Ion Exchange Biomaterials to Capture Daptomycin and Prevent Resistance Evolution in Off-Target Bacterial Populations.

Daptomycin (DAP), a cyclic anionic lipopeptide antibiotic, is among the last resorts to treat multidrug-resistant Gram-positive bacterial infections, caused by vancomycin-resistant Enterococcus faecium or methicillin-resistant Staphylococcus aureus. DAP is administered intravenously, and via biliary excretion, ∼5-10% of the intravenous DAP dose arrives in the gastrointestinal (GI) tract where it drives resistance evolution in the off-target populations of E. faecium bacteria. Previously, we have shown in vivo that the oral administration of cholestyramine, an ion exchange biomaterial (IXB) sorbent, prevents DAP treatment from enriching DAP resistance in the populations of E. faecium shed from mice. Here, we investigate the biomaterial-DAP interfacial interactions to uncover the antibiotic removal mechanisms. The IXB-mediated DAP capture from aqueous media was measured in controlled pH/electrolyte solutions and in the simulated intestinal fluid (SIF) to uncover the molecular and colloidal mechanisms of DAP removal from the GI tract. Our findings show that the IXB electrostatically adsorbs the anionic antibiotic via a time-dependent diffusion-controlled process. Unsteady-state diffusion-adsorption mass balance describes the dynamics of adsorption well, and the maximum removal capacity is beyond the electric charge stoichiometric ratio because of DAP self-assembly. This study may open new opportunities for optimizing cholestyramine adjuvant therapy to prevent DAP resistance, as well as designing novel biomaterials to remove off-target antibiotics from the GI tract.

An orally administered drug prevents selection for antibiotic-resistant bacteria in the gut during daptomycin therapy.

Background and objectives: Previously, we showed proof-of-concept in a mouse model that oral administration of cholestyramine prevented enrichment of daptomycin-resistant Enterococcus faecium in the gastrointestinal (GI) tract during daptomycin therapy. Cholestyramine binds daptomycin in the gut, which removes daptomycin selection pressure and so prevents the enrichment of resistant clones. Here, we investigated two open questions related to this approach: (i) can cholestyramine prevent the enrichment of diverse daptomycin mutations emerging de novo in the gut? and (ii) how does the timing of cholestyramine administration impact its ability to suppress resistance? Methodology: Mice with GI E. faecium were treated with daptomycin with or without cholestyramine, and E. faecium was cultured from feces to measure changes in daptomycin susceptibility. A subset of clones was sequenced to investigate the genomic basis of daptomycin resistance. Results: Cholestyramine prevented the enrichment of diverse resistance mutations that emerged de novo in daptomycin-treated mice. Whole-genome sequencing revealed that resistance emerged through multiple genetic pathways, with most candidate resistance mutations observed in the clsA gene. In addition, we observed that cholestyramine was most effective when administration started prior to the first dose of daptomycin. However, beginning cholestyramine after the first daptomycin dose reduced the frequency of resistant E. faecium compared to not using cholestyramine at all. Conclusions and implications: Cholestyramine prevented the enrichment of diverse daptomycin-resistance mutations in intestinal E. faecium populations during daptomycin treatment, and it is a promising tool for managing the transmission of daptomycin-resistant E. faecium.

Genomic characterization of Staphylococcus aureus isolates causing osteoarticular infections in otherwise healthy children.

BACKGROUND: Pediatric osteoarticular infections are commonly caused by Staphylococcus aureus. The contribution of S. aureus genomic variability to pathogenesis of these infections is poorly described. METHODS: We prospectively enrolled 47 children over 3 1/2 years from whom S. aureus was isolated on culture-12 uninfected with skin colonization, 16 with skin abscesses, 19 with osteoarticular infections (four with septic arthritis, three with acute osteomyelitis, six with acute osteomyelitis and septic arthritis and six with chronic osteomyelitis). Isolates underwent whole genome sequencing, with assessment for 254 virulence genes and any mutations as well as creation of a phylogenetic tree. Finally, isolates were compared for their ability to form static biofilms and compared to the genetic analysis. RESULTS: No sequence types predominated amongst osteoarticular infections. Only genes involved in evasion of host immune defenses were more frequently carried by isolates from osteoarticular infections than from skin colonization (p = .02). Virulence gene mutations were only noted in 14 genes (three regulating biofilm formation) when comparing isolates from subjects with osteoarticular infections and those with skin colonization. Biofilm results demonstrated large heterogeneity in the isolates' capacity to form static biofilms, with healthy control isolates producing more robust biofilm formation. CONCLUSIONS: S. aureus causing osteoarticular infections are genetically heterogeneous, and more frequently harbor genes involved in immune evasion than less invasive isolates. However, virulence gene carriage overall is similar with infrequent mutations, suggesting that pathogenesis of S. aureus osteoarticular infections may be primarily regulated at transcriptional and/or translational levels.

Daptomycin treatment impacts resistance in off-target populations of vancomycin-resistant Enterococcus faecium.

The antimicrobial resistance crisis has persisted despite broad attempts at intervention. It has been proposed that an important driver of resistance is selection imposed on bacterial populations that are not the intended target of antimicrobial therapy. But to date, there has been limited quantitative measure of the mean and variance of resistance following antibiotic exposure. Here we focus on the important nosocomial pathogen Enterococcus faecium in a hospital system where resistance to daptomycin is evolving despite standard interventions. We hypothesized that the intravenous use of daptomycin generates off-target selection for resistance in transmissible gastrointestinal (carriage) populations of E. faecium. We performed a cohort study in which the daptomycin resistance of E. faecium isolated from rectal swabs from daptomycin-exposed patients was compared to a control group of patients exposed to linezolid, a drug with similar indications. In the daptomycin-exposed group, daptomycin resistance of E. faecium from the off-target population was on average 50% higher than resistance in the control group (n = 428 clones from 22 patients). There was also greater phenotypic diversity in daptomycin resistance within daptomycin-exposed patients. In patients where multiple samples over time were available, a wide variability in temporal dynamics were observed, from long-term maintenance of resistance to rapid return to sensitivity after daptomycin treatment stopped. Sequencing of isolates from a subset of patients supports the argument that selection occurs within patients. Our results demonstrate that off-target gastrointestinal populations rapidly respond to intravenous antibiotic exposure. Focusing on the off-target evolutionary dynamics may offer novel avenues to slow the spread of antibiotic resistance.

An adjunctive therapy administered with an antibiotic prevents enrichment of antibiotic-resistant clones of a colonizing opportunistic pathogen.

A key challenge in antibiotic stewardship is figuring out how to use antibiotics therapeutically without promoting the evolution of antibiotic resistance. Here, we demonstrate proof of concept for an adjunctive therapy that allows intravenous antibiotic treatment without driving the evolution and onward transmission of resistance. We repurposed the FDA-approved bile acid sequestrant cholestyramine, which we show binds the antibiotic daptomycin, as an 'anti-antibiotic' to disable systemically-administered daptomycin reaching the gut. We hypothesized that adjunctive cholestyramine could enable therapeutic daptomycin treatment in the bloodstream, while preventing transmissible resistance emergence in opportunistic pathogens colonizing the gastrointestinal tract. We tested this idea in a mouse model of Enterococcus faecium gastrointestinal tract colonization. In mice treated with daptomycin, adjunctive cholestyramine therapy reduced the fecal shedding of daptomycin-resistant E. faecium by up to 80-fold. These results provide proof of concept for an approach that could reduce the spread of antibiotic resistance for important hospital pathogens.

Antibiotics are essential for treating infections. But their use can inadvertently lead to the emergence of antibiotic-resistant bacteria that do not respond to antibiotic drugs, making infections with these bacteria difficult or impossible to treat. Finding ways to prevent antibiotic resistance is critical to preserving the effectiveness of antibiotics. Many bacteria that cause infections in hospitals live in the intestines, where they are harmless. But these bacteria can cause life-threatening infections when they get into the bloodstream. When patients with bloodstream infections receive antibiotics, the bacteria in their intestines are also exposed to the drugs. This can kill off all antibiotic-susceptible bacteria, leaving behind only bacteria that have mutations that allow them to survive the drugs. These drug-resistant bacteria can then spread to other patients causing hard-to-treat infections. To stop this cycle of antibiotic treatment and antibiotic resistance, Morley et al. tested whether giving a drug called cholestyramine with intravenous antibiotics could protect the gut bacteria. In the experiments, mice were treated systemically with an antibiotic called daptomycin, which caused the growth of daptomycin-resistant strains of bacteria in the mice's intestines. In the laboratory, Morley et al. discovered that cholestyramine can inactivate daptomycin. Giving the mice cholestyramine and daptomycin together prevented the growth of antibiotic-resistant bacteria in the mice's intestines. Moreover, cholestyramine is taken orally and is not absorbed into the blood. It therefore only inactivates the antibiotic in the gut, but not in the blood. The experiments provide preliminary evidence that giving cholestyramine with antibiotics might help prevent the spread of drug resistance. Cholestyramine is already used to lower cholesterol levels in people. More studies are needed to determine if cholestyramine can protect gut bacteria and prevent antibiotic resistance in people.

Prior evolution in stochastic versus constant temperatures affects RNA virus evolvability at a thermal extreme.

It is unclear how historical adaptation versus maladaptation in a prior environment affects population evolvability in a novel habitat. Prior work showed that vesicular stomatitis virus (VSV) populations evolved at constant 37°C improved in cellular infection at both 29°C and 37°C; in contrast, those evolved under random changing temperatures between 29°C and 37°C failed to improve. Here, we tested whether prior evolution affected the rate of adaptation at the thermal-niche edge: 40°C. After 40 virus generations in the new environment, we observed that populations historically evolved at random temperatures showed greater adaptability. Deep sequencing revealed that most of the newly evolved mutations were de novo. Also, two novel evolved mutations in the VSV glycoprotein and replicase genes tended to co-occur in the populations previously evolved at constant 37°C, whereas this parallelism was not seen in populations with prior random temperature evolution. These results suggest that prior adaptation under constant versus random temperatures constrained the mutation landscape that could improve fitness in the novel 40°C environment, perhaps owing to differing epistatic effects of new mutations entering genetic architectures that earlier diverged. We concluded that RNA viruses maladapted to their previous environment could "leapfrog" over counterparts of higher fitness, to achieve faster adaptability in a novel environment.

Factors associated with antibiotic prescribing for acute bronchitis at a university health center.

BACKGROUND: Antibiotics are not indicated for treating acute bronchitis cases, yet up to 70% of adult acute bronchitis medical visits in the USA result in an antibiotic prescription. Reducing unnecessary antibiotic prescribing for acute bronchitis is a key antibiotic stewardship goal set forth by the Centers for Disease Control and Prevention. Understanding what factors influence prescribing for bronchitis cases can inform antimicrobial stewardship initiatives. The goal of this study was to identify factors associated with antibiotic prescribing at a high-volume student health center at a large US university. The Pennsylvania State University Health Services offers on-campus medical care to a population of over 40,000 students and receives over 50,000 visits every year. METHODS: We conducted a retrospective chart review of acute bronchitis visits for the 2015-2016 academic year and used a multivariate logistic regression analysis to identify variables associated with antibiotic prescribing. RESULTS: Findings during lung exams increased the likelihood of an antibiotic prescription (rales OR 13.95, 95% CI 3.31-80.73; rhonchi OR 5.50, 95% CI 3.08-10.00; percussion abnormality OR 13.02, 95% CI 4.00-50.09). Individual clinicians had dramatically different rates of prescribing (OR range 0.03-12.3). Male patients were more likely than female patients to be prescribed antibiotics (OR 1.68, 95% CI 1.17-2.41). Patients who reported longer duration since the onset of symptoms were slightly more likely to receive prescriptions (OR 1.04 per day, 95% CI 1.03-1.06), as were patients who reported worsening symptoms (OR 1.78, 95% CI 1.03-3.10). Visits with diagnoses or symptoms associated with viral infections or allergies were less likely to result in prescriptions (upper respiratory tract infection (URI) diagnosis OR 0.33, 95% CI 0.18-0.58; sneezing OR 0.39, 95% CI 0.17-0.86; vomiting OR 0.31, 95% CI 0.10-0.83). An exam finding of anterior cervical lymphadenopathy was associated with antibiotic prescribing (tender OR 3.85, 95% CI 1.70-8.83; general OR 2.63, 95% CI 1.25-5.54). CONCLUSIONS: Suspicious findings during lung examinations (rales, rhonchi, percussion abnormality) and individual healthcare providers were important factors influencing antibiotic prescribing rates for acute bronchitis visits. Patient gender, worsening symptoms, duration of illness, symptoms associated with viral infections or allergies, and anterior cervical lymphadenopathy also influenced prescribing rates.

Bystander Selection for Antimicrobial Resistance: Implications for Patient Health.

Antimicrobial therapy promotes resistance emergence in target infections and in off-target microbiota. Off-target resistance emergence threatens patient health when off-target populations are a source of future infections, as they are for many important drug-resistant pathogens. However, the health risks of antimicrobial exposure in off-target populations remain largely unquantified, making rational antibiotic stewardship challenging. Here, we discuss the contribution of bystander antimicrobial exposure to the resistance crisis, the implications for antimicrobial stewardship, and some novel opportunities to limit resistance evolution while treating target pathogens.

Chikungunya virus evolution following a large 3'UTR deletion results in host-specific molecular changes in protein-coding regions.

The 3'untranslated region (UTR) in alphavirus genomes functions in virus replication and plays a role in determining virus host range. However, the molecular evolution of virus UTRs is understudied compared to the evolution of protein-coding regions. Chikungunya virus (CHIKV) has the longest 3'UTR among the alphaviruses (500-700 nt), and 3'UTR length and sequence structure vary substantially among different CHIKV lineages. Previous studies showed that genomic deletions and insertions are key drivers of CHIKV 3'UTR evolution. Inspired by hypothesized deletion events in the evolutionary history of CHIKV, we used experimental evolution to examine CHIKV adaptation in response to a large 3'UTR deletion. We engineered a CHIKV mutant with a 258 nt deletion in the 3'UTR (ΔDR1/2). This deletion reduced viral replication on mosquito cells, but did not reduce replication on mammalian cells. To examine how selective pressures from vertebrate and invertebrate hosts shape CHIKV evolution after a deletion in the 3'UTR, we passaged ΔDR1/2 virus populations strictly on primate cells, strictly on mosquito cells, or with alternating primate/mosquito cell passages. We found that virus populations passaged on a single host cell line increased in fitness relative to the ancestral deletion mutant on their selected host, and viruses that were alternately passaged improved on both hosts. Surprisingly, whole genome sequencing revealed few changes in the 3'UTR of passaged populations. Rather, virus populations evolved improved fitness through mutations in protein coding regions that were associated with specific hosts.

Dynamics of molecular evolution in RNA virus populations depend on sudden versus gradual environmental change.

Understanding the dynamics of molecular adaptation is a fundamental goal of evolutionary biology. While adaptation to constant environments has been well characterized, the effects of environmental complexity remain seldom studied. One simple but understudied factor is the rate of environmental change. Here we used experimental evolution with RNA viruses to investigate whether evolutionary dynamics varied based on the rate of environmental turnover. We used whole-genome next-generation sequencing to characterize evolutionary dynamics in virus populations adapting to a sudden versus gradual shift onto a novel host cell type. In support of theoretical models, we found that when populations evolved in response to a sudden environmental change, mutations of large beneficial effect tended to fix early, followed by mutations of smaller beneficial effect; as predicted, this pattern broke down in response to a gradual environmental change. Early mutational steps were highly parallel across replicate populations in both treatments. The fixation of single mutations was less common than sweeps of associated "cohorts" of mutations, and this pattern intensified when the environment changed gradually. Additionally, clonal interference appeared stronger in response to a gradual change. Our results suggest that the rate of environmental change is an important determinant of evolutionary dynamics in asexual populations.

Evolution in spatially mixed host environments increases divergence for evolved fitness and intrapopulation genetic diversity in RNA viruses.

Virus populations may be challenged to evolve in spatially heterogeneous environments, such as mixtures of host cells that pose differing selection pressures. Spatial heterogeneity may select for evolved polymorphisms, where multiple virus subpopulations coexist by specializing on a narrow subset of the available hosts. Alternatively, spatial heterogeneity may select for evolved generalism, where a single genotype dominates the virus population by occupying a relatively broader host niche. In addition, the extent of spatial heterogeneity should influence the degree of divergence among virus populations encountering identical environmental challenges. Spatial heterogeneity creates environmental complexity that should increase the probability of differing adaptive phenotypic solutions, thus producing greater divergence among replicate virus populations, relative to counterparts evolving in strictly homogeneous host environments. Here, we tested these ideas using experimental evolution of RNA virus populations grown in laboratory tissue culture. We allowed vesicular stomatitis virus (VSV) lineages to evolve in replicated environments containing BHK-21 (baby hamster kidney) cells, HeLa (human epithelial) cells, or spatially heterogeneous host cell mixtures. Results showed that generalist phenotypes dominated in evolved virus populations across all treatments. Also, we observed greater variance in host-use performance (fitness) among VSV lineages evolved under spatial heterogeneity, relative to lineages evolved in homogeneous environments. Despite measurable differences in fitness, consensus Sanger sequencing revealed no fixed genetic differences separating the evolved lineages from their common ancestor. In contrast, deep sequencing of evolved VSV populations confirmed that the degree of divergence among replicate lineages was correlated with a larger number of minority variants. This correlation between divergence and the number of minority variants was significant only when we considered variants with a frequency of at least 10 per cent in the population. The number of lower-frequency minority variants per population did not significantly correlate with divergence.

Rate of novel host invasion affects adaptability of evolving RNA virus lineages.

Although differing rates of environmental turnover should be consequential for the dynamics of adaptive change, this idea has been rarely examined outside of theory. In particular, the importance of RNA viruses in disease emergence warrants experiments testing how differing rates of novel host invasion may impact the ability of viruses to adaptively shift onto a novel host. To test whether the rate of environmental turnover influences adaptation, we experimentally evolved 144 Sindbis virus lineages in replicated tissue-culture environments, which transitioned from being dominated by a permissive host cell type to a novel host cell type. The rate at which the novel host 'invaded' the environment varied by treatment. The fitness (growth rate) of evolved virus populations was measured on each host type, and molecular substitutions were mapped via whole genome consensus sequencing. Results showed that virus populations more consistently reached high fitness levels on the novel host when the novel host 'invaded' the environment more gradually, and gradual invasion resulted in less variable genomic outcomes. Moreover, virus populations that experienced a rapid shift onto the novel host converged upon different genotypes than populations that experienced a gradual shift onto the novel host, suggesting a strong effect of historical contingency.

Genome rearrangement affects RNA virus adaptability on prostate cancer cells.

Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5' end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene order.

Test of recrudescence hypothesis for overwintering of eastern equine encephalomyelitis virus in gray catbirds.

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) epizootics are infrequent, but they can lead to high mortality in infected horses and humans. Despite the importance of EEEV to human and animal health, little is known about how the virus overwinters and reinitiates transmission each spring, particularly in temperate regions where infected adult mosquitoes are unlikely to survive through the winter. One hypothesis to explain the mechanism by which this virus persists from year to year is the spring recrudescence of latent virus in avian reservoir hosts. In this study, we tested the recrudescence hypothesis with gray catbirds (Dumatella carolinensis) captured in northern Ohio (July-August 2007). Birds were experimentally infected with EEEV on 1 October 2007. In January 2008, they were then exposed to exogenous testosterone and/or extended photoperiod to initiate reactivation of latent EEEV infection. All birds became viremic with EEEV, with mean viremia of 6.0 log10 plaque-forming units/ml serum occurring at 1 d postinoculation. One male in the testosterone, long-day treatment group had EEEV viral RNA in a cloacal swab collected on 18 January 2008. Otherwise, no other catbirds exhibited reactivated infections in cloacal swabs or blood. Antibody titers fluctuated over the course of the study, with lowest titers observed in January 2008, which corresponded with the lowest mean weight of the birds. No EEEV viral RNA was detected in the blood, kidney, spleen, brain, liver, and lower intestine upon necropsy at 19 wk postinfection.