Abstract Tools for differential abundance testing of 16S rRNA data are conventionally treated as discrete methods, and benchmarks have accordingly sought to determine which tool performs best. However, each tool offers an array of configurations based on different normalisation, transformation, reference choice, and sensitivity filtering methods, and the specific impact of these configurations on performance has rarely been systematically investigated. We benchmarked five widely used tools (MaAsLin 2, MaAsLin 3, edgeR, ALDEx2, and ANCOM-BC2) across 18 configurations, using simulated communities and human gut profiles with implanted signals, at two taxonomic resolutions and across several design factors. Configuration accounted for as much performance variation as the choice of tool itself, with the ranking of two tools depending on which of their settings are compared. Individual parameters behaved as switches between opposite error regimes rather than as graded adjustments, and the settings carrying this weight are identifiable in advance. These behaviours were reproducible across data sources and resolutions. Our results define a configuration-aware framework for matching a tool and its settings to the cohort, study design, and feature resolution, establishing that a differential abundance result is interpretable only if the configuration used for the analysis is reported. Importance Most microbiome studies rely on a single software run with default settings to identify disease-associated bacteria, yet the settings inside a tool can alter results as much as switching tools. By systematically testing eighteen combinations of normalisation, transformation, and filtering options across human gut microbiome data, we provide a practical guide that prevents false biological conclusions in clinical microbiome research. The framework is immediately applicable to inflammatory bowel disease and dysbiosis studies, where sample size, community composition, and the expected proportion of differential taxa vary widely.

The SIS-NET ICU study aimed to describe the epidemiology of severe community-acquired pneumonia (CAP) among patients admitted to Italian intensive care units (ICUs). This study also aimed to describe the clinical and microbiological characteristics, outcomes, and treatments received by the included patients. We conducted a prospective, observational, multicenter study. We included patients consecutively admitted to the ICUs of 13 participating centers during the study period for acute respiratory failure due to CAP. The study period spanned from January to November 2025. The analyses aimed to describe the epidemiological and clinical characteristics, diagnostic pathways, factors associated with ICU mortality, and type of respiratory support during the ICU stay. We included a cohort of 150 patients with a mean age of 63 years and a male predominance (61

This dataset provides the fecal metaproteome profiles of 28 celiac disease patients on a gluten-free diet, distinguished by the presence or absence of co-occurring autoimmune conditions. The resource includes raw liquid chromatography-tandem mass spectrometry (LC-MS/MS) files, database search results, protein/peptide identification outputs, and taxonomic/functional annotation outputs, along with comprehensive anthropometric, clinical, and dietary metadata for each patient. The identified proteins originate from microbial, human, and plant sources, consistent with the multi-database search strategy used. This collection is designed for reuse in meta-analyses and integrative studies exploring functional changes in the gut microbiome related to auto-immune status and dietary variables. The complete dataset is available via the ProteomeXchange Consortium with the identifier PXD069517.

Metaproteomics enables functional insight into microbial communities by identifying and quantifying proteins in complex samples. Yet, heterogeneous analytical workflows and the lack of standardization across experimental and bioinformatics stages hinder reproducibility and comparability, limiting integration with other omics data. We here present a community-developed reporting checklist tailored to the specific needs of metaproteomics. We also outline current efforts to enable structured and interoperable metadata capture, drawing on standards from proteomics and microbiome research wherever possible. By promoting transparent reporting and advancing metadata practices, our recommendations aim to align metaproteomics more closely with FAIR principles and support reproducible and interoperable research practices.

Introduction Approximately one-third of people with epilepsy (PWE) experience resistance to treatment, including pharmacological therapies, epilepsy surgery, vagus nerve stimulation (VNS) and dietary interventions such as the ketogenic diet (KD). Emerging evidence suggests that the gut microbiota may influence seizure susceptibility and treatment response through the microbiota-gut-brain axis, potentially contributing to treatment resistance. The MiCrobiota-gut-brain Axis in Resistant Epilepsy project investigates how gut microbial features and associated host epigenetic signatures affect clinical outcomes in PWE undergoing diverse treatment strategies.Methods and analysis This is a multicentre, prospective, longitudinal study involving four clinical centres in Italy and one self-financing partner. Participants aged 3–50 years will be enrolled and stratified into four intervention cohorts: newly diagnosed drug-naïve epilepsy scheduled to start anti-seizure medications, focal drug-resistant epilepsy (DRE) undergoing epilepsy surgery, DRE receiving VNS, and DRE initiating KD. Clinical assessments (including body mass index calculation, self-reported monthly seizure count, dietary evaluation, quality of life scale and gastrointestinal symptoms scale), electroencephalography, MRI and biological sample collection (stool and blood) will be obtained at baseline and longitudinally at two or three timepoints over a 12-month observation period. Gut microbiota changes over time will be assessed via metagenomics (using 16S ribosomal RNA sequencing) and metaproteomics; the associated host DNA methylation profiles will be obtained from blood using Illumina EPIC arrays. Primary endpoints include identification of microbial or host methylation changes predictive of therapeutic response (ie, reduction from baseline in monthly seizure count) to the intervention. Data will be analysed using multivariate models and mixed-effect regression. Further, omics data and corresponding metadata will be integrated using multi-omics approaches to identify molecular signatures biomarkers predictive of treatment response and prognosis in PWE.Ethics and dissemination The study received ethical approval from the Research Ethic Board (Comitato Etico Territoriale Lombardia 3, ID 4896 – parere numero 4896_17.07.2024_N_bis). All participants or their legal guardians will provide written informed consent. Results will be disseminated through peer-reviewed publications, conference presentations or lay summaries targeting patient organisations.Trial registration number ClinicalTrials.gov Identifier NCT07010445, registered on 2 May 2025.

Fermented milk products are part of the staple diet for many Mediterranean populations. Most of these traditional foods are enriched with lactobacilli and other lactic acid bacteria, as well as with metabolites resulting from lactose fermentation. Currently, there is very little scientific knowledge on how dietary supplementation with fermented milk affects the composition of the gut microbiota and its metabolic activities. We integrated 16 S rRNA gene-based taxonomic profiling with metaproteomics-based functional analysis to investigate gut microbiota changes in rats exposed to an 8-week dietary supplementation with casu axedu, a traditional fermented milk produced within rural communities in Sardinia (Italy). Several microbial taxa showed a significantly increased abundance at the end of the dietary treatment, including Phascolarctobacterium, Prevotella, Blautia glucerasea, and Lactococcus lactis, while Bacteroides dorei and Helicobacter rodentium were decreased compared to the control rats. Metaproteomic analysis highlighted a striking reshaping of the Prevotella proteome in agreement with its blooming in casu axedu-fed animals, suggesting an increase of the glycolytic activity through the Embden-Meyerhof-Parnas pathway over the Entner-Doudoroff pathway. Moreover, an increased production of enzymes involved in succinate biosynthesis was observed, which in turn significantly boosted the abundance of Phascolarctobacterium and its production of propionate. Fermented milk consumption also promoted microbial synthesis of branched chain essential amino acids L-valine and L-leucine. Finally, metaproteomic data indicated a reduction of bacterial virulence factors and host inflammatory markers, suggesting that the consumption of casu axedu can have beneficial effects on the gut mucosa health. Our integrated multi-omics approach reveals that dietary supplementation with the traditional Sardinian fermented milk, casu axedu, induces significant shifts in the rat gut microbiota composition and function, characterized by the enrichment of beneficial taxa and metabolic pathways associated with improved gut health and reduced inflammation.

Introduction: Long-acting injectable antiretroviral therapy (ART) with Cabotegravir (CAB) and Rilpivirine (RPV) offers an alternative to daily oral regimens, improving adherence and patient satisfaction. However, its impact on body composition and metabolism remains underexplored. Methods: We conducted a prospective cohort study involving 29 people with HIV initiating CAB + RPV LA at a single centre in Italy. Body composition was assessed using dual-energy X-ray absorptiometry (DXA) and bioelectrical impedance analysis (BIA) at baseline, 24 and 48 weeks. Anthropometrics, laboratory parameters and patient-reported outcomes were also collected. Statistical comparisons across time points were performed using paired tests (p <0.05 considered significant). Results: At 48 weeks, weight and BMI remained stable. Waist circumference significantly decreased (median 97 (IQR 91-102) to 94 (IQR 89-98) cm, p = 0.026), with no significant change in total fat percentage or visceral adipose tissue. A modest but statistically significant increase in trunk/limb fat ratio (mean 1.19 (SD 0.39) to 1.25 (SD 0.41), p = 0.035). Lean mass and muscle function were unchanged. BIA findings confirmed stable fat mass and body water compartments. Virologic suppression was maintained in all participants throughout follow-up. High-density lipoprotein (HDL) cholesterol increased significantly, accompanied by a rise in total cholesterol, while low-density lipoprotein (LDL) cholesterol, triglycerides and the total cholesterol/HDL ratio remained stable. Serum creatinine significantly decreased, mainly among individuals switching from bictegravir-or dolutegravir-based regimens. Glycaemia, insulin, HOMA-IR, Metabolic Score for Insulin Resistance (METS-IR), liver enzymes and hepatic steatosis and fibrosis indices remained stable. Adverse events, mostly injection-site reactions, decreased over time. Only one participant discontinued treatment. Treatment satisfaction improved throughout the study. Conclusion: CAB + RPV LA was not associated with significant weight gain, clinically relevant changes in body composition or adverse metabolic effects over 48 weeks. Virologic suppression was maintained, renal laboratory parameters improved in prior INSTI users and treatment was well tolerated with increasing satisfaction. These findings support CAB + RPV LA as a safe, effective and metabolically neutral alternative to daily oral ART.

Bariatric surgery provides effective and durable weight loss in patients with severe obesity. Both the surgical procedure and postoperative management, including dietary and behavioral changes, are known to induce long-term alterations in gut microbiota composition. However, their impact on microbial and host proteome profiles remains poorly understood. In this prospective, multicenter cohort study, we compared the fecal metaproteome profile of 45 patients with severe obesity at preoperative baseline (T0) and 2 years after surgery (T1). Patients were randomized to receive either Roux-en-Y gastric bypass or one anastomosis gastric bypass. At T1, the relative abundance of the bacterial genera Akkermansia, Anaerotignum, Desulfovibrio, Streptococcus, and Veillonella significantly increased, whereas that of Faecalibacterium and Romboutsia decreased. Furthermore, we observed a significant increase in the relative abundance of microbial enzymes involved in glycolysis, short-chain fatty acid biosynthesis, amino acid metabolism, and cofactor/vitamin biosynthesis, alongside various outer membrane proteins encoded by Enterobacterales. Conversely, several proteins involved in oxidative phosphorylation and sporulation were reduced. Moreover, 74 human proteins, primarily associated with immune functions, showed a significant increase in relative abundance at T1, while numerous digestive enzymes displayed a downward trend. Our data reveal that a distinct reshaping of the fecal proteome and metaproteome persists 2 years after bariatric surgery. Future studies are needed to elucidate the mechanistic aspects of host-microbiota interaction and to identify associations with long-term clinical outcomes.IMPORTANCEBariatric surgery is widely recognized as the most effective and durable intervention for severe obesity; however, its long-term molecular effects on gut microbiota-host interactions remain poorly understood. By applying shotgun metaproteomics to fecal samples collected before and 2 years after surgery, our study provides novel insights into the functional consequences of bariatric bypass procedures. We demonstrate sustained alterations in both microbial and host protein profiles, including metabolic enzymes, outer membrane proteins, and immune-related factors, revealing a long-lasting remodeling of gut ecosystem functions. These findings underscore the value of metaproteomics in uncovering molecular mechanisms underlying bariatric surgery outcomes and may ultimately guide the development of microbiome- or host-targeted strategies to optimize therapy and long-term patient care.

Background and aimsHigh-fat diets are established contributors to carcinogenesis through mechanisms involving altered lipid metabolism, metabolic stress, and chronic inflammation. Time-restricted feeding (TRF) has emerged as a promising non-pharmacological strategy to improve metabolic health and potentially mitigate cancer risk by optimizing glycemic control, lipid profile, and inflammatory triggers. Despite these potential benefits, the direct impact of TRF on early-stage carcinogenesis, particularly in the context of obesogenic dietary conditions, remains inadequately explored. This study aimed to investigate the effects of TRF on the development of preneoplastic lesions in the liver and colon in male and female rats exposed to high-fat (HFD) and low-fat (LFD) diets.MethodsCarcinogenesis was initiated using diethyl nitrosamine (DENA) for the liver and azoxymethane (AOM) for the colon. Rats were assigned to ad libitum feeding (AdL) or TRF (8-h feeding window) following carcinogen exposure and were euthanized at 6 or 9 months for assessment. Pre-neoplastic lesions in the liver were evaluated by glutathione S-transferase placental form (GSTP)-positive staining, while aberrant crypt foci (ACF) were analyzed in the colon. Additionally, hepatic steatosis and metabolic parameters were assessed to determine the impact of TRF.ResultsNo differences were observed in the incidence, size, or distribution of GSTP-positive lesions in the liver or ACF in the colon between TRF and AdL groups. TRF also failed to reduce hepatic steatosis or improve serum lipid profiles in animals fed an HFD.ConclusionOur findings indicate that TRF does not significantly alter the development of early preneoplastic lesions in the liver or colon, regardless of dietary fat content, and further suggest that this dietary regimen is insufficient alone to counteract metabolic dysfunctions induced by highly obesogenic diets in rats.

One Health seeks to integrate and balance the health of humans, animals, and environmental systems. These three spheres are intricately interconnected through microbiomes, which are universally present and exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences in relation to host and ecosystem health remain poorly understood to date. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we document the potential of metaproteomics for elucidating microbial effectors and their impact on microbiomes, and discuss their potential for modulating microbiomes to foster desired functions.

Background Innovative antibiotic discovery strategies are urgently needed to successfully combat infections caused by multi-drug-resistant bacteria. Methods We employed a direct screening approach to identify compounds with antimicrobial and antimicrobial helper-drug activity against Gram-positive and Gram-negative bacteria. We used this platform in two different strains of methicillin-resistant Staphylococcus aureus (MRSA) and aminoglycoside-resistant strains of Escherichia coli to screen for antimicrobials compounds, which potentiate the activity of aminoglycoside antibiotics. Screening was performed with 75 known microbial products and 880 extracts from Actinomycetes from a collection at the company Naicons. Results The antibiotics rifamycin O and thermorubin inhibited the growth of neomycin-resistant E. coli in combination with 1/8 MIC of neomycin, suggesting a potential application as adjuvant drugs for neomycin. Additionally, in the Actinomycetes extract screen, one extract with antimicrobial activity and one extract with gentamicin adjuvant activity against gentamicin-resistant E. coli were identified, demonstrating the applicability of the screening approach. Against MRSA, the paramagnetoquinones, the lantibiotic NAI-107 and the spirotetronate NAI-414 showed the most pronounced antimicrobial activity. Difference is susceptibility towards antimicrobials and extracts were observed between the two MRSA strains used for screening. Conclusion Compounds with antibacterial and helper drug activity were identified using our screening approach. The results demonstrate the importance of strain selection in antimicrobial screening and highlight the potential of natural products as a source of potential new antibacterial and adjuvant therapies against both Gram-positive and Gram-negative bacteria.

INTRODUCTION:A reliable quantification of hepatitis D virus (HDV) RNA is of paramount importance for monitoring patients under antiviral therapy. This quality control study compares the diagnostic performances of quantitative HDV-RNA assays used in clinical practice. METHODS:Two HDV-RNA sample panels were quantified in 30 centers by RoboGene (N = 9 laboratories), EurobioPlex (N = 7), RealStar (N = 4), AltoStar (N = 1), Bosphore (N = 3), Bosphore-on-InGenius (N = 1), Dia.Pro (N = 2), Nuclear-Laser-Medicine (N = 1) and 3 in-house assays. Panel A and B comprised 8 serial dilutions of WHO/HDV standard (range: 0.5-5.0 log10 IU/ml) and 20 clinical samples (range: 0.5-6.0 log10 IU/ml), respectively. The following parameters were determined: sensitivity by 95 % LOD (limit of detection), precision by intra- and inter-run CV (coefficient of variation), accuracy by the differences between expected-observed HDV-RNA, linearity by linear regression analysis. RESULTS:95 % LOD varied across assays and centers underlining heterogeneous sensitivities: AltoStar had the lowest 95 % LOD (3 IU/ml) followed by RealStar (10 [min-max: 3-316] IU/ml), Bosphore-on-InGenius (10 IU/ml), RoboGene (31 [3-316] IU/ml), Nuclear-Laser-Medicine (31 IU/ml) and EuroBioplex (100 [100-316] IU/ml). Moreover, 6 assays (RoboGene, EurobioPlex, RealStar, AltoStar, Nuclear-Laser-Medicine and In-house) showed <0.5 log10 IU/ml differences between expected and observed HDV-RNA for all dilutions while other assays had >1 log10 IU/ml underestimations. RealStar, Bosphore-on-InGenius and EurobioPlex had the highest precision (mean intra-run CV < 20 %). Inter-run CV was higher for all assays, with CVs < 25 % for RealStar, AltoStar, Nuclear-Laser-Medicine and EurobioPlex. Seven assays (RoboGene/AltoStar/RealStar/EurobioPlex/Nuclear-Laser-Medicine/In-house) showed a good linearity (R2 > 0.90), but for HDV-RNA < 1000 IU/ml only Bosphore-on-InGenius, AltoStar, RealStar and Robogene showed a R2 > 0.85. CONCLUSIONS:This study underlines heterogeneous sensitivities (inter- and intraassays), that could hamper proper HDV-RNA quantification, particularly at low viral loads. This raises the need to improve the diagnostic performance of most assays for properly identifying virological response to anti-HDV drugs.

Metaproteomics is a valuable approach to characterize the biological functions involved in the gut microbiota (GM) response to dietary interventions. Ketogenic diets (KDs) are very effective in controlling seizure severity and frequency in drug-resistant epilepsy (DRE) and in the weight loss management in obese/overweight individuals. This case study provides proof of concept for the suitability of metaproteomics to monitor changes in taxonomic and functional GM features in an individual on a short-term very low-calorie ketogenic diet (VLCKD, 4 weeks), followed by a low-calorie diet (LCD). A marked increase in Akkermansia and Pseudomonadota was observed during VLCKD and reversed after the partial reintroduction of carbohydrates (LCD), in agreement with the results of previous metagenomic studies. In functional terms, the relative increase in Akkermansia was associated with an increased production of proteins involved in response to stress and biosynthesis of gamma-aminobutyric acid. In addition, VLCKD caused a relative increase in enzymes involved in the synthesis of the beta-ketoacid acetoacetate and of the ketogenic amino acid leucine. Our data support the potential of fecal metaproteomics to investigate the GM-dependent effect of KD as a therapeutic option in obese/overweight individuals and DRE patients.

INTRODUCTION:The human gut microbiome (HGM) profoundly influences human physiology. In recent years, it has become clearer that a healthy HGM is much better defined by its functional profile than by its taxonomic composition. Metaproteomics is the optimal approach to assessing the functional profile of the HGM in a taxon-specific manner, offering a direct view of its biological activity. AREAS COVERED:First, we summarized the main wet lab and data analysis approaches used in gut metaproteomics. Next, we reviewed metaproteomic studies that have characterized the HGM of healthy adults. Lastly, we examined the functional changes induced in the HGM by specific dietary interventions. EXPERT OPINION:Current fecal metaproteomics provides an initial understanding of the roles of gut microbes in human health, revealing redundant and taxon-specific functions. Future research should prioritize standardization, large-scale studies, and integration with multi-omics to better understand HGM metabolism. Emerging technologies, advanced mass spectrometry platforms, and AI-driven analytics are expected to increase sensitivity and depth of gut metaproteomics, accelerating discovery and potential clinical applications.

Fecal samples are widely used as a proxy for studying gut microbiome composition in both human and animal research. Fecal metaproteomics provides valuable insights by tracking changes in the relative abundance of microbial taxa and their protein functions. To ensure reliable results, it is crucial to minimize alterations in the metaproteome occurring from sample collection to protein extraction. Therefore, employing effective stabilization methods is essential to preserve the integrity of the fecal metaproteome from sample collection to laboratory analysis, particularly over long distances or when rapid freezing options are not readily available. In line with these needs, the second edition of the Critical Assessment of MetaProteome Investigation (CAMPI-2) was specifically focused on testing sample stabilization protocols to be applied before metaproteomic analysis. This collaborative multicenter study assessed the ability of five different stabilization methods, based on two commercial devices and three specific reagents (acetone, lithium dodecyl sulfate, and an RNAlater-like buffer), respectively, to stabilize the fecal metaproteome during room-temperature storage (14 days) and shipment to mass spectrometry facilities. The five methods were tested simultaneously by eight different laboratories across Europe, using aliquots from the same fecal sample. After protein extraction and digestion, duplicate aliquots of the resulting peptides were analyzed independently by two mass spectrometry facilities at distinct international locations. Analysis of the mass spectrometric data using two different search engines revealed that the fecal metaproteome profile differed considerably depending on the stabilization method used in terms of richness, alpha and beta diversity, reproducibility, and quantitative distribution of main taxa and functions. Although each method showed unique strengths and weaknesses, a commercial swab-based device stood out for its remarkable reproducibility and ranked highest for most of the metrics measured. CAMPI-2 allowed a robust evaluation of five different methods for preserving fecal metaproteome samples. The present investigation provides useful data for the design of metaproteomics and multi-omics studies where fecal sampling cannot be immediately followed by long-term storage at − 80 °C. Further optimization of the tested protocols is necessary to improve stabilization efficiency and control bias in the taxonomic and functional profile of the gut microbiome.

Metaproteomics is an emerging approach for studying microbiomes, offering the ability to characterize proteins that underpin microbial functionality within diverse ecosystems. As the primary catalytic and structural components of microbiomes, proteins provide unique insights into the active processes and ecological roles of microbial communities. By integrating metaproteomics with other omics disciplines, researchers can gain a comprehensive understanding of microbial ecology, interactions, and functional dynamics. This review, developed by the Metaproteomics Initiative (www.metaproteomics.org), serves as a practical guide for both microbiome and proteomics researchers, presenting key principles, state-of-the-art methodologies, and analytical workflows essential to metaproteomics. Topics covered include experimental design, sample preparation, mass spectrometry techniques, data analysis strategies, and statistical approaches.

The ability of methicillin-resistant Staphylococcus aureus (MRSA) to adapt to environmental stressors is crucial for its survival and persistence. This study used shotgun proteomics to analyze the protein profiles of MRSA strains ST398 and JE2 under three experimental conditions: EC1 (control, 37 °C, pH 7), EC2 (35 °C, pH 6), and EC3 (35 °C, pH 6 with 5% NaCl). Proteins were extracted and digested with trypsin using filter-aided sample preparation (FASP), followed by LC-MS/MS analysis. Raw data were analyzed using Proteome Discoverer software with label-free quantification (LFQ), identifying an average of 2640 ± 70 and 2832 ± 154 proteins in ST398 and JE2, respectively. Qualitative and quantitative differences in protein expression were observed between strains and conditions. Under EC2, ABC transporters increased and arginine metabolism was upregulated, suggesting metabolic adaptation to acidic stress. Purine metabolism was downregulated, indicating a metabolic shift. Under EC3 condition, NaCl addition induced glycine-betaine biosynthesis and proteins involved in potassium limitation. JE2 exhibited a more pronounced oxidative and stress response under salt stress, indicating a strain-specific adaptation. These findings highlight strain-specific proteomic responses in MRSA, highlighting the roles of arginine, glycine-betaine, and purine metabolism, along with stress-response proteins, in coping with environmental stressors.

Introduction: A COVID-19 outbreak occurred at the end of October 2021 among pilgrims returning from Medjugorje (Bosnia and Herzegovina). Methodology: Whole genome sequencing (WGS) of SARS-CoV-2, epidemiological data, and phylogenetic analysis were used to reconstruct outbreak dynamics. Results: The results suggest that only in one case, associated with the SARS-CoV-2 sub-lineage AY.9.2, it is possible to trace back the place of contagion to Medjugorje, while the other cases were likely to be acquired in the country of origin. Conclusions: The combined use of phylogenetic data derived from WGS, and epidemiological data allowed us to study epidemic dynamics and to formulate a possible hypothesis on the place of exposure to SARS-CoV-2. The identification of different sub-lineages of the SARS-CoV-2 Delta variant also suggested that different chains of transmission contributed to the outbreak.