The complex multicellular morphology of the food spoilage bacteria Brochothrix thermosphacta strains isolated from ground chicken.

Herein we describe a highly structured, filamentous growth phenotype displayed by an isolate of the food spoilage microorganism Brochothrix thermosphacta. The growth morphology of this B. thermosphacta strain (strain BII) was dependent on environmental factors such as the growth media, incubation temperatures, and the inoculum concentration. Inoculation of cultures in highly dilute suspensions resulted in the formation of isolated, tight aggregates resembling fungal growth in liquid media. This same strain also formed stable, mesh-like structures in 6-well tissue culture plates under specific growth conditions. The complex growth phenotype does not appear to be unique to strain BII but was common among B. thermosphacta strains isolated from chicken. Light and electron micrographs showed that the filaments of multiple BII cells can organize into complex, tertiary structures resembling multistranded cables. Time-lapse microscopy was employed to monitor the development of such aggregates over 18 h and revealed growth originating from short filaments into compact ball-like clusters that appeared fuzzy due to protruding filaments or cables. This report is the first to document this complex filamentous growth phenotype in a wild-type bacterial isolate of B. thermosphacta.

Complete Genome Sequences of Two Strains of the Meat Spoilage Bacterium Brochothrix thermosphacta Isolated from Ground Chicken.

Brochothrix thermosphacta is an important meat spoilage bacterium. Here we report the genome sequences of two strains of B. thermosphacta isolated from ground chicken. The genome sequences were determined using long-read PacBio single-molecule real-time (SMRT) technology and are the first complete genome sequences reported for B. thermosphacta.

Genome amplification and promoter mutation expand the range of csgD-dependent biofilm responses in an STEC population.

Expression of the major biofilm components of E. coli, curli fimbriae and cellulose, requires the CsgD transcription factor. A complex regulatory network allows environmental control of csgD transcription and biofilm formation. However, most clinical serotype O157 : H7 strains contain prophage insertions in the csgD regulator, mlrA, or mutations in other regulators that restrict csgD expression. These barriers can be circumvented by certain compensating mutations that restore higher csgD expression. One mechanism is via csgD promoter mutations that switch sigma factor utilization. Biofilm-forming variants utilizing RpoD rather than RpoS have been identified in glycerol freezer stocks of the non-biofilm-forming food-borne outbreak strain, ATCC 43894. In this study we used whole genome sequencing and RNA-seq to study genotypic and transcriptomic differences between those strains. In addition to defining the consequences of the csgD promoter switch and identifying new csgD-controlled genes, we discovered a region of genome amplification in our laboratory stock of 43894 (designated 43894OW) that contributed to the regulation of csgD-dependent properties.

Multiple mechanisms responsible for strong Congo-red-binding variants of Escherichia coli O157:H7 strains.

High variability in the expression of csgD-dependent, biofilm-forming and adhesive properties is common among Shiga toxin-producing Escherichia coli. Although many strains of serotype O157:H7 form little biofilm, conversion to stronger biofilm phenotypes has been observed. In this study, we screened different strains of serotype O157:H7 for the emergence of strong Congo-red (CR) affinity/biofilm-forming properties and investigated the underlying genetic mechanisms. Two major mechanisms which conferred stronger biofilm phenotypes were identified: mutations (insertion, deletion, single nucleotide change) in rcsB region and stx-prophage excision from the mlrA site. Restoration of the native mlrA gene (due to prophage excision) resulted in strong biofilm properties to all variants. Whereas RcsB mutants showed weaker CR affinity and biofilm properties, it provided more possibilities for phenotypic presentations through heterogenic sequence mutations.

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

Rapid identification and classification of Campylobacter spp. using laser optical scattering technology.

Campylobacter jejuni and Campylobacter coli are the two important species responsible for most of the Campylobacter infections in humans. Reliable isolation and detection of Campylobacter spp. from food samples are challenging due to the interferences from complex food substances and the fastidious growth requirements of this organism. In this study, a novel biosensor-based detection called BARDOT (BActerial Rapid Detection using Optical scattering Technology) was developed for high-throughput screening of Campylobacter colonies grown on an agar plate without disrupting the intact colonies. Image pattern characterization and principal component analysis (PCA) of 6909 bacterial colonies showed that the light scatter patterns of C. jejuni and C. coli were strikingly different from those of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. Examination of a mixed culture of these microorganisms revealed 85% (34/40) accuracy in differentiating Campylobacter from the other three major foodborne pathogens based on the similarity to the scatter patterns in an established library. The application of BARDOT in real food has been addressed through the analysis of Campylobacter spiked ground chicken and naturally contaminated fresh chicken pieces. Combined with real-time PCR verification, BARDOT was able to identify Campylobacter isolates from retail chicken. Moreover, applying passive filtration to food samples facilitated the isolation of pure Campylobacter colonies and therefore overcame the interference of the food matrix on BARDOT analysis.

Peroxide resistance in Escherichia coli serotype O157 : H7 biofilms is regulated by both RpoS-dependent and -independent mechanisms.

In many Escherichia coli serotype O157 : H7 strains, defences against peroxide damage include the peroxiredoxin AhpCF and three catalases: KatG (catalase/peroxidase), KatE (catalase) and the plasmid-encoded KatP (catalase/peroxidase). AhpC and KatG basal expression is maintained by RpoS, and AhpC, KatG and KatP are all induced by OxyR/σ(70) in exponential phase. KatE is regulated by RpoS during stationary growth and is independent of OxyR. In a previous study we used mutant strains of ATCC 43895 (EDL933) with deletions of katG, ahpC, katE and katP in all possible combinations to characterize peroxide resistance during both exponential and 18-24 h growth in Luria-Bertani broth at 37 °C. In this study, we used triple deletion strains that isolated each catalase/peroxidase gene to investigate their role in the peroxide resistance of biofilm-forming variant 43895OR in 48 and 72 h biofilms. We also used quantitative real-time reverse transcriptase PCR and translational lacZ fusions to study gene expression. Peroxide resistance was greater (P<0.05) in biofilm cells than in planktonic cells, and full resistance required rpoS but not oxyR. In 72 h biofilms, katG and katE were the major protective genes. katG, ahpC and katE peroxide protection had both rpoS-dependent and rpoS-independent components, but katP protection was independent of rpoS. H(2)O(2) challenge induced (P<0.05) katG, ahpC and katP expression in biofilm cells, suggesting that peroxide induction of the OxyR-dependent resistance genes may contribute to the RpoS-independent protection in Shiga toxin-producing E. coli biofilms.

A method for correcting standard-based real-time PCR DNA quantitation when the standard's polymerase reaction efficiency is significantly different from that of the unknown's.

Standard-based real-time or quantitative polymerase chain reaction quantitation of an unknown sample's DNA concentration (i.e., [DNA](unk)) assumes that the concentration dependence of the standard and unknown reactions (related to reaction efficiency, E) are equivalent. In our work with background food-borne organisms which can interfere with pathogen detection, we have found that it is generally possible to achieve an acceptable E (1 ± 0.05) for standard solutions by optimizing the PCR conditions, template purity, primer sequence, and amplicon lengths. However, this is frequently not true for the solutions containing unknown amounts of target DNA inasmuch as cell extracts are more chemically complex than the standards which have been amplified (2(30)-fold) as well as undergone a purification process. When significant differences in E occur, it is not possible to accurately estimate unknown target DNA concentration from the standard solution's slope and intercept (from threshold cycle number, or C(T), versus Log[DNA] data). What is needed is a standard-mediated intercept which can be specifically coupled with an unknown solution's PCR concentration dependence. In this work, we develop a simple mathematical procedure to generate a new standard curve with a slope (∂C(T)/∂Log[Dilution](unk)) derived from at least three dilutions of the unknown target DNA solution ([DNA](unk)) and an intercept calculated from the unknown's C(T)s, DNA concentrations interpolated from the standard curve (i.e., the traditional estimate of [DNA](unk)), and ∂C(T)/∂Log[Dilution](unk). We were able to achieve this due to our discovery of the predictable way in which the observed and ideal C(T) versus Log[DNA] slopes and intercepts deviate from one another. This "correction" in the standard-based [DNA](unk) determination is typically 20-60% when the difference in the standard and unknown E is >0.1.

Keratin capped silver nanoparticles--synthesis and characterization of a nanomaterial with desirable handling properties.

We report for the first time the stabilization of silver nanoparticles in good yield, average diameter 3.5 nm, using wool keratin hydrolysates as stabilizers. The nanoparticles are extremely stable as a suspension and can be lyophilized into a powder and easily reconstituted in solvent with no change in spectral properties relative to the initial suspension. The nanoparticles interact with nitrogen and oxygen moieties of the keratin hydrolysates under the pH conditions used in the synthesis and appear to act as cross-linkers between adjacent chains. The product has excellent handling properties which we believe will make it a very attractive biocompatible coating/additive, providing prolonged antimicrobial efficacy to a wide variety of products such as textiles, plastics, paints, orthopedic devices and others.

Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni.

The antibacterial effect of zinc oxide (ZnO) nanoparticles on Campylobacter jejuni was investigated for inhibition and inactivation of cell growth. The results showed that C. jejuni was extremely sensitive to treatment with ZnO nanoparticles. The MIC of ZnO nanoparticles for C. jejuni was determined to be 0.05 to 0.025 mg/ml, which is 8- to 16-fold lower than that for Salmonella enterica serovar Enteritidis and Escherichia coli O157:H7 (0.4 mg/ml). The action of ZnO nanoparticles against C. jejuni was determined to be bactericidal, not bacteriostatic. Scanning electron microscopy examination revealed that the majority of the cells transformed from spiral shapes into coccoid forms after exposure to 0.5 mg/ml of ZnO nanoparticles for 16 h, which is consistent with the morphological changes of C. jejuni under other stress conditions. These coccoid cells were found by ethidium monoazide-quantitative PCR (EMA-qPCR) to have a certain level of membrane leakage. To address the molecular basis of ZnO nanoparticle action, a large set of genes involved in cell stress response, motility, pathogenesis, and toxin production were selected for a gene expression study. Reverse transcription-quantitative PCR (RT-qPCR) showed that in response to treatment with ZnO nanoparticles, the expression levels of two oxidative stress genes (katA and ahpC) and a general stress response gene (dnaK) were increased 52-, 7-, and 17-fold, respectively. These results suggest that the antibacterial mechanism of ZnO nanoparticles is most likely due to disruption of the cell membrane and oxidative stress in Campylobacter.

Evidence for a bimodal distribution of Escherichia coli doubling times below a threshold initial cell concentration.

BACKGROUND: In the process of developing a microplate-based growth assay, we discovered that our test organism, a native E. coli isolate, displayed very uniform doubling times (tau) only up to a certain threshold cell density. Below this cell concentration (100 CFU mL-1), the tau values were distributed unimodally (mutau = 18 +/- 0.71 min; n = 174). Inclusion of a small amount of ethyl acetate to the LB caused a collapse of the bimodal to a unimodal form. Comparable bimodal tau distribution results were also observed using E. coli cells diluted from mid-log phase cultures. Similar results were also obtained when using either an E. coli O157:H7 or a Citrobacter strain. When sterile-filtered LB supernatants, which formerly contained relatively low concentrations of bacteria(1,000-10,000 CFU mL-1), were employed as a diluent, there was an evident shift of the two populations towards each other but the bimodal effect was still apparent using either stationary or log phase cells. CONCLUSION: These data argue that there is a dependence of growth rate on starting cell density.

The relationship between purely stochastic sampling error and the number of technical replicates used to estimate concentration at an extreme dilution.

For any analytical system the population mean (µ) number of entities (e.g., cells or molecules) per tested volume, surface area, or mass also defines the population standard deviation (σ = square root(µ)). For a preponderance of analytical methods, σ is very small relative to µ due to their large limit of detection (>10(2) per volume). However, in theory at least, DNA-based detection methods (real-time, quantitative or qPCR) can detect ≈ 1 DNA molecule per tested volume (i.e., µ ≈ 1) whereupon errors of random sampling can cause sample means (mean) to substantially deviate from µ if the number of samplings (n), or "technical replicates", per observation is too small. In this work the behaviors of two measures of sampling error (each replicated fivefold) are examined under the influence of n. For all data (µ = 1.25, 2.5, 5, 7.5, 10, and 20) a large sample of individual analytical counts (x) were created and randomly assigned into N integral-valued sub-samples each containing between 2 and 50 repeats (n) whereupon N × n = 322 to 361. From these data the average µ-normalized deviation of σ from each sub-sample's standard deviation estimate (s(j), j = 1 to N; N = 7 [n = 50] to 180 [n = 2]) was calculated (Δ). Alternatively, the average µ-normalized deviation of µ from each sub-sample's mean estimate (mean(j)) was also evaluated (Δ'). It was found that both of these empirical measures of sampling error were proportional to ⁻²âˆšn . µ. Derivative (∂/∂n · Δ or Δ') analyses of our results indicate that a large number of samplings (n ≈ 33 +/- 3.1) are requisite to achieve a nominal sampling error for samples with a µ ≈ 1. This result argues that pathogen detection is most economically performed, even using highly sensitive techniques such as qPCR, when some form of organism cultural enrichment is utilized and which results in a binomial response. Thus, using a specific gene PCR-based (+ or -) most probable number (MPN) assay one could detect anywhere from 0.2 to 10(5) CFU mL(-1) using 6 to 48 reactions (i.e., 8 dilutions × 6 replicates per dilution) depending on the initial concentration of the pathogen and volume sampled.

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7.

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 x 10(5) to 1 x 10(6) of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.

Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157:H7.

E. coli O157:H7 is a pathogenic microorganism that has been implicated in numerous cases of foodborne illnesses. A variety of rapid methods exist that show promise for the presumptive detection of this pathogen without the immediate need for incubating test samples for hours to days in microbial enrichment and culture media. In recent years, highly sensitive chemiluminescence has become a more affordable and portable detection method. Chemiluminescent detection has been coupled with the selectivity of antibodies, magnetic microparticle separation/isolation, and enzymatic signal amplification in order to develop a rapid method, termed enzyme-linked immunomagnetic chemiluminescence (ELIMCL). This work presents the application of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline with a detection limit of 7.6 x 10(3) for live cells in approx. 75 min assay time. The blocking agent casein and the surfactant Tween 20 were used to lower background luminescence and thus maximize signal-to-noise ratios. After 5.5 h of enrichment culture, ELIMCL was demonstrated to detect E. coli O157:H7 inoculated in ground beef at 10 CFU/g in a total assay time of about 7 h.

A 6 x 6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli.

A protocol was developed using 96-well plates and multichannel pipettes for serial dilutions, followed by drop plating on agar in a 6 x 6 format. This protocol permits simultaneous plating of six dilutions which greatly decreases the number of plates utilized thereby saving incubator space for organisms such as Campylobacter which require unique environmental conditions.