Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30 s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

Assessing dual use research of concern (DURC)-lessons learned from the United States government institutional DURC policy.

Life science research was analyzed for potential misuse in the 2004 report "Biotechnology Research in an Age of Bioterrorism". However, it was not until 2015 that the United States Government (USG) Institutional Dual Use Research of Concern (DURC) policy went into effect. Institutions receiving USG funding for life science research are required to scan their research portfolios for research involving one of 15 agents and subsequent 7 experimental effects described in the policy. In practice, this policy was implemented in a variety of ways with varying outcomes and lessons learned. First and foremost, reviewing research for potential DURC is a highly subjective process that differs depending on the risk tolerance, experience, and training of the individuals charged with reviewing research for an institution as well as the review process itself. The information being reviewed also lends to the subjectivity of the process, that is, the experimental data provided. It is difficult to determine whether research is potential DURC without experimental data. Any review process is hypothetical until there is data. Lastly, reviewers of the research should look beyond the research proposals, like how compounding existing research information can create new risks, potential use in other organisms or systems, or the creation of a roadmap that, for example, shows how to create a concerning organism or could be used in a pathogen.

Proceedings of the Dual Use Research of Concern Panel Discussion: challenges and perspectives.

To address real and perceived emerging risks originating from the ever-accelerating breakthroughs in life science research, the Dual Use Research of Concern (DURC) Panel Discussion, organized by Synbio Canada and the Alberta RNA Research and Training Institute (ARRTI), took place on June 23rd, 2021. It brought together six stakeholders from different levels of academic research, administration, governance, and science publishing to explore the current and future challenges in addressing DURC. Technological advancements within the life sciences, especially within the field of omics technology, make it difficult to apply a simple checklist for dual-use assessment and require continuous and integrated effort. Bottom-up approaches from within the scientific community are suggested by all stakeholders to enable efficient governance and address the true risks resulting from DURC, not just the alleged risks. To address such alleged risks, open and broadscale communication of DURC and its oversight policies may be required. At the same time, any form of open communication also contains the risk of information hazards, defined as potentially creating public fear or informing malicious actors. Here, an overview of the DURC panel and its outcomes is provided.

Demographic and Salary Trends of the 2020 Biosafety Workforce.

Introduction: This study attempts to understand the demographics and salaries of the biosafety workforce worldwide. It builds upon previous surveys of biosafety professionals. Methods: Using multiple regression, this study explored what factors significantly predict salary. Moreover, this study examined whether significant differences existed regarding salary. These differences were analyzed in isolation (i.e., the variable itself) and while controlling for the variables that predicted salary. Results: In this article, eight factors significantly predicted salary: right-to-work state first, biosafety certifications, place of employment, data entry responsibilities, percentage of biosafety job responsibilities, number of direct reports, level of education, and finally the cumulative years of experience in the field. Discussion: This study highlighted certain trends that have remained consistent and new trends that have emerged over time. This research had increased international participation as compared with previous studies.

Rotavirus vaccines and health care utilization for diarrhea in the United States (2007-2011).

OBJECTIVES: To examine reductions in diarrhea-associated health care utilization after rotavirus vaccine implementation and to assess direct and indirect effectiveness of vaccination. METHODS: Retrospective cohort analysis of claims data of commercially insured US children aged <5 years. We examined annual pentavalent (RV5) and monovalent (RV1) rotavirus vaccine coverage. We compared rates of diarrhea-associated health care utilization in prevaccine (2001-2006) versus postvaccine introduction (2007-2011) years, compared rates of diarrhea-associated health care utilization in vaccinated versus unvaccinated children and compared rates in unvaccinated children in postvaccine versus prevaccine years. RESULTS: Among children aged <5 years, RV5 and RV1 rotavirus vaccine coverage rates reached 58% and 5%, respectively, by December 31, 2010. Compared with the average rate of rotavirus-coded hospitalizations in 2001-2006, rates were reduced by 75% in 2007-2008, 60% in 2008-2009, 94% in 2009-2010, and 80% in 2010-2011. Compared with unvaccinated children, in 2010-2011, the rate of rotavirus-coded hospitalizations was reduced by 92% among RV5 recipients and 96% among RV1 recipients. Rotavirus-coded hospitalization rate reductions among RV5 recipients versus unvaccinated children ranged from 87% among <1-year-olds to 81% among 4-year-olds. Compared with prevaccine rates in 2001-2006, rotavirus-coded hospitalization rates among unvaccinated children decreased by 50% in 2007-2008, 77% in 2009-2010, and 25% in 2010-2011. CONCLUSIONS: Implementation of rotavirus vaccines has substantially reduced diarrhea health care utilization in US children. Both rotavirus vaccines conferred high protection against rotavirus hospitalizations; RV5 conferred durable protection through the fourth year of life. Vaccination also conferred indirect benefits to unvaccinated children.

The Escherichia coli argW-dsdCXA genetic island is highly variable, and E. coli K1 strains commonly possess two copies of dsdCXA.

The genome sequences of Escherichia coli pathotypes reveal extensive genetic variability in the argW-dsdCXA island. Interestingly, the archetype E. coli K1 neonatal meningitis strain, strain RS218, has two copies of the dsdCXA genes for d-serine utilization at the argW and leuX islands. Because the human brain contains d-serine, an epidemiological study emphasizing K1 isolates surveyed the dsdCXA copy number and function. Forty of 41 (97.5%) independent E. coli K1 isolates could utilize d-serine. Southern blot hybridization revealed physical variability within the argW-dsdC region, even among 22 E. coli O18:K1:H7 isolates. In addition, 30 of 41 K1 strains, including 21 of 22 O18:K1:H7 isolates, had two dsdCXA loci. Mutational analysis indicated that each of the dsdA genes is functional in a rifampin-resistant mutant of RS218, mutant E44. The high percentage of K1 strains that can use d-serine is in striking contrast to our previous observation that only 4 of 74 (5%) isolates in the diarrheagenic E. coli (DEC) collection have this activity. The genome sequence of diarrheagenic E. coli isolates indicates that the csrRAKB genes for sucrose utilization are often substituted for dsdC and a portion of dsdX present at the argW-dsdCXA island of extraintestinal isolates. Among DEC isolates there is a reciprocal pattern of sucrose fermentation versus d-serine utilization. The ability to use d-serine is a trait strongly selected for among E. coli K1 strains, which have the ability to infect a wide range of extraintestinal sites. Conversely, diarrheagenic E. coli pathotypes appear to have substituted sucrose for d-serine as a potential nutrient.

Regulation of type 1 fimbriae by unlinked FimB- and FimE-like recombinases in uropathogenic Escherichia coli strain CFT073.

Genomic DNA sequence analysis of the uropathogenic Escherichia coli strain CFT073 revealed that besides the fimB and fimE recombinase genes that control the type 1 pilus fim phase switch, there are three additional fimB- and fimE-like genes: ipuA, ipuB, and ipbA. Alignment of the predicted amino acid sequences showed that the five recombinases range in sequence similarity from 63 to 70%. An epidemiological survey indicates that ipuA and ipuB are present and linked next to the dsdCXA locus in 24 of 67 uropathogenic E. coli strains but are found in only 1 of 15 normal human fecal isolates. The ipbA sequence located next to the betABIT locus was found in 42 of 67 uropathogenic isolates and 8 of 15 of the commensal strains. We show that two of these recombinases, those encoded by ipuA and ipbA, can function at the type 1 pilus fim switch. In a CFT073 deletion mutant lacking all five recombinase genes, recombinant ipuA or ipbA provided in trans inverted the fim element from the off state to the on state. When a fim OFF CFT073 DeltafimBE mutant was used to infect the urinary tracts of mice, a switch to the fim on state was detected within 24 h in bacteria recovered from urine, the bladder, and the kidneys. A fim OFF CFT073 DeltafimBE ipuB ipbA mutant also demonstrated the ability to switch from the fim off state to the on state during mouse infection. CFT073 recombinase mutants derived from isolates in either the fim on or off state showed a reciprocal relationship for motility. Switches from a nonmotile to a motile phenotype and from a fim on to off genotype were observed in fim ON CFT073 DeltafimBE ipuAB ipbA mutants when ipuA or fimB was provided in trans. Together these results indicate that ipuA has fimB-like on-to-off and off-to-on fim switching activity and that ipbA has the ability to switch fim from the off to the on orientation.

Uropathogenic Escherichia coli use d-serine deaminase to modulate infection of the murine urinary tract.

Although once thought to be unique to bacteria, d-amino acids are also produced by mammals. For example, d-serine is excreted in human urine at concentrations ranging from 3.0 to 40 micro g ml-1. An epidemiological survey demonstrated that urine isolates of E. coli are more likely to catabolise d-serine via expression of d-serine deaminase, DsdA than enteric disease isolates. The urosepsis strain, CFT073, and an isogenic dsdA mutant have similar growth kinetics in minimal or complex media. However, relative to the wild type, the dsdA mutant has a pleiomorphic cell shape and a prolonged, 4-6 h lag phase when grown in human urine. This suggests that d-serine catabolism provides a growth advantage in the urinary tract. Unexpectedly, in a direct competition model of urinary tract infection, the dsdA mutant was recovered 300-times more frequently than the wild type in the bladders of mice 48 h after infection. A new model of E. coli uropathogenesis is proposed where growth and gene expression are modulated in response to environmental d-serine levels. In support of this, the CFT073 dsdA mutant is hyperflagellated and more motile than the wild type indicating that intracellular levels of d-serine may directly or indirectly influence the expression of regulons associated with E. coli uropathogenesis.