Biology subjects

Alexander, A. M.

Publications and source records attributed to Alexander, A. M..

5 recordsLinked to original sources

bioRxiv2025

Long-term homeostasis in microbial consortia via auxotrophic cross-feeding

Synthetic microbial consortia are collections of multiple strains or species of engineered organisms living in a shared ecosystem. Because they can separate metabolic tasks among different strains, synthetic microbial consortia have myriad applications in developing biomaterials, biomanufacturing, and biotherapeutics. However, synthetic consortia often require burdensome control mechanisms to ensure that the members of the community remain at the correct proportions. This is especially true in continuous culture systems in which slight differences in growth rates can lead to extinctions. Here, we present a simple method for controlling consortia proportions using cross-feeding in continuous auxotrophic co-culture. We use mutually auxotrophic E. coli with different essential gene deletions and regulate the growth rates of members of the consortium via cross-feeding of the missing nutrients in each strain. We demonstrate precise regulation of the co-culture steady-state ratio by exogenous addition of the missing nutrients. We also model the co-cultures behavior using a system of ordinary differential equations that enable us to predict its response to changes in nutrient concentrations. Our work provides a powerful tool for consortia proportion control with minimal metabolic costs to the constituent strains.

synthetic biology↗

bioRxiv2024

Intraspecific Diversity of Staphylococcus aureus Populations Isolated from Cystic Fibrosis Respiratory Infections

Chronic bacterial infections are often polymicrobial, comprising multiple bacterial species or variants of the same species. Because chronic infections may last for decades, they have the potential to generate high levels of intraspecific variation through within-host diversification over time, and the potential for superinfections to occur through the introduction of multiple pathogen populations to the ongoing infection. Traditional methods for identifying infective agents generally involve isolating one single colony from a given sample, usually after selecting for a specific pathogen or antibiotic resistance profile. Isolating a recognized virulent or difficult to treat pathogen is an important part of informing clinical treatment and correlative research; however, these reductive methods alone, do not provide researchers or healthcare providers with the potentially important perspective on the true pathogen population structure and dynamics over time. To begin to address this limitation, in this study, we compare findings on Staphylococcus aureus single colonies versus and pools of colonies taken from fresh sputum samples from three patients with cystic fibrosis to isolates collected from the same sputum samples and processed by the clinical microbiology laboratory. Phenotypic and genotypic analysis of isolated S. aureus populations revealed coexisting lineages in two of three sputum samples as well as population structures that were not reflected in the single colony isolates. Altogether, our observations presented here demonstrate that clinically relevant diversity can be missed with standard sampling methods when assessing chronic infections. More broadly, this work outlines the potential impact that comprehensive population-level sampling may have for both research efforts and more effective treatment practices. Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article.

microbiology↗

bioRxiv2023

Experimentally Evolved Staphylococcus aureus Survives in the Presence of Pseudomonas aeruginosa by Acquiring Mutations in the Amino Acid Transporter, GltT

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacterial pathogens isolated from cystic fibrosis (CF) related lung infections. When both of these opportunistic pathogens are found in a coinfection, CF patients tend to have higher rates of pulmonary exacerbations and experience a more rapid decrease in lung function. When cultured together under standard laboratory conditions, it is often observed that P. aeruginosa effectively inhibits S. aureus growth. Previous work from our group revealed that S. aureus from CF infections have isolate-specific survival capabilities when cocultured with P. aeruginosa. In this study, we designed a serial transfer evolution experiment to identify mutations that allow S. aureus to adapt to the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa strain, PAO1. After 8 coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompete wild-type S. aureus when glutamate is absent from chemically-defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two bacteria are cultured together. ImportanceStaphylococcus aureus and Pseudomonas aeruginosa are the two most common bacterial pathogens that infect people with the genetic disease, cystic fibrosis (CF). They are often found together in CF-associated polymicrobial infections that are associated with worse patient prognosis. Understanding how these very different opportunistic pathogens influence each other in a shared environment is pertinent to improving the treatment of polymicrobial infections. While much attention has been brought to the interspecific interactions between S. aureus and P. aeruginosa, few studies have used experimental evolution methods to identify determinants of their competition and coexistence. Here, we use a serial transfer experimental evolution approach and identified a single genetic change associated with improved survival of S. aureus in the presence of P. aeruginosa. Our findings implicate metabolism of shared resources as an important factor in S. aureuss ability to survive in the presence of P. aeruginosa.

microbiology↗

bioRxiv2021

Species-wide phylogenomics of the Staphylococcus aureus agr operon reveals convergent evolution of frameshift mutations

Staphylococcus aureus is a prominent nosocomial pathogen that causes several life-threatening diseases such as pneumonia and bacteremia. S. aureus modulates expression of its arsenal of virulence factors through sensing and integrating responses to environmental signals. The agr (accessory gene regulator) quorum sensing (QS) system is a major regulator of virulence phenotypes in S. aureus. There are four agr specificity groups each with a different autoinducer peptide sequence (encoded by the agrD gene). Though agr is critical for expression of many toxins, paradoxically, S. aureus strains often have non-functional agr activity due to loss-of-function mutations in the four-gene agr operon. To understand patterns in agr variability across S. aureus, we undertook a species-wide genomic investigation. We developed a software tool (AgrVATE; https://github.com/VishnuRaghuram94/AgrVATE) for typing and detecting frameshift mutations in the agr operon. In an analysis of over 40,000 S. aureus genomes, we showed close association between agr type and S. aureus clonal complex. We also found strong linkage between agrBDC alleles (encoding the peptidase, the autoinducing peptide itself, and the peptide sensor respectively) but not agrA (encoding the -response regulator). More than five percent of genomes were found to have frameshift mutations in the agr operon. Though most mutations occur only once in the entire species, we observed a small number of recurring mutations evolving convergently across different clonal lineages. Phylogenetic patterns suggested that strains with agr frameshifts were evolutionary dead ends. Overall, genomic analysis of agr operon suggests evolution through multiple processes with functional consequences that are not fully understood. ImportanceStaphylococcus aureus is a globally pervasive pathogen that produces a plethora of toxic molecules that can degrade, evade, or inhibit the host immune cells. Production of these toxins is mainly controlled by an active agr quorum sensing system, which senses and responds to bacterial cell density. However, there are many reports of S. aureus strains with genetic changes leading to impaired agr activity, often found during chronic bloodstream infections, and may be associated with increased disease severity. We developed an open-source software called AgrVATE to type agr systems and identify putative frameshifts. We used AgrVATE for a species-wide genomic survey of S. aureus, finding that more than 5 % of strains in the public database had non-functional agr systems. We also provide new insights into the evolution of these genetic mutations in the agr system. Overall, this study contributes to our understanding of a common but relatively understudied means of virulence regulation in S. aureus.

genomics↗

bioRxiv2019

Genotypic and phenotypic diversity of Staphylococcus aureus from cystic fibrosis lung infections and their interactions with Pseudomonas aeruginosa

Pseudomonas aeruginosa and Staphylococcus aureus are the most common bacteria that infect the respiratory tract of individuals with the genetic disease cystic fibrosis (CF); in fact, S. aureus has recently overtaken P. aeruginosa to become the most common. Substantial research has been performed on the epidemiology of S. aureus in CF; however, there appears to be a gap in knowledge in regard to the pathogenesis of S. aureus in the context of CF lung infections. Most studies have focused on a few S. aureus isolates, often exclusively laboratory adapted strains, and how they are killed by P. aeruginosa. Because of this, little is known about the diversity of S. aureus CF lung isolates in both virulence and interaction with P. aeruginosa. To begin to address this gap in knowledge, we recently sequenced 65 clinical S. aureus isolates from the Emory CF Biospecimen Registry and Boston Childrens Hospital, including the reference isolate JE2, a USA300 strain. Here, we analyzed antibiotic resistance genotypes, sequence type, clonal complex, spa type, and agr type of these isolates. We hypothesized that major virulence phenotypes of S. aureus that may be associated with CF lung infections, namely toxin production and mucoid phenotype, would be retained in these isolates. To test our hypothesis, we plated on specific agars and found that most isolates can hemolyze both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), consistent with virulence retention in CF lung isolates. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus CF isolates, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health.\n\nAuthor SummaryStaphylococcus aureus is now the most frequently detected pathogen in the lungs of individuals who have cystic fibrosis (CF), followed closely by Pseudomonas aeruginosa. When these two pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus. Here we present an in-depth study of 64 S. aureus CF clinical isolates where we investigated genetic diversity utilizing whole genome sequencing, virulence phenotypes, and interactions with P. aeruginosa. We have found that S. aureus isolated from the CF lung are phylogenetically diverse, most retain known virulence factors, and they vary in interactions with P. aeruginosa from highly sensitive to completely tolerant. Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

microbiology↗