route. We investigated the predictive performance of our methodology using simulated data, demonstrating high sensitivity and specificity, particularly for rapidly mutating pathogens with low transmissibility. We then analyzed data collected during an outbreak of methicillin-resistant Staphylococcus aureus in a hospital, identifying probable transmission routes and estimating epidemiological parameters. Our approach overcomes limitations of previous methods, providing a framework with the flexibility to allow for unobserved infection times, multiple independent introductions of the pathogen, and within-host genetic diversity, as well as allowing forward simulation.

Healthcare-associated infections (HCAIs) remain a problem worldwide, and can cause severe illness and death. The increasing level of antibiotic resistance among bacteria that cause HCAIs limits infection treatment options, and is a major concern. Statistical modelling is a vital tool in developing an understanding of HCAI transmission dynamics. In this thesis, stochastic epidemic models are developed and used with the aim of investigating methicillin-resistant Staphylococcus aureus (MRSA) transmission and intervention measures in hospital wards. A detailed analysis of MRSA transmission and the effectiveness of patient isolation was performed, using data collected from several general medical wards in London. A Markov chain Monte Carlo (MCMC) algorithm was used to derive parameter estimates, accounting for unobserved transmission dynamics. A clear reduction in transmission associated with the use of patient isolation was estimated. A Bayesian framework offers considerable benefits and flexibility when dealing with missing data; however, model comparison is difficult, and existing methods are far from universally accepted. Two commonly used Bayesian model selection tools, reversible jump MCMC and the deviance information criterion (DIC), were thoroughly investigated in a transmission model setting, using both simulated and real data. The collection of whole genome sequence (WGS) data is becoming easier, faster and cheaper than ever before. With WGS data likely to become abundant in the near future, the development of sophisticated analytical tools and models to exploit such genetic information is of great importance. New methods were developed to model MRSA transmission, using both genetic and epidemiological data, allowing for the reconstruction of transmission networks and simultaneous estimation of key transmission parameters. This approach was tested with simulated data and employed on WGS data collected from two Thai intensive care units. This work offers much sc

Whole genome sequencing of pathogens from multiple hosts in an epidemic offers the potential to investigate who infected whom with unparalleled resolution, potentially yielding important insights into disease dynamics and the impact of control measures. We considered disease outbreaks in a setting with dense genomic sampling, and formulated stochastic epidemic models to investigate person-to-person transmission, based on observed genomic and epidemiological data. We constructed models in which the genetic distance between sampled genotypes depends on the epidemiological relationship between the hosts. A data augmented Markov chain Monte Carlo algorithm was used to sample over the transmission trees, providing a posterior probability for any given transmission route. We investigated the predictive performance of our methodology using simulated data, demonstrating high sensitivity and specificity, particularly for rapidly mutating pathogens with low transmissibility. We then analyzed data collected during an outbreak of methicillin-resistant Staphylococcus aureus in a hospital, identifying probable transmission routes and estimating epidemiological parameters. Our approach overcomes limitations of previous methods, providing a framework with the flexibility to allow for unobserved infection times, multiple independent introductions of the pathogen, and within-host genetic diversity, as well as allowing forward simulation.

Whole genome sequencing of pathogens from multiple hosts in an epidemic offers the potential to investigate who infected whom with unparalleled resolution, potentially yielding important insights into disease dynamics and the impact of control measures. We considered disease outbreaks in a setting with dense genomic sampling, and formulated stochastic epidemic models to investigate person-to-person transmission, based on observed genomic and epidemiological data. We constructed models in which the genetic distance between sampled genotypes depends on the epidemiological relationship between the hosts. A data augmented Markov chain Monte Carlo algorithm was used to sample over the transmission trees, providing a posterior probability for any given transmission route. We investigated the predictive performance of our methodology using simulated data, demonstrating high sensitivity and specificity, particularly for rapidly mutating pathogens with low transmissibility. We then analyzed data collected during an outbreak of methicillin-resistant Staphylococcus aureus in a hospital, identifying probable transmission routes and estimating epidemiological parameters. Our approach overcomes limitations of previous methods, providing a framework with the flexibility to allow for unobserved infection times, multiple independent introductions of the pathogen, and within-host genetic diversity, as well as allowing forward simulation.