Antibiotic resistance trends among Vibrio vulnificus and Vibrio parahaemolyticus isolated from the Chesapeake Bay, Maryland: a longitudinal study.

Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.

An agent-based framework to study forced migration: A case study of Ukraine.

The ongoing Russian aggression against Ukraine has forced over eight million people to migrate out of Ukraine. Understanding the dynamics of forced migration is essential for policy-making and for delivering humanitarian assistance. Existing work is hindered by a reliance on observational data which is only available well after the fact. In this work, we study the efficacy of a data-driven agent-based framework motivated by social and behavioral theory in predicting outflow of migrants as a result of conflict events during the initial phase of the Ukraine war. We discuss policy use cases for the proposed framework by demonstrating how it can leverage refugee demographic details to answer pressing policy questions. We also show how to incorporate conflict forecast scenarios to predict future conflict-induced migration flows. Detailed future migration estimates across various conflict scenarios can both help to reduce policymaker uncertainty and improve allocation and staging of limited humanitarian resources in crisis settings.

Building Environmental and Sociological Predictive Intelligence to Understand the Seasonal Threat of SARS-CoV-2 in Human Populations.

Current modeling practices for environmental and sociological modulated infectious diseases remain inadequate to forecast the risk of outbreak(s) in human populations, partly due to a lack of integration of disciplinary knowledge, limited availability of disease surveillance datasets, and overreliance on compartmental epidemiological modeling methods. Harvesting data knowledge from virus transmission (aerosols) and detection (wastewater) of SARS-CoV-2, a heuristic score-based environmental predictive intelligence system was developed that calculates the risk of COVID-19 in the human population. Seasonal validation of the algorithm was uniquely associated with wastewater surveillance of the virus, providing a lead time of 7-14 days before a county-level outbreak. Using county-scale disease prevalence data from the United States, the algorithm could predict COVID-19 risk with an overall accuracy ranging between 81% and 98%. Similarly, using wastewater surveillance data from Illinois and Maryland, the SARS-CoV-2 detection rate was greater than 80% for 75% of the locations during the same time the risk was predicted to be high. Results suggest the importance of a holistic approach across disciplinary boundaries that can potentially allow anticipatory decision-making policies of saving lives and maximizing the use of available capacity and resources.

Health-Damaging Climate Events Highlight the Need for Interdisciplinary, Engaged Research.

In 2023 human populations experienced multiple record-breaking climate events, with widespread impacts on human health and well-being. These events include extreme heat domes, drought, severe storms, flooding, and wildfires. Due to inherent lags in the climate system, we can expect such extremes to continue for multiple decades after reaching net zero carbon emissions. Unfortunately, despite these significant current and future impacts, funding for research in climate and health has lagged behind that for other geoscience and biomedical research. While some initial efforts from funding agencies are evident, there is still a significant need to increase the resources available for multidisciplinary research in the face of this issue. As a group of experts at this important intersection, we call for a more concerted effort to encourage interdisciplinary and policy-relevant investigations into the detrimental health effects of continued climate change.

Increased incidence of vibriosis in Maryland, U.S.A., 2006-2019.

BACKGROUND: Vibrio spp. naturally occur in warm water with moderate salinity. Infections with non-cholera Vibrio (vibriosis) cause an estimated 80,000 illnesses and 100 fatalities each year in the United States. Climate associated changes to environmental parameters in aquatic ecosystems are largely promoting Vibrio growth, and increased incidence of vibriosis is being reported globally. However, vibriosis trends in the northeastern U.S. (e.g., Maryland) have not been evaluated since 2008. METHODS: Vibriosis case data for Maryland (2006-2019; n = 611) were obtained from the COVIS database. Incidence rates were calculated using U.S. Census Bureau population estimates for Maryland. A logistic regression model, including region, age group, race, gender, occupation, and exposure type, was used to estimate the likelihood of hospitalization. RESULTS: Comparing the 2006-2012 and 2013-2019 periods, there was a 39% (p = 0.01) increase in the average annual incidence rate (per 100,000 population) of vibriosis, with V. vulnificus infections seeing the greatest percentage increase (53%, p = 0.01), followed by V. parahaemolyticus (47%, p = 0.05). The number of hospitalizations increased by 58% (p = 0.01). Since 2010, there were more reported vibriosis cases with a hospital duration ≥10 days. Patients from the upper eastern shore region and those over the age of 65 were more likely (OR = 6.8 and 12.2) to be hospitalized compared to other patients. CONCLUSIONS: Long-term increases in Vibrio infections, notably V. vulnificus wound infections, are occurring in Maryland. This trend, along with increased rates in hospitalizations and average hospital durations, underscore the need to improve public awareness, water monitoring, post-harvest seafood interventions, and environmental forecasting ability.

Persistent effects of intramammary ceftiofur treatment on the gut microbiome and antibiotic resistance in dairy cattle.

BACKGROUND: Intramammary (IMM) ceftiofur treatment is commonly used in dairy farms to prevent mastitis, though its impact on the cattle gut microbiome and selection of antibiotic-resistant bacteria has not been elucidated. Herein, we enrolled 40 dairy (Holstein) cows at the end of the lactation phase for dry-cow therapy: 20 were treated with IMM ceftiofur (Spectramast®DC) and a non-antibiotic internal teat sealant (bismuth subnitrate) and 20 (controls) received only bismuth subnitrate. Fecal grab samples were collected before and after treatment (weeks 1, 2, 3, 5, 7, and 9) for bacterial quantification and metagenomic next-generation sequencing. RESULTS: Overall, 90% and 24% of the 278 samples had Gram-negative bacteria with resistance to ampicillin and ceftiofur, respectively. Most of the cows treated with ceftiofur did not have an increase in the number of resistant bacteria; however, a subset (25%) shed higher levels of ceftiofur-resistant bacteria for up to 2 weeks post-treatment. At week 5, the antibiotic-treated cows had lower microbiota abundance and richness, whereas a greater abundance of genes encoding extended-spectrum ß-lactamases (ESBLs), CfxA, ACI-1, and CMY, was observed at weeks 1, 5 and 9. Moreover, the contig and network analyses detected associations between ß-lactam resistance genes and phages, mobile genetic elements, and specific genera. Commensal bacterial populations belonging to Bacteroidetes most commonly possessed ESBL genes followed by members of Enterobacteriaceae. CONCLUSION: This study highlights variable, persistent effects of IMM ceftiofur treatment on the gut microbiome and resistome in dairy cattle. Antibiotic-treated cattle had an increased abundance of specific taxa and genes encoding ESBL production that persisted for 9 weeks. Fecal shedding of ESBL-producing Enterobacteriaceae, which was classified as a serious public health threat, varied across animals. Together, these findings highlight the need for additional studies aimed at identifying factors associated with shedding levels and the dissemination and persistence of antibiotic resistance determinants on dairy farms across geographic locations.

Anticipatory decision-making for cholera in Malawi.

Climate change raises an old disease to a new level of public health threat. The causative agent, Vibrio cholerae, native to aquatic ecosystems, is influenced by climate and weather processes. The risk of cholera is elevated in vulnerable populations lacking access to safe water and sanitation infrastructure. Predictive intelligence, employing mathematical algorithms that integrate earth observations and heuristics derived from microbiological, sociological, and weather data, can provide anticipatory decision-making capabilities to reduce the burden of cholera and save human lives. An example offered here is the recent outbreak of cholera in Malawi, predicted in advance by such algorithms.

Genomic diversity of Vibrio spp. and metagenomic analysis of pathogens in Florida Gulf coastal waters following Hurricane Ian.

Changing climatic conditions influence parameters associated with the growth of pathogenic Vibrio spp. in the environment and, hence, are linked to increased incidence of vibriosis. Between 1992 and 2022, a long-term increase in Vibrio spp. infections was reported in Florida, USA. Furthermore, a spike in Vibrio spp. infections was reported post Hurricane Ian, a category five storm that made landfall in Florida on 28 September 2022. During October 2022, water and oyster samples were collected from three stations in Lee County in an area significantly impacted by Ian. Vibrio spp. were isolated, and whole-genome sequencing and phylogenetic analysis were done, with a focus on Vibrio parahaemolyticus and Vibrio vulnificus to provide genetic insight into pathogenic strains circulating in the environment. Metagenomic analysis of water samples provided insight with respect to human health-related factors, notably the detection of approximately 12 pathogenic Vibrio spp., virulence and antibiotic resistance genes, and mobile genetic elements, including the SXT/R391 family of integrative conjugative elements. Environmental parameters were monitored as part of a long-term time series analysis done using satellite remote sensing. In addition to anomalous rainfall and storm surge, changes in sea surface temperature and chlorophyll concentration during and after Ian favored the growth of Vibrio spp. In conclusion, genetic analysis coupled with environmental data and remote sensing provides useful public health information and, hence, constitute a valuable tool to proactively detect and characterize environmental pathogens, notably vibrios. These data can aid the development of early warning systems by yielding a larger source of information for public health during climate change. Evidence suggests warming temperatures are associated with the spread of potentially pathogenic Vibrio spp. and the emergence of human disease globally. Following Hurricane Ian, the State of Florida reported a sharp increase in the number of reported Vibrio spp. infections and deaths. Hence, monitoring of pathogens, including vibrios, and environmental parameters influencing their occurrence is critical to public health. Here, DNA sequencing was used to investigate the genomic diversity of Vibrio parahaemolyticus and Vibrio vulnificus, both potential human pathogens, in Florida coastal waters post Hurricane Ian, in October 2022. Additionally, the microbial community of water samples was profiled to detect the presence of Vibrio spp. and other microorganisms (bacteria, fungi, protists, and viruses) present in the samples. Long-term environmental data analysis showed changes in environmental parameters during and after Ian were optimal for the growth of Vibrio spp. and related pathogens. Collectively, results will be used to develop predictive risk models during climate change.

Environmental Factors Associated with Incidence and Distribution of V. parahaemolyticus and V. vulnificus in Chesapeake Bay, Maryland, USA: A three-year case study.

Members of the genus Vibrio are ecologically significant bacteria native to aquatic ecosystems globally, and a few can cause diseases in humans. Vibrio-related illnesses have increased in recent years, primarily attributed to changing environmental conditions. Therefore, understanding the role of environmental factors in the occurrence and growth of pathogenic strains is crucial for public health. Water, oyster, and sediment samples were collected between 2009 and 2012 from Chester River and Tangier Sound sites in Chesapeake Bay, Maryland, USA, to investigate the relationship between water temperature, salinity, and chlorophyll with the incidence and distribution of Vibrio parahaemolyticus (VP) and Vibrio vulnificus (VV). Odds ratio analysis was used to determine association between the likelihood of VP and VV presence and these environmental variables. Results suggested that water temperature threshold of 20°C or higher was associated with an increased risk, favoring the incidence of Vibrio spp. A significant difference in salinity was observed between the two sampling sites, with distinct ranges showing high odds ratio for Vibrio incidence, especially in water and sediment, emphasizing the impact of salinity on VP and VV incidence and distribution. Notably, salinity between 9-20 PPT consistently favored the Vibrio incidence across all samples. Relationship between chlorophyll concentrations and VP and VV incidence varied depending on sample type. However, chlorophyll range of 0-10 µg/L was identified as critical in oyster samples for both vibrios. Analysis of odds ratios for water samples demonstrated consistent outcomes across all environmental parameters, indicating water samples offer a more reliable indicator of Vibrio spp. incidence.

Environmental Factors Influencing Occurrence of Vibrio parahaemolyticus and Vibrio vulnificus.

Incidence of vibriosis is rising globally, with evidence that changing climatic conditions are influencing environmental factors that enhance growth of pathogenic Vibrio spp. in aquatic ecosystems. To determine the impact of environmental factors on occurrence of pathogenic Vibrio spp., samples were collected in the Chesapeake Bay, Maryland, during 2009 to 2012 and 2019 to 2022. Genetic markers for Vibrio vulnificus (vvhA) and Vibrio parahaemolyticus (tlh, tdh, and trh) were enumerated by direct plating and DNA colony hybridization. Results confirmed seasonality and environmental parameters as predictors. Water temperature showed a linear correlation with vvhA and tlh, and two critical thresholds were observed, an initial increase in detectable numbers (>15°C) and a second increase when maximum counts were recorded (>25°C). Temperature and pathogenic V. parahaemolyticus (tdh and trh) were not strongly correlated; however, the evidence showed that these organisms persist in oyster and sediment at colder temperatures. Salinity (10 to 15 ppt), total chlorophyll a (5 to 25 µg/L), dissolved oxygen (5 to 10 mg/L), and pH (8) were associated with increased abundance of vvhA and tlh. Importantly, a long-term increase in Vibrio spp. numbers was observed in water samples between the two collection periods, specifically at Tangier Sound (lower bay), with the evidence suggesting an extended seasonality for these bacteria in the area. Notably, tlh showed a mean positive increase that was ca. 3-fold overall, with the most significant increase observed during the fall. In conclusion, vibriosis continues to be a risk in the Chesapeake Bay region. A predictive intelligence system to assist decision makers, with respect to climate and human health, is warranted. IMPORTANCE The genus Vibrio includes pathogenic species that are naturally occurring in marine and estuarine environments globally. Routine monitoring for Vibrio species and environmental parameters influencing their incidence is critical to provide a warning system for the public when the risk of infection is high. In this study, occurrence of Vibrio parahaemolyticus and Vibrio vulnificus, both potential human pathogens, in Chesapeake Bay water, oysters, and sediment samples collected over a 13-year period was analyzed. The results provide a confirmation of environmental predictors for these bacteria, notably temperature, salinity, and total chlorophyll a, and their seasonality of occurrence. New findings refine environmental parameter thresholds of culturable Vibrio species and document a long-term increase in Vibrio populations in the Chesapeake Bay. This study provides a valuable foundation for development of predicative risk intelligence models for Vibrio incidence during climate change.

Multilevel cultural evolution: From new theory to practical applications.

Evolutionary science has led to many practical applications of genetic evolution but few practical uses of cultural evolution. This is because the entire study of evolution was gene centric for most of the 20th century, relegating the study and application of human cultural change to other disciplines. The formal study of human cultural evolution began in the 1970s and has matured to the point of deriving practical applications. We provide an overview of these developments and examples for the topic areas of complex systems science and engineering, economics and business, mental health and well-being, and global change efforts.

Genomic attributes of Vibrio cholerae O1 responsible for 2022 massive cholera outbreak in Bangladesh.

In 2022, one of its worst cholera outbreaks began in Bangladesh and the icddr,b Dhaka hospital treated more than 1300 patients and ca. 42,000 diarrheal cases from March-1 to April-10, 20221. Here, we present genomic attributes of V. cholerae O1 responsible for the 2022 Dhaka outbreak and 960 7th pandemic El Tor (7PET) strains from 88 countries. Results show strains isolated during the Dhaka outbreak cluster with 7PET wave-3 global clade strains, but comprise subclade BD-1.2, for which the most recent common ancestor appears to be that responsible for recent endemic cholera in India. BD-1.2 strains are present in Bangladesh since 2016, but not establishing dominance over BD-2 lineage strains2 until 2018 and predominantly associated with endemic cholera. In conclusion, the recent shift in lineage and genetic attributes, including serotype switching of BD-1.2 from Ogawa to Inaba, may explain the increasing number of cholera cases in Bangladesh.

Combating cholera by building predictive capabilities for pathogenic Vibrio cholerae in Yemen.

Cholera remains a global public health threat in regions where social vulnerabilities intersect with climate and weather processes that impact infectious Vibrio cholerae. While access to safe drinking water and sanitation facilities limit cholera outbreaks, sheer cost of building such infrastructure limits the ability to safeguard the population. Here, using Yemen as an example where cholera outbreak was reported in 2016, we show how predictive abilities for forecasting risk, employing sociodemographical, microbiological, and climate information of cholera, can aid in combating disease outbreak. An epidemiological analysis using Bradford Hill Criteria was employed in near-real-time to understand a predictive model's outputs and cholera cases in Yemen. We note that the model predicted cholera risk at least four weeks in advance for all governorates of Yemen with overall 72% accuracy (varies with the year). We argue the development of anticipatory decision-making frameworks for climate modulated diseases to design intervention activities and limit exposure of pathogens preemptively.

An urgent need for COP27: confronting converging crises.

The last 12 months have provided further evidence of the potential for cascading ecological and socio-political crises that were warned of 12 months ago. Then a consensus statement from the Regional Action on Climate Change Symposium warned: "the Earth's climatic, ecological, and human systems are converging towards a crisis that threatens to engulf global civilization within the lifetimes of children now living." Since then, the consequences of a broad set of extreme climate events (notably droughts, floods, and fires) have been compounded by interaction with impacts from multiple pandemics (including COVID-19 and cholera) and the Russia-Ukraine war. As a result, new connections are becoming visible between climate change and human health, large vulnerable populations are experiencing food crises, climate refugees are on the move, and the risks of water, food, and climate disruption have been visibly converging and compounding. Many vulnerable populations now face serious challenges to adapt. In light of these trends, this year, RACC identifies a range of measures to be taken at global and regional levels to bolster the resilience of these populations in the face of such emerging crises. In particular, at all scales, there is a need for globally available local data, reliable analytic techniques, community capacity to plan adaptation strategies, and the resources (scientific, technical, cultural, and economic) to implement them. To date, the rate of growth of the support for climate change resilience lags behind the rapid growth of cascading and converging risks. As an urgent message to COP27, it is proposed that the time is now right to devote much greater emphasis, global funding, and support to the increasing adaptation needs of vulnerable populations.

Cholera resurgence potentially induced by the consequences of climate in the El Niño phenomenon: an urgent call for strengthened cholera elimination in Africa.

A resurgence in cholera cases has been observed throughout Africa during the first half of 2023. Among the many factors that drive cholera transmission, the ongoing climate phenomenon El Niño is likely to continue until March to May 2024. To prevent further cholera spread, it is critical to strengthen cholera control efforts in Africa.

Gut microbiome insights from 16S rRNA analysis of 17-year periodical cicadas (Hemiptera: Magicicada spp.) Broods II, VI, and X.

Periodical cicadas (Hemiptera: Magicicada) have coevolved with obligate bacteriome-inhabiting microbial symbionts, yet little is known about gut microbial symbiont composition or differences in composition among allochronic Magicicada broods (year classes) which emerge parapatrically or allopatrically in the eastern United States. Here, 16S rRNA amplicon sequencing was performed to determine gut bacterial community profiles of three periodical broods, including II (Connecticut and Virginia, 2013), VI (North Carolina, 2017), and X (Maryland, 2021, and an early emerging nymph collected in Ohio, 2017). Results showed similarities among all nymphal gut microbiomes and between morphologically distinct 17-year Magicicada, namely Magicicada septendecim (Broods II and VI) and 17-year Magicicada cassini (Brood X) providing evidence of a core microbiome, distinct from the microbiome of burrow soil inhabited by the nymphs. Generally, phyla Bacteroidetes [Bacteroidota] (> 50% relative abundance), Actinobacteria [Actinomycetota], or Proteobacteria [Pseudomonadota] represented the core. Acidobacteria and genera Cupriavidus, Mesorhizobium, and Delftia were prevalent in nymphs but less frequent in adults. The primary obligate endosymbiont, Sulcia (Bacteroidetes), was dominant amongst core genera detected. Chryseobacterium were common in Broods VI and X. Chitinophaga, Arthrobacter, and Renibacterium were common in Brood X, and Pedobacter were common to nymphs of Broods II and VI. Further taxonomic assignment of unclassified Alphaproteobacteria sequencing reads allowed for detection of multiple copies of the Hodgkinia 16S rRNA gene, distinguishable as separate operational taxonomic units present simultaneously. As major emergences of the broods examined here occur at 17-year intervals, this study will provide a valuable comparative baseline in this era of a changing climate.

Predictive Intelligence for Cholera in Ukraine?

Cholera, an ancient waterborne diarrheal disease, remains a threat to public health, especially when climate/weather processes, microbiological parameters, and sociological determinants intersect with population vulnerabilities of loss of access to safe drinking water and sanitation infrastructure. The ongoing war in Ukraine has either damaged or severely crippled civil infrastructure, following which the human population is at risk of health disasters. This editorial highlights a perspective on using predictive intelligence to combat potential (and perhaps impending) cholera outbreaks in various regions of Ukraine. Reliable and judicious use of existing earth observations inspired mathematical algorithms integrating heuristic understanding of microbiological, sociological, and weather parameters have the potential to save or reduce the disease burden.

Genomic and evolutionary study from SARS-CoV-2 virus isolates from Bangladesh during the early stage of pandemic strongly correlate with European origin and not with China.

The goal of this study was to identify the genomic variants and determine molecular epidemiology of SARS-CoV-2 virus during the early pandemic stage in Bangladesh. Viral RNA was extracted, converted to cDNA, and amplified using Ion AmpliSeq™ SARS-CoV-2 Research Panel. 413 unique mutants from 151 viral isolates were identified. 80% of cases belongs to 8 mutants: 241C toT, 1163A toT, 3037C toT, 14408C toT, 23403A toG, 28881G toA, 28,882 G toA, and 28883G toC. Observed dominance of GR clade variants that have strong presence in Europe, suggesting European channel a possible entry route. Among 37 genomic mutants significantly associated with clinical symptoms, 3916CtoT (associated with sore-throat), 14408C to T (associated with cough-protection), 28881G to A, 28882G to A, and 28883G to C (associated with chest pain) were notable. These findings may inform future research platforms for disease management and epidemiological study.

Metagenomics of diabetic foot ulcer undergoing treatment with total contact casting: a case study.

OBJECTIVE: Diabetic foot ulcers (DFUs) are characterised by the presence of many microbes, some of which may not be identified by traditional culture techniques. Total contact casting (TCC) remains the gold-standard for offloading, yet little is known about the microbiome of wounds that progress from hard-to-heal to closed within a TCC. METHOD: A patient with a DFU underwent weekly treatment with TCC to closure. Samples for next-generation sequencing (NGS) and bioinformatics analysis of tissue samples were collected during each visit. Detection, identification, characterisation of the microbial community and abundance of microbes in each sample were compared. RESULTS: Abundance of microbes, identified by species and strain, changed with each treatment visit. By the final week of treatment, species diversity of the wound microbiome had decreased significantly, highlighted by an observed decrease in the number of total microorganisms present. Resistance genes for tetracyclines were detected in the first sample, but not in subsequent samples. CONCLUSION: The results of this study suggest dynamic microbiological changes associated with DFUs as they progress to healing within a TCC. As NGS becomes more readily available, further studies will be helpful to gain an improved understanding of the significance of the wound microbiome in patients with DFUs.