MECHANISMS OF DISSEMINATION OF INTEGRON-MEDIATED MULTIPLE ANTIBIOTIC RESISTANCE IN SALMONELLA ENTERICA

Salmonella enterica is a major cause of disease in the United States and world-wide. This pathogen's increasing antibiotic resistance over the past decades has exacerbated its public health burden. To combat the rise in antibiotic resistance, it is necessary to understand how resistance spreads through the bacterial population. This requires study of the genes that determine resistance and also of the genetic relatedness of the isolates bearing those genes. In this dissertation, three studies are presented. The aim of the first study was to examine the utility of multilocus sequence typing (MLST), which indexes sequence changes in housekeeping genes, for investigating genetic relatedness in S. enterica. MLST was shown to be a stable genetic typing method for S. enterica that is useful for long-term and global epidemiologic studies. The aim of the second study was to examine the mechanisms of dissemination of antibiotic resistance genes mediated by a genetic structure called an integron, in a genetically diverse, global collection of S. enterica. Integrons are able to create variable collections of resistance genes and may be disseminated by horizontal gene transfer or by clonal expansion. Using MLST to determine genetic relatedness, the second study indicated that both of these mechanisms are important forces in the dissemination of integron mediated antibiotic resistance in S. enterica. The investigation of integrons revealed three integrons not previously reported in S. enterica. The aim of the third study was to characterize those integrons, which contained novel aggregates of resistance genes, making them capable of conferring resistance to multiple antibiotics. One of these integrons was found in two genetically unrelated strains, reinforcing this structure's potential for contributing to horizontal gene transfer of antibiotic resistance in S. enterica.Validation of MLST as a typing method for S. enterica has importance for public health because it facilitates large scale and global studies of this organism by providing a basis for assessing genetic relatedness of diverse isolates. An understanding of the patterns of dissemination of integrons has public health significance because it enhances the ability to accomplish surveillance for changes in antibiotic resistance

Interventions in measles outbreaks: the potential reduction in cases associated with school suspension and vaccination interventions

Background: Measles is resurgent in the US, with more cases in 2019 than any year since 1992. Many of the cases were concentrated in three outbreaks in New York and Washington states, where local governments enacted intervention strategies in an attempt to limit the spread of measles. Regulations differed by location, suggesting guidance on the optimal interventions may be beneficial. Methods: We simulate the daily interactions of the populations of six metropolitan areas of Texas, US, using an agent-based model. The real-life vaccination rates of each school in these metropolitan areas are applied to simulated equivalents. A single case of measles is introduced to the population and the resulting number of cases counted. A range of public health interventions, focused on suspending unvaccinated students and mandatory vaccinations, were simulated during measles outbreaks and the reduction in the number of measles cases, relative to no intervention, recorded. Interventions were simulated only in schools with measles cases and in all schools in each metropolitan area. Results: Suspending unvaccinated students from school was associated with the greatest reduction in measles cases. In a plausible worst-case outbreak scenario, the number of cases is forecast to reduce by 68-96%. Interventions targeting all schools in a metropolitan area is not found to be associated with fewer measles cases than only targeting schools with measles cases, at 2018 vaccination rates. Targeting all schools also increases the cumulative number of school days missed by suspended students by a factor of 10-100, depending on the metropolitan area, compared to targeting only schools with measles cases. If vaccination rates drop 5% in the schools which are under-vaccinated in 2018, metropolitan area-wide interventions are forecast to be associated with fewer cases than school-specific interventions. Conclusions: Interventions that are quickly implemented and widely followed may reduce the size of measles outbreaks by up 96%. If vaccination rates continue to fall in Texas, metropolitan area-wide interventions should be considered in the event of an outbreak

Integron-mediated Multidrug Resistance in a Global Collection of Nontyphoidal Salmonella enterica Isolates

Horizontal gene transfer and clonal expansion contribute substantially to the dissemination of resistant strain

Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents

Whole genome sequencing to investigate the emergence of clonal complex 23 Neisseria meningitidis serogroup Y disease in the United States

In the United States, serogroup Y, ST-23 clonal complex Neisseria meningitidis was responsible for an increase in meningococcal disease incidence during the 1990s. This increase was accompanied by antigenic shift of three outer membrane proteins, with a decrease in the population that predominated in the early 1990s as a different population emerged later in that decade. To understand factors that may have been responsible for the emergence of serogroup Y disease, we used whole genome pyrosequencing to investigate genetic differences between isolates from early and late N. meningitidis populations, obtained from meningococcal disease cases in Maryland in the 1990s. The genomes of isolates from the early and late populations were highly similar, with 1231 of 1776 shared genes exhibiting 100% amino acid identity and an average πN = 0.0033 and average πS = 0.0216. However, differences were found in predicted proteins that affect pilin structure and antigen profile and in predicted proteins involved in iron acquisition and uptake. The observed changes are consistent with acquisition of new alleles through horizontal gene transfer. Changes in antigen profile due to the genetic differences found in this study likely allowed the late population to emerge due to escape from population immunity. These findings may predict which antigenic factors are important in the cyclic epidemiology of meningococcal disease

Capsular Switching in Invasive Neisseria meningitidis, Brazil

During the 1990s, an epidemic of B:4 Neisseria meningitidis infections affected Brazil. Subsequent increase in C:4 disease suggested B→C capsular switching. This study identified B→C switches within the sequence type 32 complex. Substantial disease related to capsular switching emphasizes the need for surveillance of circulating meningococcal strains to optimize disease control

Can a Two-Dose Influenza Vaccine Regimen Better Protect Older Adults? An Agent-Based Modeling Study

Older adults (age ≥ 65) are at high risk of influenza morbidity and mortality. This study evaluated the impact of a hypothetical two-dose influenza vaccine regimen per season to reduce symptomatic flu cases by providing preseason (first dose) and mid-season (second dose) protection to offset waning vaccine effectiveness (VE). The Framework for Reconstructing Epidemiological Dynamics (FRED), an agent-based modeling platform, was used to compare typical one-dose vaccination to a two-dose vaccination strategy. Primary models incorporated waning VE of 10% per month and varied influenza season timing (December through March) to estimate cases and hospitalizations in older adults. Additional scenarios modeled reductions in uptake and VE of the second dose, and overall waning. In seasons with later peaks, two vaccine doses had the largest potential to reduce cases (14.4% with February peak, 18.7% with March peak) and hospitalizations (13.1% with February peak, 16.8% with March peak). Reductions in cases and hospitalizations still resulted but decreased when 30% of individuals failed to receive a second dose, second dose VE was reduced, or overall waning was reduced to 7% per month. Agent-based modeling indicates that two influenza vaccine doses could decrease cases and hospitalizations in older individuals. The highest impact occurred in the more frequently observed late-peak seasons. The beneficial impact of the two-dose regimen persisted despite model scenarios of reduced uptake of the second dose, decreased VE of the second dose, or overall VE waning