Within-host Diversity Research Articles

Use of whole genome sequencing to identify low-frequency mutations in SARS-CoV-2 patients treated with remdesivir.

Remdesivir (RDV) has been shown to reduce hospitalization and mortality in COVID-19 patients. Resistance mutations caused by RDV are rare and have been predominantly reported in patients who are on prolonged therapy and immunocompromised. We investigate the effects of RDV treatment on intra-host SARS-CoV-2 diversity and low-frequency mutations in moderately ill hospitalized COVID-19 patients and compare them to patients without RDV treatment. From March 2020 to April 2022, sequential collections of nasopharyngeal and mid-turbinate swabs were obtained from 14 patients with and 30 patients without RDV treatment. Demographic and clinical data on all patients were reviewed. A total of 109 samples were sequenced and mutation analyses were performed. Previously reported drug resistant mutations in nsp12 were not identified during short courses of RDV therapy. In genes encoding and surrounding the replication complex (nsp6-nsp14), low-frequency minority variants were detected in 7/14 (50%) and 18/30 (60%) patients with and without RDV treatment, respectively. We did not detect significant differences in within-host diversity and positive selection between the RDV-treated and untreated groups. Minimal intra-host variability and stochastic low-frequency variants detected in moderately ill patients suggests little selective pressure in patients receiving short courses of RDV. The barrier to RDV resistance is high in patients with moderate disease. Patients undergoing short regimens of RDV therapy should continue to be monitored.

Intra- and interhost genomic diversity of monkeypox virus.

The impact and frequency of infectious disease outbreaks demonstrate the need for timely genomic surveillance to inform public health responses. In the largest known outbreak of mpox, genomic surveillance efforts have primarily focused on high-incidence nations in Europe and the Americas, with a paucity of data from South-East Asia and the Western Pacific. Here we analyzed 102 monkeypox virus (MPXV) genomes sampled from 56 individuals in Melbourne, Australia. All genomes fell within the 2022 MPXV outbreak lineage (B.1), with likely onward local transmission detected. We observed within-host diversity and instances of co-infection, and highlight further examples of structural variation and apolipoprotein B editing complex-driven micro-evolution in the current MPXV outbreak. Updating our understanding of MPXV emergence and diversification will inform public health measures and enable monitoring of the virus' evolutionary trajectory throughout the mpox outbreak.

Genomic analysis of Plasmodium falciparum isolates across different altitudinal zones along the slope of Mount Cameroon

BackgroundMalaria elimination is threatened by the emergence and rapid spread of drug resistance. Understanding the demographic history of Plasmodium falciparum and the genetic basis of adaptation to antimalarial treatment and host immunity is critical to elimination efforts. This study sought to characterize the diversity of P. falciparum isolates across different altitudes along the slope of Mount Cameroon.MethodologyUsing the Illumina next-generation sequencing platform P. falciparum isolates from low, intermediate and high altitudes along the slope of Mount Cameroon were studied to determine their genetic diversity, population structures, and signatures of selection in known drug resistance alleles.ResultsA total of 77,253 single-nucleotide polymorphisms were identified from 220 quality P. falciparum clinical isolates from high (24,214), intermediate (24,426), and low (28,613) altitude. About 49%, 48.2% and 30% of the parasite isolates from high, intermediate, and low altitudes, respectively had FWS values > 0.95 indicative of dominant mixed genotype infections and low population sub-structure with high potential for out-crossing. No significant difference was observed in within-host diversity while population structure analysis did not separate the isolates in the three major altitudinal groups by PCA, FST and admixtures, suggesting bidirectional gene flow among the populations. A total of 94 antigenic genes under balancing selection were detected in the area including vaccine candidate gene ama1, eba175, msp1, trap, dblmsp, and clag2. Moreover, 17 of these genes were identified to be under both recent positive directional and positive balancing selection including the prominent host immune target genes surfin 8.2, trap, and ama1. Recent directional selection analysis using integrated standardized haplotype score (iHS) did not detect any selection signatures in the Pfdhfr, Pfdhps, Pfmdr1, and PfK13 genes. Furthermore, no PfKelch13 validated mutation associated with artemisinin resistance was identified in this study and no structural divergence was noticed among the P. falciparum parasite populations across different altitudes around the Mount Cameroon region.DiscussionFindings revealed high genetic diversity and low population structure suggesting that malaria transmission remain high in the study area and parasite populations circulating around the slope of Mount Cameroon are homogenous.

A macroecological perspective on genetic diversity in the human gut microbiome.

While the human gut microbiome has been intensely studied, we have yet to obtain a sufficient understanding of the genetic diversity that it harbors. Research efforts have demonstrated that a considerable fraction of within-host genetic variation in the human gut is driven by the ecological dynamics of co-occurring strains belonging to the same species, suggesting that an ecological lens may provide insight into empirical patterns of genetic diversity. Indeed, an ecological model of self-limiting growth and environmental noise known as the Stochastic Logistic Model (SLM) was recently shown to successfully predict the temporal dynamics of strains within a single human host. However, its ability to predict patterns of genetic diversity across human hosts has yet to be tested. In this manuscript I determine whether the predictions of the SLM explain patterns of genetic diversity across unrelated human hosts for 22 common microbial species. Specifically, the stationary distribution of the SLM explains the distribution of allele frequencies across hosts and predicts the fraction of hosts harboring a given allele (i.e., prevalence) for a considerable fraction of sites. The accuracy of the SLM was correlated with independent estimates of strain structure, suggesting that patterns of genetic diversity in the gut microbiome follow statistically similar forms across human hosts due to the existence of strain-level ecology.

Selection for immune evasion in SARS-CoV-2 revealed by high-resolution epitope mapping and sequence analysis

Here, we exploit a deep serological profiling strategy coupled with an integrated, computational framework for the analysis of SARS-CoV-2 humoral immune responses. Applying a high-density peptide array (HDPA) spanning the entire proteomes of SARS-CoV-2 and endemic human coronaviruses allowed identification of B cell epitopes and relate them to their evolutionary and structural properties. We identify hotspots of pre-existing immunity and identify cross-reactive epitopes that contribute to increasing the overall humoral immune response to SARS-CoV-2. Using a public dataset of over 38,000 viral genomes from the early phase of the pandemic, capturing both inter- and within-host genetic viral diversity, we determined the evolutionary profile of epitopes and the differences across proteins, waves, and SARS-CoV-2 variants. Lastly, we show that mutations in spike and nucleocapsid epitopes are under stronger selection between than within patients, suggesting that most of the selective pressure for immune evasion occurs upon transmission between hosts.

Mixed strain pathogen populations accelerate the evolution of antibiotic resistance in patients

Antibiotic resistance poses a global health threat, but the within-host drivers of resistance remain poorly understood. Pathogen populations are often assumed to be clonal within hosts, and resistance is thought to emerge due to selection for de novo variants. Here we show that mixed strain populations are common in the opportunistic pathogen P. aeruginosa. Crucially, resistance evolves rapidly in patients colonized by multiple strains through selection for pre-existing resistant strains. In contrast, resistance evolves sporadically in patients colonized by single strains due to selection for novel resistance mutations. However, strong trade-offs between resistance and growth rate occur in mixed strain populations, suggesting that within-host diversity can also drive the loss of resistance in the absence of antibiotic treatment. In summary, we show that the within-host diversity of pathogen populations plays a key role in shaping the emergence of resistance in response to treatment.

SARS-CoV-2 within-host diversity of human hosts and its implications for viral immune evasion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously evolving, bringing great challenges to the control of the virus. In the present study, we investigated the characteristics of SARS-CoV-2 within-host diversity of human hosts and its implications for immune evasion using about 2,00,000 high-depth next-generation genome sequencing data of SARS-CoV-2. A total of 44% of the samples showed within-host variations (iSNVs), and the average number of iSNVs in the samples with iSNV was 1.90. C-to-U is the dominant substitution pattern for iSNVs. C-to-U/G-to-A and A-to-G/U-to-C preferentially occur in 5'-CG-3' and 5'-AU-3' motifs, respectively. In addition, we found that SARS-CoV-2 within-host variations are under negative selection. About 15.6% iSNVs had an impact on the content of the CpG dinucleotide (CpG) in SARS-CoV-2 genomes. We detected signatures of faster loss of CpG-gaining iSNVs, possibly resulting from zinc-finger antiviral protein-mediated antiviral activities targeting CpG, which could be the major reason for CpG depletion in SARS-CoV-2 consensus genomes. The non-synonymous iSNVs in the S gene can largely alter the S protein's antigenic features, and many of these iSNVs are distributed in the amino-terminal domain (NTD) and receptor-binding domain (RBD). These results suggest that SARS-CoV-2 interacts actively with human hosts and attempts to take different evolutionary strategies to escape human innate and adaptive immunity. These new findings further deepen and widen our understanding of the within-host evolutionary features of SARS-CoV-2. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of the coronavirus disease 2019, has evolved rapidly since it was discovered. Recent studies have pointed out that some mutations in the SARS-CoV-2 S protein could confer SARS-CoV-2 the ability to evade the human adaptive immune system. In addition, it is observed that the content of the CpG dinucleotide in SARS-CoV-2 genome sequences has decreased over time, reflecting the adaptation to the human host. The significance of our research is revealing the characteristics of SARS-CoV-2 within-host diversity of human hosts, identifying the causes of CpG depletion in SARS-CoV-2 consensus genomes, and exploring the potential impacts of non-synonymous within-host variations in the S gene on immune escape, which could further deepen and widen our understanding of the evolutionary features of SARS-CoV-2.

Transmission Dynamics of a Mycobacterium tuberculosis Complex Outbreak in an Indigenous Population in the Colombian Amazon Region.

Whole genome sequencing (WGS) has become the main tool for studying the transmission of Mycobacterium tuberculosis complex (MTBC) strains; however, the clonal expansion of one strain often limits its application in local MTBC outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis could potentially increase the resolution, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported MTBC outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March to October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct MTBC strain belonging to lineage 4.3.3. Employing a reference genome from an outbreak strain and highly confident single nucleotide polymorphisms (SNPs) in repetitive genomic regions, e.g., the proline-glutamic acid/proline-proline-glutamic-acid (PE/PPE) gene family, increased the phylogenetic resolution compared to a classical H37Rv reference mapping approach. Specifically, the number of differentiating SNPs increased from 890 to 1,094, which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 5 versus 9 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites, suggesting that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and employment of a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal MTBC populations and help elucidate within-host MTBC diversity. IMPORTANCE The Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak of Mycobacterium tuberculosis complex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods. Here, we employed a whole-genome sequencing-based outbreak investigation in order to improve the phylogenetic resolution and gain new insights into the transmission dynamics in this remote Colombian Amazon Region. The inclusion of well-supported single nucleotide polymorphisms in repetitive regions and a de novo-assembled local reference genome provided a more granular picture of the circulating outbreak strain and revealed new transmission chains. Multiple patients from different settlements were possibly infected with at least two different clones in this high-incidence setting. Thus, our results have the potential to improve molecular surveillance studies in other high-burden settings, especially regions with few clonal multidrug-resistant (MDR) MTBC lineages/clades.

Candida albicans selection for human commensalism results in substantial within-host diversity without decreasing fitness for invasive disease.

Candida albicans is a frequent colonizer of human mucosal surfaces as well as an opportunistic pathogen. C. albicans is remarkably versatile in its ability to colonize diverse host sites with differences in oxygen and nutrient availability, pH, immune responses, and resident microbes, among other cues. It is unclear how the genetic background of a commensal colonizing population can influence the shift to pathogenicity. Therefore, we examined 910 commensal isolates from 35 healthy donors to identify host niche-specific adaptations. We demonstrate that healthy people are reservoirs for genotypically and phenotypically diverse C. albicans strains. Using limited diversity exploitation, we identified a single nucleotide change in the uncharacterized ZMS1 transcription factor that was sufficient to drive hyper invasion into agar. We found that SC5314 was significantly different from the majority of both commensal and bloodstream isolates in its ability to induce host cell death. However, our commensal strains retained the capacity to cause disease in the Galleria model of systemic infection, including outcompeting the SC5314 reference strain during systemic competition assays. This study provides a global view of commensal strain variation and within-host strain diversity of C. albicans and suggests that selection for commensalism in humans does not result in a fitness cost for invasive disease.

Under-Appreciated Phylogroup Diversity of Escherichia coli within and between Animals at the Urban-Wildland Interface.

Wild animals have been implicated as reservoirs and even "melting pots" of pathogenic and antimicrobial-resistant bacteria of concern to human health. Though Escherichia coli is common among vertebrate guts and plays a role in the propagation of such genetic information, few studies have explored its diversity beyond humans nor the ecological factors that influence its diversity and distribution in wild animals. We characterized an average of 20 E. coli isolates per scat sample (n = 84) from a community of 14 wild and 3 domestic species. The phylogeny of E. coli comprises 8 phylogroups that are differentially associated with pathogenicity and antibiotic resistance, and we uncovered all of them in one small biological preserve surrounded by intense human activity. Challenging previous assumptions that a single isolate is representative of within-host phylogroup diversity, 57% of individual animals sampled carried multiple phylogroups simultaneously. Host species' phylogroup richness saturated at different levels across species and encapsulated vast within-sample and within-species variation, indicating that distribution patterns are influenced both by isolation source and laboratory sampling depth. Using ecological methods that ensure statistical relevance, we identify trends in phylogroup prevalence associated with host and environmental factors. The vast genetic diversity and broad distribution of E. coli in wildlife populations has implications for biodiversity conservation, agriculture, and public health, as well as for gauging unknown risks at the urban-wildland interface. We propose critical directions for future studies of the "wild side" of E. coli that will expand our understanding of its ecology and evolution beyond the human environment. IMPORTANCE To our knowledge, neither the phylogroup diversity of E. coli within individual wild animals nor that within an interacting multispecies community have previously been assessed. In doing so, we uncovered the globally known phylogroup diversity from an animal community on a preserve imbedded in a human-dominated landscape. We revealed that the phylogroup composition in domestic animals differed greatly from that in their wild counterparts, implying potential human impacts on the domestic animal gut. Significantly, many wild individuals hosted multiple phylogroups simultaneously, indicating the potential for strain-mixing and zoonotic spillback, especially as human encroachment into wildlands increases in the Anthropocene. We reason that due to extensive anthropogenic environmental contamination, wildlife is increasingly exposed to our waste, including E. coli and antibiotics. The gaps in the ecological and evolutionary understanding of E. coli thus necessitate a significant uptick in research to better understand human impacts on wildlife and the risk for zoonotic pathogen emergence.

Intra-Host Co-Existing Strains of SARS-CoV-2 Reference Genome Uncovered by Exhaustive Computational Search.

The COVID-19 pandemic caused by SARS-CoV-2 has had a severe impact on people worldwide. The reference genome of the virus has been widely used as a template for designing mRNA vaccines to combat the disease. In this study, we present a computational method aimed at identifying co-existing intra-host strains of the virus from RNA-sequencing data of short reads that were used to assemble the original reference genome. Our method consisted of five key steps: extraction of relevant reads, error correction for the reads, identification of within-host diversity, phylogenetic study, and protein binding affinity analysis. Our study revealed that multiple strains of SARS-CoV-2 can coexist in both the viral sample used to produce the reference sequence and a wastewater sample from California. Additionally, our workflow demonstrated its capability to identify within-host diversity in foot-and-mouth disease virus (FMDV). Through our research, we were able to shed light on the binding affinity and phylogenetic relationships of these strains with the published SARS-CoV-2 reference genome, SARS-CoV, variants of concern (VOC) of SARS-CoV-2, and some closely related coronaviruses. These insights have important implications for future research efforts aimed at identifying within-host diversity, understanding the evolution and spread of these viruses, as well as the development of effective treatments and vaccines against them.

Within-host genetic diversity of SARS-CoV-2 lineages in unvaccinated and vaccinated individuals

Viral and host factors can shape SARS-CoV-2 evolution. However, little is known about lineage-specific and vaccination-specific mutations that occur within individuals. Here, we analysed deep sequencing data from 2,820 SARS-CoV-2 respiratory samples with different viral lineages to describe the patterns of within-host diversity under different conditions, including vaccine-breakthrough infections. In unvaccinated individuals, variant of Concern (VOC) Alpha, Delta, and Omicron respiratory samples were found to have higher within-host diversity and were under neutral to purifying selection at the full genome level compared to non-VOC SARS-CoV-2. Breakthrough infections in 2-dose or 3-dose Comirnaty and CoronaVac vaccinated individuals did not increase levels of non-synonymous mutations and did not change the direction of selection pressure. Vaccine-induced antibody or T cell responses did not appear to have significant impact on within-host SARS-CoV-2 sequence diversification. Our findings suggest that vaccination does not increase exploration of SARS-CoV-2 protein sequence space and may not facilitate emergence of viral variants.

Staphylococcus aureus Carriage in the Nasotracheal Cavities of White Stork Nestlings (Ciconia ciconia) in Spain: Genetic Diversity, Resistomes and Virulence Factors.

The molecular ecology of Staphylococcus aureus in migratory birds (such as white storks) is necessary to understand their relevance in the "One Health" ecosystems. This study determined the nasotracheal carriage rates of S. aureus from white storks in Southern Spain and genetically characterized the within-host diversity. A collection of 67 S. aureus strains, previously obtained from 87 white stork nestlings (52 nasal and 85 tracheal samples) fed by their parents with food foraged in natural and landfill habitats, were tested for their antimicrobial resistance (AMR) phenotypes. Moreover, the AMR genotypes, immune evasion cluster (IEC), virulence genes and the detection of CC398 lineage were studied by PCR. The spa types and multilocus-sequencing-typing (MLST) were also determined by PCR and sequencing. Staphylococcus aureus carriage was found in 31% of storks (36.5%/11.9% in nasal/tracheal samples). All isolates were methicillin-susceptible (MSSA) and 8.8% of them were also susceptible to all tested antibiotics. The AMR phenotype/percentage/genes detected were as follows: penicillin/79.1%/blaZ; erythromycin-clindamycin-inducible/19.1%/ermA, ermT; tetracycline/11.9%/tetK; clindamycin/4.5%/lnuA and ciprofloxacin/4.5%. Twenty-one different spa types, including 2 new ones (t7778-ST15-CC15 and t18009-ST26-CC25), were detected and ascribed to 11 clonal complexes (CCs). MSSA-CC398 (8.2%), MSSA-CC15 (7.1%) and MSSA-ST291 (5.9%) were the most prevalent lineages in storks. Moreover, tst-positive (MSSA-CC22-t223 and MSSA-CC30-t1654), eta-positive (MSSA-CC9-t209) and etb-positive strains (MSSA-CC45-t015) were detected in four storks. The 18.5% of storks harboured distinct MSSA strains (with different lineages and/or AMR genes). Nestlings of storks foraging in landfills (10 CCs) had more diverse S. aureus strains than those of parents foraging in natural habitats (3 CCs). Low level of AMR was demonstrated among S. aureus strains. The predominance of MSSA-CC398 (an emergent clade) and toxigenic MSSA strains in stork nestlings highlight the need for continuous surveillance of S. aureus in wild birds.

Genomic epidemiology and transmission dynamics of recurrent Clostridioides difficile infection in Western Australia

Recurrent cases of Clostridioides difficile infection (rCDI) remain one of the most common and serious challenges faced in the management of CDI. The accurate distinction between a relapse (caused by infection with the same strain) and reinfection (caused by a new strain) has implications for infection control and prevention, and patient therapy. Here, we used whole-genome sequencing to investigate the epidemiology of 94 C. difficile isolates from 38 patients with rCDI in Western Australia. The C. difficile strain population comprised 13 sequence types (STs) led by ST2 (PCR ribotype (RT) 014, 36.2%), ST8 (RT002, 19.1%) and ST34 (RT056, 11.7%). Among 38 patients, core genome SNP (cgSNP) typing found 27 strains (71%) from initial and recurring cases differed by ≤ 2 cgSNPs, suggesting a likely relapse of infection with the initial strain, while eight strains differed by ≥ 3 cgSNPs, suggesting reinfection. Almost half of patients with CDI relapse confirmed by WGS suffered episodes that occurred outside the widely used 8-week cut-off for defining rCDI. Several putative strain transmission events between epidemiologically unrelated patients were identified. Isolates of STs 2 and 34 from rCDI cases and environmental sources shared a recent evolutionary history, suggesting a possible common community reservoir. For some rCDI episodes caused by STs 2 and 231, within-host strain diversity was observed, characterised by loss/gain of moxifloxacin resistance. Genomics improves discrimination of relapse from reinfection and identifies putative strain transmission events among patients with rCDI. Current definitions of relapse and reinfection based on the timing of recurrence need to be reconsidered.

Nasal staphylococci community of healthy pigs and pig-farmers in Aragon (Spain). Predominance and within-host resistome diversity in MRSA-CC398 and MSSA-CC9 lineages

This study investigated the diversity and carriage rate of nasal Staphylococcus spp., and within-host variability of antimicrobial resistance (AMR), virulence determinants, immune evasion cluster (IEC) types and genetic lineages of S. aureus isolates. Also, the co-carriage rate of CoNS with S. aureus in the same nasal niche of healthy pigs and pig-farmers were studied in four pig-farms (A-D) in Aragon (Spain). Nasal samples of 40 pigs (10 pigs/farm) and 10 pig-farmers (2–3/farm) were collected for staphylococci recovery and isolates (up to 9 per sample) were identified by MALDI-TOF-MS. The virulence and AMR genes and spa-types of S. aureus isolates were investigated by PCR/sequencing. Of the 243 staphylococci identified (10 different species), 142 were S. aureus and 51 distinct isolates were selected for further characterization (that corresponded to one S. aureus/sample or more than one if they showed different AMR phenotypes). The highest carriage rate in pigs was S. aureus (65%) and S. chromogenes (22.5%), whereas in the pig-farmers, S. aureus (80%) and S. epidermidis (40%). Methicillin-resistant S. aureus (MRSA) were detected in 60% of pigs and 70% of pig-farmers. Only six S. aureus isolates were methicillin-susceptible (MSSA), all from farm-C. A multidrug resistance (MDR) phenotype was detected in all MRSA and in 83.3% of the MSSA isolates. All MRSA isolates were CC398 with spa-type t011 being the predominant (92.7%), while t034, t1451 (only in pig-farmers) and t4571 (in pigs) were also found. MSSA-CC9 isolates (t191, t1430) were detected in farm-C. All S. aureus isolates were negative for luk-S/F-PV, tst, and scn genes, except one MSSA-CC45-t065-IEC-type C isolate from a pig-farmer. About 34.6% and 75.0% of the pigs and pig-farmers S. aureus carriers, respectively, harboured within-host varied spa-types or resistomes. Moreover, 40% of pigs and pig-farmers with MRSA-CC398 had no CoNS nasal co-carriage, and 23.3% had ≥2 CoNS carriage. Conversely, only 16.7% of MSSA carriers had no CoNS co-carriage, whereas 50% had ≥2 CoNS carriages. The very high MRSA level and within-host resistome diversities highlight the need for multiple samplings to account for the dynamics of AMR crisis and control of inter-host transmission of S. aureus in pig-farms using “One Health” approach.

Patterns of variation in equine strongyle community structure across age groups and gut compartments

BackgroundEquine strongyles encompass more than 64 species of nematode worms that are responsible for growth retardation and the death of animals. The factors underpinning variation in the structure of the equine strongyle community remain unknown.MethodsUsing horse-based strongyle community data collected after horse deworming (48 horses in Poland, 197 horses in Ukraine), we regressed species richness and the Gini-Simpson index upon the horse’s age, faecal egg count, sex and operation of origin. Using the Ukrainian observations, we applied a hierarchical diversity partitioning framework to estimate how communities were remodelled across operations, age groups and horses. Lastly, strongyle species counts collected after necropsy (46 horses in France, 150 in Australia) were considered for analysis of their co-occurrences across intestinal compartments using a joint species distribution modelling approach.ResultsFirst, inter-operation variation accounted for > 45% of the variance in species richness or the Gini-Simpson index (which relates to species dominance in communities). Species richness decreased with horse’s age (P = 0.01) and showed a mild increase with parasite egg excretion (P < 0.1), but the Gini-Simpson index was neither associated with parasite egg excretion (P = 0.8) nor with horse age (P = 0.37). Second, within-host diversity represented half of the overall diversity across Ukrainian operations. While this is expected to erase species diversity across communities, community dissimilarity between horse age classes was the second most important contributor to overall diversity (25.8%). Third, analysis of species abundance data quantified at necropsy defined a network of positive co-occurrences between the four most prevalent strongyle genera. This pattern was common to necropsies performed in France and Australia.ConclusionsTaken together, these results show a pattern of β-diversity maintenance across age classes combined with positive co-occurrences that might be grounded by priority effects between the major species.Graphical

Genomic description of human clinical Aspergillus fumigatus isolates, California, 2020.

Aspergillus fumigatus, an environmental mold, causes life-threatening infections. Studies on the phylogenetic structure of human clinical A. fumigatus isolates are limited. Here, we performed whole genome sequencing of 24 A. fumigatus isolates collected from 18 patients in U.S. healthcare facilities in California. Single-nucleotide polymorphism (SNP) differences between patient isolates ranged from 187 to 70 829 SNPs. For five patients with multiple isolates, we calculated the within-host diversities. Three patients had a within-host diversity that ranged from 4 to 10 SNPs and two patients ranged from 2 to 16 977 SNPs. Findings revealed highly diverse A. fumigatus strains among patients and two patterns of diversity for isolates that come from the same patient, low and extremely high diversity.

Challenges in Harnessing Shared Within-Host Severe Acute Respiratory Syndrome Coronavirus 2 Variation for Transmission Inference.

The limited variation observed among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference. We performed tiled amplicon sequencing on 307 SARS-CoV-2 samples, including 130 samples from 32 individuals in 14 households and 47 longitudinally sampled individuals, from 4 prospective studies with household membership data, a proxy for transmission linkage. Consensus sequences from households had limited diversity (mean pairwise distance, 3.06 single-nucleotide polymorphisms [SNPs]; range, 0-40). Most (83.1%, 255 of 307) samples harbored at least 1 intrahost single-nucleotide variant ([iSNV] median, 117; interquartile range [IQR], 17-208), above a minor allele frequency threshold of 0.2%. Pairs in the same household shared significantly more iSNVs (mean, 1.20 iSNVs; 95% confidence interval [CI], 1.02-1.39) than did pairs in different households infected with the same viral clade (mean, 0.31 iSNVs; 95% CI, .28-.34), a signal that decreases with increasingly stringent minor allele frequency thresholds. The number of shared iSNVs was significantly associated with an increased odds of household membership (adjusted odds ratio, 1.35; 95% CI, 1.23-1.49). However, the poor concordance of iSNVs detected across sequencing replicates (24.8% and 35.0% above a 0.2% and 1% threshold) confirms technical concerns that current sequencing and bioinformatic workflows do not consistently recover low-frequency within-host variants. Shared within-host variation may augment the information in consensus sequences for predicting transmission linkages. Improving sensitivity and specificity of within-host variant identification will improve the informativeness of within-host variation.

Within-Host Genotypic and Phenotypic Diversity of Contemporaneous Carbapenem-Resistant Klebsiella pneumoniae from Blood Cultures of Patients with Bacteremia.

It is unknown whether bacterial bloodstream infections (BSIs) are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole-genome sequences of 10 sequence type 258 (ST258) CRKP strains from blood cultures in each of 6 patients (Illumina HiSeq). Strains clustered by patient by core genome and pan-genome phylogeny. In 5 patients, there was within-host strain diversity by gene mutations, presence/absence of antibiotic resistance or virulence genes, and/or plasmid content. Accessory gene phylogeny revealed strain diversity in all 6 patients. Strains from 3 patients underwent long-read sequencing for genome completion (Oxford Nanopore) and phenotypic testing. Genetically distinct strains within individuals exhibited significant differences in carbapenem and other antibiotic responses, capsular polysaccharide (CPS) production, mucoviscosity, and/or serum killing. In 2 patients, strains differed significantly in virulence during mouse BSIs. Genetic or phenotypic diversity was not observed among strains recovered from blood culture bottles seeded with index strains from the 3 patients and incubated in vitro at 37°C. In conclusion, we identified genotypic and phenotypic variant ST258 CRKP strains from blood cultures of individual patients with BSIs, which were not detected by the clinical laboratory or in seeded blood cultures. The data suggest a new paradigm of CRKP population diversity during BSIs, at least in some patients. If validated for BSIs caused by other bacteria, within-host microbial diversity may have implications for medical, microbiology, and infection prevention practices and for understanding antibiotic resistance and pathogenesis. IMPORTANCE The long-standing paradigm for pathogenesis of bacteremia is that, in most cases, a single organism passes through a bottleneck and establishes itself in the bloodstream (single-organism hypothesis). In keeping with this paradigm, standard practice in processing positive microbiologic cultures is to test single bacterial strains from morphologically distinct colonies. This study is the first genome-wide analysis of within-host diversity of Klebsiella pneumoniae strains recovered from individual patients with bloodstream infections (BSIs). Our finding that positive blood cultures comprised genetically and phenotypically heterogeneous carbapenem-resistant K. pneumoniae strains challenges the single-organism hypothesis and suggests that at least some BSIs are caused by mixed bacterial populations that are unrecognized by the clinical laboratory. The data support a model of pathogenesis in which pressures in vivo select for strain variants with particular antibiotic resistance or virulence attributes and raise questions about laboratory protocols and treatment decisions directed against single strains.

Tracking outbreak dynamics using within-host variation.

Tracking outbreak dynamics using within-host variation.