Identification of plasmids in avian-associated Escherichia coli using nanopore and illumina sequencing.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) are the causative agents of colibacillosis in chickens, a disease which has significant economic impact on the poultry industry. Large plasmids detected in APEC are known to contribute to strain diversity for pathogenicity and antimicrobial resistance, but there could be other plasmids that are missed in standard analysis. In this study, we determined the impact of sequencing and assembly factors for the detection of plasmids in an E. coli whole genome sequencing project. RESULTS: Hybrid assembly (Illumina and Nanopore) combined with plasmid DNA extractions allowed for detection of the greatest number of plasmids in E. coli, as detected by MOB-suite software. In total, 79 plasmids were identified in 19 E. coli isolates. Hybrid assemblies were robust and consistent in quality regardless of sequencing kit used or if long reads were filtered or not. In contrast, long read only assemblies were more variable and influenced by sequencing and assembly parameters. Plasmid DNA extractions allowed for the detection of physically smaller plasmids, but when averaged over 19 isolates did not significantly change the overall number of plasmids detected. CONCLUSIONS: Hybrid assembly can be reliably used to detect plasmids in E. coli, especially if researchers are focused on large plasmids containing antimicrobial resistance genes and virulence factors. If the goal is comprehensive detection of all plasmids, particularly if smaller sized vectors are desired for biotechnology applications, the addition of plasmid DNA extractions to hybrid assemblies is prudent. Long read sequencing is sufficient to detect many plasmids in E. coli, however, it is more prone to errors when expanded to analyze a large number of isolates.

In vivo synthesis of bacterial amyloid curli contributes to joint inflammation during S. Typhimurium infection.

Reactive arthritis, an autoimmune disorder, occurs following gastrointestinal infection with invasive enteric pathogens, such as Salmonella enterica. Curli, an extracellular, bacterial amyloid with cross beta-sheet structure can trigger inflammatory responses by stimulating pattern recognition receptors. Here we show that S. Typhimurium produces curli amyloids in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. Production of curli was associated with an increase in anti-dsDNA autoantibodies and joint inflammation in infected mice. The negative impacts on the host appeared to be dependent on invasive systemic exposure of curli to immune cells. We hypothesize that in vivo synthesis of curli contributes to known complications of enteric infections and suggest that cross-seeding interactions can occur between pathogen-produced amyloids and amyloidogenic proteins of the host.

Mosaic Evolution of Beta-Barrel-Porin-Encoding Genes in Escherichia coli.

Bacterial porin-encoding genes are often found under positive selection. Local recombination has also been identified in a few of them to facilitate bacterial rapid adaptation, although it remains unknown whether it is a common evolutionary mechanism for the porins or outer membrane proteins in Gram-negative bacteria. In this study, we investigated the beta-barrel (ß-barrel) porin-encoding genes in Escherichia coli that were reported under positive Darwinian selection. Besides fhuA that was found with ingenic local recombination previously, we identified four other genes, i.e., lamB, ompA, ompC, and ompF, all showing the similar mosaic evolution patterns. Comparative analysis of the protein sequences disclosed a list of highly variable regions in each family, which are mostly located in the convex of extracellular loops and coinciding with the binding sites of bacteriophages. For each of the porin families, mosaic recombination leads to unique combinations of the variable regions with different sequence patterns, generating diverse protein groups. Structural modeling indicated a conserved global topology among the different porins, with the extracellular surface varying a lot due to individual or combinatorial variable regions. The conservation of global tertiary structure would ensure the channel activity, while the wide diversity of variable regions may represent selection to avoid the invasion of phages, antibiotics or immune surveillance factors. Our study identified multiple bacterial porin genes with mosaic evolution. We hypothesize that this could be generalized strategy for outer membrane proteins to both maintain normal life processes and evade the attack of unfavored factors rapidly. IMPORTANCE Microevolution studies can disclose more elaborate evolutionary mechanisms of genes, appearing especially important for genes with multifaceted function such as those encoding outer membrane proteins. However, in most cases, the gene is considered as a whole unit, and the evolutionary patterns are disclosed. Here, we report that multiple bacterial porin proteins follow mosaic evolution, with local ingenic recombination combined with spontaneous mutations based on positive Darwinian selection, and conservation for most structural regions. This could represent a common mechanism for bacterial outer membrane proteins. The variable regions within each porin family showed large coincidence with the binding sites of bacteriophages, antibiotics, and immune factors and therefore would represent effective targets for the development of new antibacterial agents or vaccines.

Parallel evolution leading to impaired biofilm formation in invasive Salmonella strains.

Pathogenic Salmonella strains that cause gastroenteritis are able to colonize and replicate within the intestines of multiple host species. In general, these strains have retained an ability to form the rdar morphotype, a resistant biofilm physiology hypothesized to be important for Salmonella transmission. In contrast, Salmonella strains that are host-adapted or even host-restricted like Salmonella enterica serovar Typhi, tend to cause systemic infections and have lost the ability to form the rdar morphotype. Here, we investigated the rdar morphotype and CsgD-regulated biofilm formation in two non-typhoidal Salmonella (NTS) strains that caused invasive disease in Malawian children, S. Typhimurium D23580 and S. Enteritidis D7795, and compared them to a panel of NTS strains associated with gastroenteritis, as well as S. Typhi strains. Sequence comparisons combined with luciferase reporter technology identified key SNPs in the promoter region of csgD that either shut off biofilm formation completely (D7795) or reduced transcription of this key biofilm regulator (D23580). Phylogenetic analysis showed that these SNPs are conserved throughout the African clades of invasive isolates, dating as far back as 80 years ago. S. Typhi isolates were negative for the rdar morphotype due to truncation of eight amino acids from the C-terminus of CsgD. We present new evidence in support of parallel evolution between lineages of nontyphoidal Salmonella associated with invasive disease in Africa and the archetypal host-restricted invasive serovar; S. Typhi. We hypothesize that the African invasive isolates are becoming human-adapted and 'niche specialized' with less reliance on environmental survival, as compared to gastroenteritis-causing isolates.

A SNP in the Cache 1 Signaling Domain of Diguanylate Cyclase STM1987 Leads to Increased In Vivo Fitness of Invasive Salmonella Strains.

Nontyphoidal Salmonella (NTS) strains are associated with gastroenteritis worldwide but are also the leading cause of bacterial bloodstream infections in sub-Saharan Africa. The invasive NTS (iNTS) strains that cause bloodstream infections differ from standard gastroenteritis-causing strains by >700 single-nucleotide polymorphisms (SNPs). These SNPs are known to alter metabolic pathways and biofilm formation and to contribute to serum resistance and are thought to signify iNTS strains becoming human adapted, similar to typhoid fever-causing Salmonella strains. Identifying SNPs that contribute to invasion or increased virulence has been more elusive. In this study, we identified a SNP in the cache 1 signaling domain of diguanylate cyclase STM1987 in the invasive Salmonella enterica serovar Typhimurium type strain D23580. This SNP was conserved in 118 other iNTS strains analyzed and was comparatively absent in global S Typhimurium isolates associated with gastroenteritis. STM1987 catalyzes the formation of bis-(3',5')-cyclic dimeric GMP (c-di-GMP) and is proposed to stimulate production of cellulose independent of the master biofilm regulator CsgD. We show that the amino acid change in STM1987 leads to a 10-fold drop in cellulose production and increased fitness in a mouse model of acute infection. Reduced cellulose production due to the SNP led to enhanced survival in both murine and human macrophage cell lines. In contrast, loss of CsgD-dependent cellulose production did not lead to any measurable change in in vivo fitness. We hypothesize that the SNP in stm1987 represents a pathoadaptive mutation for iNTS strains.

Evolution and Sequence Diversity of FhuA in Salmonella and Escherichia.

The fhuACDB operon, present in a number of Enterobacteriaceae, encodes components essential for the uptake of ferric hydroxamate type siderophores. FhuA acts not only as a transporter for physiologically important chelated ferric iron but also as a receptor for various bacteriophages, toxins, and antibiotics, which are pathogenic to bacterial cells. In this research, fhuA gene distribution and sequence diversity were investigated in Enterobacteriaceae, especially Salmonella and Escherichia Comparative sequence analysis resulted in a fhuA phylogenetic tree that did not match the expected phylogeny of species or trees of the fhuCDB genes. The fhuA sequences showed a unique mosaic clustering pattern. On the other hand, the gene sequences showed high conservation for strains from the same serovar or serotype. In total, six clusters were identified from FhuA proteins in Salmonella and Escherichia, among which typical peptide fragment variations could be defined. Six fragmental insertions/deletions and two substitution fragments were discovered, for which the combination of polymorphism patterns could well classify the different clusters. Structural modeling demonstrated that all the six featured insertions/deletions and one substitution fragment are located at the apexes of the long loops present as part of the FhuA external pocket. These frequently mutated regions are likely under high selection pressure, with bacterial strains balancing escape from phage infection or toxin/antibiotics attack via fhuA gene mutations while maintaining the siderophore uptake activity essential for bacterial survival. The unusual fhuA clustering suggests that high-frequency exchange of fhuA genes has occurred between enterobacterial strains after distinctive species were established.

A global survey of bacterial type III secretion systems and their effectors.

The type III secretion system (T3SS) is an important genetic determinant that mediates interactions between Gram-negative bacteria and their eukaryotic hosts. Our understanding of the T3SS continues to expand, yet the availability of new bacterial genomes prompts questions about its diversity, distribution and evolution. Through a comprehensive survey of ∼20 000 bacterial genomes, we identified 174 non-redundant T3SSs from 109 genera and 5 phyla. Many of the bacteria are environmental strains that have not been reported to interact with eukaryotic hosts, while several species groups carry multiple T3SSs. Four ultra-conserved Microsynteny Blocks (MSBs) were defined within the T3SSs, facilitating comprehensive clustering of the T3SSs into 13 major categories, and establishing the largest diversity of T3SSs to date. We subsequently extended our search to identify type III effectors, resulting in 8740 candidate effectors. Lastly, an analysis of the key transcriptional regulators and circuits for the T3SS families revealed that low-level T3SS regulators were more conserved than higher-level regulators. This comprehensive analysis of the T3SSs and their protein effectors provides new insight into the diversity of systems used to facilitate host-bacterial interactions.

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins.

Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia.

A Modular, Tn7-Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains.

UNLABELLED: Our goal was to develop a robust tagging method that can be used to track bacterial strains in vivo To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonella and Escherichia coli and Tn7 transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ(70)-dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7 system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10(-8) to 10(-10) in Salmonella enterica serovar Typhimurium and E. coli APEC O1, whereas the existing Tn7 system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivo animal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics. IMPORTANCE: This article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system in Salmonella and E. coli, and we predict that it will be widely applicable to other bacterial strains within the Enterobacteriaceae This technology will allow for improved in vivo analysis of bacterial pathogens.

Bistable expression of CsgD in Salmonella enterica serovar Typhimurium connects virulence to persistence.

Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment.

An empirical strategy to detect bacterial transcript structure from directional RNA-seq transcriptome data.

BACKGROUND: As sequencing costs are being lowered continuously, RNA-seq has gradually been adopted as the first choice for comparative transcriptome studies with bacteria. Unlike microarrays, RNA-seq can directly detect cDNA derived from mRNA transcripts at a single nucleotide resolution. Not only does this allow researchers to determine the absolute expression level of genes, but it also conveys information about transcript structure. Few automatic software tools have yet been established to investigate large-scale RNA-seq data for bacterial transcript structure analysis. RESULTS: In this study, 54 directional RNA-seq libraries from Salmonella serovar Typhimurium (S. Typhimurium) 14028s were examined for potential relationships between read mapping patterns and transcript structure. We developed an empirical method, combined with statistical tests, to automatically detect key transcript features, including transcriptional start sites (TSSs), transcriptional termination sites (TTSs) and operon organization. Using our method, we obtained 2,764 TSSs and 1,467 TTSs for 1331 and 844 different genes, respectively. Identification of TSSs facilitated further discrimination of 215 putative sigma 38 regulons and 863 potential sigma 70 regulons. Combining the TSSs and TTSs with intergenic distance and co-expression information, we comprehensively annotated the operon organization in S. Typhimurium 14028s. CONCLUSIONS: Our results show that directional RNA-seq can be used to detect transcriptional borders at an acceptable resolution of ±10-20 nucleotides. Technical limitations of the RNA-seq procedure may prevent single nucleotide resolution. The automatic transcript border detection methods, statistical models and operon organization pipeline that we have described could be widely applied to RNA-seq studies in other bacteria. Furthermore, the TSSs, TTSs, operons, promoters and unstranslated regions that we have defined for S. Typhimurium 14028s may constitute valuable resources that can be used for comparative analyses with other Salmonella serotypes.

Reverse vaccinology as an approach for developing Histophilus somni vaccine candidates.

Histophilosis of cattle is caused by the Gram negative bacterial pathogen Histophilus somni (H. somni) which is also associated with the bovine respiratory disease (BRD) complex. Existing vaccines for H. somni include either killed cells or bacteria-free outer membrane proteins from the organism which have proven to be moderately successful. In this study, reverse vaccinology was used to predict potential H. somni vaccine candidates from genome sequences. In turn, these may protect animals against new strains circulating in the field. Whole genome sequencing of six recent clinical H. somni isolates was performed using an Illumina MiSeq and compared to six genomes from the 1980's. De novo assembly of crude whole genomes was completed using Geneious 6.1.7. Protein coding regions was predicted using Glimmer3. Scores from multiple web-based programs were utilized to evaluate the antigenicity of these predicted proteins which were finally ranked based on their surface exposure scores. A single new strain was selected for future vaccine development based on conservation of the protein candidates among all 12 isolates. A positive signal with convalescent serum for these antigens in western blots indicates in vivo recognition. In order to test the protective capacity of these antigens bovine animal trials are ongoing.

Methods for Genomic Epidemiology of Bacterial Pathogens: Example Salmonella.

Genomics has revolutionized how we characterize and monitor infectious diseases for public health. The surveillance and characterization of Salmonella has improved drastically within the past decade. In this chapter, we discuss the prerequisites for good bacterial genomics studies and make note of advantages and disadvantages of this research approach. We discuss methods for outbreak detection and the evolutionary and epidemiological characterization of Salmonella spp. We provide an outline for determining the sequence type and serotype of isolates, building a core genome phylogenetic tree, and detecting antimicrobial resistance genes, virulence factors, and mobile genetic elements. These methods can be used to study other pathogenic bacterial species.

Features and algorithms: facilitating investigation of secreted effectors in Gram-negative bacteria.

Gram-negative bacteria deliver effector proteins through type III, IV, or VI secretion systems (T3SSs, T4SSs, and T6SSs) into host cells, causing infections and diseases. In general, effector proteins for each of these distinct secretion systems lack homology and are difficult to identify. Sequence analysis has disclosed many common features, helping us to understand the evolution, function, and secretion mechanisms of the effectors. In combination with various algorithms, the known common features have facilitated accurate prediction of new effectors. Ensemblers or integrated pipelines achieve a better prediction of performance, which combines multiple computational models or modules with multidimensional features. Natural language processing (NLP) models also show the merits, which could enable discovery of novel features and, in turn, facilitate more precise effector prediction, extending our knowledge about each secretion mechanism.

Hybrid Genome Assemblies of 245 Avian and Broiler Barn Environment-Associated Escherichia coli Strains Isolated from Saskatchewan Broiler Farms.

Escherichia coli infections in poultry cause significant morbidity and economic losses for producers each year. In a 3-year period, we collected and sequenced the whole genomes of E. coli disease isolates (n = 91), isolates from presumed healthy birds (n = 61), and isolates from 8 barn sites (n = 93) on broiler farms in Saskatchewan.

A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2-Bispecific Antibody for Combination Cancer Therapy.

PURPOSE: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. EXPERIMENTAL DESIGN: Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. RESULTS: Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab. CONCLUSIONS: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment. See related commentary by Kumar, p. 2570.

Purification of the Bacterial Amyloid "Curli" from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues.

Microbiologists are learning to appreciate the importance of "functional amyloids" that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell communication. Bacterial functional amyloids are also beginning to be appreciated in the context of host-pathogen interactions, where there is evidence that they can trigger the innate immune system and are recognized as non-self-molecular patterns. The characteristic three-dimensional fold of amyloids renders them similar across the bacterial kingdom and into the eukaryotic world, where amyloid proteins can be undesirable and have pathological consequences. The bacterial protein curli, produced by pathogenic Salmonella enterica and Escherichia coli strains, was one of the first functional amyloids discovered. Curli have since been well characterized in terms of function, and we are just starting to scratch the surface about their potential impact on eukaryotic hosts. In this manuscript, we present step-by-step protocols with pictures showing how to purify these bacterial surface structures. We have described the purification process from S. enterica, acknowledging that the same method can be applied to E. coli. In addition, we describe methods for detection of curli within animal tissues (i.e., GI tract) and discuss purifying curli intermediates in a S. enterica msbB mutant strain as they are more cytotoxic than mature curli fibrils. Some of these methods were first described elsewhere, but we wanted to assemble them together in more detail to make it easier for researchers who want to purify curli for use in biological experiments. Our aim is to provide methods that are useful for specialists and non-specialists as bacterial amyloids become of increasing importance.

High Affinity Iron Acquisition Systems Facilitate but Are Not Essential for Colonization of Chickens by Salmonella Enteritidis.

The roles of TonB mediated Fe3+ (ferric iron) uptake via enterobactin (involving biosynthesis genes entABCDEF) and Fe2+ (ferrous iron) uptake through the FeoABC transporter are poorly defined in the context of chicken-Salmonella interactions. Both uptake systems are believed to be the major contributors of iron supply in the Salmonella life cycle. Current evidence suggests that these iron uptake systems play a major role in pathogenesis in mammals and as such, they represent promising antibacterial targets with therapeutic potential. We investigated the role of these iron uptake mechanisms regarding the ability of Salmonella Enteritidis (SEn) strains to colonize in a chicken infection model. Further we constructed a bioluminescent reporter to sense iron limitation during gastrointestinal colonization of Salmonella in chicken via ex vivo imaging. Our data indicated that there is some redundancy between the ferric and ferrous iron uptake mechanisms regarding iron acquisition during SEn pathogenesis in chicken. We believe that this redundancy of iron acquisition in the host reservoir may be the consequence of adaptation to unique avian environments, and thus warrants further investigation. To our knowledge, this the first report providing direct evidence that both enterobactin synthesis and FeoABC mediated iron uptake contribute to the virulence of SEn in chickens.

Characterization of colonization kinetics and virulence potential of Salmonella Enteritidis in chickens by photonic detection.

The light emitting module lux operon (luxCDABE) of Photorhabdus luminescens can be integrated into a "dark" bacterium for expression under a suitable promoter. The technique has been used to monitor kinetics of infection, e.g., by studying gene expression in Salmonella using mouse models in vivo and ex vivo. Here, we applied the bioluminescence imaging (BLI) technique to track Salmonella Enteritidis (SEn) strains carrying the lux operon expressed under a constitutive promoter sequence (sigma 70) in chicken after oral challenge. Detectable photon signals were localized in the crop, small intestine, cecum, and yolk sac in orally gavaged birds. The level of colonization was determined by quantification of signal intensity and SEn prevalence in the cecum and yolk sac. Furthermore, an isogenic SEn mutant strain tagged with the lux operon allowed for us to assess virulence determinants regarding their role in colonization of the cecum and yolk sac. Interestingly, mutations of SPI-1(Salmonella Pathogenicity Island 1) and fur (ferric uptake regulator) showed significantly decreased colonization in yolk sac that was correlated with the BLI data. A similar trend was detected in a ΔtonB strain by analyzing enrichment culture data. The inherently low quantum yield, light scattering, and absorption by tissues did not facilitate detection of signals from live birds. However, the detection limit of lux operon has the potential to be improved by resonance energy transfer to a secondary molecule. As a proof-of-concept, we were able to show that sensitization of a fluorescent-bound molecule known as the lumazine protein (LumP) improved the limit of detection to a certain extent.

Biofilm Formation and Antimicrobial Susceptibility of E. coli Associated With Colibacillosis Outbreaks in Broiler Chickens From Saskatchewan.

The global poultry industry has grown to the extent that the number of chickens now well exceeds the number of humans on Earth. Escherichia coli infections in poultry cause significant morbidity and economic losses for producers each year. We obtained 94 E. coli isolates from 12 colibacillosis outbreaks on Saskatchewan farms and screened them for antimicrobial resistance and biofilm formation. Fifty-six isolates were from broilers with confirmed colibacillosis, and 38 isolates were from healthy broilers in the same flocks (cecal E. coli). Resistance to penicillins, tetracyclines, and aminoglycosides was common in isolates from all 12 outbreaks, while cephalosporin resistance varied by outbreak. Most E. coli were able to form biofilms in at least one of three growth media (1/2 TSB, M63, and BHI broth). There was an overall trend that disease-causing E. coli had more antibiotic resistance and were more likely to form biofilms in nutrient-rich media (BHI) as compared to cecal strains. However, on an individual strain basis, there was no correlation between antimicrobial resistance and biofilm formation. The 21 strongest biofilm forming strains consisted of both disease-causing and cecal isolates that were either drug resistant or susceptible. Draft whole genome sequencing indicated that many known antimicrobial resistance genes were present on plasmids, with disease-causing E. coli having more plasmids on average than their cecal counterparts. We tested four common disinfectants for their ability to kill 12 of the best biofilm forming strains. All disinfectants killed single cells effectively, but biofilm cells were more resistant, although the difference was less pronounced for the disinfectants that have multiple modes of action. Our results indicate that there is significant diversity and complexity in E. coli poultry isolates, with different lifestyle pressures affecting disease-causing and cecal isolates.