Community involvement in addressing the antibiotic crisis.

Increasing student interest and success in STEM education is a top priority for many postsecondary educational institutions. One well-documented approach to both priorities is to have students participate in a Course Undergraduate Research Experience (CURE). Faculty from several technical colleges and universities in Wisconsin teamed up with the Tiny Earth organization to offer a CURE to address the search for new antibiotics. Students enrolled in undergraduate microbiology courses engaged in research and participated in community outreach. To involve the community, faculty from various institutions joined an NFL team, the Green Bay Packers, and created the Tiny Earth in Titletown symposium. Here, students presented their work via scientific posters, to community and industry members, and networked with other scientists from around the region. The Tiny Earth in Titletown symposium started in 2018, was held again in 2019, and returned in 2022 following a 2-year hiatus due to the COVID-19 pandemic. Record attendance in 2022 suggests that community outreach and education may be helping restore trust in science that was lost during the pandemic.

The Type 9 Secretion System Is Required for Flavobacterium johnsoniae Biofilm Formation.

Flavobacterium johnsoniae forms biofilms in low nutrient conditions. Protein secretion and cell motility may have roles in biofilm formation. The F. johnsoniae type IX secretion system (T9SS) is important for both secretion and motility. To determine the roles of each process in biofilm formation, mutants defective in secretion, in motility, or in both processes were tested for their effects on biofilm production using a crystal violet microplate assay. All mutants that lacked both motility and T9SS-mediated secretion failed to produce biofilms. A porV deletion mutant, which was severely defective for secretion, but was competent for motility, also produced negligible biofilm. In contrast, mutants that retained secretion but had defects in gliding formed biofilms. An sprB mutant that is severely but incompletely defective in gliding motility but retains a fully functional T9SS was similar to the wild type in biofilm formation. Mutants with truncations of the gldJ gene that compromise motility but not secretion showed partial reduction in biofilm formation compared to wild type. Unlike the sprB mutant, these gldJ truncation mutants were essentially nonmotile. The results show that a functional T9SS is required for biofilm formation. Gliding motility, while not required for biofilm formation, also appears to contribute to formation of a robust biofilm.

Performance Against WELCOA's Worksite Health Promotion Benchmarks Across Years Among Selected US Organizations.

PURPOSE: The purpose of this study was to quantify the performance of organizations' worksite health promotion (WHP) activities against the benchmarking criteria included in the Well Workplace Checklist (WWC). DESIGN: The Wellness Council of America (WELCOA) developed a tool to assess WHP with its 100-item WWC, which represents WELCOA's 7 performance benchmarks. SETTING: Workplaces. PARTICIPANTS: This study includes a convenience sample of organizations who completed the checklist from 2008 to 2015. The sample size was 4643 entries from US organizations. MEASURES: The WWC includes demographic questions, general questions about WHP programs, and scales to measure the performance against the WELCOA 7 benchmarks. ANALYSIS: Descriptive analyses of WWC items were completed separately for each year of the study period. RESULTS: The majority of the organizations represented each year were multisite, multishift, medium- to large-sized companies mostly in the services industry. Despite yearly changes in participating organizations, results across the WELCOA 7 benchmark scores were consistent year to year. Across all years, benchmarks that organizations performed the lowest were senior-level support, data collection, and programming; wellness teams and supportive environments were the highest scoring benchmarks. CONCLUSION: In an era marked with economic swings and health-care reform, it appears that organizations are staying consistent in their performance across these benchmarks. The WWC could be useful for organizations, practitioners, and researchers in assessing the quality of WHP programs.

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium columnare.

Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes, adhesins, and proteins involved in gliding motility. The F. columnare genome has all of the genes needed to encode a T9SS. gldN, which encodes a core component of the T9SS, was deleted in wild-type strains of F. columnare The F. columnare ΔgldN mutants were deficient in the secretion of several extracellular proteins and lacked gliding motility. The ΔgldN mutants exhibited reduced virulence in zebrafish, channel catfish, and rainbow trout, and complementation restored virulence. PorV is required for the secretion of a subset of proteins targeted to the T9SS. An F. columnare ΔporV mutant retained gliding motility but exhibited reduced virulence. Cell-free spent media from exponentially growing cultures of wild-type and complemented strains caused rapid mortality, but spent media from ΔgldN and ΔporV mutants did not, suggesting that soluble toxins are secreted by the T9SS.IMPORTANCE Columnaris disease, caused by F. columnare, is a major problem for freshwater aquaculture. Little is known regarding the virulence factors produced by F. columnare, and control measures are limited. Analysis of targeted gene deletion mutants revealed the importance of the type IX protein secretion system (T9SS) and of secreted toxins in F. columnare virulence. T9SSs are common in members of the phylum Bacteroidetes and likely contribute to the virulence of other animal and human pathogens.

Novel features of the polysaccharide-digesting gliding bacterium Flavobacterium johnsoniae as revealed by genome sequence analysis.

The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the "motor" and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae.

Development of methods for the genetic manipulation of Flavobacterium columnare.

BACKGROUND: Flavobacterium columnare is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the Bacteroidetes, including members of the genus Flavobacterium, have been described, but these methods were not adapted to work with F. columnare. RESULTS: As a first step toward developing a robust set of genetic tools for F. columnare, a protocol was developed to introduce the E. coli - Flavobacterium shuttle vector pCP29 into F. columnare strain C#2 by conjugal mating at an efficiency of 1.5 x 10(-3) antibiotic-resistant transconjugants per recipient cell. Eight of eleven F. columnare strains tested were able to receive pCP29 using the protocol. pCP29 contains the cfxA and ermF genes, conferring both cefoxitin and erythromycin resistance to recipient cells. Selection for pCP29 introduction into F. columnare was dependent on cfxA, as ermF was found not to provide strong resistance to erythromycin. This is in contrast to other Flavobacterium species where ermF-based erythromycin resistance is strong. The green fluorescent protein gene (gfp) was introduced into F. columnare strains under the control of two different native Flavobacterium promoters, demonstrating the potential of this reporter system for the study of gene expression. The transposon Tn4351 was successfully introduced into F. columnare, but the method was dependent on selecting for erythromycin resistance. To work, low concentrations of antibiotic (1 microg ml(-1)) were used, and high levels of background growth occurred. These results demonstrate that Tn4351 functions in F. columnare but that it is not an effective mutagenesis tool due to its dependence on erythromycin selection. Attempts to generate mutants via homologous recombination met with limited success, suggesting that RecA dependent homologous recombination is rare in F. columnare. CONCLUSION: The conjugation protocol developed as part of this study represents a significant first step towards the development of a robust set of genetic tools for the manipulation of F. columnare. The availability of this protocol will facilitate studies aimed at developing a deeper understanding of the virulence mechanisms of this important pathogen.

Susceptibility of zebra fish Danio rerio to infection by Flavobacterium columnare and F. johnsoniae.

Flavobacterium columnare is a serious pathogen in a wide range of fish species. F. johnsoniae is an opportunistic pathogen of certain fish. Both are gliding bacteria. These species were tested for their ability to infect the zebra fish Danio rerio. Both injection and bath infection methods were tested. The results indicate that F. johnsoniae is not an effective pathogen in D. rerio, but that F. columnare is an effective pathogen. F. johnsoniae did not cause increased death rates following bath infection, but did cause increased death rates following injection, with an LD50 (mean lethal dose) of approximately 3 x 10(10) cfu (colony-forming units). Non-motile mutants of F. johnsoniae produced a similar LD50. F. columnare caused increased death rates following both injection and bath infections. There was considerable strain variation in LD50, with the most lethal strain tested producing an LD50 of 3.2 x 10(6) cfu injected and 1.1 x 10(6) cfu ml(-1) in bath experiments, including skin damage. The LD50 of F. columnare in zebra fish without skin damage was > 1 x 10(8), indicating an important effect of skin damage.

Mutations in Flavobacterium johnsoniae gldF and gldG disrupt gliding motility and interfere with membrane localization of GldA.

Flavobacterium johnsoniae moves rapidly over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Four genes that are required for F. johnsoniae gliding motility, gldA, gldB, gldD, and ftsX, have recently been described. GldA is similar to the ATP-hydrolyzing components of ATP binding cassette (ABC) transporters. Tn4351 mutagenesis was used to identify two additional genes, gldF and gldG, that are required for cell movement. gldF and gldG appear to constitute an operon, and a Tn4351 insertion in gldF was polar on gldG. pMK314, which carries the entire gldFG region, restored motility to each of the gldF and gldG mutants. pMK321, which expresses GldG but not GldF, restored motility to each of the gldG mutants but did not complement the gldF mutant. GldF has six putative membrane-spanning segments and is similar in sequence to channel-forming components of ABC transporters. GldG is similar to putative accessory proteins of ABC transporters. It has two apparent membrane-spanning helices, one near the amino terminus and one near the carboxy terminus, and a large intervening loop that is predicted to reside in the periplasm. GldF and GldG are involved in membrane localization of GldA, suggesting that GldA, GldF, and GldG may interact to form a transporter. F. johnsoniae gldA is not closely linked to gldFG, but the gldA, gldF, and gldG homologs of the distantly related gliding bacterium Cytophaga hutchinsonii are arranged in what appears to be an operon. The exact roles of F. johnsoniae GldA, GldF, and GldG in gliding are not known. Sequence similarities of GldA to components of other ABC transporters suggest that the Gld transporter may be involved in export of some material to the periplasm, outer membrane, or beyond.