Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Emergence and clonal expansion of a qacA-harbouring sequence type 45 lineage of methicillin-resistant Staphylococcus aureus.

The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6')-aph(2")) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.

The two-component system WalKR provides an essential link between cell wall homeostasis and DNA replication in Staphylococcus aureus.

Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. IMPORTANCE The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

The whole-genome molecular epidemiology of sequential isolates of Acinetobacter baumannii colonizing the rectum of patients in an adult intensive care unit of a tertiary hospital.

IMPORTANCE: Acinetobacter baumannii is a multidrug-resistant nosocomial pathogen that colonizes and infects debilitated patients in the ICU. There is very little information on the genomic characteristics of colonizing strains. This information is important to understand the evolution of lineages of A. baumannii that develop resistance while patients receive antibiotic treatment in the ICU. Our study demonstrated different patterns of colonization of the rectum of ICU patients with different STs of A. baumannii while one ST colonized all patients. Some STs carried more antibiotic resistance genes compared to others. However, there was a correlation between ST and a particular resistance gene profile. Our results further elucidate the dynamics of enteric colonization of this opportunistic pathogen.

Novel Perspectives on the Design and Development of a Long-Acting Subcutaneous Raltegravir Injection for Treatment of HIV-In Vitro and In Vivo Evaluation.

Antiretrovirals (ARVs) are a highly effective therapy for treatment and prevention of HIV infection, when administered as prescribed. However, adherence to lifelong ARV regimens poses a considerable challenge and places HIV patients at risk. Long-acting ARV injections may improve patient adherence as well as maintaining long-term continuous drug exposure, resulting in improved pharmacodynamics. In the present work, we explored the aminoalkoxycarbonyloxymethyl (amino-AOCOM) ether prodrug concept as a potential approach to long-acting ARV injections. As a proof of concept, we synthesised model compounds containing the 4-carboxy-2-methyl Tokyo Green (CTG) fluorophore and assessed their stability under pH and temperature conditions that mimic those found in the subcutaneous (SC) tissue. Among them, probe 21 displayed very slow fluorophore release under SC-like conditions (98% of the fluorophore released over 15 d). Compound 25, a prodrug of the ARV agent raltegravir (RAL), was subsequently prepared and evaluated using the same conditions. This compound showed an excellent in vitro release profile, with a half-life (t½) of 19.3 d and 82% of RAL released over 45 d. In mice, 25 extended the half-life of unmodified RAL by 4.2-fold (t½ = 3.18 h), providing initial proof of concept of the ability of amino-AOCOM prodrugs to extend drug lifetimes in vivo. Although this effect was not as pronounced as seen in vitro-presumably due to enzymatic degradation and rapid clearance of the prodrug in vivo-the present results nevertheless pave the way for development of more metabolically stable prodrugs, to facilitate long-acting delivery of ARVs.

Design, Development, and Optimisation of Smart Linker Chemistry for Targeted Colonic Delivery-In Vitro Evaluation.

Drug targeting is necessary to deliver drugs to a specific site of action at a rate dictated by therapeutic requirements. The pharmacological action of a drug can thereby be optimised while minimising adverse effects. Numerous colonic drug delivery systems have been developed to avoid such undesirable side effects; however, these systems lack site specificity, leaving room for further improvement. The objective of the present study was to explore the potential of amino-alkoxycarbonyloxymethyl (amino-AOCOM) ether prodrugs as a general approach for future colonic delivery. To circumvent inter- and intra-subject variabilities in enzyme activities, these prodrugs do not rely on enzymes but rather are activated via a pH-triggered intramolecular cyclisation−elimination reaction. As proof of concept, model compounds were synthesised and evaluated under various pH conditions, simulating various regions of the gastrointestinal tract (GIT). Probe 15 demonstrated excellent stability under simulated stomach- and duodenum-like conditions and protected 60% of the payload in a small intestine-like environment. Moreover, 15 displayed sustained release at colonic pH, delivering >90% of the payload over 38 h. Mesalamine (Msl) prodrugs 21 and 22 were also synthesised and showed better stability than probe 15 in the simulated upper GIT but relatively slower release at colonic pH (61−68% of Msl over 48 h). For both prodrugs, the extent of release was comparable to that of the commercial product Asacol. This study provides initial proof of concept regarding the use of a cyclisation-activated prodrug for colon delivery and suggests that release characteristics still vary on a case-by-case basis.

Clinical relevance of topical antibiotic use in co-selecting for multidrug-resistant Staphylococcus aureus: Insights from in vitro and ex vivo models.

Topical antibiotic preparations, such as fusidic acid (FA) or mupirocin, are used in the prevention and treatment of superficial skin infections caused by staphylococci. Previous genomic epidemiology work has suggested an association between the widespread use of topical antibiotics and the emergence of methicillin resistant Staphylococcus aureus in some settings. In this study, we provide experimental proof of co-selection for multidrug resistance in S. aureus following exposure to FA or mupirocin. Through targeted mutagenesis and phenotypic analyses, we confirmed that fusC carriage confers resistance to FA, and mupA carriage confers high-level resistance to mupirocin in multiple S. aureus genetic backgrounds. In vitro experiments demonstrated that carriage of fusC and mupA confer a competitive advantage in the presence of sub-inhibitory concentrations of FA and mupirocin, respectively. Further, we used a porcine skin colonisation model to show that clinically relevant concentrations of topical antibiotics can co-select for presence of unrelated antimicrobial resistance determinants, such as mecA, blaZ, and qacA, in fusC or mupA harbouring S. aureus These findings provide valuable insights on the role of acquired FA or mupirocin resistance in co-selecting for broader antibiotic resistance in S. aureus, prompting greater need for judicious use of topical antibiotics.

Synthesis, Antibacterial Activity, and Nephrotoxicity of Polymyxin B Analogues Modified at Leu-7, d-Phe-6, and the N-Terminus Enabled by S-Lipidation.

With the post-antibiotic era rapidly approaching, many have turned their attention to developing new treatments, often by structural modification of existing antibiotics. Polymyxins, a family of lipopeptide antibiotics that are used as a last line of defense in the clinic, have recently developed resistance and exhibit significant nephrotoxicity issues. Using thiol-ene chemistry, the facile preparation of six unique S-lipidated building blocks was demonstrated and used to generate lipopeptide mimetics upon incorporation into solid-phase peptide synthesis (SPPS). We then designed and synthesized 38 polymyxin analogues, incorporating these unique building blocks at the N-terminus, or to replace hydrophobic residues at positions 6 and 7 of the native lipopeptides. Several polymyxin analogues bearing one or more S-linked lipids were found to be equipotent to polymyxin, showed minimal kidney nephrotoxicity, and demonstrated activity against several World Health Organisation (WHO) priority pathogens. The S-lipidation strategy has demonstrated potential as a novel approach to prepare innovative new lipopeptide antibiotics.

Detection of Chimeric Cellular: HIV mRNAs Generated Through Aberrant Splicing in HIV-1 Latently Infected Resting CD4+ T Cells.

Latent HIV-1 provirus in infected individuals on suppressive therapy does not always remain transcriptionally silent. Both HIV-1 LTR and human gene promoter derived transcriptional events can contribute HIV-1 sequences to the mRNA produced in the cell. In addition, chimeric cellular:HIV mRNA can arise through readthrough transcription and aberrant splicing. Using target enrichment coupled to the Illumina Mi-Seq and PacBio RS II platforms, we show that 3' LTR activation is frequent in latently infected cells from both the CCL19-induced primary cell model of HIV-1 latency as well as ex vivo samples. In both systems of latent HIV-1 infection, we detected several chimeric species that were generated via activation of a cryptic splice donor site in the 5' LTR of HIV-1. Aberrant splicing involving the major HIV-1 splice donor sites, SD1 and SD4 disrupts post-transcriptional processing of the gene in which HIV-1 is integrated. In the primary cell model of HIV-1 latency, Tat-encoding sequences are incorporated into the chimeric mRNA transcripts through the use of SD4. Our study unravels clues to the characteristics of HIV-1 integrants that promote formation of chimeric cellular:HIV mRNA and improves the understanding of the HIV-1 RNA footprint in latently infected cells.

Development of 1,2,4-Oxadiazole Antimicrobial Agents to Treat Enteric Pathogens within the Gastrointestinal Tract.

Colonization of the gastrointestinal (GI) tract with pathogenic bacteria is an important risk factor for the development of certain potentially severe and life-threatening healthcare-associated infections, yet efforts to develop effective decolonization agents have been largely unsuccessful thus far. Herein, we report modification of the 1,2,4-oxadiazole class of antimicrobial compounds with poorly permeable functional groups in order to target bacterial pathogens within the GI tract. We have identified that the quaternary ammonium functionality of analogue 26a results in complete impermeability in Caco-2 cell monolayers while retaining activity against GI pathogens Clostridioides difficile and multidrug-resistant (MDR) Enterococcus faecium. Low compound recovery levels after oral administration in rats were observed, which suggests that the analogues may be susceptible to degradation or metabolism within the gut, highlighting a key area for optimization in future efforts. This study demonstrates that modified analogues of the 1,2,4-oxadiazole class may be potential leads for further development of colon-targeted antimicrobial agents.

Daptomycin Resistance Occurs Predominantly in vanA-Type Vancomycin-Resistant Enterococcus faecium in Australasia and Is Associated With Heterogeneous and Novel Mutations.

Healthcare associated infections caused by vancomycin-resistant Enterococcus faecium (VREfm) have a major impact on health outcomes. VREfm is difficult to treat because of intrinsic and acquired resistance to many clinically used antimicrobials, with daptomycin being one of the few last line therapeutic options for treating multidrug-resistant VREfm. The emergence of daptomycin-resistant VREfm is therefore of serious clinical concern. Despite this, the impact that daptomycin-resistant VREfm have on patient health outcomes is not clearly defined and knowledge on the mechanisms and genetic signatures linked with daptomycin resistance in VREfm remains incomplete. To address these knowledge gaps, phenotypic daptomycin susceptibility testing was undertaken on 324 E. faecium isolates from Australia and New Zealand. Approximately 15% of study isolates were phenotypically resistant to daptomycin. Whole genome sequencing revealed a strong association between vanA-VREfm and daptomycin resistance, with 95% of daptomycin-resistant study isolates harbouring vanA. Genomic analyses showed that daptomycin-resistant VREfm isolates were polyclonal and carried several previously characterised mutations in the liaR and liaS genes as well as several novel mutations within the rpoB, rpoC, and dltC genes. Overall, 70% of daptomycin-resistant study isolates were found to carry mutations within the liaR, rpoB, rpoC, or dltC genes. Finally, in a mouse model of VREfm bacteraemia, infection with the locally dominant daptomycin-resistant clone led to reduced daptomycin treatment efficacy in comparison to daptomycin-susceptible E. faecium. These findings have important implications for ongoing VREfm surveillance activities and the treatment of VREfm infections.

Genomic Insights Into Last-Line Antimicrobial Resistance in Multidrug-Resistant Staphylococcus and Vancomycin-Resistant Enterococcus.

Multidrug-resistant Staphylococcus and vancomycin-resistant Enterococcus (VRE) are important human pathogens that are resistant to most clinical antibiotics. Treatment options are limited and often require the use of 'last-line' antimicrobials such as linezolid, daptomycin, and in the case of Staphylococcus, also vancomycin. The emergence of resistance to these last-line antimicrobial agents is therefore of considerable clinical concern. This mini-review provides an overview of resistance to last-line antimicrobial agents in Staphylococcus and VRE, with a particular focus on how genomics has provided critical insights into the emergence of resistant clones, the molecular mechanisms of resistance, and the importance of mobile genetic elements in the global spread of resistance to linezolid.

Complete microbial genomes for public health in Australia and the Southwest Pacific.

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health laboratory activities. Here, we announce a BioProject (PRJNA556438) dedicated to sharing complete genomes chosen to represent a range of pathogenic bacteria with regional importance to Australia and the Southwest Pacific; enriching the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health microbiology laboratories. In this first step, we present 26 complete high-quality bacterial genomes. Additionally, we describe here a framework for reconstructing complete microbial genomes and highlight some of the challenges and considerations for accurate and reproducible genome reconstruction.

Microbe-Metabolite Associations Linked to the Rebounding Murine Gut Microbiome Postcolonization with Vancomycin-Resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VREfm) is an emerging antibiotic-resistant pathogen. Strain-level investigations are beginning to reveal the molecular mechanisms used by VREfm to colonize regions of the human bowel. However, the role of commensal bacteria during VREfm colonization, in particular following antibiotic treatment, remains largely unknown. We employed amplicon 16S rRNA gene sequencing and metabolomics in a murine model system to try and investigate functional roles of the gut microbiome during VREfm colonization. First-order taxonomic shifts between Bacteroidetes and Tenericutes within the gut microbial community composition were detected both in response to pretreatment using ceftriaxone and to subsequent VREfm challenge. Using neural networking approaches to find cooccurrence profiles of bacteria and metabolites, we detected key metabolome features associated with butyric acid during and after VREfm colonization. These metabolite features were associated with Bacteroides, indicative of a transition toward a preantibiotic naive microbiome. This study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in response to colonization with VREfm. Our results offer insights toward identifying potential nonantibiotic alternatives to eliminate VREfm through metabolic reengineering to preferentially select for Bacteroides IMPORTANCE This study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions between commensal gut bacteria and a specific pathogen. Knowledge from this research will inform gut microbiome engineering strategies, with the aim of translating observations from animal models to human-relevant therapeutic applications.

Structure-Activity Relationships of Daptomycin Lipopeptides.

Daptomycin is a calcium-dependent cyclic lipodepsipeptide derived from the soil saprotroph Streptomyces roseosporus, and its antibiotic properties make it a key agent for treatment of drug-resistant Gram-positive infections. It is most commonly used clinically for the treatment of Gram-positive skin and skin structure infections (SSSI), Staphylococcus aureus bacteremia, and right-sided endocarditis infections associated with S. aureus, including methicillin resistant S. aureus (MRSA). It has also been used "off-label" for Enterococcal infections. There has been a tremendous amount of research investigating its mode of action, resistance mechanisms, and biosynthesis of this clinically important antimicrobial agent. Although we cover the latter aspects in detail, the primary focus of this review is to provide the most comprehensive and up-to-date reference for the medicinal chemist on the structure-activity-toxicity of this important class of lipopeptide antibiotics.

Biphasic Outbreak of Invasive Group A Streptococcus Disease in Eldercare Facility, New Zealand.

A 3-month outbreak of invasive group A Streptococcus disease at an eldercare facility, in which 5 persons died, was biphasic. Although targeted chemoprophylaxis contained the initial outbreak, a second phase of the outbreak occurred after infection control processes ended. To retrospectively investigate the genomic epidemiology of the biphasic outbreak, we used whole-genome sequencing and multiple bioinformatics approaches. Analysis of isolates from the outbreak and isolates prospectively collected during the outbreak response indicated a single S. pyogenes emm81 clone among residents and staff members. Outbreak isolates differed from nonoutbreak emm81 isolates by harboring an integrative conjugative genomic element that contained the macrolide resistance determinant erm(TR). This study shows how retrospective high-resolution genomic investigations identified rapid spread of a closed-facilty clonal outbreak that was controlled, but not readily cleared, by infection control management procedures.

Mining the Methylome Reveals Extensive Diversity in Staphylococcus epidermidis Restriction Modification.

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidishsdMS, we readily overcame restriction barriers in S. epidermidis and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies.IMPORTANCEStaphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.

Genomic Analysis of Fluoroquinolone- and Tetracycline-Resistant Campylobacter jejuni Sequence Type 6964 in Humans and Poultry, New Zealand, 2014-2016.

In 2014, antimicrobial drug-resistant Campylobacter jejuni sequence type 6964 emerged contemporaneously in poultry from 3 supply companies in the North Island of New Zealand and as a major cause of campylobacteriosis in humans in New Zealand. This lineage, not previously identified in New Zealand, was resistant to tetracycline and fluoroquinolones. Genomic analysis revealed divergence into 2 major clades; both clades were associated with human infection, 1 with poultry companies A and B and the other with company C. Accessory genome evolution was associated with a plasmid, phage insertions, and natural transformation. We hypothesize that the tetO gene and a phage were inserted into the chromosome after conjugation, leaving a remnant plasmid that was lost from isolates from company C. The emergence and rapid spread of a resistant clone of C. jejuni in New Zealand, coupled with evolutionary change in the accessory genome, demonstrate the need for ongoing Campylobacter surveillance among poultry and humans.

Unstable chromosome rearrangements in Staphylococcus aureus cause phenotype switching associated with persistent infections.

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type-like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.

Arcobacter cryaerophilus Isolated From New Zealand Mussels Harbor a Putative Virulence Plasmid.

A wide range of Arcobacter species have been described from shellfish in various countries but their presence has not been investigated in Australasia, in which shellfish are a popular delicacy. Since several arcobacters are considered to be emerging pathogens, we undertook a small study to evaluate their presence in several different shellfish, including greenshell mussels, oysters, and abalone (paua) in New Zealand. Arcobacter cryaerophilus, a species associated with human gastroenteritis, was the only species isolated, from greenshell mussels. Whole-genome sequencing revealed a range of genomic traits in these strains that were known or associated virulence factors. Furthermore, we describe the first putative virulence plasmid in Arcobacter, containing lytic, immunoavoidance, adhesion, antibiotic resistance, and gene transfer traits, among others. Complete genome sequence determination using a combination of long- and short-read genome sequencing strategies, was needed to identify the plasmid, clearly identifying its benefits. The potential for plasmids to disseminate virulence traits among Arcobacter and other species warrants further consideration by researchers interested in the risks to public health from these organisms.