Genome-Scale Investigation of the Regulation of azoR Expression in Escherichia coli Using Computational Analysis and Transposon Mutagenesis.

Bacterial azoreductases are enzymes that catalyze the reduction of ingested or industrial azo dyes. Although azoreductase genes have been well identified and characterized, the regulation of their expression has not been systematically investigated. To determine how different factors affect the expression of azoR, we extracted and analyzed transcriptional data from the Gene Expression Omnibus (GEO) resource, then confirmed computational predictions by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results showed that azoR expression was lower with higher glucose concentration, agitation speed, and incubation temperature, but higher at higher culture densities. Co-expression and clustering analysis indicated ten genes with similar expression patterns to azoR: melA, tpx, yhbW, yciK, fdnG, fpr, nfsA, nfsB, rutF, and chrR (yieF). In parallel, constructing a random transposon library in E. coli K-12 and screening 4320 of its colonies for altered methyl red (MR)-decolorizing activity identified another set of seven genes potentially involved in azoR regulation. Among these genes, arsC, relA, plsY, and trmM were confirmed as potential azoR regulators based on the phenotypic decolorization activity of their transposon mutants, and the expression of arsC and relA was confirmed, by qRT-PCR, to significantly increase in E. coli K-12 in response to different MR concentrations. Finally, the significant decrease in azoR transcription upon transposon insertion in arsC and relA (as compared to its expression in wild-type E. coli) suggests their probable involvement in azoR regulation. In conclusion, combining in silico analysis and random transposon mutagenesis suggested a set of potential regulators of azoR in E. coli.

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Phenotypic characterization of phage vB_vcM_Kuja.

Bacteriophage therapy targeting the increasingly resistant Vibrio cholerae is highly needed. Hence, studying the phenotypic behavior of potential phages under different conditions is a prerequisite to delivering the phage in an active infective form. The objective of this study was to characterize phage VP4 (vB_vcM_Kuja), an environmental vibriophage isolated from River Kuja in Migori County, Kenya in 2015. The phenotypic characteristics of the phage were determined using a one-step growth curve, restriction digestion profile, pH, and temperature stability tests. The results revealed that the phage is stable through a wide range of temperatures (20-50°C) and maintains its plaque-forming ability at pH ranging from 6 to 12. The one-step growth curve showed a latent period falling between 40 and 60 min, while burst size ranged from 23 to 30 plaque-forming units/10 µl at the same host strain. The restriction digestion pattern using EcoRI, SalI, HindIII, and XhoI enzymes showed that HindIII could cut the phage genome. The phage DNA could not be restricted by the other three enzymes. The findings of this study can be used in future studies to determine phage-host interactions.

Integrating Microbiome Analysis, Metabolomics, Bioinformatics, and Histopathology to Elucidate the Protective Effects of Pomegranate Juice against Benzo-alpha-pyrene-Induced Colon Pathologies.

Polycyclic aromatic hydrocarbons, e.g., benzo[a]pyrene (BaP), are common dietary pollutants with potential carcinogenic activity, while polyphenols are potential chemopreventive antioxidants. Although several health benefits are attributed to polyphenol-rich pomegranate, little is known about its interaction with BaP. This study integrates histochemical, microbiomic, and metabolomic approaches to investigate the protective effects of pomegranate juice from BaP-induced pathologies. To this end, 48 Sprague-Dawley rats received, for four weeks, either pomegranate, BaP, both, or neither (n = 12 rats per group). Whereas histochemical examination of the colon indicated tissue damage marked by mucin depletion in BaP-fed animals, which was partially restored by administration of pomegranate juice, the fecal microbiome and metabolome retained their resilience, except for key changes related to pomegranate and BaP biotransformation. Meanwhile, dramatic microbiome restructuring and metabolome shift were observed as a consequence of the elapsed time (age factor). Additionally, the analysis allowed a thorough examination of fecal microbiome-metabolome associations, which delineated six microbiome clusters (marked by a differential abundance of Lactobacillaceae and Prevotellaceae, Rumincococcaceae, and Erysipelotrichaceae) and two major metabolome clusters (a sugar- and amino-acids-dominated metabotype vs. a cluster of fatty acids and hydrocarbons), with sugar alcohols maintaining a unique signature. In conclusion, using paired comparisons to minimize inter-individual animal variations allowed the dissection of temporal vs. treatment-derived variations. Microbiome-metabolome association clusters may be further exploited for metabotype prediction and gut-health biomarker discovery.

Bioprospecting the microbiome of Red Sea Atlantis II brine pool for peptidases and biosynthetic genes with promising antibacterial activity.

BACKGROUND: The search for novel antimicrobial agents is crucial as antibiotic-resistant pathogens continue to emerge, rendering the available antibiotics no longer effective. Likewise, new anti-cancer drugs are needed to combat the emergence of multi-drug resistant tumors. Marine environments are wealthy sources for natural products. Additionally, extreme marine environments are interesting niches to search for bioactive natural compounds. In the current study, a fosmid library of metagenomic DNA isolated from Atlantis II Deep Lower Convective Layer (ATII LCL), was functionally screened for antibacterial activity as well as anticancer effects. RESULTS: Two clones exhibited antibacterial effects against the marine Bacillus Cc6 strain, namely clones 102-5A and 88-1G and they were further tested against eleven other challenging strains, including six safe relatives of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter  spp.), a safe relative to Mycobacterium tuberculosis and four resistant clinical isolates. Clone 88-1G resulted in clear zones of inhibition against eight bacterial strains, while clone 102-5A resulted in zones of inhibition against five bacterial strains. The whole cell lysates of clone 88-1G showed 15% inhibition of Mtb ClpP protease -Mycobacterium tuberculosis drug target-, while whole cell lysates of clone 102-5A showed 19% inhibition of Mtb ClpP protease. Whole cell lysates from the selected clones exhibited anticancer effects against MCF-7 breast cancer cells (cell viability at 50% v/v was 46.2% ± 9.9 for 88-1G clone and 38% ± 7 for 102-5A clone), U2OS osteosarcoma cells (cell viability at 50% v/v was 64.6% ± 12.3 for 88-1G clone and 28.3% ± 1.7 for 102-5A clone) and 1BR hTERT human fibroblast cells (cell viability at 50% v/v was 74.4% ± 5.6 for 88-1G clone and 57.6% ± 8.9 for 102-5A clone). Sequencing of 102-5A and 88-1G clones, and further annotation detected putative proteases and putative biosynthetic genes in clones 102-5A and 88-1G, respectively. CONCLUSIONS: The ATII LCL metagenome hosts putative peptidases and biosynthetic genes that confer antibiotic and anti-cancer effects. The tested clones exhibited promising antibacterial activities against safe relative strains to ESKAPE pathogens and Mycobacterium tuberculosis. Thus, searching the microbial dark matter of extreme environments is a promising approach to identify new molecules with pharmaceutical potential use.

The PATRIC Bioinformatics Resource Center: expanding data and analysis capabilities.

The PathoSystems Resource Integration Center (PATRIC) is the bacterial Bioinformatics Resource Center funded by the National Institute of Allergy and Infectious Diseases (https://www.patricbrc.org). PATRIC supports bioinformatic analyses of all bacteria with a special emphasis on pathogens, offering a rich comparative analysis environment that provides users with access to over 250 000 uniformly annotated and publicly available genomes with curated metadata. PATRIC offers web-based visualization and comparative analysis tools, a private workspace in which users can analyze their own data in the context of the public collections, services that streamline complex bioinformatic workflows and command-line tools for bulk data analysis. Over the past several years, as genomic and other omics-related experiments have become more cost-effective and widespread, we have observed considerable growth in the usage of and demand for easy-to-use, publicly available bioinformatic tools and services. Here we report the recent updates to the PATRIC resource, including new web-based comparative analysis tools, eight new services and the release of a command-line interface to access, query and analyze data.

Novel PhoH-encoding vibriophages with lytic activity against environmental Vibrio strains.

Cholera is a devastating diarrheal disease that accounts for more than 10% of children's lives worldwide, but its treatment is hampered by a rise in antibiotic resistance. One promising alternative to antibiotic therapy is the use of bacteriophages to treat antibiotic-resistant cholera infections, and control Vibrio cholera in clinical cases and in the environment, respectively. Here, we report four novel, closely related environmental myoviruses, VP4, VP6, VP18, and VP24, which we isolated from two environmental toxigenic Vibrio cholerae strains from river Kuja and Usenge beach in Kenya. High-throughput sequencing followed by bioinformatics analysis indicated that the genomes of the four bacteriophages have closely related sequences, with sizes of 148,180 bp, 148,181 bp, 148,179 bp, and 148,179 bp, and a G + C content of 36.4%. The four genomes carry the phoH gene, which is overrepresented in marine cyanophages. The isolated phages displayed a lytic activity against 15 environmental, as well as one clinical, Vibrio cholerae strains. Thus, these novel lytic vibriophages represent potential biocontrol candidates for water decontamination against pathogenic Vibrio cholerae and ought to be considered for future studies of phage therapy.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

Thermal Stability of a Mercuric Reductase from the Red Sea Atlantis II Hot Brine Environment as Analyzed by Site-Directed Mutagenesis.

The lower convective layer (LCL) of the Atlantis II brine pool of the Red Sea is a unique environment in terms of high salinity, temperature, and high concentrations of heavy metals. Mercuric reductase enzymes functional in such extreme conditions could be considered a potential tool in the environmental detoxification of mercurial poisoning and might alleviate ecological hazards in the mining industry. Here, we constructed a mercuric reductase library from Atlantis II, from which we identified genes encoding two thermostable mercuric reductase (MerA) isoforms: one is halophilic (designated ATII-LCL) while the other is not (designated ATII-LCL-NH). The ATII-LCL MerA has a short motif composed of four aspartic acids (4D414-417) and two characteristic signature boxes that played a crucial role in its thermal stability. To further understand the mechanism behind the thermostability of the two studied enzymes, we mutated the isoform ATII-LCL-NH and found that the substitution of 2 aspartic acids (2D) at positions 415 and 416 enhanced the thermal stability, while other mutations had the opposite effect. The 2D mutant showed superior thermal tolerance, as it retained 81% of its activity after 10 min of incubation at 70°C. A three-dimensional structure prediction revealed newly formed salt bridges and H bonds in the 2D mutant compared to the parent molecule. To the best of our knowledge, this study is the first to rationally design a mercuric reductase with enhanced thermal stability, which we propose to have a strong potential in the bioremediation of mercurial poisoning.IMPORTANCE The Red Sea is an attractive environment for bioprospecting. There are 25 brine-filled deeps in the Red Sea. The Atlantis II brine pool is the biggest and hottest of such hydrothermal ecosystems. We generated an environmental mercuric reductase library from the lowermost layer of the Atlantis II brine pool, in which we identified two variants of the mercuric reductase enzyme (MerA). One is the previously described halophilic and thermostable ATII-LCL MerA and the other is a nonhalophilic relatively less thermostable enzyme, designated ATII-LCL-NH MerA. We used the ATII-LCL-NH enzyme as a parent molecule to locate the amino acid residues involved in the noticeably higher thermotolerance of the homolog ATII-LCL MerA. Moreover, we designed a novel enzyme with superior thermal stability. This enzyme might have strong potential in the bioremediation of mercuric toxicity.

Antibacterial and anticancer activities of orphan biosynthetic gene clusters from Atlantis II Red Sea brine pool.

BACKGROUND: Cancer and infectious diseases are problematic because of continuous emergence of drug resistance. One way to address this enormous global health threat is bioprospecting the unlikeliest environments, such as extreme marine niches, which have tremendous biodiversity that is barely explored. One such environment is the Red Sea brine pool, Atlantis II Deep (ATII). Here, we functionally screened a fosmid library of metagenomic DNA isolated from the ATII lower convective layer (LCL) for antibacterial and anticancer activities. RESULTS: Selected clones, 14-7E and 10-2G, displayed antibacterial effects on the marine strain Bacillus sp. Cc6. Moreover, whole cell lysates from 14-7E and 10-2G exhibited decreased cell viability against MCF-7 (39.1% ± 6.6, 42% ± 8.1 at 50% v/v) and U2OS cells (35.7% ± 1.9, 79.9% ± 5.9 at 50% v/v), respectively. By sequencing the insert DNA from 14-7E and 10-2G, we identified two putative orphan biosynthetic gene clusters. Both clusters harbored putative ATP-binding cassette (ABC) transporter permeases and S-adenosylmethionine-related genes. Interestingly, the biosynthetic gene cluster identified on 14-7E is of archaeal origin and harbors a putative transcription factor. Several identified genes may be responsible for the observed antibacterial and anticancer activities. The 14-7E biosynthetic gene cluster may be encoding enzymes producing a specialized metabolite (effect of detected genes involved in C-C bond formation and glycosylation). The bioactivity may also be due to predicted subtilases encoded by this cluster. The 10-2G cluster harbored putative glycosyltransferase and non-ribosomal peptide synthase genes; thus the observed activity of this clone could be caused by a bioactive peptide. CONCLUSIONS: The ATII LCL prokaryotic metagenome hosts putative orphan biosynthetic gene clusters that confer antibiotic and anticancer effects. Further biochemical studies should characterize the detected bioactive components, and the potential use of 14-7E metabolite for antibiosis and 10-2G metabolite as a selective anti-breast cancer drug.

Comparative genome-scale modelling of Staphylococcus aureus strains identifies strain-specific metabolic capabilities linked to pathogenicity.

Staphylococcus aureus is a preeminent bacterial pathogen capable of colonizing diverse ecological niches within its human host. We describe here the pangenome of S. aureus based on analysis of genome sequences from 64 strains of S. aureus spanning a range of ecological niches, host types, and antibiotic resistance profiles. Based on this set, S. aureus is expected to have an open pangenome composed of 7,411 genes and a core genome composed of 1,441 genes. Metabolism was highly conserved in this core genome; however, differences were identified in amino acid and nucleotide biosynthesis pathways between the strains. Genome-scale models (GEMs) of metabolism were constructed for the 64 strains of S. aureus These GEMs enabled a systems approach to characterizing the core metabolic and panmetabolic capabilities of the S. aureus species. All models were predicted to be auxotrophic for the vitamins niacin (vitamin B3) and thiamin (vitamin B1), whereas strain-specific auxotrophies were predicted for riboflavin (vitamin B2), guanosine, leucine, methionine, and cysteine, among others. GEMs were used to systematically analyze growth capabilities in more than 300 different growth-supporting environments. The results identified metabolic capabilities linked to pathogenic traits and virulence acquisitions. Such traits can be used to differentiate strains responsible for mild vs. severe infections and preference for hosts (e.g., animals vs. humans). Genome-scale analysis of multiple strains of a species can thus be used to identify metabolic determinants of virulence and increase our understanding of why certain strains of this deadly pathogen have spread rapidly throughout the world.

Insertion sequences enrichment in extreme Red sea brine pool vent.

Mobile genetic elements are major agents of genome diversification and evolution. Limited studies addressed their characteristics, including abundance, and role in extreme habitats. One of the rare natural habitats exposed to multiple-extreme conditions, including high temperature, salinity and concentration of heavy metals, are the Red Sea brine pools. We assessed the abundance and distribution of different mobile genetic elements in four Red Sea brine pools including the world's largest known multiple-extreme deep-sea environment, the Red Sea Atlantis II Deep. We report a gradient in the abundance of mobile genetic elements, dramatically increasing in the harshest environment of the pool. Additionally, we identified a strong association between the abundance of insertion sequences and extreme conditions, being highest in the harshest and deepest layer of the Red Sea Atlantis II Deep. Our comparative analyses of mobile genetic elements in secluded, extreme and relatively non-extreme environments, suggest that insertion sequences predominantly contribute to polyextremophiles genome plasticity.

Genome-scale metabolic reconstructions of multiple Escherichia coli strains highlight strain-specific adaptations to nutritional environments.

Genome-scale models (GEMs) of metabolism were constructed for 55 fully sequenced Escherichia coli and Shigella strains. The GEMs enable a systems approach to characterizing the pan and core metabolic capabilities of the E. coli species. The majority of pan metabolic content was found to consist of alternate catabolic pathways for unique nutrient sources. The GEMs were then used to systematically analyze growth capabilities in more than 650 different growth-supporting environments. The results show that unique strain-specific metabolic capabilities correspond to pathotypes and environmental niches. Twelve of the GEMs were used to predict growth on six differentiating nutrients, and the predictions were found to agree with 80% of experimental outcomes. Additionally, GEMs were used to predict strain-specific auxotrophies. Twelve of the strains modeled were predicted to be auxotrophic for vitamins niacin (vitamin B3), thiamin (vitamin B1), or folate (vitamin B9). Six of the strains modeled have lost biosynthetic pathways for essential amino acids methionine, tryptophan, or leucine. Genome-scale analysis of multiple strains of a species can thus be used to define the metabolic essence of a microbial species and delineate growth differences that shed light on the adaptation process to a particular microenvironment.

Mutual exclusivity of hyaluronan and hyaluronidase in invasive group A Streptococcus.

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection.

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.

PhiSpy: a novel algorithm for finding prophages in bacterial genomes that combines similarity- and composition-based strategies.

Prophages are phages in lysogeny that are integrated into, and replicated as part of, the host bacterial genome. These mobile elements can have tremendous impact on their bacterial hosts' genomes and phenotypes, which may lead to strain emergence and diversification, increased virulence or antibiotic resistance. However, finding prophages in microbial genomes remains a problem with no definitive solution. The majority of existing tools rely on detecting genomic regions enriched in protein-coding genes with known phage homologs, which hinders the de novo discovery of phage regions. In this study, a weighted phage detection algorithm, PhiSpy was developed based on seven distinctive characteristics of prophages, i.e. protein length, transcription strand directionality, customized AT and GC skew, the abundance of unique phage words, phage insertion points and the similarity of phage proteins. The first five characteristics are capable of identifying prophages without any sequence similarity with known phage genes. PhiSpy locates prophages by ranking genomic regions enriched in distinctive phage traits, which leads to the successful prediction of 94% of prophages in 50 complete bacterial genomes with a 6% false-negative rate and a 0.66% false-positive rate.

Pharmacomicrobiomics-Guided Precision Oncology: A New Frontier of P4 (Predictive, Personalized, Preventive, and Participatory) Medicine and Microbiome-Based Therapeutics.

Pharmacomicrobiomics is a rapidly developing field that promises to make significant contributions to predictive, personalized, preventive, and participatory (P4) medicine. This is becoming evident particularly in the field of precision (P4) oncology by taking seriously the crucial role microbiome plays in health and disease. Several studies have already shown that clinicians can harness insights from the microbiome to better predict treatment response, reduce side effects, and improve overall outcomes for cancer patients. Furthermore, pharmacomicrobiomics will undoubtedly play a crucial role in shaping the future of cancer treatment in the era of P4 oncology as we continue to unravel the intricate relationships between the microbiome and cancer. This perspective and innovation analysis discusses the emerging intersection of P4 medicine and P4 oncology, as seen through a lens of pharmacomicrobiomics. A key promise of pharmacomicrobiomics is the development of personalized microbiome-based therapeutics. In all, we suggest that optimizing cancer treatment and prevention by harnessing pharmacomicrobiomics has vast potentials for precision oncology, and personalized medicine using the right drug, at the right dose, for the right patient, and at the right time.

Tracing the evolutionary history of the pandemic group A streptococcal M1T1 clone.

The past 50 years has witnessed the emergence of new viral and bacterial pathogens with global effect on human health. The hyperinvasive group A Streptococcus (GAS) M1T1 clone, first detected in the mid-1980s in the United States, has since disseminated worldwide and remains a major cause of severe invasive human infections. Although much is understood regarding the capacity of this pathogen to cause disease, much less is known of the precise evolutionary events selecting for its emergence. We used high-throughput technologies to sequence a World Health Organization strain collection of serotype M1 GAS and reconstructed its phylogeny based on the analysis of core genome single-nucleotide polymorphisms. We demonstrate that acquisition of a 36-kb genome segment from serotype M12 GAS and the bacteriophage-encoded DNase Sda1 led to increased virulence of the M1T1 precursor and occurred relatively early in the molecular evolutionary history of this strain. The more recent acquisition of the phage-encoded superantigen SpeA is likely to have provided selection advantage for the global dissemination of the M1T1 clone. This study provides an exemplar for the evolution and emergence of virulent clones from microbial populations existing commensally or causing only superficial infection.

The curious case of Prevotella copri.

Prevotella copri is an abundant member of the human gastrointestinal microbiome, whose relative abundance has curiously been associated with positive and negative impacts on diseases, such as Parkinson's disease and rheumatoid arthritis. Yet, the verdict is still out on the definitive role of P. copri in human health, and on the effect of different diets on its relative abundance in the gut microbiome. The puzzling discrepancies among P. copri studies have only recently been attributed to the diversity of its strains, which substantially differ in their encoded metabolic patterns from the commonly used reference strain. However, such strain differences cannot be resolved by common 16S rRNA amplicon profiling methods. Here, we scrutinize P. copri, its versatile metabolic potential, and the hypotheses behind the conflicting observations on its association with diet and human health. We also provide suggestions for designing studies and bioinformatics pipelines to better research P. copri.