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Abstract 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.

Polysaccharides are often the most abundant antigens found on the extracellular surfaces of bacterial cells. These polysaccharides play key roles in interactions with the outside world, and for many bacterial pathogens, they represent what is presented to the human immune system. As a result, many vaccines have been or currently are being developed against carbohydrate antigens. In this review, we explore the diversity of capsular polysaccharides (CPS) in Salmonella and other selected bacterial species and explain the classification and function of CPS as vaccine antigens. Despite many vaccines being developed using carbohydrate antigens, the low immunogenicity and the diversity of infecting strains and serovars present an antigen formulation challenge to manufacturers. Vaccines tend to focus on common serovars or have changing formulations over time, reflecting the trends in human infection, which can be costly and time-consuming. We summarize the approaches to generate carbohydrate-based vaccines for Salmonella, describe vaccines that are in development and emphasize the need for an effective vaccine against non-typhoidal Salmonella strains.

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.

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.

Objectives: Biofilms associated with implantable medical devices and wounds are clinically relevant, often requiring repeated use of antibiotics without success. A search for non-antibiotic antimicrobial and antibiofilm solutions is warranted, in line with antimicrobial stewardship. Our study aimed to evaluate the broad-spectrum antimicrobial efficacy of tetrasodium EDTA, ethanol and chlorhexidine hydrochloride (HCl) alone and in combination against clinically relevant planktonic and biofilm cells of bacterial and fungal pathogens. Methods: MICs and MBCs were determined for tetrasodium EDTA, ethanol and chlorhexidine HCl against planktonic cells of test pathogens. The MBEC Assay® biofilm inoculator device was used to evaluate the biofilm eradication ability of test antimicrobials alone and in combination against clinically relevant pathogens. The checkerboard microbroth dilution assay was performed to analyze the synergism between test antimicrobials. Results: Against planktonic cells, the combination of tetrasodium EDTA with ethanol or chlorhexidine HCl resulted in synergistic to indifferent activity, with no antagonism observed. Against mature biofilms, all combinations were synergistic. The MBEC of each test antimicrobial was decreased from 4- to -64-fold when used in combination as compared to when agents were used alone. We optimised the concentration of antimicrobials to achieve rapid eradication of pre-formed biofilms. A triple combination of 3% tetrasodium EDTA, 20% ethanol and 2.5 μg/mL chlorhexidine HCl completely eradicated 48-h-old biofilms of all test strains within 2 h. Conclusion: All three antimicrobial agents can be used together for prevention and treatment of biofilms and biofilm-related infections. The observed in vitro efficacy should be tested further through in vivo and clinical studies.

While numerous attempts have been made to jointly parse syntax and semantics, high performance in one domain typically comes at the price of performance in the other. This trade-off contradicts the large body of research focusing on the rich interactions at the syntax–semantics interface. We explore multiple model architectures that allow us to exploit the rich syntactic and semantic annotations contained in the Universal Decompositional Semantics (UDS) dataset, jointly parsing Universal Dependencies and UDS to obtain state-of-the-art results in both formalisms. We analyze the behavior of a joint model of syntax and semantics, finding patterns supported by linguistic theory at the syntax–semantics interface. We then investigate to what degree joint modeling generalizes to a multilingual setting, where we find similar trends across 8 languages.

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.

ABSTRACT Curli, a major component of the bacterial biofilms in the intestinal tract, activates pattern recognition receptors and triggers joint inflammation after infection with Salmonella enterica serovar Typhimurium. The factors that allow S. Typhimurium to disperse from biofilms and invade the epithelium to establish a successful infection during acute inflammation remain unknown. Here, we studied S. Typhimurium biofilms in vitro and in vivo to understand how the inflammatory environment regulates the switch between multicellular and motile S. Typhimurium in the gut. We discovered that nitrate generated by the host is an environmental cue that induces S. Typhimurium to disperse from the biofilm. Nitrate represses production of an important biofilm component, curli, and activates flagella via the modulation of intracellular cyclic-di-GMP levels. We conclude that nitrate plays a central role in pathogen fitness by regulating the sessile-to-motile lifestyle switch during infection. IMPORTANCE Recent studies provided important insight into our understanding of the role of c-di-GMP signaling and the regulation of enteric biofilms. Despite an improved understanding of how c-di-GMP signaling regulates S. Typhimurium biofilms, the processes that affect the intracellular c-di-GMP levels and the formation of multicellular communities in vivo during infections remain unknown. Here, we show that nitrate generated in the intestinal lumen during infection with S. Typhimurium is an important regulator of biofilm formation in vivo.

We investigate the relationship between the frequency with which verbs are found in particular subcategorization frames and the acceptability of those verbs in those frames, focusing in particular on subordinate clause-taking verbs, such as think, want, and tell. We show that verbs’ subcategorization frame frequency distributions are poor predictors of their acceptability in those frames—explaining, at best, less than ⅓ of the total information about acceptability across the lexicon—and, further, that common matrix factorization techniques used to model the acquisition of verbs’ acceptability in subcategorization frames fare only marginally better.

ABSTRACT Many Gram-negative bacteria infect hosts and cause diseases by translocating a variety of type III secreted effectors (T3SEs) into the host cell cytoplasm. However, despite a dramatic increase in the number of available whole-genome sequences, it remains challenging for accurate prediction of T3SEs. Traditional prediction models have focused on atypical sequence features buried in the N-terminal peptides of T3SEs, but unfortunately, these models have had high false-positive rates. In this research, we integrated promoter information along with characteristic protein features for signal regions, chaperone-binding domains, and effector domains for T3SE prediction. Machine learning algorithms, including deep learning, were adopted to predict the atypical features mainly buried in signal sequences of T3SEs, followed by development of a voting-based ensemble model integrating the individual prediction results. We assembled this into a unified T3SE prediction pipeline, T3SEpp, which integrated the results of individual modules, resulting in high accuracy (i.e., ∼0.94) and >1-fold reduction in the false-positive rate compared to that of state-of-the-art software tools. The T3SEpp pipeline and sequence features observed here will facilitate the accurate identification of new T3SEs, with numerous benefits for future studies on host-pathogen interactions. IMPORTANCE Type III secreted effector (T3SE) prediction remains a big computational challenge. In practical applications, current software tools often suffer problems of high false-positive rates. One of the causal factors could be the relatively unitary type of biological features used for the design and training of the models. In this research, we made a comprehensive survey on the sequence-based features of T3SEs, including signal sequences, chaperone-binding domains, effector domains, and transcription factor binding promoter sites, and assembled a unified prediction pipeline integrating multi-aspect biological features within homology-based and multiple machine learning models. To our knowledge, we have compiled the most comprehensive biological sequence feature analysis for T3SEs in this research. The T3SEpp pipeline integrating the variety of features and assembling different models showed high accuracy, which should facilitate more accurate identification of T3SEs in new and existing bacterial whole-genome sequences.

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.

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.

ABSTRACT Central venous access devices (CVADs) are an essential component of modern health care. However, their prolonged use commonly results in microbial colonization, which carries the potential risk of hospital-acquired bloodstream infections. These infections complicate the treatment of already sick individuals and cost the existing health care systems around the world millions of dollars. The microbes that colonize CVADs typically form multicellular biofilms that are difficult to dislodge and are resistant to antimicrobial treatments. Clinicians are searching for better ways to extend the working life span of implanted CVADs, by preventing colonization and reducing the risk of bloodstream infections. In this study, we analyzed 210 bacterial and fungal isolates from colonized CVADs or human bloodstream infections from two hospitals geographically separated in the east and west of Canada and screened the isolates for biofilm formation in vitro. Twenty isolates, representing 12 common, biofilm-forming species, were exposed to 4% tetrasodium EDTA, an antimicrobial lock solution that was recently approved in Canada for use as a medical device. The EDTA solution was effective at eradicating surface-attached biofilms from each microbial species, indicating that it could likely be used to prevent biofilm growth within CVADs and to eliminate established biofilms. This new lock solution fits with antibiotic stewardship programs worldwide by sparing the use of important antibiotic agents, targeting prevention rather than the expensive treatment of hospital-acquired infections. IMPORTANCE The colonization of catheters by microorganisms often precludes their long-term use, which can be a problem for human patients that have few body sites available for new catheters. The colonizing organisms often form biofilms, and increasingly these organisms are resistant to multiple antibiotics, making them difficult to treat. In this article, we have taken microorganisms that are associated with biofilm formation in catheters from two Canadian hospitals and tested them with tetrasodium EDTA, a new antimicrobial catheter lock solution. Tetrasodium EDTA was effective at eliminating Gram-positive, Gram-negative, and fungal species and represents a promising alternative to antibiotic treatment with less chance of the organisms developing resistance. We expect that our results will be of interest to researchers and clinicians and will lead to improved patient care.

Non-typhoidal Salmonella are a major cause of gastroenteritis worldwide, as well as causing bloodstream infections in sub-Saharan Africa with a high fatality rate. No vaccine is currently available for human use. Current vaccine development strategies are focused on capsular polysaccharides (CPS) present on the surface of non-typhoidal Salmonella. This study aimed to boost the amount of CPS purified from S. Typhimurium for immunization trials. Random mutagenesis with Tn10 transposon increased the production of CPS colanic acid, by 10-fold compared to wildtype. Immunization with colanic acid or colanic acid conjugated to truncated glycoprotein D or inactivated diphtheria toxin did not induce a protective immune response in mice. However, immunization with Generalized Modules for Membrane Antigens (GMMAs) isolated from colanic acid overproducing isolates reduced Salmonella colonization in mice. Our results support the development of a GMMA-CPS-based vaccine against non-typhoidal Salmonella.

Iron is an essential micronutrient for most bacteria. Salmonella enterica strains, representing human and animal pathogens, have adopted several mechanisms to sequester iron from the environment depending on availability and source. Chickens act as a major reservoir for Salmonella enterica strains which can lead to outbreaks of human salmonellosis. In this review article we summarize the current understanding of the contribution of iron-uptake systems to the virulence of non-typhoidal S. enterica strains in colonizing chickens. We aim to address the gap in knowledge in this field, to help understand and define the interactions between S. enterica and these important hosts, in comparison to mammalian models.

Among human food-borne pathogens, gastroenteritis-causing Salmonella strains have the most real-world impact. Like all pathogens, their success relies on efficient transmission. Biofilm formation, a specialized physiology characterized by multicellular aggregation and persistence, is proposed to play an important role in the Salmonella transmission cycle. In this manuscript, we used luciferase reporters to examine the expression of csgD, which encodes the master biofilm regulator. We observed that the CsgD-regulated biofilm system responds differently to regulatory inputs once it is activated. Notably, the CsgD system became unresponsive to repression by Cpx and H-NS in high osmolarity conditions and less responsive to the addition of amino acids. Temperature-mediated regulation of csgD on agar was altered by intracellular levels of RpoS and cyclic-di-GMP. In contrast, the addition of glucose repressed CsgD biofilms seemingly independent of other signals. Understanding the fine-tuned regulation of csgD can help us to piece together how regulation occurs in natural environments, knowing that all Salmonella strains face strong selection pressures both within and outside their hosts. Ultimately, we can use this information to better control Salmonella and develop strategies to break the transmission cycle.

Salmonella are important pathogens worldwide and a predominant number of human infections are zoonotic in nature. The ability of strains to form biofilms, which is a multicellular behavior characterized by the aggregation of cells, is predicted to be a conserved strategy for increased persistence and survival. It may also contribute to the increasing number of infections caused by ingestion of contaminated fruits and vegetables. There is a correlation between biofilm formation and the ability of strains to colonize and replicate within the intestines of multiple host species. These strains predominantly cause localized gastroenteritis infections in humans. In contrast, there are salmonellae that cause systemic, disseminated infections in a select few host species; these “invasive” strains have a narrowed host range, and most are unable to form biofilms. This includes host-restricted Salmonella serovar Typhi, which are only able to infect humans, and atypical gastroenteritis strains associated with the opportunistic infection of immunocompromised patients. From the perspective of transmission, biofilm formation is advantageous for ensuring pathogen survival in the environment. However, from an infection point of view, biofilm formation may be an anti-virulence trait. We do not know if the capacity to form biofilms prevents a strain from accessing the systemic compartments within the host or if loss of the biofilm phenotype reflects a change in a strain’s interaction with the host. In this review, we examine the connections between biofilm formation, Salmonella disease states, degrees of host adaptation, and how this might relate to different transmission patterns. A better understanding of the dynamic lifecycle of Salmonella will allow us to reduce the burden of livestock and human infections caused by these important pathogens.

Cases of foodborne disease caused by Salmonella are frequently associated with the consumption of minimally processed produce. Bacterial cell surface components are known to be important for the attachment of bacterial pathogens to fresh produce. The role of these extracellular structures in Salmonella attachment to plant cell walls has not been investigated in detail. We investigated the role of flagella, fimbriae and cellulose on the attachment of Salmonella Typhimurium ATCC 14028 and a range of isogenic deletion mutants (ΔfliC fljB, ΔbcsA, ΔcsgA, ΔcsgA bcsA and ΔcsgD) to bacterial cellulose (BC)-based plant cell wall models [BC-Pectin (BCP), BC-Xyloglucan (BCX) and BC-Pectin-Xyloglucan (BCPX)] after growth at different temperatures (28°C and 37°C). We found that all three cell surface components were produced at 28°C but only the flagella was produced at 37°C. Flagella appeared to be most important for attachment (reduction of up to 1.5 log CFU/cm2) although both cellulose and fimbriae also aided in attachment. The csgD deletion mutant, which lacks both cellulose and fimbriae, showed significantly higher attachment as compared to wild type cells at 37°C. This may be due to the increased expression of flagella-related genes which are also indirectly regulated by the csgD gene. Our study suggests that bacterial attachment to plant cell walls is a complex process involving many factors. Although flagella, cellulose and fimbriae all aid in attachment, these structures are not the only mechanism as no strain was completely defective in its attachment.

Salmonella spp. are a leading cause of human infectious disease worldwide and pose a serious health concern. While we have an improving understanding of pathogenesis and the host-pathogen interactions underlying the infection process, comparatively little is known about the survival of pathogenic Salmonella outside their hosts. This review focuses on three areas: (1) in vitro evidence that Salmonella spp. can survive for long periods of time under harsh conditions; (2) observations and conclusions about Salmonella persistence obtained from human outbreaks; and (3) new information revealed by genomic- and population-based studies of Salmonella and related enteric pathogens. We highlight the mechanisms of Salmonella persistence and transmission as an essential part of their lifecycle and a prerequisite for their evolutionary success as human pathogens.

MOTIVATION: Type III Secretion Systems (T3SSs) play important roles in the interaction between gram-negative bacteria and their hosts. T3SSs function by translocating a group of bacterial effector proteins into the host cytoplasm. The details of specific type III secretion process are yet to be clarified. This research focused on comparing the amino acid composition within the N-terminal 100 amino acids from type III secretion (T3S) signal sequences or non-T3S proteins, specifically whether each residue exerts a constraint on residues found in adjacent positions. We used these comparisons to set up a statistic model to quantitatively model and effectively distinguish T3S effectors. RESULTS: In this study, the amino acid composition (Aac) probability profiles conditional on its sequentially preceding position and corresponding amino acids were compared between N-terminal sequences of T3S and non-T3S proteins. The profiles are generally different. A Markov model, namely T3_MM, was consequently designed to calculate the total Aac conditional probability difference, i.e., the likelihood ratio of a sequence being a T3S or a non-T3S protein. With T3_MM, known T3S and non-T3S proteins were found to well approximate two distinct normal distributions. The model could distinguish validated T3S and non-T3S proteins with a 5-fold cross-validation sensitivity of 83.9% at a specificity of 90.3%. T3_MM was also shown to be more robust, accurate, simple, and statistically quantitative, when compared with other T3S protein prediction models. The high effectiveness of T3_MM also indicated the overall Aac difference between N-termini of T3S and non-T3S proteins, and the constraint of Aac exerted by its preceding position and corresponding Aac. AVAILABILITY: An R package for T3_MM is freely downloadable from: http://biocomputer.bio.cuhk.edu.hk/softwares/T3_MM. T3_MM web server: http://biocomputer.bio.cuhk.edu.hk/T3DB/T3_MM.php.

Bacteria can elaborate complex patterns of development that are dictated by temporally ordered patterns of gene expression, typically under the control of a master regulatory pathway. For some processes, such as biofilm development, regulators that initiate the process have been identified but subsequent phenotypic changes such as stress tolerance do not seem to be under the control of these same regulators. A hallmark feature of biofilms is growth within a self-produced extracellular matrix. In this study we used metabolomics to compare Salmonella cells in rdar colony biofilms to isogenic csgD deletion mutants that do not produce an extracellular matrix. The two populations show distinct metabolite profiles. Even though CsgD controls only extracellular matrix production, metabolite signatures associated with cellular adaptations associated with stress tolerances were present in the wild type but not the mutant cells. To further explore these differences we examine the temporal gene expression of genes implicated in biofilm development and stress adaptations. In wild type cells, genes involved in a metabolic shift to gluconeogenesis and various stress-resistance pathways exhibited an ordered expression profile timed with multicellular development even though they are not CsgD regulated. In csgD mutant cells, the ordered expression was lost. We conclude that the induction of these pathways results from production of, and growth within, a self produced matrix rather than elaboration of a defined genetic program. These results predict that common physiological properties of biofilms are induced independently of regulatory pathways that initiate biofilm formation.