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At least 19 records

Omics-Based Comparison of Fungal Virulence Genes, Biosynthetic Gene Clusters, and Small Molecules in Penicillium expansum and Penicillium chrysogenum

Penicillium expansum is a ubiquitous pathogenic fungus that causes blue mold decay of apple fruit postharvest, and another member of the genus, Penicillium chrysogenum, is a well-studied saprophyte valued for antibiotic and small molecule production. While these two fungi have been investigated individually, a recent discovery revealed that P. chrysogenum can block P. expansum-mediated decay of apple fruit. To shed light on this observation, we conducted a comparative genomic, transcriptomic, and metabolomic study of two P. chrysogenum (404 and 413) and two P. expansum (Pe21 and R19) isolates. Global transcriptional and metabolomic outputs were disparate between the species, nearly identical for P. chrysogenum isolates, and different between P. expansum isolates. Further, the two P. chrysogenum genomes revealed secondary metabolite gene clusters that varied widely from P. expansum. This included the absence of an intact patulin gene cluster in P. chrysogenum, which corroborates the metabolomic data regarding its inability to produce patulin. Additionally, a core subset of P. expansum virulence gene homologues were identified in P. chrysogenum and were similarly transcriptionally regulated in vitro. Molecules with varying biological activities, and phytohormone-like compounds were detected for the first time in P. expansum while antibiotics like penicillin G and other biologically active molecules were discovered in P. chrysogenum culture supernatants. Our findings provide a solid omics-based foundation of small molecule production in these two fungal species with implications in postharvest context and expand the current knowledge of the Penicillium-derived chemical repertoire for broader fundamental and practical applications.

Bartholomew, Holly P. (ORCID:0000000292726399)↗

Mining the Penicillium expansum Genome for Virulence Genes: A Functional-Based Approach to Discover Novel Loci Mediating Blue Mold Decay of Apple Fruit

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434 KD ) were generated for three loci. Data show that the Δt-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.

59 BASIC BIOLOGICAL SCIENCES↗

T-Toxin Virulence Genes: Unconnected Dots in a Sea of Repeats

In 1970 a corn disease epidemic ravaged fields in the United States to great economic loss. The outbreak was caused by a never-before seen, supervirulent strain of the fungal pathogen Cochliobolus heterostrophus. This was a plant disease epidemic, however, the current COVID-19 pandemic of humans is a stark reminder that novel, highly virulent, pathogens evolve with devastating consequences, no matter what the host-animal, plant, or other organism.

59 BASIC BIOLOGICAL SCIENCES↗

The success of particular Acinetobacter baumannii clones: accumulating resistance and virulence inside a sugary shield

Abstract Background In Portugal, carbapenem-resistant Acinetobacter baumannii (CRAB) has been associated with ST98, ST103 and ST208 (Oxford Scheme, Oxf) and a clone has usually been associated with a particular period of time. These clonal shifts were primarily explained by an increased antimicrobial resistance profile. Here we explore genomic and biochemical differences among these and more recent clones, which could further explain the diversity and evolution of this species. Methods A total of 116 CRAB isolates (2010–15), together with representatives of a previously described CRAB collection (4 isolates, 2001–06) were characterized by attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR) and MLST. Representatives of different FTIR-ATR/MLST clusters were selected for WGS (n = 13), which allowed the in silico extraction of resistance and virulence genes, capsule locus and SNP analysis. Results A. baumannii clonal shifts of OXA-58-producing ST103Oxf (2001–04), OXA-40-producing ST98Oxf (2002–06), OXA-23-producing ST208Oxf (2006–10) and OXA-23-producing ST218Oxf (2010–15) were accompanied by an increase in AMR genes and virulence factors. FTIR-ATR clustering was congruent with sugar composition predicted from the capsular locus: a fucosamine cluster comprising ST98Oxf, ST103Oxf and a single ST218Oxf isolate; a pseudaminic acid cluster of ST208Oxf and ST1557Oxf isolates; and legionaminic acid, resembling the sialic acid from mammalian cells, in a cluster comprising ST218Oxf isolates. The whole-genome phylogenetic tree was congruent with MLST, with isolates presenting 5–28 938 SNPs. ST208Oxf and ST218Oxf presented ∼1900 SNPs while ST103Oxf and ST1557Oxf showed a greater number of SNPs (∼28 000). Conclusions Clonal shifts of CRAB were promoted, in our country, by consecutive virulence and AMR gene pool enlargement, together with features increasing pathogen–host adaptation. Worldwide dominance of ST218Oxf is supported by the combination of high AMR and virulence levels.

Silva, Liliana↗

Oligodeoxynucleotide Probes for Detecting Intact Cells

A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of target bacteria in samples are usually amplified by overnight pre-enrichment growth. These tests are usually performed in laboratories by skilled technicians. The present test was designed to overcome the shortcomings of the commercial hybridization tests. The figure summarizes the major steps of the test. It is important to emphasize that the hybridization process used in this test differs from that of other hybridization tests in that it does not extract target nucleic acids. This process is based on intact-cell hybridization (so-called in situ hybridization ), wherein the intact cells act as immobilizing agents. The cells are identified and enumerated by measuring the chemiluminescence emitted from alkaline phosphatase-probe (AP-probe) hybridization; the chemiluminescence is detected or measured by use of photographic film or a luminometer, respectively.

Rosson, Reinhardt A.↗

Pseudomonas aeruginosa : One Health approach to deciphering hidden relationships in Northern Portugal

Abstract Aims Antimicrobial resistance in Pseudomonas aeruginosa represents a major global challenge in public and veterinary health, particularly from a One Health perspective. This study aimed to investigate antimicrobial resistance, the presence of virulence genes, and the genetic diversity of P. aeruginosa isolates from diverse sources. Methods and results The study utilized antimicrobial susceptibility testing, genomic analysis for resistance and virulence genes, and multilocus sequence typing to characterize a total of 737 P. aeruginosa isolates that were collected from humans, domestic animals, and aquatic environments in Northern Portugal. Antimicrobial resistance profiles were analyzed, and genomic approaches were employed to detect resistance and virulence genes. The study found a high prevalence of multidrug-resistant isolates, including high-risk clones such as ST244 and ST446, particularly in hospital sources and wastewater treatment plants. Key genes associated with resistance and virulence, including efflux pumps (e.g. MexA and MexB) and secretion systems (T3SS and T6SS), were identified. Conclusions This work highlights the intricate dynamics of multidrug-resistant P. aeruginosa across interconnected ecosystems in Northern Portugal. It underscores the importance of genomic studies in revealing the mechanisms of resistance and virulence, contributing to the broader understanding of resistance dynamics and informing future mitigation strategies.

de Sousa, Telma↗

Deletion of the H108R Gene Reduces Virulence of the Pandemic Eurasia Strain of African Swine Fever Virus with Surviving Animals Being Protected against Virulent Challenge

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a devastating disease affecting domestic and wild swine and currently causing a global pandemic, severely affecting swine production. Here, we demonstrate that the deletion of the previously uncharacterized ASFV gene, H108R from the highly virulent ASFV-Georgia2007 (ASFV-G) genome strain, reduces virulence in domestic swine. ASFV-G-DH108R, a recombinant virus with the H108R gene deleted, was used to evaluate the involvement of the H108R gene for ASFV replication and virulence in swine. ASFV-G-DH108R showed a delayed replication in swine macrophage cultures. A group of five pigs, intramuscularly inoculated with 102 HAD50 of ASFV-G-DH108R, was observed over a 28-day period and compared with a similar group of animals inoculated with similar doses of the parental virulent virus. While all animals inoculated with ASFV-G developed an acute fatal disease, ASFV-G-DH108R inoculated animals, with the exception of one animal showing a protracted but fatal form of the disease, all survived the infection, remaining clinically healthy during the observational period. The surviving animals presented protracted viremias with lower virus titers compared with those of animals inoculated with the parental virus, and all of them developed a strong virus-specific antibody response. Importantly, all animals surviving ASFV-G-DAH108R infection were protected when challenged with the virulent parental strain, ASFV-G. This report constitutes the first evidence that the H108R gene is involved in ASFV virulence in swine and that the deletion of this gene may be used as a tool to increase the attenuation of currently experimental vaccines to improve their safety profiles.

59 BASIC BIOLOGICAL SCIENCES↗

Comparative Genomics Applied to Systematically Assess Pathogenicity Potential in Shiga Toxin-Producing Escherichia coli O145:H28

Shiga toxin-producing Escherichia coli (STEC) O145:H28 can cause severe disease in humans and is a predominant serotype in STEC O145 environmental isolates. Here, comparative genomics was applied to a set of clinical and environmental strains to systematically evaluate the pathogenicity potential in environmental strains. While the core genes-based tree separated all O145:H28 strains from the non O145:H28 reference strains, it failed to segregate environmental strains from the clinical. In contrast, the accessory genes-based tree placed all clinical strains in the same clade regardless of their genotypes or serotypes, apart from the environmental strains. Loss-of-function mutations were common in the virulence genes examined, with a high frequency in genes related to adherence, autotransporters, and the type three secretion system. Distinct differences in pathogenicity islands LEE, OI-122, and OI-57, the acid fitness island, and the tellurite resistance island were detected between the O145:H28 and reference strains. A great amount of genetic variation was detected in O145:H28, which was mainly attributed to deletions, insertions, and gene acquisition at several chromosomal “hot spots”. Finally, our study demonstrated a distinct virulence gene repertoire among the STEC O145:H28 strains originating from the same geographical region and revealed unforeseen contributions of loss-of-function mutations to virulence evolution and genetic diversification in STEC.

59 BASIC BIOLOGICAL SCIENCES↗

CovS inactivation reduces CovR promoter binding at diverse virulence factor encoding genes in group A Streptococcus

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗

Population Genomics of Pseudocercospora griseola Reveals New Groups in the Middle American Clade and the Presence of the Endophytic Bacterium Achromobacter xylosoxidans

Angular leaf spot (ALS), caused by Pseudocercospora griseola is an important disease of common beans. P. griseola, is highly variable and has co-evolved with its host. In this study, 48 isolates of P. griseola from Puerto Rico, Guatemala, Honduras and Tanzania were sequenced (3RADseq), resulting in the de novo assembly of 42,214 contigs. Phylogenomic, population genetic structure and principal component analyses using 1,260 SNPs divided these isolates into two populations, Andean and Middle American, while the Middle American population was further divided into three sub-populations. There were moderate to high levels of differentiation between P. griseola populations, with pairwise Fst values ranging from 0.11 to 0.95. The Andean population was composed of isolates from Tanzania, and was separated from the Middle American population (Fst = 0.95). The Middle American population was separated into 3 subpopulations including isolates from: 1. Guatemala and Honduras, 2. Tanzania, and 3. Puerto Rico. Pathogenicity testing of 27 isolates from Puerto Rico, using 12 common bean differential lines, identified ten races, but these races were not associated with SNPs found in virulence genes. DNA of an endophytic bacterium (Achromobacter xylosoxidans) was found in seven mildly virulent isolates suggesting a possible role of the bacterium in the observed virulence patterns. To understand the evolution and diversity of P. griseola, further study of the virulence genes and the interactions among the endophytic bacterium, the fungus, and the host plant is required. Such information is critical to inform breeding strategies for the development of resistant germplasm and cultivars.

Serrato-Diaz, Luz M. [U.S. Department of Agricultu↗

Genomic surveillance indicates clonal replacement of hypervirulent Klebsiella pneumoniae ST881 and ST29 lineage strains in vivo

The emergence of hypervirulent Klebsiella pneumoniae (hvKp) poses a significant public health threat, particularly regarding its carriage in the healthy population. However, the genomic epidemiological characteristics and population dynamics of hvKp within a single patient across distinct infection episodes remain largely unknown. This study aimed to investigate the clonal replacement of hvKp K2-ST881 and K54-ST29 lineage strains in a single patient experiencing multiple-site infections during two independent episodes. Two strains, designated EDhvKp-1 and EDhvKp-2, were obtained from blood and cerebrospinal fluid during the first admission, and the strain isolated from blood on the second admission was named EDhvKp-3. Whole-genome sequencing, utilizing both short-read Illumina and long-read Oxford Nanopore platforms, was conducted. In silico multilocus sequence typing (MLST), identification of antimicrobial resistance and virulence genes, and the phylogenetic relationship between our strains and other K. pneumoniae ST881 and ST29 genomes retrieved from the public database were performed. Virulence potentials were assessed through a mouse lethality assay. Our study indicated that the strains were highly susceptible to multiple antimicrobial agents. Plasmid sequence analysis confirmed that both virulence plasmids, pEDhvKp-1 (166,008 bp) and pEDhvKp-3 (210,948 bp), belonged to IncFIB type. Multiple virulence genes, including rmpA , rmpA2 , rmpC , rmpD , iroBCDN , iucABCD , and iutA , were identified. EDhvKp-1 and EDhvKp-2 showed the closest relationship to strain 502 (differing by 51 SNPs), while EDhvKp-3 exhibited 69 SNPs differences compared to strain TAKPN-1, which all recovered from Chinese patients in 2020. In the mouse infection experiment, both ST881 EDhvKp-1 and ST29 EDhvKp-3 displayed similar virulence traits, causing 90 and 100% of the mice to die within 72 h after intraperitoneal infection, respectively. Our study expands the spectrum of hvKp lineages and highlights genomic alterations associated with clonal switching between two distinct lineages of hvKP that successively replaced each other in vivo . The development of novel strategies for the surveillance, diagnosis, and treatment of high-risk hvKp is urgently needed.

Liu, Ning↗

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

NASA Discipline Plant Biology↗

Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China

Escherichia coli is considered an opportunistic pathogen and an indicator for antimicrobial resistance (AMR) monitoring. Despite many reports on its AMR monitoring, studies based on genome-based analysis of AMR genes are still insufficient. Here, 181 E. coli strains were isolated from anal swab samples collected from pigs and chickens of animal farms located in Eastern China and sequenced through the Illumina platform. The results showed that 87.85% (159/181) of the E. coli isolates were multidrug-resistant (MDR). Ampicillin (AMP)- spectinomycin (SPT)- tetracycline (TET)- florfenicol (FFC)- sulfisoxazole (SF)- trimethoprim/sulfamethoxazole (SXT) was the predominant AMR pattern. By whole-genome sequencing, we found that ST10 (10.49%, 19/181) and ST48 (7.18%, 13/181) were major sequence types. IncFIB and IncX1 were the most prevalent plasmid replicons. The AMR genes bla NDM-5 (1.10%, 2/181), mcr -1 (1.10%, 2/181), tet (X4) (1.10%, 2/181), and cfr (6.08%, 11/181) were also found in these isolates. In addition, among the 169 virulence genes detected, we identified ast A (37.02%, 67/181), hly A (1.66%, 3/181), hly B (1.66%, 3/181) and hly D (1.66%, 3/181), which were closely related to heat-stable enterotoxin 1 and α-hemolysin. In addition, there were 33 virulence genes associated with the iron uptake system, and 46 were adhesion-related genes. Our study highlighted the need for routine surveillance of AMR with advanced genomic approaches, providing up-to-date data on the prevalence of AMR for the development and execution of antimicrobial stewardship policy.

Zhou, Wei↗

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

79 ASTRONOMY AND ASTROPHYSICS↗

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

59 BASIC BIOLOGICAL SCIENCES↗

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

Ellis, Nicole A. (ORCID:0000000195928415)↗

Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as Stx2j, Stx2m, and Stx2o, in the United States

The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.

59 BASIC BIOLOGICAL SCIENCES↗

Peeling back the many layers of competitive exclusion

Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard ® ) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log 10 Salmonella genomes/gram) was assessed ex vivo using a S. typhimurium reporter strain with fluorescent YFP and CFP gene fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities’ response to Salmonella in chickens with low (≤5.85 Log 10 genomes/g) or high (≥6.00 Log 10 genomes/g) Salmonella colonization. The ex vivo experiment revealed a reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella’s SPI-1 virulence genes and LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis were central to Salmonella metabolism co-cultured with either community, and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth in the exclusive community. There were significant differences in the cecal community’s stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community, suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth through antagonism may be key to exclusion.

59 BASIC BIOLOGICAL SCIENCES↗