Rapid identification of pathogens by using surface-enhanced Raman spectroscopy and multi-scale convolutional neural network.

Salmonella is a prevalent pathogen causing serious morbidity and mortality worldwide. There are over 2600 serovars of Salmonella. Among them, Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Paratyphi were reported to be the most common foodborne pathogenic serovars in the EU and China. In order to provide a more efficient approach to detect and distinguish these serovars, a new analytical method was developed by combining surface-enhanced Raman spectroscopy (SERS) with multi-scale convolutional neural network (CNN). We prepared 34-nm gold nanoparticles (AuNPs) as the label-free Raman substrate, measured 1854 SERS spectra of these three Salmonella serovars, and then proposed a multi-scale CNN model with three parallel CNNs to achieve multi-dimensional extraction of SERS spectral features. We observed the impact of the number of iterations and training samples on the recognition accuracy by changing the ratio of the number of the training and testing sets. By comparing the calculated data with experimental one, it was shown that our model could reach recognition accuracy more than 97%. These results indicate that it was not only feasible to combine SERS spectroscopy with multi-scale CNN for Salmonella serotype identification, but also for other pathogen species and serovar identifications.

Biomarkers of oxidative damage in bacteria for the assessment of sanitation efficacy in lettuce wash water.

In the fresh produce industry, validation of sanitation efficacy is critical to prevent cross-contamination of produce. The current validation approaches are either based on time-consuming plate counting assays or indirect measurements of chemical properties of wash water. In the study, the focus was to identify biomarkers that can provide direct assessment of oxidative damage in bacteria upon exposure to sanitizers in the presence of fresh produce and correlation of these oxidative biomarkers with logarithmic inactivation of bacteria. Two endogenous bacterial biomarkers, protein carbonylation and thiol oxidation, were evaluated for assessing oxidative damage in Escherichia coli O157:H7 and Listeria innocua during sanitation of pre-cut lettuce leaves with NaOCl or H2O2. Results show that NaOCl treatment was more effective than H2O2 for oxidation of both the intracellular thiols and protein carbonylation in the selected strains. Statistical analysis of the measurements illustrates that oxidation of the intracellular thiol induced by NaOCl or H2O2 was correlated with logarithmic reduction of E. coli O157:H7 and L. innocua. In contrast, changes in the protein carbonylation content were not correlated with reduction in bacterial cell viability. In summary, these results provide a novel approach to validate sanitation efficacy for the fresh produce industry.

Food Evolution: The Impact of Society and Science on the Fermentation of Cocoa Beans.

Cocoa is part of the cultural heritage in many areas of South and Central America and has played an important role in the history of human culture there. The modern methods of cocoa bean production for the purpose of the manufacture of modern chocolate are tied to the origin and development of cocoa bean fermentation and processing methods and the science of microbiology. To date, however, there has not been a study that discusses the impacts of both science and culture on the evolution of cocoa beans and cocoa bean processing. This work provides both a detailed overview of the evolution and historical development of cocoa, from its earliest forms to modern chocolate manufacturing, an in-depth discussion of the biochemistry of cocoa bean fermentation, as well as a compilation of primary research studies with details on fermentation methods, the scientific bases of interactions in microbial fermentations, and methods for their investigation, as well as metabolites that are produced. As a result, we present here the major microorganisms among all the ones that have been identified in previous studies. This database will aid researchers seeking standardized inoculants to drive cocoa bean fermentation, as well as serve as a guide for inventorying and assessing other food evolution-related studies regarding traditional and artisanal-based food systems.

A novel type 3 secretion system effector, YspI of Yersinia enterocolitica, induces cell paralysis by reducing total focal adhesion kinase.

Some of the world's most important diseases are caused by bacterial pathogens that deliver toxic effector proteins directly into eukaryotic cells using type III secretion systems. The myriad of pathological outcomes caused by these pathogens is determined, in part, by the manipulation of host cell physiology due to the specific activities of individual effectors among the unique suite each pathogen employs. YspI was found to be an effector, delivered by Yersinia enterocolitica Biovar 1B, that inhibits host cell motility. The action of YspI comes about through its specific interaction with focal adhesion kinase, FAK, which is a fulcrum of focal adhesion complexes for controlling cellular motility. The interaction was defined by a specific domain of YspI that bound to the FAK kinase domain. Further examination revealed that YspI-FAK interaction leads to a reduction of FAK steady-state levels without altering its phosphorylation state. This collection of observations and results showed YspI displays unique functionality by targeting the key regulator of focal adhesion complexes to inhibit cellular movement.

Transcriptomic Analysis of Yersinia enterocolitica Biovar 1B Infecting Murine Macrophages Reveals New Mechanisms of Extracellular and Intracellular Survival.

Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocolitica biovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26 °C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37 °C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

CsrA impacts survival of Yersinia enterocolitica by affecting a myriad of physiological activities.

BACKGROUND: A previous study identified a Yersinia enterocolitica transposon mutant, GY448, that was unable to export the flagellar type three secretion system (T3SS)-dependent phospholipase, YplA. This strain was also deficient for motility and unable to form colonies on Lauria-Bertani agar medium. Preliminary analysis suggested it carried a mutation in csrA. CsrA in Escherichia coli is an RNA-binding protein that is involved in specific post-transcriptional regulation of a myriad of physiological activities. This study investigated how CsrA affects expression of the flagellar regulatory cascade that controls YplA export and motility. It also explored the effect of csrA mutation on Y. enterocolitica in response to conditions that cue physiological changes important for growth in environments found both in nature and the laboratory. RESULTS: The precise location of the transposon insertion in GMY448 was mapped within csrA. Genetic complementation restored disruptions in motility and the YplA export phenotype (Yex), which confirmed this mutation disrupted CsrA function. Mutation of csrA affected expression of yplA and flagellar genes involved in flagellar T3SS dependent export and motility by altering expression of the master regulators flhDC. Mutation of csrA also resulted in increased sensitivity of Y. enterocolitica to various osmolytes, temperatures and antibiotics. CONCLUSIONS: The results of this study reveal unique aspects of how CsrA functions in Y. enterocolitica to control its physiology. This provides perspective on how the Csr system is susceptible to adaptation to particular environments and bacterial lifestyles.

Yersinia enterocolitica inhibits Salmonella enterica serovar Typhimurium and Listeria monocytogenes cellular uptake.

Yersinia enterocolitica biovar 1B employs two type three secretion systems (T3SS), Ysa and Ysc, which inject effector proteins into macrophages to prevent phagocytosis. Conversely, Salmonella enterica serovar Typhimurium uses a T3SS encoded by Salmonella pathogenicity island 1 (SPI1) to actively invade cells that are normally nonphagocytic and a second T3SS encoded by SPI2 to survive within macrophages. Given the distinctly different outcomes that occur with regard to host cell uptake of S. Typhimurium and Y. enterocolitica, we investigated how each pathogen influences the internalization outcome of the other. Y. enterocolitica reduces S. Typhimurium invasion of HeLa and Caco-2 cells to a level similar to that observed using an S. Typhimurium SPI1 mutant alone. However, Y. enterocolitica had no effect on S. Typhimurium uptake by J774.1 or RAW264.7 macrophage-like cells. Y. enterocolitica was also able to inhibit the invasion of epithelial and macrophage-like cells by Listeria monocytogenes. Y. enterocolitica mutants lacking either the Ysa or Ysc T3SS were partially defective, while double mutants were completely defective, in blocking S. Typhimurium uptake by epithelial cells. S. Typhimurium encodes a LuxR homolog, SdiA, which detects N-acylhomoserine lactones (AHLs) produced by Y. enterocolitica and upregulates the expression of an invasin (Rck) and a putative T3SS effector (SrgE). Two different methods of constitutively activating the S. Typhimurium SdiA regulon failed to reverse the uptake blockade imposed by Y. enterocolitica.

New in situ capture quantitative (real-time) reverse transcription-PCR method as an alternative approach for determining inactivation of Tulane virus.

Human noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome by in situ qRT-PCR. We first demonstrated that this in situ capture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50 assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively recover and detect the viruses, as there is no need to extract viral RNA or to transfer the captured virus from magnetic beads to PCR tubes for further amplification. Therefore, ISC-qRT-PCR can be easily adapted for use in automated systems for multiple samples.

In vitro embryotoxicity and mode of antibacterial mechanistic study of gold and copper nanoparticles synthesized from Angelica keiskei (Miq.) Koidz. leaves extract.

The present study demonstrated the in vitro embryotoxicity assessment of gold nanoparticles (AuNPs) and copper nanoparticles (CuNPs) prepared from the leaves extract of Angelica keiskei (Miq.) Koidz. and addressed their mode of antibacterial mechanisms. Both AuNPs and CuNPs were rapidly synthesized and the formations were observed within 1 h and 24 h, respectively. Further the morphological images of the nanoparticles were confirmed through transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). The high-resolution X-ray diffraction (HR-XRD) analysis of the biosynthesized AuNPs and CuNPs were matched with joint committee on powder diffraction standards (JCPDS) file no of 04-0784 and 89-5899, respectively. A strong prominent Au and Cu signals were observed through energy dispersive spectroscopy (EDS) analysis. Fourier transform infrared spectroscopy (FT-IR) analysis confirmed the responsible phytochemicals for the synthesis of AuNPs and CuNPs. In order to assess the toxic effects of AuNPs and CuNPs, bactericidal activity was performed against few of the test pathogens in which the effective inhibition was observed against Gram-negative bacteria than the Gram-positive bacteria. The mode of action and interaction of nanoparticles were performed on the bacterial pathogens and the results concluded that the interaction of nanoparticles initially initiated on the surface of the cell wall adherence followed by ruptured the cells and caused the cell death. In addition to the antibacterial activity, in vitro embryotoxicity studies were performed against zebrafish embryos and the results confirmed that 200 µg/ml concentration of AuNPs showed the embryotoxicity, whereas 2 µg/ml of CuNPs resulted the embryotoxicity. Furthermore, the morphological anomalies of zebrafish embryos revealed the toxic nature of the synthesized nanoparticles.

Quantitative Imaging of Bacteriophage Amplification for Rapid Detection of Bacteria in Model Foods.

Rapid detection of bacteria in water and food samples is a critical need. The current molecular methods like real-time PCR can provide rapid detection after initial enrichment. However, these methods require significant preparation steps, specialized facilities to reduce contamination, and relatively expensive reagents. This study evaluates a novel approach for detecting bacteria based on imaging of bacteriophage amplification upon infection of the target host bacteria to mitigate some of these constraints and improve the specificity of discriminating live vs. dead bacteria. Thus, this research leverages the natural ability of lytic bacteriophages to rapidly amplify their genetic material and generate progeny phages upon infecting the host bacterium. This study uses a nucleic acid staining dye, a conventional fluorescence microscope, and quantitative image analysis for imaging the amplification of bacteriophages. The sensitivity and assay time for imaging-based quantification of phage amplification for detecting Escherichia coli were compared with RT-PCR and the standard plaque-forming assay for detection phage amplification in model systems, including coconut water and spinach wash water. The results demonstrate that the imaging approach matches both the sensitivity and speed for detecting E. coli using the RT-PCR method without requiring isolation of nucleic acids, expensive reagents, and specialized facilities. The quantitative imaging results demonstrate the detection of 10 CFU/ml of E. coli in coconut water and simulated spinach wash water with a chemical oxygen demand (COD) of 3,000 ppm within 8 h, including initial enrichment of the bacteria. In summary, the results of this study illustrate a novel phage amplification-based approach for detecting target bacteria in complex food and water samples using simple sample preparation methods and low-cost reagents.

Application of Engineered Bacteriophage T7 in the Detection of Bacteria in Food Matrices.

Detection of pathogens in a food matrix is challenging due to various constraints including complexity and the cost of sample preparation for microbial analysis from food samples, time period for the detection of pathogens, and high cost and specialized resources required for advanced molecular assays. To address some of these key challenges, this study illustrates a simple and rapid colorimetric detection of target bacteria in distinct food matrices, including fresh produce, without prior isolation of bacteria from a food matrix. This approach combines bacteriophage-induced expression of an exogenous enzyme, alkaline phosphatase, the specific colorimetric substrate that generates insoluble color products, and a simple filtration method to localize the generation of colored signal. Using this approach, this study demonstrates the specific detection of inoculated Escherichia coli in coconut water and baby spinach leaves. Without isolating bacteria from the selected food matrices and using a food sample size that is representative of industrial samples, the inoculated samples were added to the enrichment broth for a short period (5 h) and incubated with an engineered bacteriophage T7 with a phoA gene. The incubation period with the engineered bacteriophage was 30 min for liquid samples and 2 h for fresh produce samples. The samples were then filtered through a 0.2-micron polycarbonate membrane and incubated with a colorimetric substrate, i.e., nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). This substrate forms a dark purple precipitate upon interactions with the released enzyme on a filter membrane. This approach successfully detected 10 CFU/ml of E. coli in coconut water and 102 CFU/g of E. coli on baby spinach leaves with 5 h of enrichment. Success of this approach illustrates potential for detecting target bacteria in food systems using a simple visual assay and/or quantitative colorimetric measurements.

Salmonella enterica serovar Typhimurium can detect acyl homoserine lactone production by Yersinia enterocolitica in mice.

LuxR-type transcription factors detect acyl homoserine lactones (AHLs) and are typically used by bacteria to determine the population density of their own species. Escherichia coli and Salmonella enterica serovar Typhimurium cannot synthesize AHLs but can detect the AHLs produced by other bacterial species using the LuxR homolog, SdiA. Previously we determined that S. Typhimurium did not detect AHLs during transit through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during transit through turtles colonized by Aeromonas hydrophila, leading to the hypothesis that SdiA is used for detecting the AHL production of other pathogens. In this report, we determined that SdiA is activated during the transit of S. Typhimurium through mice infected with the AHL-producing pathogen Yersinia enterocolitica. SdiA is not activated during transit through mice infected with a yenI mutant of Y. enterocolitica that cannot synthesize AHLs. However, activation of SdiA did not confer a fitness advantage in Yersinia-infected mice. We hypothesized that this is due to infrequent or short interactions between S. Typhimurium and Y. enterocolitica or that the SdiA regulon members do not function in mice. To test these hypotheses, we constructed an S. Typhimurium strain that synthesizes AHLs to mimic a constant interaction with Y. enterocolitica. In this background, sdiA(+) S. Typhimurium rapidly outcompetes the sdiA mutant in mice. All known members of the sdiA regulon are required for this phenotype. Thus, all members of the sdiA regulon are functional in mice.

Contribution of BlaA and BlaB beta-lactamases to antibiotic susceptibility of Yersinia enterocolitica biovar 1B.

Highly pathogenic Yersinia enterocolitica biovar 1B produces two distinct beta-lactamases, BlaA and BlaB. Mutants of a representative biovar 1B isolate were constructed and evaluated to determine the extent of limitation of susceptibility to broad-spectrum beta-lactam antibiotics by BlaA and BlaB. The results demonstrated that BlaA, a class A enzyme, plays a significant role in limiting susceptibility to penicillins and cephalosporins. The contribution of BlaB, a class C enzyme, was less profound and was limited primarily to cephalosporin susceptibility.

Rapid detection of Escherichia coli using bacteriophage-induced lysis and image analysis.

Rapid detection of bacterial pathogens is a critical unmet need for both food and environmental samples such as irrigation water. As a part of the Food safety Modernization Act (FSMA), The Produce Safety rule has established several requirements for testing for the presence of generic Escherichia coli in water, but the current method available for testing (EPA M1603) demands specified multiple colony verification and highly trained personnel to perform these tests. The purpose of the study was to assess a phage induced bacterial lysis using quantitative image analysis to achieve rapid detection of E. coli at low concentrations within 8 hours. This study aimed to develop a simple yet highly sensitive and specific approach to detect target bacteria in complex matrices. In the study, E. coli cells were first enriched in tryptic soy broth (TSB), followed by T7 phage induced lysis, concentration, staining and fluorescent imaging. Image analysis was conducted including image pre-processing, image segmentation and quantitatively analysis of cellular morphological features (area, eccentricity and full width at half maximum). Challenge experiments using realistic matrices, including simulated fresh produce wash water, coconut water and spinach wash water, demonstrated the method can be applied for use in situations that occur in food processing facilities. The results indicated E. coli cells that are lysed by T7 phages demonstrated significantly (P < 0.05) higher extracellular DNA release, altered cellular shape (from rod to circular) and diffused fluorescent signal intensity. Using this biosensing strategy, a sensitivity to detect Escherichia coli at 10 CFU/ml within 8 hours was achieved, both in laboratory medium and in complex matrices. The proposed phage based biosensing strategy enables rapid detection of bacteria and is applicable to analysis of food systems. Furthermore, the steps involved in this assay can be automated to enable detection of target bacteria in food facilities without extensive resources.

Culturable bacteria resident on lettuce might contribute to accumulation of human noroviruses.

Human noroviruses (HuNoVs) are the primary non-bacterial pathogens causing acute gastroenteritis worldwide. Attachment and invasion of HuNoVs are thought to involve histo-blood group antigens (HBGAs). Romaine lettuce, which is usually consumed raw, is a common food-related vehicle for HuNoVs transmission. This study investigated the possibility that bacteria resident on the surface of lettuce leaves contribute to norovirus adherence to this food. To test this hypothesis, bacteria were isolated from romaine lettuce and screened to evaluate whether they produced any polysaccharides with structures resembling HBGAs. Twenty-seven bacterial isolates were screened and 18, belonging to 13 different genera, were found to produce HBGAs-like polysaccharides that were recognized by monoclonal antibodies specific to type A, B, H and Lewis a, b, x and y. One bacterial isolate, belonging to the genus Pseudomonas was further investigated because it produced polysaccharides with the widest range of HBGA types, including type B, H and Lewis a, b and x. The Pseudomonas HBGAs-like polysaccharides were found to be extracellular and their production was enhanced when the bacteria were cultured in oligotrophic medium. HuNoVs capture assays revealed that GI.1, GI.8, and GII.2, GII.3, GII.4, GII.6, GII.12, GII.17 genotypes can be bind to Pseudomonas HBGAs-like polysaccharides. The direct evidence of bacterial production HBGAs-like polysaccharides demonstrates one possible mechanism driving accumulation of HuNoVs on lettuce.

Essential role of the SycP chaperone in type III secretion of the YspP effector.

The Ysa type III secretion (T3S) system enhances gastrointestinal infection by Yersinia enterocolitica bv. 1B. One effector protein targeted into host cells is YspP, a protein tyrosine phosphatase. It was determined in this study that the secretion of YspP requires a chaperone, SycP. Genetic analysis showed that deletion of sycP completely abolished the secretion of YspP without affecting the secretion of other Ysps by the Ysa T3S system. Analysis of the secretion and translocation signals of YspP defined the first 73 amino acids to form the minimal region of YspP necessary to promote secretion and translocation by the Ysa T3S system. Function of the YspP secretion/translocation signals was dependent on SycP. Curiously, when YspP was constitutively expressed in Y. enterocolitica bv. 1B, it was recognized and secreted by the Ysc T3S system and the flagellar T3S system. In these cases, the first 21 amino acids were sufficient to promote secretion, and while SycP did enhance secretion, it was not essential. However, neither the Ysc T3S system nor the flagellar T3S system translocated YspP into mammalian cells. This supports a model where SycP confers secretion/translocation specificities for YspP by the Ysa T3S system. A series of biochemical approaches further established that SycP specifically interacts with YspP and protected YspP degradation in the cell prior to secretion. Collectively, the evidence suggests that YspP secretion by the Ysa T3S system is a posttranslational event.

Chromosome-based genetic complementation system for Xylella fastidiosa.

Xylella fastidiosa is a xylem-limited, gram-negative bacterium that causes Pierce's disease of grapevine. Here, we describe the construction of four vectors that facilitate the insertion of genes into a neutral site (NS1) in the X. fastidiosa chromosome. These vectors carry a colE1-like (pMB1) replicon and DNA sequences from NS1 flanking a multiple-cloning site and a resistance marker for one of the following antibiotics: chloramphenicol, erythromycin, gentamicin, or kanamycin. In X. fastidiosa, vectors with colE1-like (pMB1) replicons have been found to result primarily in the recovery of double recombinants rather than single recombinants. Thus, the ease of obtaining double recombinants and the stability of the resulting insertions at NS1 in the absence of selective pressure are the major advantages of this system. Based on in vitro and in planta characterizations, strains carrying insertions within NS1 are indistinguishable from wild-type X. fastidiosa in terms of growth rate, biofilm formation, and pathogenicity. To illustrate the usefulness of this system for complementation analysis, we constructed a strain carrying a mutation in the X. fastidiosa cpeB gene, which is predicted to encode a catalase/peroxidase, and showed that the sensitivity of this mutant to hydrogen peroxide could be overcome by the introduction of a wild-type copy of cpeB at NS1. Thus, this chromosome-based complementation system provides a valuable genetic tool for investigating the role of specific genes in X. fastidiosa cell physiology and virulence.

Rapid detection of Escherichia coli in beverages using genetically engineered bacteriophage T7.

Foodborne illness due to bacterial contamination is a significant issue impacting public health that demands new technology which is practical to implement by food industry. Detection of bacteria in food products and production facilities is a crucial strategy supporting food safety assessments. Bacteriophages were investigated as a tool for bacterial detection due to their ability to infect specific strain of host bacteria in order to improve sensitivity, specificity, and rapidity of bacterial detection. The results of this investigation reveal a novel method for rapid detection. The method employs a genetically engineered bacteriophage, phage T7-ALP, which expresses alkaline phosphatase. Upon infection of Escherichia coli, overexpression of alkaline phosphatase provides an opportunity for rapid sensitive detection of a signal indicative of bacterial presence in model beverage samples as low as 100 bacteria per gram. The method employs a fluorescent precipitated substrate, ELF-97, as a substrate for alkaline phosphatase activity coupled with fluorescence imaging and image analysis allowing single-cell imaging results in high detection sensitivity. The method is easily completed within less than 6 h enabling it to be deployed within most large industrial food processing facilities that have routine 8-h operational shifts.

Loss of flagellum-based motility by Listeria monocytogenes results in formation of hyperbiofilms.

Biofilm formation by the gram-positive, motile, food-borne pathogen Listeria monocytogenes was demonstrated to occur by an ordered series of stages. Biofilm development involves flagellum-based motility, which when blocked decreases initial bacterial surface attachment but subsequently leads to the formation of hyperbiofilms, surface-attached communities reaching high density.

Effect of flagellar mutations on Yersinia enterocolitica biofilm formation.

Yersinia enterocolitica biovar 1B is one of a number of strains pathogenic to humans in the genus Yersinia. It has three different type III secretion systems, Ysc, Ysa, and the flagella. In this study, the effect of flagella on biofilm formation was evaluated. In a panel of 31 mutant Y. enterocolitica strains, we observed that mutations that abolish the structure or rotation of the flagella greatly reduce biofilm formation when the bacteria are grown under static conditions. These results were further evaluated by assessing biofilm formation under continuous culture using a flow cell chamber. The results confirmed the important contribution of flagella to the initiation of biofilm production but indicated that there are differences in the progression of biofilm development between static growth and flow conditions. Our results suggest that flagella play a critical role in biofilm formation in Y. enterocolitica.